human molecular genetics 1.examples of genetic diseases in humans 2.meiosis & recombination...

HUMAN MOLECULAR GENETICS

1. Examples of genetic diseases in Humans2. Meiosis & Recombination3. Mendelian Genetics4. Modes of Heredity5. Genetic Linkage Analysis

Role of Genes in Human Disease

• Most diseases -> phenotypes result from the interaction between genes and the environment

• Some phenotypes are primarily genetically determined– Achondroplasia (-> dwarfism)

• Other phenotypes require genetic and environmental factors– Mental retardation in persons with PKU

(polyketonuria)

• Some phenotypes result primarily from the environment or chance– Lead poisoning

Genetic Diseases in Humans

Down syndrome, achondroplasia

100%Environmental

Struck by lightning

Infection

Weight

Cancer

Diabetes

Height100% Genetic

Types of Genetic Disorders:

-> Chromosomes and chromosome abnormalities (Down Syndrome)

-> Single gene disorders (Haemophilia, sickle cell anaemia)

-> Polygenic Disorders (Cancer)



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 syndromeKARYOTYPE


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


Single gene disorders

Neonatal fractures typical of osteogenesis imperfecta, an autosomal dominant disease caused by rare mutations in the type I collagen genes COL1A1 andCOL1A2

A famous carrier of haemophilia A, an X-linked disease caused by mutationin the factor VIII gene

Sickle cell anaemia,an autosomal recessivedisease caused bymutation in the β-globin gene


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


Single gene disorders - Polygenic disorders

Autosomal dominant pedigreeAutosomal dominant pedigree Polygenic disease pedigreePolygenic disease pedigree

Male, Male, affectedaffected

Female, Female, unaffectedunaffected

DNAa b c

genes

unreplicated pair of homologs

•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.

Meiosis & Genetic Recombination

Chromosomes & Genes


Chromosomes & Genes


Chromosome structure

unreplicatedchromosome

telomeres

centromere

replicatedchromosome

sisterchromatids

Each chromatid consists of a very long strand of DNA. The DNA isroughly colinear with the chromosome but is highly structured aroundhistones and other proteins which serve to condense its length and

control the activity of genes.

Centromere:A region within chromosomesthat is required for proper segregation during meiosisand mitosis.

Telomeres:Specialized structuresat chromosome endsthat are important for chromosome stability.


Chromosome structure - Homologs

Sisterchromatids

unreplicatedhomologs

replicatedhomologs

Sister chromatids are almost always IDENTICAL (prior to recombination). Homologues may carry different alleles of any given gene.


Cell Devision

Mitosis -> 2n -> 2x 2n (diploid)Goal is to produce two cells that are geneticallyidentical to the parental cell. (somatic cells)

Meiosis -> 2n -> 4x 1n (haploid)Goal is to produce haploid gametes from adiploid parental cell. Gametes are geneticallydifferent from parent and each other.


Cell Devision -> Mitosis - Meiosis

2n 4n

2n

Mitosis

In mitosis the homologs do not pair up. Rather they behave independently. Each resultant cell receives one copy of each homolog.

2n 4n

2n 1n

I

II

Cross-over

In meiosis the products are haploid gametes so two divisions are necessary. Prior to the first division, the homologs pair up (synapse -> cross-over) 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/perfect linkage

P L

p l

P L

p l

P L

p l

P L

P L

p l

p l

P L

p l

p l

P L

onlyparental-type

gametes


Meiosis/with recombination

P L

p l

P L

p l

P L

P l

p L

p l

P l

p L

p l

P L

In some meiotic divisions these recombination events between the genes will occur resulting in recombinant gametes -> contributes to variation (evolution)

Meiotic recombination in a grasshopper

Meiosis is not conservative, rather it promotes variation through segregation of chromosomes and recombination

Mendelian GeneticsThe 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

TallP

Dwarfx

F1All Tall

Phenotype

Clearly Tall is Inherited…What happened to Dwarf?

F1 x F1 = F2

F23/4 Tall1/4 Dwarf -> Phenotype: 3:1

Dwarf is not missing…just masked as “recessive” in a diploid state

1. Tall is dominant to Dwarf

2. Use D/d rather than T/t for symbolic logic

DD dd

Dd

Genotype

HomozygousDominant

HomozygousRecessive

Heterozygous

DwarfDwarfdddd

TallTallDdDddd

TallTallDdDd

TallTallDDDDDD

ddDDPunnett Square:

possible gametes

possible gametes


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.


2. The Law of Independent AssortmentMembers 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

3:1

9:3:3:1

http://en.wikipedia.org/wiki/Genetic_linkage


After rediscovery of Mendel’s principles, an early task was to show that they were true for

animals

And especially in humans


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!!


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


Standard symbols used in pedigrees:

carrier

”inbreeding”

Modes of HeredityAutosomal 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


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


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

-> 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

These are likely to be more deleterious than dominant disorders, and so are usually very rare

The usual mating is:

Aa x Aa

Autosomal RecessiveModes of Heredity

Autosomal RecessiveModes of Heredity

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)

Dominant vs. RecessiveModes of Heredity

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.

-> 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,…..

X-Linked RecessiveModes of Heredity

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

Other sex-linked diseaseModes of Heredity

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

Exceptions to Mendelian Inheritance

Modes of Heredity

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


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


Why map and characterize disease genes?Why map and characterize disease genes?

Can lead to an understanding of the molecular basis of the diseaseCan lead to an understanding of the molecular basis of the disease

May suggest new therapiesMay suggest new therapies

Allows development of DNA-based diagnosisAllows development of DNA-based diagnosis - including pre-symptomatic and pre-natal diagnosis - including pre-symptomatic and pre-natal diagnosis

Linkage analysis

The mapping of a trait on the basis of its tendency to be co-inherited with polymorphic markers

Genetic Linkage


First question to ask in a mapping exercise:

-> Are there functional or cytogenetic clues?

Functional Clues

Osteogenesis imperfecta (OI) Collagen IHaemophilia A Factor VIIIHaemophilia B Factor IX

Cytogenetic Clues (structure and function of chromosomes)

Duchenne muscular dystrophy Translocation at Xp21Polyposis coli 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


Example: Sweat Pea Purple & Long

Consider the following pair of genes from the sweet pea that are located on the same chromosome -> linked:

Trait affected Alleles Phenotype

Purple Flower color

p

P purple

red

Long Pollen length L Long

l short

Gene

Purple

Genetic Linkage



Mating type - more clearly reveals what gametes (and how many) were contributed by the F1 generation.

P/P L/L X p/p l/l -> homozygote

F1 P/p L/l X p/p l/l "tester"

F2 ?

-> result can give indication if loci are linked or not

Genetic Linkage


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



-> Calculation of map distance between the P and L genes

gametes zygote phenotype observed P L P/p L/l Purple long 1340 parental type P l P/p l/l purple short 154 recombinant p L p/p L/l red long 151 recombinant p l p/p l/l red short 1195 parental type

2840 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


Build a mapRecombination 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 unitsP-------------------------------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.

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

a

B

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

a

B

b

A

a

B

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)

Genetic LinkageMapping a disease Locus

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)

restriction sites -> markers

Gene of interest

Mapping using molecular markers


Genetic LinkageHuman linkage map

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.

Polymorphic markers -Variable number tandem repeats (VNTRs)


Changes in the numbers of repeated DNA sequences arranged in tandem arrays

ACGTGTACTC

3-repeat allele

4-repeat allele

Polymorphic markers - MicrosatellitesParticular class of VNTR with repeat units of 1-6bp in lengthAlso known as short tandem repeats (STRs) and sometimes as simple sequence repeats (SSRs)The most widely used are the CAn microsatellites

CACACACACACA

CACACACACACACACA

6 (CA) allele

8 (CA) allele

Polymorphic markers - Single nucleotide polymorphisms (SNPs)

a polymorphism due to a base substitution or insertion or deletion of a single base


Practicalities of Linkage Analysis

Chrom. 1

Determine the genotype of each family member for polymorphic markers

across the genome

The genotype for a microsatellite marker on chromosome 1

6 (CA) allele 8 (CA) allele

Paternal copy Maternal copy

* *

-> The individuals genotype for this location is (6 8)

Uninformative and informative meioses

66 66 6 86 8

6 66 6 66 8 8

66 8 8 6 86 8

6 86 8 66 8 8

66 8 8 9 109 10

8 98 9 66 10 10

UninformativeCompletely informative

66 66 6 66 6

6 66 6 66 6 6


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

1

Disease gene

An autosomal dominant

disease for which the gene resides on chromosome 1

But you don’t know that!



Marker Studies

Disease gene

5 6 4 7 2 3

Marker studied

2 3 1 5 4 4

1 5 3 5 6 7 2 4 2 5 2 7


Genotype data for the whole family

((24)4) ((25)5) ((27)7)

((23)3) (16)(16)

(14)(14) ((26)6)

(46)(46)

(34)(34) (13)(13)

(33)(33) (14)(14)

(58)(58) (1(12))

(18)(18)

(13)(13) (78)(78)

(18)(18)

((26)6) (47)(47)

((24)4)(46)(46) (67)(67)

The next step - define the maximal region of linkage

Disease geneDisease geneGene resides Gene resides herehere


And then? -> Make a list of the genes within the interval

www.ensembl.orgwww.ensembl.org


Gene content of chromosome 1



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 β-globin 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


What about mapping polygenic disorders?

Gene1

Gene 2

Gene 3

Gene 4

PHENOTYPE

Environment

SchizophreniaAsthmaHypertension (essential)OsteoarthritisType II diabetes (NIDDM)Cancer

-> Unrelated affected individuals share

ancestral risk alleles

Affected individual joining Affected individual joining the family, emphasizing the the family, emphasizing the

common nature of the disease common nature of the disease

An affected individual An affected individual with unaffected parentswith unaffected parents

A polygenic phenotype

-> 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

Genetic Linkage

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

human molecular genetics 1.examples of genetic diseases in humans 2.meiosis & recombination...

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humansgenetic diseases

linkedgenetic diseases

gene involved1

given gene

autosomal dominant disease

interaction of multiple

xlinked disease