Molecular medicine - 2

1. Molecular medicine - 2

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

3. Example of identifying a disease allele by positional cloningCystic fibrosis

4. Pathology ‘Woe to that child which when kissed on the forehead tastes salty. He is bewitched and soon must die’

5. Example of identifying a disease allele by positional cloningCystic fibrosis Severe autosomal recessive condition among Caucasians. 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).

6. CF gene encodes a cystic fibrosis transmembrane conductance regulator (CFTR Cl- channel) CFTR is a Cl- channel (defects result in either a decrease in its Cl- transport capacity or its level of cell surface expression)

7. http://www.infobiogen.fr/services/chromcancer/IntroItems/Images/CFTREnglFig2.jpghttp://www.infobiogen.fr/services/chromcancer/IntroItems/Images/CFTREnglFig2.jpg CFTR function • 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)

8. 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

9. Mutations in CFTR

10. 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 D7S8 • extensive mapping and characterisation around the candidate region by chromosome walking, chromosome jumping and microdissection (~300kbp cloned)

11. CFTR candidate region

12. Mapping of CFTR • 2 new markers identified – KM19 and XV2c – which showed strong linkage disequilibrium • 5’ end of gene located • Bovine equivalent of candidate gene isolated from genomic library • 7 cDNA libraries screened with human clone. 1 cDNA clone identified. Northern blots show 6.5 kb mRNA • Rest of the gene obtained by screening and PCR • 1989 CFTR gene eventually isolated by mutation screening

13. linkage disequilibrium Alleles at 2 or more loci that show a non-random association are said to be in linkage disequilibrium. Conclusive evidence defective cAMP-dependent chloride conductance in CFTR-/- cells was restored when CFTR cDNA was transfected and expressed in those cells.

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

15. “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)

16. ChallengesSome of the complexities of human disease traits • Phenotypic heterogeneity • Phenocopies • Variable expressivity • Incomplete penetrance • Polygenic traits

17. Phenotypic heterogeneity Same genotypic mutation causes variable phenotypes e.g. a/b-thalassemias • Caused by mutations in either the a or b-globin genes. • Similar genotype can lead to unaffected or severe phenotypes

18. Many mechanisms contribute to the phenotypic heterogeneity of thalassaemias

19. Phenocopy 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

20. Expression of a mutant trait differs in individuals Variable expressivity

21. Incomplete penetrance – when a mutant genotype does not always cause a mutant phenotype • 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

22. Polygenic traits 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

23. Alzheimer’s disease 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 similar etiology of sporadic forms unknown familial AD – mutations in APP, presenilin-1 and 2 Sporadic AD – strong association with APOe4, Apolipoprotein e4, which affects age of onset rather than susceptibility 3 major alleles (APO E2, E3, and E4) Sudden cardiac death (SCD)

24. 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

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

26. 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)

27. 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

28. Linkage versus Association 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 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

29. 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

30. 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)