Genotyping single nucleotide polymorphisms (SNPs) across species in Old World Monkeys

The development of DNA markers is becoming increasingly useful in the field of primatology for studies on paternity, population history, and biomedical research. In this study, we determine the efficacy of using cross-species amplification to identify single nucleotide polymorphisms (SNPs) in closely related species. The DNA of 93 individuals representing seven Old World Monkey species was analyzed to identify SNPs using cross-species amplification and genotyping. The loci genotyped were 653 SNPs identified and validated in rhesus macaques. Of the 653 loci analyzed, 27% were estimated to be polymorphic in the samples studied. SNPs identified at the same locus among different species (coincident SNPs) were found in six of the seven species studied with longtail macaques exhibiting the highest number of coincident SNPs (84). The distribution of coincident SNPs among species is not biased based on proximity to genes in the samples studied. In addition, the frequency of coincident SNPs is not consistent with expectations based on their phylogenetic relationships. This study demonstrates that cross-species amplification and genotyping using the Illumina Golden Gate Array is a useful method to identify a large number of SNPs in closely related species, although issues with ascertainment bias may limit the type of studies where this method can be applied.     

African American-preponderant single nucleotide polymorphisms (SNPs) and risk of breast cancer

Background: African American women more often present with more aggressive types of breast cancer than Caucasian women, but little is known whether genetic polymorphisms specific to or disproportionate in African Americans are associated with their risk of breast cancer. Methods: A population-based case-control study was conducted including 194 cases identified through the Metropolitan Detroit Cancer Surveillance System and 189 controls recruited through random digit dialing to examine polymorphisms in genes involved in estrogen metabolism and action. Results: The African American-specific CYP1A1 5639C allele was associated with an increased risk of breast cancer (odds ratio (OR) = 2.34, 95% confidence interval (CI) 1.23–4.44) and this association with the CYP1A1 5639 locus was dependent on another polymorphism in the CYP3A4 gene (P = 0.043 for the interaction). In addition, African American-predominant CYP1B1 432 Val allele was significantly more often found in the cases than in the controls overall and the HSD17B1 312 Gly allele was specifically associated with premenopausal breast cancer risk (OR = 3.00, 95%CI 1.29–6.99). Conclusion: These observations need to be confirmed in larger studies due to the limited statistical power of the study based on a small number of cases.     

Assessment of population structure by single nucleotide polymorphisms (SNPs) in goat breeds

Single nucleotide polymorphisms (SNPs) may be used in biodiversity studies and commercial tasks like traceability, paternity testing and selection for suitable genotypes. Twenty-seven SNPs were characterized and genotyped on 250 individuals belonging to eight Italian goat breeds. Multilocus genotype data were used to infer population structure and assign individuals to populations. To estimate the number of groups (K) to test in population structure analysis we used likelihood values and variance of the bootstrap samples, deriving optimal K from a drop in the likelihood and a rise in the variance plots against K.     

TLR4 single nucleotide polymorphisms (SNPs) associated with Salmonella shedding in pigs

Toll-like receptor 4 (TLR4) is a key factor in the innate immune recognition of lipopolysaccharide (LPS) from Gram-negative bacteria. Previous studies from our group identified differences in the expression profile of TLR4 and genes affected by the TLR4 signaling pathway among pigs that shed varying levels of Salmonella, a Gram-negative bacterium. Therefore, genetic variation in this gene may be involved with the host’s immune response to bacterial infections. The current study screened for single nucleotide polymorphisms (SNPs) in the TLR4 gene and tested their association with Salmonella fecal shedding. Pigs (n?=?117) were intranasally challenged at 7 weeks of age with 1?×?10⁹ CFU of S. Typhimurium χ4232 and were classified as low or persistent Salmonella shedders based on the levels of Salmonella being excreted in fecal material. Salmonella fecal shedding was determined by quantitative bacteriology on days 2, 7, 14, and 20/21 post exposure, and the cumulative levels of Salmonella were calculated to identify the low (n?=?20) and persistent (n?=?20) Salmonella shedder pigs. From those 40 animals, the TLR4 region was sequenced, and 18 single nucleotide polymorphisms (SNPs) in TLR4 were identified. Twelve SNPs have been previously described and six are novel SNPs of which five are in the 5′ untranslated region and one is in intron 2. Single marker association test identified 13 SNPs associated with the qualitative trait of Salmonella fecal shedding, and seven of those SNPs were also associated with a quantitative measurement of fecal shedding (P?<?0.05). Using a stepwise regression process, a haplotype composed of SNPs rs80787918 and rs80907449 (P?≤?4.0?×?10^?3) spanning a region of 4.9 Kb was identified, thereby providing additional information of the influence of those SNPs on Salmonella fecal shedding in pigs.     

Identification of 187 single nucleotide polymorphisms (SNPs) among 41 candidate genes for ischemic heart disease in the Japanese population

To investigate whether common variants in the human genetic background are associated with pathogenesis of ischemic heart diseases, we systematically surveyed 41 possible candidate genes for single-nucleotide polymorphisms (SNPs) by directly sequencing 96 independent alleles at each locus, derived from 48 unrelated Japanese patients with myocardial infarction, including 25.8 kb 5' flanking regions, 56.8 kb exonic and 35.4 kb intronic sequences, and 1.8 kb 3' flanking regions. In this genomic DNA of nearly 120 kb, we identified 187 SNPs: 55 in 5' flanking regions, seven in 5' untranslated regions (UTRs), 52 in coding elements, 64 in introns, eight in 3' UTRs, and one in a 3' flanking region. Among the 52 coding SNPs, 26 were non-synonymous changes. Allelic frequencies of some of the polymorphisms were significantly different from those reported in European populations. For example, the Q506R substitution in the coagulation factor V gene, the so-called "Leiden mutation", has a reported frequency of 2.3% in Europeans, but we detected the Leiden mutation in none of the Japanese genomes that we investigated. The allelic frequencies of the -33A>G SNP in the thrombomodulin gene were also very different; this allele occurred at a 12% frequency in the Japanese patients that we examined, although it had been detected in none of 82 Caucasians reported previously. These data support the hypothesis that some SNPs are specific to particular ethnic groups.     

Linkage disequilibrium grouping of single nucleotide polymorphisms (SNPs) reflecting haplotype phylogeny for efficient selection of tag SNPs

Single nucleotide polymorphisms (SNPs) have been proposed to be grouped into haplotype blocks harboring a limited number of haplotypes. Within each block, the portion of haplotypes is expected to be tagged by a selected subset of SNPs; however, none of the proposed selection algorithms have been definitive. To address this issue, we developed a tag SNP selection algorithm based on grouping of SNPs by the linkage disequilibrium (LD) coefficient r(2) and examined five genes in three ethnic populations--the Japanese, African Americans, and Caucasians. Additionally, we investigated ethnic diversity by characterizing 979 SNPs distributed throughout the genome. Our algorithm could spare 60% of SNPs required for genotyping and limit the imprecision in allele-frequency estimation of nontag SNPs to 2% on average. We discovered the presence of a mosaic pattern of LD plots within a conventionally inferred haplotype block. This emerged because multiple groups of SNPs with strong intragroup LD were mingled in their physical positions. The pattern of LD plots showed some similarity, but the details of tag SNPs were not entirely concordant among three populations. Consequently, our algorithm utilizing LD grouping allows selection of a more faithful set of tag SNPs than do previous algorithms utilizing haplotype blocks.     

Characterization of 12 single nucleotide polymorphisms (SNPs) in Pacific abalone, Haliotis discus hannai

We report here for the first time 12 polymorphic single nucleotide polymorphisms (SNPs) in a commercially important gastropod, Pacific abalone (Haliotis discus hannai) that were identified by searching expressed sequence tag database. These SNP loci (seven nuclear and five mitochondrial SNPs) were polymorphic among 37 wild abalone individuals, based on a four-primer allele-specific polymerase chain reaction analysis. All loci had two alleles and the minor allele frequency ranged from 0.027 to 0.473. For the seven nuclear SNPs, the expected and observed heterozygosities ranged from 0.053 to 0.499 and from 0.054 to 0.811, respectively.     

Tailed primer base excision sequence scanning (TP-BESS) for detection of single nucleotide polymorphisms (SNPs)

Base excision sequence scanning (BESS) is used to detect and localise point mutations in mammalian genes. The cost of BESS can be significant in large-scale projects. however, due to the requirement for end-labelling of one of the two PCR primers. This is especially true when a fluorescent label is used for detection. If a universal label could be used for all of the PCR reactions, it would reduce the cost of this technique significantly. Here we describe a TP-BESS procedure where one fluorescence-labelled primer is used as a universal label for all the PCR reactions in BESS analysis. The universal label makes BESS financially more feasible for large-scale detection of single nucleotide polymorphisms (SNPs).     

Single nucleotide polymorphisms (SNPs) in human lactoferrin gene.

The lactoferrin protein possesses antimicrobial and antiviral activities. It is also involved in the modulation of the immune response. In a normal healthy individual, lactoferrin plays a role in the front-line host defense against infection and in immune and inflammatory responses. Whether genomic variations, such as single nucleotide polymorphisms (SNPs), have an effect on the structure and function of lactoferrin protein and whether these variations contribute to the different susceptibility of individuals in response to environmental insults are interesting health-related issues. In this study, the lactoferrin gene was resequenced as part of the Environmental Genome Project of the National Institute of Environmental Health Sciences, which operates within the National Institutes of Health. Ninety-one healthy donors of different ethnicities were used to establish common SNPs in the exons of the lactoferrin gene in the general population. The data will serve as a basis from which study the association of lactoferrin polymorphism and disease.     

L-RCA (ligation-rolling circle amplification): a general method for genotyping of single nucleotide polymorphisms (SNPs)

A flexible, non-gel-based single nucleotide polymorphism (SNP) detection method is described. The method adopts thermostable ligation for allele discrimination and rolling circle amplification (RCA) for signal enhancement. Clear allelic discrimination was achieved after staining of the final reaction mixtures with Cybr-Gold and visualisation by UV illumination. The use of a compatible buffer system for all enzymes allows the reaction to be initiated and detected in the same tube or microplate well, so that the experiment can be scaled up easily for high-throughput detection. Only a small amount of DNA (i.e. 50 ng) is required per assay, and use of carefully designed short padlock probes coupled with generic primers and probes make the SNP detection cost effective. Biallelic assay by hybridisation of the RCA products with fluorescence dye-labelled probes is demonstrated, indicating that ligation-RCA (L-RCA) has potential for multiplexed assays.     

The influence of neighboring-nucleotide composition on single nucleotide polymorphisms (SNPs) in the mouse genome and its comparison with human SNPs

We analyzed the neighboring-nucleotide composition of 433,192 biallelic substitutions, representing the largest public collection of SNPs across the mouse genome. Large neighboring-nucleotide biases relative to the genome- or chromosome-specific average were observed at the immediate adjacent sites and small biases extended farther from the substitution site. For all substitutions, the biases for A, C, G, and T were 0.21, 2.63, 0.71, and -3.55%, respectively, on the immediate adjacent 5' site and -3.67, 0.75, 2.69, and 0.23%, respectively, on the immediate adjacent 3' side. Further examination of the six categories of substitution revealed that the neighboring-nucleotide patterns for transitions were strongly influenced by the hypermutability of dinucleotide CpG and the neighboring effects on transversions were complex. Probability of a transversion increased with increasing A + T content of the two immediate adjacent sites, which was similarly observed in the human and Arabidopsis genomes. Overall, the bias patterns for the neighboring nucleotides in the mouse and human genomes were essentially the same; however, the extent of the biases was notably less in mice. Our results provide the first comprehensive view of the neighboring-nucleotide effects in the mouse genome and are important for understanding the mutational mechanisms and sequence evolution in the mammalian genomes.     

Association of single nucleotide polymorphisms (SNPs) of PADI4 gene with rheumatoid arthritis (RA) in Indian population

Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease affecting ~ 1% of the population worldwide. The genome wide association studies on RA patients revealed linkage with 1p36 locus containing peptidyl arginine deiminase 4 (PADI4) genes. Case-control association studies and mRNA stability assays reported the association of PADI4 gene with RA in Korean and Japanese populations. However, such association was not found in Spanish population. Differences in the association of PADI4 with RA in different populations prompted the present study in Indian population. Anti-CCP antibodies, RF antibody, disease activity scores at 28 joints (DAS28) and mutations in three exons of PADI4 were investigated in RA patients and control group. Among the patients anti-CCP antibody levels were found to be associated with high DAS28 values (r = 0.4526, P < 0.0001). Polymorphism in exon-4 (padi4_104, [rs1748033]) of PADI4 showed significant association of 'C' allele with RA in the study population (P = 0.0008). Polymorphism in exon-3 (padi4_92, [rs874881]) also exhibited moderate association with the disease (P = 0.075). However, no association of the disease was found with the SNPs padi4_89 [rs11203366] and padi4_90 [rs11203367] in exon-2 of PADI4.     

特发性少精子症和无精子症与Pygo2基因蛋白编码区SNPs的相关性

男性不育常伴随精子数量减少。Pygo2基因在染色质重塑的伸长精细胞中表达, 其功能受损会导致精子形成阻滞和精子生成减少而引发不育。文章旨在检测引起人特发性少精子症和无精子症的Pygo2基因突变。从77例正常生育力男性和195例特发性少精子症和无精子症患者静脉血提取DNA, 采用聚合酶链式反应-测序方法对Pygo2基因3个蛋白质编码区进行测序对比, 非同义单核苷酸多态性(Single nucleotide polymorphisms, SNPs)位点分别用SIFT、Polyphen-2和 Mutation Taster软件进行诱发蛋白质结构和表型改变的检测和分析。结果表明, 195例患者中, 178例(30例轻度或中度少精子症, 57例重度少精子症和91例无精子症)基因序列分析报告完好, 无精子症中3例患者分别在2个位点(rs61758740, rs141722381)发生了非同义突变SNPs, 重度少精子症中1例患者在位点rs61758741发生了非同义突变, 3个突变位点在SNPs基因数据库都已有报道, 轻度或中度少精子症患者以及正常生育力男性中不存在SNPs。rs61758740可使PYGO2蛋白第141位蛋氨酸(M)变为异亮氨酸(I), rs61758741使PYGO2蛋白第261位碱性赖氨酸(K)变为酸性谷氨酸(E), rs141722381使PYGO2蛋白第240位亲水侧链天冬酰胺(N)变为疏水侧链异亮氨酸(I)。软件分析表明, 在所发现的3个SNP非同义突变位点中, rs141722381引起的单个氨基酸改变会导致PYGO2蛋白空间结构破坏和诱发相关疾病。因此, Pygo2基因蛋白质编码序列区SNPs可能是特发性少精子症和无精子症的诱发因素之一, 导致男性不育。     

多重等位基因特异性扩增—— 微流控芯片电泳法同时测定多个单核苷酸多态性位点

文章以微流控芯片电泳为检测平台, 建立了一种基于DNA适配器连接介导的多重等位基因特异性扩增同时测定多个单核苷酸多态性(SNP)位点的方法。以白细胞介素1β(IL1B)基因中的7个SNP位点(794C>T、1274C>T、2143T>C、2766T>del、3298G>A、5200G>A和5277C>T)为检测对象, 通过PCR预扩增得一段含该7个待测SNP位点的长片段; 用限制性内切酶MboⅠ将其消化成短片段, 再与DNA适配器(adapter)相连; 以连接产物为模板, 在两管中分别用7条等位基因特异性引物和一条公用引物进行7重等位基因特异性扩增; 最后用微流控芯片电泳法分离等位基因特异性扩增产物, 根据两管扩增产物的芯片电泳图谱中扩增片段的大小判断SNP的类型。采用本法成功测定了48名健康中国人的IL1B基因上的7个SNP位点, 与聚合酶链反应-限制性片段长度多态性法(PCR-RFLP)和测序法测定结果完全一致。本法结果准确, 可用于同时测定多个SNP位点; 以微流控芯片电泳作为检测平台, 分析速度快, 样品需要量少; 借助于自制筛分凝胶和重复使用芯片, 使得SNP分析成本大大降低。     

Identification of novel single nucleotide polymorphisms (SNPs) in deer (Odocoileus spp.) using the BovineSNP50 BeadChip

Single nucleotide polymorphisms (SNPs) are growing in popularity as a genetic marker for investigating evolutionary processes. A panel of SNPs is often developed by comparing large quantities of DNA sequence data across multiple individuals to identify polymorphic sites. For non-model species, this is particularly difficult, as performing the necessary large-scale genomic sequencing often exceeds the resources available for the project. In this study, we trial the Bovine SNP50 BeadChip developed in cattle (Bos taurus) for identifying polymorphic SNPs in cervids Odocoileus hemionus (mule deer and black-tailed deer) and O. virginianus (white-tailed deer) in the Pacific Northwest. We found that 38.7% of loci could be genotyped, of which 5% (n = 1068) were polymorphic. Of these 1068 polymorphic SNPs, a mixture of putatively neutral loci (n = 878) and loci under selection (n = 190) were identified with the F(ST)-outlier method. A range of population genetic analyses were implemented using these SNPs and a panel of 10 microsatellite loci. The three types of deer could readily be distinguished with both the SNP and microsatellite datasets. This study demonstrates that commercially developed SNP chips are a viable means of SNP discovery for non-model organisms, even when used between very distantly related species (the Bovidae and Cervidae families diverged some 25.1-30.1 million years before present).