Chapter 7
GENETICS
Sr. No. TOPIC Page No.
1 Gene 2
2 Mutation 3
3 Single gene defect 3
4 Chromosomal number- normal and abnormal 4
5 Down’s syndrome 5
6 Klinefelter’s syndrome 6
7 Turner’s syndrome 7
8 Chromosomal structure- normal and abnormal 7
9 Molecular diagnosis of genetic disorders 9
10 Important questions 12
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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 à CAG). This substitution causes termination of β-chain of
hemoglobin, leading to it’s 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
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 von Sickle cell anemia Hemophilia A and B
Willebrand disease Thalassemias Chronic
granulomatous
disease
Glucose-6-phosphate
dehydrogenase
deficiency
Skeletal Marfan syndrome Alkaptonuria Duchenne muscular
dystrophy
Metabolic Familial Cystic fibrosis Diabetes insipidus
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hypercholesterolemia Phenylketonuria Lesch-Nyhan
Homocystinuria syndrome
Lysosomal storage
diseases
α1-Antitrypsin
deficiency
Wilson disease
Hemochromatosis
Glycogen storage
diseases
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
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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)
Monosomy: 2N – 1 / 45;
Turner’s syndrome (monosomy sex chromosome)
Mechanism: non-disjunction; failure of chromosomes to separate normally during cell division.
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
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• Oblique palpebral fissure
• Epicanthic folds
Abundant neck skin
Simian crease
Gap between first and second toe
Congenital heart disease
Umbilical hernia
Intestinal stenosis
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
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
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• Lack of secondary male characteristics: deep voice, beard……..
• Gynecomastia
• Osteoporosis
Low IQ: IQ is lower with more extra X chromosomes. Mental retardation is uncommon.
TURNER’S SYNDROME
Cause
Monosomy sex chromosome
57%: loss of entire X chromosome, (45, XO). Such fetus are 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
Classification
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Classified on the basis of 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.
Deletion
Deletion of ‘p’ arm of chromosome 11: Wilm’s tumour
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Deletion of ‘q’ arm of chromosome 13: Retinoblastoma
Inversion
Rearrangement of segments in same chromosome.
Paracentic: 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.
MOLECULAR DIAGNOSIS OF GENETIC DISEASES
Testing for germ line mutations can be divided into prenatal and postnatal analysis.
Prenatal genetic analysis
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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 amplification 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
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.
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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.
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-
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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
IMPORTANT QUESTIONS
SHORT NOTES
1. Down’s syndrome
2. Klinefelter’s syndrome
SHORT ANSWERS
(Note: Answers only to those questions not addressed in the text or written scattered are given here.
In some answers are given in detail to cover relative topics.)
[Link] & Answers
1. What is chromosomal translocation?
2. Name two patterns of gene amplification.
Ans. Polymerase Chain Reaction (PCR): gene sequencing, gene chip (microarray) study
Southern blotting
Fluorescence in Situ Hybridization (FISH)
Array-Based Comparative Genomic Hybridization (Array CGH)
3. Define mutation. Give two examples.
4. What is Down’s syndrome?
5. Name any two X-linked recessive disorders.
Ans. Hemophilia A and B, chronic granulomatous disease, G6PD deficiency, diabetes
insipidus
6. Enumerate two cytogenetic (chromosomal) disorders involving autosomes.
Ans. Down syndrome (trisomy 21), Edward’s syndrome (trisomy 18), Patau syndrome
(trisomy 13), DiGeorge syndrome (microdeletion at 22q11)
7. Write four important features of Klinefelter’s syndrome.
Ans. Male hypogonadism & infertility, gynecomastia, osteoporosis, low IQ
8. What is aneuploidy?
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The End
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