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Discovery and Analysis of Rare Coding Variants for Hipoa by Exome-Sequencing

Osteoarthritis and cartilage(2014)

Erasmus MC

Cited 2|Views37
Abstract
Purpose: It is well established that Osteoarthritis (OA) is to a large extend genetically determined. Over the last years several GWAS studies have identified multiple genetic loci associated with OA. However, only a small proportion of the heritability is explained by these loci. It is anticipated that a part of the unexplained variance must be due to rare variants, possibly unique to the disease under study. These rare variants are missed in a common GWAS study, where only common variants are examined. We here present the first exome-sequencing results done for OA. We focus our analysis on the exome, since variants in the protein-coding part of the genome are more likely to have a functional consequence. Methods: The Rotterdam study is a large prospective study population of over 15.000 individuals ages 45 years and older all living in the Ommoord district of Rotterdam in the Netherlands. The first participants were recruited in 1990, and 4 follow-up visits have been done so far, while continuous monitoring through general practitioners and pharmacies is also in place. All of the participants were deeply phenotyped for osteoarthritis by radiographs and questionnaires. For 1,524 participants, whole exome-sequencing has been performed at a mean coverage of 40x per base. This allows to find novel rare variants, not identified previously. We aim to identify functional variants associated with hipOA and its endophenotype, minimal joint space width (mJSW) a proxy for cartilage thickness in the hip joint. Our approach included a candidate gene approach for all genes previously found associated with hipOA. In addition, we conducted exome-wide association analysis on all variants found (199 cases, Kellgren-Lawrence grading ≥2; 1337 controls) and mJSW (minimal cartilage thickness in mm) as the phenotypes of interest. Results: We analyzed in total 199 hipOA cases and 1337 controls, while we had in total 1242 individuals available with data for mJSW. To investigate if other coding variants associated to hipOA and mJSW might be located in the previously identified hipOA genes, we analyzed a group of 13 candidate genes in our exome sequencing data (see table 1). This list includes also 3 genes we identified to be associated to mJSW with a GWAS meta-analysis (FGFR3, RUNX2, and TGFα). We identified 761 variants in these genes, from which the majority was found only once (576 singletons). Of the remaining variants, 70 were non-synonymous, 86 synonymous and 52 intronic; these variants (excluding the singletons) were analyzed further. We next performed gene-based rare-variant tests on these hipOA genes, by comparing the burden, i.e. amount of rare variants, in OA cases compared to controls. In 3 candidate genes a nominal significant hit was found (FGFR3, TGFα and NCOA3), after Bonferroni correction only FGFR3(p = 8.68E-05) remained highly significant. The single-variant analysis yielded 3 signals with a strong association to the mJSW or hipOA in the exome sequencing data set (p < 10E-2, see table 2A). The strongest signal for mJSW was a rare variant in the FGF3 gene, the same gene also identified in the gene specific burden test. This rare variant was not associated to the GWAS signal identified before by us (r2 < 0.1). FGFR3 has previously been shown to be involved in endochondral bone formation. In addition, mutations in FGFR3 result in achondroplasia. Conclusions: We identified rare variants in the FGF3 gene to be associated with extreme values of mJSW. Currently we are working on replication of these findings. Future analysis will consist of further functional assessment of the identified variants.Table 1Variants found in OA candidate genes in the Rotterdam Study – 1 cohortCandidate OA geneVariantsSingletonsSynonymousNonsynonymousIntronicASTN2774911125DIO21813410DOT1L117897813FGFR3724711410FILIP14127671GLT8D11614101GNL325182232MCF2L12686161410NCOA3122107483PIK3R121010PTHLH1312001RUNX21412131SENP6118101395 Open table in a new tab Table 2Variants associated to mJSW and hipOA in the Rotterdam Study – I cohortA. Single-variant analysisDiscoveryVariantChrPositionReference alleleVariant alleleGeneTypePhenotypeFrequencySEPNrs3U5898†‡41807922GAFGFR3IntronicmJSW0.00990.18964.92E-061006rs1388364569119568036GAASTN2SynonymousmJSW0.00270.34355.01E-031097rs114754024†‡41806044CTFGFR3IntronichipOA*0.00830.50067.90E-031082*Kellgren-Lawrence Grading ≥2†Some intronic/intergenic regions are present in exome-sequencing data‡Variants not in linkage disequilibrium r2 < 0.1 Open table in a new tab Tabled 1B. Candidate gene Burden testGeneChrPositionVariantsPhenotypePFGFR341801219 – 180792217mJSW/HipOA*8.68E-05TFGα270677994 – 706779941mJSW0.00786678NCOA32046256424 - 462800319HipOA0.0163782*Kellgren-Lawrence Grading ≥2 Open table in a new tab *Kellgren-Lawrence Grading ≥2 †Some intronic/intergenic regions are present in exome-sequencing data ‡Variants not in linkage disequilibrium r2 < 0.1 *Kellgren-Lawrence Grading ≥2
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