Comprehensive evaluation and efficient classification of BRCA1 RING domain missense substitutions.

BRCA1 is a high-risk susceptibility gene for breast and ovarian cancer. Pathogenic protein-truncating variants are scattered across the open reading frame, but all known missense substitutions that are pathogenic because of missense dysfunction are located in either the amino-terminal RING domain or the carboxy-terminal BRCT domain. Heterodimerization of the BRCA1 and BARD1 RING domains is a molecularly defined obligate activity. Hence, we tested every BRCA1 RING domain missense substitution that can be created by a single nucleotide change for heterodimerization with BARD1 in a mammalian two-hybrid assay. Downstream of the laboratory assay, we addressed three additional challenges: assay calibration, validation thereof, and integration of the calibrated results with other available data, such as computational evidence and patient/population observational data to achieve clinically applicable classification. Overall, we found that 15%-20% of BRCA1 RING domain missense substitutions are pathogenic. Using a Bayesian point system for data integration and variant classification, we achieved clinical classification of 89% of observed missense substitutions. Moreover, among missense substitutions not present in the human observational data used here, we find an additional 45 with concordant computational and functional assay evidence in favor of pathogenicity plus 223 with concordant evidence in favor of benignity; these are particularly likely to be classified as likely pathogenic and likely benign, respectively, once human observational data become available.

Effectiveness of Contact Precautions to Prevent Transmission of Methicillin-Resistant Staphylococcus aureus and Vancomycin-Resistant Enterococci in Intensive Care Units.

BACKGROUND: Contact precautions for endemic methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE) are under increasing scrutiny, in part due to limited clinical trial evidence. METHODS: We retrospectively analyzed data from the Strategies to Reduce Transmission of Antimicrobial Resistant Bacteria in Intensive Care Units (STAR*ICU) trial to model the use of contact precautions in individual intensive care units (ICUs). Data included admission and discharge times and surveillance test results. We used a transmission model to estimate key epidemiological parameters, including the effect of contact precautions on transmission. Finally, we performed multivariate meta-regression to identify ICU-level factors associated with contact precaution effects. RESULTS: We found that 21% of admissions (n = 2194) were placed on contact precautions, with most for MRSA and VRE. We found little evidence that contact precautions reduced MRSA transmission. The estimated change in transmission attributed to contact precautions was -16% (95% credible interval, -38% to 15%). VRE transmission was higher than MRSA transmission due to contact precautions, but not significantly. In our meta-regression, we did not identify associations between ICU-level factors and estimated contact precaution effects. Importation and transmission were higher for VRE than for MRSA, but clearance rates were lower for VRE than for MRSA. CONCLUSIONS: We found little evidence that contact precautions implemented during the STAR*ICU trial reduced transmission of MRSA or VRE. We did find important differences in the transmission dynamics between MRSA and VRE. Differences in organism and healthcare setting may impact the efficacy of contact precautions.

Transmission Dynamics of Clostridioides difficile in 2 High-Acuity Hospital Units.

BACKGROUND: The key epidemiological drivers of Clostridioides difficile transmission are not well understood. We estimated epidemiological parameters to characterize variation in C. difficile transmission, while accounting for the imperfect nature of surveillance tests. METHODS: We conducted a retrospective analysis of C. difficile surveillance tests for patients admitted to a bone marrow transplant (BMT) unit or a solid tumor unit (STU) in a 565-bed tertiary hospital. We constructed a transmission model for estimating key parameters, including admission prevalence, transmission rate, and duration of colonization to understand the potential variation in C. difficile dynamics between these 2 units. RESULTS: A combined 2425 patients had 5491 admissions into 1 of the 2 units. A total of 3559 surveillance tests were collected from 1394 patients, with 11% of the surveillance tests being positive for C. difficile. We estimate that the transmission rate in the BMT unit was nearly 3-fold higher at 0.29 acquisitions per percentage colonized per 1000 days, compared to our estimate in the STU (0.10). Our model suggests that 20% of individuals admitted into either the STU or BMT unit were colonized with C. difficile at the time of admission. In contrast, the percentage of surveillance tests that were positive within 1 day of admission to either unit for C. difficile was 13.4%, with 15.4% in the STU and 11.6% in the BMT unit. CONCLUSIONS: Although prevalence was similar between the units, there were important differences in the rates of transmission and clearance. Influential factors may include antimicrobial exposure or other patient-care factors.

Novel pedigree analysis implicates DNA repair and chromatin remodeling in multiple myeloma risk.

The high-risk pedigree (HRP) design is an established strategy to discover rare, highly-penetrant, Mendelian-like causal variants. Its success, however, in complex traits has been modest, largely due to challenges of genetic heterogeneity and complex inheritance models. We describe a HRP strategy that addresses intra-familial heterogeneity, and identifies inherited segments important for mapping regulatory risk. We apply this new Shared Genomic Segment (SGS) method in 11 extended, Utah, multiple myeloma (MM) HRPs, and subsequent exome sequencing in SGS regions of interest in 1063 MM / MGUS (monoclonal gammopathy of undetermined significance-a precursor to MM) cases and 964 controls from a jointly-called collaborative resource, including cases from the initial 11 HRPs. One genome-wide significant 1.8 Mb shared segment was found at 6q16. Exome sequencing in this region revealed predicted deleterious variants in USP45 (p.Gln691* and p.Gln621Glu), a gene known to influence DNA repair through endonuclease regulation. Additionally, a 1.2 Mb segment at 1p36.11 is inherited in two Utah HRPs, with coding variants identified in ARID1A (p.Ser90Gly and p.Met890Val), a key gene in the SWI/SNF chromatin remodeling complex. Our results provide compelling statistical and genetic evidence for segregating risk variants for MM. In addition, we demonstrate a novel strategy to use large HRPs for risk-variant discovery more generally in complex traits.

Peri-implantation intercourse does not lower fecundability.

STUDY QUESTION: Does sexual intercourse in the implantation time window (5-9 days after ovulation) reduce fecundability? SUMMARY ANSWER: After adjustment for intercourse in the fecund window and clustering by couple, there was no association between intercourse in the implantation time window and fecundity. WHAT IS KNOWN ALREADY: Previous research has suggested an association between intercourse in the peri-implantation time window (5-9 days after estimated ovulation) and reduced fecundability. STUDY DESIGN, SIZE, DURATION: We used data from the FERTILI study, a prospective observational study conducted in five European countries, with data collected from 1992 to 1996. PARTICIPANTS/MATERIALS, SETTING, METHODS: Women who were experienced in fertility awareness tracking kept a daily diary of cervical mucus observations, basal body temperature measurements, coitus and clinically identified pregnancy. We estimated the day of ovulation as cycle length minus 13 days. From 661 women, 2606 cycles had intercourse during the fecund window (from 5 days before to 3 days after the estimated day of ovulation), resulting in 418 pregnancies (conception cycles). An established Bayesian fecundability model was used to estimate the fecundability ratio (FR) of peri-implantation intercourse on fecundability, while adjusting for each partner's age, prior pregnancy, the couple's probability of conception and intercourse pattern(s). We conducted sensitivity analyses estimating ovulation as cycle length minus 12 days, or alternatively, as the peak day of estrogenic cervical mucus. MAIN RESULTS AND THE ROLE OF CHANCE: There was no effect of peri-implantation intercourse on fecundability: adjusted FR for three or more acts of peri-implantation intercourse versus none: 1.00, 95% credible interval: 0.76-1.13. Results were essentially the same with sensitivity analyses. There was an inverse relationship between frequency of intercourse in the fecund window and intercourse in the peri-implantation window. LIMITATIONS, REASONS FOR CAUTION: Women with known subfertility were excluded from this study. Many couples in the study were avoiding pregnancy during much of the study, so 61% of otherwise eligible cycles in the database were not at meaningful risk of pregnancy and did not contribute to the analysis. Some couples may not have recorded all intercourse. WIDER IMPLICATIONS OF THE FINDINGS: We believe the current balance of evidence does not support a recommendation for avoiding intercourse in the peri-implantation period among couples trying to conceive. STUDY FUNDING/COMPETING INTEREST(S): No external funding. The authors have no potential competing interests. TRIAL REGISTRATION NUMBER: N/A.

Genome-wide association of familial prostate cancer cases identifies evidence for a rare segregating haplotype at 8q24.21.

Previous genome-wide association studies (GWAS) of prostate cancer risk focused on cases unselected for family history and have reported over 100 significant associations. The International Consortium for Prostate Cancer Genetics (ICPCG) has now performed a GWAS of 2511 (unrelated) familial prostate cancer cases and 1382 unaffected controls from 12 member sites. All samples were genotyped on the Illumina 5M+exome single nucleotide polymorphism (SNP) platform. The GWAS identified a significant evidence for association for SNPs in six regions previously associated with prostate cancer in population-based cohorts, including 3q26.2, 6q25.3, 8q24.21, 10q11.23, 11q13.3, and 17q12. Of note, SNP rs138042437 (p = 1.7e(-8)) at 8q24.21 achieved a large estimated effect size in this cohort (odds ratio = 13.3). 116 previously sampled affected relatives of 62 risk-allele carriers from the GWAS cohort were genotyped for this SNP, identifying 78 additional affected carriers in 62 pedigrees. A test for an excess number of affected carriers among relatives exhibited strong evidence for co-segregation of the variant with disease (p = 8.5e(-11)). The majority (92 %) of risk-allele carriers at rs138042437 had a consistent estimated haplotype spanning approximately 100 kb of 8q24.21 that contained the minor alleles of three rare SNPs (dosage minor allele frequencies <1.7 %), rs183373024 (PRNCR1), previously associated SNP rs188140481, and rs138042437 (CASC19). Strong evidence for co-segregation of a SNP on the haplotype further characterizes the haplotype as a prostate cancer predisposition locus.

Baseline Ability Makes a Larger Contribution to Race Performance in High-School Sprinters Than Race Experience or Training Exposure.

PURPOSE: There has long been a debate regarding the importance of talent versus training in athletic performance. In this study we sought to quantify their relative contributions to the race performance of high-school sprinters. METHODS: Using race results from the athletic.net website, we identified high-school athletes who participated in at least one race in both 9th and 12th grade in the 100 m, 200 m or 400 m. Athletes with a record of racing before high school were excluded from the analyses. Using separate linear regression models for each event and gender, we analyzed the effect of baseline ability, race experience and training exposure on race time in the 12th grade. RESULTS: 35,909 athletes, running a total of 1,627,652 races, contributed to the final analyses. The proportion of variance (R2) in 12th grade race times accounted for by baseline ability ranged from 40% to 51% depending on the event, and was consistently higher for females than males. Race experience explained 3.6-4.4% of the variance and training exposure explained 0.8-1.7%. CONCLUSION: Although race experience and training exposure impact high-school sprinters' performance, baseline ability is the dominant influence.

Identification of specific Y chromosomes associated with increased prostate cancer risk.

BACKGROUND: Evidence supports the possibility of a role of the Y chromosome in prostate cancer, but controversy exists. METHODS: A novel analysis of a computerized population-based resource linking genealogy and cancer data was used to test the hypothesis of a role of the Y chromosome in prostate cancer predisposition. Using a statewide cancer registry from 1966 linked to a computerized genealogy representing over 1.2 million descendants of the Utah pioneers, 1,000 independent sets of males, each set hypothesized to share the same Y chromosome as represented in genealogy data, were tested for a significant excess of prostate cancer. RESULTS: Multiple Y chromosomes representing thousands of potentially at-risk males were identified to have a significant excess risk for prostate cancer. CONCLUSIONS: This powerful and efficient in silico test of an uncommon mode of inheritance has confirmed evidence for Y chromosome involvement in prostate cancer.

Conserved Noncoding Cis-Elements Associated with Hibernation Modulate Metabolic and Behavioral Adaptations in Mice.

Our study elucidates functional roles for conserved cis-elements associated with the evolution of mammalian hibernation. Genomic analyses found topologically associated domains (TADs) that disproportionately accumulated convergent genomic changes in hibernators, including the TAD for the Fat Mass & Obesity (Fto) locus. Some hibernation-linked cis-elements in this TAD form regulatory contacts with multiple neighboring genes. Knockout mice for these cis-elements exhibit Fto, Irx3, and Irx5 gene expression changes, impacting hundreds of genes downstream. Profiles of pre-torpor, torpor, and post-torpor phenotypes found distinct roles for each cis-element in metabolic control, while a high caloric diet uncovered different obesogenic effects. One cis-element promoting a lean phenotype influences foraging behaviors throughout life, affecting specific behavioral sequences. Thus, convergent evolution in hibernators pinpoints functional genetic mechanisms of mammalian metabolic control.

Calibration of multiple in silico tools for predicting pathogenicity of mismatch repair gene missense substitutions.

Classification of rare missense substitutions observed during genetic testing for patient management is a considerable problem in clinical genetics. The Bayesian integrated evaluation of unclassified variants is a solution originally developed for BRCA1/2. Here, we take a step toward an analogous system for the mismatch repair (MMR) genes (MLH1, MSH2, MSH6, and PMS2) that confer colon cancer susceptibility in Lynch syndrome by calibrating in silico tools to estimate prior probabilities of pathogenicity for MMR gene missense substitutions. A qualitative five-class classification system was developed and applied to 143 MMR missense variants. This identified 74 missense substitutions suitable for calibration. These substitutions were scored using six different in silico tools (Align-Grantham Variation Grantham Deviation, multivariate analysis of protein polymorphisms [MAPP], MutPred, PolyPhen-2.1, Sorting Intolerant From Tolerant, and Xvar), using curated MMR multiple sequence alignments where possible. The output from each tool was calibrated by regression against the classifications of the 74 missense substitutions; these calibrated outputs are interpretable as prior probabilities of pathogenicity. MAPP was the most accurate tool and MAPP + PolyPhen-2.1 provided the best-combined model (R(2)  = 0.62 and area under receiver operating characteristic = 0.93). The MAPP + PolyPhen-2.1 output is sufficiently predictive to feed as a continuous variable into the quantitative Bayesian integrated evaluation for clinical classification of MMR gene missense substitutions.

Pathways analysis of differential gene expression induced by engrafting doses of total body irradiation for allogeneic bone marrow transplantation in mice.

A major challenge in allogeneic bone marrow (BM) transplantation is overcoming engraftment resistance to avoid the clinical problem of graft rejection. Identifying gene pathways that regulate BM engraftment may reveal molecular targets for overcoming engraftment barriers. Previously, we developed a mouse model of BM transplantation that utilizes recipient conditioning with non-myeloablative total body irradiation (TBI). We defined TBI doses that lead to graft rejection, that conversely are permissive for engraftment, and mouse strain variation with regards to the permissive TBI dose. We now report gene expression analysis, using Agilent Mouse 8x60K microarrays, in spleens of mice conditioned with varied TBI doses for correlation to the expected engraftment phenotype. The spleens of mice given engrafting doses of TBI, compared with non-engrafting TBI doses, demonstrated substantially broader gene expression changes, significant at the multiple testing-corrected P <0.05 level and with fold change ≥2. Functional analysis revealed significant enrichment for a down-regulated canonical pathway involving B-cell development. Genes enriched in this pathway suggest that suppressing donor antigen processing and presentation may be pivotal effects conferred by TBI to enable engraftment. Regardless of TBI dose and recipient mouse strain, pervasive genomic changes related to inflammation was observed and reflected by significant enrichment for canonical pathways and association with upstream regulators. These gene expression changes suggest that macrophage and complement pathways may be targeted to overcome engraftment barriers. These exploratory results highlight gene pathways that may be important in mediating BM engraftment resistance.

Fine mapping of the Bmgr5 quantitative trait locus for allogeneic bone marrow engraftment in mice.

To identify novel mechanisms regulating allogeneic hematopoietic cell engraftment, we used forward genetics and previously described identification, in mice, of a bone marrow (BM) engraftment quantitative trait locus (QTL), termed Bmgr5. This QTL confers dominant and large allele effects for engraftment susceptibility. It was localized to chromosome 16 by quantitative genetic techniques in a segregating backcross bred from susceptible BALB.K and resistant B10.BR mice. We now report verification of the Bmgr5 QTL using reciprocal chromosome 16 consomic strains. The BM engraftment phenotype in these consomic mice shows that Bmgr5 susceptibility alleles are not only sufficient but also indispensable for conferring permissiveness for allogeneic BM engraftment. Using panels of congenic mice, we resolved the Bmgr5 QTL into two separate subloci, termed Bmgr5a (Chr16:14.6-15.8 Mb) and Bmgr5b (Chr16:15.8-17.6 Mb), each conferring permissiveness for the engraftment phenotype and both fine mapped to an interval amenable to positional cloning. Candidate Bmgr5 genes were then prioritized using whole exome DNA sequencing and microarray gene expression data. Further studies are warranted to elucidate the genetic interaction between the Bmgr5a and Bmgr5b QTL and identify causative genes and underlying gene variants. This may lead to new approaches for overcoming the problem of graft rejection in clinical hematopoietic cell transplantation.

Creation of a national resource with linked genealogy and phenotypic data: the Veterans Genealogy Project.

PURPOSE: Creation of a genealogy of the United States and its ancestral populations is under way. When complete, this US genealogy will be record linked to the National Veteran's Health Administration medical data representing more than 8 million US veterans. METHODS: Genealogical data are gathered from public sources, primarily the Internet. Record linking using data from relatives is accomplished to integrate multiple data sources and then to link genealogical data to the veteran's demographic data. RESULTS: This resource currently includes genealogy for more than 22 million individuals representing the Intermountain West and the East Coast. The demographic data for more than 40,000 veteran patients using Veterans Hospital Administration services in Utah and Massachusetts have already been record linked. CONCLUSION: The resource is only in its second year of creation and already represents the largest such combination of genealogy and medical data in the world. The data sources, the creation of the genealogy, record-linking methods and results, proposed genetic analyses, and future directions are discussed.

Random allelic expression in the adult human body.

Genes are typically assumed to express both parental alleles similarly, yet cell lines show random allelic expression (RAE) for many autosomal genes that could shape genetic effects. Thus, understanding RAE in human tissues could improve our understanding of phenotypic variation. Here, we develop a methodology to perform genome-wide profiling of RAE and biallelic expression in GTEx datasets for 832 people and 54 tissues. We report 2,762 autosomal genes with some RAE properties similar to randomly inactivated X-linked genes. We found that RAE is associated with rapidly evolving regions in the human genome, adaptive signaling processes, and genes linked to age-related diseases such as neurodegeneration and cancer. We define putative mechanistic subtypes of RAE distinguished by gene overlaps on sense and antisense DNA strands, aggregation in clusters near telomeres, and increased regulatory complexity and inputs compared with biallelic genes. We provide foundations to study RAE in human phenotypes, evolution, and disease.

Germline EGFR Mutations and Familial Lung Cancer.

PURPOSE: The genomic underpinnings of inherited lung cancer risk are poorly understood. This prospective study characterized the clinical phenotype of patients and families with germline EGFR pathogenic variants (PVs). METHODS: The Investigating Hereditary Risk from T790M study (ClinicalTrials.gov identifier: NCT01754025) enrolled patients with lung cancer whose tumor profiling harbored possible germline EGFR PVs and their relatives, either in person or remotely, providing germline testing and follow-up. RESULTS: A total of 141 participants were enrolled over a 5-year period, 100 (71%) remotely. Based upon previous genotyping, 116 participants from 59 kindreds were tested for EGFR T790M, demonstrating a pattern of Mendelian inheritance with variable lung cancer penetrance. In confirmed or obligate carriers of a germline EGFR PV from 39 different kindreds, 50/91 (55%) were affected with lung cancer with 34/65 (52%) diagnosed by age 60 years. Somatic testing of lung cancers in carriers revealed that 35 of 37 (95%) had an EGFR driver comutation. Among 36 germline carriers without a cancer diagnosis, 15 had computed tomography (CT) imaging and nine had lung nodules, including a 28-year-old with >10 lung nodules. Given geographic enrichment of germline EGFR T790M in the southeast United States, genome-wide haplotyping of 46 germline carriers was performed and identified a 4.1-Mb haplotype shared by 41 (89%), estimated to originate 223-279 years ago. CONCLUSION: To our knowledge, this is the first prospective description of familial EGFR-mutant lung cancer, identifying a recent founder germline EGFR T790M variant enriched in the Southeast United States. The high prevalence of EGFR-driver lung adenocarcinomas and lung nodules in germline carriers supports effort to identify affected patients and family members for investigation of CT-based screening for these high-risk individuals.

Effect of linkage disequilibrium on the identification of functional variants.

We summarize the contributions of Group 9 of Genetic Analysis Workshop 17. This group addressed the problems of linkage disequilibrium and other longer range forms of allelic association when evaluating the effects of genotypes on phenotypes. Issues raised by long-range associations, whether a result of selection, stratification, possible technical errors, or chance, were less expected but proved to be important. Most contributors focused on regression methods of various types to illustrate problematic issues or to develop adaptations for dealing with high-density genotype assays. Study design was also considered, as was graphical modeling. Although no method emerged as uniformly successful, most succeeded in reducing false-positive results either by considering clusters of loci within genes or by applying smoothing metrics that required results from adjacent loci to be similar. Two unexpected results that questioned our assumptions of what is required to model linkage disequilibrium were observed. The first was that correlations between loci separated by large genetic distances can greatly inflate single-locus test statistics, and, whether the result of selection, stratification, possible technical errors, or chance, these correlations seem overabundant. The second unexpected result was that applying principal components analysis to genome-wide genotype data can apparently control not only for population structure but also for linkage disequilibrium.

Pairwise shared genomic segment analysis in three Utah high-risk breast cancer pedigrees.

BACKGROUND: We applied a new weighted pairwise shared genomic segment (pSGS) analysis for susceptibility gene localization to high-density genomewide SNP data in three extended high-risk breast cancer pedigrees. RESULTS: Using this method, four genomewide suggestive regions were identified on chromosomes 2, 4, 7 and 8, and a borderline suggestive region on chromosome 14. Seven additional regions with at least nominal evidence were observed. Of particular note among these total twelve regions were three regions that were identified in two pedigrees each; chromosomes 4, 7 and 14. Follow-up two-pedigree pSGS analyses further indicated excessive genomic sharing across the pedigrees in all three regions, suggesting that the underlying susceptibility alleles in those regions may be shared in common. In general, the pSGS regions identified were quite large (average 32.2 Mb), however, the range was wide (0.3 - 88.2 Mb). Several of the regions identified overlapped with loci and genes that have been previously implicated in breast cancer risk, including NBS1, BRCA1 and RAD51L1. CONCLUSIONS: Our analyses have provided several loci of interest to pursue in these high-risk pedigrees and illustrate the utility of the weighted pSGS method and extended pedigrees for gene mapping in complex diseases. A focused sequencing effort across these loci in the sharing individuals is the natural next step to further map the critical underlying susceptibility variants in these regions.

Shared genomic segment analysis: the power to find rare disease variants.

Shared genomic segment (SGS) analysis uses dense single nucleotide polymorphism genotyping in high-risk (HR) pedigrees to identify regions of sharing between cases. Here, we illustrate the power of SGS to identify dominant rare risk variants. Using simulated pedigrees, we consider 12 disease models based on disease prevalence, minor allele frequency and penetrance to represent disease loci that explain 0.2-99.8% of total disease risk. Pedigrees were required to contain ≥ 15 meioses between all cases and to be HR based on significant excess of disease (P < 0.001 or P < 0.00001). Across these scenarios, the power for a single pedigree ranged widely. Nonetheless, fewer than 10 pedigrees were sufficient for excellent power in the majority of models. Power increased with the risk attributable to the disease locus, penetrance and the excess of disease in the pedigree. Sharing allowing for one sporadic case was uniformly more powerful than sharing using all cases. Furthermore, an SGS analysis using a large attenuated familial adenomatous polyposis pedigree identified a 1.96 Mb region containing the known causal APC gene with genome-wide significance. SGS is a powerful method for detecting rare variants and a valuable complement to genome-wide association studies and linkage analysis.

A unique genome-wide association analysis in extended Utah high-risk pedigrees identifies a novel melanoma risk variant on chromosome arm 10q.

Only two genome-wide association (GWA) screens have been published for melanoma (Nat Genet 47:920-925, 2009; Nat Genet 40:838-840, 2008). Using a unique approach, we performed a genome-wide association study in 156 related melanoma cases from 34 high-risk Utah pedigrees. Genome-wide association analysis was performed on nearly 500,000 markers; we compared cases to 2,150 genotypically matched samples from Illumina's iControls database. We performed genome-wide association with EMMAX software, which is designed to account for population structure, including relatedness between cases. Three SNPs exceeded a genome-wide significance threshold of p < 5 × 10(-8) on chromosome arm 10q25.1 (rs17119434, rs17119461, and rs17119490), where the most extreme p value was 7.21 × 10(-12). This study represents a new and unique approach to predisposition gene identification; and it is the first genome-wide association study performed in related cases in high-risk pedigrees. Our approach illustrates an example of using high-risk pedigrees for the identification of new melanoma predisposition variants.

Rare, evolutionarily unlikely missense substitutions in ATM confer increased risk of breast cancer.

The susceptibility gene for ataxia telangiectasia, ATM, is also an intermediate-risk breast-cancer-susceptibility gene. However, the spectrum and frequency distribution of ATM mutations that confer increased risk of breast cancer have been controversial. To assess the contribution of rare variants in this gene to risk of breast cancer, we pooled data from seven published ATM case-control mutation-screening studies, including a total of 1544 breast cancer cases and 1224 controls, with data from our own mutation screening of an additional 987 breast cancer cases and 1021 controls. Using an in silico missense-substitution analysis that provides a ranking of missense substitutions from evolutionarily most likely to least likely, we carried out analyses of protein-truncating variants, splice-junction variants, and rare missense variants. We found marginal evidence that the combination of ATM protein-truncating and splice-junction variants contribute to breast cancer risk. There was stronger evidence that a subset of rare, evolutionarily unlikely missense substitutions confer increased risk. On the basis of subset analyses, we hypothesize that rare missense substitutions falling in and around the FAT, kinase, and FATC domains of the protein may be disproportionately responsible for that risk and that a subset of these may confer higher risk than do protein-truncating variants. We conclude that a comparison between the graded distributions of missense substitutions in cases versus controls can complement analyses of truncating variants and help identify susceptibility genes and that this approach will aid interpretation of the data emerging from new sequencing technologies.