<Emphasis Type="Italic">RET</Emphasis> gene mutations and polymorphisms in medullary thyroid carcinomas in Indian patients

Germline mutations of RET gene are pathognomonic of multiple endocrine neoplasia (MEN; MEN 2A/MEN 2B) and familial medullary thyroid carcinoma (FMTC), constituting 25% of medullary thyroid carcinomas (MTCs). We investigated RET gene mutations and polymorphisms at exons 10, 11, 13, 14, 15 and 16 in 140 samples, comprising 51 clinically diagnosed MTC patients, 39 family members of patients and 50 normal individuals. The method of choice was PCR and direct nucleotide sequencing of the PCR products. RET gene mutations were detected in 15 (29.4%) patients, with MEN 2A/FMTC in 13 patients and MEN 2B in 2 patients. Further, 39 family members of seven index cases were analysed, wherein four of the seven index cases showed identical mutations, in 13 of 25 family members. We also examined single nucleotide polymorphisms (SNPs) in RET gene exons in 101 unrelated samples. Significant differences in the allelic frequencies of SNPs at codons 691, 769, 836 and 904 between patient and control groups were not observed. However, SNP frequencies were significantly different in the Indian group as compared with other European groups. We identified two novel, rare and unique SNPs separately in single patients. Our study demonstrated presence of MEN 2A/MEN 2B/FMTC-associated mutations in accordance with the reported literature. Thus, RET gene mutations in exons 10, 11, 13, 14, 15 and 16 constitute a rapid test to confirm diagnosis and assess risk of the disease in familial MEN 2A/MEN 2B/FMTC.     

Identification of common genetic variation that modulates alternative splicing

Alternative splicing of genes is an efficient means of generating variation in protein function. Several disease states have been associated with rare genetic variants that affect splicing patterns. Conversely, splicing efficiency of some genes is known to vary between individuals without apparent ill effects. What is not clear is whether commonly observed phenotypic variation in splicing patterns, and hence potential variation in protein function, is to a significant extent determined by naturally occurring DNA sequence variation and in particular by single nucleotide polymorphisms (SNPs). In this study, we surveyed the splicing patterns of 250 exons in 22 individuals who had been previously genotyped by the International HapMap Project. We identified 70 simple cassette exon alternative splicing events in our experimental system; for six of these, we detected consistent differences in splicing pattern between individuals, with a highly significant association between splice phenotype and neighbouring SNPs. Remarkably, for five out of six of these events, the strongest correlation was found with the SNP closest to the intron–exon boundary, although the distance between these SNPs and the intron–exon boundary ranged from 2 bp to greater than 1,000 bp. Two of these SNPs were further investigated using a minigene splicing system, and in each case the SNPs were found to exert cis-acting effects on exon splicing efficiency in vitro. The functional consequences of these SNPs could not be predicted using bioinformatic algorithms. Our findings suggest that phenotypic variation in splicing patterns is determined by the presence of SNPs within flanking introns or exons. Effects on splicing may represent an important mechanism by which SNPs influence gene function.     

Molecular characterization of the equine ATP2A2 gene

The mammalian ATP2A2 gene encodes a P-type cation pump located in the sarcoplasmic or endoplasmic reticula of muscle cells. We isolated one bacterial artificial chromosome (BAC) clone containing the equine ATP2A2 gene and determined the complete coding sequence of this gene. Cloning and characterization of the equine ATP2A2 gene revealed that the equine ATP2A2 gene consists of 20 exons. In total, 32 horses out of 16 breeds were analyzed for single nucleotide polymorphisms (SNPs). A mutation scan for SNPs included ten exons and their flanking introns. We detected in total 17 SNPs, 14 of which were located in introns, one in exon 9 and two in exon 20. In this report we provide the genomic organization and the equine ATP2A2 coding sequence and an association analysis for chronic pastern dermatitis using a sample of South German draft horses.     

Genomic structure, polymorphism and expression of ACCN1 and ACCN3 genes in the horse

A category of cation gate proteins was shown to be present in sensory neurons and act as receptors of protons present in tissues such as muscles. The Amiloride-sensitive Cation Channel, Neuronal (ACCN) gene family is known to play a role in the transmission of pain through specialized pH sensitive neurons. Muscles from horses submitted to strenuous exercises produce lactic acid, which may induce variable pain through ACCN differential properties. The sequences of the equine cDNAs were determined to be 2.6 kb in length with an open reading frame of 1539 bp for ACCN1 and 2.1 kb in length with an open reading frame of 1602 bp for ACCN3. The ACCN1 gene is 990 kb long and contains 10 exons, and the ACCN3 gene is 4.2 kb long and contains 11 exons. The equine ACCN1 and ACCN3 genes have an ubiquitous expression but ACCN1 is more highly expressed in the spinal cord. We identified one alternative ACCN3 splicing variant present in various equine tissues. These mRNA variants may encode two different protein isoforms 533 and 509 amino acids long. Ten single nucleotide polymorphisms (SNPs) were detected for ACCN1; five in the coding and five in the non-coding region, with no amino acid change, while the three SNPs identified in the coding region of the ACCN3 gene introduce amino acid changes. The equine in silico promoter sequence reveals a structure similar to those of other mammalian species, especially for the ACCN1 gene.     

Trans-eQTLs reveal that independent genetic variants associated with a complex phenotype converge on intermediate genes, with a major role for the HLA

For many complex traits, genetic variants have been found associated. However, it is still mostly unclear through which downstream mechanism these variants cause these phenotypes. Knowledge of these intermediate steps is crucial to understand pathogenesis, while also providing leads for potential pharmacological intervention. Here we relied upon natural human genetic variation to identify effects of these variants on trans-gene expression (expression quantitative trait locus mapping, eQTL) in whole peripheral blood from 1,469 unrelated individuals. We looked at 1,167 published trait- or disease-associated SNPs and observed trans-eQTL effects on 113 different genes, of which we replicated 46 in monocytes of 1,490 different individuals and 18 in a smaller dataset that comprised subcutaneous adipose, visceral adipose, liver tissue, and muscle tissue. HLA single-nucleotide polymorphisms (SNPs) were 10-fold enriched for trans-eQTLs: 48% of the trans-acting SNPs map within the HLA, including ulcerative colitis susceptibility variants that affect plausible candidate genes AOAH and TRBV18 in trans. We identified 18 pairs of unlinked SNPs associated with the same phenotype and affecting expression of the same trans-gene (21 times more than expected, P<10(-16)). This was particularly pronounced for mean platelet volume (MPV): Two independent SNPs significantly affect the well-known blood coagulation genes GP9 and F13A1 but also C19orf33, SAMD14, VCL, and GNG11. Several of these SNPs have a substantially higher effect on the downstream trans-genes than on the eventual phenotypes, supporting the concept that the effects of these SNPs on expression seems to be much less multifactorial. Therefore, these trans-eQTLs could well represent some of the intermediate genes that connect genetic variants with their eventual complex phenotypic outcomes.     

Obtaining of pure transferrins D, M and R from equine serum and determination of transferrin level in relation to phenotype

By the method of precipitation with Rivanol (2-ethoxy-6,9-diaminoacridine lactate) and ammonium sulphate followed by chromatography on DEAE cellulose three genetic variants of transferrin were purified from equine serum: D, M and R. Their molecular mass determined in this study was 80 000, and it was identical for all three variants, which differed slightly in their amino acid composition. The protein level was determined in the serum of 535 two-year-old thoroughbred English horses by the method of rocket immunoelectrophoresis using antibodies obtained against three transferrins. The individual variability of the protein level in horses of the same phenotype was fairly high (variability index 9-15%). No differences were observed in the transferrin level related to sex. It was found that the presence of D, F and H alleles was connected with a higher serum transferrin level, while O and R alleles were connected with a lower level.     

Variation in the ovocalyxin-32 gene in commercial egg-laying chickens and its relationship with egg production and egg quality traits

Avian eggshell quality is an important trait for commercial egg production, as the eggshell is the primary packaging material and antimicrobial barrier for the internal food resource. Strong eggshells are essential to ensure that eggs can reach their final destination without damage. Ovocalyxin-32 (OCX32) is a matrix protein found within the outer layers of the eggshell and in the cuticle. Numerous reports in the literature have identified association between variants in the gene encoding this protein, OCX32, and various eggshell quality traits. Thus, OCX32 is a candidate gene for selection for eggshell traits in commercial poultry populations. Sequencing of exons 2-6 of the OCX32 gene in eight elite brown and white eggshell commercial egg-laying lines revealed 28 SNPs and one SNP/indel. Eighteen of these SNPs were predicted to alter the amino acid sequence of the protein. Clusters of SNPs in complete linkage disequilibrium were found in both exons 2 and 6. A total of 19 different versions or protein-sequence haplotypes of the OCX32 protein were inferred, revealing considerable variation within commercial lines. Genotypes for 13 of the SNPs were determined for 330-1819 individuals per line. Trait association studies revealed a significant effect of OCX32 on shell color in white egg lines and line-specific significant effects on albumen height, early egg weight, puncture score, and yolk weight. Three of the lines showed a significant change in OCX32 frequency over time, indicating selection pressure for certain variants of this gene during the breeding program.     

Haplotypic variation and characteristics across the toll-like receptor 3 locus in chickens

Toll-like receptor 3 (TLR3) has an important protective function against viral infection. The ability of an individual to respond properly to TLR ligands may be impaired by variants located in the TLR genes. By directly PCR sequencing four exons and their flanking sequence of chicken TLR3, a total of 50 nucleotide variants were identified from five breeds. Tibetan chickens and Silkies exhibited more abundant variation sites and rare alleles. Thirty haplotypes were reconstructed, with 31 variants whose minor allelic frequency was above 5% in five breeds, which revealed four divergent clades. Chicken TLR3 was partitioned into three haplotype blocks by the htSNPer program, and six tag SNPs could be used to distinguish these 30 haplotypes. Thirty variants were located in the coding sequence of chicken TLR3, and 16 of them were non-synonymous substitutions. It is predicted that p.Ser180Gly amino substitution could form an N-myristoylation site; the p.Lys240Thr amino substitution in chicken TLR3 could result in the loss of one protein kinase C phosphorylation site. These data provide a basic understanding of chicken TLR3 sequence variation and provide haplotypic markers for disease association studies.     

Performance of genotype imputation for rare variants identified in exons and flanking regions of genes

Genotype imputation has the potential to assess human genetic variation at a lower cost than assaying the variants using laboratory techniques. The performance of imputation for rare variants has not been comprehensively studied. We utilized 8865 human samples with high depth resequencing data for the exons and flanking regions of 202 genes and Genome-Wide Association Study (GWAS) data to characterize the performance of genotype imputation for rare variants. We evaluated reference sets ranging from 100 to 3713 subjects for imputing into samples typed for the Affymetrix (500K and 6.0) and Illumina 550K GWAS panels. The proportion of variants that could be well imputed (true r²>0.7) with a reference panel of 3713 individuals was: 31% (Illumina 550K) or 25% (Affymetrix 500K) with MAF (Minor Allele Frequency) less than or equal 0.001, 48% or 35% with 0.0010.05. The performance for common SNPs (MAF>0.05) within exons and flanking regions is comparable to imputation of more uniformly distributed SNPs. The performance for rare SNPs (0.01相似文献   

Analysis of Starch Gene Diversity in the Wild Relatives of Oryza sativa

Genetic loci influencing traits important to humans have been selected during crop domestication. The starch properties of rice influence the ease of cooking and attractiveness of rice as a human food. Starch biosynthesis genes likely to influence starch properties in the grain were compared in wild and domesticated rice genotypes. Sequence variation was investigated in starch biosynthesis gene exons that have been reported to have a direct influence on rice amylose content, gelatinization temperature, and amylopectin chain length. Exons 6 and 10 of GBSSI, exon 8 of SSIIa and exons 11, 13, 14 and 16 of SBEIIb were amplified and sequenced from 13 wild Oryza species encompassing genome types AA to HHJJ. Thirty two single nucleotide polymorphisms (SNPs) were identified in the exons of GBSSI; 176 in exon 8 of SSIIa, and 43 in SBEIIb, giving a total of 251 SNPs among the species. Eighty six of these SNP caused changes in the encoded amino acid, of which 28 were missense mutations that affected highly conserved amino acids within the protein sequence of GBSSI, SSIIa or SBEIIb. Two indels were identified in Potamophila parviflora, a close relative of Zizania palustris, a North American native wild rice. Most of the nucleotide variations and non-conservative changes were observed in the genomes other than the AA genome species. This represents a genetic resource for use in rice starch manipulation. The impact of human selection at these loci can be deduced by comparison of modern cultivated genotypes with their wild progenitors.     

Multivariate eQTL mapping uncovers functional variation on the X-chromosome associated with complex disease traits

Very few studies have investigated the associations between genetic polymorphisms and gene expression on the X-chromosome. This is a major bottleneck when conducting functional follow-up studies of trait-associated variants, as those identified in genome-wide association studies (GWAS). We used a multivariate approach to test the association between individual single nucleotide polymorphisms (SNPs) and exon expression levels measured in 356 Epstein–Barr virus-transformed lymphoblastoid cell lines (LCLs) from the Geuvadis RNA sequencing project to identify SNPs associated with variation in gene expression on the X-chromosome, which we refer to as eSNPs. At an FDR of 5 %, we discovered 548 independent [linkage disequilibrium (LD) r ² < 0.1] eSNPs on the X-chromosome. Of these, 35 were in LD (r ² > 0.8) with previously published disease- or trait-associated variants identified through GWAS. One of the strongest eSNPs identified was rs35975601, which was associated with F8A1 expression (p value = 3 × 10^?20) and was in LD with a type 1 diabetes risk variant. Additionally, we identified a number of genes for which eSNPs were in LD with multiple diseases or traits, including DNASE1L1 which was mapped to bilirubin levels, type 1 diabetes and schizophrenia. Our results also indicate that multivariate exon-level analysis provides a more powerful approach than univariate gene-level analysis, particularly when SNPs influence the expression of different exons with different magnitude and/or direction of effect. The associations identified in our study may provide new insights into the molecular process by which gene expression may contribute to trait variation or disease risk in humans.     

Characterization of a single nucleotide polymorphism in the coding sequence of the bovine transferrin gene.

A single nucleotide polymorphism was identified in the coding sequence of the bovine transferrin gene. Two alleles (SSCP1 and SSCP2) were detected by SSCP analysis and the mutation point was identified and confirmed by direct sequencing of the PCR products. The relationship between protein and DNA polymorphism was established. Protein variants A, D1 and E correspond to SSCP allele 1 and variant D2 corresponds to SSCP allele 2. DNA sequences from genotypes AA, AE, AD2, D1E, D2E and D2D2 reveal an A/G substitution at position 1455 of the cDNA which causes a Gly/Glu substitution which could be responsible for the mobility difference between D1 and D2 variants. Because of the number of variants, this suggests that other SNPs exist in the bovine transferrin gene. A linkage analysis between the SSCPs and two microsatellites (UWCA46 and CSSM019) mapped the transferrin gene to BTA1. Two-point analysis revealed a tight linkage within the transferrin protein variants and the SSCPs.     

Association analysis of 9,560 prostate cancer cases from the International Consortium of Prostate Cancer Genetics confirms the role of reported prostate cancer associated SNPs for familial disease

Previous GWAS studies have reported significant associations between various common SNPs and prostate cancer risk using cases unselected for family history. How these variants influence risk in familial prostate cancer is not well studied. Here, we analyzed 25 previously reported SNPs across 14 loci from prior prostate cancer GWAS. The International Consortium for Prostate Cancer Genetics (ICPCG) previously validated some of these using a family-based association method (FBAT). However, this approach suffered reduced power due to the conditional statistics implemented in FBAT. Here, we use a case–control design with an empirical analysis strategy to analyze the ICPCG resource for association between these 25 SNPs and familial prostate cancer risk. Fourteen sites contributed 12,506 samples (9,560 prostate cancer cases, 3,368 with aggressive disease, and 2,946 controls from 2,283 pedigrees). We performed association analysis with Genie software which accounts for relationships. We analyzed all familial prostate cancer cases and the subset of aggressive cases. For the familial prostate cancer phenotype, 20 of the 25 SNPs were at least nominally associated with prostate cancer and 16 remained significant after multiple testing correction (p ≤ 1E ^?3) occurring on chromosomal bands 6q25, 7p15, 8q24, 10q11, 11q13, 17q12, 17q24, and Xp11. For aggressive disease, 16 of the SNPs had at least nominal evidence and 8 were statistically significant including 2p15. The results indicate that the majority of common, low-risk alleles identified in GWAS studies for all prostate cancer also contribute risk for familial prostate cancer, and that some may contribute risk to aggressive disease.     

Rare variants in ischemic stroke: an exome pilot study

The genetic architecture of ischemic stroke is complex and is likely to include rare or low frequency variants with high penetrance and large effect sizes. Such variants are likely to provide important insights into disease pathogenesis compared to common variants with small effect sizes. Because a significant portion of human functional variation may derive from the protein-coding portion of genes we undertook a pilot study to identify variation across the human exome (i.e., the coding exons across the entire human genome) in 10 ischemic stroke cases. Our efforts focused on evaluating the feasibility and identifying the difficulties in this type of research as it applies to ischemic stroke. The cases included 8 African-Americans and 2 Caucasians selected on the basis of similar stroke subtypes and by implementing a case selection algorithm that emphasized the genetic contribution of stroke risk. Following construction of paired-end sequencing libraries, all predicted human exons in each sample were captured and sequenced. Sequencing generated an average of 25.5 million read pairs (75 bp×2) and 3.8 Gbp per sample. After passing quality filters, screening the exomes against dbSNP demonstrated an average of 2839 novel SNPs among African-Americans and 1105 among Caucasians. In an aggregate analysis, 48 genes were identified to have at least one rare variant across all stroke cases. One gene, CSN3, identified by screening our prior GWAS results in conjunction with our exome results, was found to contain an interesting coding polymorphism as well as containing excess rare variation as compared with the other genes evaluated. In conclusion, while rare coding variants may predispose to the risk of ischemic stroke, this fact has yet to be definitively proven. Our study demonstrates the complexities of such research and highlights that while exome data can be obtained, the optimal analytical methods have yet to be determined.