Genetic testing for BRCA1 and BRCA2 is offered typically to selected women based on age of onset and family history of cancer. However, current internationally accepted genetic testing referral guidelines are built mostly on data from cancer genetics clinics in women of European descent. To evaluate the appropriateness of such guidelines in Asians, we have determined the prevalence of germ line variants in an unselected cohort of Asian patients with breast cancer and healthy controls.

Testicular germ cell tumor (TGCT) is the most common cancer in young men and is notable for its high familial risks. So far, six loci associated with TGCT have been reported. From genome-wide association study (GWAS) analysis of 307,291 SNPs in 986 TGCT cases and 4,946 controls, we selected for follow-up 694 SNPs, which we genotyped in a further 1,064 TGCT cases and 10,082 controls from the UK. We identified SNPs at nine new loci (1q22, 1q24.1, 3p24.3, 4q24, 5q31.1, 8q13.3, 16q12.1, 17q22 and 21q22.3) showing association with TGCT (P < 5 × 10(-8)), which together account for an additional 4-6% of the familial risk of TGCT. The loci include genes plausibly related to TGCT development. PRDM14, at 8q13.3, is essential for early germ cell specification, and DAZL, at 3p24.3, is required for the regulation of germ cell development. Furthermore, PITX1, at 5q31.1, regulates TERT expression and is the third TGCT-associated locus implicated in telomerase regulation.

Associations between single nucleotide polymorphisms (SNPs) at 5p15 and multiple cancer types have been reported. We have previously shown evidence for a strong association between prostate cancer (PrCa) risk and rs2242652 at 5p15, intronic in the telomerase reverse transcriptase (TERT) gene that encodes TERT. To comprehensively evaluate the association between genetic variation across this region and PrCa, we performed a fine-mapping analysis by genotyping 134 SNPs using a custom Illumina iSelect array or Sequenom MassArray iPlex, followed by imputation of 1094 SNPs in 22 301 PrCa cases and 22 320 controls in The PRACTICAL consortium. Multiple stepwise logistic regression analysis identified four signals in the promoter or intronic regions of TERT that independently associated with PrCa risk. Gene expression analysis of normal prostate tissue showed evidence that SNPs within one of these regions also associated with TERT expression, providing a potential mechanism for predisposition to disease.

Breast cancer is a heterogeneous disease and may be characterized on the basis of whether estrogen receptors (ER) are expressed in the tumour cells. ER status of breast cancer is important clinically, and is used both as a prognostic indicator and treatment predictor. In this study, we focused on identifying genetic markers associated with ER-negative breast cancer risk.

The cDNA-mediated Annealing, extension, Selection and Ligation (DASL) assay has become a suitable gene expression profiling system for degraded RNA from paraffin-embedded tissue. We examined assay characteristics and the performance of the DASL 502-gene Cancer Panel v1 (1.5K) and 24,526-gene panel (24K) platforms at differentiating nine human epidermal growth factor receptor 2- positive (HER2+) and 11 HER2-negative (HER2-) paraffin-embedded breast tumors.

Several genetic risk models for breast and ovarian cancer have been developed, but their applicability to specific populations has not been evaluated. We used data from French-Canadian families to evaluate the mutation predictions given by the BRCAPRO and BOADICEA models. We also used this data set to estimate the age-specific risks for breast and ovarian cancer in BRCA1 and BRCA2 mutation carriers.

About two-thirds of the excess familial risk associated with breast cancer is still unaccounted for and may be explained by multiple weakly predisposing alleles. A gene thought to be involved in low-level predisposition to the disease is ERBB2 (HER2). This gene is involved in cell division, differentiation, and apoptosis and is frequently amplified in breast tumours. Its amplification correlates with poor prognosis. Moreover, the coding polymorphism I655V has previously been associated with an increased risk of breast cancer.

The risk of breast cancer to first degree relatives of breast cancer patients is approximately twice that of the general population. Breast cancer, however, is a heterogeneous disease and it is plausible that the familial relative risk (FRR) for breast cancer may differ by the pathological subtype of the tumour. The contribution of genetic variants associated with breast cancer susceptibility to the subtype-specific FRR is still unclear.

Several studies have examined the effect of genetic variants in genes involved in the estrogen metabolic pathway on mammographic density, but the number of loci studied and the sample sizes evaluated have been small and pathways have not been evaluated comprehensively. In this study, we evaluate the association between mammographic density and genetic variants of the estrogen metabolic pathway.

Several methods have been proposed to impute genotypes at untyped markers using observed genotypes and genetic data from a reference panel. We used the Genetic Analysis Workshop 16 rheumatoid arthritis case-control dataset to compare the performance of four of these imputation methods: IMPUTE, MACH, PLINK, and fastPHASE. We compared the methods' imputation error rates and performance of association tests using the imputed data, in the context of imputing completely untyped markers as well as imputing missing genotypes to combine two datasets genotyped at different sets of markers. As expected, all methods performed better for single-nucleotide polymorphisms (SNPs) in high linkage disequilibrium with genotyped SNPs. However, MACH and IMPUTE generated lower imputation error rates than fastPHASE and PLINK. Association tests based on allele "dosage" from MACH and tests based on the posterior probabilities from IMPUTE provided results closest to those based on complete data. However, in both situations, none of the imputation-based tests provide the same level of evidence of association as the complete data at SNPs strongly associated with disease.

Genome-wide association studies (GWASs) assess correlation between traits and DNA sequence variation using large numbers of genetic variants such as single nucleotide polymorphisms (SNPs) distributed across the genome. A GWAS produces many trait-SNP associations with low p-values, but few are replicated in subsequent studies. We sought to determine if characteristics of the genomic loci associated with a trait could be used to identify initial associations with a higher chance of replication in a second cohort. Data from the age-related eye disease study (AREDS) of 100,000 SNPs on 395 subjects with and 198 without age-related macular degeneration (AMD) were employed. Loci highly associated with AMD were characterized based on the distribution of genotypes, level of significance, and clustering of adjacent SNPs also associated with AMD suggesting linkage disequilibrium or multiple effects. Forty nine loci were highly associated with AMD, including 3 loci (CFH, C2/BF, LOC387715/HTRA1) already known to contain important genetic risks for AMD. One additional locus (C3) reported during the course of this study was identified and replicated in an additional study group. Tag-SNPs and haplotypes for each locus were evaluated for association with AMD in additional cohorts to account for population differences between discovery and replication subjects, but no additional clearly significant associations were identified. Relying on a significant genotype tests using a log-additive model would have excluded 57% of the non-replicated and none of the replicated loci, while use of other SNP features and clustering might have missed true associations.

MicroRNAs (miRNAs) are a class of approximately 22 nucleotide long, widely expressed RNA molecules that play important regulatory roles in eukaryotes. To investigate miRNA function, it is essential that methods to quantify their expression levels be available.

In a systematic sequencing screen of the coding exons of the X chromosome in 250 families with X-linked mental retardation (XLMR), we identified two nonsense mutations and one consensus splice-site mutation in the AP1S2 gene on Xp22 in three families. Affected individuals in these families showed mild-to-profound mental retardation. Other features included hypotonia early in life and delay in walking. AP1S2 encodes an adaptin protein that constitutes part of the adaptor protein complex found at the cytoplasmic face of coated vesicles located at the Golgi complex. The complex mediates the recruitment of clathrin to the vesicle membrane. Aberrant endocytic processing through disruption of adaptor protein complexes is likely to result from the AP1S2 mutations identified in the three XLMR-affected families, and such defects may plausibly cause abnormal synaptic development and function. AP1S2 is the first reported XLMR gene that encodes a protein directly involved in the assembly of endocytic vesicles.

The nuclear factor-kappaB (NF-kappaB) family of transcription factors regulates the expression of a variety of genes involved in apoptosis and immune response. We examined relationships between genotypes at five NF-kappaB subunits (NFKB1, NFKB2, REL, RELA, and RELB) and variable expression levels of 15 NF-kappaB regulated proteins with heritability greater than 0.40: BCL2A1, BIRC2, CD40, CD44, CD80, CFLAR, CR2, FAS, ICAM1, IL15, IRF1, JUNB, MYC, SLC2A5, and VCAM1. SNP genotypes and expression phenotypes from pedigrees of Utah residents with ancestry from northern and western Europe were provided by Genetic Analysis Workshop 15 and supplemented with additional genotype data from the International HapMap Consortium. We conducted association, linkage, and family-based association analyses between each candidate gene and the 15 heritable expression phenotypes. We observed consistent results in association and linkage analyses of the NFKB1 region (encoding p50) and levels of FAS and IRF1 expression. FAS is a cell surface protein that also belongs to the TNF-receptor family; signals through FAS are able to induce apoptosis. IRF1 is a member of the interferon regulatory transcription factor family, which has been shown to regulate apoptosis and tumor-suppression. Analyses in the REL region (encoding c-Rel) revealed linkage and association with CD40 phenotype. CD40 proteins belong to the tumor necrosis factor (TNF)-receptor family, which mediates a broad variety of immune and inflammatory responses. We conclude that variation in the genes encoding p50 and c-Rel may play a role in NF-kappaB-related transcription of FAS, IRF1, and CD40.

Several methods to identify tagging single-nucleotide polymorphisms (SNPs) are in common use for genetic epidemiologic studies; however, there may be loss of information when using only a subset of SNPs. We sought to compare the ability of commonly used pairwise, multimarker, and haplotype-based tagging SNP selection methods to detect known associations with quantitative expression phenotypes. Using data from HapMap release 21 on unrelated Utah residents with ancestors from northern and western Europe (CEPH-Utah, CEU), we selected tagging SNPs in five chromosomal regions using ldSelect, Tagger, and TagSNPs. We found that SNP subsets did not substantially overlap, and that the use of trio data did not greatly impact SNP selection. We then tested associations between HapMap genotypes and expression phenotypes on 28 CEU individuals as part of Genetic Analysis Workshop 15. Relative to the use of all SNPs (n = 210 SNPs across all regions), most subset methods were able to detect single-SNP and haplotype associations. Generally, pairwise selection approaches worked extremely well, relative to use of all SNPs, with marked reductions in the number of SNPs required. Haplotype-based approaches, which had identified smaller SNP subsets, missed associations in some regions. We conclude that the optimal tagging SNP method depends on the true model of the genetic association (i.e., whether a SNP or haplotype is responsible); unfortunately, this is often unknown at the time of SNP selection. Additional evaluations using empirical and simulated data are needed.

A three-stage genome-wide association study recently identified single nucleotide polymorphisms (SNPs) in five loci (fibroblast growth receptor 2 (FGFR2), trinucleotide repeat containing 9 (TNRC9), mitogen-activated protein kinase 3 K1 (MAP3K1), 8q24, and lymphocyte-specific protein 1 (LSP1)) associated with breast cancer risk. We investigated whether the associations between these SNPs and breast cancer risk varied by clinically important tumor characteristics in up to 23,039 invasive breast cancer cases and 26,273 controls from 20 studies. We also evaluated their influence on overall survival in 13,527 cases from 13 studies. All participants were of European or Asian origin. rs2981582 in FGFR2 was more strongly related to ER-positive (per-allele OR (95%CI) = 1.31 (1.27-1.36)) than ER-negative (1.08 (1.03-1.14)) disease (P for heterogeneity = 10(-13)). This SNP was also more strongly related to PR-positive, low grade and node positive tumors (P = 10(-5), 10(-8), 0.013, respectively). The association for rs13281615 in 8q24 was stronger for ER-positive, PR-positive, and low grade tumors (P = 0.001, 0.011 and 10(-4), respectively). The differences in the associations between SNPs in FGFR2 and 8q24 and risk by ER and grade remained significant after permutation adjustment for multiple comparisons and after adjustment for other tumor characteristics. Three SNPs (rs2981582, rs3803662, and rs889312) showed weak but significant associations with ER-negative disease, the strongest association being for rs3803662 in TNRC9 (1.14 (1.09-1.21)). rs13281615 in 8q24 was associated with an improvement in survival after diagnosis (per-allele HR = 0.90 (0.83-0.97). The association was attenuated and non-significant after adjusting for known prognostic factors. Our findings show that common genetic variants influence the pathological subtype of breast cancer and provide further support for the hypothesis that ER-positive and ER-negative disease are biologically distinct. Understanding the etiologic heterogeneity of breast cancer may ultimately result in improvements in prevention, early detection, and treatment.

Clinical testing of BRCA1 and BRCA2 began over 20 years ago. With the expiration and overturning of the BRCA patents, limitations on which laboratories could offer commercial testing were lifted. These legal changes occurred approximately the same time as the widespread adoption of massively parallel sequencing (MPS) technologies. Little is known about how these changes impacted laboratory practices for detecting genetic alterations in hereditary breast and ovarian cancer genes. Therefore, we sought to examine current laboratory genetic testing practices for BRCA1/BRCA2. We employed an online survey of 65 questions covering four areas: laboratory characteristics, details on technological methods, variant classification, and client-support information. Eight United States (US) laboratories and 78 non-US laboratories completed the survey. Most laboratories (93%; 80/86) used MPS platforms to identify variants. Laboratories differed widely on: (1) technologies used for large rearrangement detection; (2) criteria for minimum read depths; (3) non-coding regions sequenced; (4) variant classification criteria and approaches; (5) testing volume ranging from 2 to 2.5 × 105 tests annually; and (6) deposition of variants into public databases. These data may be useful for national and international agencies to set recommendations for quality standards for BRCA1/BRCA2 clinical testing. These standards could also be applied to testing of other disease genes.

Most non-BRCA1/2 breast cancer families have no identified genetic cause. We used linkage and haplotype analyses in familial and sporadic breast cancer cases to identify a susceptibility locus on chromosome 6q. Two independent genome-wide linkage analysis studies suggested a 3 Mb locus on chromosome 6q and two unrelated Swedish families with a LOD >2 together seemed to share a haplotype in 6q14.1. We hypothesized that this region harbored a rare high-risk founder allele contributing to breast cancer in these two families. Sequencing of DNA and RNA from the two families did not detect any pathogenic mutations. Finally, 29 SNPs in the region were analyzed in 44,214 cases and 43,532 controls from BCAC, and the original haplotypes in the two families were suggested as low-risk alleles for European and Swedish women specifically. There was also some support for one additional independent moderate-risk allele in Swedish familial samples. The results were consistent with our previous findings in familial breast cancer and supported a breast cancer susceptibility locus at 6q14.1 around the PHIP gene.

Genome-wide studies of patients carrying pathogenic variants (mutations) in BRCA1 or BRCA2 have reported strong associations between single-nucleotide polymorphisms (SNPs) and cancer risk. To conduct the first genome-wide association analysis of copy-number variants (CNVs) with breast or ovarian cancer risk in a cohort of 2500 BRCA1 pathogenic variant carriers, CNV discovery was performed using multiple calling algorithms and Illumina 610k SNP array data from a previously published genome-wide association study. Our analysis, which focused on functionally disruptive genomic deletions overlapping gene regions, identified a number of loci associated with risk of breast or ovarian cancer for BRCA1 pathogenic variant carriers. Despite only including putative deletions called by at least two or more algorithms, detection of selected CNVs by ancillary molecular technologies only confirmed 40% of predicted common (>1% allele frequency) variants. These include four loci that were associated (unadjusted P<0.05) with breast cancer (GTF2H2, ZNF385B, NAALADL2 and PSG5), and two loci associated with ovarian cancer (CYP2A7 and OR2A1). An interesting finding from this study was an association of a validated CNV deletion at the CYP2A7 locus (19q13.2) with decreased ovarian cancer risk (relative risk=0.50, P=0.007). Genomic analysis found this deletion coincides with a region displaying strong regulatory potential in ovarian tissue, but not in breast epithelial cells. This study highlighted the need to verify CNVs in vitro, but also provides evidence that experimentally validated CNVs (with plausible biological consequences) can modify risk of breast or ovarian cancer in BRCA1 pathogenic variant carriers.

Purpose BRCA1/2 mutations increase the risk of breast and prostate cancer in men. Common genetic variants modify cancer risks for female carriers of BRCA1/2 mutations. We investigated-for the first time to our knowledge-associations of common genetic variants with breast and prostate cancer risks for male carriers of BRCA1/ 2 mutations and implications for cancer risk prediction. Materials and Methods We genotyped 1,802 male carriers of BRCA1/2 mutations from the Consortium of Investigators of Modifiers of BRCA1/2 by using the custom Illumina OncoArray. We investigated the combined effects of established breast and prostate cancer susceptibility variants on cancer risks for male carriers of BRCA1/2 mutations by constructing weighted polygenic risk scores (PRSs) using published effect estimates as weights. Results In male carriers of BRCA1/2 mutations, PRS that was based on 88 female breast cancer susceptibility variants was associated with breast cancer risk (odds ratio per standard deviation of PRS, 1.36; 95% CI, 1.19 to 1.56; P = 8.6 × 10-6). Similarly, PRS that was based on 103 prostate cancer susceptibility variants was associated with prostate cancer risk (odds ratio per SD of PRS, 1.56; 95% CI, 1.35 to 1.81; P = 3.2 × 10-9). Large differences in absolute cancer risks were observed at the extremes of the PRS distribution. For example, prostate cancer risk by age 80 years at the 5th and 95th percentiles of the PRS varies from 7% to 26% for carriers of BRCA1 mutations and from 19% to 61% for carriers of BRCA2 mutations, respectively. Conclusion PRSs may provide informative cancer risk stratification for male carriers of BRCA1/2 mutations that might enable these men and their physicians to make informed decisions on the type and timing of breast and prostate cancer risk management.