Large-scale identification and characterization of genetic variants in asthma candidate genes

Asthma is a chronic inflammatory disorder of the airways, and a number of genetic loci are associated with the disease. Candidate gene association studies have been regarded as effective tools to study complex traits. Knowledge of the sequence variation and structure of the candidate genes is required for association studies. Thus, we investigated the genetic variants of 32 asthma candidate genes selected by colocalization of positional and functional candidate genes. We screened all exons and promoter regions of those genes using 12 healthy individuals and 12 asthma patients and identified a total of 418 single nucleotide polymorphisms (SNPs), including 270 known SNPs and 148 novel SNPs. Levels of nucleotide diversity varied from gene to gene (0.72×10⁻⁴–14.53×10⁻⁴), but the average nucleotide diversity between coding SNPs (cSNPs) and noncoding SNPs was roughly equivalent (4.63×10⁻⁴ vs 4.69×10⁻⁴). However, nucleotide diversity of cSNPs was strongly correlated to codon degeneracy. Nucleotide diversity was much higher at fourfold degenerate sites than at nondegenerate sites (9.42×10⁻⁴ vs 3.14×10⁻⁴). Gene-based haplotype analysis of asthma-associated genes in this study revealed that common haplotypes (frequency >5%) represented 90.5% of chromosomes, and they could be uniquely identified with five or fewer haplotype-tagging SNPs per gene. Therefore, our results may have important implications for the selection of asthma candidate genes and SNP markers for comprehensive association studies using large sample populations.     

Validation of in silico-predicted genic SNPs in white clover (<Emphasis Type="Italic">Trifolium repens</Emphasis> L.), an outbreeding allopolyploid species

White clover (Trifolium repens L.) is an obligate outbreeding allotetraploid forage legume. Gene-associated SNPs provide the optimum genetic system for improvement of such crop species. An EST resource obtained from multiple cDNA libraries constructed from numerous genotypes of a single cultivar has been used for in silico SNP discovery and validation. A total of 58 from 236 selected sequence clusters (24.5%) were fully validated as containing polymorphic SNPs by genotypic analysis across the parents and progeny of several two-way pseudo-testcross mapping families. The clusters include genes belonging to a broad range of predicted functional categories. Polymorphic SNP-containing ESTs have also been used for comparative genomic analysis by comparison with whole genome data from model legume species, as well as Arabidopsis thaliana. A total of 29 (50%) of the 58 clusters detected putative ortholoci with known chromosomal locations in Medicago truncatula, which is closely related to white clover within the Trifolieae tribe of the Fabaceae. This analysis provides access to translational data from model species. The efficiency of in silico SNP discovery in white clover is limited by paralogous and homoeologous gene duplication effects, which are resolved unambiguously by the transmission test. This approach will also be applicable to other agronomically important cross-pollinating allopolyploid plant species. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. N.O.I. Cogan and M.C. Drayton contributed equally to this work.     

Identification, characterization and interpretation of single-nucleotide sequence variation in allopolyploid crop species

An understanding of nature and extent of nucleotide sequence variation is required for programmes of discovery and characterization of single nucleotide polymorphisms (SNPs), which provide the most versatile class of molecular genetic marker. A majority of higher plant species are polyploids, and allopolyploidy, because of hybrid formation between closely related taxa, is very common. Mutational variation may arise both between allelic (homologous) sequences within individual subgenomes and between homoeologous sequences among subgenomes, in addition to paralogous variation between duplicated gene copies. Successful SNP validation in allopolyploids depends on differentiation of the sequence variation classes. A number of biological factors influence the feasibility of discrimination, including degree of gene family complexity, inbreeding or outbreeding reproductive habit, and the level of knowledge concerning progenitor diploid species. In addition, developments in high-throughput DNA sequencing and associated computational analysis provide general solutions for the genetic analysis of allopolyploids. These issues are explored in the context of experience from a range of allopolyploid species, representing grain (wheat and canola), forage (pasture legumes and grasses), and horticultural (strawberry) crop. Following SNP discovery, detection in routine genotyping applications also presents challenges for allopolyploids. Strategies based on either design of subgenome-specific SNP assays through homoeolocus-targeted polymerase chain reaction (PCR) amplification, or detection of incremental changes in nucleotide variant dosage, are described.     

Comprehensive functional network analysis and screening of deleterious pathogenic variants in non-syndromic hearing loss causative genes

Hearing loss (HL) is a significant public health problem and causes the most frequent congenital disability in developed societies. The genetic analysis of non-syndromic hearing loss (NSHL) may be considered as a complement to the existent plethora of diagnostic modalities available. The present study focuses on exploring more target genes with respective non-synonymous single nucleotide polymorphisms (nsSNPs) involved in the development of NSHL. The functional network analysis and variant study have successfully been carried out from the gene pool retrieved from reported research articles of the last decade. The analyses have been done through STRING. According to predicted biological processes, various variant analysis tools have successfully classified the NSHL causative genes and identified the deleterious nsSNPs, respectively. Among the predicted pathogenic nsSNPs with rsIDs rs80356586 (I515T), rs80356596 (L1011P), rs80356606 (P1987R) in OTOF have been reported in NSHL earlier. The rs121909642 (P722S), rs267606805 (P722H) in FGFR1, rs121918506 (E565A) and rs121918509 (A628T, A629T) in FGFR2 have not been reported in NSHL yet, which should be clinically experimented in NSHL. This also indicates this variant’s novelty as its association in NSHL. The findings and the analyzed data have delivered some vibrant genetic pathogenesis of NSHL. These data might be used in the diagnostic and prognostic purposes in non-syndromic congenitally deaf children.     

Haplotypes of nine single nucleotide polymorphisms on chromosome 19q13.2-3 associated with susceptibility of lung cancer in a Chinese population

To evaluate the joint effect of nine single nucleotide polymorphisms for three DNA repair genes in the region of chromosome 19q13.2-3 on susceptibility of lung cancer in a Chinese population, we conducted a hospital-based case–control study consisting of 247 lung cancer cases and 253 cancer-free controls matched on age, gender and ethnicity. Associations between the haplotypes and susceptibility of lung cancer were tested. The global test of haplotype association revealed a statistically significant difference in the haplotype distribution between cases and controls (global test: χ² = 60.45, d.f. = 15, P = 2.11E−07). The two haplotypes were underrepresented among cases (Hap5 defined by ERCC1118^A–ERCC2156^C–ERCC2312^G–ERCC2751^A–XRCC1194^T–XRCC1206^A–XRCC1280^G–XRCC1399^G–XRCC1632^G and Hap12 defined by ERCC1118^G–ERCC2156^C–ERCC2312^G–ERCC2751^A–XRCC1194^C–XRCC1206^A–XRCC1280^G–XRCC1399^A–XRCC1632^G). Three of the haplotypes were overrepresented among cases (Hap3 defined by ERCC1118^A–ERCC2156^C–ERCC2312^G–ERCC2751^A–XRCC1194^C–XRCC1206^A–XRCC1280^G–XRCC1399^G–XRCC1632^G, Hap4 defined by ERCC1118^A–ERCC2156^C–ERCC2312^G–ERCC2751^A–XRCC1194^C–XRCC1206^G–XRCC1280^G–XRCC1399^G–XRCC1632^A, and Hap10 defined by ERCC1118^G–ERCC2156^A–ERCC2312^G–ERCC2751^A–XRCC1194^T–XRCC1206^A–XRCC1280^G–XRCC1399^G–XRCC1632^G). Haplotypes 3 and 10 (cases = 5.7%, controls = 1.0%, OR = 6.56, 95%CI = 1.83–23.54, P = 0.001; cases = 13.3%, controls = 5.6%, OR = 2.73, 95%CI = 1.51–4.94, P = 0.0006) were the most strongly associated with increased lung cancer risk. There was considerable linkage disequilibrium exists between SNPs both within genes and between genes in the region. The two blocks for solid spine of LD and six htSNPs were found. The haplotype analysis suggested that the biologically effective polymorphisms co-segregate with some of the haplotypes. This result supports the hypothesis that the sub-region is important for lung cancer susceptibility. Haplotype studies using larger study groups will be required to obtain conclusive results.     

Identification of Swedish mosquitoes based on molecular barcoding of the COI gene and SNP analysis

Mosquito‐borne infectious diseases are emerging in many regions of the world. Consequently, surveillance of mosquitoes and concomitant infectious agents is of great importance for prediction and prevention of mosquito‐borne infectious diseases. Currently, morphological identification of mosquitoes is the traditional procedure. However, sequencing of specified genes or standard genomic regions, DNA barcoding, has recently been suggested as a global standard for identification and classification of many different species. Our aim was to develop a genetic method to identify mosquitoes and to study their relationship. Mosquitoes were captured at collection sites in northern Sweden and identified morphologically before the cytochrome c oxidase subunit I (COI) gene sequences of 14 of the most common mosquito species were determined. The sequences obtained were then used for phylogenetic placement, for validation and benchmarking of phenetic classifications and finally to develop a hierarchical PCR‐based typing scheme based on single nucleotide polymorphism sites (SNPs) to enable rapid genetic identification, circumventing the need for morphological characterization. The results showed that exact phylogenetic relationships between mosquito taxa were preserved at shorter evolutionary distances, but at deeper levels, they could not be inferred with confidence using COI gene sequence data alone. Fourteen of the most common mosquito species in Sweden were identified by the SNP/PCR‐based typing scheme, demonstrating that genetic typing using SNPs of the COI gene is a useful method for identification of mosquitoes with potential for worldwide application.     

Identification of polymorphism and association analysis with reproductive traits in the porcine RNF4 gene

The ring finger protein 4 gene (RNF4), which might play a role in fetal germ cell development as well as in oocyte and granulosa cell maturation, was one of the potential candidate genes for reproductive traits. In the present work, we isolated the complete coding sequence of porcine RNF4 gene, identified a single nucleotide polymorphism (SNP: T/C) in intron5, and developed a PCR-SacII-RFLP genotyping assay. Association of this SNP with reproductive traits was assessed in three populations with diverse genetic backgrounds. One was Chinese Qingping sows. Another was consisted of crossbred sows derived from Landrace, Large White, Chinese Tongcheng and/or Chinese Meishan (Line DIV). The third is Large White × Meishan (LW × M) F₂ slaughtered population. Statistical analysis demonstrated that, in the first parity, the difference between RNF4 genotypes and reproductive traits of both Qingping and Line DIV sows was not significant. In the second and subsequent litters, CC animals in Qingping population had more piglets born (+1.74 piglets) and piglets born alive (+2.02 piglets) than sows with the TT genotype (P < 0.05). Line DIV sows inheriting the CC genotype had additional 0.69 piglets born compared to the TC animals (P < 0.05) in second and subsequent litters. No significant difference was observed between genotypes and reproductive tracts components in F₂ animals. In addition, we found RNF4 gene has a significant additive effect on both piglet born and piglet born alive in Qingping animals (P < 0.05). Results here suggested that the RNF4 SNP was significantly associated with litter size in two populations and could be useful in selection for increasing litter size in pigs. Further studies were needed to confirm these preliminary researches.     

Association and Haplotype Analysis of purH Gene with Inosine Monophosphate Content in Chickens

The current study was designed to investigate the effects of the purH gene on chicken muscle inosine monophosphate (IMP) content. Muscle IMP content was measured in five chicken breeds. Single nucleotide polymorphisms (SNPs) were detected by PCR-SSCP and DNA sequencing. Two SNPs were detected, A/T substitution at position 8023 in exon 9, and T/C substitution at position 17446 in exon 16. The results indicated that only T17446C polymorphism was associated with IMP content. The haplotype effect was higher than the single genotype effect. We tentatively conclude that purH gene is a candidate locus or linked to a major gene that affects muscle IMP content. Haplotypes are superior to single genotypes as potential molecular markers for meat quality traits in chicken.     

Seasonal effects of the UCP3 and the RPTOR gene polymorphisms on obesity traits in Japanese adults

Background

Non-shivering thermogenesis (NST) involves a substantial amount of energy expenditure in humans and, thus, contributes to reducing the risk for obesity. Molecular evolutionary studies have reported that SNPs in/near the uncoupling protein 3 gene (UCP3) and the regulatory associated protein of mTOR complex 1 gene (RPTOR) might influence NST and confer adaptive advantages for modern human dispersal into cold environments. In the present study, the impact of these SNPs on obesity-related traits was investigated.

Methods

Study subjects consisted of 2,834 Japanese adults (percentage of female: 46%, mean age: 51.5). Associations of the UCP3-55C/T and the RPTOR-26934C/T - the 2 potential genetic variations involved in cold adaptation and thermogenic mechanisms in mammals, with quantitative obesity-related traits including body mass index (BMI), waist circumference, visceral fat area (VFA), VFA adjusted for BMI, and selected blood parameters - were tested using multiple linear regression models. Sliding windowsampling analysis was applied to depict seasonal effects of the SNPs on the obesity-related phenotypes.

Results

UCP3-55C/T and the RPTOR-26934C/T did not show any association with obesity traits and blood chemical parameters in multiple linear regression models consisting of the whole subjects. Moreover, sliding window sampling-based association analyses involving seasonality also failed to find associations between these two SNPs and obesity-related traits.

Conclusions

UCP3-55C/T and the RPTOR-26934C/T may only have subtle effects on the development of obesity-related traits in the present humans. These two SNPs might be irrelevant to inter-individual variations in energy metabolism and efficiency of NST.     

Molecular identification of the strains of the domestic silkworm,Bombyx mori (Lepidoptera: Bombycidae), which are endemic to Korea,based on single nucleotide polymorphisms in mitochondrial genome sequences

The domestic silkworm, Bombyx mori (Lepidoptera: Bombycidae), has been diversified into various strains over a long period. However, methods to distinguish silkworm strains remain limited partially owing to the genetic similarity caused by the long history of domestication. In this study, we developed molecular identification methods to distinguish three domestic silkworm strains, which are endemic to Korea. By comparing publicly available complete mitochondrial genome (mitogenome) sequences of five endemic strains and 34 stock silkworm strains analyzed in a previous study, we detected 15 single nucleotide polymorphisms (SNPs; SNP1–SNP15), which distinguished the following three endemic strains: Sun7ho (SN7), Sandongsammyeon (SDS), and Sammyeonhonghoeback (SMH). We used two SNPs for each strain to identify the three endemic strains. To distinguish each SN7 and SDS from the remaining four endemic and 34 stock strains, the PCR-restriction fragment length polymorphism method was employed using Acu I and Hpa I restriction enzymes, which recognize SNP1 and SNP8, respectively. Additionally, the tetra-primer amplification refractory mutation system PCR method was used to determine the regions containing SNP3, SNP11, and both SNP14 and SNP15 to distinguish SN7, SDS, and SMH, respectively, from the remaining strains. A validation test with additional individuals showed that each target strain was clearly recognized, suggesting that mitogenome SNP-based methods can be used to identify three endemic silkworm strains during culture and breeding.     

Genetic variation in TLR10 is not associated with chronic Q fever,despite the inhibitory effect of TLR10 on Coxiella burnetii-induced cytokines in vitro

Coxiella burnetii, the causative agent of Q fever, is recognized by TLR2. TLR10 can act as an inhibitory receptor on TLR2-derived immune responses. Therefore, we investigated the role of TLR10 on C. burnetii-induced cytokine production and assessed whether genetic polymorphisms in TLR10 influences the development of chronic Q fever. HEK293 cells, transfected with TLR2, TLR10 or TLR2/TLR10, and human peripheral blood mononuclear cells (PBMCs) in the presence of anti-TLR10, were stimulated with C. burnetii. In both assays, the absence of TLR10 resulted in increased cytokine responses after C. burnetii stimulation. In addition, the effect of single nucleotide polymorphisms (SNPs) in TLR10 was examined in healthy volunteers whose PBMCs were stimulated with C. burnetii Nine Mile or the Dutch outbreak isolate C. burnetii 3262. Individuals bearing SNPs in TLR10 displayed increased cytokine production upon C. burnetii 3262 stimulation. Furthermore, 139 chronic Q fever patients and 220 controls were genotyped for TLR10 N241H, I775V and I369L. None of these polymorphisms were associated with increased susceptibility to chronic Q fever. In conclusion, TLR10 has an inhibitory effect on in vitro cytokine production by C. burnetii, but the presence of TLR10 polymorphisms does not lead to an increased risk of developing chronic Q fever.     

Nucleotide diversity and demographic history of Pinus bungeana,an endangered conifer species endemic in China

Orographic and climatic oscillations have played crucial roles in shaping the nucleotide diversity and evolutionary history of many species across the Northern Hemisphere. In this study, based on 10 nuclear loci and a chloroplast DNA marker, we analyzed the nucleotide polymorphisms and demographic history of the endangered conifer species Pinus bungeana in Northwest China and investigated the phylogenetic relationships between P. bungeana and two related species, that is, Pinus gerardiana and Pinus squamata. We found that P. bungeana exhibited an extremely low level of nucleotide diversity (π_sil = 0.00159). Demographic simulations based on DIYABC analysis showed that P. bungeana underwent demographic expansion and contraction during the Miocene. According to ecological niche modeling, we found that this species survived in situ during the glacial period and was not restricted to southern refugia. We speculate that P. bungeana may have experienced widespread population shrinkage from the Last Interglacial to the Last Glacial Maximum due to geological or climatic events. Isolation‐with‐migration analysis revealed that the divergence (~2.4–4.2 Ma) among P. bungeana and its related species was significantly associated with the Qinghai–Tibetan Plateau uplift events in the mid‐to‐late Tertiary period. Species tree analyses suggested that these three related Pinus species formed a monophyletic clade with high bootstrap support. These results suggest that the Miocene–Pliocene and Pleistocene geological and climatic fluctuations might have profoundly affected the nucleotide diversity and demography of this psychrotolerant conifer species in western China.     

Identification and characterization of polymorphic microsatellite loci in the blue shark Prionace glauca, and cross-amplification in other shark species

Two to 14 alleles were found to be segregating per locus (mean 5·2), with observed and expected heterozygosities ranging from 0·08 to 0·78 and 0·08 to 0·94, respectively. Cross-amplification of six of these microsatellite loci indicated that they are also polymorphic in three species of Carcharhiniformes and two species of Lamniformes. The newly developed primers reported here constitute a useful tool for genetic population analyses on Prionace glauca and, potentially, other related species.     

Genomic analysis of Fas and FasL genes and absence of correlation with disease progression in AIDS

Apoptosis has been suggested as a major mechanism for the CD4⁺ T-lymphocyte depletion observed in patients infected with human immunodeficiency virus 1 (HIV-1). To evaluate the impact of genetic variations to apoptosis during progression of acquired immunodeficiency syndrome (AIDS), we have performed an extensive genetic analysis of Fas and Fas ligand (FasL) genes. The coding regions and promoters of these genes were resequenced in a cohort of 212 HIV-1-seropositive patients presenting extreme disease phenotypes and 155 healthy controls of Caucasian origin. Overall, 33 single nucleotide polymorphisms (SNPs) with an allele frequency >1% were identified and evaluated for their association with disease progression. Among them, 14 polymorphisms were newly characterized. We did not find any statistically significant association of Fas and FasL polymorphisms and haplotypes with AIDS progression.     

Species delimitation with gene flow: A methodological comparison and population genomics approach to elucidate cryptic species boundaries in Malaysian Torrent Frogs

Accurately delimiting species boundaries is a nontrivial undertaking that can have significant effects on downstream inferences. We compared the efficacy of commonly used species delimitation methods (SDMs) and a population genomics approach based on genomewide single‐nucleotide polymorphisms (SNPs) to assess lineage separation in the Malaysian Torrent Frog Complex currently recognized as a single species (Amolops larutensis). First, we used morphological, mitochondrial DNA and genomewide SNPs to identify putative species boundaries by implementing noncoalescent and coalescent‐based SDMs (mPTP, iBPP, BFD*). We then tested the validity of putative boundaries by estimating spatiotemporal gene flow (fastsimcoal2 , ABBA‐BABA) to assess the extent of genetic isolation among putative species. Our results show that the A. larutensis complex runs the gamut of the speciation continuum from highly divergent, genetically isolated lineages (mean F_st = 0.9) to differentiating populations involving recent gene flow (mean F_st = 0.05; N_m > 5). As expected, SDMs were effective at delimiting divergent lineages in the absence of gene flow but overestimated species in the presence of marked population structure and gene flow. However, using a population genomics approach and the concept of species as separately evolving metapopulation lineages as the only necessary property of a species, we were able to objectively elucidate cryptic species boundaries in the presence of past and present gene flow. This study does not discount the utility of SDMs but highlights the danger of violating model assumptions and the importance of carefully considering methods that appropriately fit the diversification history of a particular system.