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 66Family Affected/normal members115001 8 affected + 1 normal115021 3 affected + 1 normal115027 9 affected + 1 normal115028 4 affected + 2 normal116003 5 affected + 2 normal117005 4 affected + 1 normalTable 2. Numbers of individuals for whom exomes have been sequenced. *Some normals arerelatives and others are married in.To date, we have performed discrete filtering on family 115027. We focused our efforts on thisfamily because of the large numbers of affected members. Our filtering process considered totalSNPs and indels and contained the following stages:(1) Inclusion of all variants present in an exome but absent from the reference human genome(hg19)(2) Inclusion of all variants present in all affected members and exclusion of all variants present inone normal relative and one unrelated normal.(3) Exclusion of all variants with allele frequencies greater than 5% or 15%. (Given an autosomaldominant mode of inheritance, an affected genotype frequency of 5% (AA and Aa) and HardyWeinberg equilibrium, the mutant allele frequency would be ~2.5%.)In the filration process, we assumed that (1) the trait was transmitted as an autosomal dominantwith complete penetrance, (2) all affected members have carry the mutation, and (3) the hg19reference genome does not have the FCH mutation. After stage (1), we found an average of231,213 SNPs and 12,273 indels per family member. Retention of SNPs present in all affectedmembers and removal of SNPs present in the two normals reduced the number of SNPs to 624and indels to 47 (stage 2). When we eliminate SNPs whose allele frequency is less than 5% or15%, total SNPS are reduced to 29 or 70 SNPS, respectively (stage 3). (Indel allele frequencieshave been obtained at this time. Table 3 shows the number of shared variants after each stageVariants per stage Filter SNPS IndelsStage 1 Total (mean per subject) 231,213 12,273Stage 2 Filtered by affected-normal 624 47Stage 3 Filtered by MAF = 15% 70 NATable 3. Variants present after each filtration stage.In order to identify pathogenic SNPs, we categorized the variants as shown in Table 4. Many of thevariants were located in intronic regions or unknown (uncharacterized) loci. At the 15% minor allelefrequency cutoff, two missense variants mapped to uncharacterized open reading frames onchromosomes 1 and 17.Variant Type Minor Allele Frequency5% 15%synonymous coding SNPs 1 4intronic 8 30 (1 in ACLY in splice site)missense 0 2 (uncharacterized ORFs)Near 3’ end of gene 1 2Near 5’ end of gene 1 1Unknown (1 on Y chromosome)11 133’ untranslated region 2 13PHS 2590 (Rev. 06/09)Continuation Format Page
Program Director/Principal Investigator (Last, First, Middle): Rankin, Gary O 675’ untranslated region 2 2novel SNPs 3 3total SNPs 29 70Table 4. Types of shared variants with allele frequencies 5% or 15%. SNP counts include those forwhich an allele frequency has not been determined. Novel SNPs are those that do not appear in thedbSNP database.Examination of the post stage 2 set of variants showed that none of the 625 variants mapped togenes that are known to cause either FH or FCH (Table 5).Gene Chromosome location (bps) Family 115027Variants Present in Gene Interval?FHLDLR 19 11,200,038-11,244,508 NOapoB 2 21,224,301-21,266,945 NOPCSK9 1 55,505,149-55,530,526 NOFCHUSF1 1 161,009,041-161,015,757 NOLPL 8 19,796,582-19,824,778 NOapoA1 11 116,706,469-116,708,338 NOTable 5. Presence of Variants in Known FH and FCH Causal Genes.6. Discussion:We have pursued a whole exome strategy to identify genes that confer susceptibility to FCH. Wehave successfully sequenced the whole exomes of 41 individuals and tested a variant discoverystrategy on one FCH Family (1150027). Our strategy at a 15% allele frequency cutoff reduced thenumber of possible variants to 70 which 45 correspond to known genes. We have reviewed thefunctions of genes from variants which result in missense or possible changes in conserved splicesites. The missense mutations correspond to uncharacterized open reading frames onchromosomes 1 and 17. Severity of these missense mutations and potential biological function ofthese ORFs must be assessed. One variant in the ATP-citrate lyase (ACLY) gene is located near asplice donor site. This enzyme catalyzes the formation of acetyl-CoA and oxaloacetate from citrateand CoA with a concomitant hydrolysis of ATP to ADP and phosphate. The product, acetyl-CoA,serves several important biosynthetic pathways, including lipogenesis and cholesterogenesis. ACLYexpression and activity is known to be suppressed by exogenous lipids (GeneCard database).Although effect of this mutation on ACLY mRNA splicing is unknown, ACLY may play a role inregulating the abundance of serum cholesterol and triglycerides.No SNP or indel variants mapped to the 3 known FH and 3 known FCH gene. This reduces thelikelihood that defects in these genes could be the cause of FCH in Family 11502 but does noteliminate it completely since deleterious mutations may be located in non-exonic regions of thesegenes. A more rigorous analysis of variants from all FCH families is under way.7. Literature Cited1. D Lloyd-Jones, RJ Adams, TM Brown, M Carnethon, S Dai, G DeSimone, TB Ferguson, E Ford,K Furie, C Gillespie, A Go, K Greenlund, N Haase, S Hailpern, PM Ho, V Howard, B Kissela, SKittner, D Lackland, L Lisabeth, A Marelli, MM McDermott, J Meigs, D Mozaffarian, M Mussolino, GNichol, VL Roger, W Rosamond, R Sacco, P Sorlie, R Stafford, T Thom, S, Wasserthiel-Smoller,ND Wong, J Wylie-Rosett and on behalf of the American Heart Association Statistics Committeeand Stroke Statistics Subcommittee. Heart Disease and Stroke Statistics 2010 Update: A Reportfrom the American Heart Association. Circulation 121: e46-e215 (2010)2. A Gaddi, AFG Cicero, FO Odoo, A Poli, R Paoletti. Practical Guidelines for familial combinedPHS 2590 (Rev. 06/09)Continuation Format Page
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