It has been shown that, for women aged 50 years or older, the discriminatory accuracy of the Breast Cancer Risk Prediction Tool (BCRAT) can be modestly improved by the inclusion of information on common single nucleotide polymorphisms (SNPs) that are associated with increased breast cancer risk. We aimed to determine whether a similar improvement is seen for earlier onset disease. We used the Australian Breast Cancer Family Registry to study a population-based sample of 962 cases aged 35 to 59 years and 463 controls frequency matched for age and for whom genotyping data was available. Overall, the inclusion of data on seven SNPs improved the area under the receiver operating characteristic curve (AUC) from 0.58 (95% confidence interval [CI]=0.55–0.61) for BCRAT alone to 0.61 (95% CI=0.58–0.64) for BCRAT and SNP data combined (p<0.001). For women aged 35 to 39 years at interview, the corresponding improvement in AUC was from 0.61 (95% CI=0.56–0.66) to 0.65 (95% CI=0.60–0.70; p=0.03), while for women aged 40 to 49 years at diagnosis, the AUC improved from 0.61 (95% CI=0.55–0.66) to 0.63 (95% CI=0.57–0.69; p=0.04). Using previously used classifications of low, intermediate and high risk, 2.1% of cases and none of the controls aged 35 to 39 years, and 10.9% of cases and 4.0% of controls aged 40 to 49 years were classified into a higher risk group. Including information on seven SNPs associated with breast cancer risk improves the discriminatory accuracy of BCRAT for women aged 35 to 39 years and 40 to 49 years. Given the low absolute risk for women in these age groups, only a small proportion are reclassified into a higher category for predicted 5-year risk of breast cancer.

VTRNA2-1 is a metastable epiallele with accumulating evidence that methylation at this region is heritable, modifiable and associated with disease including risk and progression of cancer. This study investigated the influence of genetic variation and other factors such as age and adult lifestyle on blood DNA methylation in this region. We first sequenced the VTRNA2-1 gene region in multiple-case breast cancer families in which VTRNA2-1 methylation was identified as heritable and associated with breast cancer risk. Methylation quantitative trait loci (mQTL) were investigated using a prospective cohort study (4500 participants with genotyping and methylation data). The cis-mQTL analysis (334 variants ± 50 kb of the most heritable CpG site) identified 43 variants associated with VTRNA2-1 methylation (p < 1.5 × 10−4); however, these explained little of the methylation variation (R2 < 0.5% for each of these variants). No genetic variants elsewhere in the genome were found to strongly influence VTRNA2-1 methylation. SNP-based heritability estimates were consistent with the mQTL findings (h2 = 0, 95%CI: −0.14 to 0.14). We found no evidence that age, sex, country of birth, smoking, body mass index, alcohol consumption or diet influenced blood DNA methylation at VTRNA2-1. Genetic factors and adult lifestyle play a minimal role in explaining methylation variability at the heritable VTRNA2-1 cluster.

Because of its geographic proximity to the major drug production centres, there is easy access to narcotic drugs in the Islamic Republic of Iran despite efforts by governmental and nongovernmental organizations. Using a structured questionnaire as a basis for conversation, local health workers interviewed 310 residents of a rural area in Babol province about opium use. The self-reported rate of opium use, adjusted due to a bias in the sex ratio of the sample, was 8.9%. All the 42 opium users reported opium use at least 2-3 times per week in the previous 3 months. Opium was smoked by 95.2% and taken orally by 4.8%; there was no injecting use. There was no reported use of other substances, including alcohol. There was a statistically significant relationship between opium use and male sex, unemployment and cigarette smoking. ‫اإلسالمية‬ ‫إيران‬ ‫مجهورية‬ ‫من‬ ‫الريفية‬ ‫املناطق‬ ‫يف‬ ‫األفيون‬ ‫تعاطي‬

BackgroundMassively parallel sequencing (MPS) has revolutionised biomedical research and offers enormous capacity for clinical application. We previously reported Hi-Plex, a streamlined highly-multiplexed PCR-MPS approach, allowing a given library to be sequenced with both the Ion Torrent and TruSeq chemistries. Comparable sequencing efficiency was achieved using material derived from lymphoblastoid cell lines and formalin-fixed paraffin-embedded tumour.MethodsHere, we report high-throughput application of Hi-Plex by performing blinded mutation screening of the coding regions of the breast cancer susceptibility gene PALB2 on a set of 95 blood-derived DNA samples that had previously been screened using Sanger sequencing and high-resolution melting curve analysis (n = 90), or genotyped by Taqman probe-based assays (n = 5). Hi-Plex libraries were prepared simultaneously using relatively inexpensive, readily available reagents in a simple half-day protocol followed by MPS on a single MiSeq run.ResultsWe observed that 99.93% of amplicons were represented at ≥10X coverage. All 56 previously identified variant calls were detected and no false positive calls were assigned. Four additional variant calls were made and confirmed upon re-analysis of previous data or subsequent Sanger sequencing.ConclusionsThese results support Hi-Plex as a powerful approach for rapid, cost-effective and accurate high-throughput mutation screening. They further demonstrate that Hi-Plex methods are suitable for and can meet the demands of high-throughput genetic testing in research and clinical settings.

A study was done of the bionomics, insecticide susceptibility and irritability status of Anopheles culicifacies in Sistan va Baluchestan province. Sampling was performed to determine the following parameters: species identification, seasonal activity, adult and larval susceptibility tests, irritability tests, anthropophily index and sporozoite rate. An. culicifacies adults were susceptible to all tested pyrethroid insecticides. An. culicifacies started to appear indoors in late May, showing 2 peaks in June and September. Fenitrothion, cyfluthrin and permethrin had the least irritancy effect and DDT the highest. Only 2/860 females tested were infected with Plasmodium spp. (sporozoite rate: 0.25%). ELISA testing of 250 blood meals derived from night-biting collections of female mosquitoes from humans and cows revealed that only 12.5% were human-fed.

Although per-base sequencing costs have decreased during recent years, library preparation for targeted massively parallel sequencing remains constrained by high reagent cost, limited design flexibility, and protocol complexity. To address these limitations, we previously developed Hi-Plex, a polymerase chain reaction (PCR) massively parallel sequencing strategy for screening panels of genomic target regions. Here, we demonstrate that Hi-Plex applied with hybrid adapters can generate a library suitable for sequencing with both the Ion Torrent and the TruSeq chemistries and that adjusting primer concentrations improves coverage uniformity. These results expand Hi-Plex capabilities as an accurate, affordable, flexible, and rapid approach for various genetic screening applications.

Loss-of-function mutations in PALB2 are associated with an increased risk of breast cancer, with recent data showing that female breast cancer risks for PALB2 mutation carriers are comparable in magnitude to those for BRCA2 mutation carriers. This study applied targeted massively parallel sequencing to characterize the mutation spectrum of PALB2 in probands attending breast cancer genetics clinics in the USA. The coding regions and proximal intron–exon junctions of PALB2 were screened in probands not known to carry a mutation in BRCA1 or BCRA2 from 1,250 families enrolled through familial cancer clinics by the Breast Cancer Family Registry. Mutation screening was performed using Hi-Plex, an amplicon-based targeted massively parallel sequencing platform. Screening of PALB2 was successful in 1,240/1,250 probands and identified nine women with protein-truncating mutations (three nonsense mutations and five frameshift mutations). Four of the 33 missense variants were predicted to be deleterious to protein function by in silico analysis using two different programs. Analysis of tumors from carriers of truncating mutations revealed that the majority were high histological grade, invasive ductal carcinomas. Young onset was apparent in most families, with 19 breast cancers under 50 years of age, including eight under the age of 40 years. Our data demonstrate the utility of Hi-Plex in the context of high-throughput testing for rare genetic mutations and provide additional timely information about the nature and prevalence of PALB2 mutations, to enhance risk assessment and risk management of women at high risk of cancer attending clinical genetic services.

Few genetic risk factors have been demonstrated to be specifically associated with aggressive prostate cancer (PrCa). Here, we report a case‐case study of PrCa comparing the prevalence of germline pathogenic/likely pathogenic (P/LP) genetic variants in 787 men with aggressive disease and 769 with nonaggressive disease. Overall, we observed P/LP variants in 11.4% of men with aggressive PrCa and 9.8% of men with nonaggressive PrCa (two‐tailed Fisher's exact tests, P = .28). The proportion of BRCA2 and ATM P/LP variant carriers in men with aggressive PrCa exceeded that observed in men with nonaggressive PrCa; 18/787 carriers (2.3%) and 4/769 carriers (0.5%), P = .004, and 14/787 carriers (0.02%) and 5/769 carriers (0.01%), P = .06, respectively. Our findings contribute to the extensive international effort to interpret the genetic variation identified in genes included on gene‐panel tests, for which there is currently an insufficient evidence‐base for clinical translation in the context of PrCa risk.