Genome-wide association study of Tourette's syndrome.

Tourette's syndrome (TS) is a developmental disorder that has one of the highest familial recurrence rates among neuropsychiatric diseases with complex inheritance. However, the identification of definitive TS susceptibility genes remains elusive. Here, we report the first genome-wide association study (GWAS) of TS in 1285 cases and 4964 ancestry-matched controls of European ancestry, including two European-derived population isolates, Ashkenazi Jews from North America and Israel and French Canadians from Quebec, Canada. In a primary meta-analysis of GWAS data from these European ancestry samples, no markers achieved a genome-wide threshold of significance (P<5 × 10(-8)); the top signal was found in rs7868992 on chromosome 9q32 within COL27A1 (P=1.85 × 10(-6)). A secondary analysis including an additional 211 cases and 285 controls from two closely related Latin American population isolates from the Central Valley of Costa Rica and Antioquia, Colombia also identified rs7868992 as the top signal (P=3.6 × 10(-7) for the combined sample of 1496 cases and 5249 controls following imputation with 1000 Genomes data). This study lays the groundwork for the eventual identification of common TS susceptibility variants in larger cohorts and helps to provide a more complete understanding of the full genetic architecture of this disorder.

The complex global pattern of genetic variation and linkage disequilibrium at catechol-O-methyltransferase.

Genetic variation at the catechol-O-methyltransferase (COMT) gene has been significantly associated with risk for various neuropsychiatric conditions such as schizophrenia, panic disorder, bipolar disorders, anorexia nervosa and others. It has also been associated with nicotine dependence, sensitivity to pain and cognitive dysfunctions especially in schizophrenia. The non-synonymous single nucleotide polymorphism (SNP) in exon 4--Val108/158Met--is the most studied SNP at COMT and is the basis for most associations. It is not, however, the only variation in the gene; several haplotypes exist across the gene. Some studies indicate that the haplotypic combinations of alleles at the Val108/158Met SNP with those in the promoter region and in the 3'-untranslated region are responsible for the associations with disorders and not the non-synonymous SNP by itself. We have now studied DNA samples from 45 populations for 63 SNPs in a region of 172 kb across the region of 22q11.2 encompassing the COMT gene. We focused on 28 SNPs spanning the COMT-coding region and immediately flanking DNA, and found that the haplotypes are from diverse evolutionary lineages that could harbor as yet undetected variants with functional consequences. Future association studies should be based on SNPs that define the common haplotypes in the population(s) being studied.

Global patterns of variation in allele and haplotype frequencies and linkage disequilibrium across the CYP2E1 gene.

Cytochrome P450 2E1, gene symbol CYP2E1, is one of a family of enzymes with a central role in activating and detoxifying xenobiotics and endogenous compounds. Genetic variation at this gene has been reported in different human populations, and some association studies have reported increased risk for cancers and other diseases. To the best of our knowledge, multi-single-nucleotide polymorphism haplotypes and linkage disequilibrium (LD) have not been systematically studied for CYP2E1 in multiple populations. Haplotypes can greatly increase the power both to identify patterns of genetic variation relevant for gene expression as well as to detect disease-related susceptibility mutations. We present frequency and LD data and analyses for 11 polymorphisms and their haplotypes that we have studied on over 2600 individuals from 50 human population samples representing the major geographical regions of the world. The diverse patterns of haplotype variation found in the different populations we have studied show that ethnicity may be an important variable helping to explain inconsistencies that have been reported by association studies. More studies clearly are needed of the variants we have studied, especially those in the 5' region, such as the variable number of tandem repeats, as well as studies of additional polymorphisms known for this gene to establish evidence relating any systematic differences in gene expression that exist to the haplotypes at this gene.

Understanding human DNA sequence variation.

Over the past century researchers have identified normal genetic variation and studied that variation in diverse human populations to determine the amounts and distributions of that variation. That information is being used to develop an understanding of the demographic histories of the different populations and the species as a whole, among other studies. With the advent of DNA-based markers in the last quarter century, these studies have accelerated. One of the challenges for the next century is to understand that variation. One component of that understanding will be population genetics. We present here examples of many of the ways these new data can be analyzed from a population perspective using results from our laboratory on multiple individual DNA-based polymorphisms, many clustered in haplotypes, studied in multiple populations representing all major geographic regions of the world. These data support an "out of Africa" hypothesis for human dispersal around the world and begin to refine the understanding of population structures and genetic relationships. We are also developing baseline information against which we can compare findings at different loci to aid in the identification of loci subject, now and in the past, to selection (directional or balancing). We do not yet have a comprehensive understanding of the extensive variation in the human genome, but some of that understanding is coming from population genetics.

Indications of linkage and association of Gilles de la Tourette syndrome in two independent family samples: 17q25 is a putative susceptibility region.

Gilles de la Tourette syndrome (GTS) is characterized by multiple motor and phonic tics and high comorbidity rates with other neurobehavioral disorders. It is hypothesized that frontal-subcortical pathways and a complex genetic background are involved in the etiopathogenesis of the disorder. The genetic basis of GTS remains elusive. However, several genomic regions have been implicated. Among them, 17q25 appears to be of special interest, as suggested by various independent investigators. In the present study, we explored the possibility that 17q25 contributes to the genetic component of GTS. The initial scan of chromosome 17 performed on two large pedigrees provided a nonparametric LOD score of 2.41 near D17S928. Fine mapping with 17 additional microsatellite markers increased the peak to 2.61 (P=.002). The original families, as well as two additional pedigrees, were genotyped for 25 single-nucleotide polymorphisms (SNPs), with a focus on three genes in the indicated region that could play a role in the development of GTS, on the basis of their function and expression profile. Multiple three-marker haplotypes spanning all three genes studied provided highly significant association results (P<.001). An independent sample of 96 small families with one or two children affected with GTS was also studied. Of the 25 SNPs, 3 were associated with GTS at a statistically significant level. The transmission/disequilibrium test for a three-site haplotype moving window again provided multiple positive results. The background linkage disequilibrium (LD) of the region was studied in eight populations of European origin. A complicated pattern was revealed, with the pairwise tests producing unexpectedly high LD values at the telomeric TBCD gene. In conclusion, our findings warrant the further investigation of 17q25 as a candidate susceptibility region for GTS.

COMT haplotypes suggest P2 promoter region relevance for schizophrenia.

A recent study found, in a large sample of Ashkenazi Jews, a highly significant association between schizophrenia and a particular haplotype of three polymorphic sites in the catechol-O-methyl transferase, COMT, gene: an IVS 1 SNP (dbSNP rs737865), the exon 4 functional SNP (Val158Met, dbSNP rs165688), and a downstream SNP (dbSNP rs165599). Subsequently, this haplotype was shown to be associated with lower levels of COMT cDNA derived from normal cortical brain tissue, most likely due to cis-acting element(s). As a first step toward evaluating whether this haplotype may be relevant to schizophrenia in populations other than Ashkenazi Jews, we have studied this haplotype in 38 populations representing all major regions of the world. Adding to our previous data on four polymorphic sites in the COMT gene, including the Val158Met polymorphism, we have typed the IVS 1 rs737865 and 3' rs615599 sites and also included a novel IVS 1 indel polymorphism, yielding seven-site haplotype frequencies for normal individuals in the 38 globally distributed populations, including a sample of Ashkenazi Jews. We report that the schizophrenia-associated haplotype is significantly heterogeneous in populations worldwide. The three-site, schizophrenia-associated haplotype frequencies range from 0% in South America to 37.1% in Southwest Asia, despite the fact that schizophrenia occurs at roughly equal frequency around the world. Assuming that the published associations found between the exon 4 Val158Met SNP and schizophrenia are due to linkage disequilibrium, these new haplotype data support the hypothesis of a relevant cis variant linked to the rs737865 site, possibly just upstream in the P2 promoter driving transcription of the predominant form of COMT in the brain. The previously described HindIII restriction site polymorphism, located within the P2 promoter, varies within all populations and may provide essential information in future studies of schizophrenia.

Possible epistatic role of ADH7 in the protection against alcoholism.

In recent studies of the role of the alcohol dehydrogenase genes (ADH) in alcoholism the ADH1B Arg47His polymorphism appears to affect risk via a protective effect associated with the ADH1B*47His. Here we present evidence for an additional effect from outside the Class I ADH genes, presumably from functional variation at the ADH7 gene. The protective effect is restricted to one of two haplotypes identical at ADH1B but differing at an intronic SNP at ADH7 suggesting epistasis or strong linkage disequilibrium (LD).

ALFRED: the ALelle FREquency Database. Update.

Elaboration of ALFRED (http://alfred.med.yale.edu) is being continued in two directions. One of which is developing tools for efficiently annotating the entries and checking the integrity of the data already in the database while the other is to increase the quantity and accessibility of data. Information contained in ALFRED such as, polymorphic sites, number of populations and frequency tables (one sample typed for one site) has significantly increased.

ALFRED: an allele frequency database for diverse populations and DNA polymorphisms--an update.

ALFRED (the ALelle FREquency Database) is designed to store and disseminate frequencies of alleles at human polymorphic sites for multiple populations, primarily for the population genetics and molecular anthropology communities. Currently ALFRED has information on over 180 polymorphic sites for more than 70 populations. Since our initial release of the database we have focussed on increasing the quantity and quality of data, making reciprocal links between ALFRED and other related databases, and providing useful tools to make the data more comprehensible to the end user. ALFRED is accessible from the Kidd Lab home page (http://info.med.yale. edu/genetics/kkidd/) or from ALFRED directly (http://alfred.med.yale. edu/alfred/index.asp).

Short tandem-repeat polymorphism/alu haplotype variation at the PLAT locus: implications for modern human origins.

Two dinucleotide short tandem-repeat polymorphisms (STRPs) and a polymorphic Alu element spanning a 22-kb region of the PLAT locus on chromosome 8p12-q11.2 were typed in 1,287-1,420 individuals originating from 30 geographically diverse human populations, as well as in 29 great apes. These data were analyzed as haplotypes consisting of each of the dinucleotide repeats and the flanking Alu insertion/deletion polymorphism. The global pattern of STRP/Alu haplotype variation and linkage disequilibrium (LD) is informative for the reconstruction of human evolutionary history. Sub-Saharan African populations have high levels of haplotype diversity within and between populations, relative to non-Africans, and have highly divergent patterns of LD. Non-African populations have both a subset of the haplotype diversity present in Africa and a distinct pattern of LD. The pattern of haplotype variation and LD observed at the PLAT locus suggests a recent common ancestry of non-African populations, from a small population originating in eastern Africa. These data indicate that, throughout much of modern human history, sub-Saharan Africa has maintained both a large effective population size and a high level of population substructure. Additionally, Papua New Guinean and Micronesian populations have rare haplotypes observed otherwise only in African populations, suggesting ancient gene flow from Africa into Papua New Guinea, as well as gene flow between Melanesian and Micronesian populations.

ALFRED: a Web-accessible allele frequency database.

We present a Web-accessible database (ALFRED) that allows public access to gene frequency data for a diverse set of population samples and genetic systems. The data in ALFRED are modeled based on the experience and needs of a single laboratory, but with the expectation that the database will meet the needs of a much broader scientific community that needs population-specific gene frequency estimates. Our database currently contains data on more than 40 populations representing most major regions of the world and data on more than 150 genetic systems including SNPs, STRPs, and insertion-deletion polymorphisms. While data are not available for all population-genetic system combinations, over 2000 allele frequency tables already exist. In this paper, we enumerate the broad needs in the scientific domain, describe their significance, and describe how we have designed the database to meet those needs. We compare our database with dbSNP, the NCBI database that has a broader but overlapping purpose.

The accuracy of statistical methods for estimation of haplotype frequencies: an example from the CD4 locus.

Haplotype analysis has become increasingly important for the study of human disease as well as for reconstruction of human population histories. Computer programs have been developed to estimate haplotype frequencies statistically from marker phenotypes in unrelated individuals. However, there currently are few empirical reports on the accuracy of statistical estimates that must infer linkage phase. We have analyzed haplotypes at the CD4 locus on chromosome 12 that consist of a short tandem-repeat polymorphism and an Alu insertion/deletion polymorphism located 9.8 kb apart, in 398 individuals from 10 geographically diverse sub-Saharan African populations. Haplotype frequency estimates obtained using gene counting based on molecularly haplotyped (phase-known) data were compared with haplotype frequency estimates obtained using the expectation-maximization algorithm. We show that the estimated frequencies of common haplotypes do not differ significantly with the use of phase-known versus phase-unknown data. However, rare haplotypes are occasionally miscalled when their presence/absence must be inferred. Thus, for those research questions for which the common haplotypes are most important, frequency estimates based on the phase-unknown marker-typing results from unrelated individuals will be sufficient. However, in cases where knowledge of rare haplotypes is critical, molecular haplotyping will be necessary to determine linkage phase unambiguously.

Haplotypes and linkage disequilibrium at the phenylalanine hydroxylase locus, PAH, in a global representation of populations.

Because defects in the phenylalanine hydroxylase gene (PAH) cause phenylketonuria (PKU), PAH was studied for normal polymorphisms and linkage disequilibrium soon after the gene was cloned. Studies in the 1980s concentrated on European populations in which PKU was common and showed that haplotype-frequency variation exists between some regions of the world. In European populations, linkage disequilibrium generally was found not to exist between RFLPs at opposite ends of the gene but was found to exist among the RFLPs clustered at each end. We have now undertaken the first global survey of normal variation and disequilibrium across the PAH gene. Four well-mapped single-nucleotide polymorphisms (SNPs) spanning approximately 75 kb, two near each end of the gene, were selected to allow linkage disequilibrium across most of the gene to be examined. These SNPs were studied as PCR-RFLP markers in samples of, on average, 50 individuals for each of 29 populations, including, for the first time, multiple populations from Africa and from the Americas. All four sites are polymorphic in all 29 populations. Although all but 5 of the 16 possible haplotypes reach frequencies >5% somewhere in the world, no haplotype was seen in all populations. Overall linkage disequilibrium is highly significant in all populations, but disequilibrium between the opposite ends is significant only in Native American populations and in one African population. This study demonstrates that the physical extent of linkage disequilibrium can differ substantially among populations from different regions of the world, because of both ancient genetic drift in the ancestor common to a large regional group of modern populations and recent genetic drift affecting individual populations.

ALFRED: an allele frequency database for diverse populations and DNA polymorphisms.

We have developed a publicly accessible database (ALFRED, the ALlele FREquency Database) that catalogues allele frequency data for a wide range of population samples and DNA polymorphisms. This database is web-accessible through our laboratory (Kidd Lab) Web site: http://info.med.yale.edu/genetics/kkidd. ALFRED currently contains data on 60 populations and 156 genetic systems including single nucleotide polymorphisms (SNPs), short tandem repeat polymorphisms (STRPs), variable number of tandem repeats (VNTRs) and insertion-deletion polymorphisms. While data are not available for all population-DNA polymorphism combinations, over 2000 allele frequency tables have been entered. Our database is designed (i) to address our specific research requirements as well as broader scientific objectives; (ii) to allow researchers and interested educators to easily navigate and retrieve data of interest to them; and (iii) to integrate links to other related public databases such as dbSNP, GenBank and PubMed.

Analyses of the COGA data set in one ethnic group with examinations of alternative definitions of alcoholism.

We used GENEHUNTER and GENEHUNTER-PLUS to search for linkage with the markers in the Collaborative Study on the Genetics of Alcoholism (COGA) data set in a single ethnic group. Analyses of a complex disorder such as alcoholism depend on the definition of affection status. The COGA study provides two definitions of alcoholism (variables ALDX1 and ALDX2). To identify more severely affected alcoholics that might be more homogeneous genetically, we developed two other ways of characterizing subjects as alcohol dependent: (1) by combining the symptom variable values equally into a 24-point scale and (2) by weighting optimally the symptoms and other descriptive variables into a single score using logistic regression. We applied these definitions within a single ethnic group to map alcoholism-related loci. We found two regions on chromosome 1 that have adjacent markers significant at p-values < or = 0.05. ALDX1 provided the highest Z-scores compared to the alternatives.

A more powerful method to evaluate p-values in GENEHUNTER.

The determination of statistical significance in genetic linkage studies is complicated by many factors, such as missing individuals or uninformative markers, and the validity of theoretical results is often questionable. Although many simulation-based methods have been proposed to determine empirically the statistical significance, they are either not generally applicable to complex pedigree structures, or not able to preserve the observed genetic information content at each locus in the pedigrees. We have developed and implemented a general and computationally efficient randomization procedure in GENEHUNTER that applies to arbitrary pedigree structure and preserves the observed information content at each locus. We applied this method to the Problem 1 data set of the Genetic Analysis Workshop 11. The performance of this new method was similar to the method implemented in GENEHUNTER-PLUS, and both outperformed the conservative approach in GENEHUNTER.

Genome scan for linkage to Gilles de la Tourette syndrome.

Gilles de la Tourette Syndrome (TS) is a neuropsychiatric disorder characterized by both motor and vocal tics. Despite clear evidence for a genetic predisposition to TS from family, twin, and adoption studies, there have been no confirmed linkage findings. In this article we test for linkage to TS in multigenerational families segregating TS using a panel of 386 markers with the largest interval between any two markers being 28 cM and an average distance between markers of 10 cM. We tested for linkage using an autosomal dominant model with reduced penetrance and using nonparametric methods. No significant evidence for linkage was found with parametric analysis. A logarithm of the odds (LOD) score of greater or equal to one under the autosomal dominant model was observed in 24 of these markers in at least one of the families tested. No LOD scores greater than two were observed with any of the markers. For the nonparametric analysis, eight markers were observed with a P-value less than 0.00005 for significance evidence of linkage in at least one family. However caution should be used in the interpretation of the nonparametric analyses as this statistic (the affected-pedigree-member method) is know to have a high false-positive rate. Further support for linkage in these regions is required before linkage can be assumed.

No association between DRD2 locus and alcoholism after controlling the ADH and ALDH genotypes in Chinese Han population.

BACKGROUND: Recent studies on the genetics of alcoholism have examined the association between alcoholism and the dopamine D2 receptor locus (DRD2); our study of Chinese Han gave negative results (Lu et al., 1996). The different genotypes at the genes encoding the enzymes involved in alcohol metabolism, class one alcohol dehydrogenase (ADH2 and ADH3) and mitochondrial aldehyde dehydrogenase (ALDH2), have previously been shown to confer different predispositions to the development of alcoholism in Chinese Han males (Thomasson et al., 1991; Chen WJ et al., 1996; Chen CC et al., unpublished data). Therefore, association studies of alcoholism in Chinese Han might be more sensitive if controlled for the genotypes of ADH2,ADH3, and ALDH2, when other loci, such as DRD2, are examined. This study employs such controls to evaluate the evidence for an association between alcoholism and TaqI-A and TaqI-B genotypes and haplotypes at DRD2 in the Chinese Han population. METHODS: We studied 213 Chinese Han subjects (128 alcoholics and 85 nonalcoholics) with alcohol dependence defined according to DSM-III-R criteria. RESULTS: Significant linkage disequilibrium was observed between the TaqI-A and TaqI-B sites at the DRD2 locus, as previously seen in smaller samples, but no significant association was observed between these genetic variants at the DRD2 locus and alcoholism in Chinese Han. Several different stratifications by ADH and ALDH2 genotypes were examined; no genotypes or haplotypes at DRD2 differ between alcoholics and nonalcoholics except for a small number of nominally significant p-values which do not constitute significant results given the many tests done, some of which are not independent of one another due to linkage disequilibrium. These tests included considering the high risk (ADH2*1/*1; *1/*2; ADH3*1/*2; *2/*2; and ALDH2*1/*1) and the low risk (ADH2*2/*2; ADH3*1/*1; and ALDH2*1/*2; *2/*2) groups of alcoholics, as well as nonalcoholic controls. CONCLUSIONS: After stratification by the relevant genotypes of ADH2, ADH3, and ALDH2 no significant association exists between the genetic variants at the DRD2 locus and alcoholism in the Chinese Han population.

Linkage disequilibrium at the ADH2 and ADH3 loci and risk of alcoholism.

Two of the three class I alcohol dehydrogenase (ADH) genes (ADH2 and ADH3) encode known functional variants that act on alcohol with different efficiencies. Variants at both these genes have been implicated in alcoholism in some populations because allele frequencies differ between alcoholics and controls. Specifically, controls have higher frequencies of the variants with higher Vmax (ADH2*2 and ADH3*1). In samples both of alcoholics and of controls from three Taiwanese populations (Chinese, Ami, and Atayal) we found significant pairwise disequilibrium for all comparisons of the two functional polymorphisms and a third, presumably neutral, intronic polymorphism in ADH2. The class I ADH genes all lie within 80 kb on chromosome 4; thus, variants are not inherited independently, and haplotypes must be analyzed when evaluating the risk of alcoholism. In the Taiwanese Chinese we found that, only among those chromosomes containing the ADH3*1 variant (high Vmax), the proportions of chromosomes with ADH2*1 (low Vmax) and those with ADH2*2 (high Vmax) are significantly different between alcoholics and controls (P<10-5). The proportions of chromosomes with ADH3*1 and those with ADH3*2 are not significantly different between alcoholics and controls, on a constant ADH2 background (with ADH2*1, P=.83; with ADH2*2, P=.53). Thus, the observed differences in the frequency of the functional polymorphism at ADH3, between alcoholics and controls, can be accounted for by the disequilibrium with ADH2 in this population.