Genetic Copy Number Variation and General Cognitive Ability

Differences in genomic structure between individuals are ubiquitous features of human genetic variation. Specific copy number variants (CNVs) have been associated with susceptibility to numerous complex psychiatric disorders, including attention-deficit-hyperactivity disorder, autism-spectrum disorders and schizophrenia. These disorders often display co-morbidity with low intelligence. Rare chromosomal deletions and duplications are associated with these disorders, so it has been suggested that these deletions or duplications may be associated with differences in intelligence. Here we investigate associations between large (≥500kb), rare (<1% population frequency) CNVs and both fluid and crystallized intelligence in community-dwelling older people. We observe no significant associations between intelligence and total CNV load. Examining individual CNV regions previously implicated in neuropsychological disorders, we find suggestive evidence that CNV regions around SHANK3 are associated with fluid intelligence as derived from a battery of cognitive tests. This is the first study to examine the effects of rare CNVs as called by multiple algorithms on cognition in a large non-clinical sample, and finds no effects of such variants on general cognitive ability.     

No association between genetic variants in angiogenesis and inflammation pathway genes and breast cancer survival among Chinese women

Background: Angiogenesis and inflammation are implicated in breast cancer prognosis; however, the role of individual germline variation in related genes is unknown. Methods: A two-stage candidate pathway association study was conducted among 6983 Chinese women. Stage 1 included 2884 women followed for a median of 5.7 years; Stage 2 included 4099 women followed for a median of 4.0 years. Cox proportional hazards regression was used to estimate the effects of genetic variants on disease-free survival (DFS) and overall survival (OS). Results: Stage 1 included genotyping of 506 variants in 22 genes; analysis was conducted for 370 common variants. Nominally significant associations with DFS and/or OS were found for 20 loci in ten genes in Stage 1; variants in 19 loci were successfully genotyped and evaluated in Stage 2. In analyses of both study stages combined, nominally significant associations were found for nine variants in seven genes; none of these associations surpassed a significance threshold level corrected for the total number of variants evaluated in this study. Conclusions: No association with survival was found for 370 common variants in 22 angiogenesis and inflammation pathway genes among Chinese women with breast cancer. Impact: Our data do not support a large role for common genetic variation in 22 genes in breast cancer prognosis; research on angiogenesis and inflammation genes should focus on common variation in other genes, rare host variants, or tumor alterations.     

Whole Exome Sequencing in Atrial Fibrillation

Atrial fibrillation (AF) is a morbid and heritable arrhythmia. Over 35 genes have been reported to underlie AF, most of which were described in small candidate gene association studies. Replication remains lacking for most, and therefore the contribution of coding variation to AF susceptibility remains poorly understood. We examined whole exome sequencing data in a large community-based sample of 1,734 individuals with and 9,423 without AF from the Framingham Heart Study, Cardiovascular Health Study, Atherosclerosis Risk in Communities Study, and NHLBI-GO Exome Sequencing Project and meta-analyzed the results. We also examined whether genetic variation was enriched in suspected AF genes (N = 37) in AF cases versus controls. The mean age ranged from 59 to 73 years; 8,656 (78%) were of European ancestry. None of the 99,404 common variants evaluated was significantly associated after adjusting for multiple testing. Among the most significantly associated variants was a common (allele frequency = 86%) missense variant in SYNPO2L (rs3812629, p.Pro707Leu, [odds ratio 1.27, 95% confidence interval 1.13–1.43, P = 6.6x10^-5]) which lies at a known AF susceptibility locus and is in linkage disequilibrium with a top marker from prior analyses at the locus. We did not observe significant associations between rare variants and AF in gene-based tests. Individuals with AF did not display any statistically significant enrichment for common or rare coding variation in previously implicated AF genes. In conclusion, we did not observe associations between coding genetic variants and AF, suggesting that large-effect coding variation is not the predominant mechanism underlying AF. A coding variant in SYNPO2L requires further evaluation to determine whether it is causally related to AF. Efforts to identify biologically meaningful coding variation underlying AF may require large sample sizes or populations enriched for large genetic effects.     

Assessing the prevalence of PINK1 genetic variants in South African patients diagnosed with early- and late-onset Parkinson’s disease

Mutations in the PINK1 gene are the second most common cause after parkin of autosomal recessive early-onset Parkinson’s disease (PD). PINK1 is a protein kinase that is localized to the mitochondrion and is ubiquitously expressed in the human brain. Recent studies aimed at elucidating the function of PINK1, have found that it has neuroprotective properties against mitochondrial dysfunction and proteasomally-induced apoptosis. In the present study, we aimed to investigate the prevalence of PINK1 genetic variants in 154 South African PD patients from all ethnic groups. Mutation screening was performed using the High-Resolution Melt technique and direct sequencing. A total of 16 sequence variants were identified: one known homozygous mutation (Y258X), two heterozygous missense variants (P305A and E476K), and 13 polymorphisms of which five were novel. No homozygous exonic deletions were detected. The novel P305A variant was found in a female patient of Black Xhosa ethnicity who has a positive family history of the disease and an age at onset of 30 years. This variant has the potential to modulate enzymatic activity due to its location in the kinase domain. This is the first report on mutation screening of PINK1 in the South African population. Results from the present study showed that point mutations and homozygous exonic deletions in PINK1 are not a common cause of PD in the South African population.     

Defective pre-mRNA splicing in PKD1 due to presumed missense and synonymous mutations causing autosomal dominant polycystic disease

Autosomal dominant polycystic kidney disease is the most common human monogenic disorder and is caused by mutations in the PKD1 or PKD2 genes. Most patients with the disease present mutations in PKD1, and a considerable number of these alterations are single base substitutions within the coding sequence that are usually predicted to lead to missense or synonymous mutations. There is growing evidence that some of these mutations can be detrimental by affecting the pre-mRNA splicing process. The aim of our study was to test PKD1 mutations, described as missense or synonymous in the literature or databases, for their effects on exon inclusion. Bioinformatics tools were used to select mutations with a potential effect on pre-mRNA splicing. Mutations were experimentally tested using minigene assays. Exons and adjacent intronic sequences were PCR-amplified and cloned in the splicing reporter minigene, and selected mutations were introduced by site-directed mutagenesis. Minigenes were transfected into kidney derived cell lines. RNA from cultured cells was analyzed by RT-PCR and DNA sequencing. Analysis of thirty-three PKD1 exonic mutations revealed three mutations that induce splicing defects. The substitution c.11156G > A, previously predicted as missense mutation p.R3719Q, abolished the donor splice site of intron 38 and resulted in the incorporation of exon 38 with 117 bp of intron 38 and skipping of exon 39. Two synonymous variants, c.327A > T (p.G109G) and c.11257C > A (p.R3753R), generated strong donor splice sites within exons 3 and 39 respectively, resulting in incorporation of incomplete exons. These three nucleotide substitutions represent the first PKD1 exonic mutations that induce aberrant mRNAs. Our results strengthen the importance to evaluate the consequences of presumed missense and synonymous mutations at the mRNA level.     

Exome Sequencing in 53 Sporadic Cases of Schizophrenia Identifies 18 Putative Candidate Genes

Schizophrenia (SCZ) is a severe, debilitating mental illness which has a significant genetic component. The identification of genetic factors related to SCZ has been challenging and these factors remain largely unknown. To evaluate the contribution of de novo variants (DNVs) to SCZ, we sequenced the exomes of 53 individuals with sporadic SCZ and of their non-affected parents. We identified 49 DNVs, 18 of which were predicted to alter gene function, including 13 damaging missense mutations, 2 conserved splice site mutations, 2 nonsense mutations, and 1 frameshift deletion. The average number of exonic DNV per proband was 0.88, which corresponds to an exonic point mutation rate of 1.7×10⁻⁸ per nucleotide per generation. The non-synonymous-to-synonymous mutation ratio of 2.06 did not differ from neutral expectations. Overall, this study provides a list of 18 putative candidate genes for sporadic SCZ, and when combined with the results of similar reports, identifies a second proband carrying a non-synonymous DNV in the RGS12 gene.     

Re-sequencing Expands Our Understanding of the Phenotypic Impact of Variants at GWAS Loci

Genome-wide association studies (GWAS) have identified >500 common variants associated with quantitative metabolic traits, but in aggregate such variants explain at most 20–30% of the heritable component of population variation in these traits. To further investigate the impact of genotypic variation on metabolic traits, we conducted re-sequencing studies in >6,000 members of a Finnish population cohort (The Northern Finland Birth Cohort of 1966 [NFBC]) and a type 2 diabetes case-control sample (The Finland-United States Investigation of NIDDM Genetics [FUSION] study). By sequencing the coding sequence and 5′ and 3′ untranslated regions of 78 genes at 17 GWAS loci associated with one or more of six metabolic traits (serum levels of fasting HDL-C, LDL-C, total cholesterol, triglycerides, plasma glucose, and insulin), and conducting both single-variant and gene-level association tests, we obtained a more complete understanding of phenotype-genotype associations at eight of these loci. At all eight of these loci, the identification of new associations provides significant evidence for multiple genetic signals to one or more phenotypes, and at two loci, in the genes ABCA1 and CETP, we found significant gene-level evidence of association to non-synonymous variants with MAF<1%. Additionally, two potentially deleterious variants that demonstrated significant associations (rs138726309, a missense variant in G6PC2, and rs28933094, a missense variant in LIPC) were considerably more common in these Finnish samples than in European reference populations, supporting our prior hypothesis that deleterious variants could attain high frequencies in this isolated population, likely due to the effects of population bottlenecks. Our results highlight the value of large, well-phenotyped samples for rare-variant association analysis, and the challenge of evaluating the phenotypic impact of such variants.     

Canonical Correlation Analysis for Gene-Based Pleiotropy Discovery

Genome-wide association studies have identified a wealth of genetic variants involved in complex traits and multifactorial diseases. There is now considerable interest in testing variants for association with multiple phenotypes (pleiotropy) and for testing multiple variants for association with a single phenotype (gene-based association tests). Such approaches can increase statistical power by combining evidence for association over multiple phenotypes or genetic variants respectively. Canonical Correlation Analysis (CCA) measures the correlation between two sets of multidimensional variables, and thus offers the potential to combine these two approaches. To apply CCA, we must restrict the number of attributes relative to the number of samples. Hence we consider modules of genetic variation that can comprise a gene, a pathway or another biologically relevant grouping, and/or a set of phenotypes. In order to do this, we use an attribute selection strategy based on a binary genetic algorithm. Applied to a UK-based prospective cohort study of 4286 women (the British Women''s Heart and Health Study), we find improved statistical power in the detection of previously reported genetic associations, and identify a number of novel pleiotropic associations between genetic variants and phenotypes. New discoveries include gene-based association of NSF with triglyceride levels and several genes (ACSM3, ERI2, IL18RAP, IL23RAP and NRG1) with left ventricular hypertrophy phenotypes. In multiple-phenotype analyses we find association of NRG1 with left ventricular hypertrophy phenotypes, fibrinogen and urea and pleiotropic relationships of F7 and F10 with Factor VII, Factor IX and cholesterol levels.     

Genetic association of marbling score with intragenic nucleotide variants at selection signals of the bovine genome

Selection signals of Korean cattle might be attributed largely to artificial selection for meat quality. Rapidly increased intragenic markers of newly annotated genes in the bovine genome would help overcome limited findings of genetic markers associated with meat quality at the selection signals in a previous study. The present study examined genetic associations of marbling score (MS) with intragenic nucleotide variants at selection signals of Korean cattle. A total of 39 092 nucleotide variants of 407 Korean cattle were utilized in the association analysis. A total of 129 variants were selected within newly annotated genes in the bovine genome. Their genetic associations were analyzed using the mixed model with random polygenic effects based on identical-by-state genetic relationships among animals in order to control for spurious associations produced by population structure. Genetic associations of MS were found (P<3.88×10⁻⁴) with six intragenic nucleotide variants on bovine autosomes 3 (cache domain containing 1, CACHD1), 5 (like-glycosyltransferase, LARGE), 16 (cell division cycle 42 binding protein kinase alpha, CDC42BPA) and 21 (snurportin 1, SNUPN; protein tyrosine phosphatase, non-receptor type 9, PTPN9; chondroitin sulfate proteoglycan 4, CSPG4). In particular, the genetic associations with CDC42BPA and LARGE were confirmed using an independent data set of Korean cattle. The results implied that allele frequencies of functional variants and their proximity variants have been augmented by directional selection for greater MS and remain selection signals in the bovine genome. Further studies of fine mapping would be useful to incorporate favorable alleles in marker-assisted selection for MS of Korean cattle.     

Association and Mutation Analyses of 16p11.2 Autism Candidate Genes

Background

Autism is a complex childhood neurodevelopmental disorder with a strong genetic basis. Microdeletion or duplication of a ∼500–700-kb genomic rearrangement on 16p11.2 that contains 24 genes represents the second most frequent chromosomal disorder associated with autism. The role of common and rare 16p11.2 sequence variants in autism etiology is unknown.

Methodology/Principal Findings

To identify common 16p11.2 variants with a potential role in autism, we performed association studies using existing data generated from three microarray platforms: Affymetrix 5.0 (777 families), Illumina 550 K (943 families), and Affymetrix 500 K (60 families). No common variants were identified that were significantly associated with autism. To look for rare variants, we performed resequencing of coding and promoter regions for eight candidate genes selected based on their known expression patterns and functions. In total, we identified 26 novel variants in autism: 13 exonic (nine non-synonymous, three synonymous, and one untranslated region) and 13 promoter variants. We found a significant association between autism and a coding variant in the seizure-related gene SEZ6L2 (12/1106 autism vs. 3/1161 controls; p = 0.018). Sez6l2 expression in mouse embryos was restricted to the spinal cord and brain. SEZ6L2 expression in human fetal brain was highest in post-mitotic cortical layers, hippocampus, amygdala, and thalamus. Association analysis of SEZ6L2 in an independent sample set failed to replicate our initial findings.

Conclusions/Significance

We have identified sequence variation in at least one candidate gene in 16p11.2 that may represent a novel genetic risk factor for autism. However, further studies are required to substantiate these preliminary findings.     

Genome-wide studies of copy number variation and exome sequencing identify rare variants in BAG3 as a cause of dilated cardiomyopathy

Dilated cardiomyopathy commonly causes heart failure and is the most frequent precipitating cause of heart transplantation. Familial dilated cardiomyopathy has been shown to be caused by rare variant mutations in more than 30 genes but only ~35% of its genetic cause has been identified, principally by using linkage-based or candidate gene discovery approaches. In a multigenerational family with autosomal dominant transmission, we employed whole-exome sequencing in a proband and three of his affected family members, and genome-wide copy number variation in the proband and his affected father and unaffected mother. Exome sequencing identified 428 single point variants resulting in missense, nonsense, or splice site changes. Genome-wide copy number analysis identified 51 insertion deletions and 440 copy number variants > 1 kb. Of these, a 8733 bp deletion, encompassing exon 4 of the heat shock protein cochaperone BCL2-associated athanogene 3 (BAG3), was found in seven affected family members and was absent in 355 controls. To establish the relevance of variants in this protein class in genetic DCM, we sequenced the coding exons in BAG3 in 311 other unrelated DCM probands and identified one frameshift, two nonsense, and four missense rare variants absent in 355 control DNAs, four of which were familial and segregated with disease. Knockdown of bag3 in a zebrafish model recapitulated DCM and heart failure. We conclude that new comprehensive genomic approaches have identified rare variants in BAG3 as causative of DCM.     

Comprehensive Evaluation of the Association of APOE Genetic Variation with Plasma Lipoprotein Traits in U.S. Whites and African Blacks

Although common APOE genetic variation has a major influence on plasma LDL-cholesterol, its role in affecting HDL-cholesterol and triglycerides is not well established. Recent genome-wide association studies suggest that APOE also affects plasma variation in HDL-cholesterol and triglycerides. It is thus important to resequence the APOE gene to identify both common and uncommon variants that affect plasma lipid profile. Here, we have sequenced the APOE gene in 190 subjects with extreme HDL-cholesterol levels selected from two well-defined epidemiological samples of U.S. non-Hispanic Whites (NHWs) and African Blacks followed by genotyping of identified variants in the entire datasets (623 NHWs, 788 African Blacks) and association analyses with major lipid traits. We identified a total of 40 sequence variants, of which 10 are novel. A total of 32 variants, including common tagSNPs (≥5% frequency) and all uncommon variants (<5% frequency) were successfully genotyped and considered for genotype-phenotype associations. Other than the established associations of APOE*2 and APOE*4 with LDL-cholesterol, we have identified additional independent associations with LDL-cholesterol. We have also identified multiple associations of uncommon and common APOE variants with HDL-cholesterol and triglycerides. Our comprehensive sequencing and genotype-phenotype analyses indicate that APOE genetic variation impacts HDL-cholesterol and triglycerides in addition to affecting LDL-cholesterol.     

Maternal coding variants in complement receptor 1 and spontaneous idiopathic preterm birth

Preterm birth (PTB) is a major global public health concern. However, little is known about the pathophysiology of spontaneous idiopathic PTB. We tested the hypothesis that rare variants in families would target specific genes and pathways that contribute to PTB risk in the general population. Whole-exome sequencing was performed on 10 PTB mothers from densely affected families including two mother–daughter pairs. We identified novel variants shared between the two mother–daughter pairs when compared to a 1000 Genomes Project background exome file and investigated these genes for pathway aggregation using the Kyoto Encyclopedia of Genes and Genomes (KEGG). Genes in enriched pathways were then surveyed in the other six PTB exomes and tested for association in a larger number of nuclear families. The KEGG complement and coagulation cascade was one of the most enriched pathways in our two mother–daughter pairs. When the six genes found in this pathway (CFH, CR1, F13B, F5, CR2, and C4BPA) were examined for novel missense variants, half of all the exomes harbored at least one. Association analysis of variants in these six gene regions in nuclear families from Finland (237 cases and 328 controls) found statistically significant associations after multiple test corrections in three CR1 SNPs; the strongest in an exonic missense SNP, rs6691117, p value = 6.91e?5, OR = 1.71. Our results demonstrate the importance of the complement and coagulation cascades in the pathophysiology of PTB, and suggest potential screening and intervention approaches to prevent prematurity that target this pathway.     

Distribution and Medical Impact of Loss-of-Function Variants in the Finnish Founder Population

Exome sequencing studies in complex diseases are challenged by the allelic heterogeneity, large number and modest effect sizes of associated variants on disease risk and the presence of large numbers of neutral variants, even in phenotypically relevant genes. Isolated populations with recent bottlenecks offer advantages for studying rare variants in complex diseases as they have deleterious variants that are present at higher frequencies as well as a substantial reduction in rare neutral variation. To explore the potential of the Finnish founder population for studying low-frequency (0.5–5%) variants in complex diseases, we compared exome sequence data on 3,000 Finns to the same number of non-Finnish Europeans and discovered that, despite having fewer variable sites overall, the average Finn has more low-frequency loss-of-function variants and complete gene knockouts. We then used several well-characterized Finnish population cohorts to study the phenotypic effects of 83 enriched loss-of-function variants across 60 phenotypes in 36,262 Finns. Using a deep set of quantitative traits collected on these cohorts, we show 5 associations (p<5×10⁻⁸) including splice variants in LPA that lowered plasma lipoprotein(a) levels (P = 1.5×10⁻¹¹⁷). Through accessing the national medical records of these participants, we evaluate the LPA finding via Mendelian randomization and confirm that these splice variants confer protection from cardiovascular disease (OR = 0.84, P = 3×10⁻⁴), demonstrating for the first time the correlation between very low levels of LPA in humans with potential therapeutic implications for cardiovascular diseases. More generally, this study articulates substantial advantages for studying the role of rare variation in complex phenotypes in founder populations like the Finns and by combining a unique population genetic history with data from large population cohorts and centralized research access to National Health Registers.     

Common Genetic Variants in FOXP2 Are Not Associated with Individual Differences in Language Development

Much of our current knowledge regarding the association of FOXP2 with speech and language development comes from singleton and small family studies where a small number of rare variants have been identified. However, neither genome-wide nor gene-specific studies have provided evidence that common polymorphisms in the gene contribute to individual differences in language development in the general population. One explanation for this inconsistency is that previous studies have been limited to relatively small samples of individuals with low language abilities, using low density gene coverage. The current study examined the association between common variants in FOXP2 and a quantitative measure of language ability in a population-based cohort of European decent (n = 812). No significant associations were found for a panel of 13 SNPs that covered the coding region of FOXP2 and extended into the promoter region. Power analyses indicated we should have been able to detect a QTL variance of 0.02 for an associated allele with MAF of 0.2 or greater with 80% power. This suggests that, if a common variant associated with language ability in this gene does exist, it is likely of small effect. Our findings lead us to conclude that while genetic variants in FOXP2 may be significant for rare forms of language impairment, they do not contribute appreciably to individual variation in the normal range as found in the general population.     

Exome-Wide Association Study Identified New Risk Loci for Hirschsprung’s Disease

Hirschsprung disease (HSCR) is a rare congenital disease caused by impaired proliferation and migration of neural crest cells. In this study, we aimed to investigate the genetic loci involved in the pathogenesis of HSCR. The exome-wide scan was performed to screen the genetic variants with minor allele frequency (MAF)?<?0.05 in exonic regions. Candidate mutation type and the wild type were overexpressed to investigate the affection on cell proliferation and migration. We found that ten variants were associated with HSCR at P?<?10^?4 in the single-variant analysis while ten genes were also associated with HSCR at P?<?10^?4 in the optimized sequence kernel association test (SKAT-O) test analysis. Among these SNPs, the missense variants catechol-O-methyltransferase (COMT) (rs6267) and armadillo repeat gene deleted in velocardiofacial syndrome (ARVCF) (rs80068543) indicated an ectopic expression in colon tissues of HSCR patients. The Ala72Ser variant in COMT induced proliferation suppression through NOTCH signal pathway, while the ARVCF affected cell migration via the downregulating of RHOA and ROC. In conclusion, this exome array study identified the COMT and ARVCF missense coding variants as candidate loci for HSCR. The finding implies the abnormal variant of COMT and ARVCF may account for the pathogenesis of HSCR.     

De novo mutations in ARID1B associated with both syndromic and non-syndromic short stature

Background

Human height is a complex trait with a strong genetic basis. Recently, a significant association between rare copy number variations (CNVs) and short stature has been identified, and candidate genes in these rare CNVs are being explored. This study aims to evaluate the association between mutations in ARID1B gene and short stature, both the syndromic and non-syndromic form.

Results

Based on a case-control study of whole genome chromosome microarray analysis (CMA), three overlapping CNVs were identified in patients with developmental disorders who exhibited short stature. ARID1B, a causal gene for Coffin Siris syndrome, is the only gene encompassed by all three CNVs. A following retrospective genotype-phenotype analysis based on a literature review confirmed that short stature is a frequent feature in those Coffin-Siris syndrome patients with ARID1B mutations. Mutation screening of ARID1B coding regions was further conducted in a cohort of 48 non-syndromic short stature patients,andfour novel missense variants including two de novo mutations were found.

Conclusion

These results suggest that haploinsufficient mutations of ARID1B are associated with syndromic short stature including Coffin-Siris syndrome and intellectual disability, while rare missense variants in ARID1B are associated with non-syndromic short stature. This study supports the notion that mutations in genes related to syndromic short stature may exert milder effect and contribute to short stature in the general population.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1898-1) contains supplementary material, which is available to authorized users.     

GSTM1, GSTT1, GSTP1, and GSTA1 genetic variants are not associated with coronary artery disease in Taiwan

Background and objective

The genetic variants of xenobiotic-metabolizing enzymes, such as those encoded by glutathione-S-transferase (GST) genes, may be associated with the risk of coronary artery disease (CAD). To investigate the genetic factors for CAD, we examined the GSTM1, GSTT1, GSTP1, and GSTA1 genotypes in a CAD cohort in Taiwan.

Methods

Our study included 458 CAD participants and 209 control participants who received coronary angiography to assess CAD. The severity of CAD was defined as the number of coronary vessels with 50% or greater stenosis. Sequence variation of the GSTM1 and GSTT1 genes was determined using a polymerase chain reaction (PCR). The GSTP1 (Ile105Val), and GSTA1 (-69C > T) genetic variants were identified using a combination of PCR and restriction fragment length polymorphism analysis. Logistic regression analysis was used to calculate the odds ratios (ORs) and 95% confidence intervals.

Results

Among the GST genetic variants examined, the GSTT1 null genotype was more prevalent in CAD participants with 3 stenosed vessels than in control participants (OR = 1.64, P = .02). This association was no longer observed after adjusting for age, sex, smoking, alcohol use, diabetes mellitus, and serum levels of total cholesterol and high-density lipoprotein cholesterol (OR = 1.28, P = .40). Both univariate and multivariate logistic regression analyses found no significant associations between CAD and the other genetic variants, either separately or in combination. In addition, no effects of interactions between the genotypes and environmental factors, such as cigarette smoking, were significantly associated with the risk of CAD.

Conclusion

The GST genetic variants examined were not associated with susceptibility to CAD in our Taiwanese cohort. This null association requires further confirmation with larger samples.     

Differential expression of alternative splice variants of CTLA4 in Kuwaiti autoimmune disease patients

Cytotoxic T lymphocyte associated antigen4 (CTLA4) is a candidate susceptibility gene for the study of autoimmune diseases. The present study sought to explore the expression profile of the CTLA4 gene in autoimmune patients, such as rheumatoid arthritis (RA), systemic lupus erythematous (SLE) and Hashimoto's thyroiditis (HT), compared to healthy controls (HCs). A total of 88 (22 RA, 22 SLE 22 HT, 22 HCs) age-, gender- and ethnicity-matched individuals were recruited. The hypersensitive capillary electrophoresis method was employed to detect the CTLA4 splice variants. PCRs of the patient's cDNA using CTLA4-specific primers followed by cloning and sequencing were used to distinguish the various splice variants. The biochemical properties of all known CTLA4 variants were analysed using the ExPASy and ESEfinder programmes. Six alternatively spliced variants of the CTLA4 gene were detected in this study. These included mCTLA4-672, sCTLA4-562, N-CTLA4-292, L-CTLA4-277, ssCTLA4-214 and K-CTLA4-142 bp. K-CTLA4-142 bp and N-CTLA4-292 bp represented two novel splice variants of the CTLA4 gene. A reduction in the frequency of mCTLA4-672 bp and sCTLA4-562 bp was observed in SLE and RA patients compared to healthy controls. The shortest splice variant, K-CTLA4-142 bp, was predominantly detected in all of the tested cohorts, while the decreased expression of the N-CTLA4-292 bp variant was observed in the autoimmune subjects. The exonic splice enhancer motifs of the SRp40 protein were found exactly at the splice junction of wCTLA4 (-ACAGAGC-, 2.7) and K-CTLA4 (-TGAAAAG-, 3.37), and that of the SRp55 protein was found at the splice junction of L-CTLA4 (-TGTGTG-, 2.82). Our study highlights the discrepancies in the expression spectrum of the CTLA4 gene in autoimmune patients and healthy subjects. The abnormal expression pattern of the CTLA4 gene in autoimmune patients suggests that in addition to allelic variation, the expression pattern of CTLA4 could contribute to autoimmunity.