Molecular medicine - 2

Example of identifying a monogenic condition by positional cloning

cystic fibrosis caused by mutations in the CF gene

Most common severe autosomal recessive

condition among Caucasians.

About 5% of white Caucasians of European

descent are asymptomatic carriers.

Frequency of 1 / 2,500 affecting approximately

30,000 people

In 1985, CF locus was localized on the long arm of chromosome 7

In 1989, the gene implicated in CF was isolated (Kerem 1989; Riordan 1989; Rommens 1989).

Pathology

Woe to that child which when kissed on the

forehead tastes salty. He is bewitched and soon

must die

CF from gene to product

CF encodes a Cl- channel (is caused by defects in the CF gene which results in either a decrease in its Cl- transport capacity or its level of cell surface expression

CF gene encodes a cystic fibrosis transmembrane conductance regulator

The genetic analysis showed that this gene, which is responsible for this disorder, contains 24 exons spreading over 250 kb of chromosome 7 (7q31) and encodes an mRNA of 6.5 kb.

CFTR – cystic fibrosis transmembrane

conductance regulator

member of ATP binding cassette (ABC) membrane transporter superfamily

2 homologous halves -1480 amino acids longeach half has 6 transmembrane domains (M1-12) &1 nucleotide binding domain (NBD) which are linked bya cytoplasmic regulatory domain (R-domain) that contains

phosphorylation sites

CFTR function

http://www.infobiogen.fr/services/chromcancer/IntroItems/Images/CFTREnglFig2.jpg

epithelial Cl- transport Cl- transport rate determined by activation of CFTR which in turn depends on its state of phosphorylation.

Acts as a regulator of other channels & transporters e.g CFTR mediates cAMP regulation of amiloride sensitive epithelial Na+ channels (EnaCs)

CFTR channel

Minimum channel diameter – 5.3A

Maximum channel diameter - 10-13A

Charge selectivity: R352, cytoplasmic end of M6

Overall structure: Channel with a large

extracellular vestibule which narrows towards the cytoplasmic end where the anion selectivity filter is located.

Channel lining is formed by M1, M3, M6 & M12 segments.

J Biol Chem (2000) vol 275 No 6 pp 3729 by MH Akabas

Regulation of CFTR gating

phosphorylation: necessary to activate the channel. The R domain contains phosphorylation sites for cAMP-dependent protein kinase A (PKA), C (PKC) and type II cGMP dependent protein kinases. CFTR deactivation mediated by phosphatases PP2C & PP2A.

ATP binding & hydrolysis: Opening / closing of channel controlled by ATP binding &

hydrolysis which occurs in the NBD segment. The R domain interacts with NBD & regulates their ATP affinity.

2 processes control Cl- movement

Spectrum of CF mutations that affect function

F508

70% of CF patients show a specific deletion F508 single amino acid (F) deletion in exon 10 which codes for first portion of NBD-1 of the CFTR protein. This leads to misfolding of CFTR in the endoplasmic reticulum(ER). These immature CFTR proteins are then polyubiquinated & targeted for proteosome degradation

Mapping of CFTR

1985 gene for CF linked to enzyme paraoxanase (PON)PON mapped to chromosome 7 and CF mapped to 7q31-32

(random DNA marker D7S15)2 flanking markers established (~2x106bp apart)

proximal MET oncogene and distal D7S8extensive mapping and characterisation around the candidate

region by chromosome walking, chromosome jumping and microdissection (~300kbp cloned)

CFTR candidate region

Mapping of CFTR

2 new markers identified – KM19 and XV2c – which showed strong linkage disequilibrium5’ end of gene locatedBovine equivalent of candidate gene isolated from genomic library7 cDNA libraries screened with human clone. 1 cDNA clone identified. Northern blots show 6.5 kb mRNARest of the gene obtained by screening and PCR1989 CFTR gene eventually isolated by mutation screening

Letter to Dr. Collins. Courtesy of the National Human Genome Research Institute

The spectrum of human diseases

Cystic fibrosis thalassemia

Huntington’s

cancer

<5%

‘Mendelian’ diseases (<5%)

Autosomal dominant inheritance: e.g huntington’s disease

Autosomal codominant inheritance e.g Hb-S sickle cell disease

Autosomal recessive inheritance: e.g cystic fibrosis, thalassemias

X-linked inheritance: e.g Duchenne muscular dystrophy (DMD)

Mapping complex loci

PAF – population attributable factor:

Fraction of the disease that would be eliminated if the

risk factor were removed

High PAF for single gene conditions (>50% for CF)

Low PAF for complex disease (< 5% for Alzheimer’s)

Identifying genes involved in complex diseases

Steps

Perform family, twin or adoption studies - check for genetic component

Segregation analysis- estimate type and frequency of susceptibility alleles

Linkage analysis- map susceptibility loci

Population association- identify candidate region

Identify DNA sequence variants conferring susceptibility

Linkage versus Assocciation

Association studies compare the allele frequency of a polymorphic marker, or a set of markers, in unrelated patients (cases) and healthy controls to identify markers that differ significantly between the two groups.

Used to identify common modest-risk disease variants

Higher density of markers needed

e.g. HapMap uses association data

Linkage analyses search for regions of the genome with a higher-than-expected number of shared alleles among affected individuals within a family.

Used to identify rare high-risk disease alleles

<500 markers needed for initial genome scan

Haplotype analysis• specific combination of 2 or more DNA marker

alleles situated close together on the same chromosome (cis markers)

• SNPs most commonly used markers in haplotypes.

• series of closely linked mutations accumulate over time in the surviving generation derived from a common ancestor.

• powerful genetic tool for identifying ancient genetic relationships.

• Alleles at separate loci that are associated with each other at a frequency that is significantly higher than that expected by chance, are said to be in linkage disequilibrium

Direct versus indirect association analysis.

a, In direct association analysis,all functional variants (red arrows) are catalogued and tested for association with disease. A GeneSNPs image of the CSF2 gene is shown. Genomic features are shown as boxes along the horizontal axis (for example, blue boxes indicate exons). Polymorphisms are shown as vertical bars below the axis, with the length of the line indicating allele frequency and colour indicating context (for example, red indicates coding SNPs that change amino acids). b, For indirect association analysis, all common SNPs are tested for function by assaying a subset of tagSNPs in each gene (yellow arrows), such that all unassayed SNPs (green arrows) are correlated with one or more tagSNPs. Effects at unassayed SNPs (green arrows) would be detected through linkage disequilibrium with tagSNPs. Images adapted from GeneSNPs (http://www.genome.utah.edu/genesnps).

Formation of haplotypes over time

Ancient disease loci are associated with haplotypes

• Start with population genetically isolated for a long time such as Icelanders or Amish

• Collect DNA samples from subgroup with disease• Also collect from equal number of people without

disease• Genotype each individual in subgroups for

haplotypes throughout entire genome• Look for association between haplotype and

disease phenotype• Association represents linkage disequilibrium• If successful, provides high resolution to narrow

parts of chromosomes

Haplotype analysis provides high resolution gene mapping

Genetic heterogeneityMutations at more than one locus cause

same phenotype

e.g. thalassemias – Caused by mutations in

either the or -globin genes.

– Linkage analysis studies therefore always combine data from multiple families

Why is it still so difficult?

Variable expressivity - Expression of a mutant trait differs from person to person

• Phenocopy– Disease phenotype is not caused by any

inherited predisposing mutation – e.g. BRCA1 mutations

• 33% of women who do not carry BRCA1 mutation develop breast cancer by age 55

Incomplete penetrance – when a mutant genotype does not always cause a mutant phenotype• No environmental factor associated with

likelihood of breast cancer• Positional cloning identified BRCA1 as

one gene causing breast cancer.– Only 66% of women who carry BRCA1

mutation develop breast cancer by age 55

• Incomplete penetrance hampers linkage mapping and positional cloning– Solution – exclude all nondisease individuals

form analysis– Requires many more families for study

• Polygenic inheritance– Two or more genes interact in the

expression of phenotype• QTLs, or quantitative trait loci

– Unlimited number of transmission patterns for QTLs» Discrete traits – penetrance may increase with

number of mutant loci» Expressivity may vary with number of loci

– Many other factors complicate analysis» Some mutant genes may have large effect» Mutations at some loci may be recessive while

others are dominant or codominant

Polygenic inheritance

E.g heart attacks or cholesterol levels

Sudden cardiac death (SCD)

Breast cancer

Although a genetic basis for familial BC identified, the causes of sporadic disease still unknown

Sudden cardiac death (SCD)

Mutations in 2 loci account for 20-25% of early onset (<45 years) breast cancer cases due to inherited factors– BRCA1: mutations found in 80-90% of families

with both breast and ovarian cancer– BRCA2: mutations mainly in male breast

cancer families

Common condition – familial or sporadic forms

Alzheimer’s disease

familial AD – mutations in APP, presenilin-1 and 2Sporadic AD – strong association with APO4, Apolipoprotein 4, which

affects age of onset rather than susceptibility

Sudden cardiac death (SCD)

Affects 5% of people >65 years and 20% of people over 80 has familial (early-onset) or sporadic (late-onset) forms, although

pathologically both are similarAetiology of sporadic forms unknown

3 major alleles (APO E2, E3, and E4)

Position

112 158

ApoE2 Cys Cys

ApoE3 Arg Cys

ApoE4 Arg Arg

Epigenetics – differential imprinting

failure to thrive during infancy, hyperphagia and obesity during early childhood, mental retardation, and behavioural problems

molecular defect involves a ~2 Mb imprinted domain at 15q11–q13 that contains both paternally and maternally expressed genes

Prader-Willi syndrome Angelman syndrome

births and characteristics include mental retardation, speech impairment and behavioural abnormalities

AS defect lies within the imprinted domain at 15q11–q13

Genetic causes

70% have a deletion of the PWS/AS region on their paternal chromosome 15

25% have maternal uniparental disomy for chromosome 15 (the individual inherited both chromosomes from the mother, and none from the father)

5% have an imprintingdefect

<1% have a chromosome abnormality including the PWS/AS region

Prader-Willi syndrome Angelman syndrome70% have a deletion of the PWS/AS region on their maternal chromosome 15

7% have paternal uniparental disomy  for chromosome 15 (the individual inherited both chromosomes from the father, and none from the mother)

3% have an imprinting defect

11% have a mutation in UBE3A

1% have a chromosome rearrangement

11% have a unknown genetic cause

Reading

HMG3 by T Strachan & AP Read : Chapter 15

AND/OR

Genetics by Hartwell (2e) chapter 11 References on Cystic fibrosis: Science (1989) vol 245 pg 1059 by JM Rommens et al (CF mapping)J. Biol Chem (2000) vol 275 No 6 pp 3729 by MH Akabas (CFTR)

Optional Reading on Molecular medicine Nature (May2004) Vol 429 Insight series• human genomics and medicine pp439 (editorial)• Mapping complex disease loci in whole genome

studies by CS Carlson et al pp446-452