HUMAN MOLECULAR GENETICS
1.
2.
3.
4.
5.
Examples of genetic diseases in Humans
Meiosis & Recombination
Mendelian Genetics
Modes of Heredity
Genetic Linkage Analysis
Genetic Diseases in Humans
Role of Genes in Human Disease
•
Most diseases -> phenotypes result from
the interaction between genes and the
environment
•
Some phenotypes are primarily
genetically determined
–
100%
Environmental
Achondroplasia (-> dwarfism)
Struck by lightning
Infection
Weight
•
Other phenotypes require genetic and
environmental factors
–
•
Mental retardation in persons with PKU
(polyketonuria)
Some phenotypes result primarily from
the environment or chance
–
Cancer
Lead poisoning
Diabetes
Height
100%
Genetic
Down syndrome, achondroplasia
Genetic Diseases in Humans
Types of Genetic Disorders:
-> Chromosomes and chromosome abnormalities (Down
Syndrome)
-> Single gene disorders (Haemophilia, sickle cell anaemia)
-> Polygenic Disorders (Cancer)
Genetic Diseases in Humans
Chromosomal disorders
•
Addition or deletion of entire chromosomes or parts of chromosomes
•
Typically more than 1 gene involved
•
1% of paediatric admissions and 2.5% of childhood deaths
•
Classic example is trisomy 21 - Down syndrome
KARYOTYPE
Genetic Diseases in Humans
Single gene disorders
• Single mutant gene has a large effect on the patient
• Transmitted in a Mendelian fashion
• Autosomal dominant, autosomal recessive, X-linked, Y-linked
• Osteogenesis imperfecta - autosomal dominant
• Sickle cell anaemia - autosomal recessive
• Haemophilia - X-linked
Genetic Diseases in Humans
Single gene disorders
Neonatal fractures
typical of osteogenesis
imperfecta, an
autosomal dominant
disease caused
by rare mutations
in the type I collagen
genes COL1A1 and
COL1A2
A famous carrier
of haemophilia A,
an X-linked disease
caused by mutation
in the factor VIII gene
Sickle cell anaemia,
an autosomal recessive
disease caused by
mutation in the
β-globin gene
Genetic Diseases in Humans
Polygenic disorders
• The most common yet still the least understood of human
genetic diseases
• Result from an interaction of multiple genes, each with a minor
effect
• The susceptibility alleles are common
• Type I and type II diabetes, autism, osteoarthritis, cancer
Genetic Diseases in Humans
Single gene disorders - Polygenic disorders
Polygenic disease pedigree
Autosomal dominant pedigree
Male,
affected
Female,
unaffected
Meiosis & Genetic Recombination
Chromosomes & Genes
•Are long stable DNA strands with many genes.
•Occur in pairs in diploid organisms.
•The two chromosomes in a pair are called “homologs”
•Homologs usually contain the same genes, arranged in the same order
• Homologs often have different alleles of specific genes that differ in
part of their DNA sequence.
a
b
c
DNA
genes
unreplicated pair of homologs
Meiosis & Genetic Recombination
Chromosomes & Genes
Meiosis & Genetic Recombination
Chromosome structure
Telomeres:
Specialized structures
at chromosome ends
that are important for
chromosome stability.
sister
chromatids
telomeres
Centromere:
A region within chromosomes
that is required for proper
segregation during meiosis
and mitosis.
centromere
unreplicated
chromosome
replicated
chromosome
Each chromatid consists of a very long strand of DNA. The DNA is
roughly colinear with the chromosome but is highly structured around
histones and other proteins which serve to condense its length and
control the activity of genes.
Meiosis & Genetic Recombination
Chromosome structure - Homologs
Sister
chromatids
unreplicated
homologs
replicated
homologs
Sister chromatids are almost always IDENTICAL
(prior to recombination). Homologues may carry
different alleles of any given gene.
Meiosis & Genetic Recombination
Cell Devision
Mitosis -> 2n -> 2x 2n (diploid)
Goal is to produce two cells that are genetically
identical to the parental cell. (somatic cells)
Meiosis -> 2n -> 4x 1n (haploid)
Goal is to produce haploid gametes from a
diploid parental cell. Gametes are genetically
different from parent and each other.
Meiosis & Genetic Recombination
Cell Devision -> Mitosis - Meiosis
Mitosis
Cross-over
II
I
2n
4n
2n
2n
In mitosis the homologs do not pair
up.
Rather they behave independently.
Each resultant cell receives one copy
of each homolog.
4n
2n
1n
In meiosis the products are haploid gametes so
two divisions are necessary. Prior to the first
division, the homologs pair up (synapse -> crossover) and segregate from each other. In the
second meiotic division sister chromatids
segregate. Each cell receives a single
chromatid from only one of the two homologs.
-> contributes to evolutionary variations
Meiosis & Genetic Recombination
Meiosis/perfect linkage
PL
P L
P L
p l
P L
PL
P L
P L
p l
p l
p l
p l
p l
p l
only
parental-type
gametes
Meiosis & Genetic Recombination
Meiosis/with recombination
PL
P L
p l
P L
P L
Pl
P l
p l
p L
p L
p l
p l
Meiotic recombination in a grasshopper
In some meiotic divisions these
recombination events between the genes
will occur resulting in recombinant
gametes -> contributes to variation
(evolution)
Meiosis is not conservative, rather it promotes variation through segregation of chromosomes
and recombination
Mendelian Genetics
The laws of heridity
Gregor Mendel (1822-1884): “Father of Genetics”
Augustinian Monk at Brno Monastery in
Austria (now Czech Republic)
-> well trained in math, statistics, probability, physics, and
interested in plants and heredity.
Mountains with short, cool growing season meant pea (Pisum
sativum) was an ideal crop plant.
• Work lost in journals for 50 years!
• Rediscovered in 1900s independently by 3 scientists
• Recognized as landmark work!
One Example of Mendel’s Work
P
Tall
Dwarf
x
DD
dd
Homozygous
Dominant
Homozygous
Recessive
Dd
Heterozygous
Punnett Square:
possible
gametes
Genotype
Clearly Tall is Inherited…
What happened to Dwarf?
All Tall
F1
F2
Phenotype
1.
Tall is dominant to Dwarf
2.
Use D/d rather than T/t for
symbolic logic
F1 x F1 = F2
possible gametes
D
d
D
Tall
DD
Tall
Dd
d
Tall
Dd
Dwarf
dd
3/
Tall
1/ Dwarf -> Phenotype: 3:1
4
4
Dwarf is not missing…just masked as
“recessive” in a diploid state
Mendelian Genetics
The laws of heridity
1. The Law of Segregation:
Genes exist in pairs and alleles segregate from each other
during gamete formation, into equal numbers of gametes.
Progeny obtain one determinant from each parent.
-> Alternative versions of genes account for variations in inherited
characteristics (alleles)
-> For each characteristic, an organism inherits two alleles, one from each
parent. (-> homozygote/heterozygote)
-> If the two alleles differ, then one, the allele that encodes the
dominant trait, is fully expressed in the organism's appearance; the other,
the allele encoding the recessive trait, has no noticeable effect on the
organism's appearance (dominant trait -> phenotype)
-> The two alleles for each characteristic segregate during gamete
production.
Mendelian Genetics
The laws of heridity
2. The Law of Independent Assortment
Members of one pair of genes (alleles) segregate
independently of members of other pairs.
-> The emergence of one trait will not
affect the emergence of another.
-> mixing one trait always resulted in a
3:1 ratio between dominant and recessive
phenotypes
-> mixing two traits (dihybrid cross) showed
9:3:3:1 ratios
-> only true for genes that are not linked to
each other
9:3:3:1
3:1
Mendelian Genetics
The laws of heridity
After rediscovery of Mendel’s principles, an early
task was to show that they were true for animals
And especially in humans
Mendelian Genetics
The laws of heridity
Problems with doing human genetics:
-> Can’t make controlled crosses!
-> Long generation time
-> Small number of offspring per cross
So, human genetics uses different methods!!
Mendelian Genetics
The laws of heridity
Major method used in human genetics is -> pedigree analysis
(method for determining the pattern of inheritance of any trait)
Pedigrees give information on:
-> Dominance or recessiveness of alleles
-> Risks (probabilities) of having affected offspring
Mendelian Genetics
The laws of heridity
Standard symbols used in pedigrees:
carrier
”inbreeding”
Modes of Heredity
Autosomal Dominant
Most dominant traits of clinical significance are rare
So, most matings that produce affected individuals are of the form:
Aa x aa
-> Affected person can be heterozygote (Aa) or homozygote (AA)
-> Every affected person must have at least 1 affected parent
-> expected that 50% are affected /50% are uneffected
-> No skipping of generations
-> Both males and females are affected and capable of transmitting the trait
-> No alternation of sexes: we see father to son, father to daughter, mother to son, and mother
to daughter
Modes of Heredity
Autosomal Dominant
Examples:
Tuberous sclerosis (tumor-like growth in multiple organs, clinical
manifestations include epilepsy, learning difficulties, behavioral problems, and
skin lesions)
and many other cancer causing mutations such as retinoblastoma
Brachydactyly
Modes of Heredity
Autosomal Dominant
Examples: Achondroplasia
-> short limbs, a normal-sized head and body, normal intelligence
-> Caused by mutation (Gly380Arg mutation in
transmembrane domain) in the FGFR3 gene
-> Fibroblast growth factor receptor 3 (Inhibits
endochondral bone growth by inhibiting
chondrocyte proliferation and differentiation
Mutation causes the receptor to signal even in
absence of ligand -> inhibiting bone growth
Modes of Heredity
Autosomal Recessive
These are likely to be more deleterious than dominant disorders, and so
are usually very rare
The usual mating is:
Aa x Aa
-> Affected person must be homozygote (aa) for disease allele
-> Both parents are normal, but may see multiple affected individuals in the sibship, even though the
disease is very rare in the population
-> Usually see “skipped” generations. Because most matings are with homozygous normal individuals and
no offspring are affected
-> inbreeding increases probablility that offspring are affected
-> unlikely that affected homozygotes will live to reproduce
Modes of Heredity
Autosomal Recessive
Examples:
Sickle-Cell Anaemia (sickling occurs because of a mutation in the
hemoglobin gene -> affects O2 transport; occurs more commonly in
people (or their descendants) from parts of tropical and sub-tropical
regions where malaria is common -> people with only one of the two
alleles of the sickle-cell disease are more resistant to malaria)
Cystic fibrosis (also known as CF, mucovoidosis, or mucoviscidosis;
disease of the secretory glands, including the glands that make
mucus and sweat; excess mucus production -> causing multiple chest
infections and coughing/shortness of breath; especially Pseudomonas
infections are difficult to treat -> resistance to antibiotica)
Modes of Heredity
Dominant vs. Recessive
Is it a dominant pedigree or a recessive pedigree?
1. If two affected people have an unaffected child, it must be a
dominant pedigree: A is the dominant mutant allele and a is the
recessive wild type allele. Both parents are Aa and the normal
child is aa.
2. If two unaffected people have an affected child, it is a
recessive pedigree: A is the dominant wild type allele and a is
the recessive mutant allele. Both parents are Aa and the
affected child is aa.
3. If every affected person has an affected parent it is a dominant
pedigree.
Modes of Heredity
X-Linked Recessive
-> Act as recessive traits in females (XX) -> females express it only if they get a copy from both
parents)
-> dominant traits in males (XY)
-> An affected male cannot pass the trait on to his sons, but passes the allele on to all his daughters,
who are unaffected carriers
-> A carrier female passes the trait on to 50% of her sons
Examples: About 70 pathological traits known in humans -> Hemophilia A, Duchenne muscular dystrophy,
color blindness,…..
Modes of Heredity
Other sex-linked disease
X-linked dominant:
-> caused by mutations in genes on the X chromosome
-> very rare cases
-> Males and females are both affected in these disorders, with males typically being more severely
affected than females.
-> Some X-linked dominant conditions such as Rett syndrome, Incontinentia Pigmenti type 2 and Aicardi
Syndrome are usually fatal in males
Y-linked (dominant):
-> mutations on the Y chromosome.
-> very rare cases -> Y chromosme is small
-> Because males inherit a Y chromosome from their fathers -> every son of an affected father will be
affected.
-> Because females inherit an X chromosome from their fathers -> female offspring of affected
fathers are never affected.
-> diseases often include symptoms like infertility
Modes of Heredity
Exceptions to Mendelian Inheritance
Mitochondrial inheridance:
Mitochondrial DNA is inherited only through the egg, sperm mitochondria never contribute to the
zygote population of mitochondria. There are relatively few human genetic diseases caused by
mitochondrial mutations.
-> All the children of an affected female but none of the children of an affected male will inherit
the disease.
-> Note that only 1 allele is present in each individual, so dominance is not an issue
Summary of mutations which can cause a
disease
• Three principal types of mutation
– Single-base changes
– Deletions/Insertions
– Unstable repeat units
• Two main effects
– Loss of function
– Gain of function
Genetic Linkage
Mapping a disease Locus
Linkage
Although Mendel's Law of Independent Assortment applies well
to genes that are on different chromosomes. It does not apply
well to two genes that are close to each other on the same
chromosome.
Such genes are said to be “linked” and tend to segregate
together in crosses.
Genetic Linkage
Mapping a disease Locus
Basic rules of linkage
•
Loci on different chromosomes will not be co-inherited
– i.e. locus A on chromosome 1 will not be co-inherited with locus B on chromosome 2
•
Loci on the same chromosome may be co-inherited
•
The closer two loci are on the same chromosome the greater the
probability that they will be co-inherited
–
i.e the likelyhood of recombination is small
Genetic Linkage
Mapping a disease Locus
Linkage analysis
The mapping of a trait on the basis of its tendency to be coinherited with polymorphic markers
Why map and characterize disease genes?

Can lead to an understanding of the molecular basis of the disease

May suggest new therapies

Allows development of DNA-based diagnosis
- including pre-symptomatic and pre-natal diagnosis
Genetic Linkage
Mapping a disease Locus
First question to ask in a mapping exercise:
-> Are there functional or cytogenetic clues?
Functional Clues
Osteogenesis imperfecta
Haemophilia A
Haemophilia B
(OI)
Collagen I
Factor VIII
Factor IX
Cytogenetic Clues (structure and function of chromosomes)
Duchenne muscular dystrophy
Polyposis coli
Translocation at Xp21
Deletions in 5q
-> If there are clues, then one can target a particular gene or a particular
chromosomal region
-> If there are no clues, then one needs to conduct a genome-wide linkage scan
Genetic Linkage
Mapping a disease Locus
Example: Sweat Pea Purple & Long
Consider the following pair of genes from the sweet pea that are located on the
same chromosome -> linked:
Gene
Trait affected
Purple
Flower color
Long
Pollen length
Alleles
Phenotype
P
purple
p
red
L
Long
l
short
Genetic Linkage
Mapping a disease Locus
Example: Sweat Pea Purple & Long
Mating type - more clearly reveals what gametes
(and how many) were contributed by the F1 generation.
P/P L/L
F1
F2
P/p L/l
-> homozygote
X
p/p l/l
X
p/p l/l "tester"
?
-> result can give indication if loci are linked or not
Genetic Linkage
Mapping a disease Locus
Calculation of Recombination Frequency
Recombination frequency is a direct measure of the distance between genes.
The higher the frequency of recombination (assortment) between two
genes the more distant the genes are from each other.
A map distance can be calculated using the formula:
# recombinant progeny /total progeny X 100 = map distance (% recombination)
1 map unit = 1% recombination = 1 centimorgan (cM)
1 cM (Thomas Hunt Morgan) is the unit of genetic distance

Loci 1cM apart have a 1% probability of recombination during meiosis

Loci 50cM apart are unlinked
-> LOD Score - a method to calculate linkage distances (to
determine the distance between genes)
Genetic Linkage
Mapping a disease Locus
Example: Sweat Pea Purple & Long
-> Calculation of map distance between the P and L genes
gametes
P L
P l
p L
p l
zygote
P/p L/l
P/p l/l
p/p L/l
p/p l/l
phenotype
Purple long
purple short
red long
red short
observed
1340
154
151
1195
2840
parental type
recombinant
recombinant
parental type
TOTAL
# recombinant progeny /total progeny X 100 = map distance
305 were recombinants (154 P l + 151 p L)
305/2840 X 100 = 10.7 map units or 10.7% recombination frequency
Genetic Linkage
Mapping a disease Locus
Build a map
Recombination frequencies for a third gene (X) were determined using the same type of cross as
that used for P and L.
P to L
10.7 map units
P to X
13.1 map units
X to L
2.8 map units
.
Map
13.1 units
P-------------------------------L--------------X
10.7 units
2.8 units
We can deduce from this that L is between P and X and is
closer to L than it is to P. Thus it is possible to generate a
recombination map for an entire chromosomes.
Genetic Linkage
Mapping a disease Locus
Chromosomes and Linkage
The maximum frequency of observed recombinants between two genes is 50%. At this frequency
the genes are assorting independently (as if they were on two different chromosomes).
A
B
a
b
50% parental gametes (AB, ab)
50% non-parental gametes (aB, Ab)
If on the same chromosome, but greater than 50 map units apart, crossovers will
actually occur > 50% of the time but multiples will cancel each other out.
A
B
a
b
A
B
parental gametes (AB, ab)
-> Two genes can be on the SAME
chromosome but will behave as if they are
unlinked in a test cross.
non-parental gametes (aB, Ab)
a
b
Genetic Linkage
Mapping a disease Locus
Mapping using molecular markers
Molecular markers are most often variations in DNA sequence that do not manifest a phenotype in the
organism. However they can be used to map genes in the same way that markers affecting visible
phenotypes are. An example of this would be a restriction fragment length polymorphism (RFLP)
Gene of interest
restriction sites -> markers
Genetic Linkage
Human linkage map
Genetic Linkage
Mapping a disease Locus
Polymorphic markers
-> A marker that is frequently heterozygous in the population
-> One can therefore distinguish the two copies of a gene that an individual inherits
-> They are not themselves pathological - they simply mark specific points in the
genome
Technique used for mapping with markers:
Primers are made to the unique DNA sequence to each side of a given repeat, and these primers
are used to amplify the repeat using the polymerase chain reaction (PCR).
-> copies of the repeat are either radioactively or fluorescently labeled and then run on a gel to
separate the different sizes from one another.
-> The size of each sequence, which correlates with the number of repetitive sequences within
it, can then be assessed.
Genetic Linkage
Mapping a disease Locus
Polymorphic markers -Variable number tandem repeats (VNTRs)
Changes in the numbers of repeated DNA sequences arranged in tandem arrays
3-repeat allele
4-repeat allele
ACGTGTACTC
Polymorphic markers - Microsatellites
Particular class of VNTR with repeat units of 1-6bp in length
Also known as short tandem repeats (STRs) and sometimes as simple sequence repeats (SSRs)
The most widely used are the CAn microsatellites
6 (CA) allele
CACACACACACA
8 (CA) allele
CACACACACACACACA
Polymorphic markers - Single nucleotide polymorphisms (SNPs)
a polymorphism due to a base substitution or insertion or deletion of a single base
Genetic Linkage
Mapping a disease Locus
Practicalities of Linkage Analysis
The genotype for a microsatellite
marker on chromosome 1
Maternal copy
Paternal copy
6 (CA) allele
*
8 (CA) allele
*
Chrom. 1
Determine the genotype of
each family member
for polymorphic markers
across the genome
-> The individuals genotype for this location is (6 8)
Genetic Linkage
Mapping a disease Locus
Uninformative and informative meioses
66
66
66
68
68
68
68
9 10
66
66
66
68
68
68
89
6 10
Uninformative
Completely
informative
A lab technique used to determine whether two genetic markers are linked to each
other and how closely linked they are. It uses sexual reproduction which produces
offspring in which the two markers may have crossed over during DNA
recombination.
Informative -> if repetitive sequences (markers) are different at the same location
Genetic Linkage
Mapping a disease Locus
An autosomal
dominant
disease for which the
gene resides on
chromosome 1
But you don’t
know that!
Disease gene
1
Genetic Linkage
Mapping a disease Locus
Marker Studies
Marker studied
Disease gene
56
15
47
35
23
67
23
24
15
44
25
27
Genetic Linkage
Mapping a disease Locus
Genotype data for the whole family
(23)
(14)
(26)
(34)
(13)
(58)
(24)
(46)
(33)
(14)
(18)
(12)
(25)
(16)
(13)
(78)
(18)
(26)
(27)
(46)
(47)
(67)
(24)
Genetic Linkage
Mapping a disease Locus
The next step - define the maximal region of linkage
Gene resides
here
Disease gene
Genetic Linkage
Mapping a disease Locus
And then? -> Make a list of the genes within the interval
www.ensembl.org
Genetic Linkage
Mapping a disease Locus
Gene content of chromosome 1
Genetic Linkage
Mapping a disease Locus
And finally-> Find the mutation!
Target candidate genes within the interval by DNA sequencing
Two important considerations for single-gene disorders:
•
Allelic heterogeneity
– different mutations at the same locus (or gene) cause the same disorder.
-> β-thalassemia may be caused by several different mutations in the βglobin gene
•
Locus heterogeneity
– Determination of the same disease or phenotype by mutations at different
loci (or genes)
-> medullary cystic kidney disease (ADMCKD; synonym: medullary cystic
disease, MCD); maybe huntington disease
Genetic Linkage
Mapping a disease Locus
What about mapping polygenic disorders?
Environment
Gene1
Gene 2
Gene 3
PHENOTYPE
Schizophrenia
Asthma
Hypertension (essential)
Osteoarthritis
Type II diabetes (NIDDM)
Cancer
Gene 4
-> Unrelated affected individuals share
ancestral risk alleles
Genetic Linkage
A polygenic phenotype
An affected individual
with unaffected parents
Affected individual joining
the family, emphasizing the
common nature of the disease
-> No clear inheritance pattern
Genetic Linkage
Summary
• Mapping single gene disorders
– Use clues
– If none, genome-wide linkage analysis
– DNA sequence analysis of linked region
• Mapping polygenic disorders
– Model-free genome-wide linkage analysis
– Functional analysis of associated polymorphisms within the refined genomic
interval
Conclusions
•
For a single gene disease identifying the causal mutation is now relatively
straightforward
•
Technological and analytical advances are also making polygenic diseases
tractable
•
Genetics is going to play an ever increasing role in medical diagnosis and in the
development of improved treatment regimes