HPV16 E7 Genetic Conservation Is Critical to Carcinogenesis.

Although most cervical human papillomavirus type 16 (HPV16) infections become undetectable within 1-2 years, persistent HPV16 causes half of all cervical cancers. We used a novel HPV whole-genome sequencing technique to evaluate an exceptionally large collection of 5,570 HPV16-infected case-control samples to determine whether viral genetic variation influences risk of cervical precancer and cancer. We observed thousands of unique HPV16 genomes; very few women shared the identical HPV16 sequence, which should stimulate a careful re-evaluation of the clinical implications of HPV mutation rates, transmission, clearance, and persistence. In case-control analyses, HPV16 in the controls had significantly more amino acid changing variants throughout the genome. Strikingly, E7 was devoid of variants in precancers/cancers compared to higher levels in the controls; we confirmed this in cancers from around the world. Strict conservation of the 98 amino acids of E7, which disrupts Rb function, is critical for HPV16 carcinogenesis, presenting a highly specific target for etiologic and therapeutic research.

Insights into the mechanisms and structure of breakage-fusion-bridge cycles in cervical cancer using long-read sequencing.

Cervical cancer is caused by human papillomavirus (HPV) infection, has few approved targeted therapeutics, and is the most common cause of cancer death in low-resource countries. We characterized 19 cervical and four head and neck cancer cell lines using long-read DNA and RNA sequencing and identified the HPV types, HPV integration sites, chromosomal alterations, and cancer driver mutations. Structural variation analysis revealed telomeric deletions associated with DNA inversions resulting from breakage-fusion-bridge (BFB) cycles. BFB is a common mechanism of chromosomal alterations in cancer, and our study applies long-read sequencing to this important chromosomal rearrangement type. Analysis of the inversion sites revealed staggered ends consistent with exonuclease digestion of the DNA after breakage. Some BFB events are complex, involving inter- or intra-chromosomal insertions or rearrangements. None of the BFB breakpoints had telomere sequences added to resolve the dicentric chromosomes, and only one BFB breakpoint showed chromothripsis. Five cell lines have a chromosomal region 11q BFB event, with YAP1-BIRC3-BIRC2 amplification. Indeed, YAP1 amplification is associated with a 10-year-earlier age of diagnosis of cervical cancer and is three times more common in African American women. This suggests that individuals with cervical cancer and YAP1-BIRC3-BIRC2 amplification, especially those of African ancestry, might benefit from targeted therapy. In summary, we uncovered valuable insights into the mechanisms and consequences of BFB cycles in cervical cancer using long-read sequencing.

Targeted long-read sequencing of the Ewing sarcoma 6p25.1 susceptibility locus identifies germline-somatic interactions with EWSR1-FLI1 binding.

Ewing sarcoma (EwS) is a rare bone and soft tissue malignancy driven by chromosomal translocations encoding chimeric transcription factors, such as EWSR1-FLI1, that bind GGAA motifs forming novel enhancers that alter nearby expression. We propose that germline microsatellite variation at the 6p25.1 EwS susceptibility locus could impact downstream gene expression and EwS biology. We performed targeted long-read sequencing of EwS blood DNA to characterize variation and genomic features important for EWSR1-FLI1 binding. We identified 50 microsatellite alleles at 6p25.1 and observed that EwS-affected individuals had longer alleles (>135 bp) with more GGAA repeats. The 6p25.1 GGAA microsatellite showed chromatin features of an EWSR1-FLI1 enhancer and regulated expression of RREB1, a transcription factor associated with RAS/MAPK signaling. RREB1 knockdown reduced proliferation and clonogenic potential and reduced expression of cell cycle and DNA replication genes. Our integrative analysis at 6p25.1 details increased binding of longer GGAA microsatellite alleles with acquired EWSR-FLI1 to promote Ewing sarcomagenesis by RREB1-mediated proliferation.

Sickle cell allele HBB-rs334(T) is associated with decreased risk of childhood Burkitt lymphoma in East Africa.

Burkitt lymphoma (BL) is an aggressive B-cell lymphoma that significantly contributes to childhood cancer burden in sub-Saharan Africa. Plasmodium falciparum, which causes malaria, is geographically associated with BL, but the evidence remains insufficient for causal inference. Inference could be strengthened by demonstrating that mendelian genes known to protect against malaria-such as the sickle cell trait variant, HBB-rs334(T)-also protect against BL. We investigated this hypothesis among 800 BL cases and 3845 controls in four East African countries using genome-scan data to detect polymorphisms in 22 genes known to affect malaria risk. We fit generalized linear mixed models to estimate odds ratios (OR) and 95% confidence intervals (95% CI), controlling for age, sex, country, and ancestry. The ORs of the loci with BL and P. falciparum infection among controls were correlated (Spearman's ρ = 0.37, p = .039). HBB-rs334(T) was associated with lower P. falciparum infection risk among controls (OR = 0.752, 95% CI 0.628-0.9; p = .00189) and BL risk (OR = 0.687, 95% CI 0.533-0.885; p = .0037). ABO-rs8176703(T) was associated with decreased risk of BL (OR = 0.591, 95% CI 0.379-0.992; p = .00271), but not of P. falciparum infection. Our results increase support for the etiological correlation between P. falciparum and BL risk.

Genome-wide Association Study Identifies HLA-DPB1 as a Significant Risk Factor for Severe Aplastic Anemia.

Severe aplastic anemia (SAA) is a rare disorder characterized by hypoplastic bone marrow and progressive pancytopenia. The etiology of acquired SAA is not understood but is likely related to abnormal immune responses and environmental exposures. We conducted a genome-wide association study of individuals with SAA genetically matched to healthy controls in discovery (359 cases, 1,396 controls) and validation sets (175 cases, 1,059 controls). Combined analyses identified linked SNPs in distinct blocks within the major histocompatibility complex on 6p21. The top SNP encodes p.Met76Val in the P4 binding pocket of the HLA class II gene HLA-DPB1 (rs1042151A>G, odds ratio [OR] 1.75, 95% confidence interval [CI] 1.50-2.03, p = 1.94 × 10-13) and was associated with HLA-DP cell surface expression in healthy individuals (p = 2.04 × 10-6). Phylogenetic analyses indicate that Val76 is not monophyletic and likely occurs in conjunction with different HLA-DP binding groove conformations. Imputation of HLA-DPB1 alleles revealed increased risk of SAA associated with Val76-encoding alleles DPB1∗03:01, (OR 1.66, p = 1.52 × 10-7), DPB1∗10:01 (OR 2.12, p = 0.0003), and DPB1∗01:01 (OR 1.60, p = 0.0008). A second SNP near HLA-B, rs28367832G>A, reached genome-wide significance (OR 1.49, 95% CI 1.22-1.78, p = 7.27 × 10-9) in combined analyses; the association remained significant after excluding cases with clonal copy-neutral loss-of-heterozygosity affecting class I HLA genes (8.6% of cases and 0% of controls). SNPs in the HLA class II gene HLA-DPB1 and possibly class I (HLA-B) are associated with SAA. The replacement of Met76 to Val76 in certain HLA-DPB1 alleles might influence risk of SAA through mechanisms involving DP peptide binding specificity, expression, and/or other factors affecting DP function.

In utero exposure to diethylstilbestrol and blood DNA methylation in adult women: Results from a meta-analysis of two cohort studies.

BACKGROUND: Prenatal exposure to diethylstilbestrol (DES) is associated with several adverse health outcomes. Animal studies have shown associations between prenatal DES exposure and DNA methylation. OBJECTIVE: The aim of this study was to explore blood DNA methylation in women exposed and unexposed to DES in utero. METHODS: Sixty women (40 exposed and 20 unexposed) in the National Cancer Institute's Combined DES Cohort Study and 199 women (99 exposed and 100 unexposed women) in the Sister Study Cohort were included in this analysis. Within each study, robust linear regression models were used to assess associations between DES exposure and blood DNA methylation. Study-specific associations were combined using fixed-effect meta-analysis with inverse variance weights. Our analysis focused on CpG sites located within nine candidate genes identified in animal models. We further explored whether in utero DES exposure was associated with age acceleration. RESULTS: Blood DNA methylation levels at 10 CpG sites in six of the nine candidate genes were statistically significantly associated with prenatal DES exposure (P < 0.05) in this meta-analysis. Genes included EGF, EMB, EGFR, WNT11, FOS, and TGFB1, which are related to cell proliferation and differentiation. The most statistically significant CpG site was cg19830739 in gene EGF, and it was associated with lower methylation levels in women prenatally exposed to DES compared with those not exposed (P < 0.0001; false discovery rate<0.05). The association between prenatal DES exposure in utero and age acceleration was not statistically significant (P = 0.07 for meta-analyzed results). CONCLUSIONS: There are few opportunities to investigate the effects of prenatal DES exposure. These findings suggest that in utero DES exposure may be associated with differential blood DNA methylation levels, which could mediate the increased risk of several adverse health outcomes observed in exposed women. Our findings need further evaluation using larger data sets.

The genetic structure and adaptation of Andean highlanders and Amazonians are influenced by the interplay between geography and culture.

Western South America was one of the worldwide cradles of civilization. The well-known Inca Empire was the tip of the iceberg of an evolutionary process that started 11,000 to 14,000 years ago. Genetic data from 18 Peruvian populations reveal the following: 1) The between-population homogenization of the central southern Andes and its differentiation with respect to Amazonian populations of similar latitudes do not extend northward. Instead, longitudinal gene flow between the northern coast of Peru, Andes, and Amazonia accompanied cultural and socioeconomic interactions revealed by archeology. This pattern recapitulates the environmental and cultural differentiation between the fertile north, where altitudes are lower, and the arid south, where the Andes are higher, acting as a genetic barrier between the sharply different environments of the Andes and Amazonia. 2) The genetic homogenization between the populations of the arid Andes is not only due to migrations during the Inca Empire or the subsequent colonial period. It started at least during the earlier expansion of the Wari Empire (600 to 1,000 years before present). 3) This demographic history allowed for cases of positive natural selection in the high and arid Andes vs. the low Amazon tropical forest: in the Andes, a putative enhancer in HAND2-AS1 (heart and neural crest derivatives expressed 2 antisense RNA1, a noncoding gene related to cardiovascular function) and rs269868-C/Ser1067 in DUOX2 (dual oxidase 2, related to thyroid function and innate immunity) genes and, in the Amazon, the gene encoding for the CD45 protein, essential for antigen recognition by T and B lymphocytes in viral-host interaction.

Rare germline variants in PALB2 and BRCA2 in familial and sporadic chordoma.

Chordoma is a rare bone tumor with genetic risk factors largely unknown. We conducted a whole-exome sequencing (WES) analysis of germline DNA from 19 familial chordoma cases in five pedigrees and 137 sporadic chordoma patients and identified 17 rare germline variants in PALB2 and BRCA2, whose products play essential roles in homologous recombination (HR) and tumor suppression. One PALB2 variant showed disease cosegregation in a family with four affected people or obligate gene carrier. Chordoma cases had a significantly increased burden of rare variants in both genes when compared to population-based controls. Four of the six PALB2 variants identified from chordoma patients modestly affected HR function and three of the 11 BRCA2 variants caused loss of function in experimental assays. These results, together with previous reports of abnormal morphology and Brachyury expression of the notochord in Palb2 knockout mouse embryos and genomic signatures associated with HR defect and HR gene mutations in advanced chordomas, suggest that germline mutations in PALB2 and BRCA2 may increase chordoma susceptibility. Our data shed light on the etiology of chordoma and support the previous finding that PARP-1 inhibitors may be a potential therapy for some chordoma patients.

Genetics and geography of leukocyte telomere length in sub-Saharan Africans.

Leukocyte telomere length (LTL) might be causal in cardiovascular disease and major cancers. To elucidate the roles of genetics and geography in LTL variability across humans, we compared LTL measured in 1295 sub-Saharan Africans (SSAs) with 559 African-Americans (AAms) and 2464 European-Americans (EAms). LTL differed significantly across SSAs (P = 0.003), with the San from Botswana (with the oldest genomic ancestry) having the longest LTL and populations from Ethiopia having the shortest LTL. SSAs had significantly longer LTL than AAms [P = 6.5(e-16)] whose LTL was significantly longer than EAms [P = 2.5(e-7)]. Genetic variation in SSAs explained 52% of LTL variance versus 27% in AAms and 34% in EAms. Adjustment for genetic variation removed the LTL differences among SSAs. LTL genetic variation among SSAs, with the longest LTL in the San, supports the hypothesis that longer LTL was ancestral in humans. Identifying factors driving LTL variation in Africa may have important ramifications for LTL-associated diseases.

Genetic signatures of gene flow and malaria-driven natural selection in sub-Saharan populations of the "endemic Burkitt Lymphoma belt".

Populations in sub-Saharan Africa have historically been exposed to intense selection from chronic infection with falciparum malaria. Interestingly, populations with the highest malaria intensity can be identified by the increased occurrence of endemic Burkitt Lymphoma (eBL), a pediatric cancer that affects populations with intense malaria exposure, in the so called "eBL belt" in sub-Saharan Africa. However, the effects of intense malaria exposure and sub-Saharan populations' genetic histories remain poorly explored. To determine if historical migrations and intense malaria exposure have shaped the genetic composition of the eBL belt populations, we genotyped ~4.3 million SNPs in 1,708 individuals from Ghana and Northern Uganda, located on opposite sides of eBL belt and with ≥ 7 months/year of intense malaria exposure and published evidence of high incidence of BL. Among 35 Ghanaian tribes, we showed a predominantly West-Central African ancestry and genomic footprints of gene flow from Gambian and East African populations. In Uganda, the North West population showed a predominantly Nilotic ancestry, and the North Central population was a mixture of Nilotic and Southern Bantu ancestry, while the Southwest Ugandan population showed a predominant Southern Bantu ancestry. Our results support the hypothesis of diverse ancestral origins of the Ugandan, Kenyan and Tanzanian Great Lakes African populations, reflecting a confluence of Nilotic, Cushitic and Bantu migrations in the last 3000 years. Natural selection analyses suggest, for the first time, a strong positive selection signal in the ATP2B4 gene (rs10900588) in Northern Ugandan populations. These findings provide important baseline genomic data to facilitate disease association studies, including of eBL, in eBL belt populations.

HPV+ oropharyngeal squamous cell carcinomas from patients with two tumors display synchrony of viral genomes yet discordant mutational profiles and signatures.

Human papillomavirus (HPV) positive oropharyngeal squamous cell carcinoma (HPV + OPSCC) is increasing in prevalence in the USA, as are cases of patients with multiple HPV + OPSCCs (mHPV + OPSCC). mHPV + OPSCCs present a unique opportunity to examine HPV + OPSCC mutation acquisition and evolution. We performed sequencing of the viral genome, somatic exome and somatic transcriptome from 8 patients each with 2 spatially distinct HPV + OPSCCs, and 37 'traditional' HPV + OPSCCs to first address if paired tumors are caused by the same viral isolate and next, if acquired alterations, and the underlying processes driving mutagenesis, are shared within pairs. All tumor pairs contained viral genomes from the same HPV type 16 sublineage and differed by 0-2 clonal single nucleotide polymorphisms (SNPs), suggesting infection with the same viral isolate. Despite this, there was significant discordance in expression profiles, mutational burden and mutational profiles between tumors in a pair, with only two pairs sharing any overlapping mutations (3/3343 variants). Within tumor pairs there was a striking discrepancy of mutational signatures, exemplified by no paired tumors sharing high APOBEC mutational burden. Here, leveraging mHPV + OPSCCs as a model system to study mutation acquisition in virally mediated tumors, in which the germline, environmental exposures, immune surveillance and tissue/organ type were internally controlled, we demonstrate that despite infection by the same viral isolate, paired mHPV + OPSCCs develop drastically different somatic alterations and even more strikingly, appear to be driven by disparate underlying mutational processes. Thus, despite a common starting point, HPV + OPSCCs evolve through variable mutational processes with resultant stochastic mutational profiles.

Origins, Admixture Dynamics, and Homogenization of the African Gene Pool in the Americas.

The Transatlantic Slave Trade transported more than 9 million Africans to the Americas between the early 16th and the mid-19th centuries. We performed a genome-wide analysis using 6,267 individuals from 25 populations to infer how different African groups contributed to North-, South-American, and Caribbean populations, in the context of geographic and geopolitical factors, and compared genetic data with demographic history records of the Transatlantic Slave Trade. We observed that West-Central Africa and Western Africa-associated ancestry clusters are more prevalent in northern latitudes of the Americas, whereas the South/East Africa-associated ancestry cluster is more prevalent in southern latitudes of the Americas. This pattern results from geographic and geopolitical factors leading to population differentiation. However, there is a substantial decrease in the between-population differentiation of the African gene pool within the Americas, when compared with the regions of origin from Africa, underscoring the importance of historical factors favoring admixture between individuals with different African origins in the New World. This between-population homogenization in the Americas is consistent with the excess of West-Central Africa ancestry (the most prevalent in the Americas) in the United States and Southeast-Brazil, with respect to historical-demography expectations. We also inferred that in most of the Americas, intercontinental admixture intensification occurred between 1750 and 1850, which correlates strongly with the peak of arrivals from Africa. This study contributes with a population genetics perspective to the ongoing social, cultural, and political debate regarding ancestry, admixture, and the mestizaje process in the Americas.

Admixture/fine-mapping in Brazilians reveals a West African associated potential regulatory variant (rs114066381) with a strong female-specific effect on body mass and fat mass indexes.

BACKGROUND/OBJECTIVES: Admixed populations are a resource to study the global genetic architecture of complex phenotypes, which is critical, considering that non-European populations are severely underrepresented in genomic studies. Here, we study the genetic architecture of BMI in children, young adults, and elderly individuals from the admixed population of Brazil. SUBJECTS/METHODS: Leveraging admixture in Brazilians, whose chromosomes are mosaics of fragments of Native American, European, and African origins, we used genome-wide data to perform admixture mapping/fine-mapping of body mass index (BMI) in three Brazilian population-based cohorts from Northeast (Salvador), Southeast (Bambuí), and South (Pelotas). RESULTS: We found significant associations with African-associated alleles in children from Salvador (PALD1 and ZMIZ1 genes), and in young adults from Pelotas (NOD2 and MTUS2 genes). More importantly, in Pelotas, rs114066381, mapped in a potential regulatory region, is significantly associated only in females (p = 2.76e-06). This variant is rare in Europeans but with frequencies of ~3% in West Africa and has a strong female-specific effect (95% CI: 2.32-5.65 kg/m2 per each A allele). We confirmed this sex-specific association and replicated its strong effect for an adjusted fat mass index in the same Pelotas cohort, and for BMI in another Brazilian cohort from São Paulo (Southeast Brazil). A meta-analysis confirmed the significant association. Remarkably, we observed that while the frequency of rs114066381-A allele ranges from 0.8 to 2.1% in the studied populations, it attains ~9% among women with morbid obesity from Pelotas, São Paulo, and Bambuí. The effect size of rs114066381 is at least five times higher than the FTO SNPs rs9939609 and rs1558902, already emblematic for their high effects. CONCLUSIONS: We identified six candidate SNPs associated with BMI. rs114066381 stands out for its high effect that was replicated and its high frequency in women with morbid obesity. We demonstrate how admixed populations are a source of new relevant phenotype-associated genetic variants.

Association of HPV35 with cervical carcinogenesis among women of African ancestry: Evidence of viral-host interaction with implications for disease intervention.

HPV35 has been found in only ∼2% of invasive cervical cancers (ICC) worldwide but up to 10% in Sub-Saharan Africa, warranting further investigation and consideration of impact on preventive strategies. We studied HPV35 and ethnicity, in relation to the known steps in cervical carcinogenesis, using multiple large epidemiologic studies in the U.S. and internationally. Combining five U.S. studies, we measured HPV35 positivity and, in Northern California, observed HPV35 type-specific population prevalence and estimated 5-year risk of developing precancer when HPV35-positive. HPV35 genetic variation was examined for differences in carcinogenicity in 1053 HPV35+ cervical specimens from a U.S. cohort and an international collection. African-American women had more HPV35 (12.1% vs 5.1%, P < .001) and more HPV35-associated precancers (7.4% vs 2.1%, P < .001) compared to other ethnicities. Precancer risks after HPV35 infection did not vary by ethnicity (global P = .52). The HPV35 A2 sublineage showed an increased association with precancer/cancer in African-Americans (OR = 5.6 vs A1, 95% CI = 1.3-24.8) and A2 was more prevalent among ICC in Africa than other world regions (41.9% vs 10.4%, P < .01). Our analyses support a strong link between HPV35 and cervical carcinogenesis in women of African ancestry. Current HPV vaccines cover the majority of cervical precancer/cancer across all ethnic groups; additional analyses are required to determine whether the addition of HPV35 to the already highly effective nine-valent HPV vaccine would provide better protection for women in Africa or of African ancestry.

Field Study of the Possible Effect of Parental Irradiation on the Germline of Children Born to Cleanup Workers and Evacuees of the Chornobyl Nuclear Accident.

Although transgenerational effects of exposure to ionizing radiation have long been a concern, human research to date has been confined to studies of disease phenotypes in groups exposed to high doses and high dose rates, such as the Japanese atomic bomb survivors. Transgenerational effects of parental irradiation can be addressed using powerful new genomic technologies. In collaboration with the Ukrainian National Research Center for Radiation Medicine, the US National Cancer Institute, in 2014-2018, initiated a genomic alterations study among children born in selected regions of Ukraine to cleanup workers and/or evacuees exposed to low-dose-rate radiation after the 1986 Chornobyl (Chernobyl) nuclear accident. To investigate whether parental radiation exposure is associated with germline mutations and genomic alterations in the offspring, we are collecting biospecimens from father-mother-offspring constellations to study de novo mutations, minisatellite mutations, copy-number changes, structural variants, genomic insertions and deletions, methylation profiles, and telomere length. Genomic alterations are being examined in relation to parental gonadal dose, reconstructed using questionnaire and measurement data. Subjects are being recruited in exposure categories that will allow examination of parental origin, duration, and timing of exposure in relation to conception. Here we describe the study methodology and recruitment results and provide descriptive information on the first 150 families (mother-father-child(ren)) enrolled.

Evaluation of TypeSeq, a Novel High-Throughput, Low-Cost, Next-Generation Sequencing-Based Assay for Detection of 51 Human Papillomavirus Genotypes.

BACKGROUND: Human papillomaviruses (HPV) cause over 500 000 cervical cancers each year, most of which occur in low-resource settings. Human papillomavirus genotyping is important to study natural history and vaccine efficacy. We evaluated TypeSeq, a novel, next-generation, sequencing-based assay that detects 51 HPV genotypes, in 2 large international epidemiologic studies. METHODS: TypeSeq was evaluated in 2804 cervical specimens from the Study to Understand Cervical Cancer Endpoints and Early Determinants (SUCCEED) and in 2357 specimens from the Costa Rica Vaccine Trial (CVT). Positive agreement and risks of precancer for individual genotypes were calculated for TypeSeq in comparison to Linear Array (SUCCEED). In CVT, positive agreement and vaccine efficacy were calculated for TypeSeq and SPF10-LiPA. RESULTS: We observed high overall and positive agreement for most genotypes between TypeSeq and Linear Array in SUCCEED and SPF10-LiPA in CVT. There was no significant difference in risk of precancer between TypeSeq and Linear Array in SUCCEED or in estimates of vaccine efficacy between TypeSeq and SPF10-LiPA in CVT. CONCLUSIONS: The agreement of TypeSeq with Linear Array and SPF10-LiPA, 2 well established standards for HPV genotyping, demonstrates its high accuracy. TypeSeq provides high-throughput, affordable HPV genotyping for world-wide studies of cervical precancer risk and of HPV vaccine efficacy.

Viral coinfection analysis using a MinHash toolkit.

BACKGROUND: Human papillomavirus (HPV) is a common sexually transmitted infection associated with cervical cancer that frequently occurs as a coinfection of types and subtypes. Highly similar sublineages that show over 100-fold differences in cancer risk are not distinguishable in coinfections with current typing methods. RESULTS: We describe an efficient set of computational tools, rkmh, for analyzing complex mixed infections of related viruses based on sequence data. rkmh makes extensive use of MinHash similarity measures, and includes utilities for removing host DNA and classifying reads by type, lineage, and sublineage. We show that rkmh is capable of assigning reads to their HPV type as well as HPV16 lineage and sublineages. CONCLUSIONS: Accurate read classification enables estimates of percent composition when there are multiple infecting lineages or sublineages. While we demonstrate rkmh for HPV with multiple sequencing technologies, it is also applicable to other mixtures of related sequences.

The influence of obesity-related factors in the etiology of renal cell carcinoma-A mendelian randomization study.

BACKGROUND: Several obesity-related factors have been associated with renal cell carcinoma (RCC), but it is unclear which individual factors directly influence risk. We addressed this question using genetic markers as proxies for putative risk factors and evaluated their relation to RCC risk in a mendelian randomization (MR) framework. This methodology limits bias due to confounding and is not affected by reverse causation. METHODS AND FINDINGS: Genetic markers associated with obesity measures, blood pressure, lipids, type 2 diabetes, insulin, and glucose were initially identified as instrumental variables, and their association with RCC risk was subsequently evaluated in a genome-wide association study (GWAS) of 10,784 RCC patients and 20,406 control participants in a 2-sample MR framework. The effect on RCC risk was estimated by calculating odds ratios (ORSD) for a standard deviation (SD) increment in each risk factor. The MR analysis indicated that higher body mass index increases the risk of RCC (ORSD: 1.56, 95% confidence interval [CI] 1.44-1.70), with comparable results for waist-to-hip ratio (ORSD: 1.63, 95% CI 1.40-1.90) and body fat percentage (ORSD: 1.66, 95% CI 1.44-1.90). This analysis further indicated that higher fasting insulin (ORSD: 1.82, 95% CI 1.30-2.55) and diastolic blood pressure (DBP; ORSD: 1.28, 95% CI 1.11-1.47), but not systolic blood pressure (ORSD: 0.98, 95% CI 0.84-1.14), increase the risk for RCC. No association with RCC risk was seen for lipids, overall type 2 diabetes, or fasting glucose. CONCLUSIONS: This study provides novel evidence for an etiological role of insulin in RCC, as well as confirmatory evidence that obesity and DBP influence RCC risk.

Characterization of breakpoint regions of large structural autosomal mosaic events.

Recent studies have reported a higher than anticipated frequency of large clonal autosomal mosaic events >2 Mb in size in the aging population. Mosaic events are detected from analyses of intensity parameters of linear stretches with deviations in heterozygous probes of single nucleotide polymorphism microarrays. The non-random distribution of detected mosaic events throughout the genome suggests common mechanisms could influence the formation of mosaic events. Here we use publicly available data tracks from the University of California Santa Cruz Genome Browser to investigate the genomic characteristics of the regions at the terminal ends of two frequent types of large structural mosaic events: telomeric neutral events and interstitial losses. We observed breakpoints are more likely to occur in regions enriched for open chromatin, increased gene density, elevated meiotic recombination rates and in the proximity of repetitive elements. These observations suggest that detected mosaic event breakpoints are preferentially recovered in genomic regions that are observed to be active and thus more accessible to environmental exposures and events related to gene transcription. We propose that errors in DNA repair pathways, such as non-homologous end joining and homologous recombination, may be important cellular mechanisms that lead to the formation of large structural mosaic events such as interstitial losses and copy neutral events that include telomeres. Further studies using next generation sequencing technologies should be instrumental in mapping the specific junctions of mosaic events to the nucleotide and provide insights into the molecular mechanisms responsible for clonal somatic structural events.

Rare germline variants in known melanoma susceptibility genes in familial melanoma.

Known high-risk cutaneous malignant melanoma (CMM) genes account for melanoma risk in <40% of melanoma-prone families, suggesting the existence of additional high-risk genes or perhaps a polygenic mechanism involving multiple genetic modifiers. The goal of this study was to systematically characterize rare germline variants in 42 established melanoma genes among 144 CMM patients in 76 American CMM families without known mutations using data from whole-exome sequencing. We identified 68 rare (<0.1% in public and in-house control datasets) nonsynonymous variants in 25 genes. We technically validated all loss-of-function, inframe insertion/deletion, and missense variants predicted as deleterious, and followed them up in 1, 559 population-based CMM cases and 1, 633 controls. Several of these variants showed disease co-segregation within families. Of particular interest, a stopgain variant in TYR was present in five of six CMM cases/obligate gene carriers in one family and a single population-based CMM case. A start gain variant in the 5'UTR region of PLA2G6 and a missense variant in ATM were each seen in all three affected people in a single family, respectively. Results from rare variant burden tests showed that familial and population-based CMM patients tended to have higher frequencies of rare germline variants in albinism genes such as TYR, TYRP1, and OCA2 (P < 0.05). Our results suggest that rare nonsynonymous variants in low- or intermediate-risk CMM genes may influence familial CMM predisposition, warranting further investigation of both common and rare variants in genes affecting functionally important pathways (such as melanogenesis) in melanoma risk assessment.