Quantitative biophysical analysis defines key components modulating recruitment of the GTPase KRAS to the plasma membrane.

The gene encoding the GTPase KRAS is frequently mutated in pancreatic, lung, and colorectal cancers. The KRAS fraction in the plasma membrane (PM) correlates with activation of the mitogen-activated protein kinase (MAPK) pathway and subsequent cellular proliferation. Understanding KRAS's interaction with the PM is challenging given the complexity of the cellular environment. To gain insight into key components necessary for KRAS signal transduction at the PM, we used synthetic membranes such as liposomes and giant unilamellar vesicles. Using surface plasmon resonance (SPR) spectroscopy, we demonstrated that KRAS and Raf-1 proto-oncogene Ser/Thr kinase (RAF1) domains interact with these membranes primarily through electrostatic interactions with negatively charged lipids reinforced by additional interactions involving phosphatidyl ethanolamine and cholesterol. We found that the RAF1 region spanning RBD through CRD (RBDCRD) interacts with the membrane significantly more strongly than the isolated RBD or CRD domains and synergizes KRAS partitioning to the membrane. We also found that calmodulin and phosphodiesterase 6 delta (PDE6δ), but not galectin3 previously proposed to directly interact with KRAS, passively sequester KRAS and prevent it from partitioning into the PM. RAF1 RBDCRD interacted with membranes preferentially at nonraft lipid domains. Moreover, a C-terminal O-methylation was crucial for KRAS membrane localization. These results contribute to a better understanding of how the KRAS-membrane interaction is tuned by multiple factors whose identification could inform drug discovery efforts to disrupt this critical interaction in diseases such as cancer.

Correction to: Low-cost HPV testing and the prevalence of cervical infection in asymptomatic populations in Guatemala.

Following publication of the original article [1], an error was reported in the tagging of Joël Fokom Domgue in the author group. The tagging in this correction article has been fixed.

Low-cost HPV testing and the prevalence of cervical infection in asymptomatic populations in Guatemala.

BACKGROUND: A low cost and accurate method for detecting high-risk (HR) human papillomavirus (HPV) is important to permit HPV testing for cervical cancer prevention. We used a commercially available HPV method (H13, Hybribio) which was documented to function accurately in a reduced volume of cervical specimen to determine the most prevalent HPV types and the distribution of HPV infections in over 1795 cancer-free women in Guatemala undergoing primary screening for cervical cancer by cytology. METHODS: HR-HPV detection was attempted in cervical samples from 1795 cancer-free women receiving Pap smears using the Hybribio™ real-time PCR assay of 13 HR types. The test includes a globin gene internal control. HPV positive samples were sequenced to determine viral type. Age-specific prevalence of HPV was also assessed in the study population. RESULTS: A total of 13% (226/1717) of women tested HPV+, with 78 samples (4.3%) failing to amplify the internal control. The highest prevalence was found in younger women (< 30 years, 22%) and older ones (≥60 years, 15%). The six most common HR-HPV types among the 148 HPV+ typed were HPV16 (22%), HPV18 (11%), HPV39 (11%), HPV58 (10%), HPV52 (8%), and HPV45 (8%). CONCLUSIONS: In this sample of cancer free women in Guatemala, HPV16 was the most prevalent HR type in Guatemala and the age-specific prevalence curve peaked in younger ages. Women in the 30-59-year age groups had a prevalence of HR-HPV of 8%, however, larger studies to better describe the epidemiology of HPV in Guatemala are needed.

BRCA1 Circos: a visualisation resource for functional analysis of missense variants.

BACKGROUND: Inactivating germline mutations in the tumour suppressor gene BRCA1 are associated with a significantly increased risk of developing breast and ovarian cancer. A large number (>1500) of unique BRCA1 variants have been identified in the population and can be classified as pathogenic, non-pathogenic or as variants of unknown significance (VUS). Many VUS are rare missense variants leading to single amino acid changes. Their impact on protein function cannot be directly inferred from sequence information, precluding assessment of their pathogenicity. Thus, functional assays are critical to assess the impact of these VUS on protein activity. BRCA1 is a multifunctional protein and different assays have been used to assess the impact of variants on different biochemical activities and biological processes. METHODS AND RESULTS: To facilitate VUS analysis, we have developed a visualisation resource that compiles and displays functional data on all documented BRCA1 missense variants. BRCA1 Circos is a web-based visualisation tool based on the freely available Circos software package. The BRCA1 Circos web tool (http://research.nhgri.nih.gov/bic/circos/) aggregates data from all published BRCA1 missense variants for functional studies, harmonises their results and presents various functionalities to search and interpret individual-level functional information for each BRCA1 missense variant. CONCLUSIONS: This research visualisation tool will serve as a quick one-stop publically available reference for all the BRCA1 missense variants that have been functionally assessed. It will facilitate meta-analysis of functional data and improve assessment of pathogenicity of VUS.

The polygenic nature of inhibitors in hemophilia A: results from the Hemophilia Inhibitor Genetics Study (HIGS) Combined Cohort.

Studies of determinants of development of inhibitory Abs to factor VIII in people with hemophilia A indicate a complex process involving multiple factors. The Hemophilia Inhibitor Genetics Study (HIGS) Combined Cohort was formed to extend our understanding of the genetic background of risk. The study group contains 833 subjects from 3 independent cohorts: brother pairs and singletons with and without a history of inhibitors, as well as 104 brother pairs discordant for inhibitor status. Using an Illumina iSelect platform, 13 331 single-nucleotide polymorphisms from 1081 genes, primarily immune response and immune modifier genes, were typed. Each cohort was analyzed separately with results combined using a meta-analytic technique. After adjustment for potential confounders, 53 single-nucleotide polymorphisms were found to be significant predictors of inhibitor status using the criteria of odds ratios in the same direction in all cohorts or allowing for a 20% interval around an odds ratio = 1 in 1 of the 3 and significant in at least 2. Of the 53 markers, 13 had meta P < .001. Eight of the 53 were significant predictors among the discordant pairs. Results support the complexity of the immune response and encourage further research with the goal of understanding the pathways involved.

The principal genetic determinants for nasopharyngeal carcinoma in China involve the HLA class I antigen recognition groove.

Nasopharyngeal carcinoma (NPC) is an epithelial malignancy facilitated by Epstein-Barr Virus infection. Here we resolve the major genetic influences for NPC incidence using a genome-wide association study (GWAS), independent cohort replication, and high-resolution molecular HLA class I gene typing including 4,055 study participants from the Guangxi Zhuang Autonomous Region and Guangdong province of southern China. We detect and replicate strong association signals involving SNPs, HLA alleles, and amino acid (aa) variants across the major histocompatibility complex-HLA-A, HLA -B, and HLA -C class I genes (P(HLA-A-aa-site-62) = 7.4 × 10(-29); P (HLA-B-aa-site-116) = 6.5 × 10(-19); P (HLA-C-aa-site-156) = 6.8 × 10(-8) respectively). Over 250 NPC-HLA associated variants within HLA were analyzed in concert to resolve separate and largely independent HLA-A, -B, and -C gene influences. Multivariate logistical regression analysis collapsed significant associations in adjacent genes spanning 500 kb (OR2H1, GABBR1, HLA-F, and HCG9) as proxies for peptide binding motifs carried by HLA- A*11:01. A similar analysis resolved an independent association signal driven by HLA-B*13:01, B*38:02, and B*55:02 alleles together. NPC resistance alleles carrying the strongly associated amino acid variants implicate specific class I peptide recognition motifs in HLA-A and -B peptide binding groove as conferring strong genetic influence on the development of NPC in China.

A molecular phylogeny of living primates.

Comparative genomic analyses of primates offer considerable potential to define and understand the processes that mold, shape, and transform the human genome. However, primate taxonomy is both complex and controversial, with marginal unifying consensus of the evolutionary hierarchy of extant primate species. Here we provide new genomic sequence (~8 Mb) from 186 primates representing 61 (~90%) of the described genera, and we include outgroup species from Dermoptera, Scandentia, and Lagomorpha. The resultant phylogeny is exceptionally robust and illuminates events in primate evolution from ancient to recent, clarifying numerous taxonomic controversies and providing new data on human evolution. Ongoing speciation, reticulate evolution, ancient relic lineages, unequal rates of evolution, and disparate distributions of insertions/deletions among the reconstructed primate lineages are uncovered. Our resolution of the primate phylogeny provides an essential evolutionary framework with far-reaching applications including: human selection and adaptation, global emergence of zoonotic diseases, mammalian comparative genomics, primate taxonomy, and conservation of endangered species.

DNA repair gene variants in relation to overall cancer risk: a population-based study.

The hypothesis that germ-line polymorphisms in DNA repair genes influence cancer risk has previously been tested primarily on a cancer site-specific basis. The purpose of this study was to test the hypothesis that DNA repair gene allelic variants contribute to globally elevated cancer risk by measuring associations with risk of all cancers that occurred within a population-based cohort. In the CLUE II cohort study established in 1989 in Washington County, MD, this study was comprised of all 3619 cancer cases ascertained through 2007 compared with a sample of 2296 with no cancer. Associations were measured between 759 DNA repair gene single nucleotide polymorphisms (SNPs) and risk of all cancers. A SNP in O(6)-methylguanine-DNA methyltransferase, MGMT, (rs2296675) was significantly associated with overall cancer risk [per minor allele odds ratio (OR) 1.30, 95% confidence interval (CI) 1.19-1.43 and P-value: 4.1 × 10(-8)]. The association between rs2296675 and cancer risk was stronger among those aged ≤54 years old than those who were ≥55 years at baseline (P-for-(interaction) = 0.021). OR were in the direction of increased risk for all 15 categories of malignancies studied (P < 0.0001), ranging from 1.22 (P = 0.42) for ovarian cancer to 2.01 (P = 0.008) for urinary tract cancers; the smallest P-value was for breast cancer (OR 1.45, P = 0.0002). The results indicate that the minor allele of MGMT SNP rs2296675, a common genetic marker with 37% carriers, was significantly associated with increased risk of cancer across multiple tissues. Replication is needed to more definitively determine the scientific and public health significance of this observed association.

Deep sequencing and Circos analyses of antibody libraries reveal antigen-driven selection of Ig VH genes during HIV-1 infection.

The vast diversity of antibody repertoires is largely attributed to heavy chain (V(H)) recombination of variable (V), diversity (D) and joining (J) gene segments. We used 454 sequencing information of the variable domains of the antibody heavy chain repertoires from neonates, normal adults and an HIV-1-infected individual, to analyze, with Circos software, the VDJ pairing patterns at birth, adulthood and a time-dependent response to HIV-1 infection. Our comparative analyses of the Ig VDJ repertoires from these libraries indicated that, from birth to adulthood, VDJ recombination patterns remain the same with some slight changes, whereas some V(H) families are selected and preferentially expressed after long-term infection with HIV-1. We also demonstrated that the immune system responds to HIV-1 chronic infection by selectively expanding certain HV families in an attempt to combat infection. Our findings may have implications for understanding immune responses in pathology as well as for development of new therapeutics and vaccines.

A population-based study of DNA repair gene variants in relation to non-melanoma skin cancer as a marker of a cancer-prone phenotype.

For unknown reasons, non-melanoma skin cancer (NMSC) is associated with increased risk of other malignancies. Focusing solely on DNA repair or DNA repair-related genes, this study tested the hypothesis that DNA repair gene variants contribute to the increased cancer risk associated with a personal history of NMSC. From the parent CLUE II cohort study, established in 1989 in Washington County, MD, the study consisted of a cancer-free control group (n 5 2296) compared with three mutually exclusive groups of cancer cases ascertained through 2007: (i) Other (non-NMSC) cancer only (n 5 2349); (ii) NMSC only (n 5 694) and (iii) NMSC plus other cancer (n 5 577). The frequency of minor alleles in 759 DNA repair gene single nucleotide polymorphisms (SNPs) was compared in these four groups. Comparing those with both NMSC and other cancer versus those with no cancer, 10 SNPs had allelic trend P-values <0.01. The two top-ranked SNPs were both within the thymine DNA glycosylase gene (TDG). One was a non-synonymous coding SNP (rs2888805) [per allele odds ratio (OR) 1.40, 95% confidence interval (CI) 1.16-1.70; P-value 5 0.0006] and the other was an intronic SNP in high linkage disequilibrium with rs2888805 (rs4135150). None of the associations had a P-value <6.6310(-5), the threshold for statistical significance after correcting for multiple comparisons. The results pinpoint DNA repair genes most likely to contribute to the NMSC cancer-prone phenotype. A promising lead is genetic variants in TDG, important not only in base excision repair but also in regulating the epigenome and gene expression, which may contribute to the NMSC-associated increase in overall cancer risk.

Genome-wide association study implicates PARD3B-based AIDS restriction.

BACKGROUND: Host genetic variation influences human immunodeficiency virus (HIV) infection and progression to AIDS. Here we used clinically well-characterized subjects from 5 pretreatment HIV/AIDS cohorts for a genome-wide association study to identify gene associations with rate of AIDS progression. METHODS: European American HIV seroconverters (n = 755) were interrogated for single-nucleotide polymorphisms (SNPs) (n = 700,022) associated with progression to AIDS 1987 (Cox proportional hazards regression analysis, co-dominant model). RESULTS: Association with slower progression was observed for SNPs in the gene PARD3B. One of these, rs11884476, reached genome-wide significance (relative hazard = 0.3; P =3. 370 × 10(-9)) after statistical correction for 700,022 SNPs and contributes 4.52% of the overall variance in AIDS progression in this study. Nine of the top-ranked SNPs define a PARD3B haplotype that also displays significant association with progression to AIDS (hazard ratio, 0.3; P = 3.220 × 10(-8)). One of these SNPs, rs10185378, is a predicted exonic splicing enhancer; significant alteration in the expression profile of PARD3B splicing transcripts was observed in B cell lines with alternate rs10185378 genotypes. This SNP was typed in European cohorts of rapid progressors and was found to be protective for AIDS 1993 definition (odds ratio, 0.43, P = .025). CONCLUSIONS: These observations suggest a potential unsuspected pathway of host genetic influence on the dynamics of AIDS progression.

Genetic associations of variants in genes encoding HIV-dependency factors required for HIV-1 infection.

BACKGROUND: High-throughput genome-wide techniques have facilitated the identification of previously unknown host proteins involved in cellular human immunodeficiency virus (HIV) infection. Recently, 3 independent studies have used small interfering RNA technology to silence each gene in the human genome to determine the importance of each in HIV infection. Genes conferring a significant effect were termed HIV-dependency factors (HDFs). METHODS: We assembled high-density panels of 6380 single-nucleotide polymorphisms (SNPs) in 278 HDF genes and tested for genotype associations with HIV infection and AIDS progression in 1633 individuals from clinical AIDS cohorts. RESULTS: After statistical correction for multiple tests, significant associations with HIV acquisition were found for SNPs in 2 genes, NCOR2 and IDH1. Weaker associations with AIDS progression were revealed for SNPs within the TM9SF2 and EGFR genes. CONCLUSIONS: This study independently verifies the influence of NCOR2 and IDH1 on HIV transmission, and its findings suggest that variation in these genes affects susceptibility to HIV infection in exposed individuals.

Multistage genomewide association study identifies a locus at 1q41 associated with rate of HIV-1 disease progression to clinical AIDS.

BACKGROUND: A mean of 9-10 years of human immunodeficiency virus type 1 (HIV-1) infection elapse before clinical AIDS develops in untreated persons, but this rate of disease progression varies substantially among individuals. To investigate host genetic determinants of the rate of progression to clinical AIDS, we performed a multistage genomewide association study. METHODS: The discovery stage comprised 156 individuals from the Multicenter AIDS Cohort Study, enriched with rapid and long-term nonprogressors to increase statistical power. This was followed by replication tests of putatively associated genotypes in an independent population of 590 HIV-1-infected seroconverters. RESULTS: Significant associations with delayed AIDS progression were observed in a haplotype located at 1q41, 36 kb upstream of PROX1 on chromosome 1 (relative hazard ratio, 0.69; Fisher's combined P = 6.23 X 10(-7)). This association was replicated further in an analysis stratified by transmission mode, with the effect consistent in sexual or mucosal and parenteral transmission (relative hazard ratios, 0.72 and 0.63, respectively; combined P = 1.63 X 10(-6)). CONCLUSIONS: This study identified and replicated a locus upstream of PROX1 that is associated with delayed progression to clinical AIDS. PROX1 is a negative regulator of interferon-gamma expression in T cells and also mitigates the advancement of vascular neoplasms, such as Kaposi sarcoma, a common AIDS-defining malignancy. This study adds to the cumulative polygenic host component that effectively regulates the progression to clinical AIDS among HIV-1-infected individuals, raising prospects for potential new avenues for therapy and improvements in AIDS prognosis.

Accounting for multiple comparisons in a genome-wide association study (GWAS).

BACKGROUND: As we enter an era when testing millions of SNPs in a single gene association study will become the standard, consideration of multiple comparisons is an essential part of determining statistical significance. Bonferroni adjustments can be made but are conservative due to the preponderance of linkage disequilibrium (LD) between genetic markers, and permutation testing is not always a viable option. Three major classes of corrections have been proposed to correct the dependent nature of genetic data in Bonferroni adjustments: permutation testing and related alternatives, principal components analysis (PCA), and analysis of blocks of LD across the genome. We consider seven implementations of these commonly used methods using data from 1514 European American participants genotyped for 700,078 SNPs in a GWAS for AIDS. RESULTS: A Bonferroni correction using the number of LD blocks found by the three algorithms implemented by Haploview resulted in an insufficiently conservative threshold, corresponding to a genome-wide significance level of α = 0.15 - 0.20. We observed a moderate increase in power when using PRESTO, SLIDE, and simpleℳ when compared with traditional Bonferroni methods for population data genotyped on the Affymetrix 6.0 platform in European Americans (α = 0.05 thresholds between 1 × 10(-7) and 7 × 10(-8)). CONCLUSIONS: Correcting for the number of LD blocks resulted in an anti-conservative Bonferroni adjustment. SLIDE and simpleℳ are particularly useful when using a statistical test not handled in optimized permutation testing packages, and genome-wide corrected p-values using SLIDE, are much easier to interpret for consumers of GWAS studies.

Examination of disease-based selection, demographic history and population structure in European Y-chromosome haplogroup I.

We attempted to refine the understanding of an association of Y-chromosomal haplogroup I (hg-I) with enhanced AIDS progression that had been previously reported. First, we compared the progression phenotype between hg-I and its phylogenetically closest haplogroup J. Then, we took a candidate gene approach resequencing DDX3Y, a crucial autoimmunity gene, in hg-I and other common European Y-chromosome haplogroups looking for functional variants. We extended the genetic analyses to CD24L4 and compared and contrasted the roles of disease-based selection, demographic history and population structure shaping the contemporary genetic landscape of hg-I chromosomes. Our results confirmed and refined the AIDS progression signal to hg-I, though no gene variant was identified that can explain the disease association. Molecular evolutionary and genetic analyses of the examined loci suggested a unique evolutionary history in hg-I, probably shaped by complex interactions of selection, demographic history and high geographical differentiation leading to the formation of distinct hg-I subhaplogroups that today are associated with HIV/AIDS onset. Clearly, further studies on Y-chromosome candidate loci sequencing to discover functional variants and discern the roles of evolutionary factors are warranted.

Hypothesis-driven candidate gene association studies: practical design and analytical considerations.

Candidate gene association studies (CGAS) are a useful epidemiologic approach to drawing inferences about relations between genes and disease, especially when experimental data support the involvement of specific biochemical pathways. The value of CGAS is apparent when allele frequencies are low, effect sizes are small, or the study population is limited or unique. CGAS is also valuable for validating previous reports of genetic associations with disease in different populations. Despite the many advantages, the information generated from CGAS is sometimes compromised because of either inefficient study design or suboptimal analytical approaches. Here the authors discuss issues related to the study design and statistical analyses of CGAS that can help to optimize their usefulness and information content. These issues include judicious hypothesis-driven selection of biochemical pathways, genes, and single nucleotide polymorphisms, as well as appropriate quality control and analytical procedures for measuring main effects and for evaluating environmental exposure modifications and interactions. A study design algorithm using the example of DNA repair genes and cancer is presented for purposes of illustration.

Genetic factors leading to chronic Epstein-Barr virus infection and nasopharyngeal carcinoma in South East China: study design, methods and feasibility.

Nasopharyngeal carcinoma (NPC) is a complex disease caused by a combination of Epstein-Barr virus chronic infection, the environment and host genes in a multi-step process of carcinogenesis. The identity of genetic factors involved in the development of chronic Epstein-Barr virus infection and NPC remains elusive, however. Here, we describe a two-phase, population-based, case-control study of Han Chinese from Guangxi province, where the NPC incidence rate rises to a high of 25-50 per 100,000 individuals. Phase I, powered to detect single gene associations, enrolled 984 subjects to determine feasibility, to develop infrastructure and logistics and to determine error rates in sample handling. A microsatellite screen of Phase I study participants, genotyped for 319 alleles from 34 microsatellites spanning an 18-megabase region of chromosome 4 (4p15.1-q12), previously implicated by a linkage analysis of familial NPC, found 14 alleles marginally associated with developing NPC or chronic immunoglobulin A production (p=0.001-0.03). These associations lost significance after applying a correction for multiple tests. Although the present results await confirmation, the Phase II study population has tripled patient enrollment and has included environmental covariates, offering the potential to validate this and other genomic regions that influence the onset of NPC.

Tumor RAS Gene Expression Levels Are Influenced by the Mutational Status of RAS Genes and Both Upstream and Downstream RAS Pathway Genes.

The 3 human RAS genes play pivotal roles regulating proliferation, differentiation, and survival in normal cells and become mutated in 15% to 20% of all human tumors and amplified in many others. In this report, we examined data from The Cancer Genome Atlas to investigate the relationship between RAS gene mutational status and messenger RNA expression. We show that all 3 RAS genes exhibit increased expression when they are mutated in a context-dependent manner. In the case of KRAS, this increase is manifested by a larger proportional increase in KRAS4A than KRAS4B, although both increase significantly. In addition, the mutational status of RAS genes can be associated with expression changes in other RAS genes, with most of these cases showing decreased expression. The mutational status associations with expression are recapitulated in cancer cell lines. Increases in expression are mediated by both copy number variation and contextual differences, including mutational status of epidermal growth factor receptor (EGFR) and BRAF. These findings potentially reveal an adaptive response during tumor evolution that is dependent on the mutational status of proximal genes in the RAS pathway and cellular context. Cell contextual differences in these adaptations may influence therapeutic responsiveness and alternative resistance mechanisms.

[Association study of chromosome 4 STRs polymorphisms with nasopharyngeal carcinoma].

Nasopharyngeal carcinoma (NPC) is a complex disease caused by an interaction of EBV chronic infection, environment and host genes, in a multi-step process of carcinogenesis. However, which genetic factors play an important role in the development of chronic EBV infection and NPC remain elusive. The objective of this study is to identify genetic variations associated with two key clinical stages of NPC development: persistent Epstein-Barr virus (EBV) infection of nasopharyngeal epithelia and progression to NPC. We inspected a NPC-associated region on the short arm of chromosome 4 previously implicated by a genome-wide linkage analysis of familial NPC. We determined genotypes for 319 alleles in 34 microsatellite markers spanning an 18 Mb region in 350 NPC cases, 288 individuals with IgA antibodies to EBV capsid antigen (IgA/VCA+) and 346 controls seronegative for IgA antibodies to EBV capsid antigen (IgA/VCA-). The cases and controls were Han Chinese from Wuzhou city and Cangwu County, Guangxi province where the incidence of NPC is as high as 25-50 per 100,000 individuals. Comparing NPC cases to IgA/VCA+ subjects, we found 9 alleles marginally associated with developing NPC from IgA+ status, 5 for risk (OR=1.51-5.36, P=0.01-0.03) and 4 for restrictive (OR=0.3-0.71, P=0.02-0.045). Comparing IgA/VCA+ subjects and IgA/VCA- controls, and comparing all IgA seropositives with and without NPC to IgA seronegatives revealed 12 significant and 3 highly significant (P<0.01) alleles associated with IgA+ serostatus in the two comparing groups. Alleles D4S3241-136 (P=0.004, OR=1.91, 95% CI=1.2-3.0) and D4S3347-213 (P=0.001, OR=1.6, 95% CI=1.2-2.1) were for risk. Allele D4S174-202 (P=0.001, OR=0.5, 95%CI=0.3-0.7) was restrictive. However, statistical significance was lost for all when corrected for multiple comparisons test. Our study could not affirm the genetic association within this region with NPC as did another pedigree study, but provide an opportunity for further gene discovery in this highly endemic NPC population and suggest that this region warrants further study.

A population-based study of hedgehog pathway gene variants in relation to the dual risk of basal cell carcinoma plus another cancer.

INTRODUCTION: A personal history of basal cell carcinoma (BCC) is associated with increased risk of other malignancies, but the reason is unknown. The hedgehog pathway is critical to the etiology of BCC, and is also believed to contribute to susceptibility to other cancers. This study tested the hypothesis that hedgehog pathway and pathway-related gene variants contribute to the increased risk of subsequent cancers among those with a history of BCC. METHODS: The study was nested within the ongoing CLUE II cohort study, established in 1989 in Washington County, Maryland, USA. The study consisted of a cancer-free control group (n=2296) compared to three different groups of cancer cases ascertained through 2007, those diagnosed with: (1) Other (non-BCC) cancer only (n=2349); (2) BCC only (n=534); and (3) BCC plus other cancer (n=446). The frequencies of variant alleles were compared among these four groups for 20 single nucleotide polymorphisms (SNPs) in 6 hedgehog pathway genes (SHH, IHH, PTCH2, SMO, GLI1, SUFU), and also 22 SNPs in VDR and 8 SNPs in FAS, which have cross-talk with the hedgehog pathway. RESULTS: Comparing those with both BCC and other cancer versus those with no cancer, no significant associations were observed for any of the hedgehog pathway SNPs, or for the FAS SNPs. One VDR SNP was nominally significantly associated with the BCC cancer-prone phenotype, rs11574085 [per minor allele odds ratio (OR) 1.38, 95% confidence interval (CI) 1.05-1.82; p-value=0.02]. CONCLUSION: The hedgehog pathway gene SNPs studied, along with the VDR and FAS SNPs studied, are not strongly associated with the BCC cancer-prone phenotype.