Molecular tumor profiling: translating genomic insights into clinical advances

Molecular profiling of the transcripts or proteins within an individual tumor may in future provide important prognostic and therapeutic clinical information both for the affected individual and for their extended family, but for the time being traditional genetics and pathology retain their place in the clinic

Investigation into the potential of tissue-specific promoters for gene supplementation therapy

Ornithine transcarbamylase (OTC) is the second enzyme in the hepatic urea cycle. As such, it is important in the metabolism of neurotoxic waste products into a non-toxic, water-soluble compound, urea. In humans deficiency of ornithine transcarbamylase is an X-linked single gene disorder associated with a high mortality and morbidity due to a severe metabolic disturbance. There are two naturally occuring allelic mouse models for OTC deficiency, the sparse fur (Spf) and sparse furASH (SpfASH) mice. In this study, the focus was on the use of tissue specific promoters in driving recombinant gene expression. The main question addressed was whether expression of the OTC gene in a tissue which did not normally express this gene, namely skeletal muscle, would correct the phenotype of the deficient mouse models. The approach taken was generation of conventional transgenic mice using a creatine kinase driven OTC gene construct. In addition as a positive control, supplementation of the endogenous hepatic gene expression was also undertaken by means of hepatic-specific albumin promoter. Three indices were used as a measure of correction of OTC deficiency by transgenesis in the mouse models; a) phenotype of the mice, b) plasma ammonia levels and c) direct OTC activity. Although transgenic mice expressing the OTC cDNA driven by the creatine kinase promoter showed high OTC activity in skeletal muscle, no metabolic or phenotypic correction of the mice was noted. Interestingly, the albumin driven OTC construct did not correct the phenotype of the sparse fur and sparse furASH models either, although in some transgenic lines a significant amelioration of plasma ammonia levels was noted. However, this partial metabolic correction did not correlate with a significant increase in hepatic OTC activity. In addition to these tissue-specific promoters, the potential of a novel keratin 5 minigene construct in targeting recombinant gene expression to the epidermis in a tissue and cell specific manner was assessed. The gene expression pattern of a Lac Z reporter transgenic construct driven by the regulatory sequences of the keratin 5 gene was investigated both in adult mice and during embryogenesis

human cytochrome P450 2E1導入ヒト肝癌細胞株(HLE/2E1)の樹立と薬物毒性に対する特徴

C1 - Journal Articles RefereedMutations in RAD51D have been associated with an increased risk of hereditary ovarian cancer and although they have been observed in the context of breast and ovarian cancer families, the association with breast cancer is unclear. The aim of this current study was to validate the reported association of RAD51D with ovarian cancer and assess for an association with breast cancer. We screened for RAD51D mutations in BRCA1/2 mutation-negative index cases from 1,060 familial breast and/or ovarian cancer families (including 741 affected by breast cancer only) and in 245 unselected ovarian cancer cases. Exons containing novel non-synonymous variants were screened in 466 controls. Two overtly deleterious RAD51D mutations were identified among the unselected ovarian cancers cases (0.82%) but none were detected among the 1,060 families. Our data provide additional evidence that RAD51D mutations are enriched among ovarian cancer patients, but are extremely rare among familial breast cancer patients

Clinical impact of genomic testing in patients with suspected monogenic kidney disease

Purpose: To determine the diagnostic yield and clinical impact of exome sequencing (ES) in patients with suspected monogenic kidney disease. Methods: We performed clinically accredited singleton ES in a prospectively ascertained cohort of 204 patients assessed in multidisciplinary renal genetics clinics at four tertiary hospitals in Melbourne, Australia. Results: ES identified a molecular diagnosis in 80 (39%) patients, encompassing 35 distinct genetic disorders. Younger age at presentation was independently associated with an ES diagnosis (p < 0.001). Of those diagnosed, 31/80 (39%) had a change in their clinical diagnosis. ES diagnosis was considered to have contributed to management in 47/80 (59%), including negating the need for diagnostic renal biopsy in 10/80 (13%), changing surveillance in 35/80 (44%), and changing the treatment plan in 16/80 (20%). In cases with no change to management in the proband, the ES result had implications for the management of family members in 26/33 (79%). Cascade testing was subsequently offered to 40/80 families (50%). Conclusion: In this pragmatic pediatric and adult cohort with suspected monogenic kidney disease, ES had high diagnostic and clinical utility. Our findings, including predictors of positive diagnosis, can be used to guide clinical practice and health service design

Development and validation of a targeted gene sequencing panel for application to disparate cancers

Next generation sequencing has revolutionised genomic studies of cancer, having facilitated the development of precision oncology treatments based on a tumour’s molecular profile. We aimed to develop a targeted gene sequencing panel for application to disparate cancer types with particular focus on tumours of the head and neck, plus test for utility in liquid biopsy. The final panel designed through Roche/Nimblegen combined 451 cancer-associated genes (2.01 Mb target region). 136 patient DNA samples were collected for performance and application testing. Panel sensitivity and precision were measured using well-characterised DNA controls (n = 47), and specificity by Sanger sequencing of the Aryl Hydrocarbon Receptor Interacting Protein (AIP) gene in 89 patients. Assessment of liquid biopsy application employed a pool of synthetic circulating tumour DNA (ctDNA). Library preparation and sequencing were conducted on Illumina-based platforms prior to analysis with our accredited (ISO15189) bioinformatics pipeline. We achieved a mean coverage of 395x, with sensitivity and specificity of >99% and precision of >97%. Liquid biopsy revealed detection to 1.25% variant allele frequency. Application to head and neck tumours/cancers resulted in detection of mutations aligned to published databases. In conclusion, we have developed an analytically-validated panel for application to cancers of disparate types with utility in liquid biopsy

Copy number variants as modifiers of breast cancer risk for BRCA1/BRCA2 pathogenic variant carriers

The risk of germline copy number variants (CNVs) in BRCA1 and BRCA2 pathogenic variant carriers in breast cancer is assessed, with CNVs overlapping SULT1A1 decreasing breast cancer risk in BRCA1 carriers.The contribution of germline copy number variants (CNVs) to risk of developing cancer in individuals with pathogenic BRCA1 or BRCA2 variants remains relatively unknown. We conducted the largest genome-wide analysis of CNVs in 15,342 BRCA1 and 10,740 BRCA2 pathogenic variant carriers. We used these results to prioritise a candidate breast cancer risk-modifier gene for laboratory analysis and biological validation. Notably, the HR for deletions in BRCA1 suggested an elevated breast cancer risk estimate (hazard ratio (HR) = 1.21), 95% confidence interval (95% CI = 1.09-1.35) compared with non-CNV pathogenic variants. In contrast, deletions overlapping SULT1A1 suggested a decreased breast cancer risk (HR = 0.73, 95% CI 0.59-0.91) in BRCA1 pathogenic variant carriers. Functional analyses of SULT1A1 showed that reduced mRNA expression in pathogenic BRCA1 variant cells was associated with reduced cellular proliferation and reduced DNA damage after treatment with DNA damaging agents. These data provide evidence that deleterious variants in BRCA1 plus SULT1A1 deletions contribute to variable breast cancer risk in BRCA1 carriers.Peer reviewe

Breast and Prostate Cancer Risks for Male BRCA1 and BRCA2 Pathogenic Variant Carriers Using Polygenic Risk Scores

Background: Recent population-based female breast cancer and prostate cancer polygenic risk scores (PRS) have been developed. We assessed the associations of these PRS with breast and prostate cancer risks for male BRCA1 and BRCA2 pathogenic variant carriers. Methods: 483 BRCA1 and 1318 BRCA2 European ancestry male carriers were available from the Consortium of Investigators of Modifiers of BRCA1/2 (CIMBA). A 147-single nucleotide polymorphism (SNP) prostate cancer PRS (PRSPC) and a 313-SNP breast cancer PRS were evaluated. There were 3 versions of the breast cancer PRS, optimized to predict overall (PRSBC), estrogen receptor (ER)-negative (PRSER-), or ER-positive (PRSER+) breast cancer risk. Results: PRSER+ yielded the strongest association with breast cancer risk. The odds ratios (ORs) per PRSER+ standard deviation estimates were 1.40 (95% confidence interval [CI] =1.07 to 1.83) for BRCA1 and 1.33 (95% CI = 1.16 to 1.52) for BRCA2 carriers. PRSPC was associated with prostate cancer risk for BRCA1 (OR = 1.73, 95% CI = 1.28 to 2.33) and BRCA2 (OR = 1.60, 95% CI = 1.34 to 1.91) carriers. The estimated breast cancer odds ratios were larger after adjusting for female relative breast cancer family history. By age 85 years, for BRCA2 carriers, the breast cancer risk varied from 7.7% to 18.4% and prostate cancer risk from 34.1% to 87.6% between the 5th and 95th percentiles of the PRS distributions. Conclusions: Population-based prostate and female breast cancer PRS are associated with a wide range of absolute breast and prostate cancer risks for male BRCA1 and BRCA2 carriers. These findings warrant further investigation aimed at providing personalized cancer risks for male carriers and informing clinical management.Peer reviewe

A case-only study to identify genetic modifiers of breast cancer risk for BRCA1/BRCA2 mutation carriers.

Breast cancer (BC) risk for BRCA1 and BRCA2 mutation carriers varies by genetic and familial factors. About 50 common variants have been shown to modify BC risk for mutation carriers. All but three, were identified in general population studies. Other mutation carrier-specific susceptibility variants may exist but studies of mutation carriers have so far been underpowered. We conduct a novel case-only genome-wide association study comparing genotype frequencies between 60,212 general population BC cases and 13,007 cases with BRCA1 or BRCA2 mutations. We identify robust novel associations for 2 variants with BC for BRCA1 and 3 for BRCA2 mutation carriers, P < 10-8, at 5 loci, which are not associated with risk in the general population. They include rs60882887 at 11p11.2 where MADD, SP11 and EIF1, genes previously implicated in BC biology, are predicted as potential targets. These findings will contribute towards customising BC polygenic risk scores for BRCA1 and BRCA2 mutation carriers