Identification and in silico analysis of functional SNPs of the BRCA1 gene

Single-nucleotide polymorphisms (SNPs) play a major role in the understanding of the genetic basis of many complex human diseases. Also, the genetics of human phenotype variation could be understood by knowing the functions of these SNPs. It is still a major challenge to identify the functional SNPs in a disease-related gene. In this work, we have analyzed the genetic variation that can alter the expression and the function of the BRCA1 gene using computational methods. Of the total 477 SNPs, 65 were found to be nonsynonymous (ns) SNPs. Among the 14 SNPs in the untranslated region, 4 were found in the 5' and 10 were found in the 3' untranslated region (UTR). It was found that 16.9% of the nsSNPs were damaging, by both the SIFT and the PolyPhen servers. The UTR Resource tool suggested that 2 of 4 SNPs in the 5' UTR and 3 of 10 SNPs in the 3' UTR might change the protein expression levels. We identified major mutations from proline to serine at positions 1776 and 1812 of the native protein of the BRCA1 gene. From a comparison of the stabilizing residues of the native and mutant proteins, we propose that an nsSNP (rs1800751) could be an important candidate for the breast cancer caused by the BRCA1 gene.     

Computational detection of deleterious SNPs and their effect on sequence and structural level of the <Emphasis Type="Italic">VHL</Emphasis> gene

In this work we have analyzed the genetic variation that can alter the expression and the function of the VHL gene using computational methods. Of 110 single nucleotide polymorphisms (SNPs), 33 were found to be nonsynonymous (nsSNPs) and 23 SNPs were found in untranslated regions. Of the 33 nsSNPs investigated, 36.3% were found to be deleterious by both SIFT and PolyPhen servers. An untranslated region (UTR) resource tool suggested that two SNPs in the 5' UTR region and six SNPs in the 3' UTR region might change the protein expression levels. It was found by both SIFT and PolyPhen servers that a mutation from histidine to arginine at position 115 of the native protein of the VHL gene was most deleterious. A structural analysis of this mutated protein and the native protein was performed and had a root mean square deviation (RMSD) of 2.78 A. Based on this work, we propose that the nsSNP with a SNPid of rs5030812 is an important candidate for the cause of von Hippel-Lindau syndrome via the VHL gene.     

Serotonin Transporter Genotype Modulates Social Reward and Punishment in Rhesus Macaques

Background

Serotonin signaling influences social behavior in both human and nonhuman primates. In humans, variation upstream of the promoter region of the serotonin transporter gene (5-HTTLPR) has recently been shown to influence both behavioral measures of social anxiety and amygdala response to social threats. Here we show that length polymorphisms in 5-HTTLPR predict social reward and punishment in rhesus macaques, a species in which 5-HTTLPR variation is analogous to that of humans.

Methodology/Principal Findings

In contrast to monkeys with two copies of the long allele (L/L), monkeys with one copy of the short allele of this gene (S/L) spent less time gazing at face than non-face images, less time looking in the eye region of faces, and had larger pupil diameters when gazing at photos of a high versus low status male macaques. Moreover, in a novel primed gambling task, presentation of photos of high status male macaques promoted risk-aversion in S/L monkeys but promoted risk-seeking in L/L monkeys. Finally, as measured by a “pay-per-view” task, S/L monkeys required juice payment to view photos of high status males, whereas L/L monkeys sacrificed fluid to see the same photos.

Conclusions/Significance

These data indicate that genetic variation in serotonin function contributes to social reward and punishment in rhesus macaques, and thus shapes social behavior in humans and rhesus macaques alike.     

Regulatory elements of copia retrotransposons, controlling the level of its expression in Drosophila melanogaster testes

Expression of the lacZ reporter gene under the control of five deletion derivatives of the copia regulatory region including the 5' long terminal repeat (LTR) and the 5' untranslated region (UTR) was assayed in the testes of transgenic Drosophila melanogaster males (larvae and imago). The full-length copia regulatory region (LTR + UTR) ensured expression of the reporter gene in testes of both larvae and adult males. Deletion of UTR or 3' end of LTR increased lacZ expression in the testes, whereas deletion of the 5' end of LTR increased it. This indicated that a positive regulator of copia expression is at the 5' end of LTR and that negative regulators are at the 3' end of LTR and in UTR. The effects of the fragments of the copia regulatory region on reporter gene expression in the testes in vivo did not completely coincide with the effects observed earlier in cultured cells. We suggest that this difference is due to different regulation of expression of the fusion constructs integrated into chromatin as compared to their transient expression.     

Association of single nucleotide polymorphisms in the endothelial differentiation sphingolipid G-protein-coupled receptor 1 gene with marbling in Japanese Black beef cattle

Marbling defined by the amount and distribution of intramuscular fat, so-called Shimofuri , is an economically important trait of beef cattle in Japan. The endothelial differentiation sphingolipid G-protein-coupled receptor 1 ( EDG1 ) gene, involved in blood vessel formation, has been previously shown to be expressed at different levels in musculus longissimus muscle between low-marbled and high-marbled steer groups. It is located within the genomic region of a quantitative trait locus for marbling, and thus was considered as a positionally functional candidate for the gene responsible for marbling. In this study, two single nucleotide polymorphisms (SNPs) in the 5' untranslated region (UTR) and the 3' UTR of EDG1 , referred to as c. - 312A>G and c.*446G>A , respectively, were detected between the two steer groups. The two SNPs were associated with the predicted breeding value for beef marbling standard number by analyses using a population of Japanese Black beef cattle. The effect of genotypes at each of the SNPs on the predicted breeding value for subcutaneous fat thickness was not statistically significant ( P  >   0.05). Reporter gene assays revealed no significant differences in gene expression between alleles at each of the SNPs. These findings suggest that EDG1 SNPs, although they may not be regarded as a causal mutation, may be useful for effective marker-assisted selection to increase the levels of marbling in Japanese Black beef cattle.     

Applications of computational algorithm tools to identify functional SNPs in cytokine genes

Understanding the functions of single nucleotide polymorphisms (SNPs) can greatly help to understand the genetics of the human phenotype variation and especially the genetic basis of human complex diseases. However, how to identify functional SNPs from a pool containing both functional and neutral SNPs is challenging. In this study, we analyzed the genetic variations that can alter the expression and function of a group of cytokine proteins using computational tools. As a result, we extracted 4552 SNPs from 45 cytokine proteins from SNPper database. Of particular interest, 828 SNPs were in the 5'UTR region, 961 SNPs were in the 3' UTR region, and 85 SNPs were non-synonymous SNPs (nsSNPs), which cause amino acid change. Evolutionary conservation analysis using the SIFT tool suggested that 8 nsSNPs may disrupt the protein function. Protein structure analysis using the PolyPhen tool suggested that 5 nsSNPs might alter protein structure. Binding motif analysis using the UTResource tool suggested that 27 SNPs in 5' or 3'UTR might change protein expression levels. Our study demonstrates the presence of naturally occurring genetic variations in the cytokine proteins that may affect their expressions and functions with possible roles in complex human disease, such as immune diseases.     

Mining the LIPG allelic spectrum reveals the contribution of rare and common regulatory variants to HDL cholesterol

Genome-wide association studies (GWAS) have successfully identified loci associated with quantitative traits, such as blood lipids. Deep resequencing studies are being utilized to catalogue the allelic spectrum at GWAS loci. The goal of these studies is to identify causative variants and missing heritability, including heritability due to low frequency and rare alleles with large phenotypic impact. Whereas rare variant efforts have primarily focused on nonsynonymous coding variants, we hypothesized that noncoding variants in these loci are also functionally important. Using the HDL-C gene LIPG as an example, we explored the effect of regulatory variants identified through resequencing of subjects at HDL-C extremes on gene expression, protein levels, and phenotype. Resequencing a portion of the LIPG promoter and 5' UTR in human subjects with extreme HDL-C, we identified several rare variants in individuals from both extremes. Luciferase reporter assays were used to measure the effect of these rare variants on LIPG expression. Variants conferring opposing effects on gene expression were enriched in opposite extremes of the phenotypic distribution. Minor alleles of a common regulatory haplotype and noncoding GWAS SNPs were associated with reduced plasma levels of the LIPG gene product endothelial lipase (EL), consistent with its role in HDL-C catabolism. Additionally, we found that a common nonfunctional coding variant associated with HDL-C (rs2000813) is in linkage disequilibrium with a 5' UTR variant (rs34474737) that decreases LIPG promoter activity. We attribute the gene regulatory role of rs34474737 to the observed association of the coding variant with plasma EL levels and HDL-C. Taken together, the findings show that both rare and common noncoding regulatory variants are important contributors to the allelic spectrum in complex trait loci.     

Serotonin transporter: gene, genetic disorders, and pharmacogenetics

The highly evolutionarily conserved serotonin transporter (SERT) regulates the entire serotoninergic system and its receptors via modulation of extracellular fluid serotonin concentrations. Differences in SERT expression and function produced by three SERT genes and their variants show associations with multiple human disorders. Screens of DNA from patients with autism, ADHD, bipolar disorder, and Tourette's syndrome have detected signals in the chromosome 17q region where SERT is located. Parallel investigations of SERT knockout mice have uncovered multiple phenotypes that identify SERT as a candidate gene for additional human disorders ranging from irritable bowel syndrome to obesity. Replicated studies have demonstrated that the SERT 5'-flanking region polymorphism SS genotype is associated with poorer therapeutic responses and more frequent serious side effects during treatment with antidepressant SERT antagonists, namely, the serotonin reuptake inhibitors (SRIs).     

Computational and Structural Investigation of Deleterious Functional SNPs in Breast Cancer BRCA2 Gene

In this work, we have analyzed the genetic variation that can alter the expression and the function in BRCA2 gene using computational methods. Out of the total 534 SNPs, 101 were found to be non synonymous (nsSNPs). Among the 7 SNPs in the untranslated region, 3 SNPs were found in 5′ and 4 SNPs were found in 3′ un-translated regions (UTR). Of the nsSNPs 20.7% were found to be damaging by both SIFT and PolyPhen server among the 101 nsSNPs investigated. UTR resource tool suggested that 2 SNPs in the 5′ UTR region and 4 SNPs in the 3′ UTR regions might change the protein expression levels. The mutation from asparagine to isoleucine at the position 3124 of the native protein of BRCA2 gene was most deleterious by both SIFT and PolyPhen servers. A structural analysis of this mutated protein and the native protein was made which had an RMSD value of 0.301 nm. Based on this work, we proposed that this most deleterious nsSNP with an SNPid rs28897759 is an important candidate for the cause of breast cancer by BRCA2 gene.     

A Large‐Scale SNP‐Based Genomic Admixture Analysis of the Captive Rhesus Macaque Colony at the California National Primate Research Center

Some breeding facilities in the United States have crossbred Chinese and Indian rhesus macaque (Macaca mulatta) founders either purposefully or inadvertently. Genetic variation that reflects geographic origins among research subjects has the potential to influence experimental outcomes. The use of animals from different geographic regions, their hybrids, and animals of varying degrees of kinship in an experiment can obscure treatment effects under study because high interanimal genetic variance can increase phenotypic variance among the research subjects. The intent of this study, based on a broad genomic analysis of 2,808 single nucleotide polymorphisms (SNPs), is to ensure that only animals estimated to be of pure Indian or Chinese ancestry, based on both demographic and genetic information, are used as sources of infants for derivation and expansion of the California National Primate Research Center's (CNPRC) super‐Specific Pathogen Free (SSPF) rhesus macaque colony. Studies of short tandem repeats (STRs) in Indian and Chinese rhesus macaques have reported that heterozygosity of STRs is higher in Chinese rhesus macaques than in Indian rhesus macaques. The present study shows that heterozygosity of SNPs is actually higher in Indian than in Chinese rhesus macaques and that the Chinese SSPF rhesus macaque colony is far less differentiated from their founders compared to the Indian‐origin animals. The results also reveal no evidence of recent gene flow from long‐tailed and pig‐tailed macaques into the source populations of the SSPF rhesus macaques. This study indicates that many of the long‐tailed macaques held in the CNPRC are closely related individuals. Most polymorphisms shared among the captive rhesus, long‐tailed, and pig‐tailed macaques likely predate the divergence among these groups. Am. J. Primatol. 74:747‐757, 2012. © 2012 Wiley Periodicals, Inc.     

Allelic variation in gene expression identified through computational analysis of the dbEST database

Differential expression between the two alleles of an individual and between people with different genotypes has been commonly observed. Quantitative differences in gene expression between people may provide the genetic basis for the phenotypic difference between individuals and may be the primary cause of complex diseases. In this paper, we developed a computational method to identify genes that displayed allelic variation in gene expression in human EST libraries. To model allele-specific gene expression, we first identified EST libraries in which both A and B alleles were expressed and then identified allelic variation in gene expression based on the EST counts for each allele using a binomial test. Among 1107 SNPs that had a sufficient number of ESTs for the analysis, 524 (47%) displayed allelic variation in at least one cDNA library. We verified experimentally the allelic variation in gene expression for 6 of these SNPs. The frequency of allelic variation observed in EST libraries was similar to the previous studies using the SNP chip and primer extension method. We found that genes that displayed allelic variation were distributed throughout the human genome and were enriched in certain chromosome regions. The SNPs and genes identified in this study will provide a rich source for evaluating the effects of those SNPs and associated haplotypes in human health and diseases.     

Promoter and 3'-untranslated-region haplotypes in the vitamin d receptor gene predispose to osteoporotic fracture: the rotterdam study

Polymorphisms of the vitamin D receptor gene (VDR) have been shown to be associated with several complex diseases, including osteoporosis, but the mechanisms are unknown and study results have been inconsistent. We therefore determined sequence variation across the major relevant parts of VDR, including construction of linkage disequilibrium blocks and identification of haplotype alleles. We analyzed 15 haplotype-tagging SNPs in relation to 937 clinical fractures recorded in 6,148 elderly whites over a follow-up period of 7.4 years. Haplotype alleles of the 5' 1a/1e, 1b promoter region and of the 3' untranslated region (UTR) were strongly associated with increased fracture risk. For the 16% of subjects who had risk genotypes at both regions, their risk increased 48% for clinical fractures (P = .0002), independent of age, sex, height, weight, and bone mineral density. The population-attributable risk varied between 1% and 12% for each block and was 4% for the combined VDR risk genotypes. Functional analysis of the variants demonstrated 53% lower expression of a reporter construct with the 1e/1a promoter risk haplotype (P = 5 x 10(-7)) in two cell lines and 15% lower mRNA level of VDR expression constructs carrying 3'-UTR risk haplotype 1 in five cell lines (P = 2 x 10(-6)). In a further analysis, we showed 30% increased mRNA decay in an osteoblast cell line for the construct carrying the 3'-UTR risk haplotype (P = .02). This comprehensive candidate-gene analysis demonstrates that the risk allele of multiple VDR polymorphisms results in lower VDR mRNA levels. This could impact the vitamin D signaling efficiency and might contribute to the increased fracture risk we observed for these risk haplotype alleles.     

Functional expression of milligram quantities of the synthetic human serotonin transporter gene in a tetracycline-inducible HEK293 cell line

The serotonin transporter (SERT), a member of the solute carrier 6 family, is responsible for reuptake of the monoamine neurotransmitter serotonin (5-hydroxytryptamine) from the synaptic cleft on the neural cells, and a vital target for several antidepressants. To investigate biophysical studies of this pharmacologically relevant transporter, we developed a mammalian expression system with tetracycline-inducible HEK293 cells using synthetic human SERT genes produced by PCR-based self-assembly method. Codon-optimization of this de novo constructed genes and construction of stable cell lines improved expression 3.5-fold and single-step immunoaffinity purification with FLAG-epitope tag yielded around one milligram functional SERT per liter culture medium assessed by [(3)H] imipramine ligand binding. Some characterizations including electrospray ionization MS/MS analysis, subcellular localization and cellular-uptake assay demonstrated that expressed human SERT was properly expressed, folded and fully functional. The long cytosolic N-terminal of SERT was predicted as containing 'intrinsically disordered region (IDR)' (～85 residues) by DISOPRED2 program. We engineered this salient region by step-wise truncation and ligand binding assay determined that dissociation constant for a series of de novo designed truncation constructs was close to the one for full-length wild type SERT. Our expression platform using synthetic codon-optimized gene and mammalian stable cell lines is feasible to produce milligram-scale functional membrane transporter for further biophysical and biochemical studies.