Cost-effective procedures for genotyping of human FCN2 gene single nucleotide polymorphisms

L-ficolin (ficolin-2) is a complement-activating pattern-recognition lectin taking part in the innate immune response. Both its serum concentration and sugar binding capacity are influenced by single nucleotide polymorphisms (SNP) of the corresponding FCN2 gene. Cost-effective and simple procedures, based on polymerase chain reaction (PCR) or PCR-restriction fragment length polymorphism for an investigation of four FCN2 SNPs are proposed: ?64 A?>?C (rs7865453), ?4 A?>?G (rs17514136; both located in the promoter region), +6359 C?>?T (rs17549193), +6424 G?>?T (rs7851696; both in exon 8). Variant alleles of ?64 and +6424 (in strong linkage disequlibrium) are known to be associated with low L-ficolin level or activity. In contrast, variant alleles at positions ?4 and +6359 (also in strong linkage disequlibrium) correspond to higher values. Since several L-ficolin clinical associations have been reported, FCN2 genotyping seems to be a valuable tool for disease association studies.     

Single nucleotide polymorphisms in an intron of the ovine calpastatin gene

Calpastatin is the specific inhibitor of the ubiquitous calcium-dependent proteases mu-calpain and m-calpain. Enzyme assay data from sheep and cattle inversely correlates post-mortem muscle calpastatin levels with ultimate meat tenderness. Genetic markers of meat quality may therefore be found linked to the calpastatin gene (CAST). A three-allele system detected by polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) has been observed in the ovine CAST. The three allele amplimers have been fully nucleotide sequenced and their differences in terms of single nucleotide polymorphism (SNPs) in the intron region of the amplimer are reported and compared to a consensus sequence of the orthologous region of the cattle CAST. A PCR-RFLP for more rapid CAST genotyping of all three ovine alleles was also developed.     

Haplotype frequency distribution and linkage disequilibrium analysis of single nucleotide polymorphisms at the human FMO3 gene locus

We analyzed flavin-containing monooxygenase 3 (FMO3) polymorphisms, haplotype structure, and linkage disequilibrium (LD) in 256 Han Chinese and 50 African-American individuals to compare their haplotype frequencies and LD with other world populations. For the Han Chinese, genotyping of three haplotype tag single nucleotide polymorphisms (E158K, V257M, and E308G) was performed by polymerase chain reaction (PCR)-restriction fragment length polymorphism. For the African-Americans, genotyping of all coding exons was performed by modified PCR-single strand conformational polymorphism. Haplotype frequencies, LD, and evolutionary rates were inferred and estimated computationally. There were significant differences in haplotype frequency distribution and LD pattern among Han Chinese, African-Americans, and other world populations. Four major haplotypes of Han Chinese were EVE, KVE, EME, and EVG. Two major haplotypes of African-Americans were EVE and KVE. We found that sites 158 and 257 are in significant LD in both populations. This is the first report comparing FMO haplotypes and LD of Han Chinese with African-Americans. The data presented here justify further pharmacogenetic studies for potentially optimizing recommended drug dosages and evaluating relationships with disease processes.     

The human XPG gene: gene architecture, alternative splicing and single nucleotide polymorphisms

Defects in the XPG DNA repair endonuclease gene can result in the cancer-prone disorders xeroderma pigmentosum (XP) or the XP-Cockayne syndrome complex. While the XPG cDNA sequence was known, determination of the genomic sequence was required to understand its different functions. In cells from normal donors, we found that the genomic sequence of the human XPG gene spans 30 kb, contains 15 exons that range from 61 to 1074 bp and 14 introns that range from 250 to 5763 bp. Analysis of the splice donor and acceptor sites using an information theory-based approach revealed three splice sites with low information content, which are components of the minor (U12) spliceosome. We identified six alternatively spliced XPG mRNA isoforms in cells from normal donors and from XPG patients: partial deletion of exon 8, partial retention of intron 8, two with alternative exons (in introns 1 and 6) and two that retained complete introns (introns 3 and 9). The amount of alternatively spliced XPG mRNA isoforms varied in different tissues. Most alternative splice donor and acceptor sites had a relatively high information content, but one has the U12 spliceosome sequence. A single nucleotide polymorphism has allele frequencies of 0.74 for 3507G and 0.26 for 3507C in 91 donors. The human XPG gene contains multiple splice sites with low information content in association with multiple alternatively spliced isoforms of XPG mRNA.     

The human BARX2 gene: genomic structure, chromosomal localization, and single nucleotide polymorphisms

The BARX genes 1 and 2 are Bar class homeobox genes expressed in craniofacial structures during development. In this report, we present the genomic structure, chromosomal localization, and polymorphic markers in BARX2. The gene has four exons, ranging in size from 85 to 1099 bp. BARX2 is localized on human chromosome 11q25, as determined by radiation hybrid mapping. In the mouse, Barx2 is coexpressed with Pitx2 in several tissues. Based on the coexpression, BARX2 was assumed to be a candidate gene for those cases of Rieger syndrome that cannot be associated with mutations of PITX2. Mutations in PITX2 cause some cases of Rieger syndrome, an autosomal dominant disorder affecting eyes, teeth, and umbilicus. DNA from Rieger patients was subjected to single-strand conformation polymorphism screening of the BARX2 coding region. Three single nucleotide polymorphisms were found in a normal population, although no etiologic mutations were detectable in over 100 cases of Rieger syndrome or in individuals with related ocular disorders.     

Single nucleotide polymorphisms in the human E-cadherin gene

We report four DNA variants in the gene coding for the cell adhesion molecule E-cadherin. The polymorphisms affect codons 115, 133, 582 and the 3-noncoding region.     

Short communication: Single nucleotide polymorphisms in an intron of the ovine calpastatin gene

Abstract

Calpastatin is the specific inhibitor of the ubiquitous calcium‐dependent proteases μ‐calpain and m‐calpain. Enzyme assay data from sheep and cattle inversely correlates post‐mortem muscle calpastatin levels with ultimate meat tenderness. Genetic markers of meat quality may therefore be found linked to the calpastatin gene (CAST). A three‐allele system detected by polymerase chain reaction ‐ single strand conformational polymorphism (PCR‐SSCP) has been observed in the ovine CAST. The three allele amplimers have been fully nucleotide sequenced and their differences in terms of single nucleotide polymorphism (SNPs) in the intron region of the amplimer are reported and compared to a consensus sequence of the orthologous region of the cattle CAST. A PCR‐RFLP for more rapid CAST genotyping of all three ovine alleles was also developed.     

miRNA相关单核苷酸多态性

miRNA相关单核苷酸多态性(miRNA-related single nucleotide polymorphisms或mirSNP)是可以导致miRNA基因调控功能缺失或紊乱的一类功能型SNP的总称。不论是miRNA靶基因结合位点,还是miRNA基因或miRNA加工基因上的mirSNP,都有可能影响miRNA对靶基因的调控。miRNA基因及miRNA加工基因上的mirSNP主要通过阻碍miRNA的生物合成而发挥功能,而靶基因结合位点上的mirSNP主要通过导致自由能的改变或功能构象的消失,影响miRNA与靶序列结合而丧失其原有的调控功能。mirSNP大多位于人类基因组基因间区和内含子区,与包括肿瘤在内的众多复杂性疾病密切关联。mirSNP不论对于复杂性疾病发病机制研究还是诊疗预后分子标志的确定都具有极其重要的研究价值。     

Identification of single nucleotide polymorphisms in the ovine acetyl-CoA carboxylase-alpha gene

Acetyl-CoA carboxylase (ACACA) is the rate-limiting enzyme in the biosynthesis of palmitic acid and long-chain fatty acids. The dietary intake of palmitic acid, which represents approximately 22% of sheep milk fatty acids, increases low-density lipoprotein (LDL) levels and the risk of developing human cardiovascular diseases. Following the candidate gene approach for improving sheep milk composition, and as a first step in assessing the possible influence of the ovine ACACA gene on milk fatty acid composition and its potential use as an animal genetic model of human atherosclerosis disease, we present here an investigation into the genetic variability of the ovine ACACA gene. We sequenced approximately 6.6 kb of ovine ACACA cDNA, including most of the coding sequence of the protein (except 348 bp), in Spanish Churra sheep. A total of 22 synonymous single nucleotide polymorphisms (SNPs) were identified in the analysed sequence, which were genotyped in a set of eight sheep breeds with different productive aptitudes (dairy, meat and double aptitudes). Two of the SNPs identified, SNP03 (c.1450T>C) and SNP15 (c.5134T>C), which appeared to be breed-specific variations, were situated in the gene sequence coding for the biotin-carboxylase (BC) and acetyl-CoA carboxyltransferase (ACCT) domains of the protein, respectively. Particularly interesting is SNP12 (c.4579G>A), which displayed higher frequencies in the dairy-specialised breeds relative to the meat-producing breeds. Moreover, in the dairy breeds studied, the frequency of this SNP showed a positive correlation with the degree of dairy specialisation. A previously described alternative splicing site (Ser-1200) affecting an important regulatory region of the enzyme was observed in one of the Churra animals. Despite the high genetic variability observed in this gene, none of the identified SNPs caused an amino acid change. However, these polymorphisms could be in linkage disequilibrium with other mutations showing a functional effect on the ACACA enzyme. Hence, the characterisations of the allelic variants reported herein lay the groundwork for evaluation of the potential use of these SNPs as genetic markers of fat content and fatty acid composition in sheep dairy products.     

Analysis of association at single nucleotide polymorphisms in the APOE region

The discussion of the prospects of using a dense map of single nucleotide polymorphisms (SNPs) to identify disease genes with association analysis has been extensive. However, there is little empiric evidence to support this strategy. To begin to examine the practical issues surrounding this methodology, we identified 10 SNPs in the region immediately surrounding the apolipoprotein E locus (APOE), an established susceptibility gene for Alzheimer disease. Our goal was to examine patterns of allelic association to begin to investigate the question of whether APOE could have been identified using SNPs. Our strongest evidence of association was at the 2 SNPs immediately flanking APOE.     

Detection of single nucleotide polymorphisms

Single nucleotide polymorphism (SNP) detection technologies are used to scan for new polymorphisms and to determine the allele(s) of a known polymorphism in target sequences. SNP detection technologies have evolved from labor intensive, time consuming, and expensive processes to some of the most highly automated, efficient, and relatively inexpensive methods. Driven by the Human Genome Project, these technologies are now maturing and robust strategies are found in both SNP discovery and genotyping areas. The nearly completed human genome sequence provides the reference against which all other sequencing data can be compared. Global SNP discovery is therefore only limited by the amount of funding available for the activity. Local, target, SNP discovery relies mostly on direct DNA sequencing or on denaturing high performance liquid chromatography (dHPLC). The number of SNP genotyping methods has exploded in recent years and many robust methods are currently available. The demand for SNP genotyping is great, however, and no one method is able to meet the needs of all studies using SNPs. Despite the considerable gains over the last decade, new approaches must be developed to lower the cost and increase the speed of SNP detection.     

Interferon-gamma and interleukin-4 single nucleotide gene polymorphisms in Paracoccidioidomycosis

The gene polymorphisms interferon-gamma (IFN-γ) +874 T/A and interleukin (IL)-4 −590 C/T have been associated with the altered production of cytokines. Therefore, they might be indicative of the occurrence of Paracoccidioidomycosis (PCM) caused by Paracoccidioides brasiliensis. The analysis of single nucleotide polymorphism (SNP) at position +874 IFN-γ showed an increase occurrence of A/T genotype in both PCM patients and healthy individuals as control (HIC) (56% and 45%, respectively), while the allelic distribution showed 82% of A allele in the patients and 80% in the controls. The SNP of −590 IL-4 showed that C/T genotype was significantly (p < 0.05) more prevalent (39%) in PCM group compared to the HIC group (19%), while IL-4 C/C genotype was significantly less frequent (59%) in the patient group compared to the control group (81%). Otherwise, 41% of PCM patients and 19% of HIC individuals carried the IL-4 T allele. Stimulation of peripheral blood mononuclear cells (PBMC) from PCM patients with cell extract antigenic preparations (PbAg) as well as secreted and surface antigens (MEXO) of P. brasiliensis evidenced that there is no difference in the IFN-γ production related to A and T alleles between PCM and HIC individuals. However, with IL-4 production, PCM patients classified as C phenotype showed two times more IL-4 production than PCM patients classified as T phenotype and HIC controls. In conclusion, our results suggest that functional genetic variants in the IL-4 promoter could influence the production of IL-4 in PCM.     

绵羊H-FABP基因单核苷酸多态性的研究

以小尾寒羊(48只)、宁夏滩羊(121只)、滩寒杂交羊F1(23只)、无角陶塞特(48只)、萨福克(24只)5个绵羊群体为实验材料, 利用PCR-SSCP和DNA测序技术对心脏型脂肪酸结合蛋白(H-FABP)基因(GenBank登录号: AY157617)外显子2和内含子2部分序列进行单核苷酸多态性(SNPs)检测及遗传多态性分析。结果表明: (1)引物2的PCR扩增产物中存在981(G/A)、1014(A/C)、1019(T/C) 和1058 (-/G ) 4个SNP位点, 表现为AA、BB、CC、AB、AC、BC、AD、CD和BD 9种基因型, 其中AA为优势基因型。经χ2检验后, 除滩羊和萨福克羊外, 其他群体的基因频率和基因型频率均处于Hardy-Weinberg平衡状态。群体遗传多态性分析表明: 宁夏滩羊、小尾寒羊和无角陶塞特羊3个群体中的多态信息含量(PIC)均处于0.25和0.50之间, 为中度多态, 萨福克羊和滩寒杂交羊F1为低度多态(PIC<0.25), 表明脂肪酸结合蛋白基因在不同绵羊品种中具有单核苷酸多态性, 可以进一步作为候选基因来分析其与肌内脂肪含量性状的关联性。(2)引物4的PCR扩增产物中检测到1个SNP多态位点为2407(T/C), 表现为HH、Hh和 hh 3种基因型, 基因型频率大小为HH＞Hh＞hh, 经χ2检验后, 在滩羊和无角陶塞特羊中均为达到Hardy-Weinberg平衡状态, 其多态信息含量均为低度多态(PIC<0.25), 而在小尾寒羊、滩寒杂交羊F1和萨福克羊均没有多态出现。     

Computational refinement of functional single nucleotide polymorphisms associated with ATM gene

Background

Understanding and predicting molecular basis of disease is one of the major challenges in modern biology and medicine. SNPs associated with complex disorders can create, destroy, or modify protein coding sites. Single amino acid substitutions in the ATM gene are the most common forms of genetic variations that account for various forms of cancer. However, the extent to which SNPs interferes with the gene regulation and affects cancer susceptibility remains largely unknown.

Principal findings

We analyzed the deleterious nsSNPs associated with ATM gene based on different computational methods. An integrative scoring system and sequence conservation of amino acid residues was adapted for a priori nsSNP analysis of variants associated with cancer. We further extended our approach on SNPs that could potentially influence protein Post Translational Modifications in ATM gene.

Significance

In the lack of adequate prior reports on the possible deleterious effects of nsSNPs, we have systematically analyzed and characterized the functional variants in both coding and non coding region that can alter the expression and function of ATM gene. In silico characterization of nsSNPs affecting ATM gene function can aid in better understanding of genetic differences in disease susceptibility.