Intragenic Single Nucleotide Polymorphisms Research Articles

JAM2 variants can be more common in primary familial brain calcification (PFBC) cases than those appear; may be due to a founder mutation.

Mutations in JAM2 have been linked to ~ 2% of primary familial brain calcification (PFBC) cases. PFBC is a rare neurological disorder characterized by excessive calcium deposition in the brain. It causes movement disorders and psychiatric problems. Six other genes were identified as causing PFBC. However, the genetic basis of ~ 50% of PFBC cases remains unknown. This study presented the results of a comprehensive analysis of five unrelated Iranian PFBC families. Clinical and paraclinical features of all patients were recorded. Whole-exome sequencing (WES) was done on the DNAs of probands. Data was analyzed, and haplotypes were determined. WES identified two homozygous variants in JAM2 across four families: a novel variant, c.426dup:p.Ser143Leufs*23, in one family and a known mutation, c.685C > T:p.Arg229*, in the remaining three families. Haplotype analysis using six intragenic single-nucleotide polymorphisms (SNPs) in JAM2 revealed an identical haplotype in probands who carried the same mutation, whereas two other probands presented diverse haplotypes. Based on our results, p.Arg229* may be a founder mutation in the Iranian population. The variant has been detected in two out of seven other reported JAM2-related families who may originate from the Middle East and exhibit an identical haplotype. Even though this particular mutation may not be classified as a founder mutation, it does appear to be a hotspot, given that it has been observed in 45% of the 11 JAM2-associated families. Our study expanded the clinical features and mutation spectrum of JAM2 and revealed that mutations in JAM2 may be more common than previously reported.

The Anti-Müllerian Hormone as Endocrine and Molecular Marker Associated with Reproductive Performance in Holstein Dairy Cows Exposed to Heat Stress.

Anti-Müllerian hormone (AMH) is proposed as a biomarker for fertility in cattle, yet this associative relationship appears to be influenced by heat stress (HS). The objective was to test serum AMH and AMH-related single nucleotide polymorphisms (SNPs) as markers potentially predictive of reproductive traits in dairy cows experiencing HS. The study included 300 Holstein cows that were genotyped using BovineSNP50 (54,000 SNP). A genome-wide association study was then executed. Nine intragenic SNPs within the pathways that influence the AMH gene were found important with multiple comparisons adjustment tests (p < 1.09 × 10-6). A further validation study was performed in an independent Holstein cattle population, which was divided into moderate (MH; n = 152) and severe heat-stressed (SH; n = 128) groups and then subjected to a summer reproductive management program. Serum AMH was confirmed as a predictor of fertility measures (p < 0.05) in MH but not in the SH group. Cows were genotyped, which revealed four SNPs as predictive markers for serum AMH (p < 0.01), reproductive traits (p < 0.01), and additional physiological variables (p < 0.05). These SNPs were in the genes AMH, IGFBP1, LGR5, and TLR4. In conclusion, serum AMH concentrations and AMH polymorphisms are proposed as predictive markers that can be used in conjunction with genomic breeding value approaches to improve reproductive performance in Holstein cows exposed to summer HS conditions.

Epigenomic and Transcriptomic Prioritization of Candidate Obesity-Risk Regulatory GWAS SNPs.

Concern about rising rates of obesity has prompted searches for obesity-related single nucleotide polymorphisms (SNPs) in genome-wide association studies (GWAS). Identifying plausible regulatory SNPs is very difficult partially because of linkage disequilibrium. We used an unusual epigenomic and transcriptomic analysis of obesity GWAS-derived SNPs in adipose versus heterologous tissues. From 50 GWAS and 121,064 expanded SNPs, we prioritized 47 potential causal regulatory SNPs (Tier-1 SNPs) for 14 gene loci. A detailed examination of seven loci revealed that four (CABLES1, PC, PEMT, and FAM13A) had Tier-1 SNPs positioned so that they could regulate use of alternative transcription start sites, resulting in different polypeptides being generated or different amounts of an intronic microRNA gene being expressed. HOXA11 and long noncoding RNA gene RP11-392O17.1 had Tier-1 SNPs in their 3′ or promoter region, respectively, and strong preferences for expression in subcutaneous versus visceral adipose tissue. ZBED3-AS1 had two intragenic Tier-1 SNPs, each of which could contribute to mediating obesity risk through modulating long-distance chromatin interactions. Our approach not only revealed especially credible novel regulatory SNPs, but also helped evaluate previously highlighted obesity GWAS SNPs that were candidates for transcription regulation.

Enrichment of SARM1 alleles encoding variants with constitutively hyperactive NADase in patients with ALS and other motor nerve disorders.

SARM1, a protein with critical NADase activity, is a central executioner in a conserved programme of axon degeneration. We report seven rare missense or in-frame microdeletion human SARM1 variant alleles in patients with amyotrophic lateral sclerosis (ALS) or other motor nerve disorders that alter the SARM1 auto-inhibitory ARM domain and constitutively hyperactivate SARM1 NADase activity. The constitutive NADase activity of these seven variants is similar to that of SARM1 lacking the entire ARM domain and greatly exceeds the activity of wild-type SARM1, even in the presence of nicotinamide mononucleotide (NMN), its physiological activator. This rise in constitutive activity alone is enough to promote neuronal degeneration in response to otherwise non-harmful, mild stress. Importantly, these strong gain-of-function alleles are completely patient-specific in the cohorts studied and show a highly significant association with disease at the single gene level. These findings of disease-associated coding variants that alter SARM1 function build on previously reported genome-wide significant association with ALS for a neighbouring, more common SARM1 intragenic single nucleotide polymorphism (SNP) to support a contributory role of SARM1 in these disorders. A broad phenotypic heterogeneity and variable age-of-onset of disease among patients with these alleles also raises intriguing questions about the pathogenic mechanism of hyperactive SARM1 variants.

Genome-wide association study of a thermo-tolerance indicator in pregnant ewes exposed to an artificial heat-stressed environment.

Environmental heat stress negatively influences sheep production in warm semi-arid regions. An animal's ability to tolerate warm weather is difficult to measure naturally due to environmental variability and genetic variation between animals. In this study we developed a thermo-tolerance indicator (TTI) to define heat stress tolerance in pregnant sheep in a controlled environment. Next, we performed a genome-wide association study (GWAS) to identify genomic regions and target genes associated with thermo-tolerance in sheep. Pregnant Columbia-Rambouillet crossbred ewes (n=127) were heat-stressed inside a climate-controlled chamber for 57 days by increasing the temperature-humidity index to ≥30. Rectal temperature (RT) and feed intake (FI) data were collected daily and used for the predictive TTI analysis. After the tenth day of heat stress, the regression analyses revealed that FI was stable; however, when the ewe's RT exceeded 39.8°C their FI was less than thermo-tolerant ewes. This average predicted temperature was used to classify each ewe as heat stress tolerant (≤39.8°C) and non-heat stress tolerant (>39.8°C). A GWAS analysis was performed and genomic regions were compared between heat stress tolerant and non-tolerant ewes. The single-marker genomic analysis detected 16 single nucleotide polymorphisms (SNP) associated with heat stress tolerance (P<0.0001), whereas the multi-marker Bayesian analysis identified 8 overlapped 1-Mb chromosomal regions accounting for 11.39% of the genetic variation associated with tolerance to heat stress. Four intragenic SNP showed a remarkable contribution to thermo-tolerance, and these markers were within the genes FBXO11 (rs407804467), PHC3 (rs414179061), TSHR (rs418575898) and STAT1 (rs417581105). In conclusion, genomic regions harboring four intragenic SNP were associated with heat stress tolerance, and these candidate genes are proposed to influence heat tolerance in pregnant ewes subjected to an artificially induced warm climate. Moreover, these genetic markers could be suitable for use in further genetic selection programs in sheep managed in semi-arid regions.

Integrative multiomics analysis of the acid stress response of Oenococcus oeni mutants at different growth stages

BackgroundAcid stress is one of the most important environmental stresses that adversely affect the growth of lactic acid bacteria (LAB), such as Oenococcus oeni which was isolated from grape-berries and mainly used in wine fermentation. The aim of this paper is to comprehensively characterize the mechanisms of acid stress regulation in O. oeni and to provide a viable theoretical basis for breed and improvement of existing LAB. MethodFirst, six O. oeni mutants with acid-sensitive (strains b2, a1, c2) and acid-tolerant (strains b1, a3, c1) phenotypes were screened from three wild-type O. oeni, and then their genome (sequencing), transcriptome and metabolome (LC-MS/MS) were examined. ResultsA total of 459 genes were identified with one or more intragenic single nucleotide polymorphisms (SNPs) in these mutants, and were extensively involved in metabolism and cellular functions with a high mutation rates in purine (46%) and pyrimidine (48%) metabolic pathways. There were 210 mutated genes that cause significant changes in expression levels. In addition, 446 differentially accumulated metabolites were detected, and they were consistently detected at relatively high levels in the acid-tolerant O. oeni mutant. The levels of intracellular differentially expressed genes and differential metabolites changed with increasing culture time. ConclusionThe integrative pathways analysis showed that the intracellular response associated with acid regulation differed significantly between acid-sensitive and acid-tolerant O. oeni mutants, and also changed at different growth stages.

Effect of the p53 P72R Polymorphism on Mutant TP53 Allele Selection in Human Cancer.

TP53 mutations occur in more than 50% of cancers. We sought to determine the effect of the intragenic P72R single nucleotide polymorphism (SNP; rs1042522) on the oncogenic properties of mutant p53. P72R allelic selection in tumors was determined from genotype calls and a Gaussian distributed mixture model. The SNP effect on mutant p53 was determined in p53-negative cancer cell lines. RNA-sequencing, chromatin immunoprecipitation, and survival analysis were performed to describe the SNP effect. All statistical tests were 2-sided. Among 409 patients with germline heterozygous P72R SNP who harbored somatic mutations inTP53, we observed a selection bias against missenseTP53mutants encoding the P72 SNP (P = 1.64 x 10-13). Exogenously expressed hotspotp53mutants with the P72 SNP were negatively selected in cancer cells. Gene expression analyses showed the enrichment of p53 pathway genes and inflammatory genes in cancer cells transduced with mutants encoding P72 SNP. Immune gene signature is enriched in patients harboring missense TP53 mutations with homozygous P72 SNP. These patients have improved overall survival as compared with those with the R72 SNP (P = .04). This is the largest study demonstrating a selection against the P72 SNP. Missense p53 mutants with the P72 SNP retain partial wild-type tumor-suppressive functions, which may explain the selection bias against P72 SNP across cancer types. Ovarian cancer patients with the P72 SNP have a better prognosis than with the R72 SNP. Our study describes a previously unknown role through which thers1042522SNP modifies tumor suppressor activities of mutantp53in patients.

Antithrombin p.Thr147Ala: The First Founder Mutation in People of African Origin Responsible for Inherited Antithrombin Deficiency.

Hereditary antithrombin deficiency is a rare autosomal-dominant disorder predisposing to recurrent venous thromboembolism (VTE). To date, only two founder mutations have been described. We investigated the antithrombin p.Thr147Ala variant, found in 12 patients of African origin. This variant is known as rs2227606 with minor allele frequency of 0.5% in Africans and absent in Europeans. A possible founder effect was investigated. Phenotypical characterization was established through immunological and functional methods, both under basal and stress conditions. Recombinant antithrombin molecules were constructed by site-directed mutagenesis and expressed in HEK-293T cells. Secreted antithrombin was purified and functionally characterized. Structural modeling was performed to predict the impact of the mutation on protein structure. A novel nanopore sequencing approach was used for haplotype investigation. Ten patients experienced VTE, stroke, or obstetric complications. Antithrombin antigen levels and anti-IIa activity were normal or slightly reduced while anti-Xa activity was reduced with only one commercial assay. On crossed immunoelectrophoresis, an increase of antithrombin fractions with reduced heparin affinity was observed under high ionic strength conditions but not under physiological conditions. The recombinant p.Thr147Ala protein displayed a reduced anti-Xa activity. Structural modeling revealed that residue Thr147 forms three hydrogen bonds that are abolished when mutated to alanine. The investigated patients shared a common haplotype involving 13 SERPINC1 intragenic single nucleotide polymorphisms. Antithrombin p.Thr147Ala, responsible for antithrombin type II heparin binding site deficiency, is the first founder mutation reported in people of African ancestry. This study further emphasizes the limitations of commercial methods to diagnose this specific subtype.

Evaluating the Association Between Dental Fluorosis and Polymorphisms in Bone Development and Mineralization Genes Among Population from a Fluoride Endemic Region of Eastern India.

Close to 12 million people in India are affected by more than the desirable level of fluoride in drinking water that could lead to dental, skeletal, and non-skeletal fluorosis. Dental fluorosis is a developmental defect that results in hypo-mineralization and pronounced porosity of enamel in the affected individuals. As estrogen receptor 1 (ESR1), collagen type 1 alpha 2 (COL1A2), bone γ-carboxyglutamic acid protein (BGLAP), and secreted protein acidic and cysteine rich (SPARC) genes are involved in bone development and mineralization, polymorphisms in these genes could be determining factors in influencing the risk to fluorosis among the exposed individuals in fluoride endemic areas. A case-control study was carried out among a total of 87 individuals (case = 36, control = 51) to examine the association between selected polymorphisms in the ESR1, COL1A2, BGLAP, and SPARC genes and risk of dental fluorosis from a fluoride endemic region of Eastern India. Altogether, 10 single nucleotide polymorphisms (SNPs) in ESR1 (rs2234693, rs2228480, rs3798577, rs2077647, and rs9340799), COL1A2 (rs42524, rs412777), BGLAP (rs1800247), and SPARC (rs6579885, rs4958278) genes were genotyped through PCR-RFLP in these subjects. The association of the SNPs for disease risk estimation was measured by odds ratio with 95% confidence interval. The risk genotypes of none of the 10 SNPs showed statistically significant association with risk of dental fluorosis. Frequencies of the haplotypes in the intragenic SNPs of the ESR1, COL1A2, and SPARC genes did not reveal any statistically significant difference between the case and control groups. The present study is the first of its kind from India that has attempted to investigate possible involvement of genetic factors in influencing the risk to fluorosis among the population from a fluoride endemic region.

Down-regulated RGS5 by genetic variants impairs endothelial cell function and contributes to coronary artery disease.

Genetic contribution to coronary artery disease (CAD) remains largely unillustrated. Although transcriptomic profiles have identified dozens of genes that are differentially expressed in normal and atherosclerotic vessels, whether those genes are genetically associated with CAD remains to be determined. Here, we combined genetic association studies, transcriptome profiles and in vitro and in vivo functional experiments to identify novel susceptibility genes for CAD. Through an integrative analysis of transcriptome profiles with genome-wide association studies for CAD, we obtained 18 candidate genes and selected one representative single nucleotide polymorphism (SNP) for each gene for multi-centred validations. We identified an intragenic SNP, rs1056515 in RGS5 gene (odds ratio = 1.17, 95% confidence interval =1.10-1.24, P = 3.72 × 10-8) associated with CAD at genome-wide significance. Rare genetic variants in linkage disequilibrium with rs1056515 were identified in CAD patients leading to a decreased expression of RGS5. The decreased expression was also observed in atherosclerotic vessels and endothelial cells treated by various cardiovascular risk factors. Through siRNA knockdown and adenoviral overexpression, we further showed that RGS5 regulated endothelial inflammation, vascular remodelling, as well as canonical NF-κB signalling activation. Moreover, CXCL12, a specific downstream target of the non-canonical NF-κB pathway, was strongly affected by RGS5. However, the p100 processing, a well-documented marker for non-canonical NF-κB pathway activation, was not altered, suggesting an existence of a novel mechanism by which RGS5 regulates CXCL12. We identified RGS5 as a novel susceptibility gene for CAD and showed that the decreased expression of RGS5 impaired endothelial cell function and functionally contributed to atherosclerosis through a variety of molecular mechanisms. How RGS5 regulates the expression of CXCL12 needs further studies.

Integration of genome wide association study with gene expression profiling identifies ROR1 as a novel susceptibility gene for coronary artery disease

Objective To identify new susceptibility genes for coronary artery disease(CAD)by integrating genome wide association study and gene expression profiling. Methods On account of our data of genome wide association study(GWAS)on CAD, our integrative analysis of gene expression profiles and GWAS for CAD was conducted, and we obtained candidate genes and single nucleotide polymorphisms(SNPs)for a multicenter case-control genetic association study.Using a DNA ligase chain reaction-based genotyping method, we validated these candidate loci in two independent populations. Results Firstly, we selected 21 SNPs for 21 candidate genes.Using a DNA ligase chain reaction-based genotyping method, we validated these candidate loci in two independent populations, including population 1(495 cases and 492 control)and population 2(810 cases and 853 control). And we identified an intragenic SNP rs11208367(ROR1), which was significantly associated with CAD.Meta confluence analysis in combination of GWAS and two validation populations showed that the P value of rs11208367 was 2.00×10-4, which was still significant after Bonferroni multiple test correction(0.05/21=2.38×10-3). Conclusions The rs11208367 SNP of ROR1gene is significantly associated with CAD, indicating that ROR1is a novel susceptibility gene for CAD. Key words: Genomics; Coronary disease; Genetic predisposition to disease

C6orf10 Low-Frequency and Rare Variants in Italian Multiple Sclerosis Patients.

In light of the complex nature of multiple sclerosis (MS) and the recently estimated contribution of low-frequency variants into disease, decoding its genetic risk components requires novel variant prioritization strategies. We selected, by reviewing MS Genome Wide Association Studies (GWAS), 107 candidate loci marked by intragenic single nucleotide polymorphisms (SNPs) with a remarkable association (p-value ≤ 5 × 10-6). A whole exome sequencing (WES)-based pilot study of SNPs with minor allele frequency (MAF) ≤ 0.04, conducted in three Italian families, revealed 15 exonic low-frequency SNPs with affected parent-child transmission. These variants were detected in 65/120 Italian unrelated MS patients, also in combination (22 patients). Compared with databases (controls gnomAD, dbSNP150, ExAC, Tuscany-1000 Genome), the allelic frequencies of C6orf10 rs16870005 and IL2RA rs12722600 were significantly higher (i.e., controls gnomAD, p = 9.89 × 10-7 and p < 1 × 10-20). TET2 rs61744960 and TRAF3 rs138943371 frequencies were also significantly higher, except in Tuscany-1000 Genome. Interestingly, the association of C6orf10 rs16870005 (Ala431Thr) with MS did not depend on its linkage disequilibrium with the HLA-DRB1 locus. Sequencing in the MS cohort of the C6orf10 3′ region revealed 14 rare mutations (10 not previously reported). Four variants were null, and significantly more frequent than in the databases. Further, the C6orf10 rare variants were observed in combinations, both intra-locus and with other low-frequency SNPs. The C6orf10 Ser389Xfr was found homozygous in a patient with early onset of the MS. Taking into account the potentially functional impact of the identified exonic variants, their expression in combination at the protein level could provide functional insights in the heterogeneous pathogenetic mechanisms contributing to MS.

Crohn's disease IRGM risk alleles are associated with altered gene expression in human tissues.

Crohn's disease (CD) is a chronic inflammatory gastrointestinal disorder. Genetic association studies have implicated dysregulated autophagy in CD. Among risk loci identified are a promoter single nucleotide polymorphism (SNP)( rs13361189 ) and two intragenic SNPs ( rs9637876 , rs10065172 ) in immunity-related GTPase family M ( IRGM) a gene that encodes a protein of the autophagy initiation complex. All three SNPs have been proposed to modify IRGM expression, but reports have been divergent and largely derived from cell lines. Here, analyzing RNA-Sequencing data of human tissues from the Genotype-Tissue Expression Project, we found that rs13361189 minor allele carriers had reduced IRGM expression in whole blood and terminal ileum, and upregulation in ileum of ZNF300P1, a locus adjacent to IRGM on chromosome 5q33.1 that encodes a long noncoding RNA. Whole blood and ileum from minor allele carriers had altered expression of multiple additional genes that have previously been linked to colitis and/or autophagy. Notable among these was an increase in ileum of LTF (lactoferrin), an established fecal inflammatory biomarker of CD, and in whole blood of TNF, a key cytokine in CD pathogenesis. Last, we confirmed that risk alleles at all three loci associated with increased risk for CD but not ulcerative colitis in a case-control study. Taken together, our findings suggest that genetically encoded IRGM deficiency may predispose to CD through dysregulation of inflammatory gene networks. Gene expression profiling of disease target tissues in genetically susceptible populations is a promising strategy for revealing new leads for the study of molecular pathogenesis and, potentially, for precision medicine. NEW & NOTEWORTHY Single nucleotide polymorphisms in immunity-related GTPase family M ( IRGM), a gene that encodes an autophagy initiation protein, have been linked epidemiologically to increased risk for Crohn's disease (CD). Here, we show for the first time that subjects with risk alleles at two such loci, rs13361189 and rs10065172 , have reduced IRGM expression in whole blood and terminal ileum, as well as dysregulated expression of a wide array of additional genes that regulate inflammation and autophagy.

Association of Desmin Gene Variant rs1058261 with Cardiovascular Disease, the TAMRISK Study.

Since desmin expression is diminished in vascular smooth muscle cells during reparative processes, we wanted to study whether a common intragenic single nucleotide polymorphism at nucleotide position 828 (rs1058261) of the DES gene associates with hypertension, cerebrovascular complications, and all cardiovascular events in the Tampere adult population cardiovascular risk (TAMRISK) study. A Finnish periodic health examination cohort of 336 subjects with diagnosed hypertension and 473 controls were analyzed. Samples were genotyped for polymorphism using TaqMan techniques. Prevalence of ischemic heart diseases, incidence of cerebrovascular diseases, and transient cerebral ischemic attacks (TIAs) were obtained by self-report and the National Hospital Discharge Registry (HILMO). There was no association of any of the rs1058261 genotypes with hypertension at the age of 50. When the subjects were followed to the age of 60, after adjustment for gender and body mass index, subjects with the genotype CC had higher incidence of cerebrovascular events (cerebrovascular diseases and TIA) (4.1%) compared with the T allele (1.6%) (p = 0.046). In addition, those with CC genotype had a higher incidence of all combined cardiovascular events (12.8%) compared with subjects with the T allele (8.5%) (p = 0.028). Our findings suggest that variations in the DES gene may be involved in cardiovascular disease.

Mutation Analysis of Families with Autosomal Dominant Congenital Cataract: A Recurrent Mutation in the CRYBA1/A3 Gene Causing Congenital Nuclear Cataract

ABSTRACTPurpose: To identify the CRYBA1/A3 mutation spectrum and analyze the genotype-phenotype correlations in Chinese families with congenital cataract.Methods: Family history and clinical data of 47 unrelated families with autosomal dominant congenital cataract (ADCC) were recorded. CRYBA1/A3 gene sequencing was applied to identify the causative mutation. Haplotypes were constructed using closely linked microsatellite markers and intragenic single-nucleotide polymorphisms (SNPs) to compare the affected haplotype in three families.Results: Nuclear cataract was the most common type of ADCC in Chinese families, accounting for 42.6% (20/47). A recurrent CRYBA1/A3 deletion mutation (ΔG91) was identified in three families (6.4%) with nonprogressive nuclear congenital cataract. Different haplotypes segregated with the mutation in each family.Conclusions: A recurrent ΔG91CRYBA1/A3 mutation occurs independently in 6.4% of the Chinese families with autosomal dominant nuclear cataracts and most likely represents a mutational hot spot, which underscores the relations between nonprogressive nuclear cataract and CRYBA1/A3.

Identification of Genetic Interaction with Risk Factors Using a Time-To-Event Model.

Background: Certain diseases can occur with and without a trigger. We use Venous Thromboembolism (VTE) as our example to identify genetic interaction with pregnancy in women with VTE during pre- or postpartum. Pregnancy is one of the major risk factors for VTE as it accounts for 10% of maternal deaths. Methods: We performed a whole genome association analysis using the Cox Proportional Hazard (CoxPH) model adjusted for covariates to identify genetic variants associated with the time-to-event of VTE related to pre- or postpartum during the childbearing age of 18–45 years using a case-only design in a cohort of women with VTE. Women with a VTE event after 45 years of age were censored and contributed only follow-up time. Results: We identified two intragenic single nucleotide polymorphisms (SNPs) at genome-wide significance in the PURB gene located on chromosome 7, and two additional intragenic SNPs, one in the LINGO2 gene on chromosome 9 and one in RDXP2 on chromosome X. Conclusions: We showed that the time-to-event model is a useful approach for identifying potential hazard-modification of the genetic variants when the event of interest (VTE) occurs due to a risk factor (pre- or post-partum).

Sharing of Genes and Pathways Across Complex Phenotypes: A Multilevel Genome-Wide Analysis.

Evidence from genome-wide association studies (GWAS) suggest that pleiotropic effects on human complex phenotypes are very common. Recently, an atlas of genetic correlations among complex phenotypes has broadened our understanding of human diseases and traits. Here, we examine genetic overlap, from a gene-centric perspective, among the same 24 phenotypes previously investigated for genetic correlations. After adopting the multilevel pipeline (freely available at http://grass.cgs.hku.hk/limx/kgg/), which includes intragenic single nucleotide polymorphisms (SNPs), genes, and gene-sets, to estimate genetic similarities across phenotypes, a large amount of sharing of several biologically related phenotypes was confirmed. In addition, significant genetic overlaps were also found among phenotype pairs that were previously unidentified by SNP-level approaches. All these pairs with new genetic links are supported by earlier epidemiological evidence, although only a few of them have pleiotropic genes in the GWAS Catalog. Hence, our gene and gene-set analyses are able to provide new insights into cross-phenotype connections. The investigation on genetic sharing at three different levels presents a complementary picture of how common DNA sequence variations contribute to disease comorbidities and trait manifestations.

A Trans-acting Factor May Modify Age at Onset in Familial Amyloid Polyneuropathy ATTRV30M in Portugal.

Although all familial amyloid polyneuropathy (FAP) ATTRV30M patients carry the same causative mutation, early (<40) and late-onset forms (≥50years) of FAP may coexist in the same family. However, this variability in age at onset is still unexplained. To identify modifiers closely linked to the TTR locus that may in part be associated with age at onset of FAP ATTRV30M, in particular in a group of very early-onset patients (≤30years) when compared with late-onset individuals. A clinical genetic study at a referral center comprising a sample of 910 Portuguese individuals includes 589 Val30Met carriers, 102 spouses, and 189 controls from the general population. Haplotype analysis was performed, using eight intragenic single nucleotide polymorphisms (SNPs) at the TTR locus. We compared haplotypes frequency in FAP samples and controls and in parent-offspring pairs using appropriated statistical analysis. Haplotype A was the most common in the general population. Noteworthy, haplotype C was more frequent in early-onset (<40) than in late-onset patients (≥50years) (p=0.012). When comparing allelic frequencies of each SNP within haplotype C between "very early" (≤30years) and late-onset (≥50years) cases, the A allele of rs72922947 was associated with an earlier onset (p=0.009); this remained significant after a permutation-based correction. Also, the heterozygous genotype (GA) for this SNP was associated with a decrease in mean age at onset of 8.6years (p=0.014). We found a more common haplotype (A) linked to the Val30Met variant and a possible modulatory trans effect on age at onset. These findings may lead to potential therapeutical targets.

Rapid selection of BRCA1-proficient tumor cells during neoadjuvant therapy for ovarian cancer in BRCA1 mutation carriers

Ovarian carcinomas (OC) often demonstrate rapid tumor shrinkage upon neoadjuvant chemotherapy (NACT). However, complete pathologic responses are very rare and the mechanisms underlying the emergence of residual tumor disease remain elusive. We hypothesized that the change of somatic BRCA1 status may contribute to this process. The loss-of-heterozygosity (LOH) at the BRCA1 locus was determined for 23 paired tumor samples obtained from BRCA1 germ-line mutation carriers before and after NACT. We observed a somatic loss of the wild-type BRCA1 allele in 74% (17/23) of OCs before NACT. However, a retention of the wild-type BRCA1 copy resulting in a reversion of LOH status was detected in 65% (11/17) of those patients after NACT. Furthermore, we tested 3 of these reversion samples for LOH at intragenic BRCA1 single nucleotide polymorphisms (SNPs) and confirmed a complete restoration of the SNP heterozygosity in all instances. The neoadjuvant chemotherapy for BRCA1-associated OC is accompanied by a rapid expansion of pre-existing BRCA1-proficient tumor clones suggesting that continuation of the same therapy after NACT and surgery may not be justified even in patients initially experiencing a rapid tumor regression.

Identification of unique venous thromboembolism-susceptibility variants in African-Americans

To identify novel single nucleotide polymorphisms (SNPs) associated with venous thromboembolism (VTE) in African-Americans (AAs), we performed a genome-wide association study (GWAS) of VTE in AAs using the Electronic Medical Records and Genomics (eMERGE) Network, comprised of seven sites each with DNA biobanks (total ~39,200 unique DNA samples) with genome-wide SNP data (imputed to 1000 Genomes Project cosmopolitan reference panel) and linked to electronic health records (EHRs). Using a validated EHR-driven phenotype extraction algorithm, we identified VTE cases and controls and tested for an association between each SNP and VTE using unconditional logistic regression, adjusted for age, sex, stroke, site-platform combination and sickle cell risk genotype. Among 393 AA VTE cases and 4,941 AA controls, three intragenic SNPs reached genome-wide significance: LEMD3 rs138916004 (OR=3.2; p=1.3E-08), LY86 rs3804476 (OR=1.8; p=2E-08) and LOC100130298 rs142143628 (OR=4.5; p=4.4E-08); all three SNPs validated using internal cross-validation, parametric bootstrap and meta-analysis methods. LEMD3 rs138916004 and LOC100130298 rs142143628 are only present in Africans (1000G data). LEMD3 showed a significant differential expression in both NCBI Gene Expression Omnibus (GEO) and the Mayo Clinic gene expression data, LOC100130298 showed a significant differential expression only in the GEO expression data, and LY86 showed a significant differential expression only in the Mayo expression data. LEMD3 encodes for an antagonist of TGF-β-induced cell proliferation arrest. LY86 encodes for MD-1 which down-regulates the pro-inflammatory response to lipopolysaccharide; LY86 variation was previously associated with VTE in white women; LOC100130298 is a non-coding RNA gene with unknown regulatory activity in gene expression and epigenetics.