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

COMPLEXO: identifying the missing heritability of breast cancer via next generation collaboration

Linkage analysis, positional cloning, candidate gene mutation scanning and genome-wide association study approaches have all contributed significantly to our understanding of the underlying genetic architecture of breast cancer. Taken together, these approaches have identified genetic variation that explains approximately 30% of the overall familial risk of breast cancer, implying that more, and likely rarer, genetic susceptibility alleles remain to be discovered.</p

Case report: hypoglycemia due to a novel activating glucokinase variant in an adult – a molecular approach

We present a case of an obese 22-year-old man with activating GCK variant who had neonatal hypoglycemia, re-emerging with hypoglycemia later in life. We investigated him for asymptomatic hypoglycemia with a family history of hypoglycemia. Genetic testing yielded a novel GCK missense class 3 variant that was subsequently found in his mother, sister and nephew and reclassified as a class 4 likely pathogenic variant. Glucokinase enables phosphorylation of glucose, the rate-limiting step of glycolysis in the liver and pancreatic β cells. It plays a crucial role in the regulation of insulin secretion. Inactivating variants in GCK cause hyperglycemia and activating variants cause hypoglycemia. Spleen-preserving distal pancreatectomy revealed diffuse hyperplastic islets, nuclear pleomorphism and periductular islets. Glucose stimulated insulin secretion revealed increased insulin secretion in response to glucose. Cytoplasmic calcium, which triggers exocytosis of insulin-containing granules, revealed normal basal but increased glucose-stimulated level. Unbiased gene expression analysis using 10X single cell sequencing revealed upregulated INS and CKB genes and downregulated DLK1 and NPY genes in β-cells. Further studies are required to see if alteration in expression of these genes plays a role in the metabolic and histological phenotype associated with glucokinase pathogenic variant. There were more large islets in the patient’s pancreas than in control subjects but there was no difference in the proportion of β cells in the islets. His hypoglycemia was persistent after pancreatectomy, was refractory to diazoxide and improved with pasireotide. This case highlights the variable phenotype of GCK mutations. In-depth molecular analyses in the islets have revealed possible mechanisms for hyperplastic islets and insulin hypersecretion

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

Bayesian approach to determining penetrance of pathogenic SDH variants.

BACKGROUND: Until recently, determining penetrance required large observational cohort studies. Data from the Exome Aggregate Consortium (ExAC) allows a Bayesian approach to calculate penetrance, in that population frequencies of pathogenic germline variants should be inversely proportional to their penetrance for disease. We tested this hypothesis using data from two cohorts for succinate dehydrogenase subunits A, B and C (SDHA-C) genetic variants associated with hereditary pheochromocytoma/paraganglioma (PC/PGL). METHODS: Two cohorts were 575 unrelated Australian subjects and 1240 unrelated UK subjects, respectively, with PC/PGL in whom genetic testing had been performed. Penetrance of pathogenic SDHA-C variants was calculated by comparing allelic frequencies in cases versus controls from ExAC (removing those variants contributed by The Cancer Genome Atlas). RESULTS: Pathogenic SDHA-C variants were identified in 106 subjects (18.4%) in cohort 1 and 317 subjects (25.6%) in cohort 2. Of 94 different pathogenic variants from both cohorts (seven in SDHA, 75 in SDHB and 12 in SDHC), 13 are reported in ExAC (two in SDHA, nine in SDHB and two in SDHC) accounting for 21% of subjects with SDHA-C variants. Combining data from both cohorts, estimated lifetime disease penetrance was 22.0% (95% CI 15.2% to 30.9%) for SDHB variants, 8.3% (95% CI 3.5% to 18.5%) for SDHC variants and 1.7% (95% CI 0.8% to 3.8%) for SDHA variants. CONCLUSION: Pathogenic variants in SDHB are more penetrant than those in SDHC and SDHA. Our findings have important implications for counselling and surveillance of subjects carrying these pathogenic variants

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

iPrevent®: a tailored, web-based, decision support tool for breast cancer risk assessment and management

We aimed to develop a user-centered, web-based, decision support tool for breast cancer risk assessment and personalized risk management. Using a novel model choice algorithm, iPrevent&reg; selects one of two validated breast cancer risk estimation models (IBIS or BOADICEA), based on risk factor data entered by the user. Resulting risk estimates are presented in simple language and graphic formats for easy comprehension. iPrevent&reg; then presents risk-adapted, evidence-based, guideline-endorsed management options. Development was an iterative process with regular feedback from multidisciplinary experts and consumers. To verify iPrevent&reg;, risk factor data for 127 cases derived from the Australian Breast Cancer Family Study were entered into iPrevent&reg;, IBIS (v7.02), and BOADICEA (v3.0). Consistency of the model chosen by iPrevent&reg; (i.e., IBIS or BOADICEA) with the programmed iPrevent&reg; model choice algorithm was assessed. Estimated breast cancer risks from iPrevent&reg; were compared with those attained directly from the chosen risk assessment model (IBIS or BOADICEA). Risk management interventions displayed by iPrevent&reg; were assessed for appropriateness. Risk estimation model choice was 100% consistent with the programmed iPrevent&reg;logic. Discrepant 10-year and residual lifetime risk estimates of &gt;1% were found for 1 and 4 cases, respectively, none was clinically significant (maximal variation 1.4%). Risk management interventions suggested by iPrevent&reg; were 100% appropriate. iPrevent&reg; successfully integrates the IBIS and BOADICEA risk assessment models into a decision support tool that provides evidence-based, risk-adapted risk management advice. This may help to facilitate precision breast cancer prevention discussions between women and their healthcare providers