Genome-wide association study as a method to analyze the genome architecture in polygenic diseases,with the example of multiple sclerosis

Genome-wide association study (GWAS) provides a powerful tool for investigating the genetic architecture of human polygenic diseases and is generally used to identify the genetic factors of disease susceptibility, clinical phenotypes, and treatment response. The differences in allele frequencies of single nucleotide polymorphisms (SNPs) distributed throughout the genome are analyzed with a microarray technique or other technologies that allow simultaneous genotyping at several tens of thousands to several millions of SNPs per sample. Owing to its power to find out highly reliable differences between patients and controls, GWAS became a common approach to identification of the genetic susceptibility factors in complex diseases of a polygenic nature. Using multiple sclerosis (MS) as a prototype complex disease, the review considers the main achievements and challenges of using GWAS to identify the genes involved in the disease and, therefore, to better understand the pathogenetic molecular mechanisms and genetic risk factors.     

Genome-wide single nucleotide polymorphisms and insertion–deletions of Oryza sativa L. subsp. japonica cultivars grown near the northern limit of rice cultivation

Single nucleotide polymorphisms (SNPs) and insertions–deletions (InDels) are valuable molecular markers for molecular breeding among genetically closely related cultivars. Rice (Oryza sativa L. subsp. japonica) cultivars grown in Hokkaido (45–42°N), the northernmost region of rice paddy cultivation in Japan, have been bred for over 100 years for adaptation to low summer temperatures together with high yield and good eating quality. In this study, for 10 closely related rice cultivars released in Hokkaido and cultivar Koshihikari, we identified genome-wide SNPs and InDels by next-generation sequencing. More than 29 million reads from the Hokkaido cultivars, each 101 nucleotides long, were uniquely mapped to the Nipponbare reference genome. The average of the total nucleotide length of all uniquely mapped reads corresponded to 10.9 times (3,978 Mb with genome coverage of 90.7 %) the Nipponbare reference genome. An average of 99,955 putative SNPs (1.8 times the number in Koshihikari) and 14,617 putative InDels (also 1.8 times the number in Koshihikari) were detected in Hokkaido cultivars relative to the Nipponbare genome, which enabled analyses of the inheritance of pedigree haplotypes of four cultivars, SNPs and InDels among closely related Hokkaido cultivars, and haplotype blocks unique to Hokkaido cultivars. The comprehensive SNP and InDel data provide DNA marker resources and will facilitate quantitative trait locus analysis of biparental mapping of very closely related Hokkaido cultivars. Furthermore, the haplotype blocks unique to Hokkaido cultivars represent ideal genetic regions for improvement of cultivars to be grown near the northern and southern limits of rice cultivation.     

SNPs in MicroRNA-Binding Sites in the ITGB1 and ITGB3 3′-UTR Increase Colorectal Cancer Risk

The purpose of the study was to investigate the potential associations between single-nucleotide polymorphisms (SNPs) in microRNA (miRNA)-binding sites in the integrin beta-1 (ITGB1) gene and integrin beta-3 (ITGB3) gene 3′-untranslated regions, and colorectal cancer (CRC) susceptibility in a Chinese population. A hospital-based case–control study was performed in 200 patients with CRC and 200 matched healthy donors. Two SNPs in miRNA binding of ITGB1 and ITGB3 genes (rs17468 and rs2317676) were genotyped by polymerase chain reaction-restrict fragment length polymorphism assay. The association between genotypes and CRC risk was evaluated by computing the odds ratio (OR) and 95 % confidence interval (CI) from multivariate unconditional logistic regression analyses. The frequency of the T genotype in ITGB1 rs17468 and G genotype in ITGB3 rs2317676 occurred more frequently in CRC patients than in controls (P < 0.05). We found that CT and TT genotypes of rs17468 were associated with a significantly increased risk of CRC (OR = 1.67, 95 % CI = 1.090–2.559 for CT + TT vs. CC), also the AG and GG genotype in ITGB3 rs2317676 (OR = 1.65, 95 % CI = 1.114–2.458 for AG + GG vs. AA). In conclusion, our results showed that both the ITGB1 rs17468 SNP and ITGB3 rs2317676 SNP were associated with an increased risk of CRC, which suggests that these 2 SNPs might contribute to CRC risk in a Chinese population.     

Common vitamin D pathway gene variants reveal contrasting effects on serum vitamin D levels in African Americans and European Americans

Vitamin D deficiency is more common among African Americans (AAs) than among European Americans (EAs), and epidemiologic evidence links vitamin D status to many health outcomes. Two genome-wide association studies (GWAS) in European populations identified vitamin D pathway gene single-nucleotide polymorphisms (SNPs) associated with serum vitamin D [25(OH)D] levels, but a few of these SNPs have been replicated in AAs. Here, we investigated the associations of 39 SNPs in vitamin D pathway genes, including 19 GWAS-identified SNPs, with serum 25(OH)D concentrations in 652 AAs and 405 EAs. Linear and logistic regression analyses were performed adjusting for relevant environmental and biological factors. The pattern of SNP associations was distinct between AAs and EAs. In AAs, six GWAS-identified SNPs in GC, CYP2R1, and DHCR7/NADSYN1 were replicated, while nine GWAS SNPs in GC and CYP2R1 were replicated in EAs. A CYP2R1 SNP, rs12794714, exhibited the strongest signal of association in AAs. In EAs, however, a different CYP2R1 SNP, rs1993116, was the most strongly associated. Our models, which take into account genetic and environmental variables, accounted for 20 and 28 % of the variance in serum vitamin D levels in AAs and EAs, respectively.     

Genotyping and development of single-nucleotide polymorphism (SNP) markers associated with blast resistance genes in rice using GoldenGate assay

Development and large-scale genotyping of single-nucleotide polymorphism (SNP) is required to use identified sequence variation in the alleles of different genes to determine their functional relevance to the candidate gene(s). In the present study, Illumina GoldenGate assay was used to validate and genotype SNPs in a set of six major rice blast resistance genes, viz. Pi-ta, Piz(t), Pi54, Pi9, Pi5(1) and Pib, distributed over five chromosomes, to understand their functional relevance and study the population structure in rice. All the selected SNPs loci (96) of six blast (Magnaporthe oryzae) resistance genes were genotyped successfully in 92 rice lines with an overall genotype call rate of 92.0 % and minimum GenTrain cutoff score of ≥0.448. The highest genotyped SNPs were found in japonica type (97.1 %) rice lines, followed by indica (92.12 %), indica basmati (91.84 %) and minimum in case of wild species (82.0 %). Among the genotyped loci, the highest score (98.68 %) was observed in case of Piz(t), followed by Pi-ta, Pi5(1), Pib, Pi54 and Pi9. Polymorphism was obtained in 87.5 % SNPs loci producing 7,728 genotype calls. Minor allele frequency ranged from 0.01 to 0.49 and has good differentiating power for distinguishing different rice accessions. Population structure analysis revealed that a set of genotypes from four rice subpopulations had “admix” ancestry (>26 %) with more than one genetic background of indica, japonica and wild types. SNPs markers were validated in a set of 92 rice lines and converted into CAPS markers which can be used in blast resistance breeding programme.     

KIF1B polymorphisms associated with the risk of inflammatory demyelinating disease in Korean population

In the present study, we explored the possible association between KIF1B polymorphisms and inflammatory demyelinating disease (IDD) susceptibility. Eleven single nucleotide polymorphisms (SNPs) were selected for the present study based on the literature, as well as linkage disequilibrium, minor allele frequency, and location. The SNPs were genotyped in 178 IDD subjects consisting of 99 neuromyelitis optica subjects, 79 multiple sclerosis subjects, and 237 healthy controls (Total N = 415). We next preformed logistic analysis to validate associations between the KIF1B polymorphisms and the risk of IDD. Statistical analyses revealed that rs17396382 and ht4 were significantly associated with IDD susceptibility with odds ratios of 2.22 and 2.17 (P = 0.001 and 0.004; P ^corr  = 0.01 and 0.03, respectively). In addition, although P values for six variants (rs3748576, rs7520935, rs2275424, rs11576866, rs17411502, and rs11121552) and one haplotype (ht1) did not reach the threshold of significance after correction for multiple testing, the SNPs showed a nominal association in primary analysis (P = 0.02 ~ 0.04). Our results suggest that rs17396382 and ht4 might be involved in IDD pathogenesis.     

Development of ARMS-PCR assay for genotyping of Pro12Ala SNP of PPARG gene: a cost effective way for case–control studies of type 2 diabetes in developing countries

Type 2 diabetes (T2D) is a prevalent metabolic disorder across the globe. Research is underway on various aspects including genetics to understand and control the global epidemic of diabetes. Recently, several SNPs in various genes have been associated with T2D. These association studies are mainly carried out in the developed countries through Genome Wide Association Scans, with follow-up replication/validation studies by high-throughput genotyping techniques (e.g. Taqman Technology). Although, similar studies could be conducted in developing countries, however, the limiting factors are the associated cost and expertise. These factors hamper research into the genetic association and replication studies from low-income countries to figure out the role of putatively associated SNPs in diabetes. Although, there are several SNP detection methods (e.g. Taqman assay, Dot-blot, PCR-RFLP, DGGE, SSCP) but these are either expensive or labor intensive or less sensitive. Hence, our aim was to develop a low-cost method for the validation of PPARG (Pro12Ala, CCA>GCA) SNP (rs1801282) for its association with T2D. Here, we developed a cost-effective and rapid amplification refractory mutation specific-PCR (ARMS-PCR) method for this SNP detection. We successfully genotyped PPARG SNPs (Pro12Ala) in human samples and the validity of this method was confirmed by DNA sequencing of a few representative samples for the three different genotypes. Furthermore, ARMS-PCR was applied to T2D patients and control samples for the screening of this SNP.     

Cytogenetic and immunogenetic analysis of recurrent pregnancy loss in women

Karyotyping of 366 couples (732 individuals) with early recurrent pregnancy losses in anamnesis revealed chromosomal anomalies in 4.09% (30 cases)—within them 2.05% carry reciprocal translocations, in 0.82%-Robertsonian translocations, 0.55% carry numerical and structural gonosomal anomalies and in 0.27%—marker chromosome of unknown origin. The risk of early reproductive losses in women after excluding the cytogenetic component increases three fold if SNPs 1082GG, 592CC, 819CC of IL-10 gene and IFN-γ + 874AT or 874AA genotypes are present. ELISA-mediated detection of serum IL-10 and IFN-γ showed a possibly significant increase of IFN-γ in women with the history of early reproductive losses when compared to reproductively healthy women. We are proposing a complex cyto- and immunogenetic investigation in cases of early reproductive losses in women. One of the important issues of reproduction are the immunological mechanisms of pregnancy maintenance, where the disbalance in the genetically determined Th1- and Th2-cytokine levels may be one of the causes of early fetus elimination.     

Association mapping of grain hardness,polyphenol oxidase,total phenolics,amylose content,and β-glucan in US barley breeding germplasm

A renewed interest in breeding barley specifically for food end-uses is being driven by increased consumer interest in healthier foods. We conducted association mapping on physicochemical properties of barley that play a role in food quality and processing including grain hardness, polyphenol oxidase activity, total phenolics, amylose content, and β-glucan. We used 3,069 elite two-row and six-row spring barley breeding lines from eight US breeding programs and 2,041 SNP markers for association mapping. Marker–trait associations were identified using a mixed model that incorporated population structure and kinship. We detected two previously identified QTL for grain hardness on chromosome 2H in the telomeric region of 5H along with two novel regions on 4H and 6H. For amylose content, we detected marker–trait associations on 7H from 0.63 to 30 cM. We detected four regions on chromosomes 1H, 2H, 3H, and 4H associated with polyphenol oxidase activity. The chromosome 2H region co-localized with the two previously mapped polyphenol oxidase genes PPO1 and PPO2, and the regions on chromosomes 1H, 3H, and 4H QTL were novel. For total phenolics, we identified three significant regions on 3H, 4H, and 5H. Two regions on 2H and 7H were associated with β-glucan. Both previously identified and novel QTL are segregating in elite US breeding germplasm. Only three of the 24 SNPs that were associated with traits using either the two-row or six-row mapping panel were identified in both panels. Nine SNPs were detected in the individual two-row or six-row panels that were not detected in the analysis using the complete panel and accounting for population structure. The distribution of favorable alleles at these loci that underpin food quality across the breeding programs suggests several strategies to use markers to improve barley for food uses.     

SNP characteristics predict replication success in association studies

Successful independent replication is the most direct approach for distinguishing real genotype–disease associations from false discoveries in genome-wide association studies (GWAS). Selecting SNPs for replication has been primarily based on P values from the discovery stage, although additional characteristics of SNPs may be used to improve replication success. We used disease-associated SNPs from more than 2,000 published GWASs to identify predictors of SNP reproducibility. SNP reproducibility was defined as a proportion of successful replications among all replication attempts. The study reporting association for the first time was considered to be discovery and all consequent studies targeting the same phenotype replications. We found that ?Log(P), where P is a P value from the discovery study, is the strongest predictor of the SNP reproducibility. Other significant predictors include type of the SNP (e.g., missense vs intronic SNPs) and minor allele frequency. Features of the genes linked to the disease-associated SNP also predict SNP reproducibility. Based on empirically defined rules, we developed a reproducibility score (RS) to predict SNP reproducibility independently of ?Log(P). We used data from two lung cancer GWAS studies as well as recently reported disease-associated SNPs to validate RS. Minus Log(P) outperforms RS when the very top SNPs are selected, while RS works better with relaxed selection criteria. In conclusion, we propose an empirical model to predict SNP reproducibility, which can be used to select SNPs for validation and prioritization.     

An EST-based linkage map reveals chromosomal translocation in Capsicum

To facilitate marker-assisted breeding and analysis of the structure and/or organization of Capsicum (pepper) genomes, this study utilized expressed sequence tags (ESTs) to develop single-nucleotide polymorphism (SNP) markers. Three different types of PCR-based markers derived from pepper ESTs were developed: intron-based polymorphic markers (IBPs), conserved ortholog sets (COSIIs), and eSNPs (EST–SNPs). For scanning and detection of SNPs, high-resolution melting analysis was performed and the resultant markers were used for linkage analysis. A total of 512 markers, comprising 214 IBP, 143 COSII, 48 eSNP, and 107 previously reported markers, were mapped on 12 linkage groups (LGs) of the “AC99” F2 population. This newly constructed interspecific map (AC2) covered 2,335.6 cM with an average marker interval distance of 4.5 cM and was aligned directly with another interspecific map (AF) for validation. Most LGs showed collinear relationships, except for the alignment of chromosomes 1 and 8 of the AC2 map to LG P1 of the AF map. Using our newly developed SNP markers, we generated chromosome-specific markers, and the previously predicted reciprocal translocation event between chromosomes 1 and 8 was revealed between wild and cultivated Capsicum by fluorescent in situ hybridization analysis. The results from this study will promote subsequent evolutionary studies of Capsicum species.     

Association between CLPTM1L polymorphisms (rs402710 and rs401681) and lung cancer susceptibility: evidence from 27 case–control studies

Genome-wide association studies have identified two SNPs (rs402710 and rs401681) of CLPTM1L at chromosome 5p15.33 as a new lung cancer (LC) susceptibility locus in populations of European descent. Since then, the relationship between these SNPs and LC has been reported in various ethnic groups; however, these studies have yielded inconsistent results. To investigate this inconsistency, we performed a meta-analysis of 27 studies involving a total of 60,828 cases and 109,135 controls for the two polymorphisms to evaluate its effect on genetic susceptibility for LC. An overall random-effects per-allele odds ratio of 1.14 (95 % CI 1.11–1.16, P < 10^?5) and 1.15 (95 % CI 1.12–1.19, P < 10^?5) was found for the rs401681 and rs402710 polymorphism, respectively. Significant results were also observed for under dominant and recessive genetic models. After stratified by ethnicity, significant associations were found among Caucasians and East Asians. In the subgroup analysis by sample size, significantly increased risks were found for these polymorphisms in all genetic models. In addition, we find both rs402710 and rs401681 conferred significantly greater risks for adenocarcinoma and squamous cell carcinoma when stratified by histological type of tumors. Furthermore, associations of these polymorphisms with LC risk were observed among current smokers and former smokers, as well as never smokers. Our findings demonstrated that rs402710 and rs401681 are risk-conferring factors for the development of lung cancer.     

QTL mapping in autotetraploids using SNP dosage information

Key message

Dense linkage maps derived by analysing SNP dosage in autotetraploids provide detailed information about the location of, and genetic model at, quantitative trait loci.

Abstract

Recent developments in sequencing and genotyping technologies enable researchers to generate high-density single nucleotide polymorphism (SNP) genotype data for mapping studies. For polyploid species, the SNP genotypes are informative about allele dosage, and Hackett et al. (PLoS ONE 8:e63939, 2013) presented theory about how dosage information can be used in linkage map construction and quantitative trait locus (QTL) mapping for an F₁ population in an autotetraploid species. Here, QTL mapping using dosage information is explored for simulated phenotypic traits of moderate heritability and possibly non-additive effects. Different mapping strategies are compared, looking at additive and more complicated models, and model fitting as a single step or by iteratively re-weighted modelling. We recommend fitting an additive model without iterative re-weighting, and then exploring non-additive models for the genotype means estimated at the most likely position. We apply this strategy to re-analyse traits of high heritability from a potato population of 190 F₁ individuals: flower colour, maturity, height and resistance to late blight (Phytophthora infestans (Mont.) de Bary) and potato cyst nematode (Globodera pallida), using a map of 3839 SNPs. The approximate confidence intervals for QTL locations have been improved by the detailed linkage map, and more information about the genetic model at each QTL has been revealed. For several of the reported QTLs, candidate SNPs can be identified, and used to propose candidate trait genes. We conclude that the high marker density is informative about the genetic model at loci of large effects, but that larger populations are needed to detect smaller QTLs.     

Analysis on the association between PPARα/γ polymorphisms and lipoprotein(a) in a Chinese Han population

Lipoprotein(a) [Lp(a)], a low-density lipoprotein-like particle, is recognized as an independent risk factor for atherosclerosis, cardiovascular diseases, and diabetic vascular diseases. Our recent studies revealed that the single nucleotide polymorphisms (SNPs) of peroxisome proliferator-activated receptors (PPARα/δ/γ) gene are involved in the regulation of lipid storage and metabolism. In order to investigate the relationships between the SNPs of PPARα/γ gene and plasma levels of Lp(a), 644 participants were randomly selected from Chinese Han population in the present study. As the results shown, Lp(a) was significantly associated with L162V (rs1800206) in PPARα. Compared with those subjects with widetype (LL), significantly higher Lp(a) concentration was determined in the individuals with mutant (LV + VV) (mean difference: 49.07 mg/l, 95 % CI 23.32–74.82 mg/l, p = 0.0002). Moreover, with generalized multifactor dimensionality reduction analysis, our present results indicated that there was a significant association between plasma Lp(a) level and gene–gene interaction among the polymorphisms rs1800206, rs135539 in PPARα and rs10865710, rs1805192, and rs4684847 in PPARγ. Therefore, our presented study indicated that PPARα/γ polymorphisms should be involved in the regulation of plasma Lp(a) in independently and/or in an interactive manner, suggesting that PPARα/γ gene may influence the risk of hypertension, cardiovascular diseases, and dyslipidemia by regulating Lp(a) level.     

A high-resolution linkage map of the Rfd1, a restorer-of-fertility locus for cytoplasmic male sterility in radish (Raphanus sativus L.) produced by a combination of bulked segregant analysis and RNA-Seq

Key message

We utilized a combination of BSA and RNA-Seq to identify SNPs linked to the Rfd1 locus, a restorer-of-fertility gene in radish. A high-density linkage map was constructed using this approach.

Abstract

Male fertility of cytoplasmic male sterility conditioned by the Dongbu cytoplasmic and genic male-sterility cytoplasm can be restored by a restorer-of-fertility locus, Rfd1, in radish. To construct a high-density linkage map and to identify a candidate gene for the Rfd1 locus, bulked segregant analysis and RNA-seq approaches were combined. A total of 26 and 28 million reads produced from male-fertile and male-sterile bulked RNA were mapped to the radish reference unigenes. After stringent screening of SNPs, 327 reliable SNPs of 109 unigenes were selected. Arabidopsis homologs for 101 of the 109 genes were clustered around the 4,000 kb region of Arabidopsis chromosome 3, which was syntenic to the Rfd1 flanking region. Since the reference unigene set was incomplete, the contigs were de novo assembled to identify 134 contigs harboring SNPs. Most of SNP-containing contigs were also clustered on the same syntenic region in Arabidopsis chromosome. A total of 21 molecular markers positioned within a 2.1 cM interval including the Rfd1 locus were developed, based on the selected unigenes and contigs. A segregating population consisting of 10,459 individuals was analyzed to identify recombinants containing crossovers within this interval. A total of 284 identified recombinants were then used to construct a high-density map, which delimited the Rfd1 locus into an 83-kb syntenic interval of Arabidopsis chromosome 3. Since no candidate gene, such as a pentatricopeptide repeat (PPR)-coding gene, was found in this interval, 231 unigenes and 491 contigs containing putative PPR motifs were analyzed further, but no PPR gene in linkage disequilibrium with the Rfd1 locus could be found.     

Evaluation of juvenile drought stress tolerance and genotyping by sequencing with wild barley introgression lines

Drought is a major stress which can seriously limit yield in many crops including barley. Wild barley introgression lines (ILs) like the S42IL library may enhance drought stress tolerance of barley cultivars through the introduction of exotic alleles. The S42IL library was already characterized with 636 Illumina SNPs. New approaches like genotyping by sequencing (GBS) are available for barley to enhance the characterization of ILs. We generated an improved genetic map of the S42IL library, consisting of 4,201 SNPs by adding GBS data. The new map with a total length of 989.2 cM confirmed the extent of wild barley introgressions. Adding GBS data increased the resolution of the S42IL map tenfold from 0.4 to 4.2 markers/cM. This may assist to select possible candidate genes that improve drought tolerance. In four greenhouse experiments, juvenile drought stress response of 52 barley S42ILs was tested to identify quantitative trait loci (QTL). Thirteen S42ILs showed effects for plant biomass and leaf senescence. Subsequently, two verification experiments were conducted with these S42ILs. Nine out of eleven QTL were verified, and 22 additional QTL were detected. For 21 QTL, the Hsp allele increased trait performance, indicating the value of wild barley introgressions. For example, S42IL-107 and S42IL-123 produced more biomass under drought. Two different water-saving strategies were observed. S42IL-143 and S42IL-129 both revealed increased relative water content under drought. While S42IL-143 reduced biomass under drought, S42IL-129 maintained a high biomass production. We recommend using S42IL-107, S42IL-123 and S42IL-129 in barley breeding programs to enhance drought tolerance.     

Eight SNPs of the Myf5 gene and diplotypes associated with growth and reproductive traits in Jinghai yellow chicken

The objective of this study was to analyze possible associations between single nucleotide polymorphisms (SNPs) in the Myf5 gene with chicken growth and reproductive traits. SNPs in Myf5 of the Jinghai yellow chicken were detected by the polymerase chain reaction single-strand conformation polymorphism method and the haplotypes were analyzed. Eight SNPs were identified in the exons of Myf5. Nine haplotypes were established in a group of 379 Jinghai yellow chickens. In terms of growth traits, least square analysis showed that haplotype H1H5 had significant effects on weight at weeks 8 and 12 (P < 0.05). Haplotype H2H6 had significant effects on weight at weeks 12 and 14 (P < 0.05). For reproductive traits, H1H5 had higher body weight for the first egg than H1H4 and H2H4 (P < 0.05), and H1H3 (P < 0.01). H1H3 had a poor performance in average egg weight at 300 days. On the other hand, H1H3 had an advantage in egg number at 300 days. The results showed that SNPs of Myf5 have certain effects on growth and reproductive traits in Jinghai yellow chickens, which can be used in marker-assisted selection to accelerate chicken genetic progress.