Molecular diagnosis for heterogeneous genetic diseases with high-throughput DNA sequencing applied to retinitis pigmentosa

BACKGROUND:
The genetic heterogeneity of many Mendelian disorders, such as retinitis pigmentosa which results from mutations in over 40 genes, is a major obstacle to obtaining a molecular diagnosis in clinical practice. Targeted high-throughput DNA sequencing offers a potential solution and was used to develop a molecular diagnostic screen for patients with retinitis pigmentosa.
METHODS:
A custom sequence capture array was designed to target the coding regions of all known retinitis pigmentosa genes and used to enrich these sequences from DNA samples of five patients. Enriched DNA was subjected to high-throughput sequencing singly or in pools, and sequence variants were identified by alignment of up to 10 million reads per sample to the normal reference sequence. Potential pathogenicity was assessed by functional predictions and frequency in controls.
RESULTS AND CONCLUSIONS:
Known homozygous PDE6B and compound heterozygous CRB1 mutations were detected in two patients. A novel homozygous missense mutation (c.2957A?T; p.N986I) in the cyclic nucleotide gated channel ß1 (CNGB1) gene predicted to have a deleterious effect and absent in 720 control chromosomes was detected in one case in which conventional genetic screening had failed to detect mutations. The detection of known and novel retinitis pigmentosa mutations in this study establishes high-throughput DNA sequencing with DNA pooling as an effective diagnostic tool for heterogeneous genetic diseases.

Murine model of autosomal dominant retinitis pigmentosa generated by targeted deletion at codon 307 of the rds-peripherin gene

We introduced a targeted single base deletion at codon 307 of the rds-peripherin gene in mice, similar mutations being known to cause autosomal dominant retinitis pigmentosa (RP) in man. Histopathological and electroretinographic analysis indicate that the retinopathy in mice homozygous for the codon 307 mutation appears more rapid than that in the naturally occurring null mutant, the rds(-/-) mouse, suggesting that the rds-307 mutation displays a dominant negative phenotype in combination with that due to haplosufficiency. RP is the most prevalent cause of registered visual handicap in those of working age in developed countries, the 50 or so mutations so far identified within the RDS-peripherin gene accounting for up to 10% of dominant cases of the disease. Given the sequence homologies that exist between the murine rds-peripherin and the human RDS-peripherin gene, this disease model, the first to be generated for peripherin-based RP using gene targeting techniques, should in principle be of value in the work-up in mice of therapeutics capable of targeting transcripts derived from the human gene.

Common Breast Cancer Susceptibility Alleles and the Risk of Breast Cancer for BRCA1 and BRCA2 Mutation Carriers: Implications for Risk Prediction

The known breast cancer susceptibility polymorphisms in FGFR2, TNRC9/TOX3, MAP3K1, LSP1, and 2q35 confer increased risks of breast cancer for BRCA1 or BRCA2 mutation carriers. We evaluated the associations of 3 additional single nucleotide polymorphisms (SNPs), rs4973768 in SLC4A7/NEK10, rs6504950 in STXBP4/COX11, and rs10941679 at 5p12, and reanalyzed the previous associations using additional carriers in a sample of 12,525 BRCA1 and 7,409 BRCA2 carriers. Additionally, we investigated potential interactions between SNPs and assessed the implications for risk prediction. The minor alleles of rs4973768 and rs10941679 were associated with increased breast cancer risk for BRCA2 carriers (per-allele HR - 1.10, 95% CI: 1.03-1.18, P - 0.006 and HR - 1.09, 95% CI: 1.01-1.19, P = 0.03, respectively). Neither SNP was associated with breast cancer risk for BRCA1 carriers, and rs6504950 was not associated with breast cancer for either BRCA1 or BRCA2 carriers. Of the 9 polymorphisms investigated, 7 were associated with breast cancer for BRCA2 carriers (FGFR2, TOX3, MAP3K1, LSP1, 2q35, SLC4A7, 5p12, P 7 = 10 x (11) - 0.03), but only TOX3 and 2q35 were associated with the risk for BRCA1 carriers (P = 0.0049, 0.03, respectively). All risk-associated polymorphisms appear to interact multiplicatively on breast cancer risk for mutation carriers. Based on the joint genotype distribution of the 7 risk-associated SNPs in BRCA2 mutation carriers, the 5% of BRCA2 carriers at highest risk (i.e., between 95th and 100th percentiles) were predicted to have a probability between 80% and 96% of developing breast cancer by age 80, compared with 42%

Evaluation of a candidate breast cancer associated SNP in ERCC4 as a risk modifier in BRCA1 and BRCA2 mutation carriers. Results from the Consortium of Investigators of Modifiers of BRCA1/BRCA2 (CIMBA)

BACKGROUND: In this study we aimed to evaluate the role of a SNP in intron I of the ERCC4 gene (rs744154), previously reported to be associated with a reduced risk of breast cancer in the general population, as a breast cancer risk modifier in BRCA1 and BRCA2 mutation carriers.

Predicting the likelihood of carrying a BRCA1 or BRCA2 mutation: validation of BOADICEA, BRCAPRO, IBIS, Myriad and the Manchester scoring system using data from UK genetics clinics

Objectives: Genetic testing for the breast and ovarian cancer susceptibility genes BRCA1 and BRCA2 has important implications for the clinical management of people found to carry a mutation. However, genetic testing is expensive and may be associated with adverse psychosocial effects. To provide a cost-efficient and clinically appropriate genetic counselling service, genetic testing should be targeted at those individuals most likely to carry pathogenic mutations. Several algorithms that predict the likelihood of carrying a BRCA1 or a BRCA2 mutation are currently used in clinical practice to identify such individuals.

The RET mutation E768D confers a late-onset Familial medullary thyroid carcinoma - only phenotype with incomplete penetrance: Implications for screening and management of carrier status

Objective: We describe a 4-generation family with familial medullary thyroid carcinoma (FMTC) - a variant of multiple endocrine neoplasia type 2 (MEN 2) without extra-thyroid features. RET mutation analysis confirmed an E768D mutation in exon 13 in 8 family members, 3 affected with medullary thyroid cancer alone while the other 5 were detected to be mutation carriers. This mutation has been described in very few families worldwide and the spectrum of disease and natural history is unclear. Results: Three affected members had medullary thyroid cancer (MTC) confirmed histologically at ages 25, 50 and 56 years, respectively. The E768D mutation appears to have a less aggressive clinical course compared to other high risk RET mutations with no evidence of clinical recurrence up to I I years after initial therapy. Of five gene carriers identified, two are asymptomatic at the age of 70 and 61, and three had raised calcitonin levels at 46, 39, and 45 years. Following total thyroidectomy, one gene carrier had a histologically normal thyroid at age 46, following a mildly elevated calcitonin, one had C-cell hyperplasia at the age of 39, and one had a frank focus of carcinoma in the left thyroid lobe at the age of 45. No members had evidence of phaeochromocytoma or parathyroid disease on screening. Conclusion: The RET E768D mutation is associated with MTC with a later age at presentation, incomplete penetrance and less aggressive course compared with other high risk RET mutations. To date in this family the E768D mutation has not been associated with either phaeochromocytoma or hyperparathyroidism. The appropriate screening strategy for and management of E768D carriers is difficult reflecting the phenotypic heterogeneity.

Enhanced lymphocyte interferon (IFN)-γ responses in a PTEN mutation-negative Cowden disease kindred.

Identification of immune modifiers of inherited cancer syndromes may provide a rationale for preventive therapy. Cowden disease (CD) is a genetically heterogeneous inherited cancer syndrome that arises predominantly from germline phosphatase and tensin homologue deleted on chromosome 10 (PTEN) mutation and increased phosphoinositide 3-kinase/mammalian target of rapamycin (PI3K/mTOR) signalling. However, many patients with classic CD diagnostic features are mutation-negative for PTEN (PTEN M-Neg). Interferon (IFN)-gamma can modulate the PI3K/mTOR pathway, but its association with PTEN M-Neg CD remains unclear. This study assessed IFN-gamma secretion by multi-colour flow cytometry in a CD kindred that was mutation-negative for PTEN and other known susceptibility genes. Because IFN-gamma responses may be regulated by killer cell immunoglobulin-like receptors (KIR) and respective human leucocyte antigen (HLA) ligands, KIR/HLA genotypes were also assessed. Activating treatments induced greater IFN-gamma secretion in PTEN M-Neg CD peripheral blood lymphocytes versus healthy controls. Increased frequency of activating KIR genes, potentially activating KIR/HLA compound genotypes and reduced frequency of inhibitory genotypes, were found in the PTEN M-Neg CD kindred. Differences of IFN-gamma secretion were observed among PTEN M-Neg CD patients with distinct KIR/HLA compound genotypes. Taken together, these findings show enhanced lymphocyte secretion of IFN-gamma that may influence the PI3K/mTOR CD causal molecular pathway in a PTEN mutation-negative CD kindred.