Chapter 7

GENETICS
Sr. No. TOPIC Page No.
1 Gene 2

2 Mutation 3

3 Single gene defect 3

4 Cytogenetic disorders 4

5 Chromosomal number- normal and abnormal 5

6 Down’s syndrome 6

7 Klinefelter’s syndrome 7

8 Turner’s syndrome 8

9 Chromosomal structure- normal and abnormal 8

10 Molecular diagnosis of genetic disorders 11

11 Important questions 13

Second Edition (2022) (CBME);

Notes based on: Robbins & Cotran, Pathologic Basis of Diseases, South Asia Edition, 2014.
Robbins Basic Pathology, 10th edition, 2018.

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 GENE

Structure

DNA of chromosome consists of two parallel strands (double helix).

Each strand consists of nucleotides arranged in chain.

Nucleotide of opposite strands are linked through bases (adenine-thymine; guanine-cytosine).

Proteins are made up of polypeptide chains, which in turn are made of amino acids. Each amino
acid is represented in DNA by a sequence of 3 bases (triple codes). Part of chromosomal DNA
that bears the code for a complete polypeptide chain (protein) is called gene. Thus genes are
structural units of chromosomes & each chromosome carries on it a large number of genes.

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 MUTATION

Definition

A mutation is defined as a permanent change in the DNA. Mutations that affect germ cells give
rise to inherited diseases. Mutations that arise in somatic cells cause cancer and some congenital
malformations.

Types of mutation
Point mutation: substitution of a single nucleotide base with a different base.
1. Point mutations within coding sequences (missense mutation): is point mutation altering
the code in a triplet of bases, leading to replacement of one amino acid by another.

E.g., sickle cell disease, in which nucleotide triplet CTC (thymine), which encodes
glutamic acid, is changed to CAC (adenine), which encodes valine in the 6th codon of β-
chain of hemoglobin.

E.g., in β0-thalassemia, in the β-chain of hemoglobin, in coding glutamine, U is

substituted for C (UAG → CAG). This substitution causes termination of β-chain of
hemoglobin, leading to its rapid degradation. Termination of chain due to mutation is
called stop codon (nonsense mutation).

2. Point mutations within noncoding sequences

3. Deletions and insertions (frameshift mutation): one or two base pairs may be deleted or
inserted
4. Trinucleotide-repeat mutations

Short answer question

1. Define mutation. Give two examples.

SINGLE GENE DEFECT

System Autosomal Autosomal recessive X-linked

dominant disorder disorder recessive disorder
Nervous Neurofibromatosis Fragile-X syndrome
Urinary Polycystic kidney disease
GIT Familial polyposis coli
Hematopoietic • Hereditary spherocytosis • Sickle cell anemia • Hemophilia A and
• von Willebrand disease • Thalassemias B
• Chronic
granulomatous
disease
• Glucose-6-

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 phosphate
dehydrogenase
deficiency
Skeletal Marfan syndrome Alkaptonuria Duchenne muscular
dystrophy
Metabolic Familial • Cystic fibrosis • Diabetes insipidus
hypercholesterolemia • Phenylketonuria • Lesch-Nyhan
• Homocystinuria syndrome
• Lysosomal storage
diseases
• α1-Antitrypsin
deficiency
• Wilson disease
• Hemochromatosis
• Glycogen storage
diseases

Short answer question

2. Name FOUR autosomal dominant disorders.

3. Name FOUR autosomal recessive disorders.
4. Give FOUR examples for sex linked disorders.

CYTOGENETIC DISORDERS

Definition

Cytogenetic disorders result from alterations in the number or structure of chromosomes and
may affect autosomes or sex chromosomes.

Cytogenetic disorders involving autosomes:

Trisomy 21 (Down Syndrome)
Trisomy 18: Edwards syndrome
Trisomy 13: Patau syndrome
DiGeorge syndrome (22q11.2 Deletion Syndrome)

Cytogenetic disorders involving sex chromosomes:

Klinefelter Syndrome
Turner Syndrome

Short answer question

1. Give four examples of cytogenetic disorders affecting autosomes.

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 CHROMOSOMAL NUMBER- NORMAL & ABNORMAL

Normal chromosomal number (euploid)

Somatic cells
23 pairs of chromosomes (46/2N/diploid):
22 pairs (44) : autosomes
1 pair : sex chromosomes
Females : 46 (44, XX)
Males : 46 (44, XY)

Germ cells
23 chromosomes (N/haploid):
22 : autosomes
1 : sex chromosome
Ova : 23 (22, X)
Sperm : 23 (22, Y)

Chromosomal (cytogenetic) numerical abnormalities

Polyploidy Aneuploidy

Autosomes Germ cells Autosomes Germ cells

Incompatible with life Down’s syn. Klinefelter’s syn.

Turner’s syndrome

Polyploidy- 3N, 4N… set

Definition: Number of chromosomes in multiple of haploid, other than diploid. E.g.:

triploid (3N, 69), tetraploid (4N, 92).

Consequence: Spontaneous abortion

Aneuploidy- 2N±1… part

Definition: Chromosomal number is not exactly the double of haploid. E.g.:

Trisomy: 2N +1 / 47
Down’s syndrome (trisomy autosome 21), Klinefelter’s syn. (trisomy sex chromosome)

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Monosomy: 2N – 1 / 45
Turner’s syndrome (monosomy sex chromosome)

Mechanism: non-disjunction; failure of chromosomes to separate normally during cell division.

Short answer question

1. What is aneuploidy? Give two examples.

DOWN’S SYNDROME

Numerical chromosomal abnormality

It is the most common chromosomal disorder, resulting from Trisomy of autosome 21.

Cause

• In 95% cases parents have normal karyotype. It is associated with increased maternal age.
Paternal age has no effect.

1 in 25 live births with mothers over 45 years

1 in 1550 live births with mothers under 20 years
Mechanism: Meiotic non-disjunction in ovum, thus maternal in origin

• In 5% cases, it is familial. One parent (more commonly mother) is carrier to Robertsonian

translocation of long arm of chromosome 21 to chromosome 22/14.

Clinical features
Affected can be a boy or a girl.

Major cause of mental retardation; IQ: 25-50 (mental age of 8–9-year-old)

Mongolism, former name is now considered offensive. Name was derived from ‘old &
unfortunate look’ of these patients, which resemble to members of the Mongol race of Asia:
• Flat facial profile
• Oblique palpebral fissure
• Epicanthic folds

Abundant neck skin

Simian crease
Gap between first and second toe
Congenital heart disease
Umbilical hernia
Intestinal stenosis

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Hypotonia
Predisposition to leukemia (particularly AML, megakaryoblastic and ALL)

Survival
Without congenital heart disease:
80% survive up to 30 years
May develop Alzheimer’s disease or dementia

With congenital heart disease:

survival is less favorable
die of cardiac disorder or severe infections

Short answer question

1. Give four clinical features of Down’s syndrome.

KLINEFELTER’S SYNDROME

Numerical chromosomal abnormality

Trisomy sex chromosome
Two or more X chromosomes with one or more Y chromosome
Most common abnormality is trisomy (47, XXY)
Resulting into male hypogonadism & infertility

Cause
Extra X chromosome can be of maternal / paternal origin
Increased risk with advanced maternal age or irradiation of either parent

Clinical features
Affects males
Rarely diagnosed before puberty: patient has disproportionately long arms & legs

At puberty:
Male hypogonadism & infertility characterized by:
• Atrophic testis
• Low sperm count, can be azoospermic
• Testicular biopsy shows hyalinized seminiferous tubules with hyperplastic Leydig cells
• Lack of secondary male characteristics: deep voice, beard……..
• Gynecomastia
• Osteoporosis

Low IQ: IQ is lower with more extra X chromosomes. Mental retardation is uncommon.

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Short answer question

1. Give four clinical features of Klinefelter’s syndrome.

TURNER’S SYNDROME

Cause
Monosomy sex chromosome

57%: loss of entire X chromosome, (45, XO). Such fetus is severely affected; only 1% fetuses
survive to birth

43%: Partial or complete loss of short arm of X chromosome

Clinical features

• Affects females
• Can be suspected at birth or in early childhood:
o Edema of dorsum of hand & feet
o Swelling of neck due to cystic hygroma
• Associated with female hypogonadism, called “menopause before menarche”

CHROMOSOMAL STRUCTURE- NORMAL & ABNORMAL

Normal chromosomal structure

Double helix
Each helix is called chromatid
Two chromatids cross at centromere

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Classification

Classified based on length and centromeric location:

1. Length:
‘A-G’ (7 groups)
‘A’ being longest
‘G’ being shortest

2. Centrometric location:
a. Metacentric: centromere is in center, dividing chromosomes equally
b. Sub-metacentric: centromere is off center, dividing chromosome into 2 short & 2
long arms. Short arm called ‘p’ (‘petit’ in French means short) and long arm
called ‘q’ (next alphabet).
c. Acrocentric: centromere located eccentrically, dividing chromosome into 2 very
small & 2 very long arms

Metacentric Sub-metacentric Acrocentric

Chromosomal structural abnormalities

Translocation

There is exchange of fragments of chromosome. It is classified into:

1. Balanced reciprocal translocation: A segment of one chromosome is transferred to
another chromosome with no loss of genetic material.
E.g.: Philadelphia chromosome in CML t (9 : 22)

2. Robertsonian translocation: Seen in acro-centeric chromosomes. Results in

formation of one chromosome with major part of both long arms & other with major
part of both short arms. Short arms are lost.

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Deletion

Deletion of ‘p’ arm of chromosome 11: Wilms tumor

Deletion of ‘q’ arm of chromosome 13: Retinoblastoma

Inversion
Rearrangement of segments in same chromosome.
Paracentric: rearrangement on one side of centromere
Pericentric: rearrangement across centromere

Ring chromosome

Break occurs at terminal ends of chromosome with loss of terminal segments and fusion of
broken ends.

Isochromosome
Centromere rather than dividing parallel to long axis, divides transversely, forming 2 short arms
on one side and 2 long arms on other.

Short answer question

1. What is chromosomal translocation? Give example.

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 MOLECULAR DIAGNOSIS OF GENETIC DISEASES

Testing for germ line mutations can be divided into prenatal and postnatal analysis.

Prenatal genetic analysis

It is offered to patients who are at risk of having cytogenetically abnormal progeny. It can be
performed on cells obtained by
1. amniocentesis
2. chorionic villus biopsy
3. umbilical cord blood

Indications for prenatal diagnosis:

1. Mother of advanced age (>35 years)
2. A parent who is a carrier of a balanced reciprocal translocation, Robertsonian
translocation, or inversion
3. A parent with a previous child with chromosomal abnormality
4. A fetus with ultrasound-detected abnormalities
5. A parent who is a carrier of an X-linked genetic disorder (to determine fetal sex)
6. Abnormal levels of AFP, β-HCG and estriol performed as the triple test

Postnatal genetic analysis

It is usually performed on peripheral blood lymphocytes. Indications are as follows:

1. Multiple congenital anomalies

2. Unexplained mental retardation and/or developmental delay
3. Suspected aneuploidy (e.g., features of Down syndrome)
4. Suspected sex chromosomal abnormality (e.g., Turner syndrome)
5. Infertility (to rule out sex chromosomal abnormality)
6. Multiple spontaneous abortions

Following are the various methods for detection of germ-line mutations:

Polymerase Chain Reaction (PCR)

PCR allows several million-fold amplifications of DNA or RNA. Thus, cells as few as 1 or 100
are sufficient for analysis, which can be obtained from 0.1 μl of blood or cells scraped from
buccal mucosa. Techniques of PCR include:

1. Direct method (Gene Sequencing): DNA is sequenced to obtain a read out of nucleotides
of test sample, and by comparison with a normal sequence, mutations can be identified.

Limitation: difficulty and high cost of analyzing genetic diseases with large number of
gene defects. E.g., Duchenne muscular dystrophy possesses 79 exons, Marfan syndrome
possesses 65 exons.

For such diseases, gene chips (microarrays) are used, which allows large-scale germ line
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 sequencing. In this, short sequences of DNA (oligonucleotides) of wild-type (most
common type allele in normal population) and of known mutation are “tiled” adjacent to
each other on a gene chip. The DNA sample to be tested is labeled with fluorescent dyes
and hybridized to the array. Fluorescent signal emitted by oligonucleotide, which is not
mutated will be strongest, while the presence of a mutation will show reduced signals.
Computerized algorithms rapidly decode the signals and identify the mutations.

2. Indirect method: These techniques allow detection of DNA mutations without direct
sequencing. E.g., Digestion of DNA using restriction enzymes. The bands are then made
to appear on agarose gel electrophoresis.

Polymorphic markers

In genome-wide association studies (GWAS), large cohorts of patients with and without a
disease (rather than families) are examined across the entire genome for genetic variants or
polymorphisms that are over-represented in patients with the disease.

Hybridization based techniques

Genetic lesions that involve large deletions, duplications, or more complex rearrangements that
are not easily assayed using PCR can be studied by hybridization-based techniques.

1. Southern Blotting: With the advent of FISH and microarray technology, Southern
blotting is rarely used except in cases of fragile- X syndrome and some lymphoma.

2. Fluorescence in Situ Hybridization: FISH uses DNA probes that recognize sequences
specific to particular chromosomal regions. As part of the Human Genome Project, large
libraries of bacterial artificial chromosomes that span the entire human genome were
created. These DNA clones are labeled with fluorescent dyes and applied to metaphase
spreads or interphase nuclei. The probe hybridizes to its homologous genomic sequence
and thus labels a specific chromosomal region that can be visualized under a fluorescent
microscope.

Chromosome painting is an extension of FISH, whereby probes are prepared for entire
chromosomes. Another method is spectral karyotyping (multicolor FISH), which uses
five fluorochromes and appropriate computer-generated signals, to visualize the entire
human genome.

3. Array-Based Comparative Genomic Hybridization (Array CGH): FISH requires

prior knowledge of the one or few specific chromosomal regions suspected of being
altered in the test sample. This limitation of FISH is overcome by array CGH. In array
CGH the test DNA and a reference (normal) DNA are labeled with two different
fluorescent dyes (most commonly Cy5 and Cy3, which fluoresce red and green,
respectively). If the contributions of both samples are equal for a given chromosomal
region, then all spots on the array will fluoresce yellow (the result of an equal admixture
of green and red dyes). If the test sample shows an excess of DNA, there will be a
corresponding excess of signal from the dye. The reverse will be true in the event of a
deletion.
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Epigenetic alterations (chemical modifications)

Epigenetics is defined as the study of heritable chemical modification of DNA or chromatin that
does not alter the DNA sequence itself. E.g., study of methylation of DNA, and the methylation
and acetylation of histones. The techniques available are Sanger sequencing and methylation-
specific PCR.

RNA analysis

Changes in DNA lead to alterations in mRNA expression, thus mRNA expression analysis can
be used in the diagnosis of genetic diseases. However, RNA is less stable than DNA. The most
preferred areas for use of RNA analysis are:
1. Detection of RNA viruses like HIV and HCV
2. In molecular diagnosis of certain tumors, which have translocations at scattered locations.
Amplification of such defect is beyond the capacity of PCR. E.g., BCR-ABL fusion in
CML

Short answer question

1. Name two patterns of gene amplification.

IMPORTANT QUESTIONS

SHORT NOTES
1. Down’s syndrome
2. Klinefelter’s syndrome

The End

Please refer text for further reading.

Mistakes are regretted.

Dr. Neena Doshi

Professor, Pathology, GMERS Medical College, Gotri, Vadodara, Gujarat (Email: neenapdoshi@gmail.com)

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