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  • 2. ACKNOWLEDGEMENTSIt is a matter of great privilege for me to work under the able and highly exceptionalguidance of Dr. R. Sreekumar, former Head of the department, Department of RogaNidana, Govt. Ayurveda College Kannur, who has nurtured my capabilities andalways gave me ample freedom. His energetic smile, optimistic attitude, criticaljudgments and above all trust in my abilities kept me going and ultimately towardsthe attainment of this goal. No less had been the incessant help and constant encouragement providedby my co-guide Dr. Moinak Banerjee, Scientist – E1, Human Molecular Genetics lab,Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram during the course of mystudy. His scholarly guidance, experienced words, meticulous practical training,disciplined and virtuous nature, unlimited sincerity and affection towards studentshad always inspired me and I feel extremely fortunate to be his student. Words are not enough to express my sincere and heartfelt thanks to Dr. V.N.Parameswaran Potty and Dr. K.V. Subhadra Antherjanam andDr. P.K.V. Anand for their immense help in my studies. Their devotion and dedicationto work, passion for knowledge, inquisitive mind and scientific attitude have made aneverlasting impact on me and boosted my confidence. My special thanks to Dr. K.Sankaran, Director, Ayurveda Medical Education, Dr. John K George, Head of thedepartment, Dr. S. Jayadevan Asst. Professor, department of social and preventivemedicine, Academy of Medical sciences, Pariyaram, Dr. V.K. Ajithkumar, Readerand Dr. S. Gopakumar, Tutor of my department for their kind concern, criticalcomments, wise counseling and timely tips which are invaluable.I adore and respect them for their ‘Never say no’ attitude and magnanimous natureand I owe them a lot. 2
  • 3. Former Director Dr. R.V. Thampan, Director Dr. M. Radhakrishna Pillaiand Scientists from other departments of RGCB and especially to research scholars,Mrs. Resmi Thomas, Miss. Neetha Vijayan, Mrs. Linda Koshi, Mr. Chandrashekharand technical assistants Mrs. Sudha B. Nair, Mrs. Bindu Ashokan, Mr. Antony K.Pand the summer trainee students who attended the HMG lab from variousbiotechnology colleges, for their timely assistance in the lab and their invaluable helpand moral support. I also extend my sincere thanks to my batch mates Dr. Mukesh,Dr. Viswanath, Dr. Sudarkkodi, Dr. Surabhi Mishra, Dr. Senthil Nathan,Dr. Surama Mishra, Dr. Anjali, Dr. Abhilash and Dr. Bindu Mary Mathew and otherP.G scholars of various Ayurveda Colleges for being there, whenever I was in needthem the most and for their constant support and encouragement. Nevertheless, I am not missing out this opportunity to acknowledge andappreciate some of my colleagues and very good friends like Dr. Nandalal,Dr. Sunil Babu, Dr. Madhu, Dr. Prabha Manish, Dr. Gopikrishna, Dr. Annie Yohanan,Dr. Vijay, Dr. Prasanth Kekuda, Dr. Brijula, Dr. Anupama,Dr. Yogeswar Pawale, Dr. Arbind Kumar Jha, Dr. Bindu, Dr. Sudhagopal,Dr. Eby Abraham, Dr. Vibhu Khanna, Dr. Namratha, Dr. Pradeep, Dr. Rejitha, Dr.Vidya Menon, Dr. Sarita Mohanta, Dr. Manas Ranjan Debta, Dr. Priya,Dr. Ananthalekshmi, Dr. Nandakumar Sahni, Dr. Shambhu Sharan, Dr. Sijin, and Dr.Pramod whose infinite love and affection had always strengthened my determination. Words are meaningless to express my unaccountable feelings to my fatherSri. K.V. Neelakandan Namboothiri (late) and mother Smt. C.N. Arya Devi (late);sisters, Mrs. K.N. Anjana Sree, Mrs. K.N. Archana Sree andMs. V.P Aparna; brother in laws Mr. Sivanandan and Mr. Narayanan Namboothitri, 3
  • 4. and nephew Mr. Vijay Sankar and niece Ms. Swathy for their belief in me, kindunderstanding, patience and perseverance and above all in making me what I amtoday. I owe them more than anything else in the world. Special thanks are also dueto my beloved wife Mrs. Shreeja Devi and daughter Ms. Devi Nanda without whosehelp and support, none of my dreams would have ever come true and without whomlife would have never been the same for me. The wealth of information provided by our college library also deservesspecial thanks. Dr. K.N.AJITHKUMAR 4
  • 5. INTRODUCTIONH IS EXCELLENCY THE PRESIDENT OF INDIA Dr. A.P.J. ABDUL KALAM in his Address at the Inauguration of the Govt. Ayurveda College HospitalComplex and Interaction with the Students at Govt. Ayurveda College, Trippunithura,Ernakulam, Kerala on 17th December 2005 talked on the topic “Traditional Medicine:Our Strength”.He pointed out that the traditional system of medicine like Ayurveda, Siddha etc.have advocated and practiced preventive and curative medicinal recipes specific toindividuals. The body, mind, food and environment were looked at holistically tosuggest a preventive or curative approach to health. He also stressed the need forcollaborative research of Ayurveda and biotechnology. Dr. Kalam’s focusing onpersonalized medicine of Ayurvedic system of medicine has invited our attention tothe fact that in this post genomic era, the emerging concept of personalized therapyon the basement of Pharmacogenomics is a re-definition of diseases on themolecular level so that diagnotics and therapeutics can be targeted to specificpatient populations sub-typed on the basis of genetic make-up.India is on a path breaking research and development activities in various areas tocombine the strength of Ayurveda and the power of Biotechnology so as to place thenation in global leadership position in the matter of providing quality traditional healthcare. The National Biotechnology development strategy addresses the utilization ofBiotechnology to add value to our traditional knowledge especially Ayurveda,Siddha, and Unani systems as well as tribal and folk medicine. Medicinal plants arealso prime targets of bioprospecting. Besides, the tool of biotechnology can be usedfor conservation and characterization of plants. Development of plant basedmedicine and mechanism based screening of herbal drugs known in traditional 5
  • 6. Indian system are included in the strategic action of the National Biotechnologypolicy to get value added drugs quickly. For the Inaugural Address at the “DELHISUSTAINABLE DEVELOPMENT SUMMIT 2003” Vigyan Bhavan, New Delhi,February 6, 2002, His Excellency the President of India Dr. A.P.J. Abdul Kalamselected the topic ‘PROSPEROUS – PEACEFUL – SAFE HUMAN HABITAT’. Whilenarrating the utilization of Biotechnology for wealth generation, he also stressed onintegrative research in Ayurveda and biotechnology and stated that newtechnologies as evidenced by human genome sequencing, proteomics,chemogenomics, ultra high throughput screening is revolutionizing drug discovery1.Medicinal plants offer enormous scope for development of drugs and he highlightedthe need to create database of traditional medicine for specific bioactivity and leadfor development of new drugs. He explained that the North Eastern States of Indiahave got tremendous opportunities for herbal farming and research and that there isan integrated relationship between science, technology, environment, manufacturingand the society.Dr. M.S.Valiyathan has also advocated research on valuable traditional knowledgeand basic principles of Ayurveda, and not just on herbal drug development2. Use of modern technology and Biotechnology has been identified byGovt. of India, Department of AYUSH as a thrust area for 10th plan period forstrengthening the base for sustained propagation of Ayurveda. The objectives andgoals of Kerala Biotechnology policy is also designed to catalyze the development ofAyurveda. The policy, among others, aim to apply biotechnology tools to “ boost thestates renowned health care practice of Ayurveda by synergising traditionalknowledge with the scientific validation and technical product profiling and clinicaldata base and by evolving means to conserve and substantially use one of worlds 6
  • 7. most valued biodiversity treasures located in the state.” It also aims at creation of anadvanced multipurpose analytical testing and standardization laboratory approved bythe National Accreditation Board for testing Calibration of Laboratories to cater theneeds of Ayurveda and pharmaceutical industry for meeting international standards.Pharmacogenomics – the study of how an individual’s genetic make up affects thebody’s response to treatment or drug - is a rapidly growing science after thecompletion of Human Genome Project was celebrated in April 2003 and it is adeveloping research field still in it’s infancy. Pharmacogenomics hold the view thatdrugs might one day be “tailor- made” for individuals or small populations based ongenetic make up. Modern medicine has developed based on general principles ofphysiology, pathology diagnotics and treatment were as Ayurveda upholds individualphysiology, pathology, diagnostic and personalized therapy. In this post genomicera, the emerging concept of personalized therapy on the basement ofPharmacogenomics is infact, a re-definition of diseases on the molecular level sothat diagnotics and therapeutics can be targeted to specific patient populations sub-typed on the basis of genetic make-up and there by offer the right treatment for rightpatient population individual. The emerging personalized medicine holds the view that environment, diet,age, lifestyle and state of health all can influence a person’s response to health andthat due to genetic variations a disease may manifest itself slightly differently indifferent types of patients. Understanding the genetic make up of an individual is the key to personalizedtherapy. As, at present, there is only limited knowledge of which genes are involvedin each drug response and that many genes are likely to influence a response andobtaining a correct picture on the effect of genetic polymorphisms are complicated, 7
  • 8. time consuming and expensive, a high speed data mining of both genotypic andphenotypic information is essential for the development of personalized therapy. Inshort, identification and analysis of millions of single gene nucleotide polymorphisms(SNPs) to determine which drug and specific diet are more suitable to a patient isquite impractical. Hence it has become necessary to categorize individuals on thebasis of board phenotypic clusters. Consideration of racial, ethnic and geographicalfactors for classifying phenotypes to be considered collectively for genotyping iscontroversial. Here, Ayurvedic concept of Prakrithi comes in to play. According to Ayurveda, every individual is different from another and henceshould be considered as a different entity. But for diagnostic, prognostic andtherapeutic purposes Ayurveda categorizes human population in to sub-populationssuch as Vatha Prakrithi, Pitha Prakrithi, Kapha Prakrithi or their combinations on thebasis of physical, physiological and psychological characteristics (phenotypes) andcompletely avoid racial, ethnic and geographical considerations. In fact, theaforesaid concept that due to genetic variations, a disease may manifest itselfslightly differently in different types of patients and the same therapy and drug willnot produce equal response in different individuals has well developed thousands ofyears ago in Ayurveda. After analyzing and correlating the varying responses ofindividuals to various factors like drug, food, sleep, sex, exercise, climate,environment, and assessing various physical, physiological and psychologicalcharacteristics (phenotypes) and sub typing the individuals showing similarities in theabove responses and characteristics, the human population is grouped in Ayurvedain to smaller populations and designated as a particular Prakrithi such as VathaPrakrithi, Pitha Prakrithi, Kapha Prakrithi or combinations of either two or three ofthem. Similarly, the body functions, tissues and organs are also sub-typed on the 8
  • 9. same lines. For example, on the basis of the characteristics the pulse (nadi) iscategorized into Vatha, Pitha, Kapha or their combinations. Based on the smallvariations in signs and symptoms exhibited in the above sub-typed populations,diseases especially metabolic disorders are also sub grouped in to Vathapredominant, Pitha predominant, Kapha predominant or combinations of either twoor three of them. Due to various factors, a disease may exhibit signs and symptomsof Vatha/ Pitha/ Kapha/ their combination predominance in an individual irrespectiveof the type of Prakrithi. One of the basic concepts of Ayurveda is that, if a particular disease havingthe symptamotology of Vatha predominant type is occurring to anindividual/population sub-typed as Vatha Prakrithi and dwelling in a place havingVatha predominance and if the disease is occurring in an organ /tissue of Vathapredominance at a time/season having Vatha predominance, the disease isconsidered of having bad prognosis .The Prakrithi predominance of the tissue, place,time and individual is assessed by specific criteria provided under the basicprinciples of Ayurveda. An individual/population is sub-typed into a particularPrakrithi on analyzing the phenotypic characteristics of that individual/population;and a place by analyzing the nature of micro-climatic conditions, elevation, soilconditions, vegetation, water, air and the phenotypic characteristics itself of theplants and animals of that place, and that of time/season is determined by analyzingthe seasonal/timely climatic factors such as solar radiation, atmospheric pressures(direction of wind), humidity, characteristics of clouds, etc, and the body’s responseto the above factors. Modern biotechnology upholds the view that the collectiveinteraction of about 30000 genes give us life and their normal function keeps the 3trillion – cell human being healthy by producing the right proteins where as their 9
  • 10. altered function by mutation due to exposure to harmful external and internalenvironments produce wrong proteins that make disease. In short, if the etiologicalfactors provided by place (geopathological), time (climatic) and organism (metabolicand genetic) are highly potential, co-existing and correlating in terms of Ayurvedicprinciples for manifestation of a disease, the severity of the disease thus manifestedis the most, and the disease is most difficult to cure. The genetic make up of thereceptors or its potential and the receptor sensitivity of various factors pertaining totissue, time and place is an important factor that determines the severity of thedisease. Modern western medicine has developed under the influence ofreductionism-breaking down human organism into smaller levels of such as organ,tissue, cell and gene and focus on the diseased part and often treat to suppress thesymptom to get an immediate result rather than a long term process of correcting themetabolic derangement. As a result, a drug therapy or gene therapy targeting onmodification of activities of a cell or group of cells produces toxicity to others and it isestimated that even in most developed countries out of 3.5 billion annualprescriptions 3 billion produces adverse results and the treatment adopted to onedisease give rise to another disease. With a holistic approach, Ayurveda advocatesthe need for micro analysis of the tissue morbidity, the geopathological factors of thedwelling place, the immunological status, the micro climatic elements and how theyinfluence the metabolic activities, the state of digestive and metabolic fire, thegenetic make-up by analyzing the phenotypic characteristics, the state of body thatdepends on the course of time, the state of psyche, life style, food habits etc of apatient for personalized diagnosis and therapy. 10
  • 11. If the phenotype (outward appearance of an individual as a result ofgenetic make up and environment, i.e. dosha Prakrithi lakshanas) and thecorresponding genotype are correlated by the tools of bio technology, it will not onlyenable to highlight the ever modern principles of the oldest health science, Ayurveda– Puranam cha Punarnavam - but also to generate data to the modern scientificcommunity to classify patients with same phenotypic characteristics into smaller subpopulations defined by genetic variations associated with disease, drug and foodresponse. Such studies will also enable the development of Pharmacogenomics andpersonalized therapy from infancy to child hood more securely and safely in thecradle of Mother Ayurveda. Many scholars and institutions in the field of medicine,biotechnology and policy making are up in arms for research in the field of Ayurvedain the light of modern technology and biotechnology.Dr M S Valiyathan has pointed out the possibility of having genomic counter parts for‘dosha Prakrithi’ mentioned in Ayurveda3. The National Bio-technology Policy stress that the emerging disciplineof Pharmacogenomics combines both InfoTech and BioTech skills in augmentinghigh speed data mining of both phenotypic and genotypic information from welldifferentiated patient populations with a view to evolving new forms of diagnotics andtherapies of personalized medicine. Institute of Genomics and Integrated Biology,one of the premier laboratories under the Council of Scientific and IndustrialResearch, Govt. of India is currently working on the project: “Genotype PhenotypeCorrelation based on the principles of Ayurveda with special focus on Prakrithi”. A perusal of the observations and opinions of profound scholars,research project initiated and proposed to initiate at various national and inter-national institutions in the fields of medicine, bio technology and policy- making 11
  • 12. attests that my present study which aims to analyze the role of DNA in determiningPrakrithi by conducting Polymerize Chain Reaction studies, to understand thegenetic correlation of various forms of Prakrithi and to develop standards for auniform guide line in analyzing Prakrithi is relevant in the Post Genomic Era and itwill add flavour to all the on going research process for the correlation of Ayur Tech,Bio Tech and Info Tech. 12
  • 13. PRAKRITHI - A MOLECULAR APPROACHMicrobes developed in toxins become resistant to a considerable extend to thattoxin1. Similarly, zygote developed in a particular environment also become resistantto a considerable extends to that environment. Here the environment of the zygote isan energic template – dosha sthithi2 – emitted and maintained unchanged throughout the span of life of an individual (Janma maranaantharala varthini nirvikarini) bythe interaction of the germ cell genotype (Sukla – Sonitha Prakrithi), climatic anduterine environment (Kaala garbhasaya Prakrithi), the nature of mothers food andpsychophysical activities (Mathraahara vihara Prakrithi) and the nature of Panchabhoothas of the biosphere (Maha bhootha vikara Prakrithi) that are in operation atthe time of fertilization3. The energic template – dosha sthithi – is termed as‘Prakrithi’ that transmits various physical, physiological and psychological phenotypiccharacteristics considered as normal, when compared in relation with the abnormalcharacteristics that constitute the hereditary diseases or Aadi bala pravritha roga.The energic template – dosha sthithi – has two states – normal (Praakritha) andabnormal (Vaikritha). Dalhana in Garbha vyakarana sareera of Susrutha samhitha(verse 63) states that the normal energic template is the cause for Prakrithi whereasthe abnormal energic template causes genetic disorders. The effect of abnormalenergic template in causing deformity is explained on the analogy of the effect of theforce of flood. During rainy season a system of over flowing river water, theembedded woods, rocks and surrounding land is set into action. What variations theresultant force of flood will cause to the shape and size of a tree on the bank that fallinto the river cannot be predicted even if the general behavior of the river, the natureof the embedded woods and rocks and the flood plain are well assessed. Similarly,even though the nature of interacting factors such as the germ cell genotype (Sukla 13
  • 14. – Sonitha Prakrithi), climatic and uterine environment (Kaala garbhasaya Prakrithi),the nature of mothers food and psychophysical activities (Mathraahara viharaPrakrithi) and the nature of Pancha bhoothas of the biosphere (Maha bhootha vikaraPrakrithi) are well assessed, what variation the resultant abnormal energic template(Vaikritha dosha sthithi) will cause to the phenotypic characteristics of the zygotecannot be predicted, says Charaka in Athulya Gothreeya sareera (verse 30).Charaka considers the zygote as ‘Chatushpadi4’ because it is developed from thefirst four among Pancha bhoothas – Prithwi, Ap, Thejus and Vayu derived from foursources – 1. Sperm (sukra) 2. Ovum (sonitha) 3. Nutrient fluid (rasa) and 4.‘Athma’.Abnormal state of any of the four factors derived from the aforesaid four differentsources may render the zygote abnormal the effect of which will be reflected in theoffspring. “Bija” (Sperm & Ovum) is a collection of functional units, which byrepresenting the structures and features of all parts and organs of an individual iscapable of ensuring the formation of offspring resembling the parents4a. Suchfunctional units are called “Bijabhagas”, which can be more or less similar to the term“Gene” in modern genetics. Hairs on the head, mustaches, hairs on the body, bone,nails, teeth, veins, ligaments, arteries, semen etc, which are stable, have paternaldominance. Muscles, blood, fat, bone marrow, heart, umbilicus, liver, spleen,intestines, anus etc that are soft have maternal dominance. Growth of the body,strength, colour of the body, existence of the body in normalcy and its destruction(decay) are mainly determined by factors derived from rasa dhathu (nutrients frommothers diet). The four factors derived from Athma, are in a very subtle state so thatonly the yogis can perceive it5. Vagbhata explains the transmission of such factorsassociated with Athma to the zygote in the uterus on the analogy of transmission ofenergy from solar radiation through a magnifying glass to the target object (cotton, 14
  • 15. paper, firewood etc.)6. The abnormalities in the transmitted factors associated withAthma may cause derangement mainly in sense perception and mental activities inviolation of cause and effect theory and is designated as ‘Karmaja vyadhi’. Thepattern of inheritance of the factors associated with Athma is indriyas, theirreceptivity, general knowledge, special knowledge, Ayus (life span), happiness,misery etc. Indriyas are the effect of interaction of the factors associated with Athmawith those of gametes and nutrients. Indriyas and their channels are in the form ofenergy in the brain and are explained on the analogy of the radiations emitted fromthe sun7. Hence a disease can be manifested as Poorvaparaadhaja (Karmaja), orDrishtaparaadhaja (known etiology such as Sahaja/ Garbhaja/ Jathaja/ Peedakrithaetc) or by the combined effect of both (Sankara roga). Hence diseases manifestedwithout a traceable etiological factor of genetic, nutritional, climatic, traumatic originis to be considered as of Karmaja vyadhi caused by the effect of deranged state ofthe four factors associated with Athma. It is noteworthy to mention that these factorscan induce heritable changes in Bija bhagas (genes). Piles can be seen as ahereditary disorder (Sahaja Arsas)8. Here a heritable change occurs in the gene thatdetermines the development of rectal layers. Vagbhata says that this change can beinduced by two factors:1. Psychophysical and nutritional defects of parents transmitted through gametes(MathaPithraapacharatha) 2. By ‘daivam’, which means that, the invisible factors(Susookshma bhoothas) transmitted through Athma. Charaka points out thepossibility of occurring discordance in monozygotic twins due to the effect of athmajabhava in the distribution of genetic material at the time of duplication and zygotic celldivision9. The both factors are simultaneously considered in Ayurveda for diagnotics,prognotics and therapeutics. 15
  • 16. If the 16 factors10 derived from Sperm (sukra) Ovum (sonitha) Nutrient fluid (rasa)and ‘Athma’ are of normal state, the zygote develops into a normal child. Chart 1: DETERMINANTS OF PRAKRITHI 1 - Mathrja 2 - Pithrja 3 - Rasaja 4 - Athmaja P1 P2 P3 P4 A1 A2 A3 A4 T1 T2 T3 T4 V1 V2 V3 V4 ZYGOTEAgain, the characteristics which we consider as normal and transmitted by differentforms of energic templates can be considered as abnormal, if compared with thecharacteristics of an ideal personality, which infact, is only conceptual as such apersonality can never be seen due to our inability to provide the superiorenvironmental conditions essential for the emergence of such a trait11. In thiscontext, Charaka considers Prakrithi (normal) as Vikrithi (abnormal) and use the termVathala, Pithala, Sleshmala instead of Vatha Prakrithi, Pitha Prakrithi and KaphaPrakrithi. In short, the superior germ cell genotype (Sama Prakritihi) provides healthto the developing embryo (Garbha) and its variations such as Vathala, Pithala,Sleshmala and combinations of either two of them cause varying degrees ofprownness to disease (sadaathura)12. Hence Vatha Prakrithi is considered as Heena 16
  • 17. (bad), Pitha Prakrithi as Madhyama (better) and Kapha Prakrithi as Uthama (best)and combinations of either two of them (dwantwa Prakrithis) as Nindya (worst)13. So,such individuals are always more prown to diseases, and adverse environmentalfactors can easily trigger corresponding diseases to the embryo or to the individualdepending upon the nature of factors involved. Considering the varying states ofgerm cell genotype, the effect of which is reflected in the physical, physiological andpsychological characteristics of the individual (Phenotype), Ayurvedic genomicsmandate that every individual is different from another and hence should beconsidered as a different entity14. But for diagnostic, prognostic and therapeuticpurposes, Ayurveda categorizes human population on the basis of relativesimilarities in the aforesaid phenotypic characteristics in to 7 sub-populations suchas Vatha Prakrithi, Pitha Prakrithi, Kapha Prakrithi or their combinations of two or all.Infact on microanalysis, Vatha (V), Pitha (P) and Kapha (K) have at least 62 stateson the basis of the percentage of degree to which they combine. Suppose 25%increase = +1, 50% increase = +2, 100% increase = +3 and equilibrium state = 0;whereas 25% decrease = -1, 50% decrease = -2, and 100% decrease = -3,combinations of either two of the doshas renders 18 (9+ and 9-) states and three ofthe doshas renders 26 (13+ and 13-) states and 12 +, =, and – combination states100% increase or decrease of any one of the dosha constitute 6 {3 (+3) and 3 (-3)}other states as detailed in table 1 below: V+1P+2 P+1K+2 V+1P+1 V-1P-2 P-1K-2 V-1P-1 V+1K+2 K+1V+2 V+1K+1 V-1K-2 K-1V-2 V-1K-1 P+1K+2 K+1P+2 K+1P+1 P-1K-2 K-1P-2 K-1P-1 K+1V+2P+2 K+2V+1P+1 V+1K+2P+2 K-1V-2P-2 K-2V-1P-1 V-1K-2P-2 P+2K+1V+1 P+1K+2V+2 P+2K+1V+1 P-2K-1V-1 P-1K-2V-2 P-2K-1V-1 17
  • 18. V+1P+2K+3 P+1K+2V+3 V+1K+2P+3 V-1P-2K-3 P-1K-2V-3 V-1K-2P-3 K+1P+2V+3 P+1V+2K+3 K+1V+2P+3 K-1P-2V-3 P-1V-2K-3 K-1V-2P-3 V+3 P+3 K+3 V-3 P-3 K-3 V+P0K- P+V0K- V+K0P- K+P0V- P+K0V- K+V0P- V+K+P- V+P-K- P+K+V- P+V-K- V+P+K- K+V-P- V+3P+3K+3 V-3P-3K-3(Charaka samhitha Su. 17/ 40 to 44)In Sama dosha Prakrithi, all the three doshas are in a state of balance essential toprovide superior intra zygotic environment inevitable for its healthy growth andmultiplication. A state of simultaneous provocation of all the three doshas will causedisintegration of the zygote and hence such a Prakrithi does not occur15. Where as,such a state in the somatic cells of the embryo or of the individual will manifest into adisease of bad prognosis (Janma bala pravritha roga)16.A correct perusal of the ancient and modern concepts of hereditary disease revealsthat autosomal recessive inheritance and sex linked dominant inheritance involvesboth Mathrja and Pithrja bija dushti, whereas sex linked recessive inheritance isexclusively Mathrja and all these can be included in Aadi bala pravritha roga.If a woman’s diet and psychophysical practices provokes tridoshas is reflected in thewhole body and also in ovum and uterus. If the dosha provocation is severe, thezygote only disintegrates whereas the provocation is mild, and fertilization occurs,muscles, blood, fat, bone marrow, heart, umbilicus, liver, spleen, intestines, anus etcthat are soft and are derived from the mother, become defective in the zygote as therespective bija bhagas render defective (gene mutation), either completely orpartially. When the bija bhaga that determines the development of uterus and / or 18
  • 19. ovum of the offspring is subjected to vitiation, it will give rise to a sterile female(Vandhya). If the defect is in garbhasaya bija bhaga avayava, puthi praja (still birth)or neonates with loose body tissues (klinnanga prathyanga)17. It is understood thatDystrophin is required inside muscle cells for structural support; it is thought tostrengthen muscle cells by anchoring elements of the internal cytoskeleton to thesurface membrane. Without it, the cell membrane becomes permeable, so thatextracellular components enter the cell, increasing the internal pressure until themuscle cell "explodes" and dies. The gene that encodes dystrophin is found on the Xchromosome. Duchenne muscular dystrophy (DMD) is one of the most prevalenttypes of muscular dystrophy and is characterized by rapid progression of muscledegeneration that occurs early in life. All are X-linked and affect mainly males — anestimated 1 in 3500 boys worldwide18.When the genes that determine development of uterus, ovum, and that of thesecondary sex characteristics like breast, vagina, linea alba, etc. are rendereddefective super females (sthree akrithi bhooyishtam) called Vaartha19 is born.Vaartha and super females or Trisomy X mentioned in modern genetics showssimilarity. It affects females who have three X chromosomes, instead of the usualtwo. It is the most common X-chromosome disorder in females. Triple X is arandom mutation, usually inherited from the mother. Parents who have a daughterwith Triple X usually do not have to worry about their later children having thesyndrome. The mutation occurs in one in every 1,000 to 3,000 newborn girls, but itis often not diagnosed until later in life. Many girls and women with Triple X have nosigns or symptoms. Signs and symptoms vary a lot between individuals, but caninclude: Increased space between the eyes, Vertical skin folds that may cover theinner corners of the eyes (epicanthal folds), Tall stature (height), Small head, Speech 19
  • 20. and language delays and learning disabilities. Delayed development of certain motorskills, behavioral problems, seizures, delayed puberty, infertility, rarely mentalretardation20.If a man’s diet and psychophysical practices provokes tridoshas it is reflected in thewhole body and also in genes that determines sperm and testis. If the doshaprovocation is severe, the zygote only disintegrates, whereas the provocation is mild,and fertilization occurs, hairs on the head, mustaches, hairs on the body, bone, nails,teeth, veins, ligaments, arteries, semen etc, which are stable, and are derived fromthe father, become defective in the zygote as the respective bija bhagas renderdefective (gene mutation), either completely or partially. Charaka depicts sterilemale, in relation with sperm and / or testis determining gene. If the defect is insukraroopa bija bhaga avayava, puthi praja (still birth) or neonates with highly firmbody tissues (sthiraanga prathyanga)21.Fibrodysplasia Ossificans Progressiva (FOP) is an extremely rare genetic diseasethat causes muscle to be turned into bone. FOP is an autosomal dominant condition,but most cases are sporadic. FOP patients have a genetic fault, which means thattheir bodies cannot switch off the mechanism that grows the skeleton in the womb.Any small injury to connective tissue (muscles, ligaments, and tendons) can result inthe formation of hard bone around the damaged site. Children are born with acharacteristic malformation of the great toes and begin to develop heterotopic (extra)bone formation during early childhood. Eventually, a second skeleton begins to formthat severely restricts mobility. FOP affects 1 of 2 million people. Because of the verysmall numbers of patients, identifying the mutation(s) causing FOP is difficult. Thereare several genes that have been implicated in the disease process. For example,when the Noggin gene (NOG) is deleted in mice, the mice are unable to stop the 20
  • 21. deposition of bone, causing an FOP-like disease. Another gene of interest is theBone Morphogenic Protein gene (BMP), which Noggin regulates. Proteins encodedby BMP induce bone formation, and one of their roles is to stimulate the formation ofthe fetal skeleton. In FOP, lymphocytes deliver BMP4 to areas of damaged muscle,and so initiate bone growth rather than aid tissue repair. It is hoped that futurestudies will pinpoint the mutation(s) occurring in FOP and lead to a betterunderstanding of the disease’s mechanism22.When the gene that determine development of testis, sperm, and that of thesecondary sex characteristics like beard, penis, moustache, etc. are rendereddefective super males (purusha akrithi bhooyishtam) called Trina puthrika23 is born.Trina puthrika and super males or XYY syndrome mentioned in modern geneticsshows similarity. An early observation that males with a second Y chromosome, XYYwere found in excess numbers in a maximum-security prison was greatly overinterpreted. Most boys and men with a second Y are normal. The origin of the XYY ispaternal non-disjunction at the second meiotic division, which produces YY sperm.XYY syndrome is a genetic condition in males with extra Y chromosome (in 1 in1000 male births). Symptoms: tall stature (over 6), may including sterility,developmental delay, learning problems 24. Our acharyas have mentioned variousenvironmental factors that induce genetic disorders by causing bijopatapthi andwarns not to indulge such regimns or to avoid such causes for getting a meritoriouschild. It is also adviced to use factors to make beneficial effects in bija bhaga to havean offspring of desired qualities also. Psychic factors like worry, anxiety andemotional upsets etc of the parents may produce changes in bija bhagas the effectsof which are manifested in later generations or results in reduction of phenocopies inthe next generations. Even the mental state of mother at the time of coitus and also 21
  • 22. during the child bearing period can influence the psychogenesis and even themorphogenesis of the fetus. Positive thoughts and mental wellbeing of the parentsare capable of endowing the gamates with factors essential for the formation of anoffspring with superior health of the body, mind and spirit. Thoughts related tovarious catagories can induce changes in corresponding genes and it will produceharmful or beneficial effects in the physical and mental functions of the offspring. Ifthe parents are constantly given to grief the progeny may become fearful, thin andshort lived. If they think ill of others that may make the offspring antisocial, envious,and subjugated to women. If they indulge in the feeling of stealing that makes theoffspring exceedingly lazy, malicious, and of inactive disposition. If they constantlyresort to anger that makes the offspring fierceful, deceitful, and jelous25. Vagbhataeven dare to say that at the time of coitus if orgasm is occuring to the female first,her mental functions may provoke Vatha and that will induce changes in the spermthat immidiately enters vagina i.e; it will induce changes in the genes that determinethe development of testis and / or sperm, leads to disgenesis or malformation ofseminiferous tubules and vas deferens and will give birth to a child havingazoospermia or aspermia (Vathendriya)26.Sleeping in open air and moving at night alone may result in production of insaneprogeny whereas resorting to abuses and physical hazards may make the progenyepileptic. Alcoholic addicts may make the offspring constantly thirsty, short ofmemory and fickle minded. Intake of inguana fish may make the offspring suffer fromdiabetes, stone in bladder and dribbling of urine. Intake of pork flesh may induceheretable changes that produce redness in eyes, certain obstruction of respirationand excessive roughness of hair of the offspring. Habitual intake of fish may causedelayed closure or non closure of eyelids. Intake of sweet things may produce 22
  • 23. offsprings suffering from diabetes, obesity, and dumpness. Habitual intake of sourfoods may render the offspring suffer from disorders of liver and spleen (RekthaPitha). Increase intake of salt may result in early onset of wringles, graying of hairand baldness in the child27. It is difficult to depict all such food materials and lifestyles that may produce unfavourable effects in the next generation and this doesnot fall within the scope of this treatise. Most of the diseases having a geneticcomponent are difficult to cure and certain diseases such as cleft pallete and hairlipswere corrected by surgical measures even in ancient times. Some of the inbornerrors of metabolism can be controlled by regular replacement of the missing factorsor by preventing accumulation of toxic metabolites by appropriate manipulation ofdiet and treatment. Stressing the point, prevention is better than cure to avoidgenetic abnormalities, Ayurveda priscribes several regimns if followed strictlyenables to get meritorious offsprings and thus a better and fittest species.Dalhana coments that swabhava bala pravritha rogas are due to the effects ofPrakrithi and indicates the natural or spontaneous occurance of disease. Naturalorganic phenomina such as death, ageing, hunger, thirst, and sleep are included inthis chatagory. They are subdivided into kaalakritha (timely) and akaalakritha(untimely). Since most of the species seen to have a built in survival programmePrakrithi is maintained unchanged through out the span of life. A change in Prakrithiindicates deterioration of survival programme that leads to death. The synonym ofsareera – kaaya and its nirukthi, ‘cheeyathe annadibhi:’ also implies this fact.Cheeyathe by anna means anabolism, in the same way the aadi sabda may includethe inbuilt genetic survival programme. The congenital state of dosha which interactwith genetic material (bija bhagas) for the expression of a particular Prakrithiimposes restriction upon the span of life of an individual. Vatha Prakrithi have short 23
  • 24. span iof life while Pitha Prakrithi have moderate span of life and Kapha Prakrithihave long span of life.Body of Vatha Prakrithi individuals have lean and tall shape, rough rigid, un-proportionate, asymmetrical, marked with large number of veins and bulged calveswith dusky, cracked and lusterless hair, sole of feet and hands cold to touch.Produce sound from joints while walking with unsteady and swift gait (movements).Eyes are small, round, brown, lusterless, dry, and unpleasant, unattractive with thin,partially open lid while sleeping. They are talkative and use irrelevant, obstructed,interrupted words, in harsh voice. They are fond of music, humor and gambling.Fond of foods – like sweet, sour, salty and hot items. They are cruel, thievish, andungrateful and are non-magnanimous, jealousy, unsteady in courage, memory,thinking, movements, friendship and observation. Their dreams are fearful, anddreams roaming on mountains, dwelling on trees, and moving in the sky.Body of Pitha Prakrithi individual is of medium built with medium strength,symmetrical and proportionate with very loose joints and muscles, agitatedmovements, coppery red palms, soles, tongue, lips and face, hot to touch. Skin iswhitish or yellow with wrinkles, spots or pimples and scanty grey or brown hair. Eyesare thin, small, unsteady, brown or coppery red in colour with thin, few eyelashes,becoming red very quickly by anger, alcohol and exposure to sunlight. The voice isharsh, irritable and high-pitched. They are fond of garlands, perfumeries, and havemoderate sexual and physical strength. Fond of foods – like sweet, astringent, bitter,cold items. They are jealousy, highly intelligent, good behaviour, clean andaffectionate, brave, proud; with helping disposition and quick tempered, but do not 24
  • 25. attracted by women. Their dreams are frightening, and dreams red coloured flowerslike palasa, forest fire, meteor, lightening, thunderbolt, sunrays and fireBody of Kapha Prakrithi individual is soft, symmetrical, well-defined, good lookingwith long arms, elevated chest, wide fore head, deep-seated, strong, unctuous, well-knit joints and bones and walks with steadiness and firm stepping. The skin colourslike shining of weapons, gorochana (yellowish), padma (reddish), suvarna (golden)with thick curly blue – black hair, slightly hot to touch. Eyes are red at angles,unctuous, wide, and long, with well-designed white and black spheres more withdense and dark eyelashes. Talks less, gentle, and clear with deep sounding voiceslike roaring clouds, ocean, drums or lion. Speaks relevant, not harsh or abusive orharbour enimity. Fond of foods and drinks of all tastes especially sweet, astringent,bitter, hot items, and extra curricular activities like music, dance, drawing, sex, etc.They are mild in nature, very sleepy, patient, magnanimous, courageous, intelligent,truthfulness, foresight, steady memory, thinking, movements and friendship,forgiveness, straight forwardness, obedience, and faithfulness. They dreamreservoirs of water with full of lotus, rows of birds and clouds.Vatha Prakrithi, Pitha Prakrithi and Kapha Prakrithi are known as ‘eka doshajaPrakrithi’ and are otherwise termed Heena (bad), Madhyama (better) and Uthama(best) Prakrithis respectively and combinations of either two of them seen in thesame individual (dwantwa Prakrithis) is considered as Nindya (worst). Combinationsof all the characteristics of the entire first three Prakrithis seen in one individual istermed as Sama Prakrithi and is considered as super, rare or conceptual. Theconstitutional, temperamental, psychological, emotional and social characteristics ofhuman personality are considered in detail in Ayurveda for accurate diagnosis, 25
  • 26. prognosis and treatment. Hence among under the ten investigation methods, Charaka considers assessment of Prakrithi first. Ayurveda mandates that, the study of Prakrithi should be conducted with special reference to religion (Jati prasakta), family (Kula prasakta), and also on the basis of the influence of geopathological factors (Desanupatini), climatic elements (Kalanupatini), age factors (Vayonupatini), and inividual developmental traits (Pratyatma niyata)28.Prakrithi with reference to religion (Jati prasakta Prakrithi) Seers, sages and visioneers from ancient to modern times have stressed the significance of our thinking and a strong belief in what we think has an influence in molding our character. Religion encompasses a group of individuals having an orchestrated belief system modulated through generations having specific thinking pattern, life style, food habits, recreation etc. It is a common belief that identical twins have exactly the same characteristics through out their lives. Monozygotic twins brought up under different orchestrated belief system of two different religions will exhibit remarkable differences in genomic distribution affecting their gene expression portrait at an older age, though they were epigenetically indistinguishable during the early years of life. All beliefs are basically a thought. It may be our name or an experience, belief is something we have accepted and stored in memory. If the acceptance is unverified and blind, it is a dogma. A set of beliefs becomes a belief system. That such a system has remarkable influence on our bodily and mental health. Ayurveda point out that thought pattern of the parents can influence the morphogenesis and psychogenesis of the fetus. To give birth to a child resembling the individual of a particular race of a particular place, Charaka and Vagbhata advises, that the parents should willfully follow up intentional activity of the mind of 26
  • 27. that group of individuals having a specific orchestrated belief system and also followthe life style, food habits, recreation etc of that group of individuals. Chakrapaniestablishes the existence of influence of mind upon matter with examples such asejaculations due to sexual feelings, fetal repercussions caused by the emotions ofthe mother, which centers around the non fulfilled desires during the child bearingperiod, oja kshaya (low immunological status) and sukra kshaya (oligospermia) dueto grudge, fear, etc29. Dr. Benson30 writes that, “The potentially destructive stressresponse typically occurs automatically when an outside stress or – such aspressure as work, fear, or anxiety – causes the body and brain to go on full alert.Animals and humans share this stress-response. The relaxation response (by “lettinggo”) is peculiarly human, in that it tends to arise from a specific act of volition or aconscious relaxation strategy.” Placebo effect when combined with relaxationresponse magnifies the beneficial impact on health.The effect, meditation and firm self-confidence have on our health to the extent ofeven affecting the genes is being documented through collaborative research byneuroscientists, psychiatrists and meditation practitioners. Environmental factorsincluding directed thinking appear to have an ability to modify the proteins that act asgates in activating or turning off the genes, thus controlling the gene expression. It isestablished that thought is a form of energy. Directed thought and meditation areshown to have demonstrable influence in changing the neural circuits in the brainoverriding genetic disposition. Confident positive and intense thinking within acarefully orchestrated belief system (not blind dogma) appears to have the potentialof bringing about a transformation in an individual superseding the genetic effects.Individual’s thoughts, whether one is consciously aware or unaware, also effect thegene expression. By creating a facilitating and enabling environment (Satsangatya), 27
  • 28. it can be possible to alter the program in the genes to the extent that their self-perpetuating character is curbed. Charaka, in Vimana staana 8th chapter - Jati-soothreeya (genetic equations) - proposes performance of ‘Puthrakaameshti’ for thispurpose, where as Susrutha proposes the same in the chapter of Sukla - Sonithasudhi sareera.Current brain research is establishing that brain has tremendous plasticity. This is incontrast to the earlier view of the neuroscientists that the neural circuits, when onceformed in childhood, do not change. “Even though we are born with a set ofinstructions and neuro-signatures, our brains perpetually recruit new nerve cells andnerve-cell activation patterns to handle its daily inputs”.Buddhist meditation technique of “Mindful Attention,” Schwartz (2003)31 could showthrough brain scans the changes that came about in the activity of parts of brain(particularly the caudate nucleus) when his four step therapy was followed by thepatients of Obsessive Compulsive Disorder. He established the plasticity of brain tolearn new things (change the neuronal connections) under the intentional activity ofthe mind. Dr. Schwartz says in Chapter X:“It seems that neuroscience has tiptoed up to a conclusion that would be right athome in the canon of some of the eastern philosophies: introspection, willedattention, subjective state – pick your favorite description of an internal mental state– can redraw the contours of the mind, and in so doing can rewire the circuits of thebrain, for it is attention that makes neuro-plasticity possible. The role of attentionthrows into stark relief the power of mind over brain, for it is a mental state (attention)that has the ability to direct neuro-plasticity. Confident, positive and intense thinkingwithin a carefully orchestrated belief system (not blind dogma) and scientific lifestyles and food habits and other instructions of any religion intended to bringing 28
  • 29. about a transformation in an individual superseding the bad genetic effects are now a days rarely followed by its followers and therefore the concept of assessment of Prakrithi with special reference to religion has no relevance.Prakrithi with reference to family (Kula prasakta) As in the case of religion, in olden days families were also having scientific life styles, food habits, concept of physical and mental cleanliness, etc. the effect of which are reflected in hereditary and somatic mutations. Hence assessment of Prakrithi with reference to familial practices was also included in the strategy of the olden times. Individual’s born in the family of alcoholic addicts will have the ability to withstand intoxication produced by alcohol when compared to others32. Numerous examples are provided in Ayurvedic literature to support the above view.Prakrithi with reference to geopathological factors (Desanupatini) Study of geopathological factors (Desanupata) is considered as an important factor for the pathophysiological activities of the body. Bhumidesa is classified into three according to its particular features and ecological status – Jangala, Anupa, and Sadharana33. The saint Parasara, in Vrikshayurveda, an ancient treatise on plant science in Sanskrit developed parallel with Ayurveda, also depict land into the above three classes. Instead of Sadharana desa he uses the term Misra desa. Jangala desa is an arid land with thick evergreen forests, huge trees, dry soil, with full of rough and hard granites and gravels; where birds such as quil, francolin partridge, Greek partridge etc inhabits. According to Vrikshayurveda, in Jangala desa, the tract is almost like a desert with scanty vegetation and limited water resources. The soil is sodic (Usavantha) with abundance of gravel and sand. Kadara (Acacia sp. with black heart wood), Khadira (Acacia catechu), Asana (Terminalia tomentosa), Aswakarna (Shorea dalbergioides), Sallaki (Boswellia serrata), Dhava 29
  • 30. (Anogeissus latifolia), Tinisa (Ougeinia dalbergioides), Saala (Shorea robusta),Badara (Zizyphus jujuba), Somavalka (Ficus dalhousia), Kimsuka (buteamonosperma), Amalaka (Phylanthus embilica), Vata (Ficus bengalenesis), Aswatha(Ficus religiosa), Sami (Leguminous sp.), Kakubha (Terminalia arjuna), Simsapa(Dalbergia sissoo), Yava (Hordeum vulgare), Godhuma (Triticium aestivum), Bajjira(Pennisetum americanum) and various types of legumes as well as annual grows inJangala desa. Due to extremely dry condition of the soil and the natural aridenvironment, among the panchabhoothas, the biosphere is predominant with agni,vayu and prithwi and hence it result in producing plants containing kashaya(astringent), katu (pungent), and thiktha (bitter) sap. Caharaka says that in such aplace Vatha dosha will be predominant, the people are generally strong built andhard hearted. The predominance of Vatha dosha influences the herbs, birds,animals, and human beings and it is reflected in their Prakrithi.Anupa desa is comparatively low place near the sea level with full of trees, coconutgardens etc. the place is surrounded by lakes and rivers with frequent cold winds.The atmosphere is very moist, and herbs and bushes show a very good seasonalflowering tendency. Birds such as flamingo, ruddy goose, cranes, woodpeckers,cuckoos, etc richly inhabit the place. Description of Anupa desa is more or less samein Vrikshayurveda also. Here the land is green grassy and has clusters of reedyplants (Nala), Nelumbo sp. (Kumuda), calamus rotung (vethasa) the soil is clayey.Strong storm like wind laden with heavy moisture keeps blowing. Rows of Hindala(Phoenix paludosa), Tamala (Garcinia morella), Kadali (Musa sp.) and Narikela(Cocos nucifera), Venu (Bamboosa sp.), Vanira (Calamus roxburghii), bordering theriverside present a scenic view. The forestland there appears beautiful withassemblage of various types of trees, shrubs with blossoming young branches. Due 30
  • 31. to the moist nature of soil and influence of Prithwi and Ap bhoothas, the herbs, creepers and annuals flourishing in the area generally bear sap that tastes sweet or sour. People living there are beautiful, and the place tends to produce predominance of Kapha dosha and it is reflected in the Prakrithi of the Anupa desa inhabitants. Sadharana desa or Misra desa is having mixed characteristics of the above two. The people of this place are fair, strong, and most healthy. In Misra desa the soil is gray, red or black in colour and the place is neither too dry nor too moist; neither it has an abundance of rock particles or sand. The land is fertile sustaining all kinds of trees and crops. Here dosha is generally maintained in a balanced state and is reflected in the Prakrithi of the inhabitants.Prakrithi with reference to climatic elements (Kalanupatini) Seasonal variations of climatic factors such as solar radiation, humidity, temperature, atmospheric pressure, cloud, etc. provides specific environment for gene expression to constitute a particular Prakrithi. Biology tells us that we are what our genes are. But current research on how environment can influence the gene expression has given rise to the new science of Epigenetics. Prenatal research findings demonstrate the influence the environment present in the mother’s womb has on the fetus and the way the child’s mental and physical health get affected for life. ‘Kalagarbhasaya Prakrithi’ – climatic and intra uterine environment have tremendous influence in the expression of genetic material and have influence in the determination of Prakrithi of an individual. Temperature effects the changes in penitrance of genes. The general temperature at which normal phenotypes are produced is referred to as permissive temperature and that which produces mutant phenotypes is called restrictive temperature. Environmental factors such as temperature, light, humidity, radiation etc. appear to have an ability to modify the proteins that act as gates in activating or 31
  • 32. turning off the genes, thus controlling the gene expression which is reflected in the Prakrithi of the individual.Prakrithi with reference to age factors (Vayonupatini) Age factor has also an important role in the physiological and psychological development of human beings. Age, according to Ayurveda, is a state of body, which depends on the course of time. Human beings have three state of body – age – Baala (childhood, 1-16yrs), Madhya (adolescence, 17-60yrs), Vridha (old age, above 60yrs). The effects of predominance of Kapha, Pitha, and Vatha have most and respective influences in the metabolic state of body during these periods34. Charaka point out that, during childhood, the growth rate of body is comparatively high and the tissues are not fully developed and differentiated. The individual will be tender hearted, easily distressed by mild hurt and insult and is not at all hot tempered with physical and mental dispositions yet to be developed. Softness, complexion, tenderness and unctuousness of the body are in maximum in this period. In adolescence, strength, energy, man hood, bravery etc. reaches its peak; the capacity to receive, preserve and recollect information are maximized and the individual masters the art of communication by speech. The body tissues are fully developed and differentiated in this period and are maintained undisturbed up to the age of 60. Due to dominance of Pitha during this period, the individual is more or less quick tempered and his actions may reflect rebellion against traditions of the society. During senility (old age) degenerative changes occurs in the tissues. Higher mental activities, sensory and motoric potentialities, immunological status, libido and bravery gradually decline. The capacity to receive, preserve and recollect information and 32
  • 33. communication skills gradually begin to blunt and Vatha shows dominance during the period.Prakrithi with reference to individual developmental traits(Pratyatma niyata) The Prakrithi of an individual can be assessed from the physical, physiological, psychological characteristics of an individual that are determined by germinal mutations (Praakritha doshaja). Any variation to the above characteristics will endanger the very existence of the individual/ organism, as it will influence the metabolic functions of the body as a whole. Mutations occurring in a somatic cell only produce phenotypic changes in the organ to which the mutant cell belong (Vaikritha doshaja). Prakrithi is determined by Praakritha dosha. Hereditary diseases due to defect in Sukla (sperm) or Arthava (ovum) of the parents such as Kushta, Arsas, Prameha, and Kshaya etc. are called Aadi bala pravritha (dushta sukra sonitha bala jata) as put it by Dalhana, the commentator of Susrutha samhitha35. Though the basic body structure, color of the skin etc are defined by the genes at the time of conception (Sukra Sonitha Prakrithi), what we are in our health and personality depend on the environment including our biological relationship in the womb (garbhaasaya Prakrithi) biosocial relationships in the world and how we perceive our own position relative to the world around. Prenatal research36 shows that it is not merely the post-birth environment that influences an individual. The environment within the mother’s womb too has very significant effect on the health and personality of an individual. The coded genetic information accumulated through the millennia of years of evolution is undoubtedly stored in the DNA. What appears to be more significant is the mechanism of gene expression rather than the presence or absence of a specific gene. Whether a particular gene expresses itself or not or 33
  • 34. how a protein can form through multiple pathways of instructions of several genes isbeing now understood. The view that mutations and recombinations in DNAdetermine the phenotypic traits is getting modified with the emerging science ofEpigenetics. Epigenetics is the study of heritable changes in gene function that occurwithout a change in the DNA sequence. It provides a handle to understand themechanisms in phenotype transmission and development through gene activationand inactivation without necessarily changing the genes. There are critical periodsduring prenatal development when the environment in the womb is more importantthan the genes on the health we enjoy throughout our life. “Chronic maternal stressduring pregnancy – both emotional and physical – can interfere with how the fetusutilizes nutrients and can affect how well or poorly a child functions psychologicallythroughout life.” The mother’s food habits and life style during pregnancy(mathuraahara vihara Prakrithi) have a significant influence on the child’spersonality, health and disease - janmabala pravritha as put it by Susrutha, such asKubjata, Vamana, Jada (with Vathala food) Khalati, Pinga (with Pithala food) SwitraAnd Pandu (with Sleshmala food) by Vagbhata in Ashtanga Hridayam Sareera. Inshort, the normal characters such as stature, facies, colour of eye, type of hair,fertility, vigor, longevity, etc. transmitted from parent to offspring enable to assess thePrakrithi of an individual where as the abnormal characteristics manifested help tounderstand the hereditary disease (Vikrithi).The physical, physiological, psychological characteristics of an individual that arefixed at the time of fertilization (garbhaadi pravritha) by germinal mutations(Praakritha doshaja) that will enable to determine the Prakrithi of an individual istabulated (table no 2) below: 34
  • 35. 1. General body built and other features.In Vatha Prakrithi In Pitha Prakrithi In Kapha Prakrithi1. Ugly 1. Ugly 1. Beautiful, delightful and lovely.2. Emaciated and lean 2. Body parts weak and 2. Thick feeble3. Rough and rigid 3. Loosen 3. Greasy and tender body body4. Prominent tendons 4. Loosen and delicate 4. Muscular body, smooth musculature musculature5. ---- 5. Whitish or yellow 5. White and yellow complexion complexion6. Net work of vessels 6. ------ 6. ------ prominent7. ------ 7. Joint tender and 7. Joints lubricated and loosen smooth in working2. Body formation.In Vatha Prakrithi In Pitha Prakrithi In Kapha Prakrithi1. Un proportionate and 1. Symmetrical 1. Balanced andasymmetrical. proportionate2. ------- 2. Strong and round body 2. Strong and round body3. ------ 3. Soft and loose 3. Easily movable ligaments ligaments4. ------ 4. ------- 4. Joints, body parts and muscles are well covered or concealed. 35
  • 36. 3. Skin and Temperature.In Vatha Prakrithi In Pitha Prakrithi In Kapha Prakrithi1. Dry, cracked, wrinkled 1. Early wrinkled, spotted/ 1. Smooth, oily and luster(hands & feet) pimpled/ boils.2. Cold in touch, 2. Warm in touch 2. Low and regulartemperature irregular and temperature.low.3. Intolerable to cold, 3. Intolerable to heat and 3. Tolerable to heat.prefers hot weather. prefers cold.4. ------ 4. Excessive perspiration 4. -------5. ----- 5. ------- 5. Unguent etc. are dried lately6. Body odourless 6. ------ 6. -----4. ColourIn Vatha Prakrithi In Pitha Prakrithi In Kapha Prakrithi1. Black and Dusty 1. White, yellowish, pale 1. Golden yellow, clear red, saffron and pigmented2. ------- 2. White coloured teeth 2. -------5. JointsIn Vatha Prakrithi In Pitha Prakrithi In Kapha Prakrithi1. Swift (rapid) movement 1. Movements are agitated 1. Slow in movementsof joints with a feeling of uneasiness while moving.2. Loose joints 2. Relaxed joints; 2. Strong and steady joints ligaments loose.3. Sound produced in the 3. ------ 3. ------ 36
  • 37. movements of the joints4. -------- 4. ------ 4. Concealed and symmetrical joints5. -------- 5. -------- 5. Walks steady & firm6. Dietetic preferences.In Vatha Prakrithi In Pitha Prakrithi In Kapha Prakrithi1. Likes diet of sweet, 1. Dislikes acidic 1. Prefers sweets.acidic and saltish tastes. substances.2. Prefers hot (warm) 2. Prefers cold 2. Prefers hot preparationspreparations preparations3. ------- 3. ------- 3. Takes light but maintains strong structure4. Prefers fat rich diet 4. ------- 4. Prefers dry diet and it suits him.7. Food habitsIn Vatha Prakrithi In Pitha Prakrithi In Kapha Prakrithi1. Moderate in diet 1. Excessive diet 1. Very moderate in diet2. Eats swiftly 2. ------- 2. Eats slowly3. Irregular in diet habits 3. Takes edibles many 3. Moderate in taking food. times (frequently)8. DigestionIn Vatha Prakrithi In Pitha Prakrithi In Kapha Prakrithi1. Irregular digestion 1. Quick digestion 1. Weak in digestion2. ------- 2. Intense hunger and 2. --------- thirst 37
  • 38. 9. BowelIn Vatha Prakrithi In Pitha Prakrithi In Kapha Prakrithi1. Hard bowel 1. Soft bowel 1. Moderate bowel2. Requires strong 2. Requires light 2. Requires mediumpurgatives for removing purgatives purgativesconstipation3. Constipation 3. ------ 3. -------10. Excretion and Perspiration tendencyIn Vatha Prakrithi In Pitha Prakrithi In Kapha Prakrithi1. Less in quantity 1. More in quantity 1. Less in quantity2. ----- 2. Passes urine and 2. -------- excreta in more quantity.11. Physical strengthIn Vatha Prakrithi In Pitha Prakrithi In Kapha Prakrithi1. Physical strength less 1 Moderate strength 1. Strong2. Endurance 2. Low endurance 2. Endurance (Incapable of bearing the troubles)3. ----- 3. Courageous and brave 3. --------4. Gets fatigued early. 4. ------- 4. -------12. SmellIn Vatha Prakrithi In Pitha Prakrithi In Kapha Prakrithi1. ------- 1. Unpleasant, foul and 1. Sweet agar like 38
  • 39. 13. HairIn Vatha Prakrithi In Pitha Prakrithi In Kapha Prakrithi1. Hard and rough 1. Soft 1. --------14. EyesIn Vatha Prakrithi In Pitha Prakrithi In Kapha Prakrithi1. Dusky 1. Coppery red color 1. Whitish2. Spherical, small in size 2. Rounded 2. Large, broad and elongated3. ------- 3. Eyelashes thin 3. Lashes dense, dark4. Thin lid 4. Thin lid 4. ------5. Unsteady brow 5. ------ 5. -----6. Fluctuating eyeball 6. ------- 6. ----7. Movement of eyelids 7. ------ 7. -----fixed8. Rapid and much 8. ------ 8. ------twinkling of the eye9. Emaciated, unattractive 9. ----- 9. -----10. ------- 10. Feels pleasant in 10. ----- mist and cold15. Lips, Tongue and PalateIn Vatha Prakrithi In Pitha Prakrithi In Kapha Prakrithi1. Unsteady and unsettled 1. Coppery 1. ------- 39
  • 40. 16. Speech and VoiceIn Vatha Prakrithi In Pitha Prakrithi In Kapha Prakrithi1. Swift in speech 1. Talks rapidly 1. Speaks gently2. Much talkative voice 2. Aggressive 2. Moderate in talk17. Mental StrengthIn Vatha Prakrithi In Pitha Prakrithi In Kapha Prakrithi1. Coward 1. Bold 1. Courageous2. Unstable 2. ------ 2. Steady and firm3. Feeble minded 3. ----- 3. -----4. Agonized with grief 4. ----- 4. -----5. Cherishing to humble 5. Haughty 5. Forgivenesspersons18. Reproductive StrengthIn Vatha Prakrithi In Pitha Prakrithi In Kapha Prakrithi1. Less children 1. Less children 1. More children2. Un-liked by women 2. Un-liked by women 2. Liked by women3. ------ 3. Less inclined in sex 3. More inclined in sex4. ---- 4. Sperm quantity less 4. Profuse quantity of sperm19. Life SpanIn Vatha Prakrithi In Pitha Prakrithi In Kapha Prakrithi1. Less 1. Moderate 1. Long 40
  • 41. 20. Positions and DignityIn Vatha Prakrithi In Pitha Prakrithi In Kapha Prakrithi1. Finance-poor 1. Moderate finance 1. Rich and wealthy2. Means and luxuries-less 2. Moderate 2. ------3. Fried circle- limited 3. ------ 3. Friendship stable4. ------ 4. Dignified 4. Fortunate, prosperous5. ----- 5. ----- 5. Attendants-abundant21. Provocation of doshasIn Vatha Prakrithi In Pitha Prakrithi In Kapha Prakrithi1. More susceptible to 1. More susceptible to 1. More susceptible toVatha ailments Pitha ailments Kapha ailments22. SleepIn Vatha Prakrithi In Pitha Prakrithi In Kapha Prakrithi1. Sleep less 1. ------- 1. Sleepy and drowsy2. Habit of teeth grinding 2. ----- 2. -----and beating of teeth insleep3. Eyes and mouth remain 3. ------ 3. -----slightly open at sleep4. Sudden sleep walking 4. ----- 4. ------23. DreamsIn Vatha Prakrithi In Pitha Prakrithi In Kapha Prakrithi1. Fearful in dreams 1. ------- 1. ------- 41
  • 42. 2. Riding on camels, 2. Dreams of gold, 2. Dreams of ponds full ofwalking on hells, climbing palasa, karnikara; lotus, Hans, rows of birds,on trees, flying in sky flames of fire chakravaka, clouds.24. Conduct and PurityIn Vatha Prakrithi In Pitha Prakrithi In Kapha Prakrithi1. ------- 1. Excess and quick in 1. ------ anger2. ----- 2. Excess in envious 2. Less envious3. ----- 3.Pious 3. Good character25. SimiliaIn Vatha Prakrithi In Pitha Prakrithi In Kapha Prakrithi1. Goat, jackal, rabbit, rat, 1. Snake, owl, 1. Brahma, rudra, varuna,camel, dog, vulture, crow gandharva, yaksha, indra, lion, horse, elephant, monkey, tiger, bear, cow, ox, leopard, swan. mongoose(Courtesy: Psycho- Pathology in Indian medicine) 42
  • 43. GENESA bird’s eye - view on genetics is essential before review of literature of the genesincluded in my present study.Gene is the fundamental physical and functional unit of heredity, which carriesinformation from one generation to the next. Genes make proteins. Proteins makecells carry out the various functions of the body. Proteins are made of 20 buildingblocks of life called amino acids. They combine in a thousand different ways to makevarious types of proteins. Billions of proteins combine to make a cell; billions of cellscombine to make a tissue; thousands of tissues combine to make an organ, andseveral organs, to make a man1.Life begins with a single cell. By adulthood the single cell will have replicated, dividedand differentiated many times to make a three trillion-cell human being. Each cellcontains about 30000 genes and their collective interactions in our body give us life.The normal functioning of genes keeps us healthy. When genes are exposed toharmful environments, they alter their functions. It is called mutation. Mutations arechemical imperfections in this process, where a base is accidentally skipped,inserted, or incorrectly copied, or the chain is trimmed, or added to; many basicmutations can be described as combinations of these accidental "operations".Mutations can also occur through chemical damage (through mutagens), light (UVdamage), or through other more complicated gene swapping events. A mutatedgene makes a wrong protein and this is what makes us sick. Hence all diseaseshave a genetic origin.There are also millions of plants on earth. Many of them have developed toxins toprotect themselves against insects and environment. Those plants that producedtoxins were able to survive. These toxins act as active molecules in and have the 43
  • 44. unique ability to bind to human genes too and are able to switch a gene on or off. Ifthe switching on and off of a gene is desirable, it will lead to cure of disease whengenes are switched on it produces a protein. Once produced, the protein interactswith the many other proteins in the cell, according to the cell metabolism. Thisinteraction finally produces the trait. The protein floats around in our blood streamuntil it comes in contact with a receptor site. If the protein is a bad one made by amutated gene, it can produce a disease or its simply floats around for a while andfinally is filtered out of the body.Genes are encoded in an organisms genome, composed of DNA or RNA, and directthe physical development and behavior of the organism. Most genes encodeproteins, which are biological macromolecules comprising linear chains of aminoacids that effect most of the chemical reactions carried out by the cell. Some genesdo not encode proteins, but produce non-coding RNA molecules that play key rolesin protein biosynthesis and gene regulation. Molecules that result from geneexpression, whether RNA or protein, are collectively known as gene products.Most genes contain non-coding regions, that do not code for the gene products, butoften dictate gene regulation. A critical non-coding region is the promoter, a shortDNA sequence that is required for initiation of gene expression. The genes ofeukaryotic organisms often contain non-coding regions called introns which areremoved from the messenger RNA in a process known as splicing. The regions thatactually encode the gene product, which can be much smaller than the introns, areknown as exons.The word "gene" was coined in 1909 by Danish botanist Wilhelm Johannsen for thefundamental physical and functional unit of heredity. The word was derived fromHugo De Vries term pangen, itself a derivative of the word pangenesis coined by 44
  • 45. Darwin (1868). The word pangenesis is made from the Greek words pan (a prefixmeaning "whole", "encompassing") and genesis ("birth") or genos ("origin").Although classical genetics and evolutionary biology use the term "gene" to refer to aconceptual entity or "unit of inheritance", modern molecular genetics typically usesthe term to refer to a physical molecule. Gene Ontology system, defines a gene as "alocatable region of genomic sequence, corresponding to a unit of inheritance, whichis associated with regulatory regions, transcribed regions and/or other functionalsequence regions".The word "gene" is also used in common speech to refer to the inheritance of a trait,as in "a cancer gene" or "the gene for obesity"; however, biologists rarely use theterm in this sense because it is highly unlikely that such complex and large-scalephenomena would be attributable to the influence of a single molecular gene.In molecular biology, a gene is a region of DNA (or RNA, in the case of someviruses) that determines the amino acid sequence of a protein (the coding sequence)and the surrounding sequence that controls when and where the protein will beproduced (the regulatory sequence). The genetic code determines how the codingsequence is converted into a protein sequence. The protein-coding regions of genesare composed of a series of three-nucleotide sequences called codons. Each codonspecifies a particular amino acid to be added to the protein chain; thus genesdetermine the proteins primary structure. Most genes are expressed in a two-stageprocess: first, the DNA is transcribed by enzymes known as RNA polymerases toproduce an RNA molecule known as messenger RNA (mRNA), and second, themRNA is translated by specialized cellular machinery known as the ribosome into apolypeptide chain that then folds into a functional protein. The genetic code isessentially the same for all known life, from bacteria to humans. 45
  • 46. Through the proteins they encode, genes govern the cells in which they reside. Inmulticellular organisms, they control the development of the individual from thefertilized egg and the day-to-day functions of the cells that make up tissues andorgans. The roles of their protein products range from mechanical support of the cellstructure to the transportation and manufacture of other molecules and to theregulation of other proteins activities.Due to rare, spontaneous errors (e.g. in DNA replication), mutations in the sequenceof a gene may arise. Once propagated to the next generation, this mutation may leadto variations within a species population. Variants of a single gene are known asalleles, and differences in alleles may give rise to differences in traits, for exampleeye colour. A genes most common allele is called the wild type allele, and rarealleles are called mutants.All the genes and intervening DNA together make up the genome of an organism,which in many species is divided among several chromosomes and typically presentin two or more copies. The location (or locus) of a gene and the chromosome onwhich it is situated is in a sense arbitrary. Genes that appear together on thechromosomes of one species, such as humans, may appear on separatechromosomes in another species, such as mice. Two genes positioned near oneanother on a chromosome may encode proteins that figure in the same cellularprocess or in completely unrelated processes. As an example of the former, many ofthe genes involved in spermatogenesis reside together on the Y chromosome.Many species carry more than one copy of their genome within each of their somaticcells. These organisms are called diploid if they have two copies or polyploid if theyhave more than two copies. In such organisms, the copies are practically neveridentical. With respect to each gene, the copies that an individual possesses are 46
  • 47. liable to be distinct alleles, which may act synergistically or antagonistically togenerate a trait or phenotype. The ways that gene copies interact are explained bychemical dominance relationshipsThe existence of genes was first suggested by Gregor Mendel, who, in the 1860s,studied inheritance in pea plants and hypothesized a factor that conveys traits fromparent to offspring. Although he did not use the term gene, he explained his resultsin terms of inherited characteristics. Mendel was also the first to hypothesizeindependent assortment, the distinction between dominant (An allele that determinesphenotype even when heterozygous. Also the trait controlled by that allele) andrecessive (a gene that is phenotypically manifest in the homozygous state but ismasked in the presence of a dominant allele) traits, the distinction between aheterozygote (A diploid or polyploid with different alleles at a particular locus) andhomozygote (A diploid or polyploid with identical alleles at a particular locus), and thedifference between what would later be described as genotype and phenotype.Mendels concept was finally named when Wilhelm Johannsen coined the word genein 1909.In the early 1900s, Mendels work received renewed attention from scientists. In1910, Thomas Hunt Morgan showed that genes reside on specific chromosomes. Helater showed that genes occupy specific locations on the chromosome. With thisknowledge, Morgan and his students began the first chromosomal map of the fruit flyDrosophila. In 1928, Frederick Griffith showed that genes could be transferred. Inwhat is now known as Griffiths experiment, injections into a mouse of a deadly strainof bacteria that had been heat-killed transferred genetic information to a safe strainof the same bacteria, killing the mouse. 47
  • 48. In 1941, George Wells Beadle and Edward Lawrie Tatum showed that mutations ingenes caused errors in certain steps in metabolic pathways. This showed thatspecific genes code for specific proteins, leading to the "one gene, one enzyme"hypothesis. Oswald Avery, Collin Macleod, and Maclyn McCarty showed in 1944 thatDNA holds the genes information. In 1953, James D. Watson and Francis Crickdemonstrated the molecular structure of DNA. Together, these discoveriesestablished the central dogma of molecular biology, which states that proteins aretranslated from RNA which is transcribed from DNA. This dogma has since beenshown to have exceptions, such as reverse transcription in retroviruses.Richard Roberts and Phillip Sarp discovered in 1977 that genes can be split intosegments. This leads to the idea that one gene can make several proteins. Recently(as of 2003-2006), biological results let the notion of gene appear more slippery. Inparticular, genes do not seem to sit side by side on DNA like discrete beads. Instead,regions of the DNA producing distinct proteins may overlap, so that the ideaemerges that "genes are one long continuum". (Pearson, 2006)Genes under studyMolecular typing of the following genes was carried out using the polymerase chainreaction in the present study.1. Dopamine Receptor D2 Taq 1 D DRD2 Taq 1 D2. Dopamine Receptor D2 Taq 1 B DRD2 Taq 1 B3. Dopamine Receptor D2 Taq 1 A DRD2 Taq 1 A4. Dopamine Receptor D2 Serine 311 Cysteine DRD2 S311C5. Dopamine Receptor D2 Histidine 313 H DRD2 His 313H6. Dopamine Receptor D3 Serine 9Glycine DRD3 S9G 48
  • 49. 7. 5-Hydroxy Tryptamine Receptor 1 B 5HTR 1B8. Serotonin 1438 Ser 14389. Serotonin 102 Ser 10210. Human Leukocyte Antigen B HLA B11. Human Leukocyte Antigen A HLA ADopamine Receptor D2 Taq 1D, Taq 1B, Taq 1AHuman dopaminergic neurons are involved in the control of hormone secretion,voluntary movement, and emotional behavior. Mediating these effects are thedopamine D1 and D2 receptors. These macromolecules belong to a large family ofrelated sequences known as the G protein-coupled receptors. The D2 receptorshave been of special interest because they bind, with high affinity and specificity,many of the commonly prescribed antipsychotic drugs.1. Taq 1DKidd, Kenneth K. in a study of "A global survey of haplotype frequencies and linkagedisquilibrium at the DRD2 locus." Reported in Human Genetics Aug., 1998; 103 (2)211-227, is the only reference that I could get about Taq 1D. He has reported thatthe coding region of DRD2 gene contains four RSPs (restriction site polymorphisms),Taq1A, Taq1B, and Taq1D. It also includes a one-dinucleotide STRP or shorttandem repeat polymorphism. DRD2, coded for by locus 11q23 in the humanchromosome, is one receptor for the neurotransmitter dopamine. Thistransmembrane receptor is a member of the rhodopsin family2. As researchmaterials regarding the study of DRD2 Taq 1D is very rare, I have included it in mypresent study. 49
  • 50. 2. Taq 1 BA B polymorphism at the CETP (cholesteryl ester protein transfer) locus that isdetectable with the restriction enzyme TaqI is a genetic determinant of the plasmaHDL cholesterol concentration. Cholestery ester transfer protein (CETP) transferscholesteryl esters between lipoproteins. CETP may effect susceptibility toatherosclerosis3.3. Taq 1 AThe catecholamine dopamine, a precursor of noradrenaline and adrenaline, is anendogenous neurotransmitter, which modulates a wide variety of physiologicfunctions including behavior, ion transport, vascular tone, and blood pressure. Thereare reports of a deficiency in renal dopamine synthesis and/or secretion in variousforms of human hypertension. As endogenous renal dopamine plays an importantrole in maintaining body sodium homeostasis, renal dopaminergic deficiency maycontribute to the development and maintenance of high blood pressure, at least in aproportion of subjects with essential hypertension Suppression of dopaminergicactivity has been observed in young normotensive subjects with a family history ofhypertension before the development of hypertension emerged. Dopamine alsoplays a major role in the regulation of appetite. Dopaminergic agonist drugs, such asdextroamphetamine, have been shown to suppress appetite and subsequently toreduce weight, whereas a major side effect of dopamine D2 receptor (DRD2)antagonists, such as haloperidol, is marked weight gain. The dopaminergic systeminvolves the interaction of dopamine with several specific dopamine receptors, whichbelong to a large family of G-protein-coupled receptors. Biochemical andpharmacological studies have shown that the physiological actions of dopamine aremediated by interaction with two basic types of G-protein-coupled receptors, D1-like 50
  • 51. and D2-like, which stimulate and inhibit, respectively, the enzyme adenyl cyclase.The human DD2R gene contains eight exons, spans at least 50 kilobases (kb), andhas the unusual feature of a large (greater than 25 kb) intron (intron 1) separatingthe presumed promoter region from the protein-coding region. The D2 receptor locushas been localised to the 11q22 to 11q23 region of the human genome. . Previousstudies showed that the allelic variants of the DD2R gene play a role in theregulation of body weight. A genome wide scan provided strong evidence onchromosome 11q of a locus influencing susceptibility to obesity. It has been reportedthat 1A polymorphism to be associated with both obesity and blood pressure innormoglycaemic subjects, but in diabetics only the relationship with obesity wasevident4.4. Dopamine Receptor D2 Serine 311 CysteineDRD2 gene has been suggested to be one missense nucleotide mutation from C toG resulting in a substitution of serine with cystein at the codon 311 located in thethird intracellular loop of the DRD2. Variants of the DRD2 S311C play a major role inconferring susceptibility to major psychoses, connected with disorganized anddelusional symptomatology. The schizophrenics with Cys311 tended to have a lowerage of onset and a positive family history of schizophrenia5.5. Dopamine Receptor D2 Histidine 313 HDysfunction of the dopamine D2 receptor signaling has been associated with theillness, schizophrenia. This study investigates the association of synonymouspolymorphisms (His313 and Pro319) in the dopamine D2 receptor gene may beassociated with schizophrenia. The results demonstrated that genotype distributionfor the His313 polymorphism was significantly different between schizophreniapatients and control subjects6. 51
  • 52. 6. Dopamine Receptor D3 S9GDRD3 S9G gene encodes the D3 subtype of the dopamine receptor. The D3 subtypeinhibits adenylyl cyclase through inhibitory G-proteins. This receptor is expressed inphylogenetically older regions of the brain, suggesting that this receptor plays a rolein cognitive and emotional functions7.Dopamine8 is a chemical naturally produced in the body mainly in nervous tissueand adrenal glands. In the brain, dopamine functions as a neurotransmitter,activating dopamine receptors. Dopamine is also a neurohormone released by thehypothalamus. Its main function as a hormone is to inhibit the release of prolactinfrom the anterior lobe of the pituitary.Dopamine can act on the sympathetic nervous system, producing effects such asincreased heart rate and blood pressure. However, since dopamine cannot cross theblood-brain barrier, it does not directly affect the central nervous system. To increasethe amount of dopamine in the brains of patients with diseases such as Parkinsonsdisease and Dopa-Responsive Dystonia, a synthetic precursor to dopamine such asL-DOPA can be given, since this will cross the blood-brain barrierFunctions of dopamine in the brainRole in movementDopamine is critical to the way the brain controls our movements and is a crucial partof the basal ganglia motor loop. Shortage of dopamine, particularly the death ofdopamine neurons in the nigrostriatal pathway, causes Parkinsons disease, in whicha person loses the ability to execute smooth, controlled movements.Role in cognition and frontal cortex function 52
  • 53. In the frontal lobes, dopamine controls the flow of information from other areas of thebrain. Dopamine disorders in this region of the brain can cause a decline inneurocognitive functions, especially memory, attention and problem-solving.Reduced dopamine concentrations in the prefrontal cortex are thought to contributeto attention deficit disorder and negative schizophrenia.Role in regulating prolactin secretionDopamine is the primary neuroendocrine regulator of the secretion of prolactin fromthe anterior pituitary gland. Dopamine produced by neurons in the arcuate nucleus ofthe hypothalamus is secreted into the hypothalamo-hypophysial blood vessels of themedian eminence, which supply the pituitary gland. The lactotrope cells that produceprolactin, in the absence of dopamine, secrete prolactin continuously; dopamineinhibits this secretion.Role in pleasure and motivationDopamine is commonly associated with the pleasure system of the brain, providingfeelings of enjoyment and reinforcement to motivate us to do certain activities.Dopamine is released (particularly in areas such as the nucleus accumbens andstriatum) by naturally-rewarding experiences such as food, sex, use of certain drugsand neutral stimuli that become associated with them. This theory is often discussedin terms of drugs (such as cocaine and amphetamines), which seem to be directly orindirectly related to the increase of dopamine in these areas, and in relation toneurobiological theories of chemical addiction, which argue that these dopaminepathways are pathologically altered in addicted persons. However, cocaine andamphetamine have different mechanisms of action. Cocaine is a dopaminetransporter blocker: it competitively inhibits dopamine uptake to increase the lifetimeof dopamine. On the other hand, amphetamines act as dopamine transporter 53
  • 54. substrates to competitively inhibit dopamine uptake and increase dopamine efflux viaa dopamine transporter.However, the idea that dopamine is the reward chemical of the brain, a view held bymany during early stages of its research, now seems too simple. Dopamine isreleased when unpleasant or aversive stimuli are encountered, so it is notassociated only with rewards or pleasure. Recent research has begun to examinewhether or not the firing of dopamine neurons might function as a reward-predictionerror signal, based on evidence that, when a reward is greater than expected, thereis an increase in the firing of certain dopamine neurons (in contrast to when there isa lesser-than-expected reward, and there is a marked decrease in the firing of thesame neurons). Some argue that dopamine may be involved in desire rather thanpleasure.Confusion of dopamines role in pleasure comes from studies performed on animals.It has been shown experimentally that when the dopaminergic system of a rat isselectively abolished it will stop eating. However when the rat is force fed food it willstill display the proper facial expressions which indicate whether they like or dislike it.Conversely mutant hyperdopaminergic mice show higher wanting of food but notliking. This research was taken to mean that dopamine mediated desire andincentive salience instead of pleasure. In humans, though, drugs that reducedopamine activity (e.g., antipsychotics) have been shown to induce both amotivation(lack of desire) as well as anhedonia (inability to experience pleasure). The selectiveD2/D3 agonists pramipexole and ropinirole have anti-anhedonic and pro-motivationalproperties as measured by the Snaith-Hamilton Pleasure Scale. Opioid andcannabinoid transmission instead of dopamine is believed to be what modulates foodreward and palatability (liking). This explains why animals would still have the same 54
  • 55. liking of food independent of brain dopamine concentrations. Other pleasures arelikely dependent on dopamine. Libido can be increased by drugs that enhancedopaminergic functioning but not by ones that affect opioid peptides or otherneurotransmitters. Sociability is also closely tied to dopamine neurotransmission.Low D2 receptor binding is found in people with social anxiety. Traits common tonegative schizophrenia (social withdrawal, apathy, anhedonia) are thought to berelated to a hypodopaminergic state in certain areas of the brain. In instances ofbipolar mania subjects can become hypersocial as well as hypersexual. This is alsobelieved to be due to an increase in dopamine, because it can be alleviated withdopamine blocking antipsychotics.7. 5-Hydroxy Tryptamine Receptor 1B, or 5-HTR 1B5-HT1B receptors are expressed throughout the mammalian central nervous system.These receptors are located in the axon terminals of both serotonergic andnonserotonergic neurons, where they act as inhibitory autoreceptors orheteroreceptors, respectively. 5-HT1B receptors inhibit the release of a range ofneurotransmitters, including serotonin, GABA, acetylcholine, and glutamate. Thesereceptors have been difficult to study because of the diversity of their cellularlocalization and the absence of highly selective agonists and antagonists. There hasbeen accumulating evidence, however, that 5-HT1B receptors modulate drugreinforcement, stress sensitivity, mood, anxiety, and aggression. The general resultsof a number of studies suggest that reduced 5-HT1B heteroreceptor activities mayincrease impulsive behaviors, whereas reduced 5-HT1B autoreceptor activities mayhave an antidepressant-like effect. The 5-HT (1B) receptor has been implicated inseveral psychopathologies, including pathological aggression, alcoholism andsuicide9. 55
  • 56. 5-hydroxytryptamine (5-HT), commonly known as Serotonin10, is released atperipheral sites from activated enterochromaffin cells in the gastrointestinal tract(90%), mast cells and platelets. In the body, serotonin is synthesized from the aminoacid tryptophan by a short metabolic pathway consisting of two enzymes —TPH(1,2) and DDC. TPH1 reaction controls the flux through the pathway. Serotoninis found in three main areas of the body: the intestinal wall; large constricted bloodvessels; and the central nervous system. The activity of serotonin arises in thebrainstem from clusters of neurons known as the raphe nucleus. The most widelystudied effects have been those on the central nervous system. From the brain,serotonin neurons extend to virtually all parts of the central nervous system makingthe branching of the serotonin network the most expansive neurochemical system inthe brain. The importance of this network becomes apparent when considering eachserotonin neuron exerts an influence over as many as 500,000 target neurons. Dueto the widespread distribution of serotonin in the nervous system, it is not surprisingthat this neurotransmitter can be linked to many types of behavior. The functions ofserotonin are numerous and appear to have regulation of appetite, sleep, vomiting,memory and learning, temperature regulation, mood, behavior (including sexual andhallucinogenic behavior), cardiovascular function, muscle contraction, endocrineregulation, and depression. Recent research suggests that serotonin also plays animportant role in liver regeneration and acts as a mitogen (induces cell division)throughout the body.Serotonin taken orally does not pass into the serotonergic pathways of the centralnervous system because it does not cross the blood-brain barrier. However, theamino acid tryptophan and its metabolite 5-hydroxytryptophan (5-HTP), from which 56
  • 57. serotonin is synthesized, can and do cross the blood-brain barrier. These agents areavailable as dietary supplements and may be effective serotonergic agents.One product of serotonin breakdown is 5-hydroxyindoleacetic acid (5 HIAA) which isexcreted in the urine. Serotonin and 5HIAA are sometimes produced in excessamounts by certain cancer tumors, and levels of these substances may be measuredin the urine to test for these tumors.NeurotransmissionThe neurons of the Raphe nuclei are the principal source of 5-HT release. Theseneurons are grouped into about nine pairs, distributed along the entire length of thebrainstem. 5-HT is thought to be released from serotonergic varicosities into theextra neuronal space, in other words from swellings (varicosities) along the axon,rather than from synaptic terminal buttons (in the manner of classicalneurotransmission). From here it is free to diffuse over a relatively large region ofspace (>20µm) and activate 5-HT receptors located on the dendrites, cell bodies andpresynaptic terminals of adjacent neurons.Serotonergic action is terminated primarily via uptake of 5-HT from the synapse. Thisis through the specific monoamine transporter for 5-HT, 5-HT reuptake transporter,on the presynaptic neuron.5-Hydroxy Tryptamine Receptor 2A, or 5-HTR 2A8. SER A-1438-G9. SER T-102-CCentral serotonin-2A (5-HT(2A)) receptor dysfunction is regarded as an importantfactor in the etiology of affective disorders. The relations between some personalitytraits and the vulnerability of affective disorders are also implicated. Moreover, thereare several reports which describe the association between 5-HT(2A) receptor gene 57
  • 58. polymorphisms and mental disorders. 5-HT(2A) receptor gene have polymorphisms(A-1438-G, T102C, T516C, C1340T, C1354T). The 5-HT (2A) receptor belongs tothe G-protein super family. It plays an important role in vascular regulation. Reportsindicate an association of the T102C genetic polymorphism of the 5-HT (2A) receptorwith hypertension11.Estrogen receptors and serotonin receptors coexist in cells in a wide variety oftissues. The most active and most studied form of estrogen in mammals is 17-βestradiol (hereafter E2). A critical review of the literature suggests that many of E2seffects may be mediated by changes in the actions of serotonin (5HT). Serotonin isusually considered to be a neurotransmitter, but surprisingly, only 1% of serotonin inthe human body is found in the CNS. The remaining 99% is found in other tissues,primarily plasma, the gastro-intestinal tract, and immune tissues, where serotoninacts as a hormone regulating various physiological functions including vasodilation,clotting recruitment of immune cells, gastro-intestinal motility and initiation of uterinecontraction12.HUMAN LEUCOYTE ANTIGEN GENES1310. HLA – B11. HLA – AThe term HLA refers to the Human Leucoyte Antigen system, which is controlled bygenes on short arm of chromosome 6. The HLA loci are part of the genetic regionknown as major histocompatibility complex (MHC) (Hugh et al. 1984). The MHClocus is pleiotropic, which is best known with its role in histocompatibility andimmune regulation. The HLA loci, by virtue of their extreme polymorphism ensurethat few individuals are identical and the population at large is well equipped to dealwith immunity threats. (Mc Devitt, 1985). Some HLA antigens are recognized on all 58
  • 59. tissues of the body and thus the identification of HLA antigen is described as “tissuetyping” or “HLA TYPING”.Snell first described MHC in the mouse as the H-2 system in 1948. Jean Dausset inParis recognized the immunological origin of the agglutination of white blood cells bysera from transfused patients. He identified in 1953 the first leukocyte specificityMac, which is now called HLA-A2. Rose Payne at Stanford reported in 1958 theappearance of similar leuko-agglutinating antibodies in multiparous women.Independently, Jon van Rood (Leiden, The Netherlands) made similar observationsand he used computer programs for leukocyte antigen grouping from clusters ofleukoagglutinating antibodies.The definition 4a/4b series (Jan Van Rood, 1963) and that of HLA – AI, A2, A3 (RosePayne and Walter Bodmer, 1964) indicated a need for international standardizationand thus was born a series of international workshops, starting in 1964 (Glen, 1991).During the early 1960s, a growing group of investigators attempted to defineleukocyte antigen groups with serological techniques such as leukoagglutination andcomplement fixation on platelets. While these assays were lacking reproducibility,another problem was the extreme complexity of the genetics of leukocyte antigens.A turning point in the history of leukocyte typing was the intensive internationalcollaboration that began as the First Workshop and Conference on Histocompatibilityorganized by Bernard Amos (Durham, NC) in 1964. This was a laboratory benchstudy whereby the participants compared the reactivity of their sera with varioustechniques. The results were so discordant that they could not be published. TheSecond Workshop held the following year in Leiden, yielded more coherent resultsand several serological specificities emerged clearly. The concept was forwardedthat all of them belonged to a single, complex antigenic system analogous to the H-2 59
  • 60. system of the mouse. Paul Terasaki and John McClelland at UCLA introduced thecomplement-dependent microlymphocytotoxicity technique, which has remained thestandard serological test for HLA typing. The Third Workshop organized by RuggeroCeppelini (Torino, Italy) in 1967, clearly established the HLA system and twosegregant series of specificities (now called HLA-A and HLA-B) were recognized.The success of this international collaboration has assured the continuation of thehistocompatibility workshops (Los Angeles,1970; Evian, France, 1972; Aarhus,Denmark, 1975; Oxford, 1977; Los Angeles, 1980; Munich, 1984; New York, 1987;Yokohama, 1991, and St Malo/Paris, 1996). After each workshop, a nomenclaturecommittee has incorporated salient findings towards the definition of HLApolymorphisms including the identification of additional class I loci such as HLA-C(Thorsby, 1970), and the class II loci HLA-DR (1977) and a few years later HLA-DQ(formerly called MB) and HLA-DP (formerly SB). The latter comprise the HLA-Dregion, which was first recognized as the MLC locus by Amos and Yunis in 1970 asa genetic system responsible for T-cell activation in the mixed leukocyte culture. Inthe eighties the introduction of new molecular biology techniques revolutionizedHLA-typing (Wake, 1982) (Owerbach, 1983). Today, with the use of various DNA-typing techniques (Bidwell, 1994) (Begovich, 1995), many new polymorphic lociwithin the MHC have been identified and new alleles are discovered continuously(Bodmer, 1995).Specialists use IMGT/HLA Sequence database to locate sequences of the humanmajor histocompatibility complex (HLA). It includes the official sequences for theWorld Health Organization (WHO) HLA Nomenclature Committee for Factors of theHLA System. The IMGT/HLA Database is part of the international ImMunoGeneTicsproject (IMGT). There are currently over 1600 officially recognized HLA alleles and 60
  • 61. these sequences are made available to the scientific community through theIMGT/HLA database. In 1998 the IMGT/HLA database was publicly released. Sincethis time the database has grown and is the primary source of information for thestudy of sequences of the human major histocompatibilty complex. The numbers ofantigens and alleles named since the HLA Nomenclature Committee was firstformed in 1968, up to the end of December 2004 is given in figure 2.Within the last years, by the extensive use of the molecular biology methods thenumber of the HLA-polymorphism has been increased for 2 to 3 times, bypassing therequirement for viable lymphocytes that express the proteins. The modern molecularbiology techniques allows more accurate typing of the HLA class I and class IIantigens at the DNA level, enabling determination of DNA sequence of each HLAallele. Back in the 1980’s, HLA genotyping has been introduced in routine tissuetyping laboratories and for precise allele determination the main available approachwas the use of Restriction Fragment-Length Polymorphism (RFLP) protocols.However, this method have many drawbacks such as: minimum of two weeks periodnecessary to achieve a complete HLA class II typing and the need of radioactivelabeling of probes which have restricted these analyses to a reduced number oflaboratories. Introduction of the Polymerase Chain Reaction (PCR) technology addssensitivity and specificity to HLA typing. Most PCR based methods include: DNAamplification with sequence specific primers (SSP) followed by hybridization withsequence specific oligonucleotide probes (SSOP). Sequencing is taking moreimportance as the number of HLA alleles are increasing and making more difficultiesin the specific accession of every allele. So far, among the most advancedapproaches of the future of HLA typing is DNA chip technology, which uses 61
  • 62. thousands of probes attached to silicon chips. Hybridization is conducted on thesurface of the chips and the results are obtained after a fluorescence reading, inseconds (Eggers et al, 1994).The HLA system constituting the human MHC has gene cluster spans around aregion of about 4000 k.b on short arm of chromosome six in the distal portion of 6p21.3 band (Lamm and Olaisen, 1985). Large number of genes with variableexpression are arranged in form of three regions in MHC as class I (36 genes), classII (27 genes) and class III (39 genes) as first proposed by Jan Klein in1977(Campbell And Trowsdale, 1993) as given in figure 1. Moving from telomere tocentromere, class I genes are placed first followed by class III and finally class II.The Class I region which is the most telomeric part of the complex contains genesfor the classical HLA antigens: HLA-A, B and C, as well as four non-classical Class Igenes: HLA-E, F, G and H. The central region contains more than 20 genes. Genesof this region encode structurally and functionally diverse molecules such as: serumcomplement proteins (C2, C4A, C4B, BF), tumor necrosis factors (TNF), heat shockproteins (HSP) and adrenal 21-hydroxylase enzyme (CYP 21B). The Class II regionencodes both alpha and beta chains of the three types of class II molecules: HLA-DR, DQ and DP.The Class-I and Class-II genes are among the most polymorphic genetic systemsknown. While class I products play a role in determining viral specificity and MHCrestriction (Zinkernagel And Doherty, 1997; Koopmann et al. 1997), Class II areinvolved in peptide presentation and cell to cell interaction during and immuneresponse (Pieter, 1997). The products of class III genes are components of humoral 62
  • 63. immune response (Baltimore D, 2001). The function of MHC is to present antigenicpeptides to the antigen-specific T-cell receptors. The Major HistocompatibilityComplex controls potent transplantation antigens that elicit the rejection process.FUNCTIONS OF MHCThe normal physiological function of the MHC remained largely unknown until theseventies. The control of immune responses to polypeptide antigens had beenlocalized to the H-2 complex in 1969 (Mc Devitt, 1969) and the identity betweenMHC genes and immune response (Ir) genes was confirmed in 1972 (Shevach,1972). The immune reactions were shown to involve interactions between MHCmolecules and T-lymphocytes (Rosenthal, 1973) and the T-cell response wassubsequently shown to be restricted not only by the antigen, but also by the MHCmolecule (Zinkernagel, 1974). It was furthermore found that MHC molecules wereinvolved in the production of antibodies and that this process also was MHCrestricted (Kindred, 1972) by gene products from the class II region (Katz, 1973).Today we know how MHC molecules function in detail and from crystallographicstudies also the structure of MHC class I (Bjorkman, 1987) and II molecules (Brown,1993).HLA - CLASS I GENESThe cell surface glycopeptide antigens of HLA – A, -B and -C series are called HLAclass I antigens (Roitt et al, 1998). They play a key role in the recognition anddestruction of cells carrying foreign antigens by cytotoxic t- Lymphocytes. The X–raycrystallography study of HLA class I molecules (Browning And Mc Michael, 1996)has demonstrated that the molecule has a cleft on its outermost surface for peptide 63
  • 64. binding. The proteins are expressed on the surface of the antigen presenting cells asheterodimeric molecules. Class I molecules are composed of heavy α - chain and alight β2 microglobulin chain. The β2 chain encoded by genes on chromosome 15 isneeded for transporting α chain (MHC encoded) to the cell surface. Immunologicalstudies indicate that HLA –B which is highly polymorphic is the most significant HLA– class I locus followed by HLA-A and then HLA –C. Nucleic acid-based methods forallele identification have revealed more than 470 polymorphic variants at the HLA-Blocus. Screening of potential bone marrow donors with sequence specific primerpolymerase chain reactions and sequence specific oligonucleotide probehybridization assays revealed apparent variants within the B*58, *44, *15, and *48allele groups.(Williams et al ,2002). Majority of differences among alleles are foundin exons 2 and 3. DNA based HLA typing focuses on these polymorphic exons toidentify HLA alleles. Genes in the MHC are important determinants of host responseto microbial infections (Hart. T.C., 1996) and also has a profound effect on theimmunogenetic susceptibility to various disease that are influenced by immunereactions (Firatli et al, 1996)CLINICAL RELEVANCES OF HLAThe determination of HLA alles has been extensively utilized in tissue typing fortransplantations, paternity determination and disease association. The determinationof HLA alleles, or HLA typing, has been extensively utilized in tissue typing fortransplantation, paternity determination and disease association. Upon theapproval of the idea of a link between longevity and MHC in animals, it became afield of interest for a human longevity studies. The idea of a link between longevityand MHC was first supported in animals in the research of Smith and Walford in 64
  • 65. 1977 and lately was approved by other gerontologists. They have shown that thewhole MHC is a region associated with a significant effect on life span. The age-specificity may be under partial control of H-2 or HLA-linked genes (Meredith et al,1979). Major methodological problems which make confusions in this field of impactof polymorphic HLA genes on human longevity arise from insufficient number of thesamples included to reach statistically significant conclusions; missing of theappropriate criteria for the selection of the participants for immunogerontologicalstudies; non comparison of the results with well selected control group; nonconsidering the sex influence on the HLA polymorphism associated with longevityetc. The loci investigated within the HLA region differ from study to study. During thelast two decades, among the studies concerning the HLA and human longevity,many different HLA loci belonging to both antigen classes (I and II) have beenincluded. In the study of several HLA-Class I (A, B, C) and HLA DR antigens byProust et al, in 1982, no significant differences at these loci in comparing the totalyoung and elderly series were found, pointing out that the sex differences should betaken into account. Also the hypothesis that certain HLA haplotypes are associatedwith the survival advantage was supported. Having the same aim, many authorshave studied certain HLA antigens in different populations such as Dutch (Laggay etal. 1991), Chinese (Ma et al. 1997), Japanese (Akisaka et al 1997,1998), Korean(Kyuong-Ok et al, 1996), Italian (Ricci et al., 1998), Greek (Papasteriades et al,1997) and French (Rayana Ivanova et al., 1998) population showing very differentand poorly overlapping data. In the study of Ricci et al. four antigens (HLA-A31 (19),-B7, -Cw7 and –DQ1) showed a higher frequency in the elder group, whilePapasteriades et al., found an increased frequency of HLA-B16 and HLA-DR7 and adecreased frequency of HLA-B15 and HLA-DR4. Recent investigation of Ivanova et 65
  • 66. al., showed that HLA-DR13 alleles were increased in centenarians of both genders,HLA-DR7 was elevated in longevious men and HLA-DR11 higher in women fromsibships, unchanged in women centenarians. This and several other studiesprovided evidence for association of some HLA-DR alleles with human survival andlongevity. These results are in agreement with other published data and suggest thatHLA antigens are associated with longevity, either indirectly through diseaseassociations or directly through involvement in the aging mechanism.HLA typing was applied to kidney transplantations very soon after the first HLAdeterminants were characterized (Terasaki 1992; Opelz, 1985; Sanfilipo et al. 1984).During the late sixties, Terasaki, and co-workers presented early data indicating thepotential beneficial effects of HLA matching on cadaveric kidney transplant survival,although these findings were based on typing information with a limited set of rathercrude anti-HLA antisera. Furthermore, van Rood’s group, Batchelor and Joysey, andother investigators also found that matching for HLA improves kidney and skin graftsurvivals. The conflicting presentations by Terasaki’s group at the Third InternationalCongress of the Transplantation Society in The Hague in 1970 producedconsiderable controversies about the significance of histocompatibility matching inkidney transplantation. Many well-matched kidney transplants failed early andconversely, badly matched kidneys did often enough function quite well. Otherinvestigators had noted that same experience and it was not really surprising thatmany transplant surgeons chose to ignore tissue-typing results. Of course, all thesecontroversies arose when HLA matching was limited to an incomplete set of HLA-Aand HLA-B antigens; there was no typing for HLA-DR and the available serologicaltests had a rather low level of reproducibility. Because of improved serologicalprocedures and especially, the application of DNA-based techniques, HLA 66
  • 67. compatibility can now be much better defined and there remains no doubt that HLAmatching correlates with less rejection and prolonged kidney transplant survival.Histocompatibility testing for organ transplantation requires usually a cross matchtest between recipient serum and donor cells. Starzl and co-workers reported in1965 the first case of a patient with complement-dependent anti-donor antibodies.This patient rejected almost immediately a kidney transplant from this donor and thetissue pathology suggested a Shwartzmann reaction-like mechanism. Kissmeyer-Nielsen in Copenhagen reported a similar case of what he termed a hyper acuterejection. This experience established the cross match test as a major test inhistocompatibilty testing. The Panel-Reactive Antibody (PRA) test was first reportedby Terasaki in 1971 to identify presensitized patients at higher immunological risk ofrejecting their transplant. There is evidence that the cornea may survive slightlybetter if the HLA class I antigen is matched (Mayer et al, 1983). Complete HLAmatching of bone marrow donor and recipient is crucial to the success of allogenicbone marrow transplantations (Shankarkumar And Undevia, 1999; Ghosh, 1999;Shankarkumar, 2001). The vast polymorphism of HLA system makes it a mostvaluable tool in the field of paternity testing (Bryant, 1988).HLA – B GENETIC DIVERSITY - POPULATION STUDIESInitial knowledge of highly polymorphic HLA complex system that was developedover the second half of 20th century allowed us to understand its role in the immuneresponse and its unique contributions to anthropology and population genetics(Charron, 1997). Distributions of HLA antigens in various ethnic groups of the worldand India have been reported. HLA –B40 is a common B locus antigen in Asians, butthe alles underlying this antigen depend upon the population (Clayton et al, 1997). 67
  • 68. HLA – B4006 is of high frequency among north Indians and is common among otherAsian populations (Mongolians, Chinese and Japanese). HLA-B35 is a frequentantigen in Caucasians, the prevalent Allele being HLA – B3501 (Clayton et al, 1997;Satz et al, 1995). North Indians have a higher frequency of B35 than other Asianpopulation with – B3503 as the most common subtype. B3503 is also a common Ballele in south Indians (Tait et al). HLA – B61 was the most frequent allele inDravidian tribal population of south India. Some of the most frequent HLA – B allelesin the non tribal Dravidian population were similar to north Indian population andincluded HLA B 07 , B 61 , B 51 , B 35 AND B 52 ( Thomas et al , 2003 ) HLA-B44 AND HLA – B57 ( a typical Caucasian haplotype ) were found in “ Iyers ”(Brahmins ) of Madurai ( Susuki et al , 1992 ). HLA – B5 and HLA –B7 were commonamong Brahmins of western India (Shankarkumar et al, 2002). Namboothiris who arebelieved to be migrants from North India showed a higher frequency of HLA B*07,B*35, B*61and B*52 which is common among the North Indian population. HLAB*44, common among the South Indian population was of least frequency amongNamboothiris. Two alleles (HLA B*39 and HLA B*56), which were present only intribal population of Kerala was observed in Namboothiri population alsoHLA-B AND DISEASE SUSCEPTIBILITYThere are two general explanations for HLA and disease association (Mc Devitt,1985). Firstly, there may be a linkage disequilibrium between alleles at a particulardisease associated locus and the HLA antigen associated with that disease.Secondly the HLA antigen itself plays a role in disease. MHC gene products can beinvolved in the pathogenesis of, especially autoimmune, diseases. The pathologicalfunctions of certain MHC polymorphisms became known at the same time as the 68
  • 69. normal physiological functions of the MHC gene products were reveled. In 1967, itwas found that certain MHC polymorphisms were associated with a higher risk fordeveloping Hodgkins disease (Amiel, 1967) and soon thereafter many diseaseswere shown to be associated with MHC genes. Major histocompatibility complexlinked immune response or immune suppressive gene (Benacerraf, 1981) hasresulted in various disease association studies at population level to seekassociation with HLA (Tiwari and Terasaki, 1985). Studies on genome scan haveimplicated that the genes in MHC region are contributing more to diabetessusceptibility (Davies et al., 1994). HLA – B27 shows remarkable association with`ankylosing spondylitis` and related spondylarthropathies in most populations in theworld (Genzalez – Roces et al., 1997); Ramos and Lopez de Castro, 2002). Workdone in Hungarian population showed a close association between HLA –B8 and `Graves disease`. B40 and B57 are seen closely associated with `SLE`, which is anautoimmune disease (Pradhan et al., 2004). HLA –B39 allele was found significantlymore often in insulin dependent diabetes mellitus patients (Sergei Nejentsev et al.,1997) HLA – B54 is involved in the pathogenesis to diffuse Panbrochiolitis in Koreanpatients (Myoung Lee Park et al; 1999) 69
  • 70. Gene map of the human leukocyte antigen (HLA) regionFigure 1: Gene map of the human leukocyte antigen (HLA) region. The HLA regionspans 4 × 106 nucleotides on chromosome 6p21.1 to p21.3, with class II, class IIIand class I genes located from the centromeric (Cen) to the telomeric (Tel) end. HLAclass I molecules restrict CD8+ cytotoxic T lymphocyte function and mediate immuneresponses against ‘endogenous’ antigens and virally infected targets, whereas HLAclass II molecules are involved in the presentation of ‘exogenous’ antigens to Thelper cells. The HLA class III region contains many genes encoding proteins thatare unrelated to cell-mediated immunity but that nevertheless modulate or regulateimmune responses in some way, including tumour necrosis factor (TNF), heat shockproteins (Hsps) and complement proteins (C2, C4) 70
  • 71. Figure 2: The Graph indicates the numbers of antigens and alleles named since theHLA Nomenclature Committee (IMGT HLA database) was first formed in 1968, up tothe end of December 200414, 15. 71
  • 72. SYNOPSISFull title of the thesisA CONCEPTUAL STUDY ON PRAKRITHI IN CORRELATION WITH MOLECULARPROFILESIntroductionPrakrithi - Prakrithi means “swabhava” or nature of an individual. According to“Arunadutta” it means physical condition. According to “Vagbhata” the Prakrithiremains inherited throughout the life, prior to death.The state of dosha in “sukra”(sperm) and “arthava”(ovum), the dosha predominanceat the time of fertilization, doshic state of garbhasaya (uterus), the influence ofenvironment on tridosha with respect to season during the intra uterine development,the dietetics of mother, mode of living & the nature of physical & mental activities ofmother rendering the doshas susceptible to vitiation during the child bearing period,are the factors which constitute the Prakrithi of an individual. So it is highly difficult toprovide any information regarding the actual mechanism involved in the process ofsegregation of Prakrithi, but Prakrithi can be assessed from ones’ physical structure,reaction to environment, feelings, thoughts & psychological makeup. But we can seethat only hereditary factors & post conceptional factors play a deterministic role.To understand the actual mechanism involved in the formation of a particularPrakrithi, knowledge of “LAW OF HEREDITY” (Anuvamsi siddhantha) is highlyessential. According to Ayurveda, “Bija” (Sperm & Ovum) is a collection of functionalunits, which by representing the structures and features of all parts and organs of anindividual is capable of ensuring the formation of offsprings resembling the parents.Such functional units are called “Bijabhagas”, which can be more or less similar to 72
  • 73. the term “Gene” in modern genetics. So DNA can be compared to “Bijabhagaavayava”.Molecular Profiling (MP)Molecular Profiling (MP) has emerged as a dynamic new discipline, capable ofgenerating a global view of mRNA, protein patterns, and DNA alterations in variouscell types and disease processes.MP integrates the expanding genetic databases from the Human Genome Projectwith newly developed expression analysis technologies and holds great promise tohelp us: ü Understand the molecular anatomy of normal cells and cells in various stages of disease. ü Develop new diagnostic and therapeutic targets for clinical intervention. ü Explain the relationship between genotype and phenotype in humans, which is still largely unknown.More technical and theoretical details shall be provided with methodology of thestudy.Since this technique is of high precision and need enormous complicated proceduresit will be of greater convenience to study the DNA fingerprinting as well, which maycome out with satisfactory results.DNA fingerprinting in humans Genetic uniqueness of an individual is brought about by two factors,inheritance and new mutations. Genetic fingerprinting techniques are suitable foranalyzing the diversity present within or between populations. Traditionally the term 73
  • 74. ‘Fingerprint’ refers to the pattern of fingertip ridges, which are highly characteristic forany individual. For quite a few years, this term has also been used for theelectrophoretic and chromatographic characterization of proteins and more recentlyof DNA molecules. The size of the human nuclear genome is approximately 3x109 base pairs. Allgenetic differences between individuals are laid down in the primary sequence of thisgenomic DNA. An efficient strategy for identifying an individual would be to comparethose regions of the genome, which are already known to differ frequently betweenindividuals. Such regions are termed polymorphic sites and it has been observedthat repetitive sequences present in the genomes of virtually all species are highlypolymorphic. Genetic polymorphism is defined as the simultaneous occurrence in thesame population of two or more variants. The concept of genetic polymorphism isfundamental to all current methods for determining genetic identity and relatedness.The first step in characterizing a biological sample at the genome level is to purify itsgenetic material, DNA. The source of DNA can be any biological sample containingnucleated cells, such as, blood, semen, hair, bones, saliva or urine. The nuclearDNA is compacted with proteins to form chromatin, which condenses to formchromosomes. DNA is extracted from the other components mainly by the action ofenzymes, protein denaturing agents, salts and organic solvents like phenol andchloroform. The isolated DNA can then be subjected to enzymatic amplification invitro termed as Polymerase Chain Reaction (PCR). The amplification of selectivegenomic regions including single copy sequences, as well as, repetitive sequences,like micro satellites, could be employed successfully for comparison of genotypes.The advantage of PCR technique is that it works with small amounts of DNA, and 74
  • 75. functions even if DNA is degraded or not very pure. The banding patterns generatedcan later be used to analyze the extent of diversity /relatedness between individuals.NEED AND SIGNIFICANCE OF THE STUDYThe assessment of Prakrithi is of great importance in identifying the bodyconfiguration and systemic status of an individual. Prakrithi is unchangeable; itrepresents the part of human personality, which is mostly determined by thehereditary factors. Till now we have many subjective & objective parameters forassessment. The world of genetics is growing day-by-day and evolving newdimensions in medical research. Inorder to enter into the main stream of medicalworld it is necessary to have insight into the concepts derived in genetics. Hence thisis only a preliminary study to find out the correlation of innate constitution (Prakrithi)with DNA.AIMS AND OBJECTIVES OF THE STUDY1. Detailed literary study of “Prakrithi” from various texts in Ayurveda.2. To analyze the role of DNA in determining Prakrithi by conducting a fingerprintstudy.3. To understand the genetic correlation of various forms of Prakrithi4. To develop standards for a uniform guideline in analyzing PrakrithiNULL HYPOTHESISMolecular profiles cannot help in determining the Prakrithi (constitution) of anindividualALTERNATE HYPOTHESISMolecular profiles can help in determining the Prakrithi (constitution) of an individual 75
  • 76. METHODOLOGYThe Prakrithi of persons will be assessed using Ayurvedic references. The personswill be divided into different groups according to Prakrithi. Blood sample taken fromthese persons will be subjected to DNA fingerprint study (PCR technique). Finalanalysis will be done to assess any similarity in the molecular profiles of personshaving similar Prakrithi.Inclusion Criteria1. Normal healthy individuals from different geographic area2. Both sex3. Informed consentExclusion Criteria1. Individuals in the same family2. Unwilling persons 76
  • 77. Consent LetterI, Dr. K.N.Ajithkumar, II MD Scholar, Govt. Ayurveda College, Kannur propose toconduct an Ayurvedic research on Prakrithi in correlation with Molecular profiles; inthe guidance of Dr. Moinak Banerjee, Scientist, Rajiv Gandhi Centre forBiotechnology, Thiruvananthapuram.The data developed in the work will be kept confidential and will be used onlyfor research purpose and will not be used or disclosed for any otherapplications. The data is not used for any commercial purpose.I …………………………………………………….have gone through the letter and iswilling to co-operate with his work. Signature of the ConsentSignature of the InvestigatorRGCBDate 77
  • 78. DEPARTMENT OF ROGANIDANA GOVT.AYURVEDA COLLEGE KANNURCASE PROFORMA FOR A CONCEPTUAL STUDY ON PRAKRITHI INCORRELATION WITH MOLECULAR PROFILES. By Dr.K.N.Ajithkumar undersupervision of Dr.R.Sreekumar MD (Ay) & Dr.Moinak Banerjee Ph.DCase No.:1. Name: Date:2. Address : Spouse Name:3. Age (Yrs) :4. Sex : 1. Male 2. Female5. Occupation : 1. Scientist 2. Office staff 3. Others6. Religion : 1. Hindu 2. Muslim 3. Christian 4. Others7. Domicile : 1. Urban 2. Rural8. Income : 1. Rs.<5000 2. 5001-13000 3. 13001-25000 4. > 250019. Medical History Diabetic Respiratory disease Hepatic Renal disease Thyroid disease Neurological problems TB Others10. Personal history Alcohol consumption Yes No Smoking Yes No Drug abuse Yes No11. General examination a. Pulse ......................./mt b. Weight ........................Kg c. Height .......................Cms12. Systemic Examination 1. CVS : 2. CNS : 3. R.S : 4. GIT : 5. Locomotor : 6. Uro-genital : 7. Ophthalmology & ENT : 8. Dermatology : 78
  • 79. FORMAT FOR PRAKRITHI PAREEKSHA Vatha Pitha Kapha1.BODY Lean Medium Stout Weak Fleshy Large Tall/ Short Delicate Soft Unshaped Unshaped Beautiful Prominent veins and tendons Loose Cold in touch Delicate joints Symmetrical Rigid Emits bad smell Firm Rough Proportionate Compact Un proportionate Lustrous Stable Undernourished Long developed Well developed Low body temperature2. SKIN Dry Wrinkles Oily Rough Fair Soft Lusterless Warts Glossy Blackish Moles Fair Reddish / yellow White/pinkish Bluish patches Glorious Soft3. EYE Blackish Reddish/Coppery White Dry Small RedlPink angles Lusterless Rounded Wide and long Small Quickly becomes red Pleasant Fluctuating Sharp eye sight Oily Eyelids open while sleeping Pleasant Calm Desires cold Gentle look Well designed White and Black spheres4. EYEBROWS Unstable Nothing specific Plenty of hairs Thin Thick Small5. EYE LASHES Small Thin More Dry Few Firm Oily 79
  • 80. 6. HAIRS OF BODY/HEAD & MOUSTACHE Scanty Soft Strong Rough Brown/coppery Thick Cracking Small Curly lusterless Few Oily Grayish Early baldness Black Curly Early graying Proportionate7. HEAD Unstable Nothing specific Steady8. LIPS Dry Red coloured Pinkish Cracking Soft Smooth Shapeless Thin Glossy Unstable Blackish9. TEETH Coarse Medium Firm Thin With gap Strong Few Many Protuberant Healthy gums Cracking Even Irregular Glazing Straight Smooth Shining white10. TONGUE Unstable Reddish / coppery Nothing specific11. FACE Rough Fleshy Pleasant Worried look12. FOREHEAD Small With folds Large13. LOWER JAW Small Nothing specific Big14. JOINTS Unstable Loose Firm Protuberant Moderately hidden Compact Sound producing Concealed on movements15. PALMS Unstable Reddish Big Dry Oily Rough Firm Cracked16. FOOT Unstable Reddish Nothing specific Rough Cracked17. NAILS Rough Coppery/reddish Large Thin Soft Thick Small Small White Blackish Flat Firm 80
  • 81. Cracking Convex Breaking Glossy18. CHEST Small Nothing Specific Big Not well built Elevated19. GAIT Unsteady Fast Slow Light Stable Quick Foot pressing Producing sound against ground Wandering Habit of moving hands /Iegs/shoulder20. ACTIVITIES Unsteady Un beatable Slow Quick lazy Starts any work hastily21. MUSCLES Bulged calves Loose / flaccid Well build22. STRENGTH Less Moderate Good Tires Quickly Cannot withstand Does not feel physical or mental tired after Work/ exertion physical or mental Work/ exertion23. SPEECH/ VOICE Talkative Clear Gentle Irrelevantspeech loud Speaks little Obstructed Talkative Clear voice Hoarse Good and impressive Slow Fast Good at arguments Steady Diffused Speech Firm Resonant voice24. FOOD HABITS Eats much Excessive hunger Likes hot food Eats swifty Eats much Dry food Takes light food Like cold/warmfood East slowly Likes hot food Eats / drinks often less hungry / thirst Irregular diet Prefers sweet likes spicy Irreg. digestion Prefers bitter likes bitter Prefers sweet Prefers astringent likes astringent Prefers sour likes sweet Prefers salty Weak in digestion Prefers Oily25. STOOL/BOWEL Hard bowel Expels frequently Yellowish well Tenaency of Large quantity formed constipation Yellowish loose Once in a day 81
  • 82. 26. URINE Obstructed Expels frequently Nothing specific Large quantity Yellowish27. SWEATING Nothing particular Sweat very easily less sweating Foul smell28. HUNGER/ THIRST Upredictable Good less Cannot tolerate Can tolerate29. IMMUNITY less Moderate Good30. LIFE SPAN Short Medium long31. SEXUAL DESIRE less Medium More32. REPRODUCTIVE STRENGTH Less semen / Less inclined to sex Much semen / irregular regular menstruation Unlike by women/ menstruate men Poor capacity Less semen/ovum Liked by women /men Un attracted by opposite sex Sexually powerful.33. BEHAVIOUR Coward Aversion to heat Stability Stealing Afraid of discomfort Control over senses Atheis Affectionate to dependence Grateful Unstable Like being praised Humble No control over senses Proud Generous Quick attachtment Courageous Long concealed and detachment enimity Good behaviour Not greedy Noble less Adventurous Caim Sorrowful Jealousy Quiet Less intelligent Generous Obedient Ungrateful Scholar Straight forward Aversion to cold Intelligent Religious Biting nails Clean Honour the teachers Grinding teeth while slieeping Well educated Civilised34. FRIENDSHIP Few Dependable Stable Unsteady35. ANGER Quick Quick Rarely36. FEAR Quick Rarely Not specific 82
  • 83. 37. DREAMS Climbing hills Fire /lightning Water / birds / / sky clouds38. Sleep Interrupted Sound Sound < 6 hrs. 6 - 8 hrs. < 8 hrs.39. GRASPING POWER Some times quickly / slowly Always quickly Always grasp late Very clever Genius40. MEMORY Forgets,quickly Moderate Good41. HOBBIES Music Fond of garlands Talented Humour Perfumes Hunting Decorative Gaming Music42. INTOLARANCE Cold Hot Can tolerate Anger cold/heat/ angry/hunger thirst, Hunger physical urges Physical urges Thirst43. PROBLEM FACING Worrying constantly Can take right Can take firm quick decision right firm calm decision and stable mind Cannot take stable decisionTotal number of Characters of Vata - 133Total number of Characters of Pitta - 118Total number of Characters of Kapha - 131Percentage of characters of one Dosha = No. of Characters present x 100 Total No. of CharactersPrakrithi of the individualVata....................... %Pitta....................... %Kapha.................... % = % of Characters present x 100 Total No. % 83
  • 84. Assesment of Dhehaprakrithi of.........................................................................isVata Pitta Kapha Vata PittaVata Kapha Pitta Kapha Samadosha 84
  • 85. MATERIALS AND METHODSSTUDY POPULATION Blood samples (N = 40) were collected with informed consent from different communities. Due care was taken to avoid sampling from related individuals and siblings. DNA isolation was performed using standard organic extraction methods. Molecular typing was carried out using the polymerase chain reaction.The main steps carried out in this analysis include: • Preparation of DNA • Amplification of genes • Detection of amplificationPREPARATION OF DNA1 Ø SAMPLE REQUIREMENTS v Whole blood is drawn into a vial with anticoagulants containing EDTA. Ø MATERIALS AND REAGENTS v Lyses buffer I v Lyses buffer II v 2% SDS v Proteinase K v Tris – phenol (pH 8.0) v Chloroform-isoamyl alcohol (24: 1) v Absolute alcohol v Sodium acetate (3M pH5.2) v 70% Ethanol v Tris – EDTA solution 85
  • 86. EQUIPMENTS AND INSTRUMENTATION v Deep freeze refrigerator v Water bath v Centrifuge v Homogenizer PREPARATION OF BUFFER SOLUTIONS 1. Tris (1M), 500ml [NH2C (CH2OH)3 M.W=121.14] Take 60.5gm of Tris base and dissolve it first in little amount of water, add more water slowly and stir well. Adjust the pH to 8 by adding required Hydrochloric acid. 2. EDTA (0.5M), 250ml Take 46.5gm of EDTA and dissolve in 200ml of water. Stir well. While mixing, add Sodium hydroxide pellets and adjust the pH to 8. Make the final volume to 250ml. 3. NaCl (5M) Take 58.4gm of Sodium chloride in 200ml of water and dissolve it bystirring. 4. Lysis buffer I (TEN 30:5:50Mm) Tris (1M) 7.5ml EDTA (0.5M) 2.5ml NaCl (5M) 2.5ml Distilled water 250ml 5. Lysis buffer II (NE 75:2Mm) NaCl (5M) 3.75ml 86
  • 87. EDTA (0.5M) 1ml Distilled water 250ml 6. T.E buffer (10:1Mm) Tris (1M) 1ml EDTA (0.5M) 0.2ml Distilled water 100mlØ PROCEDUREThe extraction2 is first done using phenol: chloroform and then with chloroform. Inthis case, deproteinisation is more efficient. Although phenol denatures proteinsefficiently, it does not completely inhibit RNAase activity and it is a solvent for RNAmolecules that contain long tracts of poly adenyline. Both these problems can beavoided by using phenol: chloroform: isoamylalcohol. The subsequent use ofchloroform removes any trace of phenol from nucleic acid preparation. 1. To 10ml of peripheral venous blood, collected in EDTA vial, equal volume of (10ml) RBC lysis buffer was added and frozen at –80oC for minimum of 3 Hrs. (usually over night). 2. Sample was kept in 65oC water bath for rapid freeze thow. During this step RBCs were lysed. 3. RBC lysed sample was centrifuged at 10,000 rpm for 10 min at 4 oC. Supernatant containing RBC (lysed) was discarded, WBC pellets obtained for DNA isolation. 4. To the above pellet – equal volume (10ml) WBC lysis buffer was added, and homogenized in a hand homogeniser, till the pellets get thoroughly dispersed. 5. SDS (Sodium Dodecyl Sulphate) 2% of volume (10ml blood – 1ml SDS) and proteinase K (150 μg/ml working conc.) (10ml blood – 75μl from a stoke conc. 87
  • 88. of 20mg/ml) was added to the above lysate, and incubated at 37oC overnight in water bath. During this step WBC’s were ruptured by SDS and protein in the cells were digested by proteinase K.6. Equal volume (10ml) Tris Saturated Phenol (pH 7.5) was added and mixed gently. Phenol removes protein contaminants.7. Lysate was centrifuged at 10,000 rpm for 10 min. at 4oC. Phenol forms the lower layer containing denatured proteins (organic layer). The supernatant (aqueous layer) is collected in a fresh tube carefully using a micropipette, without disturbing the organic layer.8. To this aqueous layer half volume (5ml) of Tris Saturated Phenol is added and mixed gently for 2 minutes.9. To the above mixture half volume (5ml) of chloroform: iso-amyl alcohol (24:1) also added. Mix gently till proper mixing occurred (almost 10 min.). Then centrifuged the mixture at 10,000 rpm for 10 min. at 4oC.10. The aqueous layer is collected in a fresh tube carefully using a micropipette. To this, equal volume (10ml) of chloroform: iso-amyl alcohol (24:1) also added. Mix gently till proper mixing occurred (almost 10 min.). Then again centrifuged the mixture at 10,000 rpm for 10 min. at 4oC.11. The aqueous layer was collected and 1/10th volume 3M Sodium Acetate (pH 5.2) and double volume chilled absolute alcohol was added.12. Mix gently for a while, the DNA will be precipitated as a lump. Transfer the DNA to an eppendorf tube containing 70% ethanol using a micropipette. 88
  • 89. 13. Wash (centrifuge) the DNA twice with 70% alcohol and twice with absolute alcohol (5000 rpm 5 min. at room temp.). 14. The pellet was air-dried and dried pellet was suspended in Tris - EDTA buffer. It is a low ionic strength buffer. The DNA is first made to dissolve in small amounts of Tris - EDTA, and then more is added. Rinse the walls of the tubes well with the buffer 15. Store the DNA at 4oC. For prolonged storage –20oC or -70oC is used. DNA QUANTIFICATIONOne methodSPECTROPHOTOMETRY3The quality and quantity of genomic DNAwas checked in a spectrophotometer(Biospec-1601, DNA/Protein/EnzymesAnalyzer, Shimadzu). The ratio ofabsorbance at 260 nm (normal light) and 280 nm (260/280) was used to estimate thepurity of the DNA. 1. Place two blank cuvettes in the spectrophotometer filled with distilled water (100 ul) 2. Select the DNA – Protein protocol in the spectrophotometer 3. Standardize the value to ‘0’ (zero) 4. Remove the distilled water from one blank cuvette and add DNA (i.e distilled water 98 ul and DNA 2 ul) 5. Place the cuvette in the spectrophotometer and the value will be displayed in the monitor. (In Amstrong units) 89
  • 90. 6. Convert the Amstrong value to ngm/ul. 7. A ratio between 1.7-1.8 indicates good quality DNA without protein contamination. The absorption of 1 OD (A) is equivalent to approximately 50 µg/mL of double stranded DNA. The concentration of DNA in 1 µL of the DNA sample was calculated using the following equation. 50x OD A260 x Dilution factor 1000Another method AGAROSE GEL ELECTROPHORESIS4 1. 0.7 % Agarose in 0.5X TBE Buffer (Stock 5X) (0.7 gm Agarose + 100 ml 0.5X TBE) 2. Microwave heat until agarose is dissolved and solution is clear. 3. Allow the solution to cool to about 55oC before pouring. Ethidium bromide is added and mixed well before pouring. 4. Prepare the gel tray by sealing the ends with tape. 5. Place the comb in the gel tray and position the comb vertically such that the teeth are about 1-2mm above the surface of the tray. 6. Pour the gel solution into the tray to a depth of 5mm. Allow the gel to solidify for about 20 minutes at room temperature. 7. Gently remove the comb, place the tray in electrophoresis chamber and submerge it with the same buffer used to prepare the gel. 90
  • 91. 8. To prepare samples for electrophoresis add 1ul of dye to the 1ul DNA and 4 ul of sterile distilled water mix well using a centrifuge. 9. The samples are then pipetted into the sample wells. 10. 80 Volts current is applied. This can be confirmed by observing bubbles coming off the electrodes. 11. DNA will migrate towards the anode, coloured red. 12. The distance DNA has migrated in the gel can be judged by monitoring the migration of the dye. 13. After adequate migration, the gel can be viewed using a trans illuminator. The DNA quantity can be detected as a band. 14. Save the gel using a multi- Imager.TBE Buffer 5X (Stock)Tris – 54 gmBoric Acid - 27.5 gmEDTA (0.5 M) – 20 mlDistilled Water – 1000 mlpH - 8.00 Loading dye Single dye : Bromophenol blue - 0.25% Glycerol - 30% Double dye: Bromophenol blue - 0.25% (for small fragments) Xylene cyanol - 0.25% Glycerol - 30% 91
  • 92. Gel showing DNA after electrophoresis for quantificationAMPLIFICATION OF GENESMOLECULAR METHOD The PCR is used to generate a large number of copies of a gene for rapiddetection and to reduce non-specific hybridization of oligonucleotide probes. Sincethese methods are performed at genetic level, better resolution of the alleles can beachieved. Polymerised Chain Reaction (PCR)5PCR is a procedure based on the ability of DNA polymerase to copy a strand of DNAby elongation of complementary strands initiated from a pair of closely spacedoligonucleotide primers. Each cycle of the reaction doubles the amount of the targetDNA, leading to million fold levels of amplification. PCR has high sequencespecificity because the two unique primers must hybridize in relatively closeproximity to each other on the target DNA sequence under stringent temperature andreaction conditions before exponential amplification can occur.Ø PRINCIPLES OF PCR6 There are three major steps in a PCR, which are repeated for 35 or 40 cycles in anautomated cycler, which can heat and cool the tubes with the reaction mixture in avery short time (Figure 3). 92
  • 93. 1. DENATURATION AT 94 oC: During denaturation, the double strand melts open to single stranded DNA, allenzymatic reactions stops.2. ANNEALING AT 50 oC: The hydrogen bonds are formed and broken between the single strandedtemplate. The more stable bonds last longer and on that double stranded DNA madeof the template and the primer, the polymerase can attach and start copying thetemplate. Once few bases are built in, the hydrogen bond is so strong between thetemplate and the primer that it does not break anymore.3. EXTENSION AT 72oC: The primers, where there are a few bases built in, have a stronger attractionto the template, created by hydrogen bonds, than the forces breaking theseattractions. Primers having no exact contact with the template do not give anextension of the fragment. The bases are coupled to the primer on 3’ side, thepolymerase adds dNTPs from 5’ to 3’, reading the template from 3’ to 5’, and basesare added complementary to the template.Ø SAMPLE REQUIREMENTS2µl of the DNA is required for each PCR.Ø MATERIALS AND REAGENTSDeoxynucleoside triphosphate (dNTP) solutionPCR primers (forward and reverse)Taq A or 10 X polymerase bufferTaq enzyme with Mg2+ ionsSterile distilled water 93
  • 94. Ø EQUIPMENTS AND INSTRUMENTATIONThermal cycler (PCR mechine, eppendorf)Vortex mixerMicrocentrifugeØ PROCEDURE 1. Assemble the PCR mix for a single sample: 2ul of PCR buffer, 2ul of dNTP Solution, 0.2ul of each primer, about 0.3ul of Taq enzyme and water required to bring the final volume to 20ul. 2. Aliquot approximately 2ul of the DNA samples into the appropriate reaction tubes. 3. Vortex the PCR mix well, so that the components get evenly distributed. 4. Centrifuge in a micro centrifuge. 5. Place the samples in the Thermo cycler. 6. PCR amplification was carried out in eppendorf Thermal Cycler machine with heated lid option. The cycling parameters for 20 µL reactions were as follows: initial denaturation at 94°C for 5 minutes followed by 30 seconds at 94°C, 30 seconds at 50°C, 30 seconds at 72°C followed by, 5 minutes at 72°C followed by 3 seconds at 40 C comprising one cycle. 7. The time and the temperatures for denaturation, primer annealing, and extension will vary depending on the primers utilized and the Thermal cycler chosen. 8. Store the amplified DNA at 4oC. 94
  • 95. PCR is the most efficient method for the amplification of alleles. The key forthe success of PCR is Taq polymerase. This enzyme is extracted from Thermusaquaticus, a bacterium that grows at elevated temperatures. This enzyme hasextreme thermal stability and has biochemical properties quite adequate for repeatedexposures to the denaturing temperatures required in PCR reactions. This enzymerequires Mg+2 ion for optimal activity. 95
  • 96. Gel showing DNA after amplification (in wells 1, 3, 4, 5, 9 shows good amplification.11th one is marker)POST AMPLIFICATION TECHNIQUE:RESTRICTION AND DIGESTION7Ø SAMPLE REQUIREMENTS8µl of the PCR product is required for each restrictionØ MATERIALS AND REAGENTSBSA (Bovine serum albumin) solution10 X bufferSterile distilled waterEnzymes (Specified for each genes) which is listed below Genes Enzymes DRD2 Taq 1 D Taq 1 ∝ enzyme DRD2 Taq 1 B Same as above DRD2 Taq 1 A Same as above DRD2 S311C Sau 961 enzyme DRD His313 H NCO1 enzyme DRD3 S9G MSC1 enzyme 5 HTR 1 B Hin C2 enzyme Ser 1438 MSP1 enzyme 96
  • 97. Ser 102 Same as aboveØ EQUIPMENTS AND INSTRUMENTATIONVortex mixerMicrocentrifugeWater bathØ PROCEDURE 9. Assemble the PCR product for a single sample: 2ul of PCR buffer, 0.2ul of BSA Solution, about 0.8ul of enzyme and water required to bring the final volume to 20ul. 10. Aliquot approximately 8ul of the PCR product into the appropriate reaction tubes. 11. Vortex the mix well, so that the components get evenly distributed. 12. Centrifuge in a micro centrifuge. 13. Place the samples in the PCR product over night in 370 C water bath.AGAROSE GEL ELECTROPHORESISPRINCIPLE The amplified PCR products are detected using this method. When electricfield is applied to an agarose gel in presence of a buffer solution, which conductselectricity and maintains the pH, DNA fragments that are negatively charged movesthrough the gel towards the positive electrode at a rate dependent on size andshape. Small linear fragments move quickly than larger ones, which are retarded dueto entanglement with the network of agarose fibers forming the gel. As the voltageapplied to the gel is increased larger fragments migrate faster than smaller 97
  • 98. fragments. Ethidium bromide is a fluorescent dye that intercalates between bases ofnucleic acids and allows detection of DNA fragments.Ø SAMPLE REQUIREMENTS The amplified PCR products to be detectedØ MATERIALS AND REAGENTS Agarose powder (1.2%) TBE buffer (0.5X) Gel loading dye Ethidium bromideØ EQUIPMENTS AND INSTRUMENTATION Electrophoresis chamber Power supply Gel casting trays Combs to form sample wells Trans illuminator Multi-Imager Micro centrifugeØ PROCEDURE 1. To prepare 60ml of 1.2% agarose solution, measure 0.72gm agarose into a glass flask and add 60ml of 0.5X TBE. 2. Microwave heat until agarose is dissolved and solution is clear. 3. Allow the solution to cool to about 55oC before pouring. Ethidium bromide is added and mixed well before pouring. 98
  • 99. 4. Prepare the gel tray by sealing the ends with tape.5. Place the comb in the gel tray and position the comb vertically such that the teeth are about 1-2mm above the surface of the tray.6. Pour the gel solution into the tray to a depth of 5mm. Allow the gel to solidify for about 20 minutes at room temperature.7. Gently remove the comb, place the tray in electrophoresis chamber and submerge it with the same buffer used to prepare the gel.8. To prepare samples for electrophoresis add 5ul of dye to the 20ul PCR product and mix well using a centrifuge.9. The samples are then pipetted into the sample wells.10. Current is applied. This can be confirmed by observing bubbles coming off the electrodes.11. DNA will migrate towards the anode, coloured red.12. The distance DNA has migrated in the gel can be judged by monitoring the migration of the dye.13. After adequate migration the gel can be viewed using a trans illuminator. The gene amplified can be detected as a band.14. Save the gel using a multi-Imager.Some examples are given below: 99
  • 100. Positive results showing in Taq 1D reactions (wells 4 to 8 and 12)Positive results showing in Taq 1B reactions (wells 3,4,6 to 10)Positive results showing in Taq 1A reactions (wells 3 to 9 and 11) 100
  • 101. Materials and Methods HLAMaterials Chemicals and Biologicals Glassware and plastic waresIsolation of Genomic DNADNA QuantificationHLA Typing PCR amplification using sequence specific primers HLA (PCR-SSP) Amplification Conditions Agarose Gel ElectrophoresisMaterialsChemicals and Biologicals1All the chemicals used were of either molecular biology grade or extra pureanalytical-reagent grade. Ethylene diamine tetra acetic acid (EDTA), sodium dodecylsulphate (SDS), agarose, dimethyl sulfoxide (DMSO), acrylamide was obtained from(Invitrogen, Life technologies). Various chemicals like sodium chloride, chloroform,isoamyl alcohol, phenol, Orange G, glacial acetic acid, sodium hydroxide, sodiumcarbonate, isopropanol, dimethyl formamide (DMF) was obtained from Qualigens.Proteinase K, Taq DNA polymerase, (Sigma); Boric acid, Tris base, Tris-Cl, RNaseA, X-gal, from (USB); GFX PCR DNA gel band purification system from (AmershamBiosciences) were used in the study. 101
  • 102. Glassware and plastic waresGlassware was from either Borosil or Schott-Duran. Plastic ware including multi stripPCR tubes and plates were from Axygen.Isolation of Genomic DNA from whole bloodDNA was isolated from lymphocytes obtained from EDTA anti-coagulated blood. Amodified form of the standard organic extraction method was used for DNAextraction2Briefly, an equal volume of RBC lysis buffer was added to the blood sample collectedin the EDTA vial and was frozen at –80˚C for 3 hours. The sample was then freeze-thawed at 65˚C resulting in RBC lysis. The RBC lysed sample was centrifuged at4000 rpm for 10 minutes at 4˚C. The supernatant containing the lysed RBC wasdiscarded. The WBC pellet was resuspended in equal volume of WBC lysis bufferand homogenized. SDS and Proteinase K were added to the lysate at finalconcentrations of 2.0% and 150 ug/mL respectively and incubated at 37˚C for 8hours. During this step, the SDS ruptures the WBCs and the proteins in the cells isdigested by the action of Proteinase K. Equal volume of tris-saturated phenol (pH7.5) was added to the sample and mixed gently. The sample was centrifuged at10000 rpm for 10 minutes at 4˚C. The aqueous layer was collected and the organiclayer containing phenol and denatured proteins was discarded. To the aqueouslayer, equal volumes of a mixture of tris-saturated phenol /chloroform/isoamyl alcohol(25:24:1) was added and mixed gently. The sample was centrifuged at 10000 rpmfor 10 minutes at 4˚C. The aqueous layer was collected and the organic layercontaining carbohydrates and lipids was discarded. To the sample equal volume ofchloroform/isoamyl alcohol (24:1) was added and mixed gently. The sample wascentrifuged at 10000 rpm for 10 minutes at 4˚C. The aqueous layer was transferred 102
  • 103. into a fresh tube and the organic layer was discarded. To the sample, 1/10th volumesodium acetate (3 M, pH 5.2) and twice the volume chilled absolute ethanol wereadded and mixed gently. The precipitated lump of DNA was spooled out into amicrofuge tube. The DNA was washed twice in 70% ethanol. The pellet was air-driedand resuspended in TE buffer. The DNA samples were stored in –20˚C.DNA QuantificationThe quality and quantity of genomic DNA was checked in a spectrophotometer3(Biospec-1601, DNA/Protein/Enzymes Analyzer, Shimadzu). The ratio of absorbanceat 260 nm and 280 nm (260/280) was used to estimate the purity of the DNA. A ratiobetween 1.7-1.8 indicates good quality DNA without protein contamination. Theabsorption of 1 OD (A) is equivalent to approximately 50 µg/mL of double strandedDNA. The concentration of DNA in 1 µL of the DNA sample was calculated using thefollowing equation. 50x OD A260 x Dilution factor 1000HLA TypingPCR amplification using sequence specific primersMolecular HLA typing8 was carried out using the polymerase chain reaction withsequence specific primers (PCR-SSP) method for the HLA-A, & -B loci. Amplificationprimers for HLA-B were synthesized based on published literature by Bunce et al1995 while HLA-A primers were based on a revised paper by Tonks et al 1999. Thebasic principle of the PCR-SSP typing utilizes the same PCR protocol andparameters for typing HLA-A, & -B alleles. Most of the alleles are detected in group-specific amplifications corresponding to serological specificities although some allele 103
  • 104. specific detection is possible. Thirty-four primers were used for analyzing HLA-Alocus, 59 primers for HLA-B loc. The primers were purchased as crudeoligonucleotides from Sigma-Genosys. Amplification control primers giving rise to a796 base pair fragment from the third intron of HLA-DRB1 (Olerup and Zetterquist1992) were included in all PCR reactions as internal control. The controlamplification primer sequences are listed below: Forward: CP 63 5-TgCCAAgTggAgCACCCAA Reverse: CP 64 5-gCATCTTgCTCTgTgCAgATAll primers were resuspended in sterile distilled water at stock concentrations of 100pmoles/µL and stored at –20˚C. The primers were diluted to working concentrationsof 10 pmoles/µL.HLA (PCR-SSP) Amplification ConditionsThe final volume of all PCR reactions is 15µL. The PCR reaction mixture9 consistedof 10 X PCR buffer (67 mM Tris Base pH 8.8, 16.6 mM ammonium sulphate, 0.01%(v/v) Tween 20), 2.0 mM magnesium chloride, 200 µM of each deoxynucleotidephosphate (dNTP), 0.5 pmole of each allele- specific primer, 0.5 pmole of eachcontrol primer (CP 63 and CP 64) and 0.5 Units of Taq Polymerase (BangaloreGenei, India). Around 50-100 ng of genomic DNA was used each PCR reaction. Thedifferent primer mixes are shown in table 2.5.PCR amplification was carried out in 8 well PCR strips (Axygen) in a MJ ResearchThermal Cycler (PTC 200) machine with heated lid option. The cycling parametersfor 15 µL reactions were as follows: initial denaturation at 96°C for 1 minute followedby 5 cycles of 25 seconds at 96°C, 45 seconds at 70°C, 45 seconds at 72°C followedby 21 cycles of 25 seconds at 96°C, 50 seconds at 65°C, 45 seconds at 72°C 104
  • 105. followed by 4 cycles of 25 seconds at 96°C, 60 seconds at 55°C, 120 seconds at72°C.Table 2.5 Phototype primer mix composition for PCR-SSP reactions10 Sense Anti- Antigens Alleles Primer MixNumber bp size Primer sense Primer1A 01A AL16 ALZ A01 A*0101-04N 5752A 02A AL16 ALV A36 A*3601 5643A 03A AL37 ALAW A*02 A*0201-0227,68032 9404A 04A AL7 ALD A*03 A*0301-0304 6285A 05A AL8 ALQ A23, A24 A*2301,2413 5576A 06A AL8 ALR A24 A*2402-05, 2407, 2409N-11N, 2414 5577A 07A AL11 ALC A25 A*2501, 2502 4008A 08A AL34 ALC A26, A43 A*2601-02,2604,2607-09,2611N, 4301 4029A 09A AL4 ALC A25, 26 A*2501-02,2601-09, 2611N, 440 3401-02, 6601-0210A 10A AL6 ALC A25, 34,66 A*2502, 3402, 6601-02 41911A 11A AL17 ALC A43 A*4301 44212A 12A AL6 ALI A11 A*1101-2, 1104 52013A 13A AL6 ALH A68, 69 A*6801-02, 6806, 6808, 6901 44715A 14A AL4 ALAM A34, 68 A*3402, 6801-05, 6808 42716A 15A AL25 ALH A68 A*6802 62517A 16A AL6 ALY A69 A*6901 38318A 17A AL35 ALF A29 A*2901-03 44219A 18A AL12 ALG A30 A*3001-04, 3006 56120A 19A AL10 ALF A31 A*31012 48121A 20A AL11 ALF A32 A*3201-02 42322A 21A AL4 ALF A33 A*3301, 3303 46323A 22A AL24 ALAR A32, 74 A*3201-02, 7401-03 49224A 23A AL32 ALF A29, 31,33,74 A*2901-3, 31012, 3301, 412 3303, 7401-0225A 24A AL54 ALBK A80 A*8001 543 105
  • 106. 26A 25A AL30, ALH A02, 23,24,68,69 A*0201-22, 0225-27, 446 AL31 2301, 2402-0427A 26A AL30, ALL A01, A02, A25, A*0101-04,0224, 2501, 2601-09, 2611, 445 AL31 A26, A30, A31, 3002-04, 3006, 31012, 3201- A32, A33, 02,3301,3303,3601,4301, 6603,7401- 03,8001,0301-04,1101-04,2502,2901- 03,3001,3401-02,6601-021B 34 193 221 B7, 8101 B*0702-0705,8101 6192B 35 312 221 B703 B*0703 6003B 36 195 212 B8, 4406 B*0801,0802,4406 5434B 155 195 220 B8 B*0801-02 6065B 37 280 225 B49, 50,4005, B*4901,5001,4005,2704,2706 635 2706,27046B 38 208 215 B49, 59 B*4901,5901 3857B 39 246 215 B45, 49,50 B*4501,4901,5001 6008B 40 207 219 B45, 76 B*4501,1514 5369B 160 202, 393, B44 B*4402-06 546/ 272 285 48110B 42 192 220 B41 B*4101-02 60511B 43 272 276 B49, 50,4005, B*4002-4006,4008,4101-02, 566 61,41, 44,45, 47 4501,4901,5001,4402-5,470112B 44 192 247 B60, 61,4005, B*4801,40011-4006 465 41, 480113B 45 272 218 B60, 61,47 B*40011-4004,4006-8,4701 62715B 46 280 229 B60 B*40011,40012,4007 60716B 47 243 215 B13 B*1301,1302,1303# 48617B 48 197 127 B64, 65 B*1401,1402 38918B 49 205 232 B65, 3904 B*1402,3904 18719B 50 207 217 B39, 67 B*39011-3908,6701 50720B 51 206 217 B67 B*67011,67012 54821B 172 208, 217 B38 B*3801,3802 498/ 435 50822B 53 209 217 B38, 39,67 B*3801,3802,3901-8,67011,67012 612 106
  • 107. 23B 54 208 223 B5801, 77, B*5801,5104,5301,1513 319 5104, 5324B 56 194 213 B58 B*5801-3 37425B 55 194 224 B57 B*5701,5702,5703 35126B 166 243 438 B57, 63,13,1524 B*5701-03,1513,1516-17,1524 14327B 57 187 214 B18 B*1801-02 45828B 58 209 236 B55, 56,73, B*5501,5502,5601,5602,7301, 422 3906 39061-21C 59 242 215 B54, 55,56,45,50 B*5401,5501,5502,5601,4501,5001 3832C 60 203 238 B56 B*5601,5602 5513C 61 395 236 B54 B*5401 4214C 62 280 281,282 B27 B*2701,2709 149/1505C 63 188 212 B37, 4406 B*3701,B*4406 6066C 64 192 392 B37, 3902,3908 B*37,3902,3908 4227C 65 192 228 B47 B*4701 4148C 66 367 236 B73 B*7301 2899C 67 203 220 B42 B*4201 59410C 68 209 229 B4801, B8101 B*4801,8101 56711C 69 194 225 B63 B*1516,1517 51612C 70 240 241 B46 B*4601 45913C 72 243 250 B62, 62V,75, B*1501-02,1504-08,1511-12, 124 76,1521 1514-15,1519-21,1525,1526N, 152815C 73 192 214 B62, 72,76, B*1501,1503-07,1512,1514, 421 4802, 4003 1519-20,1524-25,4802,400316C 156 271 223 B4802 B*4802 48717C 74 209 214 B72, 72V, B*1503,1518,1523,1529, 486 71,4802,3907, 4802,3907 Cw070318C 75 271 238 B4802, B*4802,1503,1509-10,1518,1523,1529 691 71,70,72,72V19C 76 189 238 B71,70 (NOT B*1509-10,1518,1521,1523 562 B72), 1521,152320C 77 243 219, B76 B*1512,1514,1519 636- 107
  • 108. 244 63721C 78 193 377 B62 Variants B*1508,1511,1515,1522, 553 A*68, 2501, 2601-5,3401,6601-222C 79 193 223 B35, 53,75,77, B*3501-04,3506-09,3511, 369 5104,1521,4406 3512,5301,1502, 1513,5104,1521,440623C 80 188 237 B35, 18,78,1522 B*3501-13,18,78012, 1522 12824C 81 195 213, B35,53 B*3501-09,3511,5301 389/ 277 34025C 82 207 216 B78, 1509 B*7801-2,1509 40026C 83 208 216 B51,52 B*5101-05,52011,52012 40127C 84 193 216 B51, 78,1509 B*5101-05,7801-2,1509 45128C 85 192 216 B52 B*52011,52012 440Agarose Gel ElectrophoresisPCR products were electrophoresed in 1.2% agarose gels containing 0.5µg/mlethidium bromide after the addition of 5 µL of loading buffer consisting of 0.25%Orange G, 30% v/v glycerol and 0.5X TBE buffer. Orange G is an excellentelectrophoresis marker as it migrates with the primer band on gel electrophoresis.The gels were run for 68 minutes till the Orange G migrated 5 cm. The gels werevisualized using UV illumination and documented in a multi-imager system (Biorad).A 100 bp ladder was run in all the gels as a standard for checking the sizes of thedifferent amplicons11. 108
  • 109. Positive reactions for PM 03 (HLA A*02) in wells 2,3 and 12,13. 7th one is markerPositive reactions for PM 25 (HLA A*02, 23,24,39,69) in wells 2,3,4,6,8,9,11,12,13.7th is markerReactions for PM 12 and 21 (HLA A*11 and 33) 109
  • 110. Reactions for PM 26 and 04 (HLA A*01, 02, 25,26, 30, 31, 32, 33) and A*03Reactions for PM 155 (HLA B*8) 7th well is markerReactions shown for PM 69 (HLA B*63). 7th well is marker 110
  • 111. OBSERVATIONS AND RESULTSObservation of the data and analysis of the results are essential for a properlyconducted research. Totally 40 healthy individuals from different geographic areaand communities belonging to both the sex were selected for the study with informedconsent. Due care was taken to avoid sampling from related individuals and siblingsand unwilling and non-cooperative persons.Blood samples were collected and DNA isolation was performed using standardorganic extraction methods. Molecular typing of the following genes were carried outusing the polymerase chain reaction (PCR), table no. 1Table no.1 Genes under study 1. Dopamine Receptor D2 Taq 1 D DRD2 Taq 1 D 2. Dopamine Receptor D2 Taq 1 B DRD2 Taq 1 B 3. Dopamine Receptor D2 Taq 1 A DRD2 Taq 1 A 4. Dopamine Receptor D2 Serine 311 Cysteine DRD2 S311C 5. Dopamine Receptor D2 Histidine 313 H DRD2 His 313H 6. Dopamine Receptor D3 Serine 9 Glycine DRD3 S9G 7. 5-Hydroxy Tryptamine Receptor 1 B 5HTR 1B 8. Serotonin 1438 Ser 1438 9. Serotonin 102 Ser 102 10. Human Leukocyte Antigen B HLA B 11. Human Leukocyte Antigen A HLA AFollowing are the observations of the analysis done. Out of the 40 individualsselected the age varies from 25 to 45 111
  • 112. Chart no.1 Distribution according to gender 30% Male 70% FemaleIn this study maximum individuals were male (70%) and remaining 30% femalesGraph 1 Distribution of Prakrithi according to gender Gender and Prakrithi 12 Male 10 10 Female 8 8 6 6 6 4 3 3 2 2 2 0 0 0 Pitha Kapha Vatha Pitha Pitha Kapha Vatha Kapha 112
  • 113. Chart no.3 Distribution according to RELIGION 15% 5% Hindu Muslim Christian 80%In this maximum individuals were Hindus (80%) Christians (15%), Muslims (5%)Graph 2 Distribution of Prakrithi according to RELIGION Religion and Prakrithi Hindu 14 Muslim 12 12 Christian 10 9 8 8 6 4 2 2 2 2 2 1 1 0 0 0 0 0 0 Pitha Kapha Vatha Pitha Pitha Kapha Vatha Kapha 113
  • 114. Table no.2, Graph 3 Distribution according to Prakrithi (Phenotype) Frequency Percent Vatha 0 0 Pitha 9 22.5 Kapha 13 32.5 Vatha Pitha 2 5.0 Pitha Kapha 14 35.0 Vatha Kapha 2 5.0 Sama 0 0 Total 40 100.016 1414 131210 9 8 6 4 2 2 2 0 Pitha Kapha Vatha Pitha Vatha Pitha Kapha KaphaPitha Kapha Prakrithi is found more frequent 35% (14), Kapha Prakrithi 32.5% (13),Pitha Prakrithi 22.5% (9), Vatha Pitha (2) and Vatha Kapha (2) 5% each. It wasobserved that there were no Vatha Prakrithi phenotypes and Sama dosha Prakrithi(combination of the three doshas) phenotypes. 114
  • 115. Table no.3, Graph 4 Genotype DRD2 Taq1D distribution in the population Frequency Percent D1/D1 7 17.5 D2/D1 20 50.0 D2/D2 13 32.5 Total 40 100.0In this heterozygous genotype (D2/D1) individuals were found more (50%) Taq 1 D 20 20 18 16 14 13 12 10 8 7 6 4 2 0 D 1 /D 1 D 2 /D 1 D 2 /D 2 115
  • 116. Table no.4, Graph 5 Genotype DRD2 Taq1B distribution in the population Frequency Percent B1/B1 6 15.0 B2/B1 10 25.0 B2/B2 24 60.0 Total 40 100.0In this dominant homozygous genotype (B2/B2) individuals were found frequent(60%) Taq 1 B 24 25 20 15 10 10 6 5 0 B1/B1 B2/B1 B2/B2 116
  • 117. Table no.5, Graph 6 Genotype DRD2 Taq1A distribution in the population Frequency Percent A1/A1 9 22.5 A2/A1 13 32.5 A2/A2 18 45.0 Total 40 100.0In this dominant homozygous genotype (A2/A2) individuals were found frequent(45%) Taq 1 A 18 18 16 14 13 12 10 9 8 6 4 2 0 A 1 /A 1 A 2 /A 1 A 2 /A 2 117
  • 118. Table no.6, Chart 3 Genotype DRD2 S311C distribution in the population Frequency Percent G/G 32 80.0 G/C 8 20.0 Total 40 100.0 S 311C 20% G /G G /C 80%In this dominant homozygous genotype (G/G) individuals were found frequent (80%) 118
  • 119. Table no.7, Graph 7 Genotype DRD2 His 313H distribution in the population Frequency Percent T/T 15 37.5 C/T 19 47.5 C/C 6 15.0 Total 40 100.0In this heterozygous genotype (C/T) individuals were found frequent (47.5%) H is 3 1 3 H 20 19 18 16 15 14 12 10 8 6 6 4 2 0 T /T C /T C /C 119
  • 120. Table no.8, Graph 8 Genotype DRD3 S9G distribution in the population Frequency Percent G/G 13 32.5 S/G 17 42.5 S/S 10 25.0 Total 40 100.0In this heterozygous genotype (S/G) individuals were found frequent (42.5%) S9G 18 17 16 14 13 12 10 10 8 6 4 2 0 G /G S /G S /S 120
  • 121. Table no.9, Graph 9 Genotype 5 HTR 1B distribution in the population Frequency Percent G/G 19 47.5 C/G 16 40.0 C/C 5 12.5 Total 40 100.0In this recessive homozygous genotype (G/G) individuals were found frequent(47.5%) 5 HTR 1 B 20 19 18 16 16 14 12 10 8 6 5 4 2 0 G/G C/G C/C 121
  • 122. Table no.10; Graph 10 Genotype Ser 1438 distribution in the population Frequency Percent G/G 10 25.0 A/G 23 57.5 A/A 7 17.5 Total 40 100.0In this heterozygous genotype (A/G) individuals were found frequent (57.5%) Ser 1438 25 23 20 15 10 10 7 5 0 G/G A/G A/A 122
  • 123. Table no.11, Graph 11 Genotype Ser 102 distribution in the population Frequency Percent T/T 6 15.0 C/T 24 60.0 C/C 10 25.0 Total 40 100.0In this heterozygous genotype (C/T) individuals were found frequent (60%) Ser 102 30 25 24 20 15 10 10 6 5 0 T/T C/T C/C 123
  • 124. Graph 12- % of Genotype distribution in the population % of genotype with various Prakrithis Pitha Kapha120.00 Pitha Kapha Vatha Pitha Vatha Kapha100.00 80.00 60.00 40.00 20.00 0.00 B2/B1 B1/B1 A2/A1 A1/A1 S/S A/A C/C C/C C/C C/C D2/D1 D1/D1 G/G G/G G/G G/G T/T T/T Table no.12, Graph 13 Correlation between Phenotype (Prakrithi type) and HLA B alleles Prakrithi HLA B alleles 7 8 13 15 35 37 38 39 40 44 51 52 55 56 57 58 Pitha 1 1 2 1 3 2 4 1 1 2 Kapha 3 1 3 2 1 7 2 2 2 3 Vatha Pitha 1 1 2 Pitha Kapha 3 1 3 1 2 6 5 1 1 2 1 2 Vatha Kapha 1 1 1 1 It is observed that there is a genetic basis for the three major constitutions (Prakrithi) described in Ayurveda. The Prakrithi classification is based on 124
  • 125. differences in physical, physiological, and psychological characteristicsand is independent of racial, ethnic, or geographical considerations. Itmay provide an appropriate means of classifying phenotypes to beconsidered collectively for genotyping. Of the evaluated 40 subjects bothfor their Prakrithi and human leukocyte antigen (HLA) B types, weobserved that a reasonable correlation between HLA B type and Prakrithitype. HLA B *07, *08, *13, *15, *35, *37, *38, *39, *40, *44, *51, *52, *55,*56, *57, and *58 were observed with varying frequencies in differentPrakrithi populations. The complete absence of the HLA B*07 allele inthe Vatha Pitha and Vatha Kapha type, and of HLA B*08 and B*38 in allthe four Prakrithis except Kapha, HLA B*13 in Pitha, Kapha and VathaPitha Prakrithis, HLA B*15 in the Kapha, Vatha Pitha and Vatha KaphaPrakrithis. Interestingly, absence of HLA B*35 alleles were found only inVatha Pitha Prakrithi. HLA B*37 were completely absent in all the dualconstitutions of Vatha Pitha, Pitha Kapha and Vatha Kapha Prakrithis.HLA B*39 was absent in Pitha, Kapha, Vatha Kapha. HLA B*40 andB*44 were absent in Vatha Pitha and Vatha Kapha. HLA B*51 werepresent in all the Prakrithis. HLA B*52 was absent only in Vatha Kapha.HLA B*55 was absent in Kapha, Vatha Pitha, and Vatha Kapha. HLAB*56 was present only in Pitha Kapha and B*57 was only in VathaKapha. HLA B*58 was absent in Vatha Pitha and Vatha Kapha. A correctperusal of table no 12 of correlation between Phenotype (Prakrithi type)and Genotype of HLA B attest that, of the 16 alleles only B*39, B*51 andB*52 were present in Vatha Pitha Prakrithi and B*13, B*35, B*51, B*57were only present in Vatha Kapha Prakrithi. In Pitha Kapha Prakrithi all 125
  • 126. the 12 alleles were present except B*08, B*37, B*38, and B*57. Among the 16 alleles studied, the allele frequency of HLA B*40 was seen highest as 16 and its distribution was highest in Kapha Prakrithi population and Pitha Kapha Prakrithi population. The lowest allelic frequency of HLA B was observed in B*08 & B*38 (in Kapha) B*56 (in Pitha Kapha), and B*57 (in Vatha Kapha). HLA B & Prakrithi Pitha8 Kapha 7 Vatha Pitha7 Pitha Kapha 6 Vatha Kapha6 55 44 3 3 3 3 3 33 2 2 2 22 2 22 2 2 22 1 1 11 1 11 1 1 1 111 1 1 1 1 110 7 8 13 15 35 37 38 39 40 44 51 52 55 56 57 58 Table no. 13, Correlation between Phenotype (Prakrithi type) and HLA A alleles Prakrithi HLA A alleles 1 2 3 11 24 26 31 32 33 68 Pitha 1 3 1 1 4 2 3 3 Kapha 1 6 3 3 2 2 1 5 3 126
  • 127. Vatha Pitha 1 1 1 1Pitha Kapha 1 2 2 3 7 4 3 2 4Vatha Kapha 1 1 1 1Of the evaluated 40 subjects both for their Prakrithi and human leukocyteantigen (HLA) A types, we observed that a reasonable correlation betweenHLA A type and Prakrithi type. HLA A *01, *02, *03, *24, *26, *31, *32, *33,*68 were observed with varying frequencies in different Prakrithi populations.The presence of HLA A*01and HLA A*24 alleles were observed in all the fivePrakrithi populations. Complete absence of the HLA A*02 and HLA A*03allele in the Vatha Pitha and Vatha Kapha type, and of A*11 in all the fourPrakrithis except Vatha Kapha, HLA A*26 in Kapha and Vatha KaphaPrakrithis, HLA A*31 in the Vatha Pitha and Pitha Kapha Prakrithis. HLA A*32absent in Pitha and Vatha Pitha where as A*33 was absent in Vatha Pitha andVatha Kapha. HLA A*68 was absent in Pitha, Vatha Pitha, and Vatha Kapha.A correct perusal of table no….. of correlation between Phenotype (Prakrithitype) and Genotype of HLA A attest that, of the 10 alleles only A*01, A*11 andA*24 and A*26 were present in Vatha Pitha Prakrithi and A*01, A*24, A*31,A*32 were present in Vatha Kapha Prakrithi. Among the 10 alleles studied,the allele frequency of HLA A*24 was seen highest as 15 and its distributionwas highest in Pitha Kapha Prakrithi population and Pitha Prakrithi population.The lowest allelic frequency of HLA A was observed in A*01 and itsdistribution were equal in all the 5 Prakrithis under study. 127
  • 128. Graph 14 HLA A & Prakrithi Pitha Kapha Vatha Pitha 8 Pitha Kapha 7 7 Vatha Kapha 6 6 5 5 4 4 4 4 3 3 3 3 3 3 3 3 3 2 2 2 2 2 2 2 11 1 11 1 1 1 1 1 1 1 1 1 1 0 1 2 3 11 24 26 31 32 33 68 Table no.14 Correlation between Phenotypes (Prakrithi types) and various alleles DRD2 DRD2 DRD2 DRD2 DRD2 DRD2 DRD2 DRD2 DRD2 DRD2 DRD3 DRD3 Taq1 Taq 1 Taq1 Taq1 Taq1 Taq1 S311C S311C His313H His313H S9G S9G 5HTR1B 5HTR1B Ser1438 Ser1438 Ser102 Ser102Prakrithi D1 D2 B1 B2 A1 A2 C G C T S G C G A G C TKapha 13 13 5 21 10 16 3 23 8 18 10 16 11 15 12 14 14 12Pitha 8 10 8 10 8 10 1 17 5 13 11 7 5 13 9 9 9 9Pitha Kapha 11 17 8 20 12 16 3 25 14 14 14 14 9 19 12 16 17 11Vatha Kapha 1 3 1 3 1 3 0 4 2 2 0 4 1 3 2 2 2 2Vatha Pitha 1 3 0 4 0 4 1 3 2 2 2 2 0 4 2 2 2 2 128
  • 129. Graph.15 Correlation between Phenotypes (Prakrithi types) and various alleles % of allele frequency with various Prakrithis Pitha Kapha120.00 Pitha Kapha Vatha Pitha100.00 Vatha Kapha 80.00 60.00 40.00 20.00 0.00 D1 D2 B1 B2 A1 A2 G C T C S G G C G A T C 129
  • 130. Table no.15 Allele frequencies (%) in various PrakrithisTest Allele Pitha Kapha Pitha Kapha Vatha Pitha Vatha KaphaTaq 1 D1 44.44 50.00 39.29 25.00 25.00 D2 55.56 50.00 60.71 75.00 75.00Taq 1 B1 44.44 19.23 28.57 0.00 25.00 B2 55.56 80.77 71.43 100.00 75.00Taq 1 A1 44.44 38.46 42.86 0.00 25.00 A2 55.56 61.54 57.14 100.00 75.00DRD2 S311C G 94.44 88.46 89.29 75.00 100.00 C 5.56 11.54 10.71 25.00 0.00DRD HIS313 H T 72.22 69.23 50.00 50.00 50.00 C 27.78 30.77 50.00 50.00 50.00DRD3 S9G S 61.11 38.46 50.00 50.00 0.00 G 38.89 61.54 50.00 50.00 100.005HTR 1B G 72.22 57.69 67.86 100.00 75.00 C 27.78 42.31 32.14 0.00 25.00SER 1438 G 50.00 53.85 57.14 50.00 50.00 A 50.00 46.15 42.86 50.00 50.00SER 102 T 50.00 46.15 39.29 50.00 50.00 C 50.00 53.85 60.71 50.00 50.00 130
  • 131. The response to Prakrithi is presumed to differ with the individual; thisvariation might be due to genetic differences. Nine-receptor polymorphismswere screened in four receptor genes. These receptor genes includeDopamine receptor D2 and D3 genes and two Serotonin receptor gene e.g.HTR1B and HTR2A.A total of 40 healthy individuals participated in this study. The subjectsunderwent Prakrithi analysis. We observe an increased incidence of KaphaPrakrithi individuals. Among mixed characters we observe an increasedincidence of Pitha Kapha Prakrithi. In our sample 70% of the individuals weremales. When we compared the Prakrithis in males and females we observealmost similar trend of Prakrithi distribution, however, males had higherfrequency of Kapha Prakrithi in compared their female counterpart. The entirepopulation was further classified into their religion. 80% of the individuals wereHindus. We do not observe any significant shift in the trend of Prakrithisbased on religion; however, Hindus display lower tendencies of Vatha PithaPrakrithi.DRD2 TaqI D, Taq 1A, Taq 1B, S311C, His313H, DRD3 S9G, 5HTR 1B,5HTR 2A (Ser 1438 and Ser 102) restriction enzyme fragment lengthpolymorphism (RFLP) study examined genomic DNA extracted from bloodsamples.The proportions in Pitha, Kapha, Pitha Kapha, Vatha Pitha, and Vatha Kaphaof both the alleles were observed as shown in the table no.15The RFLP study showed that the distribution of the DRD2*Taq 1D genotypeand allele frequencies differed significantly between the Prakrithis, and that 131
  • 132. Vatha Pitha and Vatha Kapha Prakrithis had the highest and equal (75%)DRD2*Taq 1D2 allele frequency than other Prakrithis. Equal distribution offrequency of D2 and D1 alleles was observed in Kapha Prakrithi.The RFLP study showed that the distribution of the DRD2*Taq 1B genotypeand allele frequencies differed significantly between the Prakrithis, and thatVatha Pitha Prakrithi had the highest (100%) DRD2*Taq 1B2 allele frequencythan other Prakrithis.The RFLP study showed that the distribution of the DRD2*Taq 1A genotypeand allele frequencies differed significantly between the Prakrithis, and thatVatha Pitha Prakrithi had the highest (100%) DRD2*Taq 1A2 allele frequencythan other Prakrithis.The RFLP study showed that the distribution of the DRD2*S311C genotypeand allele frequencies differed significantly between the Prakrithis, and thatVatha Kapha Prakrithi had the highest (100%) DRD2*S311C G allelefrequency, in Pitha, in Pitha Kapha, and in Kapha Prakrithis it was 94.44%,89.29%, 88.46% respectively.The RFLP study showed that the distribution of the DRD2*His 313H genotypeand allele frequencies differed significantly between the Prakrithis, and thatPitha Prakrithi had the highest (72.22%) DRD2*His 313H T allele frequency,and in all the three dual - Pitha Kapha, Vatha Pitha and Vatha Kapha -Prakrithis it was equal for both the alleles (T and C) and were observed as50%.The RFLP study showed that the distribution of the DRD3*S9G genotype andallele frequencies differed significantly between the Prakrithis, and that VathaKapha Prakrithi had the highest (100%) DRD3*S9G G allele frequency, and in 132
  • 133. the two dual - Pitha Kapha, Vatha Pitha - Prakrithis it was equal for both thealleles (S and G) and were observed as 50%.The RFLP study showed that the distribution of the 5HTR 1B genotype andallele frequencies differed significantly between the Prakrithis, and that VathaPitha Prakrithi had the highest (100%) 5HTR 1B G allele frequency than otherPrakrithis.The RFLP study showed that the distribution of the 5HTR 2A (Ser 1348)genotype and allele frequencies differed significantly between the Prakrithis,and that Pitha Kapha Prakrithi had the highest (57.14%) 5HTR 2A G allelefrequency than other Prakrithis and in the two dual - Vatha Pitha and VathaKapha – Prakrithis and in Pitha Prakrithi, it was equal for both the alleles (Gand A) and were observed as 50%.The RFLP study showed that the distribution of the 5HTR 2A (Ser 102)genotype and allele frequencies differed significantly between the Prakrithis,and that Pitha Kapha Prakrithi had the highest (60.71%) 5HTR 2A C allelefrequency than other Prakrithis and in the two dual - Vatha Pitha and VathaKapha – Prakrithis and in Pitha Prakrithi, it was equal for both the alleles (Cand T) and were observed as 50%.Out of nine SNPs selected we observed increased frequency of one allele in 5polymorphisms screened in all the Prakrithis while in remaining fourpolymorphisms more or less similar pattern of distribution of alleles in allPrakrithis was observed. When individual Prakrithis and individualspolymorphism were assessed we observe a different pattern of distribution,however, HTR2A did not display any significant differences in frequency ofdistribution of alleles among different Prakrithis. 133
  • 134. When individuals polymorphism were compared in different Prakrithis weobserve increased DRD2 Taq1 D2 allele in all mixed Prakrithis, while DRD2Taq1 B2, DRD2 Taq1 A2, DRD2 311 G and HTR1B G alleles were observedto be increased in all Prakrithi in compared to their alternate allele.In Pitha Prakrithi significant differences were observed in DRD2 S311C à Gallele, DRD2 313 Cà T allele and HTR1B CàG allele in compared to itscounterpart allele.In Kapha Prakrithi significant differences were observed in DRD2 Taq1 B1àB2, DRD2 Taq A2 à A1, DRD2 311 CàG, DRD2 313 Cà T allele.ANALYSISOn analysis of the results of the study of the various dopaminergic receptorgenes with respect to various Prakrithi populations a significant increase ofthe allele frequencies in the Prakrithis having an association of Vatha is found.The allele frequency of Taq 1D2 in Vatha Pitha and Vatha Kapha Prakrithishad the highest and equal (75%). Similarly, the allele frequency of otherdopaminergic receptor genes Taq 1B2, Taq 1A2, S311C G, and His 313H (Tand C) and DRD3 S9G also shows an increasing tendency in all the Prakrithipopulations having the association of Vatha, when compared to otherPrakrithis that are having no influence of Vatha. Hence it is understood thatassociation of Vatha increases the allele frequency in dopaminergic receptorgenes under my study.Similarly, serotoninergic receptor gene 5HTR 1B with respect to variousPrakrithi populations shows a significant increase of the allele frequencies inthe Prakrithis having an association of Vatha. But in the case of serotoninergic 134
  • 135. receptor gene 5HTR 2A (Ser 1438 and Ser 102) shows a significant increaseof the allele frequency in the Prakrithi having an association of Kapha.DRD2*B2 allele and DRD2*A2 and 5HTR 1B G allele are influenced by Vatha.DRD2 S311C and DRD3 S9G are influenced by Kapha.Dopamine is a chemical naturally produced in the body mainly in nervoustissue and adrenal glands. In the brain, dopamine functions as aneurotransmitter, activating dopamine receptors. Dopamine is also aneurohormone released by the hypothalamus. Dopamine is critical to the waythe brain controls our movements and is a crucial part of the basal gangliamotor loop. All the functions atributed to dopamine receptors can be includedin the functions of Vatha in Ayurveda, and the allele frequency ofdopaminergic receptor genes Taq 1B2, Taq 1A2, S311C G, and His 313H (Tand C) and DRD3 S9G shows an increasing tendency in all the Prakrithipopulations having the association of Vatha.We have also noticed that Serotoninergic 5-HT1B receptors are expressedthroughout the mammalian central nervous system and, only 1% of serotoninin the human body is found in the CNS and also the receptor gene 5HTR 1Bshows a significant increase of the allele frequencies in the Prakrithis havingan association of Vatha. But surprisingly 99% of serotonin is found in othertissues, primarily plasma, the gastro-intestinal tract, and immune tissues andserotoninergic receptor gene 5HTR 2A (Ser 1438 and Ser 102) shows asignificant increase of the allele frequency in the Prakrithis having anassociation of Kapha. 135
  • 136. Graph 16 Allele frequency of Dopamine Receptor D2 Taq 1 D 80.00 75.00 75.00 70.00 D2 60.00 60.71 55.56 50.00 50.00 44.44 40.00 39.29 D1 30.00 25.00 25.00 20.00 10.00 0.00 Pitha Kapha Pitha Kapha Vatha Pitha Vatha Kapha Graph 17 Genotypic frequency of Taq 1D in various Prakrithis Dopamine Receptor D2 Taq 1D60.00 Heterozygous 53.8550.00 D2/D1 50.00 50.00 50.00 44.4440.00 D2/D2 35.71 33.3330.00 22.22 23.0820.00 D1/D1 14.2910.00 0.00 0.00 0.00 Pitha Kapha Pitha Kapha Vatha Pitha Vatha Kapha 136
  • 137. Graph 18 Allele frequency of Dopamine Receptor D2 Taq 1 B120.00100.00 100.00 80.00 80.77 75.00 B2 71.43 60.00 55.56 44.44 40.00 28.57 25.00 B1 20.00 19.23 0.00 0.00 Pitha Kapha Pitha Kapha Vatha Pitha Vatha Kapha Graph 19 Genotypic frequency of Taq 1B in various Prakrithis Dopamine Recepto D2 Taq 1B120.00100.00 100.00 80.00 B2/B2 69.23 64.29 60.00 50.00 44.44 40.00 33.33 Heterozygous 20.00 22.22 23.08 21.43 14.29 B2/B1 B1/B1 7.69 0.00 0.00 0.00 Pitha Kapha Pitha Kapha Vatha Pitha Vatha Kapha 137
  • 138. Graph 20 Allele frequency of Dopamine Receptor D2 Taq 1 A120.00100.00 100.00 80.00 75.00 A2 60.00 61.54 55.56 57.14 44.44 42.86 40.00 38.46 A1 25.00 20.00 0.00 0.00 Pitha Kapha Pitha Kapha Vatha Pitha Vatha Kapha Graph 21 Genotypic frequency of Taq 1A in various Prakrithis Dopamine Receptor D2 Taq 1A 120.00 A2/A2 100.00 100.00 80.00 60.00 50.00 44.44 46.15 42.86 40.00 38.46 Heterozygous 33.33 28.57 A2/A1 20.00 22.22 15.38 A1/A1 0.00 0.00 0.00 Pitha Kapha Pitha Kapha Vatha Pitha Vatha Kapha 138
  • 139. Graph 22 Allele frequency of Dopamine Receptor D2 Serine 311 Cysteine 120.00 100.00 100.00 94.44 88.46 89.29 80.00 G 75.00 60.00 40.00 25.00 20.00 C 11.54 10.71 5.56 0.00 0.00 Pitha Kapha Pitha Kapha Vatha Pitha Vatha Kapha Graph 23 Genotypic frequency of S311C in various Prakrithis Dopamine Receptor D2 S311C120.00100.00 100.00 G/G 88.89 80.00 78.57 76.92 60.00 50.00 40.00 G/C Heterozygous 20.00 23.08 21.43 11.11 C/C 0.00 0.00 0.00 0.00 0.00 0.00 Pitha Kapha Pitha Kapha Vatha Pitha Vatha Kapha 139
  • 140. Graph 24 Allele frequency of Dopamine Receptor D2 Histidine 313 H 80.00 70.00 T 72.22 69.23 60.00 50.00 50.00 50.00 50.00 40.00 30.00 C 27.78 30.77 20.00 10.00 0.00 Pitha Kapha Pitha Kapha Vatha Pitha Vatha Kapha Graph 25 Genotypic frequency of His 313H in various Prakrithis Dopamine Receptor D2 His 313H120.00 Heterozygous T/C100.00 100.00 80.00 T/T 71.43 66.67 60.00 50.00 46.15 40.00 C/C 20.00 22.22 11.11 14.29 7.69 0.00 0.00 0.00 Pitha Kapha Pitha Kapha Vatha Pitha Vatha Kapha 140
  • 141. Graph 26 Allele frequency of Dopamine Receptor D3 S9G120.00100.00 100.00 80.00 G 60.00 61.11 61.54 50.00 50.00 40.00 38.89 38.46 20.00 S 0.00 0.00 Pitha Kapha Pitha Kapha Vatha Pitha Vatha Kapha Graph 27 Genotypic frequency of DRD3 S9G in various Prakrithis Dopamine Receptor D3 S9G120.00 Heterozygous100.00 100.00 100.00 80.00 S/G 60.00 55.56 S/S 46.15 40.00 38.46 33.33 35.71 28.57 20.00 G/G 15.38 11.11 0.00 0.00 0.00 Pitha Kapha Pitha Kapha Vatha Pitha Vatha Kapha 141
  • 142. Graph 28 Allele frequency of 5-Hydroxy Tryptamine Receptor 1 B120.00100.00 100.00 80.00 72.22 75.00 67.86 60.00 G 57.69 40.00 42.31 C 32.14 27.78 25.00 20.00 0.00 0.00 Pitha Kapha Pitha Kapha Vatha Pitha Vatha Kapha Graph 29 Genotypic frequency of 5-HTR 1B in various Prakrithis 5 Hydroxy Tryptamine Receptor 1B 120.00 100.00 100.00 80.00 Heterozygous 60.00 C/G 55.56 53.85 57.14 50.00 G/G 44.44 40.00 30.77 20.00 C/C 21.43 15.38 0.00 0.00 0.00 0.00 Pitha Kapha Pitha Kapha Vatha Pitha Vatha Kapha 142
  • 143. Graph 30 Allele frequency of Serotonin 143860.00 G 57.14 53.8550.00 50.00 50.00 50.00 46.15 A 42.8640.0030.0020.0010.00 0.00 Pitha Kapha Pitha Kapha Vatha Pitha Vatha KaphaGraph 31 Genotypic frequency of Ser 1438 in various Prakrithis Serotonin 1438120.00 Heterozygous100.00 100.00 100.00 80.00 A/G 60.00 55.56 57.14 G/G 46.15 40.00 30.77 28.57 20.00 22.22 23.08 A/A 14.29 0.00 0.00 0.00 Pitha Kapha Pitha Kapha Vatha Pitha Vatha Kapha 143
  • 144. Graph 32 Allele frequency of Serotonin 10270.0060.00 C 60.71 53.8550.00 50.00 50.00 50.00 46.1540.00 T 39.2930.0020.0010.00 0.00 Pitha Kapha Pitha Kapha Vatha Pitha Vatha Kapha Graph 33 Genotypic frequency of Ser 102 in various Prakrithis Serotonin 102 120.00 Heterozygous 100.00 100.00 100.00 80.00 T/C 64.29 60.00 55.56 T/T 46.15 40.00 30.77 28.57 20.00 22.22 23.08 C/C 7.14 0.00 0.00 0.00 Pitha Kapha Pitha Kapha Vatha Pitha Vatha Kapha 144
  • 145. DISCUSSIONBefore entering to the discussion proper, I think it is proper to discuss oncertain aspects of Prakrithi, such as the importance of study of Prakrithi, theviews of various Acharyas regarding the influence of Vatha, Pitha and Kaphaon various physical, physiological and pshycological functions of humanorganism such as digestion, metabolism, sense perception, thought processand content of thinking.Understanding how tridosha work is a big challenge. To reduce the complexityof the problem Ayurvedic Acharyas have broken it into smaller pieces andhave discussed it in various levels and analyzed. They have discussed theworking of tridosha at molecular level by analyzing the zygote and explainedthe role of Panch bhoothas and there by tridoshas in the development ofzygote to a human organism. They have also discussed how all the particlesof Pancha bhootha work together to give a cell of man, animal and plant itsspecial properties. How do the 16 types of bhoothas derived from fourdifferent sources constitute the zygote, and how they function as tridoshas,controlling its structural and functional activities are well discussed. Acharyashave also discussed the role of tridoshas in sensing information fromperception of external world to make decisions and execute movements, inphysiological and pathological state. A review of the phenomina of visualsense perception on the basis of Pancha bhootha / Tridosha theory willenable to understand the effect of Prakrithi on sense perception and thecharacteristics of sensory apparatus specific to the individuals of differentPrakrithis and thereby design the treatment to various diseases of the sensoryapparatus (Kaarana Dourbalya) and also to adopt personalised preventive 145
  • 146. measures for better visual, olfactory , auditory, tactile and gestatorysensations essential for a more pleasurable and healthy life.Among the Pancha bhoothas, Thejus has a dominant role in perception ofvisual sense. Theja Paramanus (Photons?) are made up of 50% Thejus andthe other 50% by the other Bhoothas in equal percentage (12.5%). Thenature of light according to Indian Metaphysics is in the form of particles,which radiate retlinially in all directions at very high speed. When lightparticles pass through a transparent medium, it penetrate through the inter-atomic spaces with vibrations (Parispandana) of the nature of deflection(Thiryaggamana) or reflections (Vibhaga)1. Dalhana, while explaining theword ‘Vivarakrithi’ depicted by Susrutha to denote the nature of the outermostlayer of the eye (Cornea) explains that Cornea is highly transparent(Athyanthaachatwa) and have inter atomic spaces (Vivarantharathwa) andhence it does not obstruct the entry of Theja paramanus and thereby enablesto perceive the properties of objects that can be perceived by alochaka thejus(Energy of ocular apparatus)2. The state of energy in ocular apparatus is ofapanchikritha form (100% thejus) and hence our receptors of the ocularapparatus (or that of any electronic equipment?) is insensitive to such formsof energy (Atheendriya) and their limitations restrict our knowledge in the field.Acharya Nimi also considers that visual sense perception is brought about bythe interaction of two types of energies (Theja samyoga) – that emitted fromluminous objects and the Alochakathejus (energy present in the ocularapparatus)3. The interaction of these two types of energies is designated asIndriya-Indriyaardha Sannikarsha. By the ‘hitting’ and Samyoga, light energyhaving high speed may alter the nature of energy causing visual impulse 146
  • 147. through Netranaadi (Optic Nerve)4. In fact, the pattern of inheritance ofMahabhoothas derived from four different sources at the time of fertilisationalso differs and is reflected to the Prakrithi of an Individual. The percentage ofPanchabhoothas that make up various parts of the eye also varies. TheSuklamandala (Schlera and bulbar conjunctiva) have a dominance ofJalabhootha derived from father whereas the Krishna Mandala (Cornea & Iris)have a dominance of Vayu Mahabhootha from mother. The Mamsamandala(mascular parts) have a dominance of Prithvi Mahabhootha derived frommother and the Drishtimandala (Pupil, Lens and retina) have a dominance ofAgni and Jala Mahabhoothas derived equally from father and mother.However the Nethra naadi (Optic Nerve?) and the nerve centre have adominance of Agni Mahabhootha derived mainly from Athmajabhaava.Hence, any minute change in the Prakrithi may reflect in the eye of theoffspring as tabulated in Table No.2.A Thejaparamanu (Photon) having a structure of 50% Agni and 12.5% each ofother Bhoothas produces various effects on the energy system of eye in tunewith the Prakrithi of an Individual. Or, the sensitivity of the receptors ofvarious Individuals have minute varations in accordance with their Prakrithi.However, a more sharp vision is for Pitha Prakrithi Individuals and the mentalimagery of different Prakrithis at the time of dreams and hallucinations mayalso vary according to the genetic make-up examples of which are providedas itm 23 of Table No2.While narrating the process of segregation of Prakrithi, Dalhana clearlymentions about ‘Drishtyaarmbhaka bijabhaga’ or gene that determine thedevelopment of lens/retina. The action of vitiated dosha on 147
  • 148. drishtiaarambhaka bijabhaga can produce unfavourable and potent changesin bijabhagas (Mutations) the effect of which is manifested as Jaathyandhathaor congenital blindness5. But if the changes occurring in the genes are notsufficient to produce any disturbance to the general functions of the eye of theoffspring, the changes are accepted as of in normal limits but its effects will bereflected in the development of various parts of the ocular apparatus in tunewith the nature of doshas involved. The varying characteristics of eye ofindividual belonging to different Prakrithi as enumerated in the item 14 ofTable No.2. Pitha Prakrithi Individuals are more prone to Pitha predominanteye diseases. But it does not mean that only the Pitha predominant diseaseswill affect a Pitha Prakrithi Individual. Proneness to diseases of variousIndividuals belonging to other Prakrithis may also be considered on the samelines. But a correct assessment of the Prakrithi of Individuals by analysingthe Phenotypic characteristics will enable to foresight the chances for theoccurrence of eye diseases, its nature and diseases of other systems as well,so that we can take preventive measures by adopting specific life styles, foodhabits and medicines according to the Prakrithi of Individual and therebytechnologies of personalised preventive medicine of Ayurveda may bebrought in to practice.Acharyas have also discussed the role of tridosha/ Pancha bhoothas inproducing integrated behaviors, process of dream and higher levels of humanmental activity such as self-awareness, mental imagery, and language.Mental activity influences tridosha activity and that in turn influence the brainactivity, activities of the various systems of the body and even the geneactivity. The role of tridoshas, in the formation, association and dissociation of 148
  • 149. molecules or paramanus for the formation of zygote, development of the bodyand process of death are discussed in detail in our science of life. Afteranalyzing the functions of tridoshas at molecular, cellular, systems, behavioraland cognitive levels, the ancient sages have developed a holistic approach inunderstanding the state of human organism in health and disease and cameto a conclusion that, these two states are two sides of the same coin oftridosha and postulated the concept that, the living and non living and life anddeath are nothing but two sides of the same coin of Pancha bhootha.Study of Prakrithi and assessment of Prakrithi of an individual is of supremeimportance in Ayurveda. It enables to understand the nature of variouscategories of diseases classified on the basis of different criteria. It also helpsto predict whether a factor responsible for aggravation of Dosha is potent toproduce a disease in a person/ population assessed of having a particularPrakrithi and to understand the state of Agni (factors or enzymes responsiblefor digestion and metabolism) and maintenance of their normalcy. Withoutassessing Prakrithi it is not possible to maintain health of various groups ofindividuals designated as a particular Prakrithi because the preventivemeasures to be adapted to various Prakrithi’s differ.Diseases are classified into two groups, each on the basis of five differentcriteria – Prognosis (curable/ incurable), Intensity (mild/ severe), Location(mental/ physical), Nature of causative factors (endogenous/ exogenous) andSite of origin (amasaya/ pakwasaya)6.One substance may vitiate many Doshas, e.g. Substance having sour, saline,and pungent taste vitiates Pitha; but those of sour taste vitiate Pitha as well asKapha, those of saline taste Kapha as well as Pitha and those of pungent 149
  • 150. taste vitiate Pitha as well as Vatha. Similarly, spring season, which normallyaggravates Kapha, also aggravates Pitha and Vatha because of its ‘aadaana’nature (property of absorbing water from earth). Pitha accumulated in therainy season gets aggravated during autumn, but there is simultaneousaggravation of Kapha also. Similarly in summer, due to ununctuousness,Vatha gets aggravated in view of the ununctuousness of the season andsimultaneously there is slight accumulation of Pitha due to seasonal heat. Tounderstand the effect of food and climate having different characteristics onthe metabolic activities of an individual, a correct knowledge of Prakrithi is ofhigh importance.The metabolic fire is the most important factor that determines the state ofbody. Increase or decrease of it may produce disease and its state ofequilibrium maintains health. In short, if the anabolism and catabolism are in abalanced state the body will be in a state of health. But the state of metabolicfire (Agni) defers according to the Prakrithi of the individual – that can becatagorised into intense (Theekshna), mild (Manda), regular (Sama) andirregular (Vishama). In Sama Prakrithi individuals, Agni is regular. In VathaPrakrithi individuals, Agni becomes irregular as the site of Agni is subdued byVatha. Similarly, in Pitha Prakrithi individuals, Agni is intense and in KaphaPrakrithi individuals, it becomes mild. These varying states of Agni inindividuals having different Prakrithi denotes that digestion, absorption,assimilation and other biological activities varies in different individuals by theinfluence of tridoshas and it affects the body’s response to treatment, food ordrug. Intense metabolic fire tolerates all sorts of improper food while the mildone got a contrary character. Improper diet easily affects the regular 150
  • 151. metabolic fire but otherwise remain normal, the irregular fire got the charactercontrary to that of regular fire7.Persons of eka Dosha/ DWI Dosha Prakrithi is considered as sadaathuras, asthey are more prown to disease when they come in contact withcorresponding factors that aggravate the Dosha which have dominance intheir genetic makeup. Depending upon the nature of Doshas involved, it isnecessary to adopt food habits and life styles as would be in contradictionwith the predominating Dosha, till there is normalcy of Agni. It is only afternormalcy is attained, balanced regimens should be adopted. If an individualhaving a particular Prakrithi resorts to such thing as are aggravators of thepredominating Dosha in his body, the corresponding Dosha is aggravated andafflicts the individual by manifest of diseases resulting in impairment ofstrength, complexion, happiness and longevity. In short, in a Vatha Prakrithiindividual, Vatha gets immediately aggravated when Vatha aggravating thingsare used. But if the same individual takes things that aggravate Pitha orKapha, then Pitha and Kapha do not get so vitiated to manifest a disease as ithappens in the case of Vatha.In this connection, it is noted that certain Acharyas are of opinion, that, exceptSama Prakrithi, all other Prakrithis are to be considered as pathological, andCharaka considers Prakrithi (normal) as Vikrithi (abnormal) and use the termVathala, Pithala and Sleshmala instead of Vatha Prakrithi, Pitha Prakrithi andKapha Prakrithi. For maintenance of positive health, adopting differentmeasures to the above group of individuals are essential. In the case of SamaPrakrithi individuals who are having superior functioning of the dhathus,measures balanced in all aspects are to be adopted, whereas in the case of 151
  • 152. eka Dosha Prakrithi, measures contrary to the properties of Dosha, afterconsidering the predominance of respective Dosha, are beneficial till Agnibecomes regular. There after balanced measure should be applied. The sameis applicable in the case of psychophysical activities and treatment regimen.Food habits and life styles to be adopted by individuals/ population having aparticular Prakrithi can be summarized as follows:For Vatha Prakrithi individual 1. Proper administration of oils and fomentations 2. Food having mild laxative effect prepared by addition of fat, hot things and substances having sweet, sour and saline taste. 3. Massage, bandage, kneading, affusion, bath (hot) 4. Use of wine and asavaas (fermented drinks) 5. Fats from different sources mixed with herbs/ food materials having digestive, stimulant, carminative, Vatha alleviating and laxative properties. 6. Practice of Vasthi along with its regimens according to season.For Pitha Prakrithi individuals 1. External and internal use of ghee 2. Purgatives, food and herbs having sweet, bitter and astringent taste and cooling property. 3. Use of mild, fragrant, cooling and cordial perfumeries. 4. Use of pearls, jewels and garlands having cooling effect 5. Frequent sprinkling of cold water with cold air containing herbal perfumeries. 152
  • 153. 6. Hearing of mild, sweet, agreeable songs and music, information regarding prosperity and keeping company with friends and agreeable ladies wearing cooling garments and garlands. 7. Residence in built in cold places in mountains and riverbanks, which is cooled by moon rays, exposed to breezes from all sides, with beautiful gardens and having pleasing cold and fragrant wind. 8. Use of perfumeries made of various types of lotus, rose, etcFor Kapha Prakrithi individual 1. Foods mostly ununctuous with pungent, bitter and astringent taste. 2. Running, jumping, swimming, whirling, keeping awake late at night, wrestling, sexual intercourse, exercise, oil massage and bath. 3. Intake of strong alcohols preserved for long time, use of medicated smoking 4. Use of warm apparels 5. Proper use of slimming therapies and use of strong and hot elimination processes when required, and avoid lifestyles that provokes Kapha.Practice of drugs, foods and life styles without considering the Prakrithi ofindividuals will not enable to cure or prevent disease. Such practices ifadopted to cure a disease will give rise to a new disease and may inviteadverse effect. While discussing about the importance of the study ofPrakrithi, Bhadantha Nagarjuna, in his great work, Rasa vaisheshika soothrastates ‘Praadurbhavedanyo va’ – or may develop another disease - (verse 89).Such treatments are considered as improper. Curing of disease by treatmentwithout considering the Prakrithi of the individual is considered as accidental. 153
  • 154. He further discusses that, focusing on the disease under treatment andignoring the complications associated, when treated without consideration ofPrakrithi, cannot be accepted, as complications may sometimes endanger thevery life of the patient8. Some are of opinion that, during treatment only eitherof the metabolic fire, immunological status or the life style should beconsidered. Bhadantha Nagarjuna is of opinion that, focus is to be given onany factor only after considering the various stages of the disease and thecondition of the patient.We have already seen that, any change in the Prakrithi of an individual, whichcan be assessed by interpreting the changes in the phenotypiccharacteristics, is fetal. Change in Prakrithi is to be understood as rishta(symptoms of imminent death). Rasa vaisheshika soothra depicts rishta as‘trika thraya dwandwa dikkaala vikriya’9.Here the three triads are - Guna trika, Dosha trika and Karana trika 1. Gunatrika - Satwa, Raja, Thama 2. Dosha trika - Vatha, Pitha, Kapha 3. Karanatrika - Body, Mind and Word; Dwandwa - twin factors like heaviness &lightness, coldness & hotness, smoothness & roughness, softness &hardness, wetness & dryness, mobility & stiffness, bluntness & sharpness,mild & intense along with Dik and Kaala (orientation of place and time) are thefactors to be considered to assess weather the Prakrithi of an individual ismaintained unchanged or changes have initiated indicating imminent death(rishta)10.A person having Satwika nature, if shows the nature of Rajasa or Thamasa orvise versa is to be understood as initiation of changes in Prakrithi leading todeath. Satwa, Rajas And Thamas are the three forces of mind and are 154
  • 155. responsible for all mental activities. They are in a state of conflict. Accordingto the dominance of one of them, the individual is classified into Satwika,Rajasa And Thamasa. The characteristics of Satwika individuals are merciful,sharing, endurant, truthful, proper in words and deeds, god fearing, selfknowledge, intelligent, retentive, recollective, resolute and those with selflessservice. Rajasa individuals are unhappy, wandering, coward, haughty, lying,cruel, cheating, self-respectful, excited, sensual and quick tempered.Thamasa types are gloomy, atheistic, unrighteous, unintelligent, spirituallyignorant, evil minded, indolent and drowsy. Infact the characteristics of all thethree type are seen in one individual and on the basis of dominance themental makeup of the individual is designated as Satwika, Rajasa, orThamasa or combinations of either two or three of them.Tridosha vaikrithi is the variation in the Prakrithi of an individual and isassessed by alternations in the earlier tabulated phenotypic characteristics.Karana trika vaikrithi includes deterioration of mental faculty such asalternation of consciousness and disorientation of people/ day/ date etc isincluded in mano vikrithi. Derangement in physical properties such as gainingof weight by starvation, emaciation without any cause etc. is Sareera vikriya.Vak vikrithi (irrelevant words) pointing disorders of thought process.Dwandwa vikriya (change in twin factors) denotes alternations in senseperception, such as hot as cold, soft as hard, sweet as astringent, microscopicas macroscopic, sweet smell as foul smell etc.Dik vikrithi means disorientation of space such as feeling of north as southand vise versa etc. 155
  • 156. Kaala vikrithi is disorientation of time including the sensing of day as night, orotherwise. Expression of altered physical, physiological and psychologicalcharacteristics without any cause is considered as symptoms of alternation ofPrakrithi, which is also considered as the symptoms of imminent death.Detailed description of the phenomena is included in Ayurvedic books,especially in the 12 chapters of Indriya sthaana of Charaka samhitha. Thestudies of Prakrithi in comparison with Vikrithi (rishta) enable to predictprognosis of the disease and span of life of the individuals.In the introductory part, it was discussed that the traditional system ofmedicine like Ayurveda, Siddha etc. have advocated and practiced preventiveand curative medicinal recipes specific to individuals. The body, mind, foodand environment were looked at holistically to suggest a preventive orcurative approach to health. His Excellency the President of India has pointedout the need for collaborative research of Ayurveda and biotechnology. Dr.Kalam’s focusing on personalized medicine of Ayurvedic system of medicinehas invited our attention to the fact that in this post genomic era, the emergingconcept of personalized therapy on the basement of Pharmacogenomics is are-definition of diseases on the molecular level so that diagnotics andtherapeutics can be targeted to specific patient populations sub-typed on thebasis of genetic make-up. Use of modern technology and Biotechnology hasbeen identified by AYUSH as a thrust area for 10th plan period. The KeralaBiotechnology policy is also designed with an objective to catalyze thedevelopment of Ayurveda by collaborative research. The NationalBiotechnology development strategy addresses the utilization ofBiotechnology to add value to Ayurveda. The emerging personalized medicine 156
  • 157. upholds the views of Ayurveda that environment, diet, age, lifestyle and stateof health all can influence a person’s response to health and that due togenetic variations a disease may manifest itself slightly differently in differenttypes of patients. Ayurveda upholds individual physiology, pathology,diagnostic and personalized therapy whereas Modern medicine base ongeneral principles of physiology, pathology diagnotics and treatment.Study of the genetic make up of an individual is essential for personalizedtherapy. A high-speed data mining of both genotypic and phenotypicinformation is essential for the development of personalized therapy on thebasement of Pharmacogenomics. To categorize individuals on the basis ofboard phenotypic clusters, consideration of racial, ethnic and geographicalfactors to be considered collectively for genotyping is controversial. Hencestudy of the concept of Prakrithi is beneficial. For diagnostic, prognostic andtherapeutic purposes, Ayurveda categorizes human population in to sub-populations such as Vatha Prakrithi, Pitha Prakrithi, Kapha Prakrithi or theircombinations on the basis of physical, physiological and psychologicalcharacteristics (phenotypes) and completely avoid racial, ethnic andgeographical considerations.My present study aims to analyze the role of DNA in determining Prakrithi byconducting Polymerize Chain Reaction studies, genetic correlation of variousforms of Prakrithi and to develop standards for a uniform guide line inanalyzing Prakrithi in this study I have tried to correlate Ayur Tech, Bio Techand Info Tech by seeking guidance and assistance of experts from therespective fields. 157
  • 158. An attempt was made to analyze the molecular basis of Prakrithi. As in thecase of microbes, zygote also become resistant to the medium in which it isdeveloped, whereas the medium for microbe is a toxin and that of the zygoteis an energic template – Dosha sthithi – emitted and maintained unchangedthrough out the span of life by the interaction of the extra zygotic and intrazygotic environments at the time of fertilization. The zygote or Chatushpadi isthe sum total of the first four Maha-bhoothas – Prithwi, Ap, Thejus and Vayuderived from four different sources – 1. Sperm (sukra) 2. Ovum (sonitha) 3.Nutrient fluid (rasa) and 4. ‘Athma’ and any abnormalities of the four factorsderived from the aforesaid sources may render the zygote abnormal, theeffect of which will be reflected in the offspring. “Bija” (Sperm & Ovum) isconsidered as collection of Genes. The superior germ cell genotype (SamaPrakritihi) provides health to the developing embryo (Garbha) and itsvariations Vathala, Pithala, Sleshmala and combinations of either two of themcause varying degrees of prownness to disease (sadaathura). Hence VathaPrakrithi is considered as Heena (bad), Pitha Prakrithi as Madhyama (better)and Kapha Prakrithi as Uthama (best) and combinations of either two of them(dwantwa Prakrithis) as Nindya (worst). Diseases are mainly manifested asPoorvaparaadhaja (Karmaja), or Drishtaparaadhaja (known etiology such asSahaja/ Garbhaja/ Jathaja/ Peedakritha etc) or by the combined effect of both(Sankara roga).Autosomal recessive inheritance and sex linked dominant inheritance involvesboth Mathrja and Pithrja bija dushti, whereas sex linked recessive inheritanceis exclusively Mathrja and all these can be included in Aadi bala pravritharoga. Defect in the somatic cells of the embryo will manifest into disease 158
  • 159. called Janma bala pravritha roga. The difference between diseases of aadibala pravritha and janma bala pravritha is that the former is due to the defectin the germ cell and the later in the somatic cell due to the intra uterineenvironment. Super females (sthree akrithi bhooyishtam) called Vaartha isborn due to the defect in genes that determine development of uterus, ovum,and that of the secondary sex characteristics like breast, vagina, linea alba,etc. Vaartha and super females or Trisomy X mentioned in modern geneticsshows phenotypic similarity. A sterile female (Vandhya) is born by defectivebija bhaga (gene) that determines the development of uterus and / or ovum ofthe offspring. Defect in garbhasaya bija bhaga avayava, give rise to puthipraja (still birth) or neonates with loose body tissues (klinnanga prathyanga).Duchenne muscular dystrophy (DMD) is one of the most prevalent types ofmuscular dystrophy and shows similarity to the above condition. Similarly insuper males called trina puthrika, a sterile male (Vandhya) and puthi praja orsthiraanga prathyanga are occurring due to defect in sperm, showscorresponding similarity with XYY syndrome, Fibrodysplasia OssificansProgressiva (FOP). Charaka clearly explains the bija bhagas (genes) thatdetermines the development of uterus and/ or ovum, testis and/ or sperm andalso those of the male and female secondary sex characteristics. Theexpression of bija bhagas that determine the development of breast andbeard by adolescence is clearly explained. The influence of mental activityover gene activity and how the mental disturbances of parents and that of themother during coitus and child bearing period explained by Charaka seems tobe an important subject of research for modern geneticists. Innumerablefactors pertaining to physical, physiological, and psychological aspect of 159
  • 160. mother that renders the offspring susceptible to various diseases, enumeratedby Charaka under ‘garbhopaghathakara bhavas’ was also discussed in detailin the introductory part. The phenotypic characteristics of individualsbelonging to different Prakrithis are also explained and tabulated. The study ofPrakrithi with special reference to religion (Jati prasakta), family (Kulaprasakta), and also on the basis of the influence of geopathological factors(Desanupatini), climatic elements (Kalanupatini), age factors (Vayonupatini),and inividual developmental traits (Pratyatma niyata) are discussed in detail.A review of literature of genes was included in my study, before discussingthe nature and functions of the genes under my study. Genes are thefundamental physical and functional unit of heredity. Genes make proteins,which are made of 20 amino acids. Billions of proteins combine to make acell; billions of them to make a tissue; thousands of tissues combine to makean organ, and several organs, to make a man. The normal functioning ofgenes keeps us healthy. When genes are exposed to harmful environments,they alter their functions. It is called mutation. Plants interact with environmentand produce toxins, which act as active molecules that have the unique abilityto bind to human genes too and are able to switch a gene on or off. If theswitching on and off of a gene is desirable, it will lead to cure of disease whengenes are switched on it produces a protein. Genes are encoded in anorganisms genome, composed of DNA or RNA, and direct the physicaldevelopment and behavior of the organism. DNA does not change during anindividual’s lifetime but its functional properties like RNA and protein maychange depending on the environment. Various species carry more than onecopy of their genome within each of their somatic cells. These organisms are 160
  • 161. called diploid if they have two copies or polyploid if they have more than twocopies. In such organisms, the copies are practically never identical. Withrespect to each gene, the copies that an individual possesses are liable to bedistinct alleles, which may act synergistically or antagonistically to generate atrait or phenotype. Sir John gregor Mendel was the first to hypothesizeindependent assortment, the distinction between dominant (An allele thatdetermines phenotype even when heterozygous, and also the trait controlledby that allele) and recessive (a gene that is phenotypically manifest in thehomozygous state but is masked in the presence of a dominant allele) traits,the distinction between a heterozygote (A diploid or polyploid with differentalleles at a particular locus) and homozygote (A diploid or polyploid withidentical alleles at a particular locus), and the difference between what wouldlater be described as genotype and phenotype.Molecular typing of the following genes was carried out.1. Dopamine ReceptorD2 Taq1D2. Dopamine Receptor D2 Taq1B3. Dopamine Receptor D2 Taq1A4. Dopamine Receptor D2 S311C5. Dopamine Receptor D2 His 313H6. Dopamine Receptor D3 S9G7. 5 Hydroxy Tryptamine Receptor 1B8. Serotonin 14389. Serotonin 10210. Human Leukocyte Antigen B11. Human Leukocyte Antigen A 161
  • 162. A detailed study about each of the above genes was reviewed to understandtheir nature and function.To analyze the role of DNA in determining Prakrithi, to understand the geneticcorrelation of various forms of Prakrithi and to develop standards for a uniformguideline in analyzing Prakrithi, the study was conducted. As per criteria fixedearlier, the Prakrithi of 40 unrelated individuals were assessed with informedconsent. Blood samples were collected. DNA isolation was performed usingstandard organic extraction methods. Quantification of DNA was done by twomethods – spectrophotometry and agarose gel electrophoresis. Amplificationof genes was carried out using the polymerase chain reaction. Moleculartyping (Detection of amplification) was done by agarose gel electrophoresismethod and documented the gels using multi imager.Of the 40 individuals selected for the study, maximum individuals were male70% and remaining 30% females; Hindus (80%) Christians (15%), Muslims(5%). The distribution according to various Prakrithis was noted. KaphaPrakrithi 32.5% (13), Pitha Prakrithi 22.5% (9), Pitha Kapha Prakrithi is foundmore frequent 35% (14), Vatha Pitha (2) and Vatha Kapha (2) 5% each. Itwas observed that there were no Vatha Prakrithi phenotypes and SamaDosha Prakrithi (combination of the three Doshas) phenotypes (graph 3).The genotype distribution in the population was as follows:Dopamine ReceptorD2 Taq1D heterozygous genotype (D2/D1) individuals (50%);Dopamine Receptor D2 Taq1B dominant homozygous genotype (B2/B2) (60%);Dopamine Receptor D2 Taq1A dominant homozygous genotype (A2/A2) (45%);Dopamine Receptor D2 S311C dominant homozygous genotype (G/G) (80%);Dopamine Receptor D2 His 313H heterozygous genotype (C/T) (47.5%); DopamineReceptor D3 S9G heterozygous genotype (S/G) (42.5%); 5 Hydroxy Tryptamine 162
  • 163. Receptor 1B recessive homozygous genotype (G/G) (47.5%); Serotonin 1438heterozygous genotype (A/G) (57.5%); Serotonin 102 heterozygous genotype (C/T)(60%); Human Leukocyte Antigen B allele B*40 was frequent (40%); HumanLeukocyte Antigen A allele A*24 was frequent (37.5%)Analysis of the results of the study was conducted by statistical analysis usingthe software SPSS version 10 for windows and Microsoft excel sheet forpreparing graphs and charts.Of the various dopaminergic receptor genes with respect to various Prakrithipopulations a significant increase of the allele frequencies in the Prakrithishaving an association of Vatha is found. The allele frequency of dopaminergicreceptor genes Taq 1B2, Taq 1A2, S311C G, and His 313H (T and C) andDRD3 S9G also shows an increasing tendency in all the Vatha associatedPrakrithi populations. Hence it is understood that association of Vathaincreases the allele frequency in dopaminergic receptor genes under mystudy.Similarly, serotoninergic receptor gene 5HTR 1B shows a significant increaseof the allele frequencies in the Vatha associated Prakrithis. But in the case ofserotoninergic receptor gene 5HTR 2A (Ser 1438 and Ser 102) shows asignificant increase of the allele frequency in the Kapha associated Prakrithi.Dopamine functions as a neurotransmitter, activating dopamine receptors.Dopamine is also a neurohormone released by the hypothalamus. Dopamineis critical to the way the brain controls our movements and is a crucial part ofthe basal ganglia motor loop. All the functions attributed to dopamine receptorcan be included in the functions of Vatha in Ayurveda.Serotoninergic 5-HT1B receptors are expressed throughout the mammaliancentral nervous system and, only 1% of serotonin in the human body is found 163
  • 164. in the CNS and also the receptor gene 5HTR 1B shows a significant increaseof the allele frequencies in the Vatha associated Prakrithis. 99% of serotoninis found in other tissues, primarily plasma, the gastro-intestinal tract, andimmune tissues and serotoninergic receptor gene 5HTR 2A (Ser 1438 andSer 102) shows a significant increase of the allele frequency in the Kaphaassociated Prakrithis.Among the 16 alleles studied, the allele frequency of HLA B*40 was seenhighest in Kapha Prakrithi and Pitha Kapha Prakrithi. The lowest allelicfrequency of HLA B was observed in B*08 & B*38 (in Kapha) B*56 (in PithaKapha), and B*57 (in Vatha Kapha).Among the 10 alleles studied, the allele frequency of HLA A*24 was seenhighest in Pitha Kapha Prakrithi and Pitha Prakrithi. The lowest allelicfrequency of HLA A was observed in A*01 and its distribution were equal in allthe 5 Prakrithis.Human leukocyte antigen (MHC class I) genes are important determinants ofhost response to microbial infections and also have a profound effect on theimmunogenetic susceptibility to various diseases that are influenced byimmune reactions. It has gene cluster spans around a region of about 4000k.bps on short arm of chromosome six in the distal portion of 6p 21.3 band.Large number of genes with variable expression is arranged in form of threeregions in MHC as class I (36 genes), class II (27 genes) and class III (39genes). Class I products play a role in determining viral specificity and wereinvolved in the production of antibodies. They play a key role in therecognition and destruction of cells carrying foreign antigens by cytotoxict- Lymphocytes. 164
  • 165. The defense mechanism in the human body is associated with Kapha in termswith the doctrines of Ayurveda; oja otherwise called as bala is ‘somathmaka’;‘sleshma somah:’ so it is understood that Kapha plays an important role inmaintaining the immunological status of the body. Kapha Prakrithi isconsidered as best. Human leukocyte antigen (MHC class I) genes havingrole in host response to infections and profound effect on the immunogeneticsusceptibility to various diseases, the frequency of which is high in KaphaPrakrithi attest the scientific basis of tridosha theory.Analysis of the result shows that all genes show varying frequencies indifferent Prakrithis having predominance of Vatha, Pitha, Kapha or theircombinations. It is due to the influence of tridoshas, that genes showvariations in their frequency in different individuals with varying dominance ofVatha, Pitha and Kapha. Ayurveda clearly states that, it is the due to theinfluence of tridoshas on the genetic material that the correspondingvariations are occurring in phenotypic characteristics of different individualssub typed into different Prakrithis. When Vatha influences the innumerablegenes that determines the development of various organs and tissues of thebody, the organs and systems are developed with specific characteristics sothat, the individual can be grouped in Vatha Prakrithi. Further studies withlarge samples are essential to establish the influence of tridoshas on geneticmaterial. Infact the aim of my study was to analyze the role of DNA indetermining Prakrithi, but it is found that tridoshas influence DNA so that anindividual is developed with the specific characteristics of a particularPrakrithi. Prakrithi, which also means the ‘basic cause’, represents the Dosha 165
  • 166. sthithi – energic template – that influence the gentic material to determine thedevelopment of the specific characteristics of an individual. 166
  • 167. CONCLUSIONS$ It is observed that there is a genetic basis for the three major constitutions (Prakrithi) described in Ayurveda, and that tridoshas influence DNA at the time of fertilization, so that an individual is developed with the specific characteristics of a particular Prakrithi.$ This study enables to uphold the Ayurvedic concepts of personalized therapy by analyzing the population on the basis of phenotypic clusters of different genotypes.$ Association of Vatha increases the allele frequency in dopaminergic receptor genes and serotoninergic receptor gene 5HTR 1B under my study. All the functions atributed to dopamine receptors are included in the functions of Vatha in Ayurveda.$ Serotoninergic receptor gene 5HTR 2A (Ser 1438 and Ser 102) shows a significant increase of the allele frequency in the Kapha associated Prakrithi.$ Kapha plays an important role in maintaining the immunological status of the body. Kapha Prakrithi is considered as most healthy with high immunological status. Human leukocyte antigen (MHC class I) genes having role in host response to infections and profound effect on the immunogenetic susceptibility to various diseases, the frequency of which is high in Kapha Prakrithi attest the scientific basis of tridosha theory.$ Study on the association of Pitha on any receptor genes were not conducted due to the lack of availability of enzymes at the time of my study. 167
  • 168. $ Of the 40 individuals selected none was having Sama dosha Prakrithi and it attest the Ayurvedic concept that such a Prakrithi, though super, is rare or conceptual due to our inability to provide the superior environmental conditions essential for the emergence of such a trait.$ Absence of Vatha Prakrithi and increased frequency of Kapha or Kapha associated Prakrithi indicate the high health status of the population under study.$ When we compared the Prakrithis in males and females we observe almost similar trend of Prakrithi distribution, males had higher frequency of Kapha Prakrithi in compared their female counterpart.$ Though, 80% of the individuals were Hindus, we do not observe any significant shift in the trend of Prakrithis based on religion.$ More study is essential to develop standards for a uniform guideline in analyzing Prakrithi, with large and equal sample size of all the six types of Prakrithi.$ A repetition of the study of the individuals included in my study at their old age may enable to interpret the influence of tridoshas on DNA at the time of old age in tune with the basic concepts of tridosha theory. 168
  • 169. REFERENCESIntroduction 1. 2. Dr. M.S. Valiyathan, 70th annual meeting in Indian academy of science News, current science, vol.88 3. Dr. M.S. Valiyathan, in the book -The legacy of Charaka - 2003Prakrithi 1. Susrutha samhitha Sareera sthaana 4/ 79 2. Rasa vaisheshika soothra translated by Naarasimha 1/ 7 3. Charaka samhitha Vimana sthaana 8/ 95 4. Charaka samhitha Sareera sthaana 2/ 3 a. Charaka samhitha Sareera sthaana 3/ 7, Chakrapani 5. Charaka samhitha Sareera sthaana 2/ 31, Ibid Chakrapani 6. Ashtanga hridayam Sareera sthaana 1/ 3 7. Charaka samhitha Siddhi sthaana 9/ 4, Ibid Chakrapani 8. Ashtanga Hridayam Nidana sthaana 7/ 16 9. Charaka samhitha Sareera sthaana 2/ 16, 26, 35, Ibid Chakrapani 10. Charaka samhitha Sareera sthaana 2/ 26 11. Charaka samhitha Vimana sthaana 6/ 13, Ibid Chakrapani 12. Charaka samhitha Suthra sthaana 7/ 39, Ibid Chakrapani 13. Ashtanga hridayam Suthra sthaana 1/ 10 14. Dr. M.S. Valiyathan, in the book -The legacy of Charaka - 2003 15. Charaka samhitha Suthra sthaana 17/ 40 to 44 16. Susrutha samhitha Suthra sthaana 24/5, Ibid Dalhana 17. Charaka samhitha Sareera sthaana 4/ 30, Ibid Chakrapani 169
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  • 174. 6. Charaka samhitha Vimana sthaana 6/ 177. Charaka samhitha Vimana sthaana 6/ 128. Rasa vaisheshika soothra, soothra 91, 929. Rasa vaisheshika soothra by Bhadantha Nagarjuna, soothra 9710. Sri. Raghavan thirumulppad: Rasa vaisheshika soothra by Bhadantha Nagarjuna, soothra 97, Malayalam translation, 1977. -------- 174