introduction to genetics

1. GENETICS
DR. ASHUTOSH SINGH

2. CYTOGENETICS
• Human somatic cells are diploid and contain 46
chromosomes:
• 22 pairs of autosomes and one pair of sex chromosomes
(XX in the case of female and XY in the males)
• Gametes (sperm and ova) contain 23 chromosomes and are
called haploid cells
All ova contain 23X while sperms contain either 23X or
23Y chromosomes
Thus, the sex of the offspring is determined by paternal
chromosomal contribution i.e. If the ovum is fertilised by
X-bearing sperm, female zygote results, while an ovum
fertilised by Y-bearing sperm forms male zygote

3. Karyotyping
• Karyotype is defined as the sequence of
chromosomal alignment on the basis of size,
centromeric location and banding pattern
• Determination of karyotype of an individual is
an important tool in cytogenetic analysis

4. Broad outlines of
karyotyping
1. Cell selection
• Cells capable of growth and division are
selected for cytogenetic analysis
• These include: cells from amniotic fluid,
chorionic villus (CVS) sampling, peripheral
blood lymphocytes, bone marrow, lymph node,
solid tumours etc

5. 2. Cell culture.
• The sample so obtained is cultured in mitogen
media
• A mitogen is a substance which induces mitosis
in the cells e.g. PPD, phytohaemagglutinin
(PHA), pokeweed mitogen (PWM), phorbol ester
etc
• The dividing cells are then arrested in metaphase
by the addition of colchicine or colcemid, both of
which are inhibitory to microtubule formation
Subsequently, the cells are lysed by adding
hypotonic solution

6. 3. Staining/banding.
• When stained, chromosomes have the property of forming
alternating dark and light bands
• For this purpose, fixed metaphase preparation is stained by one of
the following banding techniques:
a) Giemsa banding or G-banding, the most commonly used.
b) Quinacrine banding or Q-banding used to demonstrate bands along
chromosomes
c) Constitutive banding or C-banding is used to demonstrate
constitutive heterochromatin.
d) Reverse staining Giemsa banding (or R-banding) gives pattern
opposite to those obtained by G-banding

7. 4. Microscopic analysis.
• Chromosomes are then photographed by examining the
preparation under the microscope
• From the photograph, chromosomes are cut and then
arranged according to their size, centromeric location
and
banding patterns
The pairs of chromosomes are identified by the arm
length of chromosomes
The centromere divides the chromosome into a short
upper arm called p arm (p for petit in French meaning
‘short’) and a long lower arm called q arm (letter q
next to p)

8. Applications
• The field of cytogenetics has widespread applications in diagnostic
pathology
i) Chromosomal numerical abnormalities e.g. Down’s
syndrome(trisomy 21 involving autosome 21), Klinefelter’s
syndrome(trisomy 46), Turner’s syndrome (monosomy 45, XO),
spontaneous abortions
ii) Chromosome structural abnormalities include translocations{e.g.
Philadelphia chromosome t(9;22), cri-du-chat (5p)syndrome,
repeated spontaneous miscarriages}, deletions,insertions,
isochromosome, and ring chromosome formation
iii) Cancer is characterised by multiple and complex chromosomal
abnormalities which include deletions, amplifications,inversions and
translocations, especially in leukaemias and lymphomas, germ cell
tumours, some sarcomas

9. DIAGNOSTIC MOLECULAR
PATHOLOGY
• All the DNA/RNA-based molecular techniques
employ hybridization (meaning joining
together)technique based on recombinant
technology
• Specific region of DNA or RNA is detected by
labelling it with a probe (Probe is a chain of
nucleotides consisting of certain number of
known base pairs)

10. • Probes are of different sizes and sources as under:
1. Genomic probes derived from a region of DNA of cells
2. cDNA probe derived from RNA by reverse
transcription.
3. Oligonucleotide probe is a synthetic probe contrary to
genomic DNA and cDNA probe both of which are
derived from cellular material
4. Riboprobe is prepared by in vitro transcription system

11. MOLECULAR METHODS
1. IN SITU HYBRIDISATION. In situ hybridisation
(ISH) is a molecular hybridisation technique which
allows localisation of nucleic acid sequence directly in
the intact cell (i.e. in situ)without DNA extraction
• ISH involves specific hybridisation of a single strand
of a labelled nucleic acid probe to a single strand of
complementary target DNA or RNA in the tissue
• The end-product of hybridisation is visualised by
radioactivelabelled probe (32P, 125I), or non-
radioactive-labelled probe(e.g. biotin, digoxigenin)

12. Applications
• ISH is used for the following:
i) In viral infections e.g. HPV, EBV, HIV, CMV,
HCV etc.
ii) In human tumours for detection of gene
expression and oncogenes.
iii) In chromosomal disorders, particularly by
use of fluorescent in situ hybridisation (FISH)

13. 2. FILTER HYBRIDISATION.
• In this method, target DNA or RNA is
extracted from the tissue, which may either be
fresh,frozen and unfixed tissue, or formalin-
fixed paraffinembedded tissue
• Extracted target DNA or RNA is then
immobilised on nitrocellulose filter or nylon
• Hybridisation of the target DNA is then done
with labelled probe.

14. i) Slot and dot blots in which the DNA sample is directly
bound to the filter without fractionation of nucleic acid
size.
ii) Southern blot which is similar to dot-blot but differs in
performing prior DNA-size fractionation by gel
electrophoresis(E.M. Southern is the name of scientist
who described Southern blot technique)
iii) Northern blot is similar to Southern blot but involves
size fractionation of RNA (Northern is, however,
opposite direction of southern and not someone’s name)
iv) Western blot is employed for protein fractionation; in
this method antibodies are used as probes.

15. Applications
• In view of high degree of specificity and sensitivity of the molecular
hybridisation techniques, these techniques have widespread
applications in diagnostic pathology:
i) In neoplasia, haematologic as well as non-haematologic.
ii) In infectious diseases for actual diagnosis of causative
agent,epidemiologic studies and identification of newer infectious
agents
iii) In inherited genetic diseases for carrier testing, prenatal diagnosis
and direct diagnosis of the genetic disease.
iv) In identity determination for tissue transplantation, forensic
pathology, and parentage testing

16. 3. POLYMERASE CHAIN
REACTION
• Polymerase chain reaction (PCR) is a
revolutionary technique for molecular genetic
purpose with widespread applications in
diagnostics and research The technique is based
on the principle that a single strand of DNA has
limitless capacity to duplicate itself to form
millions of copies
• In PCR, a single strand of DNA generates
another by DNA polymerase using a short
complementary DNA fragment; this is done using
a primer which acts as an initiating template

17. • A cycle of PCR consists of three steps:
i) Heat denaturation of DNA (at 94°C for 60-90
seconds).
ii) Annealing of the primers to their
complementary sequences (at 55°C for 30-120
seconds)
iii) Extension of the annealed primers with DNA
polymerase (at 72°C for 60-180 seconds).

18. • Repeated cycling can be done in automated
thermal cycler and yields large accumulation
of the target sequence since each newly
generated product, in turn, acts as template in
the next cycle

19. Applications
• PCR analysis has the same applications as for
filter hybridisation techniques and has many
advantages over them in being more rapid, can
be automated by thermal cyclers and requires
much lower amount of starting DNA
• However, PCR suffers from the risk of
contamination; thus extreme caution is
required in the laboratory during PCR
technique

20. GENETIC DISORDERS
• For the purpose of convenience of discussion,
genetic and paediatric diseases are covered under
the following headings:
1. Developmental defects: Errors in morphogenesis
2. Cytogenetic (Karyotypic) defects: chromosomal
abnormalities
3. Single-gene defects: Mendelian disorders
4. Multifactorial inheritance disorders
5. Other paediatric diseases

21. DEVELOPMENTAL DEFECTS
• Developmental defects are a group of
abnormalities during foetal life due to errors in
morphogenesis
• The branch of science dealing with the study of
developmental anomalies is called teratology
• Certain chemicals, drugs, physical and biologic
agents are known to induce such birth defects and
are called teratogens
• The morphologic abnormality or defect in an
organ or anatomic region of the body so produced
is called malformation

22. Pathogenesis
• The teratogens may result in one of the
following outcomes:
i) Intrauterine death
ii) Intrauterine growth retardation (IUGR)
iii) Functional defects
iv) Malformation

23. • The effects of teratogens in inducing developmental defects
are related to the following factors:
• Variable individual susceptibility to teratogen: All patients
exposed to the same teratogen do not develop birth defect
• Intrauterine stage at which patient is exposed to teratogen:
• Most teratogens induce birth defects during the first
trimester of pregnancy
• Dose of teratogen: Higher the exposure dose of teratogen,
greater the chances of inducing birth defects.
• Specificity of developmental defect for specific teratogen: A
particular teratogen acts in a particular way and induces the
same specific developmental defect

24. Classification
• Various developmental anomalies resulting from
teratogenic effects are categorised as under:
• Agenesis means the complete absence of an organ
e.g. unilateral or bilateral agenesis of kidney.
• Aplasia is the absence of development of an organ
with presence of rudiment or anlage e.g. aplasia of lung
with rudimentary bronchus.
• Hypoplasia is incomplete development of an organ
not reaching the normal adult size e.g. microglossia.
• Atresia refers to incomplete formation of lumen in
hollow viscus e.g. oesophageal atresia.

25. • Developmental dysplasia is defective
development of cells and tissues resulting in
abnormal or primitive histogenetic structures e.g.
renal dysplasia
• Dystraphic anomalies are the defects resulting
from failure of fusion e.g. spina bifida.
• Ectopia or heterotopia refers to abnormal
location of tissue at ectopic site e.g. pancreatic
heterotopia in the wall of stomach

26. Examples of Developmental Defects
• A few common clinically important examples are given
below:
1. Anencephaly-spina bifida complex. This is the group of
anomalies resulting from failure to fuse (dystraphy).
• While anencephaly results from failure of neural tube
closure, spina bifida occurs from incomplete closure of the
spinal cord and vertebral column, often in the lumbar region
• The latter results in meningocele or meningomyelocele.
2. Thalidomide malformations. Thalidomide is the best
known example of teratogenic drug which was used as a
sedative by pregnant women in 1960s in England and
Germany and resulted in high incidence of limb-reduction
anomalies (phocomelia) in the newborns

27. 3. Foetal hydantoin syndrome. Babies born to
mothers on anti-epileptic treatment with
hydantoin have characteristic facial features and
congenital heart defects.
4. Foetal alcohol syndrome. Ethanol is another
potent teratogen
• Consumption of alcohol by pregnant mother in
first trimester increases the risk of miscarriages,
stillbirths,growth retardation and mental
retardation in the newborn

28. 5. TORCH complex. Infection with TORCH group
of organisms (Toxoplasma, Others, Rubella,
Cytomegalovirusand Herpes simplex) during
pregnancy is associated with multisystem
anomalies and TORCH syndrome in the newborn
6. Congenital syphilis. Vertical transmission of
syphilis from mother to foetus is characterised by
Hutchinson’s triad: interstitial keratitis,
sensorineural deafness and deformed
Hutchinson’s teeth,along with saddle-nose
deformity

29. CYTOGENETIC (KARYOTYPIC)
ABNORMALITIES
• Human germ cells (ova and sperms) contain 23
chromosomes(haploid or 1N) while all the
nucleated somatic cells of the human body
contain 23 pairs of chromosomes(diploid or
2N)—44 autosomes and 2 sex chromosomes,
being XX in females (46, XX) and XY in males
(46, XY)
• The branch of science dealing with the study of
human chromosomal abnormalities is called
cytogenetics

30. • In a female, one of the two X chromosomes (paternal or maternal
derived) is inactivated during embryogenesis as stated in Lyon
hypothesis
• This inactivation is passed to all the somatic cells while the germ
cells in the female remain unaffected i.e. ovary will always have
active X chromosome
• Such an inactive X chromosome in the somatic cells in females lies
condensed in the nucleus and is called as sex chromatin seen
specifically in the somatic cells in females
• Nuclear sexing can be done for genetic female testing by preparing
and staining the smears of squamous cells scraped from oral cavity,
or by identifying the Barr body in the circulating neutrophils as
drumstick appendage attached to one of the nuclear lobes
• A minimum of 30% cells positive for sex chromatin is indicative of
genetically female composition

31. • Though chromosomes can be studied in any
human nucleated cells, circulating
lymphocytes are more often used for this
purpose
• The study is done by arresting the dividing
cells in metaphase by colchicine and then
spreading them on glass slide and staining
them with Giemsa stain

32. • Karyotype is the photographic representation
of the stained preparation of chromosomes
• Each chromosome is composed of a pair of
identical double helix of chromosomal DNA
called chromatids
• The chromosomes are classified based on their
length and location of the centromere;
centromere is the point where the two
chromatids cross each other

33. • Based on centromeric location, they are classified into
3 groups:
• Metacentric chromosomes (numbers 1, 3, 16, 19, 20)
are those in which the centromere is exactly in the
middle
• Submetacentric chromosomes (numbers 1, 3) in which
the centromere divides the chromosomes into short arm
(p arm; petit means short in French) and long arm (q
arm; for alphabet next to p).
• Acrocentric chromosomes (numbers 13, 14, 15, 21, 22,
and Y) have very short arm and the centromere is
eccentrically located

35. • Based on length of chromosomes, they are divided into 7
groups—A to G, called Denver classification adopted at a
meeting in Denver, Colorado in US.
• Chromosomal banding techniques are employed for study of
classes of chromosomes
• Chromosomal bands are unique alternate dark and light
staining patterns
• Banding techniques include:
i) G-banding (Giemsa stain);
ii) Q-banding (quinacrine fluorescence stain);
iii) R-banding (reverse Giemsa staining); and
iv) C-banding (constitutive heterochromatin demonstration).

36. 1. Polyploidy is the term used for the number of
chromosomes which is a multiple of haploid number
e.g. triploid or 3N (69 chromosomes), tetraploid or 4N
(92 chromosomes).
• Polyploidy occurs normally in megakaryocytes and
dividing liver cells
• Polyploidy in somatic cells of conceptus results in
spontaneous abortions
2. Aneuploidy is the number of chromosomes which is not
an exact multiple of haploid number e.g. hypodiploid or
2N-1 (45 chromosomes) monosomy, hyperdiploid or 2
N+1(47 chromosomes) trisomy

37. • The most common mechanism of aneuploidy is
nondisjunction. Nondisjunction is the failure of
chromosomes to separate normally during cell division
during first or second stage of meiosis, or in mitosis
• Nondisjunction during first meiotic division stage will
result in two gametes from both the parental
chromosomes due to failure to separate while the other
two gametes will have no chromosomes (nullisomic)
• Nondisjunction during second meiotic division stage
results in one gamete with two identical copies of the
same chromosome, one nullisomic gamete, and two
gametes with normal chromosome number.

38. • Nondisjunction during mitosis results in
mosaicism,meaning thereby that the individual
has two or more types of cell lines derived
from the same zygote
• Mosaicism of mitotic nondisjunction of
chromosomes occurs in cancers

39. • Anaphase lag is a form of nondisjunction
involving single pair of chromosomes in which
one chromosome in meiosis or a chromatid in
mitosis fails to reach the pole of dividing cell
at the same time (i.e. it lags behind) and is left
out of the nucleus of daughter cell
• This results in one normal daughter cell and
the other monosomic for the missing
chromosome.

40. • Three clinically important syndromes resulting
from numerical aberrations of chromosomes
due to nondisjunction are as under and their
main clinical features are illustrated

41. Down’s syndrome. There is trisomy 21 in about 95% cases of Down’s
syndrome due to nondisjunction during meiosis in one of the parents
Down’s syndrome is the most common chromosomal disorder and is
the commonest cause of mental retardation
• The incidence of producing offspring with Down’s syndrome rises
in mothers over 35 years of age.
Klinefelter’s syndrome. Klinefelter’s syndrome is the most important
example of sex chromosome trisomy
About 80% cases have 47, XXY karyotype while others are mosaics
• Typically, these patients have testicular dysgenesis
• In general, sex chromosome trisomies are more common than
trisomies of autosomes.
Turner’s syndrome. Turner’s syndrome is an example of monosomy
(45, X0) most often due to loss of X chromosome in paternal
meiosis.

42. Structural Abnormalities
• During cell division (meiosis as well as mitosis), certain
structural abnormalities of chromosomes may appear
• These may occur during gametogenesis and then
transmitted to all somatic cells and cause hereditary
transmissible disorders, or may produce somatic cell
mutations and result in changes varying from no effect to
some forms of cancers
• Structural abnormalities may be balanced or unbalanced.
• Balanced structural alteration means no change in total
number of genes or genetic material.
• Unbalanced structural alteration refers to gene
rearrangement resulting in loss or gain of genetic material.

43. TRANSLOCATIONS
• Translocation means crossing over or
exchange of fragment of chromosome which
may occur between non-homologous or
homologous chromosomes.
• There are two main types of translocations:
reciprocal in about two-third and Robertsonian
in one-third cases:

44. • Reciprocal translocation is the exchange of genetic material between two non-
homologous (heterologous) chromosomes without involving centromere (acentric)
• Such translocations occur due to single breaks in both the chromosomes and the
exchange is detected by banding techniques
• Reciprocal translocation may be balanced(without any loss of genetic material
during the exchange)or unbalanced (with some loss of genetic material)
i) Balanced reciprocal translocation is more common and the individual is
phenotypically normal e.g. Translocation between long arm (q) of chromosomes
22 and long arm (q)of chromosome 9 written as 46, XX, t (9;22)
ii) This translocation is termed Philadelphia chromosome seen in most cases of
chronic myeloid leukaemia
ii) Unbalanced reciprocal translocations are less common and account for repeated
abortions and malformed children

45. • Robertsonian translocation is less common than
reciprocal translocation
• In this, there is fusion of two acrocentric chromosomes
(having very short arms) at the centromere (centric
fusion) with loss of short arms
• The result of this fusion is one very large chromosome
and the other very small one
• Individuals born with Robertsonian translocation may
be phenotypically normal but suffer from infertility and
are at higher risk of producing malformed children in
the next progeny

46. DELETIONS
• Loss of genetic material from the chromosome is
called deletion
• Deletion may be from the terminal or middle
portion of the chromosome
• The examples of deletion are: cri du chat (named
after cry of infant like that of a cat)syndrome
(deletion of short arm of chromosome 5) and
several cancers with hereditary basis (e.g.
Retinoblastoma with deletion of long arm of
chromosome 13, Wilms’ tumour with deletion of
short arm of chromosome 11).

47. INVERSION.
• Inversion is a form of rearrangement involving
breaks of a single chromosome at two points.
Inversion may be pericentric or paracentric,
depending upon whether the rotation occurs at
the centromere or at the acentric portion of the
arm of chromosome
• Inversions are not associated with any
abnormality.

49. RING CHROMOSOME
• A ring of chromosome is formed by a break at
both the telomeric (terminal) ends of a
chromosome followed by deletion of the
broken fragment and then end-to-end fusion.
The consequences of ring chromosome depend
upon the amount of genetic material lost due to
break

50. ISOCHROMOSOME
• When centromere, rather than dividing parallel
to the long axis, instead divides transverse to
the long axis of chromosome, it results in
either two short arms only or two long arms
only called isochromosomes
• The example involving isochromosome of X-
chromosome is seen in some cases (15%) of
Turner’s syndrome

51. SINGLE-GENE DEFECTS
(MENDELIAN DISORDERS)
• The classic laws of inheritance of characteristics
or traits were outlined by Austrian monk Gregor
Mendel in 1866 based on his observations of
cross-breeding of red and white garden peas
Single-gene defects follow the classic mendelian
patterns of inheritance and are also called
mendelian disorders
• These disorders are the result of mutation of a
single gene of large effect.

52. MUTATIONS.
• The term mutation is applied to permanent
change in the DNA of the cell
• Mutations affecting germ cells are transmitted
to the next progeny producing inherited
diseases, while the mutations affecting somatic
cells give rise to various cancers and
congenital malformations

53. i) Point mutation is the result of substitution
of a single nucleotide base by a different base
i.e. replacement of an amino acid by another
e.g. in sickle cell anaemia there is point
mutation by substitution of glutamic acid by
valine in the polypeptide chain
ii) Stop codon or nonsense mutation refers to
a type of point mutation in which the protein
chain is prematurely terminated or truncated

54. iii) Frameshift mutation occurs when there is
insertion or deletion of one or two base pairs
in the DNA sequence e.g.in cystic fibrosis of
pancreas
iv) Trinucleotide repeat mutation is
characterised by amplification of a sequence
of three nucleotides

55. INHERITANCE PATTERN
• A dominant gene* produces its effects, whether combined with
similar dominant or recessive gene
• Recessive genes are effective only if both genes are similar.
However, when both alleles of a gene pair are expressed in
heterozygous state, it is called codominant inheritance
• A single gene may express in multiple allelic forms known as
polymorphism.
• Autosomal dominant inheritance pattern is characterised by one
faulty copy of gene (i.e. mutant allele) in any autosome and one
copy of normal allele; disease phenotype is seen in all such
individuals
• Patients having autosomal dominant inheritance disease have 50%
chance of passing on the disease to the next generation.

56. • In autosomal recessive inheritance, both copies of genes are
mutated. Usually, it occurs when both parents are carriers of the
defective gene, i.e. having one normal allele and one defective allele
in each parent, and each parent passes on their defective gene to the
next progeny causing disease.
• There is 25% chance of transmission of autosomal recessive disease
when both parents are carriers
• X-linked disorders are caused by mutations in genes on
Xchromosome, derived from either one of the two Xchromosomes in
females, or from the single X-chromosome of the male
• There are much fewer genes on Y-chromosome and are determinant
for testis; they are not known to cause any sex-linked disorder
• Therefore, all sex-linked disorders are, in fact, X-linked disorders

57. AUTOSOMAL RECESSIVE
INHERITANCE
1. β-thalassaemia
2. Sickle cell anaemia
3. Haemochromatosis
4. Cystic fibrosis of pancreas
5. Albinism
6. Wilson’s disease
7. Xeroderma pigmentosum
8. Inborn errors of metabolism (Lysosomal storage
diseases,glycogenosis, alkaptonuria,
phenylketonuria

58. AUTOSOMAL CODOMINANT
INHERITANCE
1. ABO blood group antigens
2. α 1-antitrypsin deficiency
3. HLA antigens

59. AUTOSOMAL DOMINANT
INHERITANCE
1. Familial polyposis coli
2. Adult polycystic kidney
3. Hereditary spherocytosis
4. Neurofibromatosis (von Recklinghausen’s disease)
5. Marfan’s syndrome
6. von Willebrand’s disease
7. Hereditary haemorrhagic telangiectasia
8. Acute intermittent porphyria
9. Familial hypercholesterolaemia
10.Osteogenesis imperfecta

60. SEX-(X-) LINKED RECESSIVE
INHERITANCE
1. Haemophilia A
2. G6PD deficiency
3. Diabetes insipidus
4. Chronic granulomatous disease
5. Colour blindness
6. Bruton’s agammaglobulinaemia
7. Muscular dystrophies

61. SEX-(X-) LINKED DOMINANT
INHERITANCE
1. Hypophosphataemic rickets
2. Incontinentia pigmenti

62. THANK YOU