Hi-Plex targeted sequencing is effective using DNA derived from archival dried blood spots.

Many genetic epidemiology resources have collected dried blood spots (predominantly as Guthrie Cards) as an economical and efficient means of archiving sources of DNA, conferring great value to genetic screening methods that are compatible with this medium. We applied Hi-Plex to screen the breast cancer predisposition gene PALB2 in 93 Guthrie Card-derived DNA specimens previously characterized for PALB2 genetic variants via DNA derived from lymphoblastoid cell lines, whole blood, and buffy coat. Of the 93 archival Guthrie Card-derived DNAs, 92 (99%) were processed successfully and sequenced using approximately half of a MiSeq run. From these 92 DNAs, all 59 known variants were detected and no false-positive variant calls were yielded. Fully 98.13% of amplicons (5417/5520) were represented within 15-fold of the median coverage (2786 reads), and 99.98% of amplicons (5519/5520) were represented at a depth of 10 read-pairs or greater. With Hi-Plex, we show for the first time that a High-Plex amplicon-based massively parallel sequencing (MPS) system can be applied effectively to DNA prepared from dried blood spot archival specimens and, as such, can dramatically increase the scopes of both method and resource.

Rare mutations in XRCC2 increase the risk of breast cancer.

An exome-sequencing study of families with multiple breast-cancer-affected individuals identified two families with XRCC2 mutations, one with a protein-truncating mutation and one with a probably deleterious missense mutation. We performed a population-based case-control mutation-screening study that identified six probably pathogenic coding variants in 1,308 cases with early-onset breast cancer and no variants in 1,120 controls (the severity grading was p < 0.02). We also performed additional mutation screening in 689 multiple-case families. We identified ten breast-cancer-affected families with protein-truncating or probably deleterious rare missense variants in XRCC2. Our identification of XRCC2 as a breast cancer susceptibility gene thus increases the proportion of breast cancers that are associated with homologous recombination-DNA-repair dysfunction and Fanconi anemia and could therefore benefit from specific targeted treatments such as PARP (poly ADP ribose polymerase) inhibitors. This study demonstrates the power of massively parallel sequencing for discovering susceptibility genes for common, complex diseases.

Genetic aberrations detected by comparative genomic hybridisation in vulvar cancers.

Squamous cell carcinoma of the vulva is a disease of significant clinical importance, which arises in the presence or absence of human papillomavirus. We used comparative genomic hybridisation to document non-random chromosomal gains and losses within human papillomavirus positive and negative vulvar cancers. Gain of 3q was significantly more common in human papillomavirus-positive cancers compared to human papillomavirus-negative cancers. The smallest area of gain was 3q22-25, a chromosome region which is frequently gained in other human papillomavirus-related cancers. Chromosome 8q was more commonly gained in human papillomavirus-negative compared to human papillomavirus-positive cancers. 8q21 was the smallest region of gain, which has been identified in other, non-human papillomavirus-related cancers. Chromosome arms 3p and 11q were lost in both categories of vulvar cancer. This study has demonstrated chromosome locations important in the development of vulvar squamous cell carcinoma. Additionally, taken together with previous studies of human papillomavirus-positive cancers of other anogenital sites, the data indicate that one or more oncogenes important in the development and progression of human papillomavirus-induced carcinomas are located on 3q. The different genetic changes seen in human papillomavirus-positive and negative vulvar squamous cell carcinomas support the clinicopathological data indicating that these are different cancer types.

Distinct molecular pathogeneses of early-onset breast cancers in BRCA1 and BRCA2 mutation carriers: a population-based study.

Breast cancers arising in women with and without a germline mutation in the BRCA1 or BRCA2 gene display different histological features, which suggests unique mechanisms of molecular pathogenesis: We used a molecular pathological analysis to define the genetic abnormalities relevant to these specific pathogeneses. Tumor material was studied from 40 women with breast cancer diagnosed before 40 years of age, sampled from a population-based study and stratified by BRCA1 and BRCA2 germline mutation status. Cases were not selected for family history or ethnic origin, and none were known to be genetically related. Thus, germline mutation itself is likely to impact on the molecular pathogenesis of these tumors, with no substantial influence due to modifying genetic or environmental factors. Breast cancers occurring in BRCA1 mutation carriers had significantly higher levels of p53 expression, including the preinvasive (carcinoma in situ) stage of disease, compared with cancers occurring in BRCA2 mutation carriers or women with no detectable germline mutation. These cancers also had a higher proliferation rate as measured by Ki-67 antibody. Expression of the prognostic factors c-erbB-2, cyclin D1, and estrogen receptor was significantly less common in BRCA1 mutation carriers. Lower levels of cyclin D1 were also found in cancers from BRCA2 mutation carriers compared with non-mutation carriers. Direct p53 mutation analysis revealed mutations in 18% of all of the early-onset breast cancers within the study and included rare insertion and deletional mutations in cancers from BRCA1 mutation carriers. Our data indicate that a BRCA1 breast cancer phenotype may be recognized by an exceptionally high proliferation rate and early and frequent p53 overexpression but infrequent selection for overexpression of several other prognostic factor proteins known to be involved in breast oncogenesis. In contrast, breast cancers arising in BRCA2 mutation carriers have a more heterogeneous phenotypic profile.

Expression of the P2Y6 purinergic receptor in human T cells infiltrating inflammatory bowel disease.

The human P2Y6 receptor is a member of the G-protein-coupled P2Y purinergic receptor family that responds to extracellular uridine diphosphate (UDP). In previous work, we cloned the human P2Y6 receptor from an activated T-cell library, and others have shown that it is expressed as a 1.9-kb transcript in several lymphoid tissues. This suggests a role for P2Y6 in T-cell function. However, the precise cellular expression pattern and regulation of P2Y6 in immune cells have not yet been established. In this study, we have examined the expression of P2Y6 in a range of tissues containing leukocytes by a combination of in situ hybridization (ISH), Northern blot analysis, and RT-PCR. Northern hybridization revealed that activated peripheral T cells show increased levels of P2Y6 mRNA. Furthermore, RT-PCR analysis of CD4+ and CD8+ subsets illustrated strong expression in both activated CD4+ and CD8+ T cells. Stimulation of resting and activated T cells with the P2Y6 ligand UDP caused a rise in the intracellular free calcium concentration in only the activated subset, indicating the presence of functional receptor. By ISH, P2Y6 expression was detected in the T cells of the thymic medulla and spleen, whereas no signal was detected in the bone marrow, fetal liver, or lymph nodes. T cells are thought to play an important role in the pathogenesis of inflammatory bowel disease (IBD), and because a recent finding has suggested a role for extracellular nucleotides in mediating colonic epithelial cell damage in IBD, we speculated that the P2Y6 nucleotide receptor may be expressed in the T cells infiltrating IBD. ISH results reveal that P2Y6 is highly expressed in the T cells infiltrating active IBD, whereas P2Y6 expression was absent from the T cells of unaffected bowel. These results demonstrate expression and regulation of P2Y6 expression in T cells, and suggest a role for P2Y6 in the pathogenesis of IBD-mediated intestinal damage.

Chromosomal localization of the human P2y6 purinoceptor gene and phylogenetic analysis of the P2y purinoceptor family.

The G-protein-coupled P2Y purinoceptors mediate a variety of physiological effects in response to extracellular nucleotides. With the recent discovery of several new members from a variety of species, the P2Y purinoceptor family now encompasses types P2Y1 to P2Y6. By fluorescence in situ hybridization and utilization of the National Center for Biotechnology Information (NCBI) database, the human P2Y6 gene was localized to chromosome 11q13.5, between polymorphic markers D11S1314 and D11S916. NCBI database analysis of the remaining human P2Y purinoceptor genes revealed that P2Y2 and P2Y6 mapped to within less than 4 cM, and thus constitute the first described chromosomal clustering of this gene family. Phylogenetic analysis of the P2Y purinoceptor family demonstrated the presence of five evolutionary branches and suggests the occurrence of an ancient gene duplication event.

Molecular cloning and sequencing of a novel human P2 nucleotide receptor.

A novel human P2 nucleotide receptor has been cloned from a T-cell cDNA library. The predicted amino acid sequence shows characteristics of a G-protein-coupled receptor, and shares 88% homology with a recently characterised rat P2 nucleotide receptor sequence. Distinctive features include an extremely short cytoplasmic tail with only one putative protein kinase C phosphorylation site. Northern blot analysis revealed a 1.9 kb transcript expressed in the placenta.