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.

Deep sequencing reveals predominant expression of miR-21 amongst the small non-coding RNAs in retinal microvascular endothelial cells

The retinal vascular endothelium is essential for angiogenesis and is involved in maintaining barrier selectivity and vascular tone. The aim of this study was to identify and quantify microRNAs and other small regulatory non-coding RNAs (ncRNAs) which may regulate these crucial functions. Primary bovine retinal microvascular endothelial cells (RMECs) provide a well-characterized in vitro system for studying angiogenesis. RNA extracted from RMECs was used to prepare a small RNA library for deep sequencing (Illumina Genome Analyzer). A total of 6.8 million reads were mapped to 250 known microRNAs in miRBase (release 16). In many cases, the most frequent isomiR differed from the sequence reported in miRBase. In addition, five novel microRNAs, 13 novel bovine orthologs of known human microRNAs and multiple new members of the miR-2284/2285 family were detected. Several similar to 30 nucleotide sno-miRNAs were identified, with the most highly expressed being derived from snoRNA U78. Highly expressed microRNAs previously associated with endothelial cells included miR-126 and miR-378, but the most highly expressed was miR-21, comprising more than one-third of all mapped reads. Inhibition of miR-21 with an LNA inhibitor significantly reduced proliferation, migration, and tube-forming capacity of RMECs. The independence from prior sequence knowledge provided by deep sequencing facilitates analysis of novel microRNAs and other small RNAs. This approach also enables quantitative evaluation of microRNA expression, which has highlighted the predominance of a small number of microRNAs in RMECs. Knockdown of miR-21 suggests a role for this microRNA in regulation of angiogenesis in the retinal microvasculature. J. Cell. Biochem. 113: 20982111, 2012. (C) 2012 Wiley Periodicals, Inc.

ISOLATION AND PARTIAL SEQUENCING OF A PANCREATIC POLYPEPTIDE-LIKE NEUROPEPTIDE FROM THE LIVER FLUKE, FASCIOLA-HEPATICA

1. A pancreatic polypeptide (PP)-immunoreactive neuropeptide has been isolated and partially sequenced from the liver fluke, Fasciola hepatica.

Exome sequencing of gastric adenocarcinoma identifies recurrent somatic mutations in cell adhesion and chromatin remodelling genes

Gastric cancer is a major cause of global cancer mortality. We surveyed the spectrum of somatic alterations in gastric cancer by sequencing the exomes of 15 gastric adenocarcinomas and their matched normal DNAs. Frequently mutated genes in the adenocarcinomas included TP53 (11/15 tumors), PIK3CA (3/15) and ARID1A (3/15). Cell adhesion was the most enriched biological pathway among the frequently mutated genes. A prevalence screening confirmed mutations in FAT4, a cadherin family gene, in 5% of gastric cancers (6/110) and FAT4 genomic deletions in 4% (3/83) of gastric tumors. Frequent mutations in chromatin remodeling genes (ARID1A, MLL3 and MLL) also occurred in 47% of the gastric cancers. We detected ARID1A mutations in 8% of tumors (9/110), which were associated with concurrent PIK3CA mutations and microsatellite instability. In functional assays, we observed both FAT4 and ARID1A to exert tumor-suppressor activity. Somatic inactivation of FAT4 and ARID1A may thus be key tumorigenic events in a subset of gastric cancers.

A whole-genome massively parallel sequencing analysis of BRCA1 mutant oestrogen receptor-negative and -positive breast cancers

BRCA1 encodes a tumour suppressor protein that plays pivotal roles in homologous recombination (HR) DNA repair, cell-cycle checkpoints, and transcriptional regulation. BRCA1 germline mutations confer a high risk of early-onset breast and ovarian cancer. In more than 80% of cases, tumours arising in BRCA1 germline mutation carriers are oestrogen receptor (ER)-negative; however, up to 15% are ER-positive. It has been suggested that BRCA1 ER-positive breast cancers constitute sporadic cancers arising in the context of a BRCA1 germline mutation rather than being causally related to BRCA1 loss-of-function. Whole-genome massively parallel sequencing of ER-positive and ER-negative BRCA1 breast cancers, and their respective germline DNAs, was used to characterize the genetic landscape of BRCA1 cancers at base-pair resolution. Only BRCA1 germline mutations, somatic loss of the wild-type allele, and TP53 somatic mutations were recurrently found in the index cases. BRCA1 breast cancers displayed a mutational signature consistent with that caused by lack of HR DNA repair in both ER-positive and ER-negative cases. Sequencing analysis of independent cohorts of hereditary BRCA1 and sporadic non-BRCA1 breast cancers for the presence of recurrent pathogenic mutations and/or homozygous deletions found in the index cases revealed that DAPK3, TMEM135, KIAA1797, PDE4D, and GATA4 are potential additional drivers of breast cancers. This study demonstrates that BRCA1 pathogenic germline mutations coupled with somatic loss of the wild-type allele are not sufficient for hereditary breast cancers to display an ER-negative phenotype, and has led to the identification of three potential novel breast cancer genes (ie DAPK3, TMEM135, and GATA4).

Long-range Transcriptome Sequencing Reveals Cancer Cell Growth Regulatory Chimeric mRNA

mRNA chimeras from chromosomal translocations often play a role as transforming oncogenes. However, cancer transcriptomes also contain mRNA chimeras that may play a role in tumor development, which arise as transcriptional or post-transcriptional events. To identify such chimeras, we developed a deterministic screening strategy for long-range sequence analysis. High-throughput, long-read sequencing was then performed on cDNA libraries from major tumor histotypes and corresponding normal tissues. These analyses led to the identification of 378 chimeras, with an unexpectedly high frequency of expression (˜2 x 10(-5) of all mRNA). Functional assays in breast and ovarian cancer cell lines showed that a large fraction of mRNA chimeras regulates cell replication. Strikingly, chimeras were shown to include both positive and negative regulators of cell growth, which functioned as such in a cell-type-specific manner. Replication-controlling chimeras were found to be expressed by most cancers from breast, ovary, colon, uterus, kidney, lung, and stomach, suggesting a widespread role in tumor development.