Rare SNPs Research Articles

Functional relevance of synonymous alleles reflected in allele rareness in the population

We provide theoretical evidence supporting the non-neutrality of synonymous alleles by investigating the rareness of synonymous alleles in the population. We find a significantly greater number of synonymous rare alleles than conventional neutral alleles derived from noncoding regions. A permutation experiment shows that the rareness of synonymous alleles is not a byproduct of random statistical noise. We then compare the frequencies of synonymous rare alleles and common alleles in various functional contexts in which synonymous alleles are known to be involved. Subsequently, we perform logistic regression analysis to elucidate the effect size of each independent factor contributing to the rareness of synonymous alleles. Additionally, we show that changes in optimality caused by synonymous mutations resulting in rare SNPs in the population tend to be biased toward optimality loss. We think that our study will contribute to the development of novel strategies for identifying functional synonymous mutations.

Genetic variation of the Toll-like receptors in a Swedish allergic rhinitis case population

BackgroundVariation in the 10 toll-like receptor (TLR) genes has been significantly associated with allergic rhinitis (AR) in several candidate gene studies and three large genome-wide association studies. These have all investigated common variants, but no investigations for rare variants (MAF ≤ 1%) have been made in AR. The present study aims to describe the genetic variation of the promoter and coding sequences of the 10 TLR genes in 288 AR patients.MethodsSanger sequencing and Ion Torrent next-generation sequencing was used to identify polymorphisms in a Swedish AR population and these were subsequently compared and evaluated using 1000Genomes and Exome Aggregation Consortium (ExAC) data.ResultsThe overall level of genetic variation was clearly different among the 10 TLR genes. The TLR10-TLR1-TLR6 locus was the most variable, while the TLR7-TLR8 locus was consistently showing a much lower level of variation. The AR patients had a total of 37 promoter polymorphisms with 14 rare (MAF ≤ 1%) and 14 AR-specific polymorphisms. These numbers were highly similar when comparing the AR and the European part of the 1000Genomes populations, with the exception of TLR10 where a significant (P = 0.00009) accumulation of polymorphisms were identified. The coding sequences had a total of 119 polymorphisms, 68 were rare and 43 were not present in the European part of the 1000Genomes population. Comparing the numbers of rare and AR-specific SNPs in the patients with the European part of the 1000Genomes population it was seen that the numbers were quite similar both for individual genes and for the sum of all 10 genes. However, TLR1, TLR5, TLR7 and TLR9 showed a significant excess of rare variants in the AR population when compared to the non-Finnish European part of ExAC. In particular the TLR1 S324* nonsense mutation was clearly overrepresented in the AR population.ConclusionsMost TLR genes showed a similar level of variation between AR patients and public databases, but a significant excess of rare variants in AR patients were detected in TLR1, TLR5, TLR7, TLR9 and TLR10. This further emphasizes the frequently reproduced TLR10-TLR1-TLR6 locus as being involved in the pathogenesis of allergic rhinitis.

An exome-wide analysis of low frequency and rare variants in relation to risk of breast cancer in African American Women: the AMBER Consortium.

A large percentage of breast cancer heritability remains unaccounted for, and most of the known susceptibility loci have been established in European and Asian populations. Rare variants may contribute to the unexplained heritability of this disease, including in women of African ancestry (AA). We conducted an exome-wide analysis of rare variants in relation to risk of overall and subtype-specific breast cancer in the African American Breast Cancer Epidemiology and Risk (AMBER) Consortium, which includes data from four large studies of AA women. Genotyping on the Illumina Human Exome Beadchip yielded data for 170 812 SNPs and 8287 subjects: 3629 cases (1093 estrogen receptor negative (ER-), 1968 ER+, 568 ER unknown) and 4658 controls, the largest exome chip study to date for AA breast cancer. Pooled gene-based association analyses were performed using the unified optimal sequence kernel association test (SKAT-O) for variants with minor allele frequency (MAF) ≤ 5%. In addition, each variant with MAF >0.5% was tested for association using logistic regression. There were no significant associations with overall breast cancer. However, a novel gene, FBXL22 (P = 8.2×10(-6)), and a gene previously identified in GWAS of European ancestry populations, PDE4D (P = 1.2×10(-6)), were significantly associated with ER- breast cancer after correction for multiple testing. Cases with the associated rare variants were also negative for progesterone and human epidermal growth factor receptors-thus, triple-negative cancer. Replication is required to confirm these gene-level associations, which are based on very small counts at extremely rare SNPs.

Rare SNPs in receptor tyrosine kinases are negative outcome predictors in multiple myeloma.

Multiple myeloma (MM) is a plasma cell disorder that is characterized by a great genetic heterogeneity. Recent next generation sequencing studies revealed an accumulation of tumor-associated mutations in receptor tyrosine kinases (RTKs) which may also contribute to the activation of survival pathways in MM. To investigate the clinical role of RTK-mutations in MM, we deep-sequenced the coding DNA-sequence of EGFR, EPHA2, ERBB3, IGF1R, NTRK1 and NTRK2 which were previously found to be mutated in MM, in 75 uniformly treated MM patients of the “Deutsche Studiengruppe Multiples Myelom”. Subsequently, we correlated the detected mutations with common cytogenetic alterations and clinical parameters. We identified 11 novel non-synonymous SNVs or rare patient-specific SNPs, not listed in the SNP databases 1000 genomes and dbSNP, in 10 primary MM cases. The mutations predominantly affected the tyrosine-kinase and ligand-binding domains and no correlation with cytogenetic parameters was found. Interestingly, however, patients with RTK-mutations, specifically those with rare patient-specific SNPs, showed a significantly lower overall, event-free and progression-free survival. This indicates that RTK SNVs and rare patient-specific RTK SNPs are of prognostic relevance and suggests that MM patients with RTK-mutations could potentially profit from treatment with RTK-inhibitors.

TLR2 and TLR4 polymorphisms in Southern Chinese Psoriasis Vulgaris patients

BackgroundThe toll-like receptors-(TLR)-2 and −4 play a role in the innate immune system and drive the autoimmune T cell cascade in psoriasis. But little is known about the association of the polymorphisms of TLR2/TLR4 and psoriasis vulgaris (PsV). MethodsWe performed a hospital-based association study involved in355 PsV patients and 213 controls. Genotyping of 11 SNPs rs4696480(−16934A/T), rs121917864 (2029C/T;Arg677Trp), rs5743708 (2408G/A;Arg753Gln), rs3804099 (+597T/C), rs5743699(+1349T/C) of TLR2 on chromosome 4q32; and rs1927914 (−6143A/G), rs10759932(−5724T/C), rs4986790 (896A/G; Asp299Gly), rs4986791 (1196C/T; Thr399lle); rs11536889(+7263G/C), rs11536891(+8469T/C) of TLR4 on chromosome 9q33.1 were determined using SNaPshot Multiplex Kit (Applied Biosystems Co., USA). ResultsWe did not detect the presence of any of the five rare SNPs (rs5743708, rs121917864, rs5743699, rs4986790, rs4986791). The comparison of allele distributions revealed that only one SNP (rs3804099) of 6 common SNPs(rs10759932, rs11536889, rs11536891, rs1927914, rs3804099, rs4696480) was significant associated with the risk of PsV (P<0.01; FDR p-Value). However, there were no significant differences found in other subphenotypes of family history or PASI between patients positive and those negative for a particular phenotype. ConclusionsThe SNP rs3804099 of TLR2 may have significant effects on heritability of PsV in our Chinese population. TLR2 and its pathway might take part in the pathogenesis of PsV.

Polymorphism of the LCT gene regulatory region in Turkicspeaking populations of the Altay-Sayan region (southern Siberia)

Retention of lactase activity in adulthood (lactase persistence) is one of the most important adaptive traits for human populations that consume fresh milk from domestic animals. At a molecular-genetic level, lactase persistence is determined by the presence of specific alleles of polymorphic sites in cis-regulatory elements of the LCT gene located on chromosome 2q21. Ascertainment of the molecular-genetic causes of lactase persistence has made this trait one of the most convenient for studying mechanisms of human population adaptation to environmental conditions. But the populations of many regions remain insufficiently investigated in relation to the genetic variability of the LCT loci. This paper presents the results of polymorphism analysis of loci, including the enhancer element for the LCT gene and its flanking regions, in two Turkic-speaking populations from southern Siberia, Altaian Kazakhs and Khakasses. It was found that the “European” allele LCT-13910T is the most characteristic of the Turkic-speaking populations from Altai-Sayan regions among all the polymorphic variants associated with lactase persistence. The expansion of the “European” allele LCT-13910T to the gene pool of the populations in southern Siberia could be related to migration waves of ancient herders form western Eurasia during the Bronze Age (in III – II millennium BC). A decrease of the LCT-13910T allele frequency and the total frequency of its carriers in the Turkic-speaking populations of southern Siberia in comparison with the majority of European populations and the Kazakhs from southern Central Asia can be attributed to: (1) a significant influence on the Altai- Sayan population’s gene pool by Eastern Eurasian populations, for which the LCT-13910T allele is rare; (2) a lesser adaptive significance of lactase persistence for south Siberian populations, compared to the populations of Europe. Rare and unique SNPs in the locus under consideration that were found in the Altaian Kazakhs (LCT-13895G &gt; C and LCT-13927C &gt; G) and Khakasses (LCT-14011C &gt; T) potentially play a role in regulation of LCT gene expression, because they are located within the enhancer, regulating activity of its promoter.

Exome Genotyping Links Venous Thrombosis Risk with the Skeletal Muscle Myosin Gene Cluster and Leads to Discovery of New Family of Procoagulant Factors

Venous thromboembolism (VTE) exacts a severe toll, and its toll persists even after the acute thrombotic event is resolved. Although several low frequency, racially-specific, genetic variants have been linked to VTE (e.g., factor V Leiden, prothrombin nt20210A, protein S Tokushima, Protein C R147W and del-Lys150), these polymorphisms fail to explain the majority of VTE risk. These variants are causally linked to increased VTE risk and, based on their racial-specific occurrence, they likely provided evolutionary benefits. Thus, to discover new rare missense functional variants linked to VTE, we used the new Affymetrix Axiom array to determine ~ 300,000 mostly exomic rare variant polymorphisms in the Scripps venous thrombosis registry (N=214 controls and 107 VTE cases) and gained multiple novel insights. When recurrent VTE cases were analyzed, 34 SNPs, including two F5 SNPs (rs6025 and rs6687813), were significant (p<0.05) after FDR correction. Analysis of clustering for these SNPs revealed associations of VTE recurrence risk with skeletal muscle myosin (MYH) rare SNPs (chr 17p13.1) in a highly conserved region with clustered myosin heavy chain genes (MYH1, 2, 4, 8). Among 11 rare MYH SNPs, rs111567318 was significantly linked to VTE (p=8.55x10-6, FDR p<0.05). Notably, 16 of 107 (15. %) VTE subjects had ≥ one SNP in the MYH chr17 cluster compared to only 1 of 212 (0.5 %) controls.Studies were initiated to define procoagulant or anticoagulant properties of skeletal muscle myosins which heretofore had no known function in blood clotting and which are broadly found in the body, even in plasma (e.g., at 20 nM). Remarkably, purified rabbit skeletal muscle myosin enhanced TF-induced thrombin generation in plasma. In neat plasma, anti-myosin antibodies reduced TF-induced thrombin generation, suggesting the contribution of endogenous plasma myosin to plasma thrombin generation. Skeletal muscle myosin also enhanced recalcification-induced thrombin generation in plasma. Studies to tests if prothrombinase activity is promoted by skeletal muscle myosin in purified prothrombinase (IIase) assays (factor Xa, factor Va, and II plus Ca++, with or without phospholipid added) showed that skeletal muscle myosin greatly enhanced IIase activity. When factor (F) Va was absent, myosin did not detectably enhance IIase activity, showing FVa was required. The myosin heavy chain can be split into 1 light meromyosin (LMM) and 1 heavy meromyosin (HMM) domains which can be further split into 2 globular subfragments (S1) and 1 rod-shaped subfragment (S2). HMM enhanced IIase activity whereas the myosin S1 subfragment did not, suggesting the S2 subfragment and LMM were required for the procoagulant activity. When prothrombin cleavage kinetics in the presence of skeletal muscle myosin were studied using PAGE, negligible amounts of meizothrombin were observed while fragment 1.2 and prothrombin 2 (or a-thrombin) bands were observed. This implies that skeletal muscle myosin promotes the prothrombin cleavage pathway similar to that enhanced by platelets, but not similar to that enhanced by phospholipid vesicles. The IIase activity of plasma-derived, purified extra cellular vesicles (ECVs) was markedly inhibited (> 60 %) by anti-skeletal muscle myosin antibodies, suggesting that a major fraction of procoagulant extracellular vesicle procoagulant activity involves skeletal muscle myosin. In purified systems, the apparent Kd of skeletal myosin in the presence of FVa for FXa was 9 nM.In summary, we found that MYH SNPs may be linked to VTE risk which led to the discovery of a new family of procoagulant proteins, skeletal myosins, that bind FXa, stimulate thrombin generation in plasma and in purified systems, thus paving the way for future genetic and functional VTE research. DisclosuresNo relevant conflicts of interest to declare.

Abstract LB-189: Genetic Epidemiology of Lung Cancer Consortium: genome-wide association study of familial lung cancer cases

Abstract Several studies have identified common genetic factors that influence susceptibility to lung cancer but no large studies have systematically studied familial lung cancers. The Genetic Epidemiology of Lung Cancer Consortium completed a genome-wide association study (GWAS) using samples from 757 cases and 796 controls, all of European American ancestry, and frequency matched on age and sex. Genotyping was performed by the Center for Inherited Disease Research (CIDR) using an Illumina Human OmniExpressExome-8v1 array. The most significantly associated SNPs were identified in the cases as a whole, and were also stratified by histology (adenocarcinoma and squamous cell carcinoma) and family history (0-1 and 2-5 affected family members). Analysis of all cases and controls identified a large number of SNPs around the CFTR gene that are strongly associated with lung cancer risk. Upon imputation with European Ancestry 1000 genomes data, the most significant SNP in this region is rs43034 (p = 3.89E-07, OR = 0.657) in the promoter region. In addition, an exonic missense SNP, rs213950 (p = 6.32E-07, OR = 0.663), and an intronic SNP with a possible splicing effect, rs213935 (p = 5.20E-07, OR = 0.660) were also identified as possible protective alleles. Analysis by histological subtype identified several rare and exonic SNPs that were specific by histology. We highlight two SNPs, rs114719990 (p = 1.194E-04, OR = 336.8, SETD2, c.3229A&amp;gt;G, p.Thr1077Ala) and rs199710487 (p = 1.124E-04, OR = 366, NTN5, c.656G&amp;gt;A, p.Arg219Gln), that were identified as strongly associated SNPs in cases with histologically confirmed adenocarcinoma, and two SNPs, rs144465058 (p = 1.029E-04, OR = 484.6, ANK3, c.6555G&amp;gt;T, p.Gln2185His) and rs140216112 (p = 1.087E-04, OR = 476, GRIK4, c.1013C&amp;gt;G, p.Ala338Gly), that were identified as top SNPs in cases with histologically confirmed squamous cell carcinoma. The SETD2 gene is thought to be a tumor suppressor gene and variants in this gene have previously been associated with leukemia and glioma (Zhu X et al. 2014, Fontebasso AM et al. 2013), and may now have implications in lung cancer, specifically adenocarcinoma development. In conclusion, we identified SNPs in the CFTR gene that may have a protective effect on lung cancer and several rare and exonic variants that were specific to histology subtype. SNPChrPositionGeneGenomic/ Nucleotide/ Amino acid changeOdds Ratiop-valueCFTR Protective Allelesrs430347117108449CFTRg.117108449T&amp;gt;C; 2.4kb upstream0.6573.89×10−7rs2139507117199533CFTRc.1408G&amp;gt;A; p.Val470Met0.6636.32×10−7rs2139357117178208CFTRc.869+1481A&amp;gt;G0.6605.20×10−7 Citation Format: Diana M. Toledo, Susan M. Pinney, Diptasri Mandal, Mariza de Andrade, Elena Kupert, Jennifer Franks, Colette Gaba, Claire L. Simpson, Ming You, Marshall W. Anderson, Joan E. Bailey-Wilson, Christopher I. Amos, Ann Schwartz. Genetic Epidemiology of Lung Cancer Consortium: genome-wide association study of familial lung cancer cases. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-189. doi:10.1158/1538-7445.AM2015-LB-189

Uncovering the molecular pathogenesis of congenital hyperinsulinism by panel gene sequencing in 32 Chinese patients.

Congenital hyperinsulinism (CHI) has been mostly associated with mutations in seven major genes. We retrospectively reviewed a cohort of 32 patients with CHI. Extensive mutational analysis (ABCC8,KCNJ11,GCK,GLUD1,HADH,HNF4A, and UCP2) was performed on Ion torrent platform, which could analyze hundreds of genes simultaneously with ultrahigh-multiplex PCR using up to 6144 primer pairs in a single primer pool and address time-sensitive samples with single-day assays, from samples to annotated variants, to identify the genetic etiology of this disease. Thirty-seven sequence changes were identified, including in ABCC8/KCNJ11 (n=25, 65.7%), GCK (n=2), HNF4A (n=3), GLUD1 (n=2), HADH (n=4), and UCP2 (n=1); these mutations included 14 disease-causing mutations, eight rare SNPs, 14 common SNPs, and one novel mutation. Mutations were identified in 21 of 32 patients (65.6%). Among the patients with an identified mutation, 14 had mutations in ABCC8, one of which was combined with a GLUD1 mutation. Four patients had mutations in KCNJ11, 1 had a GCK mutation, 1 had a mutation in HADH, and two had a mutation in HNF4A. Among the 32 patients, the age at the onset of hyperinsulinemia ranged from the neonatal period to 1year of age; five patients underwent a pancreatectomy due to intractable hyperinsulinemia. This study describes novel and previously identified mutations in patients with CHI. The spectrum of mutations in CHI patients represents an important tool for the diagnosis and prognosis of CHI patients in the Chinese population as well as for the genetic counseling of CHI families.

Significant association between rare IPO11-HTR1A variants and attention deficit hyperactivity disorder in Caucasians.

We comprehensively examined the rare variants in the IPO11-HTR1A region to explore their roles in neuropsychiatric disorders. Five hundred seventy-three to 1,181 rare SNPs in subjects of European descent and 1,234-2,529 SNPs in subjects of African descent (0 < minor allele frequency (MAF) < 0.05) were analyzed in a total of 49,268 subjects in 21 independent cohorts with 11 different neuropsychiatric disorders. Associations between rare variant constellations and diseases and associations between individual rare variants and diseases were tested. RNA expression changes of this region were also explored. We identified a rare variant constellation across the entire IPO11-HTR1A region that was associated with attention deficit hyperactivity disorder (ADHD) in Caucasians (T5: P = 7.9 × 10(-31) ; Fp: P = 1.3 × 10(-32) ), but not with any other disorder examined; association signals mainly came from IPO11 (T5: P = 3.6 × 10(-10) ; Fp: P = 3.2 × 1 0(-10) ) and the intergenic region between IPO11 and HTR1A (T5: P = 4.1 × 10(-30) ; Fp: P = 5.4 × 10(-32) ). One association between ADHD and an intergenic rare variant, i.e., rs10042956, exhibited region- and cohort-wide significance (P = 5.2 × 10(-6) ) and survived correction for false discovery rate (q = 0.006). Cis-eQTL analysis showed that, 29 among the 41 SNPs within or around IPO11 had replicable significant regulatory effects on IPO11 exon expression (1.5 × 10(-17) ≤P < 0.002) in human brain or peripheral blood mononuclear cell tissues. We concluded that IPO11-HTR1A was a significant risk gene region for ADHD in Caucasians.

Assessing the effects of multiple markers in genetic association studies.

Assessing the effects of multiple markers in genetic association studies.

Genetic Association Studies of Pain Phenotypes in Adult SCD Patients

Vasoocclusive episodes (VOEs) are the hallmark of sickle cell disease (SCD) responsible for > 90% of health care utilization. Several studies have elucidated the spectrum of pain syndromes in SCD and have led to the recognition of chronic, neuropathic, and centralized pain in these patients. We previously reported that adult SCD patients exhibited significant hyperalgesia in response to pressure pain algometer testing compared to age, race, and sex matched controls. While demographic and anthropometric factors were associated with self-reported pain, distress, and pressure pain threshold, genetic associations were not tested. Thus, we studied genetic polymorphisms in 167 adult patients with SCD (SS and Sb0-thal). Data were collected on number of hospitalizations, ED visits, and opioid use (morphine equivalents) in the past 12 months. Daily pain, subjective distress, and interference with daily activities were measured using daily diaries (as in the PiSCES study) over a period of 6 months. Subjects underwent mechanical pain testing with a pressure algometer at 3 anatomic sites (masseter, ulna, and trapezius) during steady state. Blood samples were obtained for ascertainment of SCD genotype and DNA extraction. Genotyping was performed at the University of Florida Genomics Facility using the Algynomics (Chapel Hill, NC) Pain Research Panel, a custom made chip utilizing the Affymetrix MegAllele technology. The panel assesses 3,295 SNPs representing 358 genes known to be involved in systems relevant to pain perception, nociceptive transmission, inflammation, mood, and affect.Individuals with >10% missing data were excluded from analysis. Hardy-Weinberg Equilibrium was checked for the genotypes at each SNP with a chi-squared test. A total of 328 rare SNPs with minor allele frequencies (<1%) were dropped. The final pain phenotypes included experimental pain, distress, interference with daily activities, opioid usage, and hospitalizations / ED visits. Linear regression was fitted to each SNP, regressing each phenotype onto genotype under an additive model, with age, gender, and BMI as covariates. The analysis was performed using Plink. False discovery rate was controlled using the Benjamini & Hochberg method.Six SNPs in 5 genes (ESR2, KCNJ11, DBH, ATP1A1, CACNA2D2) were significantly associated with pressure pain threshold. ESR2 polymorphisms have been reported to be associated with temporomandibular, synovial, and neurogenic pain. KCNJ11 has been reported to play an important role in opioid sensitivity/analgesia. DBH polymorphisms have been related to analgesia, migraines, and catecholamine and serotonin metabolism. ATP1A1 was reported in association with headaches and paresthesias. CACNA2D2 has been correlated with increased sensitivity to opioids.In terms of opioid usage, 3 SNPs (CAMK2B, PRKD1, CACNA2D4) had significant associations and have been reported to be related to stress, depression, neuronal connectivity, pain modulation, neurogenic inflammation, and pain transmission.ED visits were significantly associated with 3 SNPS (TRIM31, PLAUR, PRKG1), which have been reported to be associated with fibromyalgia, nociceptive processing, and inflammation.Two SNPs (PRL and ADRA1A) were significantly associated with hospitalizations that have previously been related to nociception and hyperalgesia as well as inflammation and allodynia.To our knowledge, this is the first detailed study of genetic associations with different pain related phenotypes in subjects with SCD. Our results show that SNPs in genes relevant to pain processing, nociception, hyperalgesia, opioid sensitivity, inflammation, and stress are significantly associated with different pain phenotypes and therefore appear functionally relevant in adults with SCD. These findings will hopefully serve as a discovery cohort, which should be validated in an independent group of SCD patients. A better understanding of pathways and networks relevant to SCD pain may ultimately lead to new therapeutic interventions. DisclosuresKutlar:NIH/NIMHD: Research Funding. Wells:NIH/NIMHD: Research Funding. Xu:NIH/NIMHD: Research Funding. Bowman:NIH/NIMHD: Research Funding. Bora:NIH/NIMHD: Research Funding. Clair:NIH/NIMHD: Research Funding. Meiler:NIH/NIMHD: Research Funding. Wang:NIH/NIMHD: Research Funding. Fillingim:NIH/NIMHD: Research Funding; Algynomics: Equity Ownership.

Genetic variation in the UGT1A locus is associated with simvastatin efficacy in a clinical practice setting.

Aim: Simvastatin is a lactone prodrug that exists in equilibrium with its active hydroxyacid through a process mediated by UGT1A enzymes. The UGT1A locus has been associated with simvastatin response and disposition in humans. Therefore, we fine-mapped the UGT1A locus to identify genetic variations contributing to simvastatin disposition and response variability. Methods: Using de-identified electronic medical records linked to a DNA biobank, we extracted information about dose and low-density lipo-protein cholesterol (LDL-C) concentrations for patients who received more than two different doses of simvastatin. Pharmacodynamic measures of simvastatin potency and efficacy were calculated from dose-response curves (E0 = baseline LDL-C, ED50 = dose yielding 50% maximum response, and Emax = maximum decrease in LDL-C) in 1100 patients. We selected 153 polymorphisms in UGT1A1 and UGT1A3 for genotyping and conducted genotype-phenotype associations using a prespecified additive model. Results: Two variants in UGT1A1 (rs2003569 and rs12052787) were associated with Emax (p = 0.0059 and 0.031, respectively; for rs2003569 the mean Emax was 59.3 ± 23.0, 62.0 ± 22.4, and 69.7 ± 24.8 mg/dl, for patients with 0, 1 or 2 copies of the minor A allele, respectively). When stratified by race, the difference in response was greater in African-Americans than in European Americans. Rs2003569 was also negatively associated with total serum bilirubin levels (p = 7.85 × 10-5). Four rare SNPs were nominally associated with E0 and ED50. Conclusion: We identified a UGT1A1 promoter variant (rs2003569) associated with simvastatin efficacy. Original submitted 26 March 2014; Revision submitted 26 August 2014.

Abstract 454: TET2 alterations facilitate progression of metastatic prostate cancer

Abstract Metastatic prostate cancer (mPCa) is a highly lethal disease and molecular markers identifying disease subtypes and/or therapeutic targets are needed. We sequenced the exomes of five metastatic tumors and healthy kidney tissue from an index patient with castration resistant mPCa to identify lesions associated with disease progression and metastasis. A somatic missense TET2 alteration predicted to be deleterious to the protein was observed at the metastatic stage of disease. We sequenced TET2 in additional mPCa tumors and PCa cell lines and detected somatic missense alterations, frequent LOH, and a frameshift truncation in PCa cell line, DU-145, derived from a mPCa brain tumor. We also observed complex combinations of frequent and rare germline missense SNPs, which may contribute to a previously described association of TET2 with PCa risk by GWAS. In this study, we confirm transcription factor binding to TET2 alleles associated with disease by GWAS. We examine TET2 binding partners in prostate cells by affinity purification, mass spectrometry, and forward and reverse immunoprecipitation. We confirmed a protein complex containing TET2, OGT, and two transcriptional co-activators/splicing factors implicated in receptor-mediated gene expression. Functional analysis shows TET2 loss alters expression of a unique set of genes, increases cell proliferation, and therefore is a driver of PCa. TET2 loss may facilitate progression to castration resistant mPCa and appears to define a subset of cases with metastatic disease. Citation Format: Michael L. Nickerson, Sudipto Das, Hong Lou, Hongchuan Li, Sevilay Turan, Kate Im, Stephen Anderson, Thorkell Andresson, Michael Dean. TET2 alterations facilitate progression of metastatic prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 454. doi:10.1158/1538-7445.AM2014-454

The functional significance of common polymorphisms in zinc finger transcription factors.

Variants that alter the DNA-binding specificity of transcription factors could affect the specificity for and expression of potentially many target genes, as has been observed in several tumor-derived mutations. Here we examined if such trans expression quantitative trait loci (trans-eQTLs) could similarly result from common genetic variants. We chose to focus on the Cys2-His2 class of zinc finger transcription factors because they are the most abundant superfamily of transcription factors in human and have well-characterized DNA binding interactions. We identified 430 SNPs that cause missense substitutions in the DNA-contacting residues. Fewer common missense SNPs were found at DNA-contacting residues compared with non-DNA-contacting residues (P = 0.00006), consistent with possible functional selection against SNPs at DNA-contacting positions. Functional predictions based on zinc finger transcription factor (ZNF) DNA binding preferences also suggested that many common substitutions could potentially alter binding specificity. However, Hardy-Weinberg Equilibrium analysis and examination of seven orthologs within the primate lineage failed to find evidence of trans-eQTLs associated with the DNA-contacting positions or evidence of a different selection pressure on a contemporary and evolutionary timescales. The overall conclusion was that common SNPs that alter the DNA-contacting residues of these factors are unlikely to produce strong trans-eQTLs, consistent with the observations by others that trans-eQTLs in humans tend to be few and weak. Some rare SNPs might alter specificity and remained rare due to purifying selection. The study also underscores the need for large-scale eQTLs mapping efforts that might provide experimental evidence for SNPs that alter the choice of transcription factor binding sites.

Resequencing and association study of the NFKB activating protein-like gene (NKAPL) in schizophrenia

ObjectivesSchizophrenia is a highly inheritable disorder, but many aspects of its etiology and pathophysiology remain poorly understood. Recently, in the Han Chinese population, a SNP rs1635 located within the exon of the NKAPL gene (encoding NFKB activating protein-like) achieved genome-wide significance in schizophrenia. MethodsIn order to find the causal variants of the NKAPL gene in schizophrenia, we searched for genetic variants in the promoter region, and exons (including both UTR ends) using direct sequencing in a sample of patients with schizophrenia (n=515) and non-psychotic controls (n=456), all Han Chinese from Taiwan, and conducted an association and rudimentary functional study. ResultsWe identified 5 common SNPs (defined as minor allele frequency (MAF)>0.01) in the NKAPL gene. In a case–control association analysis, the minor allele (A) of rs1635 was significantly more common among patients than controls (P=0.0003, OR=1.41, 95% CI=1.17–1.71). A haplotype analysis of the 5 common SNPs indicated a risk haplotype (rs12000C–rs1635A–rs9461446C–rs3734564G–rs1679709G) associated with schizophrenia (P=2.77e-005, OR=1.53, 95% CI=1.25–1.87). In addition, we identified 4 patient-specific rare SNPs (MAF<0.01) (c.137G>A, c.213G>A, c.752C>T (rs370337122), and c.844G>A (rs147161729)) within the NKAPL gene. In silico analysis demonstrated their functional impact on the protein; however, there was also 1 control-specific rare SNP (c.119G>A) detected within the NKAPL gene, indicating that the clinical relevance of these putatively pathological rare SNPs is not straightforward. ConclusionsThis study suggested that rs1635 in the NKAPL gene appeared to play a role in conferring susceptibility to schizophrenia. In addition, some rare SNPs in the NKAPL gene with possibly damaging effects may be important in our patients. Our study provides genetic clues to indicate the involvement of NKAPL in schizophrenia.

GWAS to Sequencing: Divergence in Study Design and Analysis.

The success of genome-wide association studies (GWAS) in uncovering genetic risk factors for complex traits has generated great promise for the complete data generated by sequencing. The bumpy transition from GWAS to whole-exome or whole-genome association studies (WGAS) based on sequencing investigations has highlighted important differences in analysis and interpretation. We show how the loss in power due to the allele frequency spectrum targeted by sequencing is difficult to compensate for with realistic effect sizes and point to study designs that may help. We discuss several issues in interpreting the results, including a special case of the winner's curse. Extrapolation and prediction using rare SNPs is complex, because of the selective ascertainment of SNPs in case-control studies and the low amount of information at each SNP, and naive procedures are biased under the alternative. We also discuss the challenges in tuning gene-based tests and accounting for multiple testing when genes have very different sets of SNPs. The examples we emphasize in this paper highlight the difficult road we must travel for a two-letter switch.

Functional Mutation Analysis Provides Evidence for a Role of REEP1 in Lipid Droplet Biology

Hereditary axonopathies are frequently caused by mutations in proteins that reside in the endoplasmic reticulum (ER). Which of the many ER functions are pathologically relevant, however, remains to be determined. REEP1 is an ER protein mutated in hereditary spastic paraplegia (HSP) and hereditary motor neuropathy (HMN). We found that HSP-associated missense variants at the N-terminus of REEP1 abolish ER targeting, whereas two more central variants are either rare benign SNPs or confer pathogenicity via a different mechanism. The mis-targeted variants accumulate at lipid droplets (LDs). N-terminal tagging, deletion of the N-terminus, and expression of a minor REEP1 isoform had the same effect. We also confirmed an increase in LD size upon cooverexpression of atlastins and REEP1. Neither wild-type REEP1, LD-targeted HSP variants, nor a non-LD-targeted HMN variant reproduced this effect when expressed alone. We conclude that the N-terminus of REEP1 is necessary for proper targeting to and/or retention in the ER. The protein's potential to also associate with LDs corroborates a synergistic effect with atlastins on LD size. Interestingly, LD size is also altered upon knockdown of seipin, mutations of which also cause HSP and HMN. Regulation of LDs may thus be an ER function critical for long-term axonal maintenance.

Empirical prediction of genomic susceptibilities for multiple cancer classes.

An empirical approach is presented for predicting the genomic susceptibility of an individual to the most likely one among nine traits, consisting of eight major cancer classes plus a healthy trait. We use four prediction methods by applying two supervised learning algorithms to two different descriptors of common genomic variations (the profiles of genotypes of SNPs and SNP syntaxes with low P values or low frequencies) of each individual genome from normal cells. All four methods made correct predictions substantially better than random predictions for most cancer classes, but not for some others. A combination of the four results using Bayesian inference better predicted overall than any individual method. The multiclass accuracy of the combined prediction ranges from 33% to 56% depending on cancer classes of testing sets, compared with 11% for a random prediction among nine traits. Despite limited SNP data available and the absence of rare SNPs in public databases, at present, the results suggest that the framework of this approach or its improvement can predict cancer susceptibility with probability estimates useful for making health decisions for individuals or for a population.

Rare mutations of peroxisome proliferator-activated receptor gamma: frequencies and relationship with insulin resistance and diabetes risk in the mixed ancestry population from South Africa.

Background. Genetic variants in the nuclear transcription receptor, PPARG, are associated with cardiometabolic traits, but reports remain conflicting. We determined the frequency and the clinical relevance of PPARG SNPs in an African mixed ancestry population. Methods. In a cross-sectional study, 820 participants were genotyped for rs1800571, rs72551362, rs72551363, rs72551364, and rs3856806, using allele-specific TaqMan technology. The homeostatic model assessment of insulin (HOMA-IR), β-cells function (HOMA-B%), fasting insulin resistance index (FIRI), and the quantitative insulin-sensitivity check index (QUICKI) were calculated. Results. No sequence variants were found except for the rs3856806. The frequency of the PPARG-His447His variant was 23.8% in the overall population group, with no difference by diabetes status (P = 0.215). The His447His allele T was associated with none of the markers of insulin resistance overall and by diabetes status. In models adjusted for 2-hour insulin, the T allele was associated with lower prevalent diabetes risk (odds ratio 0.56 (95% CI 0.31–0.95)). Conclusion. Our study confirms the almost zero occurrences of known rare PPARG SNPs and has shown for the first time in an African population that one of the common SNPs, His447His, may be protective against type 2 diabetes.