Mutation

INTRODUCTION
• MUTATION- Any sudden change occurring in hereditary material is called as mutation.
• They may be harmful, beneficial or neutral.
• DNA is highly stable molecule that replicates with amazing accuracy some errors of
replication do occur.
CLASSIFICATION OF MUTATION
1. Direction of Mutation:
a) Forward mutation: wild type mutant type
b) Reverse mutation: mutant type wild type

2. Cause of mutation:
c) Spontaneous mutation: natural
d) Induced mutation: mutagenic agents (physical and chemical)

3. Dominance Relationship:
e) Dominant mutation: mutant alleles are dominant eg: bar eye shape in Drosophila
f) Recessive mutation: mutant alleles are recessive (common)
4. Tissue of origin:
a) Somatic mutation
b) Germinal mutation

5. Effect on Survival:
c) Lethal mutation
d) Sub-lethal mutation
e) Vital mutation

6. Cytological Basis:
f) Chromosomal aberration – structural and numerical
g) Gene mutation
h) Cytoplasmic mutation
GENE MUTATION

• A gene mutation is defined as

an alteration in the sequence
of nucleotides in DNA.
• This change can affect a
single nucleotide pair or
larger gene segment of a
chromosome.
POINT MUTATION
• Point mutations are the most common type of gene mutation.
• Also known as base pair substitution.
• Change in a single nucleotide base pair.
• Point mutation can be categorized into three types:
• Silent mutation
• Missense mutation
• Nonsense mutation
• The change in one codon for an amino acid into another codon for that same amino acid .
Silent mutations are also referred to as synonymous mutations.
• The codon for one amino acid is changed into a codon for another amino acid. Missense
mutations are sometimes referred to as non-synonymous mutations.
• The codon for one amino acid is changed into a translation termination (stop) codon.
FRAME SHIFT MUTATIONS
• This type of mutation occurs when the addition or
loss of DNA bases changes a gene' s reading frame.
• A reading frame consists of 3 bases, each code for
one amino acid.
• A frame shift mutation shifts the grouping of these
bases and changes the code for amino acids.
• The resulting protein is usually nonfunctional.
Insertions and deletion can all be frame shift
mutations

https://ghr.nlm.nih.gov/mutationsandhealth
Structural Chromosomal Aberration
• Change in chromosome structure from its normal complement
• Result in change in gene sequence in the chromosome

Types:
1. Deletion 2. Duplication
3. Inversion 4. Translocation
Deletion
• Loss of chromosome segment
Types:
a) Terminal deletion : Loss of terminal segment of the chromosome
b) Interstitial deletion: Loss of intercalary segment
Duplication
• Addition of chromosome segment Example: Bar eye shape
Inversion
• Breakage of chromosome segment and joining in reverse orientation

Types:
a) Pericentric : inverted segment involves centromere
b) Paracentric : inverted segment does not contain centromere
Translocation
• Integration of segments into non-homologous chromosome
Types:
a) Simple: integration of terminal segment to another non-homologous chromosome
b) Shift: integration of intercalary segment to another non-homologous chromosome
c) Reciprocal translocation: exchange of chromosome segments between non-
homologous chromosomes.
Applications of Deletion

1. Crop improvement
2. Deletion mapping
3. Study of biosynthetic pathway
 Numerical Chromosomal Aberration (Ploidy)
Change in chromosome number from the normal diploid (2n) number
Types:
1. Aneuploidy
2. Euploidy

Aneuploidy: Loss or gain of one or few chromosomes

Nomenclature:
Monosomic:- 2n-1 (loss of 1 chromosome homolog)
Nullisomic:- 2n-2 (loss of 1 chromosome pair)
Double monosomic:- 2n-1-1 (loss of 2 non-homologous chromosomes)
Trisomic:- 2n+1 (gain of 1 chromosome)
Tetrasomic:- 2n+2 (gain of 2 chromosomes)
Double trisomic:- 2n+1+1 (gain of 2 non-homologous chromosomes)
Production: Non-disjunction in humans (meiotic irregularities)
Triploid plants produce aneuploids with high frequency.
Use: Establish linkage group
Mutation in Humans (Aneuploids)
Down Syndrome
• It is also known as trisomy 21, is a genetic
disorder caused by the presence of all or part of
a third copy of chromosome 21.
• It is typically associated with physical growth
delays, characteristic facial features and mild to
moderate intellectual disability.
• The average IQ of a young adult with Down
syndrome is 50, equivalent to the mental age of
an 8 or 9 year old child, but this varies widely.
Edward’s syndrome
• It is also known as Trisomy 18 [T18] is a
chromosomal disorder caused by the presence
of all or part of an extra 18th chromosome.
• This genetic condition almost always results
from nondisjunction during meiosis.
• It is named after John Hilton Edwards, who
first described the syndrome in 1960.
• It is the second most common autosomal
trisomy, after Down syndrome, that carries to
term.
• Characteristics: kidney malformations,
structural heart defects at birth, intestines
protruding outside the body (omphalocele),
intellectual disability, developmental delays,
growth deficiency, feeding difficulties,
breathing difficulties, and arthrogryposis.
 Patau syndrome
• It is a syndrome caused by a chromosomal abnormality, in which some or all of the cells
of the body contain extra genetic material from chromosome 13.
• The extra genetic material from chromosome 13 disrupts the normal course of
development, causing multiple and complex organ defects.
Monosomy 7
• It is typically characterized by early childhood onset of evidence of bone marrow
insufficiency/failure associated with increased risk for myelodysplastic syndrome (MDS)
or acute myelogenous leukemia (AML).
• Bone marrow failure/MDS/AML follows within a few months to years of identification of
a monosomy 7 cell line in peripheral blood. Nearly all individuals reported with familial
mosaic monosomy 7 have died of their disease.
Tetrasomy 18p
• It is a genetic condition that is caused by the presence of an isochromosome, composed
of two copies of the short arm of chromosome 18.
• It is characterized by multiple medical and developmental concerns.
• cryptorchidism among males
• feeding difficulties, respiratory difficulty and jaundice are also relatively frequent.
Pallister–Killian syndrome
• Pallister–Killian syndrome (also tetrasomy 12p mosaicism or Pallister mosaic aneuploidy syndrome) is an
extremely rare genetic disorder occurring in humans.
• Pallister-Killian occurs due to the presence of the anomalous extra isochromosome 12p, the short arm of
the twelfth chromosome.
• Characteristics include varying degrees of developmental disability, epilepsy, hypotonia, and both
hypopigmentation and hyperpigmentation.
• Patients also exhibit a distinctive facial structure, characterized by high foreheads, sparse hair on the
temple, a wide space between the eyes, epicanthal folds, and a flat nose.
• Vision and hearing impairments may occur. Patients may also exhibit congenital heart defects,
gastroesophageal reflux, cataracts, and supernumerary nipples.
• Diaphragm problems seen in newborns can lead to death shortly after birth.
• As patients pass into adolescence, the syndrome is characterized by a coarse and flat face, macroglossia
prognathia, inverted lower lip, and psychomotor retardation with muscular hypertonia and contractures.
KLINEFELTER SYNDROME
• 47,XXY is the most common sex chromosome aneuploidy in humans.
• Characteristic features include enlarged breasts, loss of body hair, small testis, small
prostrate gland.

TURNER SYNDROME
• This condition in females is cused due to absence of one X chromosome (45,XO)
• Characteristic features include absence of ovaries, poorly developed secondary sexual
characters.
 Euploidy
Multiples of the basic haploid(n) set
Types: 1. Autopolyploid 2. Allopolyploid

1. Autopolyploid: Multiples of the same species

Nomenclature:
3n-triploid
4n-tetraploid
5n- pentaploid

2. Allopolyploid: Multiples of different species

Nomenclature:
2n1+2n2 – Allotetraploid
2n1+2n2+2n3 – Allohexaploid
2n1+2n2+2n3 +2n4– Allooctaploid
Production: chromosome doubling-treatment of seeds with colchicines that result in production
of unreduced gametes(2n)
Use: Plant breeding
Eg:-Triticale(wheat and rye)
Pedigree Analysis
• A very important tool for studying human inherited diseases
• These diagrams make it easier to visualize relationships with in families, particularly large extended
families.
• Pedigrees are often used to determine the mode of inheritance (dominant, recessive, etc.) of genetic
diseases.

A basic family history should include three generations. To begin taking a family history, healthcare
professionals start by asking the patient about his/her health history and then ask about siblings and parents.

Questions should include:

1.General information such as names and birthdates
2.Family’s origin or racial/ethnic background
3.Health status, including medical conditions and ages at diagnoses
4.Age at death and cause of death of each deceased family member
5.Pregnancy outcomes of the patient and genetically-related relatives

It may be easier to list all the members of the nuclear family first, then go back and ask about the health
status of each one. After you have taken the family history of the patient’s closest relatives, go back one
generation at a time and ask about aunts, uncles, grandparents, and first cousins.
Symbols followed in Pedigree analysis
Categories of inheritance
• Autosomal means inherited on chromosome 1-22 while sexlinked means inherited on either
X or Y chromosome.
• Autosomal recessive
e.g., PKU, Tay-Sachs, albinism
• Autosomal dominant
e.g., Huntington’s Disease
• X-linked recessive (meaning this allele is found on only the X chromosome: can be in
males or females)
e.g., color-blindness, hemophilia
• X-linked dominant (meaning this allele is found on X chromosomes; can be in males or
females)
e.g., hypophosphatemia
• Y-linked (meaning the allele is found on the Y chromosome and can only be in males
Autosomal Recessive Pedigree
• Trait is rare in the pedigree
• Trait often skips generations (hidden
in heterozygous carriers)
• Trait affects males and females
equally
• Possible diseases include: Cystic
fibrosis, Sickle cell anemia,
Phenylketonuria (PKU), Tay-Sachs
disease
Autosomal Dominant Pedigree
• Trait is common in the pedigree
• Trait is found in every generation
• Affected individual also transmit the
trait to about 1/2 of their children
(regardless of sex).
• There are few autosomal dominant
human diseases but some rare traits have
this inheritance pattern.
• For example: achondroplasia (a sketelal
disorder causing dwarfism)
X – Linked dominant
• Trait is common in pedigree
• Affected fathers pass to ALL of their
daughters
• Males and females are equally likely to be
affected
• X-linked dominant diseases are extremely
unusual
• Often, they are lethal (before birth) in males
and only seen in females ex. incontinentia
pigmenti (skin lesions) ex. X-linked rickets
(bone lesions)
X – Linked Recessive
• Trait is rare in pedigree
• Trait skips generations
• Affected fathers DO NOT pass to
their sons
• Males are more often affected than
females
• Females are carriers (passed from
mom to son)
Y – Linked Inheritance
• Traits on the Y chromosome are only
found in males, never in females.
• The father’s traits are passed to all
sons.
• Dominance is irrelevant: there is only
1 copy of each Y -linked gene
(hemizygous).
KARYOTYPING
KARYOTYPE
Arrangement of Chromosomes in descending order based on size with
centromere in a straight line
Steps in preparation of Karyotype:
1. Preparation of metaphase spread
2. Staining of chromosomes-Banding Technique
3. Analysis of the metaphase spread.
Idiogram:
1.The diagrammatic representation of karyotype is called the idiogram.
2.While making an idiogram, the homologous pair of chromosomes are
arranged in order of decreasing lengths.
Preparation of Metaphase spread
• Venous blood is added to culture medium and phytohemagglutinin (PHA)
to induce mitotic division.

• Cultured cells are arrested in metaphase by adding colchicine

(chromosomes are most condensed and easy to identify).

• The cells are then swollen by treatment with hypotonic saline for
chromosomes to spread without any overlap.

• Metaphase chromosomes are fixed and stained by banding technique.

Banding Technique
• Identification of human chromosomes that differ morphologically.

• This technique is based on identification of chromosome segments that consists

of AT and GC rich regions by treatment with specific dyes that produces a
banded pattern.

• Chromosome banding pattern is highly specific to each chromosome of a

species.

• This is a useful tool for identification of chromosomes.

• Types of Banding: G-band, Q-band, R-band, C-band.

• G-banding (Geimsa):- Chromosomes are treated with trypsin and stained with
geimsa (AT specific dye).
• Dark bands observed under light microscope represents heterochromatin and
light bands represents euchromatin
• Q-banding (Quinacrine):- Staining of metaphase spread with quinacrine (AT
specific dye) that produces bright and dull fluorescent bands observed under
fluorescent microscope.
• Bright bands represents heterochromatin and dull bands represents euchromatin
(similar to G-band)
• R-banding (Reverse):- opposite to G-band
• Metaphase spread is heat denatured and stained with chromomycin or acridine
orange.
• R-bands are GC rich. Dark bands represent euchromatin.

a) G-band B) R-band
• C-banding (Centromeric):- Metaphase spread is treated with acid followed by
alkali and then stained with geimsa.
• Used to identify centromere
• Dark bands represents highly condensed heterochromatic regions that contain
repetitive sequences
• Used for paternity testing and gene mapping.
Chromosome Analysis
• Stained chromosomes are photographed through
microscope.
• ISCN (International System for Human
Cytogenetic Nomenclature) is used for identifying
each chromosome.
• Chromosome arms: p-short arm & q-long arm.
• Band close to the centromere is assigned the
lowest number.
• High resolution banding sub bands are given
decimal points.
• Chromosome 4 Eg: 4q23.1
• Around 30 metaphase spread are scanned in the slide and good well spread metaphase
is photographed and used for chromosome identification.
• 3 Key feature for identification includes size (start from longest to smallest), banding
pattern (similar for homologues), centromere position.
• Using these features all 23 chromosome pairs are matched with reference bands
specific for each chromosome.
• Identified chromosomes are cut from the photograph and arranged from longest to the
shortest in the standard format with centromere in a straight line.
• Karyotype is analyzed and represented. Eg. Normal female -46,XX ; Down female –
47, XX, +21
Match chromosomes in the karyotype
Applications of Karyotype
• Prenatal Diagnosis: Amniocentesis (identify chromosomal aberration in the
developing fetus), chrionic villi sampling (Prebirth diagnosis of genetic
diseases).
• Clinical Diagnosis: Identification of chromosomal aberrations in patients with
congenital malformation.
• Repeated fetal loss: chromosomal defect
• Stage in cancer
• Gene Mapping
Fluorescence-activated Cell Sorting (FACS)
• Fluorescence-activated cell sorting (FACS)
is a specialized type of flow cytometry.
• It provides a method for sorting a
heterogeneous mixture of biological cells
into two or more containers, one cell at a
time, based upon the specific light
scattering and fluorescent characteristics of
each cell.
• It is a useful scientific instrument, as it
provides fast, objective and quantitative
recording of fluorescent signals from
individual cells as well as physical
separation of cells of particular interest.
FLUORESCENCE INSITU HYBRIDIZATION (FISH)
• Cytogenetic technique that allows detection and localization of specific
nucleic acid sequences on morphologically preserved chromosomes.
Steps in FISH
1. chromosome preparation
2. Denature chromosomes
3. Denature probe
4. Insitu hybridization
5. Detection of hybridization – fluorescent staining
6. Bound probe is visualised under fluorescent microscope -Microphotography
Detection of Aneuploids
Follow Up Counseling
• Follow-up counseling is a continuation of the care that was provided to the clients in the
rehab, which can provide them effective assistance while dealing with the many
stressors that accompany everyday life. Follow-up counseling represents a stage of
treatment that comes after the successful completion of the indoor treatment phase
within the rehabilitation facility.
• This form of counseling is basically a continuation of the emotional support and
reinforcement that was made available to them during their time in the rehab. Through
treatment, clients are made aware of the actuality that the process of recovery extends
well past the confines of the treatment facility.
Amniocentesis
Chorionic Villus Sampling
• Chorionic villus sampling is a procedure performed to biopsy placental tissue between 10 to 13 weeks
gestation for prenatal genetic testing. The primary advantage of chorionic villus sampling is earlier genetic
results in pregnancy.
• This knowledge provides patients with the opportunity to seek counseling for obstetric management and
recommendations, early referral to pediatric subspecialists, or earlier and safer methods of pregnancy
termination if results are abnormal.
Prenatal genetic testing cannot identify all abnormalities, so testing should be focused on the patient’s risk,
reproductive goals, and preferences. Ideally, genetic testing should be discussed at the first obstetric visit.
• Indications for chorionic villus sampling include
• Abnormal early genetic screening on a non-invasive prenatal screening (NIPS)
• A prior child with a structural birth defect
• A prior child with autosomal trisomy or sex chromosome aneuploidy
• Advanced maternal or paternal age
• Parental carrier of a chromosomal rearrangement
• Parental aneuploidy or aneuploidy mosaicism
• Parental carrier of a genetic disorder, such as Tay Sachs,
Sickle Cell Disease, or Neurofibromatosis
Multifactorial Inheritance
• Multifactorial inheritance is when more than 1 factor causes a trait or health
problem, such as a birth defect or chronic illness. Genes can be a factor, but
other things that aren't genes can play a part, too. These may include:
• Nutrition
• Lifestyle
• Alcohol and tobacco
• Some medicines
• An illness
• Pollution
Types Of Multifactorial Traits And Disorders
• Health problems that are caused by both genes and other factors include:
• Birth defects such as neural tube defects and cleft palate
• Cancers of the breast, ovaries, bowel, prostate, and skin
• High blood pressure and high cholesterol
• Diabetes
• Alzheimer disease
• Schizophrenia
• Bipolar disorder
• Arthritis
• Osteoporosis
• Skin conditions such as psoriasis, moles, and eczema
• Asthma and allergies
• Multiple sclerosis and other autoimmune disorders
COMPARATIVE GENOMIC HYBRIDIZATION (CGH)
Molecular cytogenetic method for analysing copy number variation (ploidy) in
a DNA for a test sample compared to reference sample without cell culturing.
Method:
1. Metaphase slide preparation (Normal-reference sample)
2.DNA isolation from test and reference sample
3. DNA labelling (different colour labels for test and refrence)
4. Hybridization
5. Fluorescence visualization and imaging
Application:
• Diagnosis and prognosis of cancer
• Study chromosomal aberrations in fetal and neonatal genomes
CGH
CGH