FA 5 Progress Report WV-INBRE - Joan C. Edwards School of ...

Program Director/Principal Investigator (Last, First, Middle): Rankin, Gary O 62sequencing on affected FCH family members and apply a stepwise filtering process to identifyvariants that are most likely to confer susceptibility to the disease. From those families that have nodeleterious LDLR mutations in affected members, we will then select affected individuals for wholeexome sequencing. We will select families with the largest numbers of affected individuals and withmultiple generations of affected members. This would allow for substantial reduction in the numberof coincident variants and for a comparison of variants between the families as means of detectinggenetic heterogeneity.(2a) We will first select pathogenic variants (nonsynonymous SNPs, splice site mutations andcoding regions indels) by comparison to control genomes (e.g. HapMap and dbSNP referencegenomes) as described in [2]. This will remove common variants, i.e. those with allele frequenciesgreater than 5%. Given that the frequency of FCH affected individuals in general populations is~1%, the combined frequency of susceptibility alleles would be ~0.5% under a simple autosomaldominant model. However, since FCH is probably genetically heterogeneous, the actual allelicfrequency of individual susceptibility variants may be much less than 0.5%. Given this, it is unlikelythat exclusion of the common variants will eliminate FCH susceptibility variants.(2b) We anticipate that step 2a will reduce the number of shared variant-containing genes to5000-10,000 based on prior Mendelian studies [24,25]. We will further reduce the number ofvariants by selecting only those variants that are shared by affected members within the samefamily. By selecting more distantly related affected members in a pedigree (e.g. 2° and 3°relatives), we can more efficiently reduce the number of coincident variants that are identical bydescent. This filtering process is based on the study of Ng et al [24,25]). In their study on MillerSyndrome (under an autosomal dominant model), Ng et al [25] were able to reduce the number ofcandidate genes to 83 by filtering for pathogenic variants and for variants shared between twoaffected siblings; by filtering only for sharing among three affected members, the number of sharedgenes was reduced to between 10-26 depending on the compared affected individuals. By using 2°and 3° relatives and 3 or more affected members per family, we anticipate being able to reduce thenumber of candidates to about 10-20. Since the mode of inheritance for FCH is unknown, we willconduct filtering under both autosomal dominant and recessive models. We will assess FCHphenotype using our standard algorithm based on age, sex and race adjusted cutoff values forcholesterol and triglycerides.(2c) We will use the PolyPhen-2 program to predict variants that will be more deleterious to proteinfunction. PolyPhen is a tool which predicts possible impact of an amino acid substitution on thestructure and function of a human protein using physical and comparative considerations(http://genetics.bwh.harvard.edu/pph/). PolyPhen predictions may reduce the candidate genes byruling in variants that are most likely to have an effect on protein function. Ng et al (2000b) usedPolyphen in one of their filtering processes and found that it eliminated DHODH, their top candidategene. They advocated use of alternative filtering strategies including + Polyphen and + unrelatedaffected individualsStage 3. Resequencing of candidate genes in affected and normal relatives. At the completion of allsteps in stage 2, the total number of genes with shared variants should be reduced to between 10and 20. We will confirm the significance of these variants by showing that these variants arepresent in other affected family members and that they are largely absent from unaffected relatives.This confirmatory sequencing will be limited to the variant sequence and ~200bp of flankingsequence and can therefore be effectively conducted on all affected and normal persons from ourFCH families; currently there are 80 affected individuals and 92 normals based on our phenotypealgorithm. In this fashion, we will eliminate coincident variants and focus on the remainder forbiological relevance to FCH. Also, by expanding sequencing to unrelated affected members we cantest for allelic and locus heterogeneity.PHS 2590 (Rev. 06/09)Continuation Format Page