Download Molecular Genetics S Brown 30th May 2014

Molecular Genetics
Dr Alison Battersby
Dr Martin Evans
Chromosomes
Karyotype
• 23 derived from each parent
• Sex determined by X and Y chromosomes
• Males XY, females XX
Number, size and shape of chromosomes
arranged in standard manner
Position of centromere
Convention/Terms
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Metacentric, centromere is median
Acrocentric centromere close to one end
Telocentric centromere terminal ie 1 arm
Short arm on top (P or petit), long arm Q
Biggest first, sex chromosomes last
Banded karyotype
The FISH technique utilizes DNA probes that are specific to regions of individual chromosomes. The probe attaches to
the spread of chromosomes from a cell, then a fluorescein stain is applied. This "paints" the chromosome so that it is
visible with the aid of a fluorescent microscope. In the example diagram below the chromosome 21 pair have been
painted.
The FISH technique requires viewing many
cells to diminish artefactual problems in
counting the painted chromosomes. FISH is
good at identifying abnormal numbers of
chromosomes such as trisomies and
monosomies. FISH is also useful when probes
are available for specific regions of
chromosomes to determine if deletions,
translocations, or duplications are present. In
the above diagrams, a trisomy 21 is depicted at
the right, while a probe for chromosome 22 has
detected a translocation, probably to
chromosome 9, on the left.
DNA section showing gene ACT on the descending
strand and the gene AGT on the ascending strand
DNA double helix
Replication
Central Dogma
DNA contains codes
DNA vs RNA
DNA
• Sugar
deoxyribose
• Bonds with Adenine thymine
• # of Strands
two
RNA
ribose
uracil
one
Kinds of RNA
• Messenger RNA (mRNA)
• Ribosomal RNA (rRNA)
• Transfer RNA (tRNA)
Transcription
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DNA unwinds
RNA polymerase recognises promotor
Binds
Termination code in DNA
Processing the mRNA transcript
Translation
Mutation
• Frameshift mutation:
• THE BIG RED ANT ATE ONE FAT BUG
THB IGR EDA NTA TEO NEF ATB UG?
• Point mutation:
• THE BIG RED ANT ATE ONE FAT BUG
THA BIG RED ANT ATE ONE FAT BUG
• Silent, Missense and Nonsense Mutation
Mendelian Inheritance
• 1850’s showed independent units of heredity
• Genome: entire complement of genetic
material in a chromosome set
• Genetic polymorphism: naturally occurring
differences between individual members of a
population
TERMS
• Genotype: Genetic contribution inherited
from parents
• Phenotype: Observed variations in
physiology/morphology
• Forward Genetics: individuals of 2 distinct
phenotypes then to DNA level
• Reverse Genetics: stretch normal DNA and
insert a mutation
Single gene inheritance pattern
• Useful for gene discovery eg Cystic Fibrosis/
Tay-Sachs
• Human is a diploid organism with 2 complete
genomes and therefore 2 identical
chromosome sets ie 2n = 46
• 2 members of a chromosome pair called
homologous chromosomes/homologs
• Each gene a gene pair
Mendel’s First Law
The Law of Equal Segregation
• Garden pea, used pre-exisiting mutants
• Seven properties each had 2 contrasting
phenotypes
• All lines used were pure lines (produced
identical offspring)
• Eg seed colour of green or yellow
• Made crosses and observed results
Seed colour crosses
• Female (yellow seed) x Male (green seed)
= F1 (first filial generation)
F1 peas all yellow
• Female (green seed) x Male (yellow seed)
= F1 peas all yellow
Selfing the F1 generation
• F1 pea x F1 pea
= F2 generation
¾ yellow, ¼ green
Green phenotype had re-appeared from
yellow parents
Selfing the F1 generation
Individually selfed F2 generation
• F2 green seeded peas selfed only gave green
peas
• F2 yellow seeded peas selfed were found to
be of two types:
• 1/3 pure breeding for yellow
• 2/3 progeny ratio of ¾ yellow seeds and ¼
green seeds
More information!
• Mendel crossed an F1 plant with a greenseeded plant
• ½ yellow and ½ green
What this means in modern terms
• Gene: a hereditary factor is necessary for
producing pea colour
• Gene has 2 forms or alleles
Eg Y (yellow phenotype) y (green phenotype)
A plant can be Y/Y, Y/y or y/y
The ‘/’ denotes a pair of alleles
Dominant/Recessive
• In Y/y plants the Y allele dominates and
phenotype will be yellow
• Therefore Y allele is dominant and y allele is
recessive
• In meiosis members of a gene pair segregate
equally into the gametes ie Mendel’s 1st Law
of equal segregation
Terminology
• A plant with a pair of identical alleles is called
a homozygote
• A plant where pair of alleles differ is called a
heterozygote
• A heterozygote for one gene is also sometimes
called a monohybrid
Which means
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Individual can be:
Homozygous dominant (Y/Y)
Heterozygous (Y/y)
Homozygous recessive (y/y)
Allelic combinations underlying phenotypes
are called genotypes eg Y/Y, Y/y and y/y
Testcross
• A cross of an individual organism of unknown
genotype or a heterozygote with a tester
Message
• All 1:1, 3:1, and 1:2:1 ratios are diagnostic of
single-gene inheritance and are based on
equal segregation in a heterozygote
Sex-Linked Inheritance
• Humans 22 homologous pairs of
chromosomes and 2 sex chromosomes
• Females 2 X chromosomes
• Males X and Y (non-identical pair)
• Homolgous and differential regions
• Y chromosome SRY genes (male sexual
function)
Sex Linkage
• Genes in the differential regions show
inheritance patterns called sex linkage
• X linkage: mutant alleles in the differential
region of the X chromosome show this
• Phenotypic rations can be different in each sex
• Pseudoautosomal region
Human Pedigree Analysis
Human Pedigree Analysis
• No controlled matings
• Study medical history of family to see if
consistent with single gene inheritance
• Propositus is subject who brought the case to
attention
• Draws a family tree using standard symbols
• Difficult to use ratios as small numbers
Autosomal recessive
• Eg Phenylketonuria (or cystic fibrosis) p allele
recessive
• Sufferers p/p
• Others P/P or P/p
• Disorder usually appears in progeny of
unaffected parents
• Affected progeny includes males and females
Human pedigree of autosomal
recessive disorder
Simple Monohybrid Cross
Autosomal dominant disorders
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Defective allele is dominant
So a rare disorder can be dominant
Eg pseudoachondroplasia, Huntington’s disease
Pedigrees show affected males and females in each
generation: they also show affected men and women
transmitting the condition to equal proportions of
their sons and daughters
X-linked recessive disorders
• Pedigrees have more males than females
showing the phenotype under study
• Female would need her mother AND her
father to carry the defective allele
• None of female offspring of affected male
show the condition but are carriers
• None of sons of affected male show the
female as they inherited his Y chromsome
X-linked recessive disorders
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Red-green colour blindness
Hemophilia (Factor VIII)
Duchenne muscular dystrophy
Androgen insensitivity syndrome
X-linked dominant rare
eg hypophosphatemia
Calculating risks in pedigree analysis
• Tay-sachs disease
History
• Newly-married husband and wife find they
both had an uncle with Tay-Sachs disease
(autosomal recessive disease)
• Calculate the probability of the children’s first
child having the disease
Calculation
• Neither of the couple has the disease so each
could be normal homozygote or heterozygote
• If both are heterozygotes, they could each
pass a recessive allele to their child who
would be affected
More maths
• We calculate the probability of the couple both being
heterozygotes and then, if so, the probability of
passing the deleterious allele onto a child
• The husband’s grandparents must have both been
heterozygotes (T/t) because they had a t/t child (like
a monohybrid cross)
• The husband’s father could be T/T or T/t, but we
know the relative probabilities of these genotypes
must be ¼ and ½. Therefore 2/3 probability that the
father is a heterozygote
And again
• Husband’s mother is assumed to be T/T as
allele is rare. Thus if the father is T/t then the
mating was T/t x T/T
• Expected proportions of progeny are ½ T/T
and ½ T/t
Product Rule
• Overall probability of the husband’s being a
heterozygote is calculated using product rule
• “The probability of two independent events both
occurring is the product of the individual
probabilties”
• Gene transmission is an independent event
• Therefore probability of husband being a
heterozygote is 2/3 x ½ = 1/3
Now the wife
• Likewise the probability of her being a
heterozygote is 1/3
• If they are both heterozygotes (T/t) their
mating is again a standard monhybrid cross so
probability of their having a t/t child is 1/4
Overall
• Overall, the probability of the couple’s having an
affected child is the probability of them both being
heterozygotes and then both transmitting the
recessive allele to the child which again is an
independent event
• Therefore probability is:
• 1/3 x 1/3 x ¼ = 1/36
• Ie a 1 in 36 chance of having a child with Tay-Sachs
disease
Mendel’s Second Law:
The Law of Independent Assortment
• “Gene pairs on different chromosomes assort
independently at meiosis”
• Dihybrid crosses or 9:3:3:1
• Punnett square or 4 x 4 grid
The sum rule
• “the probability of either of two mutually
exclusive events occurring is the sum of their
individual probabilities”
The chi-square test
• A statistical test used to determine the
probability of obtaining observed proportions
by chance, under a specific hypothesis
• Or “how close to an expected result is close
enough”
Polygenic inheritance
• Most variation in natural populations take on the
form of continuous variation eg height, weight,
colour (bell-shaped distribution)
• Environment plus genes
• Interacting genes underlying hereditary continuous
variation are called polygenes or quantitative trait
loci (QTL’s)
• Often distributed throughout the genome
Recombination
• “Mapping”
• Physical maps shows the genes as segments
arranged along the DNA molecule of the
chromosome
• Recombination maps map the loci of genes
that have been identified by mutant
phenotypes showing single-gene inheritance
• Uses Linkage analysis
Recombinant frequency to recognise
linkage
• When geneticists say two genes are linked they mean
that the loci of the two genes are on the same
chromosome hence any alleles on any one homolog
are physically joined
• When 2 genes are close together on the same
chromosome pair ie linked they do not assort
independently but produce a recombinant frequency
of less than 50% ie a recombinant frequency of less
than 50% is diagnostic for linkage
How crossovers produce recombinants
for linked genes
• How are any recombinants produced?
• When homologous chromosomes pair at
meiosis the chromosomes occasionally break
and exchange parts in a process called
“crossing over”
• Two new products are crossover products
Mapping by recombinant frequency
• Recombinant frequencies for linked genes vary
between 0 to 50% depending on closeness providing
the basis for a genetic map
• One genetic map unit is the distance between genes
for which one product of meiosis in 100 is
recombinant
• A recombinant frequency of 10.7% is sometimes
called a centimorgan (cM)
Using Lod scores to assess linkage in
human pedigrees
• “log of odds” useful in humans with small numbers
• Calculates 2 different probabilities
• 1. calculate probability of occurring with independent
assortment
• 2. calculate probability of occurring with the assumption of a
specific degree of linkage
• Ratio of probabilities calculated, the logarithm of the number
taken which is the Lod value
• Can add the Lod value to those accumulated from other
researchers
• Quite a rigorous test
Gene Interaction
• How do genes in a set interact?
• Often several known mutants of a gene
• Incomplete dominance- phenotype based on
2 alleles of a single gene but the heterozygote
is of intermediate phenotype
Codominance
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Expression of both alleles in a heterozygote
Eg human ABO groups
Genotype
Blood type
IA/IA, IA/I
A
IB/IB, IB/I
B
IA/IB
AB
i/i
O
Penetrance
• Penetrance: the percentage of individuals with
a given allele who exhibit the phenotype
associated with the allele
• Why wouldn’t this be 100%?
• Environment
• Other interacting genes
• Subtlety of the mutant phenotype
Expressivity
• The degree to which a given allele is expressed
at the phenotypic level ie the intensity of the
phenotype eg brown hair
• May be due to variation in the allelic
constitution of the rest of the genome or
environment
Is a trait heritable?
• Correlations between relatives are only
evidence for genetic variation if the relatives
do not share common environments
familiality or heritability
• Twin/adoption studies: need to have no
correlation between adopting families to rule
out environment. Very difficult to meet!
Molecular Techniques
• Restriction enzymes: An endonuclease (cuts
nucleotide chain with straight or ‘sticky’ends)
that will recognise specific target nucleotide
sequences in DNA and break the DNA chain at
those points; a variety of these enzymes are
known and they are extensively used in
genetic engineering
Southern blot
Agarose gel and X ray film
Restriction Fragment Length
Polymorphisms
Some definitions
• Cloning: in recombinant DNA research is the process
of creating and amplifying specific DNA segments
• Gene therapy: the correction of a genetic deficiency
in a cell by the addition of new DNA and its insertion
into the genome. Different techniques have the
potential to carry out gene therapy only in somatic
tissues or to correct the genetic deficiency in the
zygote
Prenatal identification
• Chorionic Villus Sampling used to detect
specific known genetic disorders eg Down
syndrome. Reliable test. 11-13 weeks of
pregnancy. Cells grown in the lab for 2 weeks,
detects trisomies 13, 18 and 21 (after 72
hours). Produces a karyotype
• Amniocentesis week 16 onwards. Cells grown
and 2 weeks to results
Genetic counselling
• Tests performed on people who do not have
the disease themselves, there are a number of
issues to discuss before testing. The
counsellor discusses pros and cons of testing
eg do you want to know? What would you do
with the information and how might you
react?
Role clinical geneticist
• Diagnosis of genetic disorders affecting all ages and all body
systems, birth defects and developmental disorders
• Investigation and assessment of genetic risk
• Genetic counselling
• Follow up, support, co-ordination of health surveillance
• Offer genetic services to extended family if appropriate
• Liaison with genetic labs
• Education and training
• research
Organisation of clinical genetic
services
• Usually regionally based in a major city
• Currently 25, 100 consultants in field
• Many centres offer joint clinics with different
departments
• The interface between scientists and patients
DNA Banks
• Researchers from the UK are taking part in a global study of the link
between genetic variation and diseases.
• Scientists from the UK, US and China will work together to create the
largest DNA database in the world.
• The 1000 Genomes Project will map the DNA make-up of 1000 people
from different parts of the world to create a detailed catalogue of the
most common genome variants.
• It is hoped this will lead to a greater understanding of the relationship
between genetic variation and common diseases - benefiting both the
medical and human biology sectors.
Now for the test!
Terms to remember
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Congenital abnormalities
Genetic abnormalities
Terratogenic
Heritability, proband
Chromosomal abnormalities, including
numerical
• Deletions/ microdeletions
Terms to remember
• Autosomal versus
• X-linked: females carriers, males show the disease
• Heterozygotes/ homozygotes-who is affected Mendelian
patterns
• Alelles
• Genome
• Genotype/fenotype
• Genetic studies: family, twin, adoption- concordance rate,
monozygotic, dyzigotic
Terms to remember
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Mitosis
Meiosis
Mithocondrial DNA (always maternal, both sexes can suffer)
Linkage
Polygenic trait is one whose phenotype is influenced by more than one
gene. Traits that display a continuous distribution, such as height or skin
color. Do not show the phenotypic ratios characteristic of Mendelian
inheritance, though each of the genes contributing to the trait is inherited
as described by Gregor Mendel.
• Many polygenic traits are also influenced by the environment and are
called multifactorial.
Genetics linked with:
Schizophrenia: heritability about 82%, life time risks in
relatives of patients with schizophrenia, probably
genetically heterogenous, no single locus responsible
demonstrated so far; children of mothers with S have
13% chance of S both in adoption and twin studies
page 358 Puri Hall
Huntignton Disease
• Wilson Disease
• Prader Willi- deletion chromozome 15
• Learning disability syndromes and autism
Genetic aspects of epidemiology/ what is
involved, gender rates, ethnicity, onset
• Schizophrenia- risk if a parent, other sibling or twin brother
have schizophrenia
• Mood disorders: 26% biological: 12% adoptive parents,
complex genetic heterogeneity, overlapping sets of
susceptibility genes ie. COMT encoding for tyrosine
hydroxilase, serotonin transporter and BDNF brain derived
neurotropic factor
• Depression/ endogenous
• Bipolar affective disorder
• Anxiety disorders- spectrum of neurotic traits, prevalence is
1in 6 in general population in UK
• Personality disorder/ psychopatic traits
Example-Dementia
• Neuron loss with age but not necessarily loss of
function
• Accumulation of Tau protein resulting in
neurofibrillary tangles
• Senile plaques: aggregation of amyloid
• APP gene (amyloid precursor gene) in early onset
dementia, runs in families
• Amyloid deposition predisposed by apolipoprot E e4
allele on Chr 19
• Presenilin 1 PS1 on Chr 14 and PS2 on Chr 1