1. Molecular medicine - 2

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

3. 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).

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

5. 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

6. 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.

7. CFTR – cystic fibrosis transmembrane conductance regulator member of ATP binding cassette (ABC) membrane transporter superfamily 2 homologous halves -1480 amino acids long each half has 6 transmembrane domains (M1-12) & 1 nucleotide binding domain (NBD) which are linked by a cytoplasmic regulatory domain (R-domain) that contains phosphorylation sites

8. 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)

9. 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

10. 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

12. Spectrum of CF mutations that affect function

13.  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

14. 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 (~2x10 6 bp apart) proximal MET oncogene and distal D7S8 extensive mapping and characterisation around the candidate region by chromosome walking, chromosome jumping and microdissection (~300kbp cloned)

15. CFTR candidate region

16. 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

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

18. The spectrum of human diseases Cystic fibrosis thalassemiaHuntington’s cancer <5%

19. ‘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)

20. 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)

21. 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

22. 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

23. 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

24. 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).

25. Formation of haplotypes over time

26. 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

27. Haplotype analysis provides high resolution gene mapping

28. Genetic heterogeneity Mutations 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?

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

30. 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

31. 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

32. 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

33. Polygenic inheritance E.g heart attacks or cholesterol levels Sudden cardiac death (SCD)

34. 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

35. Alzheimer’s disease familial AD – mutations in APP, presenilin-1 and 2 Sporadic AD – strong association with APO  4, 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 similar Aetiology of sporadic forms unknown 3 major alleles (APO E2, E3, and E4)

36. 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

37. 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 imprinting defect <1% have a chromosome abnormality including the PWS/AS region Prader-Willi syndromeAngelman syndrome 70% 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

38. 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