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In the Name of Allah
The Most Merciful, The Most Beneficient
Medical Genetics
"Genetics"
Fields:
Heredity and its variation.
Subfields:
- "Human
Genetics”:
denotes the science of heredity and
its variation
in human.
- ”Medical Genetics”:
deals with human genetic variations
of medical relevance / significance .
Medical Genetics
subgroups
Molecular and biochemical
genetics the study of the
structure and function
of individual genes.
Population genetics the study of genetics
of populations.
Clinical Geneticsconcerned with
Clinical manifestation
Of genetic diseases
Cytogenetics the study of the
structure of
chromosomes.
Immunogenetics the study of the
genetics of the
immune
system
.
Genetic epidemiology the study of
epidemiology of
genetic disease.
A brief History of
Genetics
Historical
* Engravings (around 6,000 years)
-Showed pedigree documenting the transmission of certain
characteristics of some animals.
*Aristotle and Hippocrates
-Human characteristics determined by the semen
(utilising the menstrual blood as a culture medium and
uterus as an incubator).
-Semen was thought to be produced by the whole body and
hence it was explained that 'baldheaded fathers’ had
'baldheaded' sons.
* 17th century
-‘Sperm' and 'ovum' were recognised by
Dutch scientists and it was explained that female could also
transmit characteristics to her offspring.
(Contd)
Historical (Conti.)
* 18th and 19th centuries
-There was a revival of interest in heredity and
it was shown that several traits such as extra
digits (polydactyly) were inherited in different ways.
* 19th century
-Joseph Adams published
"A treatise on the
Supposed Hereditary Properties" and indicated
different mechanisms of inheritance.
-This book was intended as a basis for
genetic counselling.
Historical (Conti.)
* In 1865, Gregor Mendel
- An Austrian Monk, published his results
of breeding experiments on Garden Peas.
- His work can be considered as the discovery
of `genes' (traits) and how they are inherited.
- He put forward patterns of inheritance of various
characteristics and single gene disorders.
-These are known as ‘Mendels Laws of Inheritance.
Historical (Conti.)
* Mendel showed that some characteristics were:
-"dominant" (e.g.tall height),
- others were "recessive“ (e.g. short height).
-each characteristic was controlled by a pair of "factors".
* In 1909, a Danish botanist, Johannsen, named
the hereditary factors as ‘genes’.
- two identical genes was referred to as `homozygous',
- two different genes for the same characteristic,
were called `heterozygous'.
Historical (Conti.)
Multiple forms of the same gene that occupy the same loci
and give rise to different forms of the same characteristics
are referred to as allelomorphs"or alleles
Alleles
Homozygous
Heterozygous
Historical (Conti.)
* The 20th century ( development of genetics):
- Mendels Laws were independently
rediscovered by three workers:
- Hugo De Vries ( in Holland)
- Carl Correns (in Germany) and
- Erich Von Tschermakin (in Austria).
Historical (Conti.)
* In 1902 :
- Archibald Garrod and William Bateson
(fathers of Medical Genetics), discovered
`Alkaptonuria'
and recognized it as an inherited disorder
involving chemical processes.
- Garrod called it an "Inborn Errors of
Metabolism”
- Todate several thousand of such disorders
have been identified.
Historical (Conti.)
* In 1903 :
- Sutton and Boveri proposed that
‘chromosomes’ carry the hereditary factors.
Chromosomes( Chroma=color; soma=body)
were recognised as thread like structures,
(so called because of their affinity for certain stains).
* In 1906 :
- Bateson contributed the term "Genetics"
for this new science.
* In 1941:
- Beadle and Tatum formulated the
"one gene - one protein" theory.
* In 1956 :
- The correct number of chromosomes
was established as 46.
Historical (Conti.)
* By late 1950's :
- Excellent techniques for the study
of chromosomes were developed.
* In 1953:
- James Watson and Francis Crick ( in Britain)
described the structure of the genetic
material i.e. DNA, and were awarded Nobel
prize in 1962.
* Mid 1970's :
-The field of Medical Genetics
has been transformed and significant new
discoveries about the genes, their expression
and genetic diseases have been made.
Historical (Conti.)
* The 'Human Genome Project‘:
- An International project, to map the entire
human genome, was initiated in 1990 to
be completed by the year 2005( however,
it was completed in 2003).
* To-date:
- extensive information has been
gained about chromosomes, gene mapping,
gene sequencing, functions and genetic disorders.
The genetic knowledge is increasing exponentially and has extensive
applications in clinical medicine
* During the last three decades:
- a decrease in frequency of infectious diseases.
- improved nutrition, antibiotics and immunization.
- almost one third of the patients in paediatric
suffer from genetic defects.
It has become essential for all medical
personnel's to have a clear knowledge
of human and medical genetics.
Mendels Laws of Inheritance
Three Laws of Inheritance:
i)
The Law of Unit Inheritance.
i)
The Law of Segregation.
iii) The Law of Independent Assortment.
The Law of Unit Inheritance
The characteristics (traits i.e. genes) do not blend
( mix), but are inherited as units, which might not
be expressed in the first generation off-springs,
but may appear unaltered in later generations.
First Generation
Second Generation
TT
tt
Tt
Tt
Tt Tt
Tt
Tt
TT
Tt
Tt
tt
All tall in the first generation 75% Tall and 25% short in 2nd
(As t is recessive & does not appear)
generation.
( T= Tall, dominant gene; t = Short, recessive gene)
The Law of Segregation
- The two members of a single trait (gene)
i.e. alleles, are never found in the same gamete,
but always segregate and pass to
different gametes
Gamete
Zygote
- The failure of two alleles
to segregate due to chromosome
non-disjunction give rise to genetic
defects(e.g. in Down’s
syndrome)
Gamete
The Law of Independent Assortment
* Members of different gene pairs assort to the gametes
independently of one another
i.e. random recombination of maternal and paternal chromosomes occur in gametes.
Maternal
Paternal
Crossing-over
Gametes
The exceptions to Law of Independent Assortment
(not recognised by Mendel) are closely "linked“
genes on the same chromosome, which do not
assort independently.
Maternal
Paternal
Crossing-over
Gametes
The Genetic Material
What is the Genetic Material?
Proteins ?
RNA?
DNA?
Griffith’s Experiment
In live animal
( Smooth &Virulent)- due to polysaccharide capsule
(Non-Virulent)
(Non-Virulent)
Due to absence
of polysaccharide
capsule
Transformation
(Non-Virulent)
Of rough to smooth form
(Virulent)
(Non-Virulent)
What is the Transforming Factor?
Griffith’s Experiment
Conducted in 1928 On a bacteria that produces pneumonia:
- R(Rough) strains were non-virulent(did not produce disease)
- S(Smooth) strains were virulent (produced disease)
- Heated R and S strains were both non-virulent
_The experiment:
- R injected in rats
No disease
- Heated R injected in rats
No disease
- S injected in rats
Disease (Rat Died)
- Heated S injected in rats
No disease
- Heated S + live R injected in rats Disease (Rat Died)
Some substance in heated S transformed the R to S
What was the Transforming Principle?
Experiment of Avery, Macleod and McCarty (1944)
In culture
(Culture)
Smooth colonies
No colonies
Growth of S colonies
What is the Transforming
Factor?
The Transforming Principle
Experiment of Avery, Macleod and McCarty (1944)
1. Took extract from virulent(S) cells + R cells
S Colonies
As the bacteria was destroyed, but DNA was not.
2. Treated the extract with:
(a) Proteases---------Mixed with R cells
S Colonies
(b) Ribonuclease----Mixed with R cells
S Colonies
(c) DNase------------Mixed with R cells
No Colonies of S
Concluded that the transforming principle in the extract was DNA
(Mixing)
These and many other experiments proved
that DNA is the carrier of genetic
information
in all living organisms except RNA viruses
which have RNA as the carrier of genetic
information
Genetic Material in the Living Cells
* All living organisms are made
up of cells.
* Cells contain a nucleus
surrounded
by
a
nuclear
membrane in eukaryotic cells,
and a nuclear region in the
prokaryotic cells.
* Chromatin is made up of DNA
and
proteins
(mainly
histones(basic) and non-histone
(acidic) proteins.
Genetic material…contd
The study of chromosomes, their structure and
their inheritance is known as Cytogenetics.
Each species has a characteristic number of
chromosomes and this is known as karyotype.
Prior to 1950's it was believed that humans had
48 chromosomes but in 1956 it was confirmed
that each human cell has 46 chromosomes (Tjio
and Levan, 1956).
The genes are situated on the chromosomes in
a linear order. Each gene has a precise position
or locus.
•
•
•
•
Chromosomes
(Metaphase)
Chromatin
Chromosomes
One member of each chromosome pair is
derived from each parent.
Somatic cells have diploid complement
of chromosomes i.e. 46.
Germ cells (Gametes: sperm and ova)
have haploid complement i.e 23.
The chromosomes of dividing cells are
most readily analyzed at the `metaphase'
or prometaphase stage of mitosis .
*
*
*
*
The Normal Human Chromosomes
Normal human cells contain 23 *
pairs
of
homologous
chromosomes:
- 22 pairs of autosomes
(numbered
as
1-22
in
decreasing order of size)
- 1 pair of sex chromosomes.
Autosomes are the same in *
males and females
Sex chromosomes are: *
- XX in females
- XY in males.
* Both X are homologous. Y is
much smaller than X and has
only a few genes.
p
q
Chromosome
Structure
Telomere
p
Centromere
q
At the metaphase stage each *
chromosome consists of two
chromatids joined at the
centromere or primary
constriction
The
centromere
divides •
chromosomes into short (p i.e.
petit) and long (q e.g. g=grand)
arms.
The
tip
of
each
chromosome is called telomere.
The exact function of the •
centromere is not clear, but it is
known to be responsible for the
movement of the chromosomes
at cell division.
Chromosomes … contd
In a non-dividing cell the •
nucleus is filled with a threadlike
material
known
as
"chromatin".
Mitosis
G2
G1
S
The Cell Cycle
Go
Before
cell
division,
the •
chromatin
multiplies
(replicates), loses the relatively
homogenous appearances and
condenses to form rod like
structures .
"Genes" , •
are units of genetic information
present on the DNA.
Each species has a characteristic gene map
i.e. the chromosomal location of the genes, and
it is the same in all normal individuals of each species
Classification Of Chromosomes
• Chromosomes are classified (analysed) accordig to:
•1. Shape and
•2. Staining
1. Morphologically (shape)
According to the position of the centromere as:
(i) metacentric,
(ii) sub-metacentric,
(iii) acrocentric,
(iv) telocentric (with centromere at one end.
This occurs in other species, but not in man).
Sub-Metacentric
Chromosomes
Metacentric
Chromosomes
Telomeres
p
Centromere
q
* Acrocentric chromosomes (13, 14, 15, 21 and
22) have a small mass of chromatin known
as satellite attached to their short arm by
narrow stalks (secondary constrict).
* The stakes contain genes for 18S and 28S rRNA.
Satellite
Stalk
Staining Methods for cytogenetic
analysis of chromosomes
There are several staining methods for cytogenetic •
analysis of chromosomes.
Each stain produces specific banding patterns known as •
"Chromosome Banding"
G banding,
- Q banding,
R banding,
C banding.
The pattern is specific for each chromosome, and is the •
characteristics utilized to identify each chromosome.
Staining Methods for Cytogenetic Analysis
G Banding:
Treat with trypsin and then with Geimsa Stain.
R Banding:
Heat and then treat with Geimsa Stain.
Q Banding:
Treat with Quinicrine dye giving rise to
Fluorescent bands.
C Banding:
Staining of the Centromere.
The G-Banding
Pattern of
Chromosomes
DNA packing in the Chromosomes
Composition of Nucleosomes
DNA
Histones
2( H2A,H2B,H3,H4)
The Genetic material-Deoxyribonucleic acid (DNA)
-Double strand
5’
of polynucleotide.
-Coiled around each
other forming
double helix.
-Strands are antiParallel.
-Sugar phosphate
backbone is outside
& bases are inside.
-A=T and G=C.
-A/T=1 andG/C=1
(Cargaff Ratio)
3’
3’
5’
Nitrogenous bases in DNA and RNA
Pyrimidines
Purines
Detailed view of DNA Structure
The Central Dogma
Replicationin nucleus
Transcriptionin nucleus
Translationin cytoplasm on ribosomes
DNA Replication
-Replications occurs
before cell division.
During S Phase of
cell cycle.
-Entire DNA content
is doubled.
-Replication is
Semi-conservative.
-Requires:
-DNA polymerases
-dNTPs(N=A,T,C,G)
-RNA primer
-Mg++
-DNA ligases
- Primase
- Helicase
- SS DNA binding proteins
Major Steps in DNA Replication
Lagging strand
Leading strand,continuous
Transcription
Steps in transcription
Initiation
Binding of RNA polymerase •
causes opening of the DNA
strand and synthesis of the RNA
Elongation
RNA polymerase continues
synthesis of RNA complementary
to DNA till termination site
•
Steps in transcription
(contd)
Elongation -contd
Termination
Rho factor binds to the termination
site and when RNA polymerase
reaches this site, termination
occurs
•
Translation
On ribosomes
Ribosomes- free and attached to endoplasmic reticulum
Codons on mRNA
Structure of tRNA
Steps in Translation
i. Initiation
ii. Elongation
iii. Termination
Polysomes
Mitochondrial DNA
In the human mitochondria the chromosomes are
present as 10 circular double helices of DNA.
They are self replicative.
Contain: 16,596 bp, genes for 22 tRNAs and 2 types of
ribosomal RNA required for mitochondrial protein
synthesis.
They also have genes for 13 polypeptides, involved in
cellular oxidative phosphorylation.
Both strands of DNA are transcribed and translated.
•
•
•
•
•
Mitochondrial DNA
The genes on mitochondrial DNA have no •
introns.
The codon recognition pattern for several amino •
acids is different from the nuclear DNA.
Mitochondria are transmitted in the egg from a •
mother to all of her children. Thus mitochondrial
DNA is only maternally derived.
The Cell Cycle
G2
S
Mitosis
G1
Go
The Cell Cycle
The cell cycle consists of 2 phases: •
Mitosis and Interphase.
Mitosis (cell division) is the shortest phase. •
Interphase The period between successive mitosis. •
The G1, S and G2 phase constitute interphase. •
In a typical growing cell this lasts 16-24 hours and •
mitosis lasts 1-2 hours.
Some cells e.g. neurons and RBCs, do not divide •
and enter the Go phase. Other cells may enter Go
but eventually return to continue through the cell
cycle.
(Contd..)
The Cell Cycle (Contd..)
Immediately after mitosis, the cell enters G1 •
(Gap 1) phase, where there is no DNA
synthesis. Some cells spend a few hours
others up to years in this phase. At this phase
cells perform metabolic functions.
S phase - the phase of DNA synthesis. •
Each chromosome in G1 phase double, and
forms two chromatids joined together. By the
end of S phase the DNA content of cells is
doubled.
The Cell Cycle (Contd..)
G2 phase - The chromatin condenses •
and
forms
chromosomes.
Each
chromosome consists of two identical
sister chromatids. During this period the
DNA synthesis is restricted, RNA and
protein synthesis occur and cell enlarges,
eventually doubling its total mass before
next mitosis.
Cell Division
Cell Division
Mitosis:
Meiosis:
- Occurs in Somatic cells.
- Occur in cells of germ line.
- Division by which the body
- Only once in generation.
- Results in the formation of
haploid, reproductive cell
(gametes: ova and sperms).
- Chromosomes duplicates
followed by 2 cell divisions
resulting in cells with
half the number of
chromosomes (haploid).
grows, differentiates and repairs.
- Results in two identical daughter
Diploid cells with genes identical
to parent cells.
- Chromosomes are first doubled,
followed by cell division in which
the number in each cell is halved
(diploid).
Mitosis
At conception the human zygote consists of a •
single cell. This undergoes rapid cell division
leading ultimately to the mature human adult
body. Each adult human being has
approximately 1x1014 cells in the body.
In most organs and tissues e.g. bone marrow, •
skin etc. cells continue to divide throughout
life.
This process of somatic cell division during •
which the nucleus divides to produce two
identical daughter cells is known as Mitosis.
Mitosis
(contd..)
* Each chromosomes divides into two
daughter chromatids, one of which
segregates into each daughter cells.
- The number of chromosomes per cell
remains unchanged.
Mitosis lasts 1-2 hours. It occurs in five distinct stages : Prophase, prometaphase,
metaphase, anaphase and telophase.
Phases of Mitosis:
Prophase: The chromosome condenses and
mitotic spindle begins to form. Two centrioles
form in each cell from which microtubules
radiate as the centrioles move towards
opposite poles of the cell.
Prometaphase: The nuclear membrane begins
to disintegrate and chromosome spread around
the cells. Each chromosome becomes attached
at its centromere to a microtubule of the mitotic
spindle
by a specialised structure called
Kinetochores.
Phases of Mitosis (Contd..):
Metaphase: The Chromosomes are maximally •
contracted and most easily visible. The
Chromosomes become oriented along the
equatorial plain and each chromosome is
attached to the centriole by a microtubule
forming the mature spindle.
Anaphase:
The
centromere
of
each •
chromosome divides longitudinally and the two
daughter chromatids separate to opposite poles
of the cell.
Phases of Mitosis (Contd..):
Telophase: The chromatids separate completely
and a new nuclear membrane is formed around
each set of chromosomes. The cytoplasm
separates (cytokinesis) to form two daughter
cells.
Mitotic Cell Cycle:
Meoisis:
The type of cell division by which the diploid cells of •
the germline give rise to haploid gamets, i.e. oocytes
and sperms.
The process involves two successive meiotic •
divisions:
Meiosis I: This is the reduction division and the •
chromosome number is reduced from diploid to
haploid.
Meiosis II - follows Meiosis I without an intervening •
stage of DNA replication. The chromosomes
disjoin, and one chromatid of each chromosome
passes to each daughter cell.
Meoisis:
This stage has:
Meiosis I: •
Prophase I, Prometaphase I,
Metaphase I, Anaphase I &
Telophase I, just like mitosis.
Meiosis II: has: •
Metaphase II and telophase II
and results in formation of ova in
female and sperms in males.
Meiotic
Cell
Cycle:
Genetic Consequence of Meiosis
- Reduction of chromosome number from diploid
to haploid, the essential step in the formation of
gametes.
- Segregation of alleles, at either meiosis I or
meiosis II, in accordance with Mendel’s First Law.
- Shuffling of the genetic material by random
assortment.
- Additional shuffling of genetic material by
crossing-over mechanism substantially increasing
genetic variation.
Gametogenesis:
There are differences in female and males gametogenesis
Oogenesis: .1
Mature ova develops from oogonia by a complex series of
intermediate steps:
During the first few months of embryonic life : •
Oogonia originate from primodial germ cells
by a process involving 20-30 mitotic divisions.
At completion of embryogenesis at 3 months of intra- •
uterine life:
The oogonia mature to primary oocytes which start to
undergo meiosis.
Gametogenesis:
At birth, all primary oocytes have entered dictyotene, a •
phase of maturation arrest at which they remain
resuspended until meiosis is completed at the time of
ovulation .
At the time of ovulation, a single secondary oocyte is •
formed. Most of the cytoplasm is received by the
daughter cell from the 1st meiotic division consists largely
of a nucleus known as a polar body.
Meiosis II then commences during which fertilization can •
occur. A second polar body is formed.
Gametogenesis:
2. Spermatogenesis:
Rapid process - average duration of 60-65 days.
At puberty, spermatogonia (which have already •
undergone 30 mitotic divisions) begin to mature into
primary spermatocytes.
These enter meiosis 1 and emerge as haploid secondary •
spermatocytes.
These undergo second meiotic division to form •
spermatids, which change to mature spermatozoa
Genetic
Disorders
Mutations in the:
* Genome,
* Chromosome or
* Gene
- Decrease or increase in the amount of genetic material
- Abnormal genetic maerial
- Increase or decrease in the amount of gene products (proteins).
- Decrease in the amount of one protein.
- Defective function of the protein.
- Increased function.
- Decreased or complete loss of function.
Genetic Disease
Genetic Diseases
Classification of
Genetic Diseases
Single Gene
Disorders
Chromosomal
Disorders
Acquired Somatic
Genetic Diseases
Multifactorial
Disorders
Mitochondrial
Disorders
Single Gene Disorders
• Caused by mutation in or around a gene.
• Can lead to critical errors in the genetic information.
• Exhibit characteristic pedigree pattern of
inheritance (Mendelian Inheritance)
• Occur at a variable frequency in different
population
•Over 7,000 single gene disorders have been
identified.
• May be: - Autosomal
- Sex linked
Chromosomal Disorders
• Result from defect in the number (i.e. Numerical
disorders) or structure (i.e. Structural disorders)
of chromosomes.
• The first chromosomal disorder was Trisomy 21
(Downs syndrome) and was recognised in 1959.
• These disorders are quite common and affect about
1/800 liveborn infants.
• Account for almost half of all spontaneous
first-trimester abortions.
• Do not follow a Pedigree pattern of inheritance.
Multifactorial Disorders
• Result from interaction between environmental
and genetic factors.
• Often polygenic in nature, no single error in the
genetic information.
• Environmental factors play a significant role
in precipitating the disorder in genetically
susceptible individuals.
•Tend to cluster in families.
• Do not show characteristic pedigree pattern
of inheritance.
Multifactorial
Disorders
Congenital
malformations
Common disorders
of adult life.
Mitochondrial Disorders
* The defective gene is present on the
mitochondrial chromosomes.
* Effect generally energy metabolism.
* Effect those tissues more which require
constant supply of energy e.g muscles.
* Shows maternal inheritance:
-effected mothers transmit the disorder
equally to all their children.
-affected fathers do not transmit the
disease to their children.
Acquired Somatic Genetic Diseases
• Recent advances in Molecular Biology
techniques have shown that mutations occur
on a regular basis throughout the life of the
somatic cell.
• These somatic mutations account for
1. A large proportion of malignancy and
2. possibly involved in events such as
'senescence' and the 'ageing process'.
Single Gene Disorders
May be: - Autosomal
- Sex linked:
Y- linked , holanderic, hemizygote
X- linked , dominant or recessive
Modes of Inheritance of Single gene Disorders
Autosomal
Recessive
Sex Linked
Dominant
Y Linked
X Linked
Abnormal
homozygous
Recessive
Normal
homozygous
Heterozygous
Normal
Abnormal
Dominant
Autosomal Inheritance
- This is the inheritance of the gene present on the Autosomes.
- Both sexes have equal chance of inheriting the disorder.
- Two types:
Autosomal dominant inheritance, if the gene is dominant.
Autosomal recessive inheritance, if the gene is recessive.
Normal
homozygous
Heterozygous
Abnormal
homozygous
Autosomal Dominant Inheritance
- Autosomal dominant inheritance, if the gene is dominant.
- The trait (characteristic, disease) appears in every generation.
- The trait is transmitted by an affected person to half the children.
- Unaffected persons do not transmit the trait to their children.
- The occurrence and transmission of the trait is not affected by sex.
Normal male
Normal female
Disease male
Disease female
Examples of Autosomal dominant disorders
Disorder
Approximate
Frequency/1000
Familial hypercholesterolemia
2
Von Willebrand disease
Adult polycystic kidney disease
1
1
Huntington disease
0.5
Myotonic dystrophy
0.2
Acute intermittent porphyria
Rare
Dominant blindness
0.1
0.1
Dominant deafness
Acute Intermittent Porphyria
- AD.
- Expressed in heterozygotes and homozygotes.
- Uroporphyrinogen synthetase deficiency.
- Increased urinary excretion of 5-amino levulinic acid
and porphobilinogen (diagnostic ) .
- Characterized by neurological symptoms that include
severe abdominal pain, peripheral neuropathy and
psychosis.
Punnet Square
Affected
Mother
D
Normal
d
dD
d
dD
d
dd
Father
dd
50% Normal
50% Affected
Affected
Mother
Affected
Father
D
d
D
DD
dD
d
dD
dd
25% Normal
75% Affected
Autosomal Recessive Inheritance
- The trait (characteristic, disease) is recessive.
- The trait expresses itself only in homozygous state.
- Unaffected persons (heterozygotes) may have affected
childrens (if the other parent is heterozygote) .
- The parents of the affected child maybe consanguineous.
- Males and female are equally affected.
Punnett square showing autosomal recessive inheritance:
(1) Both Parents Heterozygous:
25% offspring affected Homozygous”
A
a
AA
Aa
Female
A
50% Trait “Heterozygous normal but
carrier”
25% Normal
a
Aa
aa
Contd.
(2) One Parent Heterozygous:
Male
A
a
A
AA
Aa
A
AA
Aa
Female
50% Off springs normal but carrier
“Heterozygous”
50% Normal
_________________________________________________________________________
(3) If one Parent Homozygous:
Male
A
A
100% of springs carriers.
Female
a
Aa
Aa
a
Aa
Aa
Family tree of an Autosomal recessive disorder
Sickle cell disease (SS)
A family with sickle cell disease -Phenotype
Hb Electrophoresis
AA
AS
SS
Examples of Autosomal Recessive Disorders
Approximate
Frequency/100
0
Cystic fibrosis
0.5
Recessive Mental
0.5
retardation
Congenital deafness
0.2
Phenyketonuria
0.1
Sickle cell anaemia
0.1-5
-Thalassaemia
0.1-5
Recessive blindness
0.1
Spinal muscular atrophy
0.1
Disease
Cystic fibrosis
- Most frequent autosomal recessive (AR) disorder (1 in 200
births in Caucasians)
- Expressed only in homozygotes.
- Heterozygote carriers are normal phenotypically
- If both parents are heterozygote to abnormal gene than
there is 1 in 4 (25%) chance of having child with cystic
fibrosis (homozygous).
- If one parent has cystic fibrosis (homo) while the other
parent is normal, then all childrens will be carriers of the
abnormal gene.
Sex – Linked Inheritance
- This is the inheritance of a gene present on the sex
chromosomes.
- The Inheritance Pattern is different from the autosomal
inheritance.
- Inheritance is different in the males and females.
X-Linked
Sex – linked
inheritance
Y- Linked
Recessive
Dominant
Y – Linked Inheritance
- The gene is on the Y chromosomes.
- Shows Holandric inheritance. i.e.
The gene is passed from fathers to sons only.
- Daughters are not affected.
e.g. Hairy ears in India.
- Male are Hemizygous, the condition exhibits itself whether dominant or recessive.
male
Female
X
Y*
X
XX
XY*
X
XX
XY*
X – Linked Inheritance
- The gene is present on the X - chromosome.
- The inheritance follows specific pattern.
- Males have one X chromosome, and are hemizygous.
- Females have 2 X chromosomes, they may be
homozygous or heterozygous.
- These disorders may be : recessive or dominant.
X – Linked Recessive Inheritance
-
The incidence of the X-linked disease is higher in male than in female.
- The trait is passed from an affected man through all his daughters to
half their sons.
- The trait is never transmitted directly from father to sons.
- An affected women has affected sons and carrier daughters.
(1) Normal female, affected male
Ova
X
X
X*
X*X
X*X
Y
XY
XY
All daughters carriers “not affected,
All sons are normal
(2)
Carrier female, normal male:
Ova
50% sons affected,
Sperm
X*
X
X
XX*
XX
Y
X*Y
XY
50% daughters carriers,
(3) Homozygous female, normal male:
- All daughters carriers.
- All sons affected.
X - Linked Recessive Disorders
- Albinism (Ocular).
- Angiokeratoma (Fabry’s disease).
- Chronic granulomutous disease.
- Ectodermal dysphasia (anhidrotic).
- Fragile X syndrome.
- Hemophilia A and B.
- Ichthyosis (steroid sulphatase deficiency).
- Lesch–Nyhan syndrome.
- Menkes’s syndrome.
- Mucopoly Sacchuridosis 11 (Hunter’s
syndrome)
- Muscular dystrophy (Duchenne and Beeker’s).
- G-6-PD
- Retinitis pigmentosa.
Lesch – Nyhan Syndrome
- X – linked recessive disease.
- Due to deficiency of hypoxanthine guanine phosphoriboyl
transferase - Purine salvage pathway is impaired.
- Symptoms include:
- Self mutilation tendency.
- Mental retardation.
- Cerebral palsy.
- Uric aciduria.
- Gout and kidney stones.
The Hemophilias
- X – linked recessive disease.
- Expressed in males, very rare in females (homozygotes)
[ 1 in 10,000 male births ].
- In this abnormality, the blood fails to clot due to
abnormality of antihemophilic globulin.
- Clinical features include severe arthritis.
X-Linked Dominant Disorders
-
The gene is on X Chromosome and is dominant.
-
The trait occurs at the same frequency in both males
and females.
-
Hemizygous male and heterozygous females express
the disease.
** Punnett square showing X – linked dominant type of Inheritance:
(1) Affected male and normal female:
OVA
All daughters affected, all sons normal.
X
X
X*
X*X
X*X
Y
XY
XY
Sperm
(2) Affected female (heterozygous) and normal male:
OVA
Sperm
X*
X
X
XX*
XX
Y
X*Y
XY
50% sons and 50% daughters are affected.
50% of either sex normal.
Contd.
(3) Affected female (homozygous) and normal male:
OVA
All children affected..
X*
X*
X
X*X
XX*
Y
X*Y
X*Y
Sperm
Chromosomal disorders
- These defects result from defects in the chromosomes.
- Two groups:
* Structural defects– defects in structure of chromosome.
* Numerical defects– Increase or decrease in number of
chromosomes
- These defects are quite common (7 in 1000 live births).
- Chromosomal defects do not obey specific pattern of
inheritance.
- These defects account for over half of all spontaneous abortions
in first trimester.
Chromosomal Disorders
Numerical
Structural
Increase or decrease in the
number of chromosomes
Euploidy
Aneuploidy
Change in the structure
of chromosomes
Euploidy
Increase in the total
set of chromosomes
e.g 3N or 4N
-Triploidy (69 chromosomes)
found in cases of
spontaneous abortions
Aneuploidy
Increase or decrease in
one or more chromosomes.
e.g 2N+1, 2N-1
-Trisomy (46+1) chromosomes
(Down Syndrome)
-Monosomy (46-1) chromosomes
(Turner Syndrome)
Non-Disjunction
Triploidy (69, XXY)
Structural Abnormalities
Duplication
Isochromosomes
Translocation
Insertion
Inversion
Ring
Chromosomes
The Philadelphia Chromosome*
* Mutation found in all cases of chronic myeloid leukemia
* The ABL & BCR fuse due to translocation and form an oncogene
Mitochondrial Disorders
* Effect generally energy metabolism.
* Effect more those tissues which require
constant supply of energy e.g muscles.
* Shows maternal inheritance:
-affected mothers transmit the disorder
equally to all their children.
-affected fathers do not transmit the
disease to their children.
Mitochondrial Disorders
Lebers hereditary optic neuropathy
Mitochondrial Inheritance
- Affected females transmit the disease to all their children.
- Affected males have normal children.
- Males cannot transmit the disease as the cytoplasm is inherited
only from the mother, and mitochondria are present in the
cytoplasm.
Multifactorial Disorders
• Result from interaction between environmental
and genetic factors.
• Often polygenic in nature, no single error in the
genetic information.
• Environmental factors play a significant role
in precipitating the disorder in genetically
susceptible individuals.
•Tend to cluster in families.
• Do not show characteristic pedigree pattern
of inheritance.
Multifactorial
Disorders
Congenital
malformations
Common disorders
of adult life.
Acquired Somatic Genetic Diseases
• Recent advances in Molecular Biology
techniques have shown that mutations occur
on a regular basis throughout the life of the
somatic cell.
• These somatic mutations account for a large
proportion of malignancy and are possibly
also involved in events such as 'senescence'
and the 'ageing process'.
Examples of Genetic
Diseases
A.Single-gene Disorders
- Adenosine deaminase deficiency
- Alpha-1-antitypsin deficiency
- Cystic fibrosis
- Duchenne muscular dystrophy
- Familial hypercholesterolemia
- Fragile X-syndrome
- Hemophilia A and B
- G-6-PD deficiency
- Phenylketonuria
- Sickle cell anaemia
- Thalassaemia
B. Examples of Numerical Chromosomal
Aberrations
Karyotype
92, XXYY
69, XXY
47, XX+21
47,XX+18
47, XX+13
47,XXY
47,XXX
45, X
Example
Tetraploidy
Triploidy
Trisomy 21(Down
Syndrome)
Trisomy 18
Trisomy 13
Klienfelter Syndrome
Trisomy X
Turners Syndrome
* Examples of significant genetic disorders:
(Chromosomal disorder):
Disorder
Down –
Syndrome
Trisomy 18 –
Trisomy 13 –
Klinefelter –
Syndrome
XXX Syndrome –
XYY Syndrome –
Defect
Trisomy 21
Trisomy 18
Trisomy 13
47, XXY
45, X
47, XXX
47, XYY
Incidence
1/800 –
1/25000 –
1/1000 (Males) –
1/5000 –
(Females)
1/1000 –
(Females)
1/1000 (Males) –
C. Multifactorial Disorders
(i) Congenital malformation
- Cleft lip and cleft palate
- Congenital heart disease
- Neural tube defects
(ii) Adult onset disease
- Cancer (some)
- Coronary artery disease
- Diabetes mellitus
Examples of Multifactorial disorder
Disorder
Incidence
Cleft lip/ Cleft palate
1/250 – 1/600
Congenital heart
disease
Neural tube defects
1/125 – 1/250
Coronary heart disease
1/15 – Variable
Diabetes mellitus
1/10 – 1/20
Cancer
variable
1/100 – 1/500
D.Mitochondrial Disorders
Lebers hereditary optic neuropathy
E. Acquired somatic genetic disorders
Some forms of cancer
Genotype-Phenotype
correlations
Genotype
- The genetic constitution (genes on the pair of
homologous chromosomes).
- The alleles present at one locus. e.g..
(a) TT or Tt or tt i.e genes for height.
Where T is the “tall” gene and t is the
gene for “short” height
(b) A  A, A S, or S S
Where A is for HbA and  S for HbS.
Phenotype
The observed biochemical, physiological and morphological
characteristics of an individual as determined by his/her
genotype and the environment in which it is expressed. e.g.
Genotype
Phenotype
TT or Tt
Tall
tt
Short
AA
HbA (normal)
Hetero
A S
HbAS
 S S
HbS (SCA)
( Homo = Identical , Hetero = different)
Dominant
* Hetero
Recessive
Genotype – Phenotype relationship
Genotype (i.e. genetic make up) determines phenotype (i.e.
appearance etc.), though environmental factors may modify
the phenotypic expression:
e.g.
TT (Proper nutrition)
TT (Poor nutrition)
Tall
Stunted growth and
poor development.
- The Genotype determines the phenotype, but is affected by
presence of Recessive or Dominant Gene, e.g.
(Conti..)
e.g:
(i) As T is dominant, it is expressed in Homozygotes and
Heterozygotes, but t is recessive and is expressed only in
Homozygotes.
TT and Tt
tall
tt
short
(ii) s is recessive, it is expressed only in Homozygotes while
Heterozygotes are carriers but normal:
A A
A S
S S
HbA – Normal
HbAS – Normal
HbS – Abnormal “Sickle cell anemia”
- Genotype differ in the degree of their expression of:
Clinical severity, onset age, or both.(Variable expressivity).
- Expression of abnormal genotype maybe modified by:
-
-
-
Other genetic loci, environmental factors or both
Reduced Penetrance: in some heterozygous individuals with a
dominant disorder, the presence of the mutation is reduced.
Non-Penetrance: when a heterozygous individuals with a
dominant disorder has no features of the disorder.
“Pleiotropy” – multiple phenotypic effects of a single basic gene
defect on multiple organs (genetic heterogeneity) e.g Tuberous
sclerosis(AD) : learning disability, epilepsy, facial rash.
New Mutations: A sudden appearance of a dominant disorder in
the offspring with normal parents.
Codominance: When two allelic traits are both expressed equally
in a heterozygote e.g ABO blood groups.
Pseudodominance: If a homozygous for AR mutation marries a
carrier for the same mutation, their children have 1 in 2 chance of
being affected (homo). This pattern is like dominant inheritance.
Genetic
Polymorphism
Mutations
Genetic diversity among individuals
Deleterious mutations
Disease
not deleterious
mutation
May effect phenotype
Over generations, the influx of new
nucleotide variations has ensured a high
degree of genetic diversity and individuality.
Genetic Variation*
Some mutations in the
gene(coding sequence)
Variant protein
Altered structure and
Altered properties
Some mutation in the gene
DNA (coding sequence)
Variant protein ,but not
critical for the function
Normal properties
Some mutations in DNA
(non-coding regions)
No effect on proteins
structure
*Polymorphisms are common, particularly in
non-coding regions of DNA
Genetic Polymorphism*
Many genetic loci are characterised by a number of relatively
common alleles, thus producing many phenotypes in normal
population
Alleles that occur at a
frequency of > 1% are said
to be polymorphic variants
Alleles that occur at a
frequency of < 1% are said
to be rare variants
If there are two or more alleles(several forms of the same genes occupy the same locus) and
the rarest occurs at a frequency of more than 1%
then this loci will be considered polymorphic.
Gene polymorphism
e.g. Gene for hair colour
Wild type
Alleles
If there are two or more alleles(several forms of the same genes occupy the same locus) and
the rarest occurs at a frequency of more than 1%
then this loci will be considered polymorphic.
Types of Polymorphisms
(Defined by the method of detection)
DNA
Polymorphism
Protein
Polymorphism
Detected by
altered DNA
sequences
Altered
physical
features
Chromosome
heteromorphisms
Contd…..
- Restriction Fragment Length Polymorphism (RFLPs):
- Inherited variations in DNA sequence,
- Results in gain or loss of a site recognised by restriction
endonuclease
- Variable number of tandem repeats (VNTRs):
- Variations in the number of short, repeated nucleotide sequences
(eg GC) between restriction sites
- VNTRs are extremely polymorphic
- Valuable in forensic medicine
Types of Polymorphisms
(Defined by the method of detection)
Contd…
Protein
Polymorphism
Altered physical
features
Chromosome
heteromorphisms
Detected by:
Electrophoresis
Altered activity,
Altered physical properties
Contd…..
- Enzyme variant: altered enzyme activity, electrophoretic
mobility, thermostability or other physical properties
e.g.G-6-PD deficiency.
- Antigenic variants: altered antigenic properties
e.g. ABO blood groups.
Protein Polymorphism
- Several proteins exist in two or more relatively common,
genetically distinct , structurally
different & functionally identical.
- The causes of polymorphic forms:
Mutation in or around gene
- Examples :
ABO Blood groups, Transferrin, Hb, 1 antitrypsin.
Not all variant proteins
have clinical consequences
Types of Polymorphisms
(Defined by the method of detection)
Contd…
Altered physical
features
Chromosome
heteromorphisms
Detected by:
Physical appearence
Altered physical features e.g. polydacytyly,
gagantism, dwarfs, hair on ears, baldness.
Types of Polymorphisms
(Defined by the method of detection)
Contd…
Chromosome heteromorphisms
Detected by:
Cytogenetic studies
FISH
 Heritable differences in chromosomal appearances from
one person to another, e.g.
 Variations in the size of the Y chromosome long arm.
 Variation in the size of the centromere .
 Variation in satellite size and structure.
 The occurrence of fragile sites.
Genetic diversity among individuals
Chromosome heteromorphisms
Protein variations
• Generally, the karyotype of
normal persons of the same sex
are quite similar.
• Almost 25% are silent
mutation with no effect on
protein structure.
•Occasional variants are seen on
staining. These are called
heteromorphisms.
• Most mutations alter amino
acid sequence but do not have
phenotypic effect (e.g. ABO
blood groups).
•These reflect difference in amount
or type of DNA sequence at a
particular location along a
chromosome.
e.g
•Rare mutations produce
severe phenotype effect or
influence survival (e.g.
phenylketonuria)
• In long arm of chromosome.
• In chromosomes 1, 9, 16.
• In short arm of acrocentric chromosomes
Uses of Polymorphism
As genetic “Markers”
- To distinguish inherited forms of a gene in a family.
- Mapping gene to individual chromosomes by likage
analysis.
- Presymptomatic and prenatal diagnosis of genetic
disease.
- Evaluation of high and low – risk persons.
- Paternity testing and forensic applications.
- Matching of donor-recipient pairs of tissue and organ
transplantation.
Advantages of Polymorphism
- Polymorphic forms are produced as result of
mutation in the genetic loci.
- The advantages are possibly:
- Production of more stable forms.
- Production of such forms that give resistance
against disease:
e.g. Hb S Trait are resistance
to malarial plasmodia.
- Natural selection for survival of the fittest.
Area of Significance of Polymorphism
- Blood transfusion.
- Tissue typing.
- Organ Transplantation.
- Treatment of Haemolytic disease of new born.
ABO System
- First identified by Landsteiner in 1900.
- Human blood can be assigned to one of four types
according to presence of two antigens, A and B, on the
surface of Red Blood Cell and
the presence of two corresponding antibodies, Anti A
and Anti B in the plasma.
* RBC Antigen Polymorphism:
- Useful marker for:
- Family and population studies.
- Linkage analysis.
- Different frequencies in different population.
Contd.
* Blood Group Substances:
- Blood group substances are encoded by allelic genes A and
B.
- Blood group substances exhibit polymorphism.
Polymorphic Chromosom
System
al Location
ABO
MNSs
9 q34
4q28 – 31
Xg
Xp 22.3
Common
Alleles
A, B and O
M and N;S
and s
Xga and Xg.
ABO Blood groups and Reaction with Antibodies
Grou
p
Geno
Type
Anti A
Anti B
Cellular
Antigen
Serum Anti
Frequenci
es
O
O/O
-
-
NO
Anti A+B
45%
A
A/A,
+
-
A
Anti B
42%
-
+
B
Anti A
10%
+
+
A+B
Neither
4%
O/A
B
B/B,
O/B
AB
A/B
Clinical Importance of Polymorphism
Some disease
genes occur with
polymorphic
frequencies
e.g.
- HbS in African, Saudi
Arabia
- Thalassaemia in
Mediterranean region
Saudi Arabia
- Cystic fibrosis in
Europeans
Genetic
polymorphisms
may produce
disease
Some polymorphisms
determine antigenic
differences
e.g.
On exposure to
drugs or
environmental factor
- G-6-PD deficiency
- Malignant
hyperthermia.
e.g.
- Blood group
- HLA antigen for
tissue typing.
Clinical Importance of Polymorphism
Contd…..
Forensic Medicine
e.g.
DNA fingerprint of each
individual differs due to
polymorphic sites in many
non-coding sequences
As genetic markers
e.g.
Predisposing to a disease
within families or
populations
Genetic Linkage
The occurrence of two or more genetic loci in
such close physical proximity on a
chromosome that they are more likely to assort
(linked) together
Crossing over does not take place between
closely situated loci – So they are said to be linked
A
B
Linked
No
C
a
b
B
c
b
Not linked
Crossing
During
meiosis
C
c
b
B
Concept of Genetic Linkage
Linkage refers to
loci, not to alleles
(which occupy
different
chromosomes
Measurement of
genetic linkage can
only take place in
family studies
Statistical method of
measuring linkage is by
calculation of
lod score
Closeness of a genetic linkage is expressed in
Cente Morgans (cM) or percent recombination
Loci separated
by crossing over
in 1% of
gametes are 1
cM apart
Loci close to each
other, so they never
separate are linked
at a genetic
distance of zero
cM
Cont
d….
Unlinked loci are separated
by a genetic distance of 50
cM at a given allele at one
locus has a 50% of being
transmitted with either allele
at an unlinked loci.
Concept of Genetic Linkage
Contd…..
Lod Score
- Lod score is a acronym for “Logarithm of the Odds”
( Logrithm of the likelihood ratio).
- Lod score of +3 or greater at recombination distance of
less than 50 cM between two loci is considered to be a
strong evidence of linkage (1000 : 1 odds for linkage.
- Lod score of 2 or less is taken as a strong evidence
there is no linkage (100: 1 odds against linkage).
Concept of Genetic Linkage
Contd…..
Linkage disequilibrium
Measure in populations,
not in families
This is the tendency for certain alleles at two linked loci to
occur together more often than expected by chance. e.g.
Mb
D
Distance=5cM
If the mutant allele at D occurs on the
same chromosome as Mb more often
than expected within a certain population
linkage disequilibrium is said to exist.
Disease locus = D
Marker = M
Alleles of Marker Ma and Mb.
centi Morgan
Defines the distance
between two gene loci
If two loci are IcM apart, there is a 1%
change of recombination between
these loci as the chromosome is
passed from parent to child
It gives a rough unit of distance
along the chromosome
- Different chromosomes have different sizes.
- Average chromosomes contain about 150 cM.
- There are about 3300 cM in the whole human genome.
This corresponds to 3x109 bp.
- On average IcM is about 1 million bp (1000 kb).
Markers tightly linked to a disease
- The marker linked to a disease
gene, must be on the same
region on the chromosome
(within < 1 cM distance).
Markers that are a long
distance away on the
same chromosome may
not appear to be linked,
because recombination
between the two loci is
high
Clinical Applications of Linkage
Linkage is clinically
useful as it may permit
Prenatal
diagnosis
More precise
determination of the
genotype at an
unidentified gene locus
on the basis of readily
identified linked markers
Used in
Carrier
detection
Presymptomatic
diagnosis
Determination of the
pattern of inheritance or
specific for disease that
exhibits genetic
heterogeneity
Elucidation of
genetic factors
in multifactorial
disorders
Gene mapping by
determining the
recombination distance
between two genes on a
chromosome
Gene Mapping
This is the assignment of genes to specific
chromosomal locations.
Mapping is done by:
Family
studies to
demonstrate
linkage
between loci
Somatic cell
genetic method
to show that two
loci are not linked
(demonstrate
synteny) or that
an unmapped
loci resides on a
chromosome
Gene dosage
studies
Cytogenetic
techniques e.g. in
situ hybridization
Indirect means of
identifying location of a
gene
Importance of Gene Mapping
The gene map is
the anantomy of the
human genome
Analysis of
heterogeneity
and segregation
of human genetic
diseases
To develop optimal
strategy for gene therapy
by improved knowledge of
genomic organization
Provides
information about
linkage
Haemoglobinopathies
and Thalassaemias
Haemoglobinopathies and
Thalassaemias
Genetic
Disorders
of
Haemoglobin
Haemoglobin
-
A conjugated protein consisting of iron-containing
heme and protein (globin).
Globin chains are of different types:
-chains and non  -chains
Each molecule is a tetramer of two - and non  chains.
Each globin binds a haem in a haem binding site.
Haemoglobin binds and transports oxygen from
lungs to the tissues, while it transports CO2 from
tissues to the lungs.
Types of Hemoglobin in adults
Globin genes
Chromosome
16
11
Gene product
(globin)
Tetramers
in RBCs
Name ofConc. in
haemoglobin
adult


, -chain
2 2
Hb A
96-97


, -chain
2 2
Hb A2
2.3-3.5


 ,-chain
2 2
Hb F
<1.0
----------------------------------------------------------------Emberyonic Hb:


 , -chain
2 2
Hb-Gower II
0


,


, -chain
 -chain  2 2
2 2
Hb-Gower I
Hb-Portland
0
0
Chromosome 11

G
5’

A


3’
Chromosome 16


2 1
2
1
5’
3’
Structure of each Globin gene
5’
3’
Exon 1 Intron 1 Exon 2
Intron 2
Exon 3
Disorders of Haemoglobin
Haemoglobinopathies
(Structural disorder
of Hb)
Thalassaemias
(Biosynthetic
disorder
of Hb)
Co-existing
structural /
biosynthetic
disorders
Constitute a major health problem in several
populations of the world
(particularly those residing in malaria
endemic region)
Haemoglobinopathies
•
•
•
•
•
Genetic structural disorder.
Due to mutation in the globin gene of haemoglobin.
Mostly autosomal recessive inheritance.
Result in haemoglobin variants with altered structure
and function.
Altered functions include:
• Reduced solubility
• Reduced stability
• Altered oxygen affinity- increased or decreased
• Methaemoglobin formation
*Types of Mutations in Haemoglobin
•
Point mutation: a change of a single nucleotide base in a DNA
giving rise to altered amino acids in the polypeptide chains
(e.g. Hb S , Hb Riyadh, Hb C)
•
Deletions and additions: Addition and deletion of one or more
bases in the globin genes
(e.g. Hb-constant spring which is associated with mild thalassaemia).
•
Unequal crossing over: as in Hb-lepore and Hb-antilepore
associated with -thalassaemias.
________________________________________________________
*Most abnormal Hbs are produced by mutations in the structural
genes which determine the amino acid sequence of the globin
chains of the Hb molecule.
Geographical distribution of common Hb variants
Variant
Occurrence predominantly in:
Hb S (6GluVal)
Africa, Arabia, Black Americans
Hb C (6Glulys)
West Africa, China
Hb E (26Glulys)
South East Asia
Hb D (121GluGln)
Hb O (121GluVal)
Asia
Turkey and Bulgury
Other examples of Haemoglobin variants
His
Lys
Tyr
His
CAC AAG UAU CAC
3’ Normal
Shorter chain
His
Lys
CAC AAG
Mutation
UAA 3’
Longer chains, e.g.
(Lys)
A
 2 gene
AUG ---
----- UAA --------- UAA
C
(Gln)
 globin
(Glu)
G
C
(Ser)
Gln
Lys
Glu
Ser
142
Sickle Cell
Haemoglobin


GAG
6

RBC


GTG
Sickle Cell
Haemolysis
Inheritance of Sickle Cell Anaemia
AR
AS
AS
AS
SS
AA
AS
Red cell sickling
Lungs
pO2
Tissues
pO2
Major abnormalities & problems in SCA
- Sickling of the red cell during deoxygenation, as the HbS
has low solubility at low O2 partial pressure and precipitates.
- Chronic haemolytic anaemia due to repeated sickling in tissues
and unsickling in the lungs.
- Plugging of microcapillaries by rigid sickled cells leading to sickle
cell crises i.e severe pain and edema. This causes significant damage
to internal organs, such as heart, kidney, lungs and endocrine glands.
- Repeated infections.
- Frequent cerebrovascular accidents.
- Hand-foot syndrome (in small,i.e.around age of 3y)
- Bone deformation – bossing of the forehead.
- Hepato-spleenomegaly.
- Growth retardation.
- Frequent blood transfusion requirements.
- Psychosocial problems.
Thalassaemias
Genetic disorders resulting from
decreased biosynthesis of globin chains
of haemoglobin.
Thalassaemias
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A group( not single identity) of Genetic defects.
Due to mutations in and around the globin genes.
Decreased production of one or more of the globin
chains.
Result in an imbalance in the relative amounts of the and non  -chains. Altered /non-  ratio.
A few rare Hb variants are effectively synthesized but
are highly unstable, and thus cause thalassaemias as
the : chain ratio is altered.
As a consequences of thalassaemias there is excess
production of the other chains, and a decreased over
all haemoglobin synthesis.
Types of Thalassaemias
- Thalassaemia*
- Thalassaemia
-Thalassaemia*
- Thalassaemia
- Thalassaemia
* Most common
- Thalassaemia
- Decreased / ratio
Hb




In - Thalassaemia
Decreased
production
of - chains
Normal = 

- Thalassaemia



Accumulation of 


Point Mutation producing - Thalassaemia
Less Frequent
Chromosome 16
5’
exon1
Introns
exon2
exon3
3’
Base Substitution
2bp del
5bp del
Chain Termination
Defect
Poly A signal
Mutation
Mutations Producing - Thalassaemia
Deletions
Most frequent:
Chromosome 16
/
Normal
-/
-thal 2
hetero
-/-
--/
-thal 2
homo
-thal 1
hetero
--/-
--/--
HbH
Disease
Hydrops
fetalis
-thalassaemia -2
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One  -gene deletion.
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-chain production is only about 75% of normal.
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May be homo- (- /- ) or heterozygous (- / )
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The patient usually shows a normal phenotypic
appearance but there might be mild thalassaemia
symptoms.
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Hypochromic-microcytic RBC’s due to partial reduction
of -chain.
-thalassaemia- 1
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Two  -genes deletion- (o )thal.
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The patient synthesizes -chain but it is decreased to about
50% of normal.
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Anaemic symptoms- hypochromic microcytic anaemia.
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May be homozygous (- -/- -) or heterozygous(--/ ). If the
patient is homozygous than there is no -chain synthesis,
and if heterozygous then there is decreased synthesis of
the -chain to half normal level.
Hb H Disease
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Three -gene deletion.
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The Hb present during foetal life is “Hb Bart’s” (4), while
during adulthood the Hb present is “Hb H” (4).
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Some of the symptoms include:
hepatosplenomegally, impairment of erythropoisis, and
hypochromoc-microcytic haemolytic anaemia.
Hydrops foetalis
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Homozygous o-thalassaemia.
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There is a complete absence of -chain (all -genes
are deleted).
•
The Hb produced at birth is Hb Barts (4).
•
Hydrops foetalis is lethal and the baby is born dead.
•
Symptoms include: Hepatosplenomegaly, severe
hypochromic- microcytic anaemia.
- Thalassaemia
Increased / ratio
Hb
 
 
Decreased
production
of - chains
In - Thalassaemia
Normal = 
- Thalassaemia



Accumulation of 



-Thalassaemia
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It is characterized by either no -chain synthesis (i.e.
o) or decreased synthesis of -chain (+).
•
Excess -chains precipitate in RBC’s causing severe
ineffective erythropoiesis and haemolysis.
•
The greater the -chains, the more severe the
anaemia.
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Production of -chains helps to remove excess chains and to improve the -thalassaemia. Often HbF
level is increased.
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Majority of -thalassaemia is due to point mutation.
o-Thalassaemia
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The -chain is totally absent.
There is increase in HbF with absence of HbA.
This is combined with ineffective erythropoisis.
In majority of the cases, -gene is present but there is complete
absence of mRNA.
Characteristics of this disorder are:
• Skeletal deformities (e.g. enlargement of upper jaw, bossing of
skull and tendency of bone fractures).
• Severe hypochromic- microcytic anaemia.
• Survival depends on regular blood transfusion.
• This leads to iron overload (iron accumulates in the blood and
tissues, causing tissue damage).
• Death usually occurs in the 2nd decade of life (i.e. at age of
about 20 years) if measures are not taken to avoid iron
overload by chelation therapy.
+-Thalassaemia
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There is a variable amount of -chain production.
There is decreased HbA level, and increased Hb A2,
level with normal or increased Hb F level (and there is
an increased number of -chains in the free form).
The -chain is present but there is decreased numbers
of mRNA or there is an abnormality in the mRNA.
Mutations affecting the -Globin gene.
Chromosome 11
1.
2.
3.
4.
5.
Mutations affecting transcription initiation
Mutations affecting RNA splicing
Mutations affecting translation initiation
Non-sense Mutations.
Mutations of polyadenylation site.
>200 -Thal
mutations reported
to-date
Worldwide
Clinical Classification of Thalassaemias
1. Thalassaemia major:
The patient depends on blood transfusions especially if he is
homozygous.
2. Thalassaemia intermediate:
• Homozygous mild +-thalassaemia.
• Co-inheritance of -thalassaemia.
• Heterozygous -thalassaemia.
• Co-inheritance of additional -globin genes.
•  -thalassaemia and hereditary persistence of foetal Hb
• Homozygous Hb lepore
• Hb H disease.
3. Thalassaemia minor (trait):
• o-thalassaemia trait.
• +-thalassaemia trait.
• Hereditary persistence of foetal Hb only.
• -thalassaemia trait.
• o- and +-thalassaemia trait.
Hb-Lepore
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This is an abnormal Hb due to unequal crossing-over of the - and
-genes to produce a polypeptide chain consisting of the - chain
at its amino end and - chain at its carboxyl end.
The -fusion(hybrid) chain is synthesized inefficiently and normal
 and -chain production is abolished.
The homozygotes show thalassaemia intermediate and
heterozygotes show thalassaemia trait.
Unequal crossing-over can be explained as crossing over between
similar DNA sequence that are misaligned resulting in sequences
with deletions or duplications of DNA segments; a cause of a
number of genetic variants.
The adjacent  and -genes differ at only 10 of their 146 a.a.
residues, if mispairing occurs followed by intergenic crossing over,
two hybrid genes result: one with a deletion of part of each locus
(lepore gene) and one with a corresponding duplication (antilepore gene).
High Persistence of Foetal Hb (HPFH)
A group of disorders due to deletions or cross over
abnormalities which affect the production of  and 
chains in non-deletion forms to point mutations
upstream from the -globin genes.
Double heterozygous indicates the
presence of combinations of the
following:
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Hb S + O-thalassaemia.
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Hb S + --thalasaemia.
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Hb S + -thalasaemia.
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Hb S + HbC disease
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Hb S + HbE disease
Diagnosis of Genetic Diseases
Diagnosis of Genetic Diseases
Family History*
Estimation of
Haematological
parameters
Clinical
Presentation*
Chromosomal
Analysis
Estimation of
Biochemical
Parameters
Determination of
Enzyme Activity
or Specific Protein
Recombinant
DNA
Technology
* Important for all genetic diseases
1. Family History
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Consanguinity of parents.
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Presence of other siblings with the same disorder.
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Occurrence of the disorder in other members of the family.
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Repeated abortions or still births,
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mother and fathers ages.
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Drawing punnet square helps to determine the mode of inheritance
of the genetic disorders.
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Autosomal or X-linked
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Dominant or recessive
2. Clinical Presentation
Certain clinical features are specific for a disease:
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Chronic anaemia:
• Haemoglobinopathies
• Thalassaemia
• Other genetic anaemias
Acute anaemia, under certain stressful conditions.
• G-6-PD deficiency
Hypoxia – sickle cell disease.
Dependence on blood transfusion - -thalassaemia (major)
Severe immune deficiency – ADA deficiency.
Emphysema - 1 anti-trypsin deficiency.
Hypercholesterolaemia – familial hypercholesterolaemia.
Delayed blood coagulation – Haemophilia (decrease in factor VIII or
IX).
Mental retardation – Fragile syndrome (in X chromosome) or
phenylketonuria (PKU).
Muscular weakness and degeneration – Duchenne muscular
dystrophy.
Recombinant DNA Technology
( Genetic Engineering)
Recombinant DNA Technology
( Genetic Engineering)
Techniques for
cutting
and joining DNA
Requirements for DNA technology
Restriction endonucleases
Primers
Vectors
NTPs
Probes
Special chemicals and
equipment
DNA
Other enzymes
e.g ligases,
Taq polymerases
Restriction Endonuclease
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Endonucleases.
Synthesized by procaryotes. Do
not restrict host DNA.
Recognize and cut specific base
sequence of 4-6 bases in
double helical DNA.
The sequence of base pairs is
palindromic i.e. it has two fold
symmetry and the sequence, if
read, from 5’ or 3’ end is the
same.
5’-GAATTC-3’
3’-CTTAAG-5’
Restriction Endonuclease
Produce either Blunt Ends or Staggered ends:
Blunt Ends
5’-GAATTC-3’
3’-CTTAAG-5’
5’-GAA
3’-CTT
TTC-3’
AAG-5’
or
Staggered Ends
5’-GAATTC-3’
3’-CTTAAG-5’
5’-G
AATTC-3’
3’-CTTAA
G-5’
Uses of Restriction Endonuclease
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Obtaining DNA fragments of interest.
Gene mapping.
Sequencing of DNA fragments.
DNA finger printing
Recombinant DNA technology
Study of gene polymorphism.
Diagnosis of disease.
Prenatal diagnosis
Sources of DNA
cDNA
Genomic
DNA
Synthesis of DNA
DNA extracted
from cells
Using DNA synthesiser
Synthesised from
mRNA using reverse
transcriptase
cDNA Synthesis
Poly A tail
AAAAAAAAA
mRNA
Viral reverse transcriptase
AAAAAA
TTTT
Hair pin loop
NaOH( Hydrolysis of RNA)
dNTP
DNA polymerase
DNA nuclease
(single-strand specific)
Double strand cDNA
Vectors
Cloning vesicles
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DNA molecules.
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Can replicate in a host e.g bacterial cells or
yeast.
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Can be isolated and re-injected in cells.
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Presence can be detected.
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Can be introduced into bacterial cells e.g. E.
coli.
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May carry antibiotic resistance genes.
Types of vectors
Type
Plasmid : circular, double
stranded cytoplasmic DNA in
procaryotic e.g. PBR 3 of Ecoli.
Insert size
• <5-10 kb.
Bacteriophage lambda: a bacterial
virus infects bacteria.
• Upto 20kb.
III. Cosmids: a large circular
cytoplasmic double stranded DNA
similar to plasmid.
• Upto 50kb.
IV. Yeast Artificial Chromosomes (YAC)
•~100-1000kb.
I.
II.
Probes
Cloned or synthetic nucleic acids used for DNA:DNA or
DNA:RNA hybridization reactions to hybridize to
DNA of interest.
•
DNA or RNA.
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cDNA.
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Labeling of probes:
3H
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Radioactive
32P
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Hybridization
Recombinant DNA Technology
Amplification of DNA
Study of DNA structure
and functions
Others
DNA cloning
Polymerase chain
reaction
DNA sequencing
ARMS
DGGE
RT PCR
Dot blot analysis
Principles of Molecular Cloning
Involves:
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Isolation of DNA sequence
of interest.
Insertion of this DNA in the
DNA of an organism that
grows rapidly and over
extended period e.g.
bacteria.
Growing of the bacteria
under appropriate condition.
Obtaining the pure form of
DNA in large quantities for
molecular analysis.
Polymerase Chain Reaction (PCR)
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Method to amplify a target sequence of DNA or RNA
several million folds.
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Developed by Saiki et al in 1985.
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Based on Enzymatic amplification of DNA fragment
flanked by primers i.e. short oligonucleotides fragments
complimentary to DNA. Synthesis of DNA initiates at
the primers.
DNA
5’ ATCAGGAATTCATGCCAAGGTTGATCGATGATCGATCGATCGATTGAT 3’
3’AGCTAGCTAGCT 5’
Primer
Application of PCR
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Diagnosis of genetic disease by amplification of the
gene of interest, followed by detection of mutation.
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Detection of infectious agent e.g. bacteria and
viruses.
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DNA sequencing.
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In forensic medicine.
Application of Recombinant
DNA Technology
1.
Clinical Chemistry:
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Diagnosis of disease e.g. sickle
cell anaemia by Mst II.
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Prenatal diagnosis,
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Premarital “
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Presymptomatic “
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Neonatal screening
Southern Blotting
Pathogenesis of -Thalassaemia
Withdraw
blood
12.5Kb
7.0Kb
14.5Kb
BglII
Extract DNA
2
BglII
Treat with BglII
Electrophoresis
Southern Blotting
Visualize
1
BamHI BglII
BamHI
L
R
2.
Human Genetics
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3.
Forensic Medicine
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4.
Detection of viral diseases e.g. hepatitis
Microbiology
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6.
Analysis of stains of blood, semin.
Virology
•
5.
Mutations in genes causing hereditary disease e.g.
diagnosis of fibrosis Channes disease.
Using specific gene probes for detection of E.coli
Cytology, Histology and Pathology
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Used in detection of tumor.
7.
Synthesis of protein in bacterial
8.
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Insulin
•
GH
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Somatostatin
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Interferon
Transgenic animal production
Genetic Counselling
Genetic Counselling for Mendelian Disorders
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Genetic disorders:
• Chromosomal
• Single gene
• Multifactorial
• Mitochondrial
• Acquired somatic
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Only single disorders follow a clearly defined pedigree pattern
of inheritance “Mendelian Pattern”.
•
During genetic counselling it is essential to establish whether
or not the disorder is Mendelian and
to calcualte the precise risk of recurrence.
Essential Components of
Genetic Counselling
Recurrence Risk
History and
pedigree
construction
Clinical
Examination
Follow-up
Confirmatory
diagnosis
- History findings
- Clinical examination findings
- Radiology findings
- Laboratory parameter results
- DNA studies results
- Others
Calculation of
recurrence risk
Counseling
Available
options
ETHICAL
PRINCIPLES
Beneficence
Fidelity
Veracity
Autonomy
Justice
Non-Maleficence
Arabic/Islamic Communities
Strong Religious
believes
Unique features
Possibility of
polygamy
Strong link to
traditions and
customs
Large family size
Religious
And cultural
cohesion
High rate of
Consanguinous
marriages
Special views on
Reproductive issues
Artificial
insemination
Family
planning
Combined family
Living style
In-vitro
Abortion
fertilization
Adoption
Fetal
rights
Establishment of Mendelian Inheritance
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Pattern of transmission judged from family tree.
For several diseases the family tree may be
conclusive even if accurate diagnosis is not made.
•
For some diseases pedigree pattern is not helpful and only
clinical diagnosis is used
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For some disorders the pattern looks complicated and
the exact diagnosis cannot be made.
•
More common by combination of clinical diagnosis
and comparable pedigree pattern.
Premarital Screening
*Man -History
-(Physical Examination)
Blood
Sample
Genetic Screening (Laboratory)
Carrier
Normal
affected
**Women –History
-(Physical examination)
Blood
Sample
Safe
Marriage
No Problem from
marriage from
any Women
Genetic Screening (Laboratory)
Carrier
Not safe
Marriage
affected
Genetic Counseling(advise no
marriage with carrier or affected)
Normal
No Problem from
marriage from
any man
Safe
Marriage
Complexities in AD Disorders
1. Late or variable onset of the disease.
How old will the family members be, to be certain of not
developing the disease, e.g.
• Huntington’s disease, adult onset polycystic kidney
disease, myotonic dystrophy.
• For some conditions life tables have been prepared.
2. Lack of penetrance
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Penetrance:
- Is the index of the proportion of individuals with the
affected gene who present the disease.
- Some disorders show lack of penetrance I.e. biochemical
defect is present, but clinical features are absent, e.g.
•
•
Huntington disease – Penetrance decreases with age.
Retinoblastoma: Lack of penetrance unrelated to age.
Complexities in AD Disorders
3. Variation in Expression:
Several AD disorders show variation in clinical expression and hence
the disorders cannot be ruled out unless careful examination is carried
out.
Mild
Moderate
Severe expression
*Problems in G.C. since those who reproduce are least
severely affected, but may have severely affected children
e.g. Tuberousclerosis, Myotonic dystrophy, Huntingtons
disease.
*Disease severity may depend on sex of the transmitting parent.
“Anticipation: refers to the state that a genetic disease worsens with
successive generation.
Factors underlying variability in AD disorders
Factors
Effect
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Genomic imprinting
Phenotype varies accordingly
•
Anticipation due to unstable
DNA
More severe phenotype in
successive generation
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Mosaicism
Mild or
non-penetran phenotype
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Modifying alleles
Influence of unaffected parent
•
Somatic mutations also
required for presentation
(e.g. familial cancers)
Variable penetrance
•
New mutations
Sudden appearance of (AD)
disorder in normal parent
II. Complexities in AR Disorders
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Difficult to confirm as homozygote born to phenotypically normal
(carrier) parents, who may not have an affected relative.
Horizontal transmission ( sudden appearance of a disorder in a generation)
Diagnosis makes the mode of inheritance certain.
Risk
Very low
Low
Problems with AR disorders
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Genetic heterogeneity.
•
Lack of penetrance and variation in expression are much less.
• If consanguinity present the risk is increased:
(a) Rare disorder
increase in the number of effected children
due to consanguinity
(c) Extensive consanguinity
Appear like AD inheritance
(pseudo AD)
Population Risk
Can be calculated from:
•
Hardy Weinberg Equilibrium
p + q = 1 [p2 + q2 = 2pq = 1]
q2 = Abnormal homozygote
p2 = Normal
2pq = Heterozygote
e.g. 2 patients of PKU in 10000 screened.
q2 = 2; q = 0.0002 = 0.014
p = 1 – q = 0.986
(hetero)2pq = 0.0276
Risk of transmitting an AR disorder in relation to disease incidences
(the spouse is healthy)
Disease
frequency
(q2)/10000
Gene
frequency
(q) (%)
Carrier
frequency
=2pq(%)
Risk for
offspring
homo. (%)
(affected sib)
Risk for
offspring
healthy
sib
100
50
20
10
8
6
5
4
2
1
0.5
0.1
10.1
7.1
4.5
3.3
2.8
2.4
2.2
2.0
1.4
1.0
0.71
0.32
18.0
13.2
8.6
6.2
5.4
4.7
4.3
3.9
2.8
2.0
1.4
0.64
9.0
6.6
4.3
3.1
2.7
2.3
2.1
2.0
1.4
1.0
0.7
0.32
3.0
2.2
1.4
1.0
0.9
0.78
0.72
0.65
0.46
0.33
0.23
0.11
0.22
0.10
0.44
0.2
0.22
0.10
0.07
0.03
0.05
0.01
X-Linked Disorders
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Occupy a prominent place in genetic counselling.
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>100 X-linked disorders recognised.
•
Majority XR; some dominant (often lethal in
hemizygous male).
•
X-chromosomes inactivation (lyonns phenomenon).
applies to almost all human X-chromosomes.
Recognition of X-Linkage
•
•
•
•
•
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No male-to-male transmission.
Affected male  All daughters carriers (XR).
 All daughters affected (XD).
Unaffected males never transmit disease to either sex.
A definite carrier women  risk ½ sons affected.
Carrier women  ½ daughters carrier (XR)
 ½ daughters affected (XD).
Homozygous affected women are few  affected
male are much more.
These guidelines will cover most genetic counseling problems.
Mitochondrial Inheritance
•
No transmission in descendents of males, affected or not.
•
Both sexes may be affected.
•
Females may be symptomless carriers.
•
All daughters of an affected or carrier female are at risk of
transmitting the disorders or of becoming affected.
•
All sons may become affected, but do not transmit it to their children
Degree of Relationship to patients
Proportion of gene shared
•
First degree…………………………………….
•
Sibs (brothers & sisters)
•
Dizygotic twins
•
Parents
•
Child
1/4
•
Second degree …….. …………………………..
•
Half sibs
•
Uncles, aunts
•
Nephew, nieces
•
Double first cousins
1/4
•
Third degree: …………………………………….
•
First cousins
•
Half uncles, aunts
•
half nephew, nieces
1/8
Degree of
Relation
Gene
Chance
shared
of Homo.
Monozygotic twin
-
1
-
Dizytotic twin
1st
1/2
1/4
Sibs
1st
1/2
1/4
Uncle-nephew
(aunt-niece)
2nd
1/4
1/8
Half-sibs
2nd
1/4
1/8
Double 1st cousin
2nd
1/4
1/8
First cousin
3rd
1/8
1/16
Consanguinity
•
Only relevant to genetic risks if it involves both parental lives not just one.
Consanguinity relevant
Not relevant
•
The rarer the disorder the higher the proportion of affected individuals from
consanguineous marriages.
•
Consanguinity must be seen in the context of particular community. An
apparent relationship of a particular disorder is much less certain if 30% cousin
marriages, compared to non-consanguineous mating.
•
Extensive consanguinity (AR) appears like AD.