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By
DR. ABDUL HASEEB KHAN
Assistant Professor
INTRODUCTION
 670 per 1000.
 1% of all newborn infants possess a gross
chromosomal abnormality,
 5% of individuals unde...
 Any two individual share 99.9% of their
DNA sequences.
 Only 0.1% show remarkable diversity
which represents about 3 mi...
Molecular basis of human disease.
 Functional cloning
◦ Approach to make a diagnosis of inborn
errors of metabolism e.g.
...
 Positional cloning.
◦ To get clue about the nature of defective
gene.
◦ It ignores by chemical clues form the
phenotype ...
Clinical Phenotype
Mapping –
linkage to a
chromosomal
region
Identify
Candidate genes
Gene Cloning
Abnormal gene
Biochemic...
 Production of human biologically
active agents:-
◦ By inserting the requisite gene into
bacteria or other suitable cell ...
 Gene Therapy:-
◦ Transfer of somatic cells transfected with
the normal gene by appropriate vector.
 Disease diagnosis:-...
 Human diseases can be classified.
1. Environmentally determined e.g. obesity
2. Genetically determined
3. Both environme...
 Hereditary disorders are derived form
one’s parents and are transmitted in the
germ line through the generations and
the...
Mutations
 Mutations is defined as permanent change
in DNA.
 Mutations effect germ cell and transmitted
to progeny givin...
 Point mutation it results due to change in
single base.
 It is also called missense mutation e.g. CTC
(GAG in messenger...
 Nonsense mutation an amino acid
substitution may change an amino acid
codon to a chain terminator is called stop
codon (...
 Mutation within non coding sequence:-
◦ Transcription of DNA is initiated and regulated by
promoter and enhancer sequenc...
 Genetic disorders can be classified as…
◦ Disorders related to mutations genes of large
effect, e.g. storage diseases an...
MENDELIAN DISORDERS
 Mendelian Genetics Definitions
 Allele - one alternative form of a given
allelic pair; tall and dwarf are the alleles
f...
 Homozygote - an individual which contains
only one allele at the allelic pair; for example
DD is homozygous dominant and...
 Mendel also hypothesized that allele pairs separate randomly, or
segregate, from each other during the production of gam...
 The genotype of an individual is made up of the many
alleles it possesses. An individual's physical appearance, or
pheno...
 Mendel's Laws of Inheritance Law
Definition Law of Segregation
During gamete formation, the alleles for
each gene segreg...
 Autosomal mutations produce partial
expression in the heterozygote and full
expression in the homozygote.
 Homozygous f...
 In the heterozygote, only proportion of Hb
is HbS ( remainder being HbA) that is way
red cell sickling take place only i...
 Gene expression is described as
dominant or recessive.
 Both alleles of a gene pair may be fully
expressed in heterozyg...
Autosomal Dominant Disorders
 Any effected parent can pass disease to
their children.
 When an effected person marry wit...
 Autosomal dominant inheritance
pattern
 express the traClinical features can be
modified by reduced penetrance and
variable expressivity.
 Some individuals inh...
 In many conditions the age at onset is
delayed symptoms and signs do not
appear until adulthood (in Huntington
disease.
...
 Mechanism:
◦ Two major categories of non enzyme protein are
effected.
1. 50% loss of LDL receptor result in secondary
el...
Autosomal recessive Disorders
 Single largest category of mendelian
disorders having mutant genes at both
alleles charact...
 Expression is uniform…
1. Complete penetrance is common.
2. Onset is frequently early in life.
3. Asymptomatic heterozyg...
X-LINKED DISORDERS
 All sex linked disorders are X-linked,
almost all X-linked recessive.
 Gene are encoded in the male
...
 The Y chromosome is not homologous to the
X so mutant genes on X are not paired with
alleles on Y that male is called he...
 Heterozygous female does not express
the full phenotypic change because of
paired normal allele.
Son of heterozygous wom...
 If in activation of one of X chromosome in
female has remote possibility of expression of
heterozygotes X-linked in fema...
Biochemical and Molecular
Basis of Single Gene
(Mendelian) Disorders
Enzyme Defects
 Accumulation of the substrate
depending on the site of block
accumulation of one or both
intermediate may...
 In galactosemia deficiency of
galactose -1- phosphate
uridyltransferate leads to
accumulation of galactose causing
tissu...
 Enzyme defect may lead to metabolic
block leading to decrease amount of end
product which is necessary for normal
functi...
Defect in receptors and transport
system
 In familial hypercholesterolemia reduced
synthesis of LDL receptor leads to
def...
Alteration in Structure, Function, or
Quantity of Nonenzyme Proteins
 Hemoglobinopathies, sickle cell disease
affect the ...
Disorders associated with defects in
receptor proteins
 Familial Hypercholesterolemia
◦ Mutation in gene encoding the rec...
Cholesterol metabolism
 1st step is secretion of VLDL by the liver
into the bloodstream which is rich in
riglycerides hav...
 50% of IDL is taken up by liver through a
receptor mediated transport which bind
IDL to liver cell membrane recognize bo...
 Fibroblast lymphocytes, smooth muscle
cells, hepatocytes and adrenocortical
cells possess high affinity LDL receptor.
 70% of plasma LDL is cleared by
liver by following mechanisms.
◦ 1st step is binding of LDL to cell surface
receptors (c...
 Here LDL dissociates and recycled to
surfae and LDL molecule is enzymatically
degraded to amino acids where as
cholester...
1. Cholesterol suppresses cholesterol
synthesis by inhibiting activity of
enzyme 3 hydoroxy-3, methylglutryl
conenzyme A (...
 In heterozygotes with familial
hypercholesterolemia possess only 50% of
normal high affinity LDL receptor leading
to two...
 In familial hypercholesterolemia, impaired
IDL transport into the liver secondarily
diverts a greater porportion fo plas...
 This increase is responsible for the
appearance of xanthomas and contributes
to the pathogenesis of premature
atheroscle...
 These can be classified into five groups.
1. Class I mutations are relatively uncommon,
and they lead to a complete fail...
4. Class IV mutations encode proteins that are
synthesized transported to the cell surface
efficiently.
5. Class V mutatio...
Disorders with multifactorial
Inheritance
 It is due to both environmental influences
and two or more mutant genes having...
Normal Karyotyping
 Study of chromosomes (Karyotyping) is
basic tool of cytogeneticist.
 By arresting mitosis in dividin...
 Chromosome comprises of short arm
designated p (for petit) and long arm
referred as q.
 Each arm is divided into region...
 Fluorescence in Situ Hybridizaiton (FISH)
 Keryotyping is only applicable in dividing
cells.
 This problem can be over...
 FISH can be used to demonstrate
microdeletions, complex translocations
and telomere alterations which are not
detectable...
 All the chromosome can not be detected
simultaneously by chromosome painting.
 This hurdle has been overcome by
spectra...
 Figure 4b demonstrates that BCR-ABL fusions can be readily
identified by FISH when a green-labeled hybridization probe
f...
 Figure 4b shows a FISH analysis that was used to detect the
presence of a chromosomal translocation in a patient sufferi...
Cytogenetic Disorder
 Any abnormality leading to an abnormal
number of chromosome or alteration in
the structure of one o...
 Nondisjunciton take place when a
homologous pair of chromosomes fails to
disjoin at the first meiotic division or two
ch...
 In anaphase lag. One homologous in
meiosis or one chromatid in mitosis lags
behind and is left out of nucleus resulting
...
 Occasionally mitotic errors in early
development in two or more population of
cells (mosaicism).
 Mosaicism can results...
 Second category of chromosomal
aberrations is associated with changes in
structure of chromosome.
 It result from chrom...
 A ring chromosome is a special form of
deletion in which a break occur at both
ends o f chromosome with fusion of
damage...
 Isochromosome formation results when one
arm of chromosome is lost and the remaining
arm is duplicated resulting in chro...
Cytogenic disorders involving
Trisomy 21 (Down Syndrome)
 Down syndrome is the most common of
the chromosomal disorders a...
 Clinical features include flat facial profile
oblique palpebral fissures, and epicanthic
fold with IQ of 25 to 50 in 80%...
Cytogenetic disorders involving sex
chromosome
 Characterized by disorders relating to
sexual development and fertility.
...
Klinefelter Syndrome
 Male hypogonadism due to two or more X
chromosome and one or more Y
chromosome.
 Gynocemastic may ...
 Reduce spermatogenesis and male
infertility with the evidence of atrophied
or prmitive tubules.
 Leyding cells are prom...
Turner Syndrome
 It results from complete or partial monosomy
of X chromosome characterized primarily by
hypogonadism in ...
 Clinically present as swelling of the nape
of the neck with distended lymphatic
channels (cystic hygroma)
 Bilateral ne...
Diagnosis Of Genetic Disease
 There are two methods.
◦ Cytogenetic analysis
◦ Molecular analysis
Cytogenetic Analysis:
 ...
◦ A parent with a previous child with a
chromosomal abnormality.
◦ Parent carrier of an X-linked genetic disorder
(determi...
Molecular diagnosis
 It is possible to indentify mutations at the
level of DNA.
 Use of recombinant DNA technology as
se...
Direct gene diagnosis
 It is also called diagnostic biopsy of
human genome, it depend upon on the
detection of an importa...
 By using DNA polymerases and thermal
cycling, DNA between the primers is
amplified producing millions of Copies of
DNA.
...
 Mutations that affect the length of DNA
(e.g. deletions) can also be detected by
PCR.
 A variety of PCR based technolog...
Indirect DNA diagnosis (linkage analysis)
 It depend upon by exploiting naturally
occurring variations or polymophisms in...
 Site polymorphisms are also called restriction
fragment length polymorphisms (RFLPs).
Examination of DNA from any two pe...
 Length polymorphisms:
◦ Human DNA contains short repetitive sequence
of noncoding DNA. Hence these stretches of
DNA can ...
Genetic
Genetic
Genetic
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Genetic
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Genetic
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Transcript of "Genetic"

  1. 1. By DR. ABDUL HASEEB KHAN Assistant Professor
  2. 2. INTRODUCTION  670 per 1000.  1% of all newborn infants possess a gross chromosomal abnormality,  5% of individuals under age 25 develop serious disease with significant genetic component.  Genetics is the study of single or a few genes and with phenotypic effects.  Genomics is the study of all the genes in the genome and their interactions.
  3. 3.  Any two individual share 99.9% of their DNA sequences.  Only 0.1% show remarkable diversity which represents about 3 million base pairs.  The most common variations in DNA is called single nucleotide polymorphism (SNP). And only 1% SNPs occurs in coding regions.  Proteomics concerns itself with the measurement of all proteins expressed in a cell tor tissue.
  4. 4. Molecular basis of human disease.  Functional cloning ◦ Approach to make a diagnosis of inborn errors of metabolism e.g. phenylketonuria and disorders of hemoglobin synthesis.
  5. 5.  Positional cloning. ◦ To get clue about the nature of defective gene. ◦ It ignores by chemical clues form the phenotype and relies on mapping the disease phenotype to particular chromosome location e.g. cystic fibrosis, using the linkage analysis in which gene is determined by linkage to known "marker genes” or SNPs.
  6. 6. Clinical Phenotype Mapping – linkage to a chromosomal region Identify Candidate genes Gene Cloning Abnormal gene Biochemical abnormality Functional Cloning Positional Cloning
  7. 7.  Production of human biologically active agents:- ◦ By inserting the requisite gene into bacteria or other suitable cell in tissue culture e.g. tissue plasminogen activator for the treatment of thrombotic state growth hormone for the treatment of deficiency states.
  8. 8.  Gene Therapy:- ◦ Transfer of somatic cells transfected with the normal gene by appropriate vector.  Disease diagnosis:- ◦ Molecular probes can be used for diagnosis of genetic and non-genetic disease e.g. recombinant DNA technology.
  9. 9.  Human diseases can be classified. 1. Environmentally determined e.g. obesity 2. Genetically determined 3. Both environmental and genetic e.g. peptic ulcer diabetes mellitus, atherosclerosis, schizophrenia, autoimmune disorders & cancers.
  10. 10.  Hereditary disorders are derived form one’s parents and are transmitted in the germ line through the generations and therefore called familial.  Congenital simply implies “born with”  All the congenital disease are not genetic e.g. congenital syphilis.  Not all genetic disease are congenital patients with Hunitngton disease, begin to manifest only after their twenties or thirties
  11. 11. Mutations  Mutations is defined as permanent change in DNA.  Mutations effect germ cell and transmitted to progeny giving rise to inherited disease  Genome mutations involve loss of gain of whole chromosome giving rise to monosomy or trisomy.  Gene mutation results due to partial or complete deletion of gene.
  12. 12.  Point mutation it results due to change in single base.  It is also called missense mutation e.g. CTC (GAG in messenger RNA) with is codes for glutamic acid, is change to CAC (or GUG in messenger RNA) which code for valine leads to change in physicochemical properties of hemoglobin resulting in skill cell anemia.
  13. 13.  Nonsense mutation an amino acid substitution may change an amino acid codon to a chain terminator is called stop codon (nonsense mutation) e.g. Glutamine (CAG) create a stop codon (UAG) leads to premature termination of β globin chains leading to β0 thalassemia.
  14. 14.  Mutation within non coding sequence:- ◦ Transcription of DNA is initiated and regulated by promoter and enhancer sequence. ◦ Mutation in these area leads to reduce a total lake of transcription e.g. hereditary hemolytic anemia.  Deletions and insertions. (frame shift mutations):- ◦ It results in abnormal protein missing one or more amino acids is synthesized.  Trinucleoide repeat mutation:- ◦ These are characterized by amplificaiton of a sequence of three nucleotides. Seen in FMR-1 gene that give rise to mental retardation.
  15. 15.  Genetic disorders can be classified as… ◦ Disorders related to mutations genes of large effect, e.g. storage diseases and inborn errors of metabolism also called the mendelian disorders. ◦ Disease with multifactorial inheritance e.g. hypertension diabetes mellitus are due to genetic and environmental factors. ◦ Chromosomal disorders due to structure changes and numeric difference. ◦
  16. 16. MENDELIAN DISORDERS
  17. 17.  Mendelian Genetics Definitions  Allele - one alternative form of a given allelic pair; tall and dwarf are the alleles for the height of a pea plant; more than two alleles can exist for any specific gene, but only two of them will be found within any individual  Allelic pair - the combination of two alleles which comprise the gene pair
  18. 18.  Homozygote - an individual which contains only one allele at the allelic pair; for example DD is homozygous dominant and dd is homozygous recessive; pure lines are homozygous for the gene of interest  Heterozygote - an individual which contains one of each member of the gene pair; for example the Dd heterozygote  Genotype - the specific allelic combination for a certain gene or set of genes
  19. 19.  Mendel also hypothesized that allele pairs separate randomly, or segregate, from each other during the production of gametes: egg and sperm. Because allele pairs separate during gamete production, a sperm or egg carries only one allele for each inherited trait. When sperm and egg unite at fertilization, each contributes its allele, restoring the paired condition in the offspring. This is called the Law of Segregation. Mendel also found that each pair of alleles segregates independently of the other pairs of alleles during gamete formation. This is known as the Law of Independent Assortment.
  20. 20.  The genotype of an individual is made up of the many alleles it possesses. An individual's physical appearance, or phenotype, is determined by its alleles as well as by its environment. The presence of an allele does not mean that the trait will be expressed in the individual that possesses it. If the two alleles of an inherited pair differ (the heterozygous condition), then one determines the organism’s appearance and is called the dominant allele; the other has no noticeable effect on the organism’s appearance and is called the recessive allele. This is known as the Law of Dominance.
  21. 21.  Mendel's Laws of Inheritance Law Definition Law of Segregation During gamete formation, the alleles for each gene segregate from each other so that each gamete carries only one allele for each gene. Law of Independent Assortment Genes for different traits can segregate independently during the formation of gametes. Law of Dominance Some alleles are dominant while others are recessive; an organism with at least one dominant allele will display the effect of the dominant allele.
  22. 22.  Autosomal mutations produce partial expression in the heterozygote and full expression in the homozygote.  Homozygous for the mutant gene, all the hemoglobin is abnormal, Hbs type and disorder is fully expressed sickle cell anemia
  23. 23.  In the heterozygote, only proportion of Hb is HbS ( remainder being HbA) that is way red cell sickling take place only in lower oxygen tension and condition is called sickle cell trait.
  24. 24.  Gene expression is described as dominant or recessive.  Both alleles of a gene pair may be fully expressed in heterozygote (codominance e.g. HLA and blood group antigen.).  A single gene may have many end defects (pleiotropism e.g. sickle cell anemia)  Mutation at several genetic loci may produce same trait (genetic heterogeneity e.g. childhood deafness.)
  25. 25. Autosomal Dominant Disorders  Any effected parent can pass disease to their children.  When an effected person marry with unaffected one, every child has one chance in two of having disease.  If patient don’t have effected parents, new mutations take place and their siblings are neither effected nor at increased risk for developing disease.
  26. 26.  Autosomal dominant inheritance pattern
  27. 27.  express the traClinical features can be modified by reduced penetrance and variable expressivity.  Some individuals inherit the mutant gene but are phenotopically normal (50% penetrance indicates that 50% of those who carry the gene it)
  28. 28.  In many conditions the age at onset is delayed symptoms and signs do not appear until adulthood (in Huntington disease. Trait is seen in all). individuals carrying the mutant gene but is expressed differently the phenomenon is called variable expressivity e.g. brownish spots on the skin to multiple skin tumors & skeletal deformities.
  29. 29.  Mechanism: ◦ Two major categories of non enzyme protein are effected. 1. 50% loss of LDL receptor result in secondary elevation of cholesterol predisposes to atherosclerosis in affected heterozygote's. 2. Collagen and cytoskeleton elements of the RBCs (spectrum), lack of three collagen chain in helical configuration in osteogenesis imprefecta.
  30. 30. Autosomal recessive Disorders  Single largest category of mendelian disorders having mutant genes at both alleles characterized by… 1. Trait does not affect the parents and sibling show disease. 2. Siblings have 25% chance of disease
  31. 31.  Expression is uniform… 1. Complete penetrance is common. 2. Onset is frequently early in life. 3. Asymptomatic heterozygote may pass mutant gene up to several generations up to a person mate with other heterozygote's and produce effected of offfspring.
  32. 32. X-LINKED DISORDERS  All sex linked disorders are X-linked, almost all X-linked recessive.  Gene are encoded in the male specific region of Y related to spermatogenesis and may having mutation are infertile and hence there is no Y-linked inheritance.
  33. 33.  The Y chromosome is not homologous to the X so mutant genes on X are not paired with alleles on Y that male is called hemizygous for X-linked mutant genes and disorders are expressed in male.  Affected male does not transmit the disorders to his sons, but all daughters are carriers.
  34. 34.  Heterozygous female does not express the full phenotypic change because of paired normal allele. Son of heterozygous women have one chance in two of receiving the mutant gene.
  35. 35.  If in activation of one of X chromosome in female has remote possibility of expression of heterozygotes X-linked in female e.g. glucose-6-phosphate dehydrogenase (G6PD) deficiency which predisposes RBCs hemolysis in patients receiving certain types of drugs is expressed principally in males, in female a perportion of RBCs with inactivation of normal allele, is susceptible to drug induced hemolytic reactions.
  36. 36. Biochemical and Molecular Basis of Single Gene (Mendelian) Disorders
  37. 37. Enzyme Defects  Accumulation of the substrate depending on the site of block accumulation of one or both intermediate may be increased.  Increased concentration of intermediate 2 may stimulate the minor pathway.
  38. 38.  In galactosemia deficiency of galactose -1- phosphate uridyltransferate leads to accumulation of galactose causing tissue damage.  In phenyleketonuria, deficiney of phenylalanine hydroxylase results in the accumulation of phenylalanine.
  39. 39.  Enzyme defect may lead to metabolic block leading to decrease amount of end product which is necessary for normal function e.g. deficiency of melanin result from lack of tyrosinase which is necessary for biosynthesis of melanin form its precursor (tyrosine) seen in albinism.  Failure to inactivate a tissue damaging substrate (α1 antitrypsin deficiency) resulting in inactivation of neutrophil elastase in the lung producing destruction of elastin leading eventuly (emphysema).
  40. 40. Defect in receptors and transport system  In familial hypercholesterolemia reduced synthesis of LDL receptor leads to defective transport of LDL and secondarily to excessive cholesterol synthesis.  In cystic fibrosis transport for chloride ions in exocrine glands is defective which leads to injury.  In thalasemia oxygen caring capacity of HB is reduced due to defects in Hb chain after mutation.
  41. 41. Alteration in Structure, Function, or Quantity of Nonenzyme Proteins  Hemoglobinopathies, sickle cell disease affect the amount of normal α & β globin chain synthesis.  Factor VIII deficiency leads to hemophilia A.  Hereditary retinoblastoma and Neurofibromatosis type I are seen when Growth Regulation is hampered after deletions of certain gene.
  42. 42. Disorders associated with defects in receptor proteins  Familial Hypercholesterolemia ◦ Mutation in gene encoding the receptor for LDL which is involved in transport and metabolism of cholesterol leading to elevated level of cholesterol inducing premature atherosclerosis. ◦ Heterozygotes show 2 to 3 times elevation of plasma cholesterol leading to xanthomas and premature atherosclerosis. ◦ Hosozygotes shows fivefold to sixfold elevaiton in plasma cholesterol.
  43. 43. Cholesterol metabolism  1st step is secretion of VLDL by the liver into the bloodstream which is rich in riglycerides having laser amounts of cholsteryl esters.  When VLDL reaches capillaries of adipose tissue or muscle, it is cleaved by lipoprotein lipase forming IDL which have reduced triglyceride content and high cholesteryl esters but it contain 2 of 3 apoproteins (B-100 and E)
  44. 44.  50% of IDL is taken up by liver through a receptor mediated transport which bind IDL to liver cell membrane recognize both apoprotein B-100 and apoprotien (LDL Receptor)  In liver IDL is recycle to generate VLDL, IDL is intermediate and major source of plasma LDL.  Two mechanisms for removal of LDL from plasma, one mediated by LDL receptor for and other by oxidized LDL (scavenger receptor)
  45. 45.  Fibroblast lymphocytes, smooth muscle cells, hepatocytes and adrenocortical cells possess high affinity LDL receptor.
  46. 46.  70% of plasma LDL is cleared by liver by following mechanisms. ◦ 1st step is binding of LDL to cell surface receptors (coated pits) ◦ After binding the coated pits are converted into coated vesicles which later on fuse with the lysosomes.
  47. 47.  Here LDL dissociates and recycled to surfae and LDL molecule is enzymatically degraded to amino acids where as cholesteryl esters are broken down to free cholesterol which is used for membrane synthesis and homeostasis by three mechanisms.
  48. 48. 1. Cholesterol suppresses cholesterol synthesis by inhibiting activity of enzyme 3 hydoroxy-3, methylglutryl conenzyme A (HMG CoA) 2. Cholesterol favoring esterificaiton by activating enzyme acyl-coenzyme A;cholesterol axcylrransferase. 3. Cholesterol suppresses the synthesis of LDL receptors.
  49. 49.  In heterozygotes with familial hypercholesterolemia possess only 50% of normal high affinity LDL receptor leading to two fold increase in cholesterol.  In hemozygotes with familial hypercholesterolemia possess no normal high affinity LDL receptor leading to five fold increase in cholesterol.
  50. 50.  In familial hypercholesterolemia, impaired IDL transport into the liver secondarily diverts a greater porportion fo plasma IDL into the precursor pool for plasma LDL.  The transport of LDL via the scavenger receptor appears to occur at least in part into the ells of the mononuclear phagocyte system.  Normally the amount of LDL transported along this scavenger receptor pathway is less than that mediated by the LDL receptor-dependent mechanisms.
  51. 51.  This increase is responsible for the appearance of xanthomas and contributes to the pathogenesis of premature atherosclerosis.  The human LDL receptor gene, located on chromosome 19, is extremely large, with 18 exons and 5 domains, including insertions, involving the LDL receptor gene have been identified.
  52. 52.  These can be classified into five groups. 1. Class I mutations are relatively uncommon, and they lead to a complete failure of synthesis of the receptor protein (null allele) 2. Class II mutations are fairly common they encode receptor proteins that accumulate in the endoplasmic reticulum. 3. Class III mutations affect the LDL-binding domain of the receptor; the encoded proteins reach the cell surface but fail to bind LDL or do so poorly.
  53. 53. 4. Class IV mutations encode proteins that are synthesized transported to the cell surface efficiently. 5. Class V mutations encode proteins that are expressed on the cell surface can bind LDL and can be internalized. Acid dependent dissociation of the receptor and the bound LDL fails to occur. Note: certain drugs (statins) are used to suppress intracellular cholesterol synthesis by inhibiting the enzyme.
  54. 54. Disorders with multifactorial Inheritance  It is due to both environmental influences and two or more mutant genes having additive effects.  It include Cleft lip or cleft palate Congenital heart disease Coronary heart disease Hypertension Gout Diabetes Mellitus Pyloric stenosis.
  55. 55. Normal Karyotyping  Study of chromosomes (Karyotyping) is basic tool of cytogeneticist.  By arresting mitosis in dividing cell in metaphase by use of mitotic spindle inhibitors and then to stain the chromosomes are major steps in karyotyping usually Giemsa stain is used (G banding).
  56. 56.  Chromosome comprises of short arm designated p (for petit) and long arm referred as q.  Each arm is divided into regions.  Each region is divided into bands and sub bands.  Xp21.2 means short arm of X chromosome, in region 2, band 1, and sub band 2.
  57. 57.  Fluorescence in Situ Hybridizaiton (FISH)  Keryotyping is only applicable in dividing cells.  This problem can be over come with DNA probes that recognize chromosome- specific sequences.  Such probes are labeled with fluorescent dyes and applied to interphase nuclei.  The probe binds to its complementary sequence labels the specific chromosome and can be visualized under a fluorescent microscope (FISH)
  58. 58.  FISH can be used to demonstrate microdeletions, complex translocations and telomere alterations which are not detectable by routine karyotyping.  FISH can be used to physically map newly isolated genes.  Chromosome paining is an extension of FISH whereby whole chromosomes can be labeled with series of fluorescent DNA probes.
  59. 59.  All the chromosome can not be detected simultaneously by chromosome painting.  This hurdle has been overcome by spectral karyotyping by using five fluorochromes and appropriate computer generated signals the entire human genome can be visualized which is called (spectral karyotyping SKY or “spectacular karyotyping”)  FISH directly compares the DNA content of differentially labeled normal and tumor cell
  60. 60.  Figure 4b demonstrates that BCR-ABL fusions can be readily identified by FISH when a green-labeled hybridization probe flanking BCR is applied together with a red-labeled probe flanking ABL. In this image, the normal copies of chromosomes 9 and 22 are detected as red and green spots, respectively.  On the other hand, the Philadelphia chromosome is visible as a complex fused spot, which appears to have a central yellow region with red and green subregions on either side. (In fluorescence microscopy, yellow is indicative of very close proximity of red and green probes, such that they appear to overlap.)
  61. 61.  Figure 4b shows a FISH analysis that was used to detect the presence of a chromosomal translocation in a patient suffering from chronic myelogenous leukemia (Tkachuk et al., 1990). In most cases of this disease, a segment of chromosome 9 that contains the ABL proto-oncogene fuses with the breakpoint cluster region (BCR) on chromosome 22 during a reciprocal translocation. The derived chromosome 22, or der(22), also known as the Philadelphia chromosome, contains a BCR-ABL fusion gene in which the powerful BCR promoter drives synthesis of the ABL oncogene transcript, leading to cancer.
  62. 62. Cytogenetic Disorder  Any abnormality leading to an abnormal number of chromosome or alteration in the structure of one or more chromosome is called cytogenetic disorder.  An exact multiple of the haploid number is called euploid.  If it is not exact multiple of 23 it is referred to as aneuploidy either due to nondisjunciton and anaphase lag.
  63. 63.  Nondisjunciton take place when a homologous pair of chromosomes fails to disjoin at the first meiotic division or two chromatids fail to separate either at second meiotic division or in somatic cell divisions resulting in two aneuploid cells.  If nondisjunciton occurs during gametogenesis the gametes formed have either an extra chromosome or one less chromosome.  Fertilizaiton of such gametes by normal gametes result in either trisomic (2n+1) or monosomic (2n – 1 ).
  64. 64.  In anaphase lag. One homologous in meiosis or one chromatid in mitosis lags behind and is left out of nucleus resulting in one normal cell and one cell with monosomy.  Monosomy or trisomy involving in sex chromosome or compatible with life with variable degree of phenotypic abnormalities.  Monosomy involving an autosome leads to loss of too much genetic informaiton to permit the life birth with the exception of trisomy 21, all die in early age.
  65. 65.  Occasionally mitotic errors in early development in two or more population of cells (mosaicism).  Mosaicism can results form mitotic errors during the cleavage of fertilized ovum or in somatic cells.  Mosaicism is common in sex chromosome e.g. 45,X/47,XXX (mosaic variant of Turner syndrome.  Autosomal mosaicism e.g. 46,XY/47,XY,+21. (trisomy 21 mosaic with variable expression of Down syndorme)
  66. 66.  Second category of chromosomal aberrations is associated with changes in structure of chromosome.  It result from chromosome breakage followed by loss or rearrangements of material.  Deletion refers to loss of a portion of a chromosome. Deletion are interstitial but terminal deletions may occur.  Interstitial deletions occur when two breaks within a chromosome followed b loss of material between the breaks, and fusion of broken ends.
  67. 67.  A ring chromosome is a special form of deletion in which a break occur at both ends o f chromosome with fusion of damaged ends.  Inversion refers to rearrangements that involves two breaks within a single chromosome with inverted reincorporation of the segment.  If it involve only one arm of chromsome is call paracentric.  If the breaks are opposite sides of the centromere, pericentric.
  68. 68.  Isochromosome formation results when one arm of chromosome is lost and the remaining arm is duplicated resulting in chromosome consisting of two short arms only or two long arms.  In translocation segment of chromosome is transferred to another.  If single breaks in each of two chromosome with exchange of material take place it is called balanced reciprocal translocation.  A Robertosnian translocation (centric fusion), a translocation between two acrocentric chromosome.
  69. 69. Cytogenic disorders involving Trisomy 21 (Down Syndrome)  Down syndrome is the most common of the chromosomal disorders and is a major cause of mental retardation.  Tirsomy 21 have normal chromosome numbers, but the extra chromosomal material is present as translocation due to meiotic nondisjunction.  Mosaicism is also seen in 1% of cases having mixture of 46 and 47 chromosome resulting from mitotic nondisjunction of chromosome 21 during an early stage of embroygenesis.
  70. 70.  Clinical features include flat facial profile oblique palpebral fissures, and epicanthic fold with IQ of 25 to 50 in 80% of cases.  40% cases have congenital heart disease, atrial septal defects, valvular malformations and ventricular septal defect and these are causes of death in early infancy.  10 to 20 fold increased risk of acute leukemia.  In a later phase neurological deficit are also seen with abnormal immune response to infections.
  71. 71. Cytogenetic disorders involving sex chromosome  Characterized by disorders relating to sexual development and fertility.  Difficult to diagnose at birth and first recognized at the time of puberty.  Higher the number of X chromosome, greater the chance of mental retardation.
  72. 72. Klinefelter Syndrome  Male hypogonadism due to two or more X chromosome and one or more Y chromosome.  Gynocemastic may be present.  IQ is < normal.  FSH is elevated and testosterone is reduced. Estradiol levels are elevated.  Ratio of estrogens and testosterone determines the degree of feminization.
  73. 73.  Reduce spermatogenesis and male infertility with the evidence of atrophied or prmitive tubules.  Leyding cells are prominent.  Classic pattern of KF syndrome is 47,XXY karyotype (82%).  15% of KF have mosaic patterns, 46,XY/47,XXY, 47, XXY/48,XXXY.
  74. 74. Turner Syndrome  It results from complete or partial monosomy of X chromosome characterized primarily by hypogonadism in phenotypic females.  57% show missing X chromosome resulting in 45,X karyotype.  14% have structural abnormalities of the X chromosome (deletion of the small arm, deletion of short arms, deletion of short and long arm. and remaining show mosaics pattern (45,X/46,XX 45,X/46,XY).
  75. 75.  Clinically present as swelling of the nape of the neck with distended lymphatic channels (cystic hygroma)  Bilateral neck webbing and persistent looseness of skin.  Congenital heart disease.  Failure to develop normal secondary sex. (breast development is inadequate)  Truner syndrome is the single most important cause of primary amenorrhea, accounting for approximately one third of the cases.
  76. 76. Diagnosis Of Genetic Disease  There are two methods. ◦ Cytogenetic analysis ◦ Molecular analysis Cytogenetic Analysis:  Prenatal chromosome analysis:  It can be performed on the cell obtained by aminocentesis, on chorionic villus biopsy, or umbilical cord blood e.g. ◦ Advanced maternal age >34 years due to risk of trisomies. ◦ Parent who is carrier of balanced reciprocal, robertsonian translocation, inversion.
  77. 77. ◦ A parent with a previous child with a chromosomal abnormality. ◦ Parent carrier of an X-linked genetic disorder (determine fetal sex)  Postnatal chromosome analysis: ◦ Multiple congenital anomalies. ◦ Unexplained mental retardation ◦ Suspected aneuploidy ◦ Suspected unbalanced autosome. ◦ Sex chromosomal abnormality ◦ Suspected fragile X-syndrome ◦ Infertility ◦ Multiple spontaneous abortions
  78. 78. Molecular diagnosis  It is possible to indentify mutations at the level of DNA.  Use of recombinant DNA technology as several distinct advantages.  It is remarkable sensitive.  By the use of PCR only 100 cells are even lesser no of cell are sufficient for diagnosis.  DNA based test or not dependent on gene product.  Two distant approaches for the diagnosis of single gene (direct detection of mutations and indirect detection)
  79. 79. Direct gene diagnosis  It is also called diagnostic biopsy of human genome, it depend upon on the detection of an important qualitative change in the DNA.  It is usually based on PCR analysis.  If RNA is used as substrate (RT-PCR) in which first reverse transcribed to obtain cDNA and then amplification by PCR.  To detect the mutant gene, two primers that bind to 3’ and 5’ prime ends of normal sequence are designed.
  80. 80.  By using DNA polymerases and thermal cycling, DNA between the primers is amplified producing millions of Copies of DNA.  The normal DNA and patients DNA are digested with the Mnl1 enzyme.  Normal DNA yields three fragments (67 base pairs, 37 base pairs, and 163 base pairs long) while patient’s DNA yields only two products (abnormal fragment of 200 base pairs and normal fragment of 67)
  81. 81.  Mutations that affect the length of DNA (e.g. deletions) can also be detected by PCR.  A variety of PCR based technologies (real time) can detect presence and absence of mutations during exponential phase of DNA amplification by using fluorophore indicators e.g. molecular beacon technology in which hairpin-shaped fluorescent oligonucleotide probes that fluoresced only on hybridization to target sequences wild-type DNA. In case of mismatch there is no fluorescence.
  82. 82. Indirect DNA diagnosis (linkage analysis)  It depend upon by exploiting naturally occurring variations or polymophisms in DNA sequences.  Such polymophisms can be grouped into two general categories site polymorphisms and length polymorphismsm.
  83. 83.  Site polymorphisms are also called restriction fragment length polymorphisms (RFLPs). Examination of DNA from any two persons reveals variations in the DNA sequences involving approximately one nucleotide in every 200 to 500 base pair stretches.
  84. 84.  Length polymorphisms: ◦ Human DNA contains short repetitive sequence of noncoding DNA. Hence these stretches of DNA can be affective to distinguish different chromosome. ◦ SNPs serve both as a physical landmark within the genome and as a genetic marker whose transmission can be followed from parent to child. ◦ It is necessary to determine the polymorphism pattern.
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