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

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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
Program Director/Principal Investigator (Last, First, Middle): Rankin, Gary O 63We note that a small fraction of affected members may not share a candidate variant becausethese individuals may be phenocopies (individuals who have the disease because of environmentalbut not genetic causes), or have inherited an alternative susceptibility variant from a married-inindividual. Further, some of the unaffected relatives could have a candidate variant because theyare nonpenetrant carriers.Outcomes: FCH is thought to be an oligogenic disorder. By sequencing the exomes of three ormore 2° or 3° individuals in a given family, we should be able to identify the major susceptibilitygenes. Further by sequencing the exomes of several unrelated families, we may find alternative oroverlapping sets of susceptibility genes.Confirmation of causality: Identification of one or more FCH candidate susceptibility genes by thisfiltering process should be viewed with some caution. Some initial confirmation can be achieved byreview of the candidate gene’s metabolic function and its relevance to the dyslipidemic phenotype.Long term studies should include replication of the FCH phenotype in animal models containing theappropriate variant transgenes. Funds for the animal studies are not budgeted and would need to bepart of a future proposal.Alternatives to whole exome sequencing approachWhole exome sequencing is an efficient strategy for discovering the genetic causes of Mendeliandisorders given that (1) most known variants disorders disrupt protein coding sequences, (2) alarge fraction of rare variants are predicted to be deleterious and (3) there are now ten Mendeliandisorders that have been solved by exome sequencing [8, 9 and J. Shendure, Illumina Webinar2010). Although a number of investigators now view whole exome approaches as viable means offinding susceptibility in polygenic and multifactorial disorders, it is possible that we will not findappropriate candidates because (1) no variants are identified that are shared by a preponderanceof the affected members or (2) we are not able to reduce the number of candidates to anappropriate size. In the former case, the failure to identify shared genes may stem from thepresence of susceptibility variants in promoters or regulatory sequencers. Whole genomesequencing could be used to identify those variants but the cost would be prohibitive (at least at thistime). Potential solutions would include seeking additional funding or finding a collaborator who iswilling to absorb the cost. In the latter case, we may employ linkage analysis on single families toidentify candidates of interest. We may also take steps to merge our exome data with other studiesin an effort to reduce the pool of variants to a manageable number.4. Materials and Methods:Human Subject Enrollment and FCH Phenotypes:We recruited FCH families who met adult inclusion criteria established by previousinvestigations or our criteria for FCH families with a juvenile proband. Human subjects wereenrolled according to approved Institutional Review Board (IRB) protocols from the following WVhospitals or clinics: Charleston Area Medical Center, the Department of Adolescent Medicine at theUniversity of Kentucky (UK), WVU Department of Pediatric Cardiology and Valley Health Systems.FCH families were recruited by two primary mechanisms: (1) The Coronary Artery Risk DetectionIn Appalachian Communities (CARDIAC) Project is a population-based and high-risk individualizedprogram designed to raise awareness of CAD risk factors in the community and identify individualsat high risk for premature CAD. Dr. William Neal, CARDIAC program director, contacted adultsfrom prospective families through the WV lipid clinics to initiate the enrollment process. Thisrecruiting mechanism mainly provided families with juvenile probands. (2) We also recruited FCHfamilies through Charleston Area Medical Center (CAMC), UK and Valley Health System (VHS).Prospective FCH probands were identified by chart review or by physician referral. This processprovided both adult and juvenile FCH families.FCH families were ascertained by identifying probands with one of two phenotypes.PHS 2590 (Rev. 06/09)Continuation Format Page
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