Rare, low frequency and common coding variants in CHRNA5 and their contribution to nicotine dependence in European and African Americans.

The common nonsynonymous variant rs16969968 in the α5 nicotinic receptor subunit gene (CHRNA5) is the strongest genetic risk factor for nicotine dependence in European Americans and contributes to risk in African Americans. To comprehensively examine whether other CHRNA5 coding variation influences nicotine dependence risk, we performed targeted sequencing on 1582 nicotine-dependent cases (Fagerström Test for Nicotine Dependence score⩾4) and 1238 non-dependent controls, with independent replication of common and low frequency variants using 12 studies with exome chip data. Nicotine dependence was examined using logistic regression with individual common variants (minor allele frequency (MAF)⩾0.05), aggregate low frequency variants (0.05>MAF⩾0.005) and aggregate rare variants (MAF<0.005). Meta-analysis of primary results was performed with replication studies containing 12 174 heavy and 11 290 light smokers. Next-generation sequencing with 180 × coverage identified 24 nonsynonymous variants and 2 frameshift deletions in CHRNA5, including 9 novel variants in the 2820 subjects. Meta-analysis confirmed the risk effect of the only common variant (rs16969968, European ancestry: odds ratio (OR)=1.3, P=3.5 × 10(-11); African ancestry: OR=1.3, P=0.01) and demonstrated that three low frequency variants contributed an independent risk (aggregate term, European ancestry: OR=1.3, P=0.005; African ancestry: OR=1.4, P=0.0006). The remaining 22 rare coding variants were associated with increased risk of nicotine dependence in the European American primary sample (OR=12.9, P=0.01) and in the same risk direction in African Americans (OR=1.5, P=0.37). Our results indicate that common, low frequency and rare CHRNA5 coding variants are independently associated with nicotine dependence risk. These newly identified variants likely influence the risk for smoking-related diseases such as lung cancer.

Characterizing allelic association in the genome era.

Whole genome data are allowing the estimation of population genetic parameters with an accuracy not imagined 50 years ago. Variation in these parameters along the genome is being found empirically where once only approximate theoretical values were available. Along with increased information, however, has come the issue of multiple testing and the realization that high values of the coefficients of variation of quantities such as relatedness measures may make it difficult to draw inferences. This review concentrates on measures of allelic association within and between individuals and within and between populations.

Chronic Periodontitis Genome-wide Association Study in the Hispanic Community Health Study / Study of Latinos.

Chronic periodontitis (CP) has a genetic component, particularly its severe forms. Evidence from genome-wide association studies (GWASs) has highlighted several potential novel loci. Here, the authors report the first GWAS of CP among a large community-based sample of Hispanics/Latinos. The authors interrogated a quantitative trait of CP (mean interproximal clinical attachment level determined by full-mouth periodontal examinations) among 10,935 adult participants (mean age: 45 y, range: 18 to 76 y) from the Hispanic Community Health Study / Study of Latinos. Genotyping was done with a custom Illumina Omni2.5M array, and imputation to approximately 20 million single-nucleotide polymorphisms was based on the 1000 Genomes Project phase 1 reference panel. Analyses were based on linear mixed models adjusting for sex, age, study design features, ancestry, and kinship and employed a conventional P < 5 × 10-8 statistical significance threshold. The authors identified a genome-wide significant association signal in the 1q42.2 locus ( TSNAX-DISC1 noncoding RNA, lead single-nucleotide polymorphism: rs149133391, minor allele [C] frequency = 0.01, P = 7.9 × 10-9) and 4 more loci with suggestive evidence of association ( P < 5 × 10-6): 1q22 (rs13373934), 5p15.33 (rs186066047), 6p22.3 (rs10456847), and 11p15.1 (rs75715012). We tested these loci for replication in independent samples of European-American ( n = 4,402) and African-American ( n = 908) participants of the Atherosclerosis Risk in Communities study. There was no replication among the European Americans; however, the TSNAX-DISC1 locus replicated in the African-American sample (rs149133391, minor allele frequency = 0.02, P = 9.1 × 10-3), while the 1q22 locus was directionally concordant and nominally significant (rs13373934, P = 4.0 × 10-2). This discovery GWAS of interproximal clinical attachment level-a measure of lifetime periodontal tissue destruction-was conducted in a large, community-based sample of Hispanic/Latinos. It identified a genome-wide significant locus that was independently replicated in an African-American population. Identifying this genetic marker offers direction for interrogation in subsequent genomic and experimental studies of CP.

GWAS Identifies New Loci for Painful Temporomandibular Disorder: Hispanic Community Health Study/Study of Latinos.

Temporomandibular disorder (TMD) is a musculoskeletal condition characterized by pain and reduced function in the temporomandibular joint and/or associated masticatory musculature. Prevalence in the United States is 5% and twice as high among women as men. We conducted a discovery genome-wide association study (GWAS) of TMD in 10,153 participants (769 cases, 9,384 controls) of the US Hispanic Community Health Study/Study of Latinos (HCHS/SOL). The most promising single-nucleotide polymorphisms (SNPs) were tested in meta-analysis of 4 independent cohorts. One replication cohort was from the United States, and the others were from Germany, Finland, and Brazil, totaling 1,911 TMD cases and 6,903 controls. A locus near the sarcoglycan alpha ( SGCA), rs4794106, was suggestive in the discovery analysis ( P = 2.6 × 106) and replicated (i.e., 1-tailed P = 0.016) in the Brazilian cohort. In the discovery cohort, sex-stratified analysis identified 2 additional genome-wide significant loci in females. One lying upstream of the relaxin/insulin-like family peptide receptor 2 ( RXP2) (chromosome 13, rs60249166, odds ratio [OR] = 0.65, P = 3.6 × 10-8) was replicated among females in the meta-analysis (1-tailed P = 0.052). The other (chromosome 17, rs1531554, OR = 0.68, P = 2.9 × 10-8) was replicated among females (1-tailed P = 0.002), as well as replicated in meta-analysis of both sexes (1-tailed P = 0.021). A novel locus at genome-wide level of significance (rs73460075, OR = 0.56, P = 3.8 × 10-8) in the intron of the dystrophin gene DMD (X chromosome), and a suggestive locus on chromosome 7 (rs73271865, P = 2.9 × 10-7) upstream of the Sp4 Transcription Factor ( SP4) gene were identified in the discovery cohort, but neither of these was replicated. The SGCA gene encodes SGCA, which is involved in the cellular structure of muscle fibers and, along with DMD, forms part of the dystrophin-glycoprotein complex. Functional annotation suggested that several of these variants reside in loci that regulate processes relevant to TMD pathobiologic processes.

The effect of an intronic polymorphism on alcohol dehydrogenase expression in Drosophila melanogaster.

Several lines of evidence indicate that natural selection controls the frequencies of an allozyme polymorphism at the alcohol dehydrogenase (Adh) locus in Drosophila melanogaster. However, because of associations among sequence polymorphisms in the Adh region, it is not clear whether selection acts directly (or solely) on the allozymic site. This problem has been approached by using in vitro mutagenesis to distinguish among the effects on Adh expression of individual polymorphisms. This study shows that a polymorphism within the first Adh intron (delta 1) has a significant effect on the level of ADH protein. Like the allozyme, delta 1 shows a geographic cline in frequency, indicating that it may also be a target of natural selection. These results suggest that multisite selection models may be required to understand the evolutionary dynamics of individual loci.

Use of in vitro mutagenesis to analyze the molecular basis of the difference in Adh expression associated with the allozyme polymorphism in Drosophila melanogaster.

In natural populations of Drosophila melanogaster, the alcohol dehydrogenase (Adh) locus is polymorphic for two allozymes, designated Slow and Fast. Fast homozygotes generally have a two- to threefold higher ADH activity level than Slow homozygotes for two reasons: they have a higher concentration of ADH protein and the Fast protein has a higher catalytic efficiency. DNA sequencing studies have shown that the two allozymes generally differ by only a single amino acid at residue 192, which must therefore be the cause of the catalytic efficiency difference. A previous P element-transformation experiment mapped the difference in ADH protein level to a 2.3-kb HpaI/ClaI restriction fragment; which contains all of the Adh coding sequences but excludes all of the 5' flanking region of the distal transcriptional unit. Here we report the results of a site-directed in vitro mutagenesis experiment designed to investigate the effects of the amino acid replacement. This replacement has the expected effect on catalytic efficiency, but there is no detectable effect on the concentration of ADH protein estimated immunologically. This result shows that the average difference in ADH protein level between the allozymic classes is due to linkage disequilibrium between the amino acid replacement and one or more other polymorphisms within the HpaI/ClaI fragment. Sequence analysis of several Fast and Slow alleles suggested that the other polymorphism might be a silent substitution at nucleotide 1443, but another in vitro mutagenesis experiment reported here shows that this is not the case. Therefore, the molecular basis of the difference in ADH protein concentration between the allozymic classes remains an open question.

Effects of a transposable element insertion on alcohol dehydrogenase expression in Drosophila melanogaster.

Variation in the DNA sequence and level of alcohol dehydrogenase (Adh) gene expression in Drosophila melanogaster have been studied to determine what types of DNA polymorphisms contribute to phenotypic variation in natural populations. The Adh gene, like many others, shows a high level of variability in both DNA sequence and quantitative level of expression. A number of transposable element insertions occur in the Adh region and one of these, a copia insertion in the 5' flanking region, is associated with unusually low Adh expression. To determine whether this insertion (called R142) causes the low expression level, the insertion was excised from the cloned R142 Adh gene and the effect was assessed by P-element transformation. Removal of this insertion causes a threefold increase in the level of ADH, clearly showing that it contributes to the naturally occurring variation in expression at this locus. Removal of all but one LTR also causes a threefold increase, indicating that the mechanism is not a simple sequence disruption. Furthermore, this copia insertion, which is located between the two Adh promoters and their upstream enhancer sequences, has differential effects on the levels of proximal and distal transcripts. Finally, a test for the possible modifying effects of two suppressor loci, su(wa) and su(f), on this insertional mutation was negative, in contrast to a previous report in the literature.

The relationship between dipeptidase activity variation and larval viability in Drosophila melanogaster.

The enzyme dipeptidase-A (DIP-A) in Drosophila melanogaster is coded by a second chromosome locus that is polymorphic for three allozymes in natural populations. DIP-A appears to be the only enzyme in D. melanogaster capable of hydrolyzing the dipeptide glycyl-L-isoleucine, since flies homozygous for null alleles at this locus have no detectable glycyl-L-isoleucine-ase activity. DIP-A activity occurs in many tissues and throughout development, but is particularly high in the larval midgut, suggesting an important role in protein digestion. These observations suggested an experimental design for investigating the adaptive significance of genetic variation in DIP-A activity. Fitness components of DIP-A variants could be estimated and compared under two environmental conditions (defined diets under axenic conditions). In the restrictive environment, the essential amino acid L-isoleucine is provided only in the form of glycyl-L-isoleucine, whereas in the permissive environment, L-isoleucine is provided in free form. We predicted that DIP-A activity would be essential in the restrictive, but not in the permissive environment. The results reported here clearly contradict this prediction. Two stocks homozygous for DIP-A null alleles from different geographic locations are each viable on the restrictive diet. Furthermore, relative viability experiments in which null allele larvae compete with larvae having DIP-A activity provide no evidence for even a partial reduction in egg to adult survival on the restrictive diet. Apparently, the null allele larvae have some alternative mechanism for obtaining L-isoleucine from the dipeptide, even though no glycyl-L-isoleucine-ase activity can be detected in vitro. These results, along with the viability of null alleles for many other enzymes, support the idea that eukaryotes have an intricate network of alternative biochemical pathways through which the same necessary function may be achieved. Such "buffering capacity" makes it very difficult to analyze the effects of enzyme variants on fitness components.

Molecular dissection of a major gene effect on a quantitative trait: the level of alcohol dehydrogenase expression in Drosophila melanogaster.

A molecular mapping experiment shows that a major gene effect on a quantitative trait, the level of alcohol dehydrogenase expression in Drosophila melanogaster, is due to multiple polymorphisms within the Adh gene. These polymorphisms are located in an intron, the coding sequence, and the 3' untranslated region. Because of nonrandom associations among polymorphisms at different sites, the individual effects combine (in some cases epistatically) to produce "superalleles" with large effect. These results have implications for the interpretation of major gene effects detected by quantitative trait locus mapping methods. They show that large effects due to a single locus may be due to multiple associated polymorphisms (or sequential fixations in isolated populations) rather than individual mutations of large effect.

Associations between DNA sequence variation and variation in expression of the Adh gene in natural populations of Drosophila melanogaster.

A large part of the genetic variation in alcohol dehydrogenase (ADH) activity level in natural populations of Drosophila melanogaster is associated with segregation of an amino acid replacement polymorphism at nucleotide 1490, which generates a difference in electrophoretic mobility. Part of the allozymic difference in activity level is due to a catalytic efficiency difference, which is also caused by the amino acid replacement, and part is due to a difference in the concentration of ADH protein. A previous site-directed in vitro mutagenesis experiment clearly demonstrated that the amino acid replacement has no effect on the concentration of ADH protein, nor does a strongly associated silent polymorphism at nucleotide 1443. Here we analyze associations between polymorphisms within the Adh gene and variation in ADH protein level for a number of chromosomes derived from natural populations. A sequence length polymorphism within the first intron of the distal (adult) transcript, 1, is in strong linkage disequilibrium with the amino acid replacement. Among a sample of 46 isochromosomal lines analyzed, all but one of the 14 Fast lines have 1 and all but one of the 32 Slow lines lack 1. The exceptional Fast line has an unusually low level of ADH protein (typical of Slow lines) and the exceptional Slow line has an unusually high level (typical of Fast lines). These results suggest that the 1 polymorphism may be responsible for the average difference in ADH protein between the allozymic classes. A previous experiment localized the effect on ADH protein to a 2.3-kb restriction fragment. DNA sequences of this fragment from several alleles of each allozymic type indicate that no other polymorphisms within this region are as closely associated with the ADH protein level difference as the 1 polymorphism.

A genome-wide survey of hybrid incompatibility factors by the introgression of marked segments of Drosophila mauritiana chromosomes into Drosophila simulans.

In hybrids between Drosophila simulans and D. mauritiana, males are sterile and females are fertile, in compliance with HALDANE's rule. The genetic basis of this phenomenon was investigated by introgression of segments of the mauritiana genome into a simulans background. A total of 87 positions throughout the mauritiana genome were marked with P-element insertions and replicate introgressions were made by repeated backcrossing to simulans for 15 generations. The fraction of hemizgyous X chromosomal introgressions that are male sterile is approximately 50% greater than the fraction of homozygous autosomal segments. This result suggests that male sterility factors have evolved at a higher rate on the X, but chromosomal differences in segment length cannot be ruled out. The fraction of homozygous autosomal introgression that are male sterile is several times greater than the fraction that are either female sterile or inviable. This observation strongly indicates that male sterility factors have evolved more rapidly than either female sterility or inviability factors. These results, combined with previous work on these and other species, suggest that HALDANE's rule has at least two causes: recessivity of incompatibility factors and differential accumulation of sterility factors affecting males and females.

Differences in crossover frequency and distribution among three sibling species of Drosophila.

Comparisons of the genetic and cytogenetic maps of three sibling species of Drosophila reveal marked differences in the frequency and cumulative distribution of crossovers during meiosis. The maps for two of these species, Drosophila melanogaster and D. simulans, have previously been described, while this report presents new map data for D. mauritiana, obtained using a set of P element markers. A genetic map covering nearly the entire genome was constructed by estimating the recombination fraction for each pair of adjacent inserts. The P-based genetic map of mauritiana is approximately 1.8 times longer than the standard melanogaster map. It appears that mauritiana has higher recombination along the entire length of each chromosome, but the difference is greates in centromere-proximal regions of the autosomes. The mauritiana autosomes show little or no centromeric recombinational suppression, a characteristic that is prominent in melanogaster. D. simulans appears to be intermediate both in terms of total map length and intensity of the autosomal centromeric effect. These interspecific differences in recombination have important evolutionary implications for DNA sequence organization and variability. In particular, mauritiana is expected to differ from melanogaster in patterns and amounts of sequence variation and transposon insertions.

An introgression analysis of quantitative trait loci that contribute to a morphological difference between Drosophila simulans and D. mauritiana.

Drosophila simulans and D. mauritiana differ markedly in morphology of the posterior lobe, a male-specific genitalic structure. Both size and shape of the lobe can be quantified by a morphometric variable, PCl, derived from principal components and Fourier analyses. The genetic architecture of the species difference in PCl was investigated previously by composite interval mapping, which revealed largely additive inheritance, with a minimum of eight quantitative trait loci (QTL) affecting the trait. This analysis was extended by introgression of marked segments of the mauritiana third chromosome into a simulans background by repeated backcrossing. The two types of experiment are consistent in suggesting that several QTL on the third chromosome may have effects in the range of 10-15% of the parental difference and that all or nearly all QTL have effects in the same direction. Since the parental difference is large (30.4 environmental standard deviations), effects of this magnitude can produce alternative homozygotes with little overlap in phenotype. However, these estimates may not reflect the effects of individual loci, since each interval or introgressed segment may contain multiple QTL. The consistent direction of allelic effects suggests a history of directional selection on the posterior lobe.

Patterns of naturally occurring restriction map variation, dopa decarboxylase activity variation and linkage disequilibrium in the Ddc gene region of Drosophila melanogaster.

Forty-six second-chromosome lines of Drosophila melanogaster isolated from five natural populations were surveyed for restriction map variation in a 65-kb region surrounding the gene (Ddc) encoding dopa decarboxylase (DDC). Sixty-nine restriction sites were scored, 13 of which were polymorphic. Average heterozygosity per nucleotide was estimated to be 0.005. Eight large (0.7-5.0 kb) inserts, two small inserts (100 and 200 bp) and three small deletions (100-300 bp) were also observed across the 65-kb region. We see no evidence for a reduction in either nucleotide heterozygosity or insertion/deletion variation in the central 26-kb segment containing Ddc and a dense cluster of lethal complementation groups and transcripts (greater than or equal to 9 genes) compared to that seen in the adjacent regions (totaling 39 kb) in which only a single gene and transcript has been detected, or to that observed for other gene regions in D. melanogaster. The distribution of restriction site variation shows no significant departure from that expected under an equilibrium neutral model. However insertions and deletions show a significant departure from neutrality in that they are too rare in frequency, consistent with them being deleterious on average. Significant linkage disequilibrium among variants exists across much of the 65-kb region. Lower regional rates of recombination combined with the influence of polymorphic chromosomal inversions, rather than epistatic selection among genes in the dense cluster, probably are sufficient explanations for the creation and/or maintenance of the linkage disequilibrium observed in the Ddc region. We have also assayed adult DDC enzyme activity in these same lines. Twofold variation in activity among lines is observed within our sample. Significant associations are observed between level of DDC enzyme activity and restriction map variants. Surprisingly, one line with a 5.0-kb insert within an intron and one line with a 1.5-kb insert near the 5' end of Ddc each show normal adult DDC activities.

Genetic analysis of a morphological shape difference in the male genitalia of Drosophila simulans and D. mauritiana.

Two closely related species of Drosophila, D. simulans and D. mauritiana, differ markedly in morphology of the posterior lobe of the male genital arch. Both size and shape aspects of lobe variation can be quantified by a morphometric descriptor based on elliptical Fourier and principal components analyses. The genetic architecture of this quantitative trait (PC1) was investigated by hybridizing inbred lines to produce two backcross populations approximately 200 individuals each, which were analyzed jointly by a composite interval mapping procedure with the aid of 18 marker loci. The parental lines show a large difference in PC1 (30.4 environmental standard deviations), and the markers account for > 80% of the phenotypic variation in backcross populations. Eight of 15 intervals analyzed show convincing evidence of quantitative trait loci (QTL), and the range of estimated QTL effects is 5.7-15.9% of the parental difference (1.7-4.8 environmental standard deviations). These estimates may represent the joint effects of multiple QTL within a single interval (which averaged 23 cM in length). Although there is some evidence of partial dominance of mauritiana alleles and for epistasis, the pattern of inheritance is largely additive.

Genetic architecture of a morphological shape difference between two Drosophila species.

The size and shape of the posterior lobe of the male genital arch differs dramatically between Drosophila simulans and D. mauritiana. This difference can be quantified with a morphometric descriptor (PC1) based on elliptical Fourier and principal components analyses. The genetic basis of the interspecific difference in PC1 was investigated by the application of quantitative trait locus (QTL) mapping procedures to segregating backcross populations. The parental difference (35 environmental standard deviations) and the heritability of PC1 in backcross populations (>90%) are both very large. The use of multiple interval mapping gives evidence for 19 different QTL. The greatest additive effect estimate accounts for 11. 4% of the parental difference but could represent multiple closely linked QTL. Dominance parameter estimates vary among loci from essentially no dominance to complete dominance, and mauritiana alleles tend to be dominant over simulans alleles. Epistasis appears to be relatively unimportant as a source of variation. All but one of the additive effect estimates have the same sign, which means that one species has nearly all plus alleles and the other nearly all minus alleles. This result is unexpected under many evolutionary scenarios and suggests a history of strong directional selection acting on the posterior lobe.

Genome-wide association scan for childhood caries implicates novel genes.

Dental caries is the most common chronic disease in children and a major public health concern due to its increasing incidence, serious health and social co-morbidities, and socio-demographic disparities in disease burden. We performed the first genome-wide association scan for dental caries to identify associated genetic loci and nominate candidate genes affecting tooth decay in 1305 US children ages 3-12 yrs. Affection status was defined as 1 or more primary teeth with evidence of decay based on intra-oral examination. No associations met strict criteria for genome-wide significance (p < 10E-7); however, several loci (ACTN2, MTR, and EDARADD, MPPED2, and LPO) with plausible biological roles in dental caries exhibited suggestive evidence for association. Analyses stratified by home fluoride level yielded additional suggestive loci, including TFIP11 in the low-fluoride group, and EPHA7 and ZMPSTE24 in the sufficient-fluoride group. Suggestive loci were tested but not significantly replicated in an independent sample (N = 1695, ages 2-7 yrs) after adjustment for multiple comparisons. This study reinforces the complexity of dental caries, suggesting that numerous loci, mostly having small effects, are involved in cariogenesis. Verification/replication of suggestive loci may highlight biological mechanisms and/or pathways leading to a fuller understanding of the genetic risks for dental caries.

Quantitative analysis of RNA produced by slow and fast alleles of Adh in Drosophila melanogaster.

The alcohol dehydrogenase (ADH) locus (Adh) of Drosophila melanogaster in polymorphic on a world-wide basis for two allozymes, Fast and Slow. This study was undertaken to determine whether the well-established difference in ADH protein concentration between the allozymes is due to a difference in mRNA levels. RNA gel blot hybridization and an RNase protection assay were used to quantify ADH mRNA levels. Each method used an Adh null mutant as an internal standard. Several Slow and Fast allele pairs of different geographic origins were analyzed. The results provide strong evidence that the ADH protein concentration difference is not accounted for by RNA level.