Xiang-Yang Lou

A Combinatorial Approach to Detecting Gene-Gene and Gene-Environment Interactions in Family Studies

Widespread multifactor interactions present a significant challenge in determining risk factors of complex diseases. Several combinatorial approaches, such as the multifactor dimensionality reduction (MDR) method, have emerged as a promising tool for better detecting gene-gene (G x G) and gene-environment (G x E) interactions. We recently developed a general combinatorial approach, namely the generalized multifactor dimensionality reduction (GMDR) method, which can entertain both qualitative and quantitative phenotypes and allows for both discrete and continuous covariates to detect G x G and G x E interactions in a sample of unrelated individuals. In this article, we report the development of an algorithm that can be used to study G x G and G x E interactions for family-based designs, called pedigree-based GMDR (PGMDR). Compared to the available method, our proposed method has several major improvements, including allowing for covariate adjustments and being applicable to arbitrary phenotypes, arbitrary pedigree structures, and arbitrary patterns of missing marker genotypes. Our Monte Carlo simulations provide evidence that the PGMDR method is superior in performance to identify epistatic loci compared to the MDR-pedigree disequilibrium test (PDT). Finally, we applied our proposed approach to a genetic data set on tobacco dependence and found a significant interaction between two taste receptor genes (i.e., TAS2R16 and TAS2R38) in affecting nicotine dependence.

A Genomewide Search Finds Major Susceptibility Loci for Nicotine Dependence on Chromosome 10 in African Americans

Epidemiological studies have demonstrated that genetic factors account for at least 50% of the liability for nicotine dependence (ND). Although several linkage studies have been conducted, all samples to date were primarily of European origin. In this study, we conducted a genomewide scan of 1,261 individuals, representing 402 nuclear families, of African American (AA) origin. We examined 385 autosomal microsatellite markers for ND, which was assessed by smoking quantity (SQ), the Heaviness of Smoking Index (HSI), and the Fagerstrom Test for ND (FTND). After performing linkage analyses using various methods implemented in the GENEHUNTER and S.A.G.E. programs, we found a region near marker D10S1432 on chromosome 10q22 that showed a significant linkage to indexed SQ, with a maximum LOD score of 4.17 at 92 cM and suggestive linkage to HSI, SQ, and log-transformed SQ. Additionally, we identified three regions that met the criteria for suggestive linkage to at least one ND measure: on chromosomes 9q31 at marker D9S1825, 11p11 between markers D11S1993 and D11S1344, and 13q13 between markers D13S325 and D13S788. Other locations on chromosomes 15p11, 17q25, and 18q12 exhibited some evidence of linkage for ND (LOD >1.44). The four regions with significant or suggestive linkage were positive for multiple ND measures by multiple statistical methods. Some of these regions have been linked to smoking behavior at nominally significant levels in other studies, which provides independent replication of the regions for ND in different cohorts. In summary, we found significant linkage on chromosome 10q22 and suggestive linkage on chromosomes 9, 11, and 13 for major genetic determinants of ND in an AA sample. Further analysis of these positive regions by fine mapping and/or association analysis is thus warranted. To our knowledge, this study represents the first genomewide linkage scan of ND in an AA sample.

Gene-Gene Interactions Among CHRNA4, CHRNB2, BDNF, and NTRK2 in Nicotine Dependence

BACKGROUND Epidemiological data indicate that nicotine dependence (ND) are influenced by genes, environmental factors, and their interactions. Although it has been documented from molecular experiments that brain-derived neurotrophic factor (BDNF) exerts its functions via neurotrophic tyrosine kinase receptor 2 (NTRK2) and both alpha 4 (CHRNA4) and beta 2 (CHRNB2) subunits are required to form functional alpha 4 beta 2-containing nicotinic receptors (nAChRs), no study is reported demonstrating that there exist gene-gene interactions among the four genes in affecting ND. METHODS To determine if gene-gene interactions exist among the four genes, we genotyped six single nucleotide polymorphisms (SNPs) for CHRNA4 and BDNF, nine SNPs for NTRK2, and four SNPs for CHRNB2 in a case-control sample containing 275 unrelated smokers with a Fagerström Test for Nicotine Dependence score of 4.0 or more and 348 unrelated nonsmokers. RESULTS By using a generalized multifactor dimensionality reduction algorithm recently developed by us, we found highly significant gene-gene interactions for the gene pairs of CHRNA4 and CHRNB2, CHRNA4 and NTRK2, CHRNB2 and NTRK2, and BDNF and NTRK2 (p < .01 for all four gene pairs) and significant gene-gene interaction between CHRNA4 and BDNF (p = .031) on ND. No significant interaction was detected for CHRNB2 and BDNF (p = .068). CONCLUSIONS Our study provides first evidence on the presence of gene-gene interaction among the four genes in affecting ND. Although CHRNB2 alone was not significantly associated with ND in several previously reported association studies on ND, we found it affects ND through interactions with CHRNA4 and NTRK2.

An improved genetic model generates high- resolution mapping of QTL for protein quality in maize endosperm

The genetic mapping of polymorphic markers in a cross between two inbred plant lines has proven to be a powerful method for detecting quantitative trait loci (QTL) underlying complex traits. However, existing methods of QTL mapping were developed for disomic inheritance of both marker and QTL loci in a diploid population. To map QTL influencing traits expressed in the endosperm, a triploid embryo-nourishing tissue resulting from double fertilization, existing QTL mapping models require modification to consider the trisomic inheritance of the endosperm and the generation difference between the mapping population and the endosperm. Such a model requires simultaneous use of two successive generations, which theoretically can lead to an increase in resolution for QTL mapping compared with the use of a single backcross or F2 generation. Using a newly developed model based on these considerations, we demonstrate the improved resolution of QTL, influencing protein quality traits in maize endosperm. The increased resolution made possible with this approach makes identified QTL accessible to positional cloning.

Practical and theoretical considerations in study design for detecting gene-gene interactions using MDR and GMDR approaches.

Detection of interacting risk factors for complex traits is challenging. The choice of an appropriate method, sample size, and allocation of cases and controls are serious concerns. To provide empirical guidelines for planning such studies and data analyses, we investigated the performance of the multifactor dimensionality reduction (MDR) and generalized MDR (GMDR) methods under various experimental scenarios. We developed the mathematical expectation of accuracy and used it as an indicator parameter to perform a gene-gene interaction study. We then examined the statistical power of GMDR and MDR within the plausible range of accuracy (0.50∼0.65) reported in the literature. The GMDR with covariate adjustment had a power of>80% in a case-control design with a sample size of≥2000, with theoretical accuracy ranging from 0.56 to 0.62. However, when the accuracy was<0.56, a sample size of≥4000 was required to have sufficient power. In our simulations, the GMDR outperformed the MDR under all models with accuracy ranging from 0.56∼0.62 for a sample size of 1000–2000. However, the two methods performed similarly when the accuracy was outside this range or the sample was significantly larger. We conclude that with adjustment of a covariate, GMDR performs better than MDR and a sample size of 1000∼2000 is reasonably large for detecting gene-gene interactions in the range of effect size reported by the current literature; whereas larger sample size is required for more subtle interactions with accuracy<0.56.

Association and interaction analysis of variants in CHRNA5/CHRNA3/CHRNB4 gene cluster with nicotine dependence in African and European Americans.

Several previous genome‐wide and targeted association studies revealed that variants in the CHRNA5–CHRNA3–CHRNB4 (CHRNA5/A3/B4) gene cluster on chromosome 15 that encode the α5, α3, and β4 subunits of the nicotinic acetylcholine receptors (nAChRs) are associated with nicotine dependence (ND) in European Americans (EAs) or others of European origin. Considering the distinct linkage disequilibrium patterns in European and other ethnic populations such as African Americans (AAs), it would be interesting to determine whether such associations exist in other ethnic populations. We performed a comprehensive association and interaction analysis of the CHRNA5/A3/B4 cluster in two ethnic samples to investigate the role of variants in the risk for ND, which was assessed by Smoking Quantity, Heaviness Smoking Index, and Fagerström test for ND. Using a family‐based association test, we found a nominal association of single nucleotide polymorphisms (SNPs) rs1317286 and rs8040868 in CHRNA3 with ND in the AA and combined AA and EA samples. Furthermore, we found that several haplotypes in CHRNA5 and CHRNA3 are nominally associated with ND in AA, EA, and pooled samples. However, none of these associations remained significant after correction for multiple testing. In addition, we performed interaction analysis of SNPs within the CHRNA5/A3/B4 cluster using the pedigree‐based generalized multifactor dimensionality reduction method and found significant interactions within CHRNA3 and among the three subunit genes in the AA and pooled samples. Together, these results indicate that variants within CHRNA3 and among CHRNA5, CHRNA3, and CHRNB4 contribute significantly to the etiology of ND through gene–gene interactions, although the association of each subunit gene with ND is weak in both the AA and EA samples.

Linkage and association studies in African- and Caucasian-American populations demonstrate that SHC3 is a novel susceptibility locus for nicotine dependence.

Our previous linkage study demonstrated that the 9q22–q23 chromosome region showed a ‘suggestive’ linkage to nicotine dependence (ND) in the Framingham Heart Study population. In this study, we provide further evidence for the linkage of this region to ND in an independent sample. Within this region, the gene encoding Src homology 2 domain-containing transforming protein C3 (SHC3) represents a plausible candidate for association with ND, assessed by smoking quantity (SQ), the Heaviness of Smoking Index (HSI) and the Fagerström Test for ND (FTND). We utilized 11 single-nucleotide polymorphisms within SHC3 to examine the association with ND in 602 nuclear families of either African-American (AA) or European-American (EA) origin. Individual SNP-based analysis indicated three SNPs for AAs and one for EAs were significantly associated with at least one ND measure. Haplotype analysis revealed that the haplotypes A-C-T-A-T-A of rs12519–rs3750399–rs4877042–rs2297313–rs1547696–rs1331188, with a frequency of 27.8 and 17.6%, and C-T-A-G-T of rs3750399–rs4877042–rs2297313–rs3818668–rs1547696, at a frequency of 44.7 and 30.6% in the AA and Combined samples, respectively, were significantly inversely associated with the ND measures. In the EA sample, another haplotype with a frequency of 10.6%, A-G-T-G of rs1331188–rs1556384–rs4534195–rs1411836, showed a significant inverse association with ND measures. These associations remained significant after Bonferroni correction. We further demonstrated the SHC3 contributed 40.1–59.2% (depending on the ND measures) of the linkage signals detected on chromosome 9. As further support, we found that nicotine administered through infusion increased the Shc3 mRNA level by 60% in the rat striatum, and decreased it by 22% in the nucleus accumbens (NA). At the protein level, Shc3 was decreased by 38.0% in the NA and showed no change in the striatum. Together, these findings strongly implicate SHC3 in the etiology of ND, which represents an important biological candidate for further investigation.

Efficient mapping of a female sterile gene in wheat ( Triticum aestivum L.)

Studies on inheritance of fertility are of great importance in wheat breeding. Although substantial progress has been achieved in molecular characterization of male sterility and fertility restoration recently, little effort has been devoted to female sterility. To identify the gene(s) controlling female sterility in wheat efficiently, an investigation was conducted for the seed setting ratio using a set of F2 populations derived from the cross between a female sterile line XND126 and an elite cultivar Gaocheng 8901. Bulked segregation analysis (BSA) method and recessive class approach were adopted to screen for SSR markers potentially linked to female fertility gene loci in 2005. Out of 1080 SSRs in wheat genome, eight markers on chromosome 2D showed a clear difference between two disparate bulks and small recombination frequency values, suggesting a strong linkage signal to the sterility gene. Based on the candidate linked markers, partial linkage maps were constructed with Mapmaker 3.0 (EXP) instead of whole genome maps, and quantitative trait locus (QTL) mapping was implemented with software QTLNetwork 2.0. A major gene locus designated as taf1, was located on chromosome 2DS. The above result was confirmed by the analysis for 2007 data, and taf1 was identified on the same chromosome 2DS with a confidence interval of 2.4 cM, which could explain 44.99% of phenotypic variation. These results provided fundamental information for fine mapping studies and laid the groundwork for wheat fertility genetic studies.

Genome-wide linkage scan for nicotine dependence in European Americans and its converging results with African Americans in the Mid-South Tobacco Family sample

Previously, we reported a genome-wide scan for nicotine dependence (ND) in the African American (AA) sample of the Mid-South Tobacco Family (MSTF) cohort. In this study, we conducted a genome-wide scan in 629 individuals representing 200 nuclear families of European American (EA) origin of the MSTF cohort with the goals of identifying vulnerability loci for ND in the EAs and determining converging regions across the ethnic groups. We examined 385 autosomal microsatellite markers for ND, which was assessed by smoking quantity (SQ), the Heaviness of Smoking Index (HSI) and the Fagerström test for ND (FTND). After performing linkage analyses using various methods implemented in the GENEHUNTER and SAGE programs, we found eight regions on chromosomes 2, 4, 9–12, 17 and 18 that met the criteria for suggestive linkage to at least one ND measure in the EA sample. Of these, the region on chromosome 4 at 43 cM showed suggestive linkage to indexed SQ, the HSI and the FTND, and the region on chromosome 9 at 24 cM showed suggestive linkage to the HSI and the FTND. To increase detection power, we analyzed a combined AA and EA sample using age, gender and ethnicity as covariates and found that the region on chromosome 12 near marker D12S372 showed significant linkage to SQ. Additionally, we found six regions on chromosomes 9–11, 13 and 18 that showed suggestive linkage to at least one ND measure in the combined sample. When we compared the linkage peaks detected for ND among the two samples and a combined sample, we found that four regions on chromosomes 9 (two regions), 11 and 18 overlapped. On the other hand, we identified five regions on chromosomes 2, 4, 10, 12 and 17 that showed linkage to ND only in the EA sample, and two regions on chromosomes 10 and 13 that showed linkage to ND only in the AA sample. For those linkages identified in only one sample, we found that the combined analysis of AA plus EA samples actually decreased the linkage signal. This indicates that some chromosomal regions may be more homogenous than others across the ethnic samples. All regions except for the one on chromosome 12 have been detected at nominally significant levels in other studies, providing independent replication of ND loci in different populations.

Association and Interaction Analyses of GABBR1 and GABBR2 with Nicotine Dependence in European- and African-American Populations

Previous studies have demonstrated that the γ-aminobutyric acid type B (GABAB) receptor plays an essential role in modulating neurotransmitter release and regulating the activity of ion channels and adenyl cyclase. However, whether the naturally occurring polymorphisms in the two GABAB receptor subunit genes interact with each other to alter susceptibility to nicotine dependence (ND) remains largely unknown. In this study, we genotyped 5 and 33 single nucleotide polymorphisms (SNPs) for GABAB receptor subunit 1 and 2 genes (GABBR1, GABBR2), respectively, in a sample of 2037 individuals from 602 nuclear families of African- American (AA) or European-American (EA) origin. We conducted association analyses to determine (1) the association of each subunit gene with ND at both the individual SNP and haplotype levels and (2) the collective effect(s) of SNPs in both GABAB subunits on the development of ND. Several individual SNPs and haplotypes in GABBR2 were significantly associated with ND in both ethnic samples. Two haplotypes in AAs and one haplotype in EAs showed a protective effect against ND, whilst two other haplotypes in AAs and three haplotypes in EAs showed a risk effect for developing ND. Interestingly, these significant haplotypes were confined to two regions of GABBR2 in the AA and EA samples. Additionally, we found two minor haplotypes in GABBR1 to be positively associated with Heaviness of Smoking Index (HSI) in the EA sample. Finally, we demonstrated the presence of epistasis between GABBR1 and GABBR2 for developing ND. The variants of GABBR1 and GABBR2 are significantly associated with ND, and the involvement of GABBR1 is most likely through its interaction with GABBR2, whereas GABBR2 polymorphisms directly alter susceptibility to ND. Future studies are needed with more dense SNP coverage of GABBR1 and GABBR2 to verify the epistatic effects of the two subunit genes.