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Background Environmental and genetic factors play an important role in the etiology of breast cancer. Several small blood-based DNA methylation studies have reported risk associations with methylation at individual CpGs and average methylation levels; however, these findings require validation in larger prospective cohort studies. To investigate the role of blood DNA methylation on breast cancer risk, we conducted a meta-analysis of four prospective cohort studies, including a total of 1663 incident cases and 1885 controls, the largest study of blood DNA methylation and breast cancer risk to date. Methods We assessed associations with methylation at 365,145 CpGs present in the HumanMethylation450 (HM450K) Beadchip, after excluding CpGs that did not pass quality controls in all studies. Each of the four cohorts estimated odds ratios (ORs) and 95% confidence intervals (CI) for the association between each individual CpG and breast cancer risk. In addition, each study assessed the association between average methylation measures and breast cancer risk, adjusted and unadjusted for cell-type composition. Study-specific ORs were combined using fixed-effect meta-analysis with inverse variance weights. Stratified analyses were conducted by age at diagnosis (< 50, ≥ 50), estrogen receptor (ER) status (+/−), and time since blood collection (< 5, 5–10, > 10 years). The false discovery rate ( q value) was used to account for multiple testing. Results The average age at blood draw ranged from 52.2 to 62.2 years across the four cohorts. Median follow-up time ranged from 6.6 to 8.4 years. The methylation measured at individual CpGs was not associated with breast cancer risk ( q value > 0.59). In addition, higher average methylation level was not associated with risk of breast cancer (OR = 0.94, 95% CI = 0.85, 1.05; P  = 0.26; P for study heterogeneity = 0.86). We found no evidence of modification of this association by age at diagnosis ( P  = 0.17), ER status ( P  = 0.88), time since blood collection ( P  = 0.98), or CpG location ( P  = 0.98). Conclusions Our data indicate that DNA methylation measured in the blood prior to breast cancer diagnosis in predominantly postmenopausal women is unlikely to be associated with substantial breast cancer risk on the HM450K array. Larger studies or with greater methylation coverage are needed to determine if associations exist between blood DNA methylation and breast cancer risk.

Chronic granulomatous disease is caused by mutations in any of the 6 components of the phagocytic NADPH oxidase complex including gp91 , p47 , p22 , p40 , p67 , or EROS. Functional assays include reactive oxygen species (ROS) production, flow cytometry, and immunoblotting for NADPH proteins. The advent of high-throughput sequencing allows genetic diagnosis for all components except (p47 ) due to two, nearly identical, pseudogenes ( , ). The majority of NCF1-CGD patients carry a 2-base deletion caused by crossover between and or . Currently, NCF1 deficiency is diagnosed functionally: a characteristic DHR with low levels of residual ROS, loss of p47 on immunoblot, or digital droplet PCR or Gene-scan to enumerate intact (GTGT) or deleted (ΔGT). While this provides patients a clinical CGD diagnosis, for the 20% of NCF1-CGD patients with a non-ΔGT mutation a definitive genetic diagnosis is still lacking. We developed a bioinformatic method using existing short or long-read sequencing data from 48 NCF1-CGD patients or carriers. We identified both ΔGT and non-ΔGT gene mutations. Additionally, we confirm that the presence of ΔGT in is due to pseudogene copy into the locus. We compare sequence from NCF1-CGD patients to cohorts of non-NCF1-CGD and healthy controls (1000Genomes), demonstrating pseudogene replacement of in NCF1-CGD as well as the reciprocal replacement of or by in some healthy controls. With this method, reanalysis of existing sequence data may provide genetic diagnosis to NCF1-CGD patients. This technique may be modified for other diagnostically relevant pseudogenes.

The domestic dog is a robust model for studying the genetics of complex disease susceptibility. The strategies used to develop and propagate modern breeds have resulted in an elevated risk for specific diseases in particular breeds. One example is that of Standard Poodles (STPOs), who have increased risk for squamous cell carcinoma of the digit (SCCD), a locally aggressive cancer that causes lytic bone lesions, sometimes with multiple toe recurrence. However, only STPOs of dark coat color are at high risk; light colored STPOs are almost entirely unaffected, suggesting that interactions between multiple pathways are necessary for oncogenesis. We performed a genome-wide association study (GWAS) on STPOs, comparing 31 SCCD cases to 34 unrelated black STPO controls. The peak SNP on canine chromosome 15 was statistically significant at the genome-wide level (P(raw) = 1.60 × 10(-7); P(genome) = 0.0066). Additional mapping resolved the region to the KIT Ligand (KITLG) locus. Comparison of STPO cases to other at-risk breeds narrowed the locus to a 144.9-Kb region. Haplotype mapping among 84 STPO cases identified a minimal region of 28.3 Kb. A copy number variant (CNV) containing predicted enhancer elements was found to be strongly associated with SCCD in STPOs (P = 1.72 × 10(-8)). Light colored STPOs carry the CNV risk alleles at the same frequency as black STPOs, but are not susceptible to SCCD. A GWAS comparing 24 black and 24 light colored STPOs highlighted only the MC1R locus as significantly different between the two datasets, suggesting that a compensatory mutation within the MC1R locus likely protects light colored STPOs from disease. Our findings highlight a role for KITLG in SCCD susceptibility, as well as demonstrate that interactions between the KITLG and MC1R loci are potentially required for SCCD oncogenesis. These findings highlight how studies of breed-limited diseases are useful for disentangling multigene disorders.

Background The recommended genomic DNA input requirements for whole genome single nucleotide polymorphism microarrays can limit the scope of molecular epidemiological studies. We performed a large-scale evaluation of whole genome amplified DNA as input into high-density, whole-genome Illumina® Infinium® SNP microarray. Results Overall, 6622 DNA samples from 5970 individuals were obtained from three distinct biospecimen sources and genotyped using gDNA and/or wgaDNA inputs. When genotypes from the same individual were compared with standard, native gDNA input amount, we observed 99.94% mean concordance with wgaDNA input. Conclusions Our results demonstrate that carefully conducted studies with wgaDNA inputs can yield high-quality genotyping results. These findings should enable investigators to consider expansion of ongoing studies using high-density SNP microarrays, currently challenged by small amounts of available DNA.

Background Few studies have examined epigenetic age acceleration (AA), the difference between DNA methylation (DNAm) predicted age and chronological age, in relation to somatic genomic features in paired cancer and normal tissue, with less work done in non-European populations. In this study, we aimed to examine DNAm age and its associations with breast cancer risk factors, subtypes, somatic genomic profiles including mutation and copy number alterations and other aging markers in breast tissue of Chinese breast cancer (BC) patients from Hong Kong. Methods We performed genome-wide DNA methylation profiling of 196 tumor and 188 paired adjacent normal tissue collected from Chinese BC patients in Hong Kong (HKBC) using Illumina MethylationEPIC array. The DNAm age was calculated using Horvath’s pan-tissue clock model. Somatic genomic features were based on data from RNA sequencing (RNASeq), whole-exome sequencing (WES), and whole-genome sequencing (WGS). Pearson’s correlation ( r ), Kruskal–Wallis test, and regression models were used to estimate associations of DNAm AA with somatic features and breast cancer risk factors. Results DNAm age showed a stronger correlation with chronological age in normal (Pearson r  = 0.78, P  < 2.2e−16) than in tumor tissue (Pearson r  = 0.31, P  = 7.8e−06). Although overall DNAm age or AA did not vary significantly by tissue within the same individual, luminal A tumors exhibited increased DNAm AA ( P  = 0.004) while HER2-enriched/basal-like tumors exhibited markedly lower DNAm AA ( P  = < .0001) compared with paired normal tissue. Consistent with the subtype association, tumor DNAm AA was positively correlated with ESR1 (Pearson r  = 0.39, P  = 6.3e−06) and PGR (Pearson r  = 0.36, P  = 2.4e−05) gene expression. In line with this, we found that increasing DNAm AA was associated with higher body mass index ( P  = 0.039) and earlier age at menarche ( P  = 0.035), factors that are related to cumulative exposure to estrogen. In contrast, variables indicating extensive genomic instability, such as TP53 somatic mutations, high tumor mutation/copy number alteration burden, and homologous repair deficiency were associated with lower DNAm AA. Conclusions Our findings provide additional insights into the complexity of breast tissue aging that is associated with the interaction of hormonal, genomic, and epigenetic mechanisms in an East Asian population.

The domestic dog exhibits greater diversity in body size than any other terrestrial vertebrate. We used a strategy that exploits the breed structure of dogs to investigate the genetic basis of size. First, through a genome-wide scan, we identified a major quantitative trait locus (QTL) on chromosome 15 influencing size variation within a single breed. Second, we examined genetic variation in the 15-megabase interval surrounding the QTL in small and giant breeds and found marked evidence for a selective sweep spanning a single gene (IGF1), encoding insulin-like growth factor 1. A single IGF1 single-nucleotide polymorphism haplotype is common to all small breeds and nearly absent from giant breeds, suggesting that the same causal sequence variant is a major contributor to body size in all small dogs.

Background Germline mutations in the BRCA2 gene have been suggested to account for about 5% of familial prostate cancer; mutations have been reported in 2% of early onset (i.e., ≤ 55 years) prostate cancer cases and a segregating founder mutation has been identified in Iceland (999del5). However, the role of BRCA2 in high risk prostate cancer pedigrees remains unclear. Findings We examined the potential involvement of BRCA2 in a set offive high-risk prostate cancer pedigrees in which all prostate cases were no more distantly related than two meioses from another case, and the resulting cluster contained at least four prostate cancer cases. We selected these five pedigrees from a larger dataset of 59 high-risk prostate cancer pedigrees analyzed in a genome-wide linkage screen. Selected pedigrees showed at least nominal linkage evidence to the BRCA2 region on chromosome 13q. We mutation screened all coding regions and intron/exon boundaries of the BRCA2 gene in the youngest prostate cancer case who carried the linked 13q segregating haplotype, as well as in a distantly related haplotype carrier to confirm any segregation. We observed no known protein truncating BRCA2 deleterious mutations. We identified one non-segregating BRCA2 variant of uncertain significance, one non-segregating intronic variant not previously reported, and a number of polymorphisms. Conclusion In this set of high-risk prostate cancer pedigrees with at least nominal linkage evidence to BRCA2 , we saw no evidence for segregating BRCA2 protein truncating mutations in heritable prostate cancer.

The domestic dog is a robust model for studying the genetics of complex disease susceptibility. The strategies used to develop and propagate modern breeds have resulted in an elevated risk for specific diseases in particular breeds. One example is that of Standard Poodles (STPOs), who have increased risk for squamous cell carcinoma of the digit (SCCD), a locally aggressive cancer that causes lytic bone lesions, sometimes with multiple toe recurrence. However, only STPOs of dark coat color are at high risk; light colored STPOs are almost entirely unaffected, suggesting that interactions between multiple pathways are necessary for oncogenesis. We performed a genome-wide association study (GWAS) on STPOs, comparing 31 SCCD cases to 34 unrelated black STPO controls. The peak SNP on canine chromosome 15 was statistically significant at the genome-wide level (Praw = 1.60×10-7; Pgenome = 0.0066). Additional mapping resolved the region to the KIT Ligand (KITLG) locus. Comparison of STPO cases to other at-risk breeds narrowed the locus to a 144.9-Kb region. Haplotype mapping among 84 STPO cases identified a minimal region of 28.3 Kb. A copy number variant (CNV) containing predicted enhancer elements was found to be strongly associated with SCCD in STPOs (P = 1.72×10-8). Light colored STPOs carry the CNV risk alleles at the same frequency as black STPOs, but are not susceptible to SCCD. A GWAS comparing 24 black and 24 light colored STPOs highlighted only the MC1R locus as significantly different between the two datasets, suggesting that a compensatory mutation within the MC1R locus likely protects light colored STPOs from disease. Our findings highlight a role for KITLG in SCCD susceptibility, as well as demonstrate that interactions between the KITLG and MC1R loci are potentially required for SCCD oncogenesis. These findings highlight how studies of breed-limited diseases are useful for disentangling multigene disorders.

The fibroblast growth factor receptor 4 ( FGFR4 ) is thought to be involved in many critical cellular processes and has been associated with prostate cancer risk. Four single nucleotide polymorphisms (SNPs) within or near FGFR4 were analyzed in a population-based study of 1458 prostate cancer patients and 1352 age-matched controls. We found no evidence to suggest that any of the FGFR4 SNP genotypes were associated with prostate cancer risk or with disease aggressiveness, Gleason score or stage. A weak association was seen between rs351855 and prostate cancer-specific mortality. Subset analysis of cases that had undergone radical prostatectomy revealed an association between rs351855 and prostate cancer risk. Although our results confirm an association between FGFR4 and prostate cancer risk in radical prostatectomy cases, they suggest that the role of FGFR4 in disease risk and outcomes at a population-based level appears to be minor.

Mammalian X and Y chromosomes share a common evolutionary origin and retain regions of high sequence similarity. This sequence homology can cause the mismapping of short sequencing reads derived from the sex chromosomes and affect variant calling and other downstream analyses. Understanding and correcting this problem is critical for medical genomics and population genomic inference. Here, we characterize how sequence homology can affect analyses on the sex chromosomes and present XYalign, a new tool that: (1) facilitates the inference of sex chromosome complement from next-generation sequencing data; (2) corrects erroneous read mapping on the sex chromosomes; and (3) tabulates and visualizes important metrics for quality control such as mapping quality, sequencing depth, and allele balance. We show how these metrics can be used to identify XX and XY individuals across diverse sequencing experiments, including low and high coverage whole genome sequencing, and exome sequencing. We also show that XYalign corrects mismapped reads on the sex chromosomes, resulting in more accurate variant calling. Finally, we discuss how the flexibility of the XYalign framework can be leveraged for other use cases including the identification of aneuploidy on the autosomes. XYalign is available open source under the GNU General Public License (version 3). Footnotes * Minor updates to the text. Updated supplemental archive.