Centre d'Etude Du Polymorphisme Humain Research Articles

Genome-wide amplification of proviral sequences reveals new polymorphic HERV-K(HML-2) proviruses in humans and chimpanzees that are absent from genome assemblies.

BackgroundTo date, the human population census of proviruses of the Betaretrovirus–like human endogenous retroviral (HERV-K) (HML-2) family has been compiled from a limited number of complete genomes, making it certain that rare polymorphic loci are under-represented and are yet to be described.ResultsHere we describe a suppression PCR-based method called genome-wide amplification of proviral sequences (GAPS) that selectively amplifies DNA fragments containing the termini of HERV-K(HML-2) proviral sequences and their flanking genomic sequences. We analysed the HERV-K(HML-2) proviral content of 101 unrelated humans, 4 common chimpanzees and three centre d’etude du polymorphisme humain (CEPH) pedigrees (44 individuals). The technique isolated HERV-K(HML-2) proviruses that had integrated in the genomes of the great apes throughout their divergence and included evolutionarily young elements still unfixed for presence/absence.ConclusionsBy examining the HERV-K(HML-2) proviral content of 145 humans we detected a new insertionally polymorphic Type I HERV-K(HML-2) provirus. We also observed provirus versus solo long terminal repeat (LTR) polymorphism within humans at a previously unreported, but ancient, locus. Finally, we report two novel chimpanzee specific proviruses, one of which is dimorphic for a provirus versus solo LTR. Thus GAPS enables the isolation of uncharacterised HERV-K(HML-2) proviral sequences and provides a direct means to assess inter-individual genetic variation associated with HERV-K(HML-2) proviruses.Electronic supplementary materialThe online version of this article (doi:10.1186/s12977-015-0162-8) contains supplementary material, which is available to authorized users.

Identifying rare variants for genetic risk through a combined pedigree and phenotype approach: application to suicide and asthma.

Suicidal behavior is a complex disorder, with evidence for genetic risk independent of other genetic risk factors including psychiatric disorders. Since 1996, over 3000 DNA samples from Utah suicide decedents have been collected and banked for research use through the Utah Medical Examiner. In addition, over 12 000 Utah suicides were identified through examination of death certificates back to 1904. By linking this data with the Utah Population Database, we have identified multiple extended pedigrees with increased risk for suicide completion. A number of medical conditions co-occur with suicide, including asthma, and this study was undertaken to identify genetic risk common to asthma and suicide. This study tests the hypothesis that a particular comorbid condition may identify a more homogeneous genetic subgroup, facilitating the identification of specific genetic risk factors in that group. From pedigrees at increased risk for suicide, we identified three pedigrees also at significantly increased familial risk for asthma. Five suicide decedents from each of these pedigrees, plus an additional three decedents not from these pedigrees with diagnosed asthma, and 10 decedents with close relatives with asthma were genotyped. Results were compared with 183 publicly available unaffected control exomes from 1000 Genomes and CEPH (Centre d'etude du polymorphisme humain) samples genotyped on the same platform. A further 432 suicide decedents were also genotyped as non-asthma suicide controls. Genotyping was done using the Infinium HumanExome BeadChip. For analysis, we used the pedigree extension of Variant Annotation, Analysis and Search Tool (pVAAST) to calculate the disease burden of each gene. The Phenotype Driven Variant Ontological Re-ranking tool (Phevor) then re-ranked our pVAAST results in context of the phenotype. Using asthma as a seed phenotype, Phevor traversed biomedical ontologies and identified genes with similar biological properties to those known to result in asthma. Our top associated genes included those related to neurodevelopment or neural signaling (brain-derived neurotrophic factor (BDNF), neutral sphingomyelinase 2 (SMPD2), homeobox b2 (HOXB2), neural cell adhesion molecule (NCAM2), heterogeneous nuclear ribonucleoprotein A0 (HNRNPA0)), inflammation (free fatty acid receptor 2 (FFAR2)) and inflammation with additional evidence of neuronal involvement (oxidized low density lipoprotein receptor 1 (OLR1), toll-like receptor 3 (TLR3)). Of particular interest, BDNF has been previously implicated in both psychiatric disorders and asthma. Our results demonstrate the utility of combining pedigree and co-occurring phenotypes to identify rare variants associated with suicide risk in conjunction with specific co-occurring conditions.

Abstract 5562: Screening of in vitro cytotoxicity of nucleoside analogs in HapMap cell lines to identify predictive markers of drug sensitivity

Abstract Nucleoside analogues (NAs) are synthetic molecules that resemble naturally occurring nucleosides. These analogs are themselves inactive and require activation by multiple phosphorylation steps to their triphosphate forms which depending on the particular agent either inhibits DNA/RNA (cytarabine, clofarabine), or inhibits, ribonucleotide reductases (clofarabine, gemcitabine) or DNA methyl transferases (decitabine). Resistance to nucleoside analogue-based chemotherapy is a major problem in the treatment of cancers. In the current study, we determined the in vitro cytotoxicity of the nucleoside analogs clofarabine, fludarabine and decitabine in 60 unrelated Epstein Barr virus (EBV)-transformed B-lymphoblastoid HapMap cell lines with European ancestry [Centre d'Etude du Polymorphisme Humain (CEU) samples] were obtained from the Coriell Institute for Medical Research. The cell lines were maintained at 37°C in RPMI1640 medium supplemented with 2mM Glutamine. In vitro cytotoxicity was determined by plating LCLs in a 96 well plate at density of 250,000cells/ml. Cell survival was determined using MTT ((3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay 48hr after exposure to varying concentrations of each nucleoside analog. Gen 5 software was used to calculate IC50 values for each cell line and agent. Our results showed wide inter-individual variation in cytotoxicity to nucleoside analogs among HapMap cell lines. Significant correlation was observed between the IC50 values of fludarabine vs. clofarabine (r = 0.732; P = 0.00001). However no significant correlation was observed with the IC50 values of decitabine. The reason for the observed relationship might be the fact that clofarabine is a 2nd generation purine nucleoside and structurally related to fludarabine. Additionally, the mechanism of action of decitabine is different than clofarabine and fludarabine with it being a hypomethylating agent. Since genome-wide genotype and gene expression data on these cell lines is available publicly, currently we are analyzing the data for identification of genomics markers predictive of cell survival and response to these nucleoside analogs. Our preliminary analysis showed that polymorphisms in candidate genes involved in drug metabolism pathway may be involved in cancer drug resistance. For example, a promoter SNP in the gene DCK was associated with both clofarabine IC50 and fludarabine IC50 while another promoter SNP in NT5C2 was associated with fludarabine IC50. These pharmacogenomic factors may then serve as predictive markers for improvement of cancer chemotherapy. Citation Format: Taraswi Mitra Ghosh, Tanya J. Feldberg, Neha Bhise, Jatinder K. Lamba. Screening of in vitro cytotoxicity of nucleoside analogs in HapMap cell lines to identify predictive markers of drug sensitivity. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5562. doi:10.1158/1538-7445.AM2014-5562

Allelic Frequency in Human SNPs Predicts the Rate of Non-Synonymous Nucleotide Substitutions between Human and Chimpanzee Genes

The combination of comparative genomics and population genetics may provide important clues regarding human evolution. We have hypothesized that the divergence between various human and chimpanzee orthologs will be reflected in the variability of single nucleotide polymorphisms (SNPs) that are localized in the vicinity of the corresponding loci in different human populations. Consistently with this notion, more diverged genes between humans and chimpanzees are more likely to be associated with human speciation and are anticipated to contain SNPs with reduced variability between different human populations. In order to test this hypothesis, we have compared the rate of non synonymous nucleotide substitutions (Ka) between 255 chimpanzee and human orthologs with the average deviation in the allelic frequencies of corresponding closely linked SNPs in two distinct human populations: The Yoruba people in Ibadan, Nigeria (YRI) and US residents with ancestry from Northern and Western Europe, collected in 1980 by the Centre d’Etude du Polymorphisme Humain (CEU). We found a significant (p

Ancestry analysis reveals a predominant Native American component with moderate European admixture in Bolivians

We have genotyped 46 Ancestry Informative Markers (AIMs) in two of the most populated areas in Bolivia, namely, La Paz (Andean region; n=105), and Chuquisaca (Sub-Andean region; n=73). Using different analytical tools, we inferred admixture proportions of these two American communities by comparing the genetic profiles with those publicly available from the CEPH (Centre d'Etude du Polymorphisme Humain) panel representing three main continental groups (Africa, Europe, and America). By way of simulations, we first evaluated the minimum sample size needed in order to obtain accurate estimates of ancestry proportions. The results indicated that sample sizes above 30 individuals could be large enough to estimate main continental ancestry proportions using the 46 AIMs panel. With the exception of a few individuals, the results also indicated that Bolivians showed a predominantly Native American ancestry with variable levels of European admixture. The proportions of ancestry were statistically different in La Paz and Chuquisaca: the Native American component was 86% and 77% (Mann-Whitney U-test: un-adjusted P-value=2.1×10(-5)), while the European ancestry was 13% and 21% (Mann-Whitney U-test: un-adjusted P-value=3.6×10(-5)), respectively. The African ancestry in Bolivians captured by the AIMs analyzed in the present study was below 2%. The inferred ancestry of Bolivians fits well with previous studies undertaken on haplotype data, indicating a major proportion of Native American lineages. The genetic differences observed in these two groups suggest that forensic genetic analysis should be better performed based on local databases built in the main Bolivian areas.

Large-scale profiling and identification of potential regulatory mechanisms for allelic gene expression in colorectal cancer cells

Allelic variation in gene expression is common in humans and this variation is associated with phenotypic variation. In this study, we employed high-density single nucleotide polymorphism (SNP) chips containing 13,900 exonic SNPs to identify genes with allelic gene expression in cells from colorectal cancer cell lines. We found 2 monoallelically expressed genes (ERAP2 and MYLK4), 32 genes with an allelic imbalance in their expression, and 13 genes showing allele substitution by RNA editing. Among a total of 34 allelically expressed genes in colorectal cancer cells, 15 genes (44.1%) were associated with cis-acting eQTL, indicating that large portions of allelically expressed genes are regulated by cis-acting mechanisms of gene expression. In addition, potential regulatory variants present in the proximal promoter regions of genes showing either monoallelic expression or allelic imbalance were not tightly linked with coding SNPs, which were detected with allelic gene expression. These results suggest that multiple rare variants could be involved in the cis-acting regulatory mechanism of allelic gene expression. In the comparison with allelic gene expression data from Centre d'Etude du Polymorphisme Humain (CEPH) family B cells, 12 genes showed B-cell specific allelic imbalance and 1 noncoding SNP showed colorectal cancer cell-specific allelic imbalance. In addition, different patterns of allele substitution were observed between B cells and colorectal cancer cells. Overall, our study not only indicates that allelic gene expression is common in colorectal cancer cells, but our study also provides a better understanding of allele-specific gene expression in colorectal cancer cells.

SNP-based large-scale identification of allele-specific gene expression in human B cells

Polymorphism and variations in gene expression provide the genetic basis for human variation. Allelic variation of gene expression, in particular, may play a crucial role in phenotypic variation and disease susceptibility. To identify genes with allelic expression in human cells, we genotyped genomic DNA and cDNA isolated from 31 immortalized B cell lines from three Centre d'Etude du Polymorphisme Humain (CEPH) families using high-density single-nucleotide polymorphism (SNP) chips containing 13,900 exonic SNPs. We identified seven SNPs in five genes with monoallelic expression, 146 SNPs in 125 genes with allelic imbalance in expression with preferentially higher expression of one allele in a heterozygous individual. The monoallelically expressed genes ( ERAP2, MDGA1, LOC644422, SDCCAG3P1 and CLTCL1) were regulated by cis-acting, non-imprinted differential allelic control. In addition, all monoallelic gene expression patterns and allelic imbalances in gene expression in B cells were transmitted from parents to offspring in the pedigree, indicating genetic transmission of allelic gene expression. Furthermore, frequent allele substitution, probably due to RNA editing, was also observed in 21 genes in 23 SNPs as well as in 48 SNPs located in regions containing no known genes. In this study, we demonstrated that allelic gene expression is frequently observed in human B cells, and SNP chips are very useful tools for detecting allelic gene expression. Overall, our data provide a valuable framework for better understanding allelic gene expression in human B cells.

Genetic Variants in Cytosolic 5′-Nucleotidase II Are Associated with Its Expression and Cytarabine Sensitivity in HapMap Cell Lines and in Patients with Acute Myeloid Leukemia

Cytosolic 5'-nucleotidase II (NT5C2) is involved in the development of 1-β-d-arabinofuranosylcytosine (ara-C) resistance and has been associated with clinical outcome in patients receiving ara-C-based chemotherapy. NT5C2 inactivates ara-C by dephosphorylating ara-C monophosphate to ara-C. In this study, we sequenced NT5C2 in genomic DNA samples from International HapMap project panels with European [Centre d'Etude du Polymorphisme Humain (CEU); n = 90] or African [Yoruba people in Ibadan, Nigeria (YRI); n = 90] ancestry. We identified 41 genetic variants [one insertion-deletion and 40 single nucleotide polymorphisms (SNPs)], including three nonsynonymous SNPs (Y3A, K47R, and Q136R). Twenty-five SNPs were novel and 16 overlapped with the HapMap data. Subjects with African ancestry had NT5C2 mRNA expression levels that was significantly higher than those with European ancestry (p = 0.005). Furthermore, there was a correlation between NT5C2 mRNA expression and ara-C sensitivity in CEU but not in YRI cell lines. None of the nonsynonymous SNPs demonstrated any effect on NT5C2 activity. The genotypes of several SNPs were significantly associated with NT5C2 mRNA expression and/or ara-C sensitivity in CEU cell lines, but very few were significant in YRI cell lines. Of most interest, SNPs (linkage disequilibrium group CEU.12) in the 5'-untranslated region were associated with NT5C2 expression and ara-C sensitivity in HapMap cell lines and with NT5C2 mRNA expression and ara-C sensitivity in diagnostic leukemic blasts from pediatric patients with acute myeloid leukemia. Functional genomics analysis demonstrated that the promoter SNP rs11191612 was associated with altered luciferase activation in reporter assays and altered DNA-protein binding in gel shift assays. These results suggest that genetic variations in NT5C2 influence its expression and, potentially, cellular responses to nucleoside analogs.

Whole-Exome Sequencing Identifies FAM20A Mutations as a Cause of Amelogenesis Imperfecta and Gingival Hyperplasia Syndrome

Amelogenesis imperfecta (AI) describes a clinically and genetically heterogeneous group of disorders of biomineralization resulting from failure of normal enamel formation. AI is found as an isolated entity or as part of a syndrome, and an autosomal-recessive syndrome associating AI and gingival hyperplasia was recently reported. Using whole-exome sequencing, we identified a homozygous nonsense mutation in exon 2 of FAM20A that was not present in the Single Nucleotide Polymorphism database (dbSNP), the 1000 Genomes database, or the Centre d'Etude du Polymorphisme Humain (CEPH) Diversity Panel. Expression analyses indicated that Fam20a is expressed in ameloblasts and gingivae, providing biological plausibility for mutations in FAM20A underlying the pathogenesis of this syndrome.

Genome-wide search for genetic modulators in gene regulatory pathways: Weighted window-based peak identification algorithm

Genome-wide gene expression and genotype data have been integratively analyzed in expression quantitative trait loci (eQTL) studies to elucidate the genetics of gene transcription. Most eQTL analyses have focused on identifying polymorphic genetic variants that influence the expression levels of individual genes, and such analyses may have limitations in explaining gene regulatory pathways that are likely to involve multiple genes and their genetic and/or non-genetic modulators. We have developed a novel two-step method for identifying potential genetic modulators of transcription processes for multiple genes in a biological pathway. We proposed a new weighted window-based peak identification algorithm to improve the detection of genetic modulators for individual genes and employed a Poisson-based test to search for master genetic modulators of multiple genes. Here, we have illustrated this two-step approach by analyzing the gene expression data in the Centre d'Etude du Polymorphisme Humain (CEPH) lymphoblast cells and single nucleotide polymorphism chip data.

In Vitro Screening for Population Variability in Chemical Toxicity

Immortalized human lymphoblastoid cell lines have been used to demonstrate that it is possible to use an in vitro model system to identify genetic factors that affect responses to xenobiotics. To extend the application of such studies to investigative toxicology by assessing interindividual and population-wide variability and heritability of chemical-induced toxicity phenotypes, we have used cell lines from the Centre d'Etude du Polymorphisme Humain (CEPH) trios assembled by the HapMap Consortium. Our goal is to aid in the development of predictive in vitro genetics-anchored models of chemical-induced toxicity. Cell lines from the CEPH trios were exposed to three concentrations of 14 environmental chemicals. We assessed ATP production and caspase-3/7 activity 24 h after treatment. Replicate analyses were used to evaluate experimental variability and classify responses. We show that variability of response across the cell lines exists for some, but not all, chemicals, with perfluorooctanoic acid (PFOA) and phenobarbital eliciting the greatest degree of interindividual variability. Although the data for the chemicals used here do not show evidence for broad-sense heritability of toxicity response phenotypes, substantial cell line variation was found, and candidate genetic factors contributing to the variability in response to PFOA were investigated using genome-wide association analysis. The approach of screening chemicals for toxicity in a genetically defined yet diverse in vitro human cell-based system is potentially useful for identification of chemicals that may pose a highest risk, the extent of within-species variability in the population, and genetic loci of interest that potentially contribute to chemical susceptibility.

JEAN B DAUSSET, 19 OCTOBER 1916–6 JUNE 2009

Professor Jean B Dausset, who shared The Nobel Prize in Physiology or Medicine, 1980, with Professors George D Snell and Baruj Benacerraf for his discovery and development of the human major histocompatibility complex (MHC, also commonly called HLA), died in Mallorca, Spain, on 6 June 2009. He was 92 years old. The landmark discoveries of Professor Dausset include agglutination of white blood cells by antibodies from patients who had received blood transfusions, which he realized was due to genetic variation, description of the first leukocyte antigen (now known as HLA-2) and of the first tissue group system (Hu-1, later named HLA), and correlation between graft survival and tissue incompatibility, which was the result of working with volunteer skin donors and recipients. He was active in finding associations of HLA with complex diseases. He was keen on using DNA polymorphisms to characterize HLA. Professors Dausset and E Carosella collaborated in research into the role of HLA-G in human physiology, with implications for immune tolerance in general and pregnancy in particular. Professor Dausset understood the importance of research collaboration and was a dedicated, leading spirit in the HLA Workshops and Conferences, in which competitors collaborate by typing shared tissue resources. Professor Dausset's research achievements were fundamental for the development of tissue typing, which improved the quality of clinical transplantation. He contributed even more to this field by founding France Transplant and France Greffe de Moelle, organizations that are dedicated to bringing donor organs and bone marrow to needy recipients in an efficient and rapid manner. In 1984, Professor Dausset created the Centre d'Etude du Polymorphisme Humain (CEPH), a laboratory, later an internationally renowned genome center, which coordinated the first international genome-mapping collaboration by making available DNA from 40 large reference families (later 61) to researchers throughout the world. By genotyping DNA from the same set of families, it was possible to map the human genome by linkage which determined order of DNA markers along all autosomes, the X chromosome and the X–Y pseudoautosomal region. Knowledge of these chromosome maps, which was made available to the scientific community worldwide, permitted researchers, some of them at the CEPH, to localize the major genes for various genetic disorders to regions of the human genome. Localization of such genes was the first, important step in cloning and identifying them, which was a breakthrough for medical genetics. Furthermore, the linkage maps provided the foundation for the International Genome Project, the physical mapping of the human genome (largely initiated at CEPH), which in turn led to determination of the DNA sequence. The CEPH reference families continue to be used for genomics research. More recently, Professor Dausset collaborated with Professor LL Cavalli-Sforza in developing, at the Foundation, a widely used DNA resource from world populations for research in human population genetics, the HGDP-CEPH Diversity Panel. In 1993, the CEPH became the Foundation Jean Dausset – CEPH, a non-profit research institute funded in part by the Republic of France. Professor Dausset retired as President of the Foundation in 2003; he remained its honorary President until his death. Professor Dausset was a member of the French National Academy of Sciences, professor at the College de France, a foreign member of the National Academy of Sciences, USA, an honorary member of the American Academy of Arts and Sciences, and a member of the founding Council and Vice President of the Human Genome Organization. He received various prestigious prizes, such as the Landsteiner Award and prizes from the Koch and Wolf Foundations. He served on the advisory boards of numerous research institutions. A CEPH collaborator wrote in a tribute to Professor Dausset, ‘He set a standard for cooperative sharing in science with the goal of helping others that has truly revolutionized the ways in which we do our work. All of the genome sharing efforts of sequence and SNPs is based on his CEPH model. It's really not possible to even measure the impact this has had on science.'

Association of thymidylate synthase variants with 5-fluorouracil cytotoxicity

Identifying relevant cytotoxicity genes using an ex-vivo lymphoblastoid cell line (LCLs) model has distinct advantages for pharmacogenomic discovery studies of cancer chemotherapy, including standardized treatment conditions, availability of large numbers of samples, and publicly available genotypic data. However, there is little proof of principal data to confirm the promise of this approach. One of the known targets of 5-fluorouracil (5-FU) treatment is thymidylate synthase (TYMS). We hypothesized that genetic variants in TYMS would alter cytotoxicity because of 5-FU treatment using a LCL model system. LCLs from the Centre d'Etude du Polymorphisme Humain (CEPH) pedigrees (N=427) were treated with eight concentrations of 5-FU for 72 h, and cytotoxicity was determined using an Alamar Blue assay. For a subset of the 30 International Haplotype Mapping project (HapMap) trios, genotype data for 46 single-nucleotide polymorphism (SNP) variants encompassing the TYMS gene were downloaded from the HapMap website. Using a mixed models approach, each SNP was tested for association to 5-FU cytotoxicity in the subset of HapMap trios. Putatively associated SNPs (P<0.01), were then genotyped in the remaining LCLs in the CEPH pedigrees and tested for association. Two intronic SNPs in TYMS (rs2847153 and rs2853533) were significantly associated (P<0.01) with 5-FU cytotoxicity in the HapMap subset using the mixed models approach. After genotyping these SNPs in the full CEPH pedigrees, the associations with cytotoxicity showed a more reliable significance (P<0.0005), as a result of the increase in sample size. These results highlight the importance of the TYMS gene variants in response to 5-FU treatment. Furthermore, they provide additional biological validation of the relevance of LCLs as a model for pharmacogenomic gene discovery in cancer chemotherapy.

Expression and Alternative Splicing of Folate Pathway Genes in HapMap lymphoblastoid Cell Lines

Folate is vital for cell growth and development through its important role in one-carbon metabolism - an essential process in the synthesis of amino acids and nucleic acids. Folate pathway genes have been considered as therapeutic targets of drugs for the treatment of cancer and other diseases. Racial and ethnic disparities of folate metabolism and outcome of antifolate therapies have been reported. In this study, we evaluate the genetic regulation for expression and alternative splicing of folate related genes in HapMap lymphoblastoid cell lines (LCLs) of individuals of European and African descent. Gene and exon level expression and alternative splicing of folate pathway genes were compared in LCLs derived from the Centre d'Etude du Polymorphisme Humain (CEPH) from Utah (CEU) and the Yoruba from Ibadan (YRI) using a permutation-based test. A genome-wide association study was performed to search for SNPs associated with folate pathway gene expressions and alternative splicing in the combined population samples. A total of 52 folate pathway genes were evaluated in the analysis of which 46 were expressed in the LCLs. There were 12 genes (26%) with differential gene-level expression and 23 genes (50%) with differential alternative splicing for exons or UTRs between the CEU and the YRI (permutation p <or= 0.05). The expression level of FPGS and the splicing indices of eight genes (ATP13A2, ASCC3L1, IFIH1, SMARCA5, SMARCA2, SETX, DDX52 and RUVBL2) were found to be associated with SNP genotypes in the combined populations (p < 3.2 x 10(-8), Bonferroni corrected p < 0.05). Our study suggests that LCLs are an in vitro system suitable to evaluate the expression levels of folate pathway genes. The differential transcript-level expressions and the differentially alternative splicing events of exons or UTRs and associated SNP markers in 2 populations will enhance our understanding of the folate pathway and, thus, facilitate research in the areas of nutrition and folate metabolism.

Fine Mapping on Chromosome 10q22-q23 Implicates Neuregulin 3 in Schizophrenia

Linkage studies have implicated 10q22-q23 as a schizophrenia (SZ) susceptibility locus in Ashkenazi Jewish (AJ) and Han Chinese from Taiwan populations. To further explore our previous linkage signal in the AJ population (NPL score: 4.27, empirical p = 2 x 10(-5)), we performed a peakwide association fine mapping study by using 1414 SNPs across approximately 12.5 Mb in 10q22-q23. We genotyped 1515 AJ individuals, including 285 parent-child trios, 173 unrelated cases, and 487 unrelated controls. We analyzed the binary diagnostic phenotype of SZ and 9 heritable quantitative traits derived from a principal components factor analysis of 73 items from our consensus diagnostic ratings and direct assessment interviews. Although no marker withstood multiple test correction for association with the binary SZ phenotype, we found strong evidence of association by using the "delusion" factor as the quantitative trait at three SNPs (rs10883866, rs10748842, and rs6584400) located in a 13 kb interval in intron 1 of Neuregulin 3 (NRG3). Our best p value from family-based association analysis was 7.26 x 10(-7). We replicated this association in the collection of 173 unrelated AJ cases (p = 1.55 x 10(-2)), with a combined p value of 2.30 x 10(-7). After performing 10,000 permutations of each of the phenotypes, we estimated the empirical study-wide significance across all 9 factors (90,000 permutations) to be p = 2.7 x 10(-3). NRG3 is primarily expressed in the central nervous system and is one of three paralogs of NRG1, a gene strongly implicated in SZ. These biological properties together with our linkage and association results strongly support NRG3 as a gene involved in SZ.

Genome-wide copy number analysis using copy number inferring tool (CNIT) and DNA pooling

Copy number variation (CNV) has become an important genomic structure element in the human population, and some CNVs are related to specific traits and diseases. Moreover, analysis of human genomes has been potentiated by the use of high-resolution microarrays that assess single nucleotide polymorphisms (SNPs). Although many programs have been designed to analyze data from Affymetrix SNP microarrays, they all have high false-positive rates (FPRs) in copy number (CN) analyses. Copy number analysis tool (CNAT) 4.0 is a recently developed program that offers improved CN estimation, but small amplifications and deletions are lost when using the smoothing procedure. Here, we propose a copy number inferring tool (CNIT) algorithm for the 100K SNP microarray to investigate CNVs at 29.6-kb resolution. CNIT estimated SNP allelic and total CN with reliable P values based on intensity data. In addition, the hidden Markov model (HMM) method was applied to predict regions having altered CN by considering contiguous SNPs. Based on a CN analysis of 23 unrelated Taiwanese and 30 HapMap Centre d'Etude du Polymorphisme Humain (CEPH) trios, CNIT showed higher accuracy and power than other programs. The FPRs and false-negative rates (FNRs) of CNIT were 0.1% and 0.16%, respectively. CNIT also showed better sensitivity for detecting small amplifications and deletions. Furthermore, DNA pooling of 10 and 30 normal unrelated individuals were applied to the 100K SNP microarray, respectively, and 12 common CN-variable regions were identified, suggesting that DNA pooling can be applied to discover common CNVs.

Genetic Variants Contributing to Daunorubicin-Induced Cytotoxicity

Identifying heritable genetic variants responsible for chemotherapeutic toxicities has been challenging due in part to its multigenic nature. To date, there is a paucity of data on genetic variants associated with patients experiencing severe myelosuppression or cardiac toxicity following treatment with daunorubicin. We present a genome-wide model using International HapMap cell lines that integrate genotype and gene expression to identify genetic variants that contribute to daunorubicin-induced cytotoxicity. A cell growth inhibition assay was used to measure variations in the cytotoxicity of daunorubicin. Gene expression was determined using the Affymetrix GeneChip Human Exon 1.0ST Array. Using sequential analysis, we evaluated the associations between genotype and cytotoxicity, those significant genotypes with gene expression and correlated gene expression of the identified candidates with cytotoxicity. A total of 26, 9, and 18 genetic variants were identified to contribute to daunorubicin-induced cytotoxicity through their effect on 16, 9, and 36 gene expressions in the combined, Centre d' Etude du Polymorphisme Humain (CEPH), and Yoruban populations, respectively. Using 50 non-HapMap CEPH cell lines, single nucleotide polymorphisms generated through our model predicted 29% of the overall variation in daunorubicin sensitivity and the expression of CYP1B1 was significantly correlated with sensitivity to daunorubicin. In the CEPH validation set, rs120525235 and rs3750518 were significant predictors of transformed daunorubicin IC(50) (P = 0.005 and P = 0.0008, respectively), and rs1551315 trends toward significance (P = 0.089). This unbiased method can be used to elucidate genetic variants contributing to a wide range of cellular phenotypes.

Chromosome 22 LD Map Comparison between Korean and Other Populations

Single nucleotide polymorphisms (SNPs) are the most abundant forms of human genetic variations and resources for mapping complex genetic traits and disease association studies. We have constructed a linkage disequilibrium (LD) map of chromosome 22 in Korean samples and compared it with those of other populations, including Yorubans in Ibadan, Nigeria (YRI), Centre d'Etude du Polymorphisme Humain (CEPH) reference families (CEU), Japanese in Tokyo (JPT) and Han Chinese in Beijing (CHB) in the HapMap database. We genotyped 4681 of 111,448 publicly available SNPs in 90 unrelated Koreans. Among genotyped SNPs, 4167 were polymorphic. Three hundred and five LD blocks were constructed to make up 18.6% (6.4 of 34.5 Mb) of chromosome 22 with 757 tagSNPs and 815 haplotypes (frequency ≥ 5.0%). Of 3430 common SNPs genotyped in all five populations, 514 were monomorphic in Koreans. The CHB + JPT samples have more than a 72% overlap with the monomorphic SNPs in Koreans, while the CEU + YRI samples have less than a 38% overlap. The patterns of hot spots an d LD blocks were dispersed throughout chromosome 22, with some common blocks among populations, highly concordant between the three Asian samples. Analysis of the distribution of chimpanzee-derived allele frequency (DAF), a measure of genetic differentiation, Fst levels, and allele frequency difference (AFD) among Koreans and the HapMap samples showed a strong correlation between the Asians, while the CEU and YRI samples showed a very weak correlation with Korean samples. Relative distance as a quantitative measurement based upon DAF, Fst, and AFD indicated that all three Asian samples are very proximate, while CEU and YRI are significantly remote from the Asian samples. Comparative genome-wide LD studies provide useful information on the association studies of complex diseases.

The feasibility of oligogenic combined segregation and linkage analysis in CEPH pedigrees

The CEPH samples are an invaluable resource for mapping genes that contribute to traits that can be measured in cell lines. With the many markers that have already been genotyped for the Centre d'Etude du Polymorphisme Humain (CEPH) pedigrees and are readily available, one need only obtain phenotypes to conduct a linkage analysis. For Genetic Analysis Workshop 15 (GAW15), over 3000 expression levels of genes in lymphoblastoid cells in 14 of the CEPH pedigrees were provided. For this study, eight of these expression levels were selected to obtain a spectrum of heritabilities, three were selected based on linkage results with traditional LOD scores >3, and one trait was selected at random. A Bayesian Monte Carlo Markov chain oligogenic segregation and linkage analysis was conducted on each of these 12 traits using the genome-wide single-nucleotide polymorphism linkage markers provided for GAW15. Our goal was to assess the ability of these methods to map genes in the CEPH pedigrees. Surprisingly, positive linkage signals were found for all 12 traits, even those with a very small traditionally calculated heritability. However, the portion of the variance attributed to genetic sources by the oligogenic segregation analysis differed substantially in some cases from the traditional heritability. It appears that genetic variance estimated from oligogenic segregation analysis may be a better indicator of whether genes can be mapped for complex traits than traditional heritability.

Artificial neural networks for linkage analysis of quantitative gene expression phenotypes and evaluation of gene × gene interactions

Using single-nucleotide polymorphism (SNP) genotypes and selected gene expression phenotypes from 14 CEPH (Centre d'Etude du Polymorphisme Humain) pedigrees provided for Genetic Analysis Workshop 15 (GAW15), we analyzed quantitative traits with artificial neural networks (ANNs). Our goals were to identify individual linkage signals and examine gene x gene interactions. First, we used classical multipoint methods to identify phenotypes having nominal linkage evidence at two or more loci. ANNs were then applied to sib-pair identity-by-descent (IBD) allele sharing across the genome as input variables and squared trait sums and differences for the sib pairs as output variables. The weights of the trained networks were analyzed to assess the linkage evidence at each locus as well as potential interactions between them. Loci identified by classical linkage analysis could also be identified by our ANN analysis. However some ANN results were noisy, and our attempts to use cross-validated training to avoid overtraining and thereby improve results were only partially successful. Potential interactions between loci with high-ranked weight measures were also evaluated, with the resulting patterns suggesting existence of both synergistic and antagonistic effects between loci. Our results suggest that ANNs can serve as a useful method to analyze quantitative traits and are a potential tool for detecting gene x gene interactions. However, for the approach implemented here, optimizing the ANNs and obtaining stable results remains challenging.