SNP-based Association Analysis Research Articles

Powerful QTL mapping and favorable allele mining in an all-in-one population: a case study of heading date.

The multiparent advanced generation intercross (MAGIC) population is characterized with great potentials in power and resolution of quantitative trait locus (QTL) mapping, but single nucleotide polymorphism (SNP)-based GWAS does not fully reach its potential. In this study, a MAGIC population of 1021 lines was developed from four Xian and four Geng varieties from five subgroups of rice. A total of 44000 genes showed functional polymorphisms among eight parents, including frameshift variations or premature stop codon variations, which provides the potential to map almost all genes of the MAGIC population. Principal component analysis results showed that the MAGIC population had a weak population structure. A high-density bin map of 24414 bins was constructed. Segregation distortion occurred in the regions possessing the genes underlying genetic incompatibility and gamete development. SNP-based association analysis and bin-based linkage analysis identified 25 significant loci and 47 QTLs for heading date, including 14 known heading date genes. The mapping resolution of genes is dependent on genetic effects with offset distances of <55kb for major effect genes and <123kb for moderate effect genes. Four causal variants and noncoding structure variants were identified to be associated with heading date. Three to four types of alleles with strong, intermediate, weak, and no genetic effects were identified from eight parents, providing flexibility for the improvement of rice heading date. In most cases, japonica rice carries weak alleles, and indica rice carries strong alleles and nonfunctional alleles. These results confirm that the MAGIC population provides the exceptional opportunity to detect QTLs, and its use is encouraged for mapping genes and mining favorable alleles for breeding.

Genetic variation in promoter region of the bovine LAP3 gene associated with estimated breeding values of milk production traits and clinical mastitis in dairy cattle.

The purpose of this study was to identify genetic variants in the promoter and 5'UTR regions of bovine leucine amino peptidase three (LAP3) gene and analysed their associations with estimated breeding values (EBVs) of milk production traits and clinical mastitis in Sahiwal and Karan Fries cattle. Eleven SNPs were identified within the region under study of the LAP3 gene, including seven promoter variants (rs717156555: C>G, rs720373055: T>C, rs715189731: A>G, rs516876447: A>G, rs461857269: C>T, rs136548163: C>T, and rs720349928: G>A) and four 5'UTR variants (rs717884982: C>T, rs722359733: C>T, rs481631804: C>T and rs462932574: T>G). Out of them, 10 SNPs variants were found in both Sahiwal and Karan Fries cattle, with one SNP variant (rs481631804: C>T) being unique to Karan Fries cattle. Seven of these identified SNPs were chosen for association analyses. Individual SNP based association analysis revealed that two SNPs (rs720373055: T>C and rs720349928: G>A) were significantly associated with EBVs of lactation milk yield (LMY), 305-day milk yield (305dMY), and one significant association of SNP rs722359733: C>T with lactation length (LL) was observed. Haplotype based association analysis indicated that diplotypes are significantly associated with EBVs of LMY, 305dMY, and LL, individuals with H1H3 (CTACGCT/GCGTACG) being linked to higher lactation performance than other diplotypes. Further logistic regression analysis revealed that, animals with diplotype H1H3 was less susceptible to the incidence of clinical mastitis than other cows, as the odds ratio for the non-incidence of clinical mastitis was found to be low. Altogether, variations in the LAP3 gene promoter could be used as a genetic marker, most notably diplotype H1H3, may greatly benefit the simultaneous improvement of mastitis resistance and milk yield traits in dairy cattle. Moreover, bioinformatics analysis predicted that the SNPs rs720373055: T>C, rs715189731:A>G and rs720349928: G>A is located in the core promoter region and in TFBs, play key role in regulation of studied phenotypes.

Association of Predisposing Haplotype Snp-Markers of HBB Gene in Thalassaemia

Most of the beta thalassaemia cases are heterogeneous at the molecular level. The genetic variation arises due to the mutation of a single beta-globin gene (HBB) on chromosome 11p15.5. Eventhough, the basis and natural history of this disorder are still not well understood and the clinical presentation in certain patients was not always correlated well with the single gene diagnosis [1].&#x0D; &#x0D; The study aimed to evaluate the predisposing effect of HBB polymorphisms and their haplotypes in conferring beta-thalassaemia in Malaysia using a bioinformatics tool. Archived genotype data for five tagging SNPs of HBB gene assigned as c.9T/C, IVS2-16G/C, IVS2-74T/G, 3'UTR +314G/A and 3'UTR +316A/C across thalassaemia patient group (n=433) and normal control group (n=794) controls were retrieved from the year 2011 to 2016 at Institute for Medical Research (IMR). The genotype data were analysed for individual SNP-based association analysis and haplotype-based association analysis using SHEsis online software.&#x0D; &#x0D; The study in all races in Malaysia showed significant association in four SNPs of the HBB gene (c.9T/C, P-value = 0.014; IVS2-16G/C, P-value = 0.017; 3'UTR+314G/A, P value = 2.08x108; 3'UTR+316A/C, P-value = 0.021). However, IVS2-74T/G did not show significant association with a P-value of 0.763 (Table 1). We further revealed two susceptible haplotypes in all races (haplotype TGTAA, P-value = 1.68x10-7, OR = 2.382 [1.707~3.325]; haplotype CCTGC, P-value = 0.003, OR = 1.300 [1.089~1.551]). Meanwhile, one protective haplotype observed was haplotype TGTGA (P-value = 2.99x10-7, OR = 0.625 [0.522~0.749]) (Table 2).&#x0D; In conclusion, this study suggests the potential susceptible and protective haplotypes of the HBB gene in predisposing to beta-thalassaemia in Malaysia

Association between CARD14 gene polymorphisms and psoriasis vulgaris in Hainan Han population based on exon sequencing: A case-control study.

Psoriasis is a serious non-communicable, chronic immune-inflammatory mediated disease affecting about 125 million people worldwide. Its effects go beyond skin manifestation. Through genome-wide association studies, the caspase recruitment domain family member 14 (CARD14) gene and other gene variants have been implicated to have an association with Psoriasis, and as we move towards individualized therapy the discovery of single nucleotide polymorphism (SNP) is of great importance. This study aimed to determine whether the CARD14 gene is a susceptible gene for psoriasis vulgaris. In this study, 101 psoriasis patients and 79 healthy controls were subjected to exome sequencing. The CARD14 gene regions upstream and downstream of 1kb were sequenced. SNP-based association analysis and haplotype-based association analysis were performed in SNPs with minimum allele frequency (MAF) greater than 1%. Bioinformatic methods were used to predict the impact of risk loci on gene function. A total of 32 polymorphisms were identified in this study, of which 3 SNPs (1 in exon and 2 in intron) were susceptible to psoriasis (P < .05, OR = 0.19~0.53, 95%CI = 0.05~0.70). Bioinformatics analysis showed that rs144475004 located on the exon led to an amino acid change from aspartate to histidine. On the other hand, results of haplotype-based association analysis showed that 2 haplotypes (CARD14-1 and CARD14-2) were protective haplotypes of the disease (P < .05, OR = 0.18~0.38, 95%CI = 0.05~0.88), the frequencies in healthy controls and patients was 6.96% and 1.49%, respectively. CARD14 gene is associated with susceptibility to psoriasis vulgaris in the Hainan Han population.

Fine-mapping and identification of candidate causal genes for tail length in the Merinolandschaf breed

Docking the tails of lambs in long-tailed sheep breeds is a common practice worldwide. But this practice is associated with pain. Breeding for a shorter tail could offer an alternative. Therefore, this study aimed to analyze the natural tail length variation in the Merinolandschaf and to identify causal alleles for the short tail phenotype segregating within long-tailed breeds. We used SNP-based association analysis and haplotype-based mapping in 362 genotyped (Illumina OvineSNP50) and phenotyped Merinolandschaf lambs. Genome-wide significant regions were capture sequenced in 48 lambs and comparatively analyzed in various long and short-tailed sheep breeds and wild sheep subspecies. Here we show a SNP located in the first exon of HOXB13 and a SINE element located in the promotor of HOXB13 as promising candidates. These results enable more precise breeding towards shorter tails, improve animal welfare by amplification of ancestral alleles and contribute to a better understanding of differential embryonic development.

Vitamin D receptor gene polymorphisms are associated with psoriasis susceptibility and the clinical response to calcipotriol in psoriatic patients.

Psoriasis is a common genetic disease characterized by hyperproliferation and disordered maturation of keratinocytes. To date, many association studies between psoriasis and VDR gene have been conducted, but the results are controversial. Furthermore, vitamin D3 analogue has anti-psoriatic activity; however, the clinical response is variable. This study was conducted to explore whether VDR gene polymorphisms are associated with psoriasis susceptibility and clinical response to calcipotriol in psoriatic patients. A total of 110 patients and 183 controls were genotyped for VDR gene polymorphisms rs2228570, rs731236, rs1544410 and rs7975232 by LDR method. SNP-based and haplotype-based association analyses were subsequently performed. Patients with PASI<3 were treated with calcipotriol ointment monotherapy. After 6weeks of therapy, the correlations between efficacy and the genotypes of each polymorphism were evaluated. The results showed that for rs7975232, allele A was significantly over-represented in psoriasis patients relative to controls (39.09% vs. 27.05%, OR (95% CI)=1.731 (1.213-2.471)), and compared with the reference CC genotype, the following ORs were observed: AA genotype OR=2.404 (95% CI: 1.085-5.328; P=.034) and GA genotype OR=2.143 (95% CI: 1.283-3.579; P=.005). Haplotype analyses showed that the rs2228570/rs731236/rs1544410/rs7975232 CTGA was significantly over-represented in psoriasis patients compared with controls (OR (95% CI)=1.907 (1.132-3.214); P=.020). Among the patients with PASI<3, the response rates to calcipotriol were significantly higher in patients with rs7975232 CC genotypes than in those with other genotypes (x2 =9.172, P=.010). These data suggest that VDR polymorphisms are associated with psoriasis susceptibility and clinical response to calcipotriol in psoriatic patients.

Suggestive evidence of protective haplotype within TGF-B1 gene region in breast density utilizing fine mapping analysis

Introduction and aimIncreased mammographic density is a strong and independent attribute factor for breast carcinoma. The density is associated with epithelial cell proliferation and stromal fibrosis development that is regulated by Transforming Growth Factor-Beta 1 (TGF-β1). This study was aimed to fine map the TGF-β1 gene cluster in predicting the role of predisposing haplotypes in breast density development using tagging SNPs. Materials and methodsGenotyping was conducted for three tagging TGF-β1 SNPs assigned as rs1800469, rs1800470 and rs4803455 in a total of 50 consented female participants as case group which involved BI-RADS 3 and 4, while the control group (n = 50) consisted of BI-RADS 1 and 2 who underwent a mammography check-up at The Department of Radiology, Hospital Sultanah Nur Zahirah of Kuala Terengganu. A series of bioinformatics analysis including individual SNP-based association analysis, haplotype-based association analysis and fine mapping analysis was conducted spanning the gene region of 20 kb utilizing Chinese Hans Beijing (CHB) and Japanese (JPT) representing the Asian dataset from the HapMap database. ResultsThe study demonstrated two tagging SNPs of TGF-β1 yielded a significant association (rs1800469, p-value = .034; rs1800470, p-value = .004). However, rs4803455 did not show a significant association with p value of 0.053. We further revealed the susceptible haplotype that was assigned as haplotype GGA with significant P value 0.003 indicated by odd ratio of 3.349 (1.482–7.568). A potential protective haplotype was observed in haplotype AGC with odd ratio 0.585 (0.316–1.080) even though with less significant (p = .085). The fine map revealed four targeted SNPs in which consist of rs12461895, rs10416269, rs7408955 and rs1549934 from Block 2 and five potential SNPs from Block 4 assigned as rs4803457, rs2317130, rs1166933, rs1982072, rs11670143 as another potential genetic variant that might contribute to breast density. DiscussionThis fine map study suggests the potential protective haplotype and genetic variants of TGF-β1 in predisposing to breast density development which is worth to be genotyped and further investigated.

Polymorphisms and genetic effects of PRLR, MOGAT1, MINPP1 and CHUK genes on milk fatty acid traits in Chinese Holstein

BackgroundOur initial genome-wide association study (GWAS) identified 20 promising candidate genes for milk fatty acid (FA) traits in a Chinese Holstein population, including PRLR, MOGAT1, MINPP1 and CHUK genes. In this study, we performed whether they had significant genetic effects on milk FA traits in Chinese Holstein.ResultsWe re-sequenced the entire exons and 3000 bp of the 5′ and 3′ flanking regions, and identified 11 single nucleotide polymorphisms (SNPs), containing four in PRLR, two in MOGAT1, two in MINPP1, and three in CHUK. The SNP-based association analyses showed that all the 11 SNPs were significantly associated with at least one milk FA trait (P = 0.0456 ~ < 0.0001), and none of them had association with C11:0, C13:0, C15:0 and C16:0 (P > 0.05). By the linkage disequilibrium (LD) analyses, we found two, one, one, and one haplotype blocks in PRLR, MOGAT1, MINPP1, and CHUK, respectively, and each haplotype block was significantly associated with at least one milk FA trait (P = 0.0456 ~ < 0.0001). Further, g.38949011G > A in PRLR, and g.111599360A > G and g.111601747 T > A in MOGAT1 were predicted to alter the transcription factor binding sites (TFBSs). A missense mutation, g.39115344G > A, could change the PRLR protein structure. The g.20966385C > G of CHUK varied the binding sequences for microRNAs. Therefore, we deduced the five SNPs as the potential functional mutations.ConclusionIn summary, we first detected the genetic effects of PRLR, MOGAT1, MINPP1 and CHUK genes on milk FA traits, and researched the potential functional mutations. These data provided the basis for further investigation on function validation of the four genes in Chinese Holstein.

Demonstration of critical role of GRIN3A in nicotine dependence through both genetic association and molecular functional studies.

Nicotine dependence (ND) is a chronic disease with catastrophic effects on individual and public health. The glutamate receptor subunit gene, ionotropic N-methyl-d-aspartate 3A (GRIN3A), encodes a crucial subunit of N-methyl-d-aspartate receptors (NMDARs), which play an essential role in synaptic plasticity in the brain. Although various variants of GRIN3A have been associated with ND in European-American and African-American samples, no study has been reported for the association between GRIN3A and ND in Chinese Han population. We performed an association study of 16 single nucleotide polymorphisms (SNPs) in GRIN3A with ND in 2616 Chinese individuals. SNP-based association analysis indicated that SNP rs1323423 was significantly associated with the Fagerström Test for Nicotine Dependence (FTND) score after correction for multiple testing (P=0.0026). Haplotype-based association analysis revealed that Block 3, formed by rs1323423-rs10989591, was significantly associated with the FTND score after correction for multiple testing (global P=0.0183). Furthermore, luciferase reporter assay demonstrated that the DNA region containing rs1323423 was an enhancer element, the activity of which was significantly impacted by rs1323423 genotype. Considering that rs1323423 is located in a potential enhancer region, we performed GRIN3A editing in HEK293T cells with CRISPR/Cas9 and found that the DNA region around rs1323423 has a regulatory function and the expression of GRIN3A affects the expression of other NMDA subunits. Moreover, we demonstrated that nicotine at a concentration of 100μM decreased expression of GRIN3A in SH-SY5Y and HEK293T cells at the RNA and protein level, respectively. This study provides novel evidence for the involvement of GRIN3A in ND.

Identification of exon skipping events associated with Alzheimer\u2019s disease in the human hippocampus

BackgroundAt least 90% of human genes are alternatively spliced. Alternative splicing has an important function regulating gene expression and miss-splicing can contribute to risk for human diseases, including Alzheimer’s disease (AD).MethodsWe developed a splicing decision model as a molecular mechanism to identify functional exon skipping events and genetic variation affecting alternative splicing on a genome-wide scale by integrating genomics, transcriptomics, and neuroimaging data in a systems biology approach. In this study, we analyzed RNA-Seq data of hippocampus brain tissue from Alzheimer’s disease (AD; n = 24) and cognitively normal elderly controls (CN; n = 50) and identified three exon skipping events in two genes (RELN and NOS1) as significantly associated with AD (corrected p-value < 0.05 and fold change > 1.5). Next, we identified single-nucleotide polymorphisms (SNPs) affecting exon skipping events using the splicing decision model and then performed an association analysis of SNPs potentially affecting three exon skipping events with a global cortical measure of amyloid-β deposition measured by [18F] Florbetapir position emission tomography (PET) scan as an AD-related quantitative phenotype. A whole-brain voxel-based analysis was also performed.ResultsTwo exons in RELN and one exon in NOS1 showed significantly lower expression levels in the AD participants compared to CN participants, suggesting that the exons tend to be skipped more in AD. We also showed the loss of the core protein structure due to the skipped exons using the protein 3D structure analysis. The targeted SNP-based association analysis identified one intronic SNP (rs362771) adjacent to the skipped exon 24 in RELN as significantly associated with cortical amyloid-β levels (corrected p-value < 0.05). This SNP is within the splicing regulatory element, i.e., intronic splicing enhancer. The minor allele of rs362771 conferred decreases in cortical amyloid-β levels in the right temporal and bilateral parietal lobes.ConclusionsOur results suggest that exon skipping events and splicing-affecting SNPs in the human hippocampus may contribute to AD pathogenesis. Integration of multiple omics and neuroimaging data provides insights into possible mechanisms underlying AD pathophysiology through exon skipping and may help identify novel therapeutic targets.

Genome Wide Association Analyses Identify Pleiotropic Variants Associated with Acute Myeloid Leukemia (AML) and Myelodysplastic Syndrome (MDS) Susceptibility

The first two authors and last two authors contributed equally.Genome-wide association studies (GWAS) have identified risk loci for Acute Lymphoblastic Leukemia (ALL), Chronic Lymphoblastic Leukemia (CLL) and Non-Hodgkin Lymphoma, however an Acute Myeloid Leukemia (AML) GWAS has not been published to date. We performed a GWAS to identify AML and Myelodysplastic Syndrome (MDS) risk loci using a nested case-control study design in the DISCOVeRY-BMT cohorts which includes almost 2000 AML and MDS patients as cases and 2813 unrelated donors as controls.Genotyping was performed using the Illumina Human OmniExpress BeadChip and imputed using the Haplotype Reference Consortium, yielding > 8 million high-quality variants for statistical analysis. Logistic regression models with AML (de novo AML with normal cytogenetics, de novo AML with abnormal cytogenetics and therapy-related AML) and MDS cases and European American healthy donor controls adjusted for age and sex were used to test the association of each SNP with disease status. To identify the strongest association signal with disease we conducted a summary statistic SNP-based association analysis (ASSET) using non-overlapping AML and MDS cases implemented in R statistical software. ASSET uses an exhaustive search for SNPs with small but common pleiotropic effects across groups of traits while accounting for the multiple tests required by the subset search, as well as any shared controls between groups. This approach allowed us to further investigate the heterogeneity within AML subtypes and to gain increased power by pooling subtypes that show pleiotropic effects. ASSET genome wide (GW) significance is defined as P<5.0x10-8, however, we present results of each subset analysis.ASSET analyses identified an association of T allele at rs12203592, Interferon Regulatory Factor 4 (IRF4), with increased risk of de novo AML and MDS (Figure 1) (OR=1.37; 95% CI, 1.25-1.51, Pmeta=1.59x10-10). The variant has been reported significant in GWAS of skin pigmentation, hair color, non-melanoma skin cancer, squamous cell carcinoma, actinic keratosis, and childhood ALL; GW case-control studies of neuroblastoma and breast cancer show suggestive association signals (P<5 x 10-6).IRF4 belongs to the IRF family of transcription factors and is a key regulator of differentiation stages in hematopoiesis. The T allele at rs12203592 is associated with significantly increased expression of IRF4 in whole blood and lung tissue and in vitro mouse studies have shown the T allele to be associated with higher levels of IRF4 transcription. rs12203592 is <80bp from an IRF4 transcription start site and in an important position within NF-κB motifs in multiple blood and immune cell lines, supporting the hypothesis that this SNP modulates NF-κB repression of IRF4 expression.Another variant in the IRF4 regulatory region, rs62389423 (A allele), showed a putative association with subsets of de novo AML and MDS (OR=1.36; 95% CI,1.21-1.52, Pmeta=1.2x10-7). Although not in linkage disequilibrium with rs12203592, the A allele has also been previously associated with susceptibility to skin cancer and melanoma in multiple GWAS. Analysis of 596 UKbiobank CLL cases and >300,000 controls, shows the A allele correlates with a 50% increased risk of CLL (95% CI, 1.32-1.73, P= 2.4x10-9).Several other GWAS also show an association between additional IRF4 variants and CLL.Analyses in individual subtypes also revealed an intronic variant, rs10098598, in RAD21 to be associated with de novo AML (OR=2.18; 95% CI, 1.60-2.97; Pmeta=8.9x10-7). RAD21, on8q24, a region known to be associated with multiple cancer types, is involved in DNA double-stranded break repair and in chromatid cohesion in mitotic cells. Loss of function would theoretically lead to chromosomal instability and tumorigenesis. Components of the cohesion complex (including RAD21) are somatically mutated in ~12% of de novo AML and MDS patients, where it is frequently an early event.We provide the first GW evidence of association between a common variant and AML susceptibility. This SNP has been shown to be associated with multiple phenotypes, suggesting that there are pleiotropic effects at work. Our RAD21 finding is consistent with the role of cohesion in leukemogenesis and provides some evidence of its role in de novo AML susceptibility. Replication and further subset analyses with genome-wide data in >2000 AML cases are ongoing. [Display omitted] DisclosuresGriffiths:Alexion Inc.: Honoraria, Research Funding; Astex/Otsuka Pharmaceuticals: Honoraria, Research Funding; Celgene, Inc: Honoraria, Research Funding; Novartis, Inc.: Research Funding; Pfizer, Inc.: Research Funding. McCarthy:Bristol Myers Squibb: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Karyopharm: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Janssen: Consultancy, Honoraria.

Association and cis-mQTL analysis of variants in serotonergic genes associated with nicotine dependence in Chinese Han smokers

Variants in serotonergic genes are implicated in nicotine dependence (ND) in subjects of European and African origin, but their involvement with smoking in Asians is largely unknown. Moreover, mechanisms underlying the ND risk-associated single-nucleotide polymorphisms (SNPs) in these genes are rarely investigated. The Fagerström Test for Nicotine Dependence (FTND) score was used to assess ND in 2616 male Chinese Han smokers. Both association and interaction analysis were used to examine the association of variants in the serotonergic genes with FTND. Further, expression and methylation quantitative trait loci (cis-mQTL) analysis was employed to determine the association of individual SNPs with the extent of methylation of each CpG locus. Individual SNP-based association analysis revealed that rs1176744 in HTR3B was marginally associated with FTND (p = 0.042). Haplotype-based association analysis found that one major haplotype, T-T-A-G, formed by SNPs rs3758987-rs4938056-rs1176744-rs2276305, located in the 5′ region of HTR3B, showed a significant association with FTND (p = 0.00025). Further, a significant genetic interactive effect affecting ND was detected among SNPs rs10160548 in HTR3A, and rs3758987, rs2276305, and rs1672717 in HTR3B (p = 0.0074). Finally, we found four CpG sites (CpG_4543549, CpG_4543464, CpG_4543682, and CpG_4546888) to be significantly associated with three cis-mQTL SNPs (i.e., rs3758987, rs4938056, and rs1176744) located in our detected haplotype within HTR3B. In sum, we showed SNP rs1176744 (Tyr129Ser) to be associated with ND. Together with the SNPs rs3758987 and rs4938056 in HTR3B, they formed a major haplotype, which had significant association with ND. We further showed these SNPs contribute to ND through four methylated sites in HTR3B. All these findings suggest that variants in the serotonergic system play an important role in ND in the Chinese Han population. More importantly, these findings demonstrated that the involvement of this system in ND is through gene-by-gene interaction and methylation.

An Exome-Wide Association Study Identifies New Susceptibility Loci for Age of Smoking Initiation in African- and European-American Populations.

Cigarette smoking is one of the largest causes of preventable death worldwide. This study aimed to identify susceptibility loci for age at smoking initiation (ASI) by performing an exome-wide association analysis. A total of 2510 smokers of either African-American (AA) or European-American (EA) origin were genotyped and analyzed at both the single nucleotide polymorphism (SNP) and gene levels. After removal of those SNPs with a minor allele frequency (<0.01), 48091 and 34933 SNPs for AAs and EAs, respectively, were used to conduct a SNP-based association analysis. Gene-based analyses were then performed for all SNPs examined within each gene. Further, we estimated the proportion of variance explained by all common SNPs included in the analysis. The strongest signals were detected for SNPs rs17849904 in the pitrilysin metallopeptidase 1 gene (PITRM1) in the AA sample (p = 9.02 × 10-7) and rs34722354 in the discoidin domain of the receptor tyrosine kinase 2 gene (DDR2) in the EA sample (p = 9.74 × 10-7). Both SNPs remained significant after Bonferroni correction for the number of SNPs tested. Subsequently, the gene-based association analysis revealed a significantly associated gene, DHRS7, in the AA sample (p = 5.00 × 10-6), a gene previously implicated in nicotine metabolism. Our study revealed two susceptibility loci for age of smoking initiation in the two ethnic samples, with the first being PITRM1 for AA smokers and the second DDR2 for EA smokers. In addition, we found DHRS7 to be a plausible candidate for ASI in the AA sample from our gene-based association analysis. PITRM1 and DHRS7 for African-American smokers and DDR2 for European-American smokers are new candidate genes for smoking initiation. These genes represent new additions to smoking initiation, an important but less studied phenotype in nicotine dependence research.

A SNP-based association analysis for plant growth habit in worldwide cowpea (Vigna unguiculata (L.) Walp) Germplasm

Cowpea is a legume widely grown in Africa, North, Central and South America, and Asia. The cowpea plant growth habits consist of erect, semi-prostrate, and prostrate types. Developing a cultivar while considering plant growth habit is essential within a breeding program since the need for a particular growth habit is region-specific, and significantly depends on the end user preference. Some cowpea growers might prefer erect types over semi-prostrate and prostrate types, while others would chose prostrate types, which provide more leaves for feed supplies to livestock. However, very little is known regarding the genetics of plant growth habit in cowpea to assist plant breeders in developing suitable cowpea cultivars having the desired growth habit plus the other required features. Therefore, the objectives of this study were to conduct an association mapping for cowpea growth habit, and to identify SNP markers associated with this trait. A total of 487 cowpea genotypes were evaluated for growth habit and a total of 1031 SNPs postulated from genotyping-by-sequencing to conduct association analysis study for cowpea growth habit. Our results showed that: (1) significant differences in cowpea growth habit were identified between countries, (2) the cowpea erect-type was prevalent, and (3) ten SNP markers, C35060651_729, C35061339_799, C35062457_1855, C35072764_1384, C35080248_2355, Scaffold2771_4351, Scaffold29522_3213, Scaffold35913_2678, Scaffold53560_188, and Scaffold58098_4297, were significantly associated with cowpea growth habit. These results could be used for enhancing marker-assisted selection (MAS) in breeding programs aimed at developing cowpea cultivars having a particular growth habit.

SNP-based association analysis for seedling traits in durum wheat (Triticum turgidum L. durum (Desf.)).

In the present study, 150 accessions of worldwide originated durum wheat germplasm (Triticum turgidum spp. durum) were observed for major seedling traits and their growth. The accessions were evaluated for major seedling traits under controlled conditions of hydroponics at the 13th, 20th, 27th and 34th day-after germination. Biomass traits were measured at the 34th day-after germination. Correlation analysis was conducted among the seedling traits and three field traits at maturity, plant height, grain weight and 1000-grain weight observed in four consecutive years. Associations of the measured seedling traits and SNP markers were analyzed based on the mixed linear model (MLM). The results indicated that highly significant genetic variation and robust heritability were found for the seedling and field mature traits. In total, 259 significant associations were detected for all the traits and four growth stages. The phenotypic variation explained (R2) by a single SNP marker is higher than 10% for most (84%) of the significant SNP markers. Forty-six SNP markers associated with multiple traits, indicating non-neglectable pleiotropy in seedling stage. The associated SNP markers could be helpful for genetic analysis of seedling traits, and marker-assisted breeding of new wheat varieties with strong seedling vigor.

Association genetics in Populus reveals the interactions between Pto-miR160a and its target Pto-ARF16.

MicroRNAs (miRNAs) play important roles in the regulation of gene expression in various biological processes. However, the interactions between miRNAs and their targets are largely unknown in plants. As a powerful tool for identification of variation associated with traits, association genetics provides another strategy for exploration of interactions between miRNAs and their targets. Here, we conducted expression analysis and association mapping to evaluate the interaction between Pto-miR160a and its target Pto-ARF16 in Populus tomentosa. By examining the expression patterns of Pto-MIR160a and Pto-ARF16, we identified a significant, negative correlation between their expression levels, indicating that Pto-miR160a may affect the expression of Pto-ARF16. Among the single nucleotide polymorphisms (SNPs) identified in this study, one common SNP in the pre-miRNA region of Pto-miR160a altered its predicted secondary structure while another common SNP in the predicted miRNA target site changed the binding affinity of Pto-miR160a. Linkage disequilibrium (LD) analysis revealed low LD levels of Pto-MIR160a and Pto-ARF16, indicating that they are suitable for candidate gene-based association analysis. Single SNP-based association analysis identified 19 SNPs (false discovery rate Q<0.05) in Pto-MIR160a and Pto-ARF16 associated with three phenotypic traits. Epistasis analysis further identified 36 SNP-SNP interactions between SNPs in Pto-MIR160a and SNPs in Pto-ARF16, reflecting the possible genetic interaction of Pto-miR160a and Pto-ARF16. Taking these results together, our study identified SNPs in Pto-MIR160a and Pto-ARF16 associated with tree growth and wood properties, providing SNPs with potential applications in marker-assisted breeding and evidence for the genetic interaction of Pto-miR160a and Pto-ARF16.

Fine mapping analysis of HLA-DP/DQ gene clusters on chromosome 6 reveals multiple susceptibility loci for HBV infection.

Recent genome-wide association studies have revealed the HLA region on chromosome 6p21 as a susceptibility locus for hepatitis B virus (HBV) infection, a finding subsequently replicated in independent samples. However, only limited single nucleotide polymorphisms (SNPs) were analyzed in most of these studies, and it remains to be determined which SNPs contribute to the detected association. After genotyping 140 SNPs within this genomic region in a total of 1657 HBV-positive patients and 1456 HBV-negative controls, we conducted a series of genetic epidemiological and bioinformatics analysis, including individual SNP-based association analysis, haplotype-based association analysis, and conditional analysis. We identified 76 SNPs and 5 LD blocks in HLA-DP/DQ clusters that are significantly associated with HBV infection, with the smallest P value being 3.88 × 10(-18) for rs9277535 in HLA-DPB1. With conditional analysis, we further revealed that the genes contributing to the effects of variants in HLA-DP/DQ on infection are independent of each other, and the LD block 5 in the 3'-UTR region of HLA-DPB1 had a predominant effect in the association of HLA-DP with HBV infection. We also found that the SNPs in the 3'-UTR region of HLA-DPB1 were significant between the subgroups of inactive HBV carrier, chronic hepatitis B, or hepatic cirrhosis from the case group and the spontaneous HBV-clearance subgroup from the control group. Finally, we did further association analysis of SNPs in this region with different subgroups from the case group, which revealed no association of these SNPs with the progression of HBV-related diseases. In sum, we showed, for the first time, that the HLA-DP/DQ clusters contribute independently to HBV infection, and the 3'-UTR region of HLA-DPB1 represents an important functional region involved in HBV infection.

Association and interaction analyses of 5-HT3 receptor and serotonin transporter genes with alcohol, cocaine, and nicotine dependence using the SAGE data.

Previous studies have implicated genes encoding the 5-HT3AB receptors (HTR3A and HTR3B) and the serotonin transporter (SLC6A4), both independently and interactively, in alcohol (AD), cocaine (CD), and nicotine dependence (ND). However, whether these genetic effects also exist in subjects with comorbidities remains largely unknown. We used 1,136 African-American (AA) and 2,428 European-American (EA) subjects from the Study of Addiction: Genetics and Environment (SAGE) to determine associations between 88 genotyped or imputed variants within HTR3A, HTR3B, and SLC6A4 and three types of addictions, which were measured by DSM-IV diagnoses of AD, CD, and ND and the Fagerström Test for Nicotine Dependence (FTND), an independent measure of ND commonly used in tobacco research. Individual SNP-based association analysis revealed a significant association of rs2066713 in SLC6A4 with FTND in AA (β = -1.39; P = 1.6E - 04). Haplotype-based association analysis found one major haplotype formed by SNPs rs3891484 and rs3758987 in HTR3B that was significantly associated with AD in the AA sample, and another major haplotype T-T-G, formed by SNPs rs7118530, rs12221649, and rs2085421 in HTR3A, which showed significant association with FTND in the EA sample. Considering the biologic roles of the three genes and their functional relations, we used the GPU-based Generalized Multifactor Dimensionality Reduction (GMDR-GPU) program to test SNP-by-SNP interactions within the three genes and discovered two- to five-variant models that have significant impacts on AD, CD, ND, or FTND. Interestingly, most of the SNPs included in the genetic interaction model(s) for each addictive phenotype are either overlapped or in high linkage disequilibrium for both AA and EA samples, suggesting these detected variants in HTR3A, HTR3B, and SLC6A4 are interactively contributing to etiology of the three addictive phenotypes examined in this study.

Advances in development of gene-gene interaction analy-sis methods based on SNP data: a review

The SNP-based association analysis has become one of the most important approaches to interpret the underlying molecular mechanisms for human complex diseases. Nevertheless, the widely-used singe-locus analysis is only capable of capturing a small portion of susceptible SNPs with prominent marginal effects, leaving the important genetic component, epistasis or joint effects, to be undetectable. Identifying the complex interplays among multiple genes in the genome-wide context is an essential task for systematically unraveling the molecular mechanisms for complex diseases. Many approaches have been used to detect genome-wide gene-gene interactions and provided new insights into the genetic basis of complex diseases. This paper reviewed recent advances of the methods for detecting gene-gene interaction, categorized into three types, model-based and model-free statistical methods, and data mining methods, based on their characteristics in theory and numerical algorithm. In particular, the basic principle, numerical implementation and cautions for application for each method were elucidated. In addition, this paper briefly discussed the limitations and challenges associated with detecting genome-wide epistasis, in order to provide some methodological consultancies for scientists in the related fields.

Meta-analysis of genome-wide association data identifies novel susceptibility loci for obesity.

Obesity is a major public health problem with strong genetic determination. Multiple genetic variants have been implicated for obesity by conducting genome-wide association (GWA) studies, primarily focused on body mass index (BMI). Fat body mass (FBM) is phenotypically more homogeneous than BMI and is more appropriate for obesity research; however, relatively few studies have been conducted on FBM. Aiming to identify variants associated with obesity, we carried out meta-analyses of seven GWA studies for BMI-related traits including FBM, and followed these analyses by de novo replication. The discovery cohorts consisted of 21 969 individuals from diverse ethnic populations and a total of over 4 million genotyped or imputed SNPs. The de novo replication cohorts consisted of 6663 subjects from two independent samples. To complement individual SNP-based association analyses, we also carried out gene-based GWA analyses in which all variations within a gene were considered jointly. Individual SNP-based association analyses identified a novel locus 1q21 [rs2230061, CTSS (Cathepsin S)] that was associated with FBM after the adjustment of lean body mass (LBM) (P = 3.57 × 10(-8)) at the genome-wide significance level. Gene-based association analyses identified a novel gene NLK (nemo-like kinase) in 17q11 that was significantly associated with FBM adjusted by LBM. In addition, we confirmed three previously reported obesity susceptibility loci: 16q12 [rs62033400, P = 1.97 × 10(-14), FTO (fat mass and obesity associated)], 18q22 [rs6567160, P = 8.09 × 10(-19), MC4R (melanocortin 4 receptor)] and 2p25 [rs939583, P = 1.07 × 10(-7), TMEM18 (transmembrane protein 18)]. We also found that rs6567160 may exert pleiotropic effects to both FBM and LBM. Our results provide additional insights into the molecular genetic basis of obesity and may provide future targets for effective prevention and therapeutic intervention.