Molecular medicine - 2

Key concepts

Positional cloning in identifying disease alleles

(an example – cystic fibrosis)

Majority of human diseases have polygenic or

multi-factorial risk factors.

Challenges and approaches in the post genomic

era

Example of identifying a disease allele by positional cloning

Cystic fibrosis

Pathology ‘Woe to that child which when kissed on the forehead tastes salty. He

is bewitched and soon must die’

Example of identifying a disease allele by positional cloning

Cystic fibrosis

cystic fibrosis caused by mutations in the CF gene

5% of Caucasians are asymptomatic carriers. Frequency of 1 / 2,500 (~ 30,000)

CF disease locus identified on chromosome 7q 31.2(Kerem 1989; Riordan 1989; Rommens 1989).

Severe autosomal recessive condition among Caucasians.

CFTR is a Cl- channel (defects result 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 (CFTR Cl- channel)

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)

Mutations in CFTR

70% of CF patients show a specific deletion F508 deletion in exon 10 (F): NBD-1 domain CFTR misfolding in the ER and targeted for proteosome degradation

Mutations in CFTR

Mapping of CF allele

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

linkage disequilibrium Alleles at 2 or more loci that show a non-random

association are said to be in linkage disequilibrium.Allelic association in cystic fibrosisMarker alleles CF Normal

chromosomes chromosomesX1,K1 3 49X1,K2 147 19X2,K1 8 70X2,K2 8 25

RFLP markers XV2C (X1,X2) and KM19 (K1,K2)

Conclusive evidence defective cAMP-dependent chloride conductance in CFTR-/- cells was restored when CFTR cDNA was transfected and expressed in those cells.

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

“For any given trait there will be few (if any) large effects, a handful of modest effects, and a substantial number of genes generating small or very small increases in disease risk.”

Nature 447, 661–678 (2007)

ChallengesSome of the complexities of

human disease traits

• Phenotypic heterogeneity • Phenocopies • Variable expressivity• Incomplete penetrance• Polygenic traits

Same genotypic mutation causes variable phenotypes

e.g. thalassemias – Caused by mutations in either the or -globin

genes. – Similar genotype can lead to unaffected or severe

phenotypes

Phenotypic heterogeneity

GENOTYPE PHENOTYPE a+ a+ a+ a+ Normal a+ a a+ a+ Silent carrier asymptomatic condition. thalassaemia - 2 a+ a a+ a -thalassaemia trait minor anaemic conditions a+ a+ a a a+ a a a HbH mild – moderate anaemia a a a a Hydrops foetalis foetus survives until around birth

Many mechanisms contribute to the phenotypic heterogeneity of

thalassaemias

Disease phenotype is not caused by any known inherited predisposing mutation e.g. BRCA1 mutations

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

Phenocopy

Variable expressivityExpression of a mutant trait differs in individuals

Incomplete penetrance

• 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

– when a mutant genotype does not always cause a mutant phenotype

Two or more genes interact in the expression of phenotype e.g. cancer• QTLs, or quantitative trait loci

– Penetrance / expressivity may vary with number of mutant loci

– Some mutant genes may have large effect– Mutations at some loci may be recessive while

others may be dominant or codominant

Polygenic traits

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 similaretiology of sporadic forms unknown

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

Position

112 158

ApoE2 Cys Cys

ApoE3 Arg Cys

ApoE4 Arg Arg

Apart from SNPs, structural variants such as CNVs may explain some of these

complexities

• Changes in copy number may directly affect risk factor

• Rearrangements / fusion may alter expression

• CNVs could increase risk of secondary pathogenic rearrangements

• CNVs could indirectly affect environmental interaction leading to different phenotypes

What approaches should be used in the post-genomic era?

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

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 Association

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 Map (HapMap)• Haplotype: specific combination of 2 or more DNA marker alleles situated

close together on the same chromosome (cis markers). E.g. SNPs• HapMap - catalog of common genetic variants in populations• International HapMap Project - identify common haplotypes in four

populations with African, Asian, and European ancestry• provide information to link genetic variants to the risk of disease

Reading

HMG3 by T Strachan & AP Read : Chapter 14

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)

Nature Vol 437|27 pp1241-42 October 2005 (HapMap Project)

Nature (Oct 2007) VOLUME 82 NUMBER 4 pp 366-70 (CNVs)