Prioritizing single-nucleotide variations that potentially regulate alternative splicing

Recent evidence suggests that many complex diseases are caused by genetic variations that play regulatory roles in controlling gene expression. Most genetic studies focus on nonsynonymous variations that can alter the amino acid composition of a protein and are therefore believed to have the highest impact on phenotype. Synonymous variations, however, can also play important roles in disease pathogenesis by regulating pre-mRNA processing and translational control. In this study, we systematically survey the effects of single-nucleotide variations (SNVs) on binding affinity of RNA-binding proteins (RBPs). Among the 10,113 synonymous SNVs identified in 697 individuals in the 1,000 Genomes Project and distributed by Genetic Analysis Workshop 17 (GAW17), we identified 182 variations located in alternatively spliced exons that can significantly change the binding affinity of nine RBPs whose binding preferences on 7-mer RNA sequences were previously reported. We found that the minor allele frequencies of these variations are similar to those of nonsynonymous SNVs, suggesting that they are in fact functional. We propose a workflow to identify phenotype-associated regulatory SNVs that might affect alternative splicing from exome-sequencing-derived genetic variations. Based on the affecting SNVs on the quantitative traits simulated in GAW17, we further identified two and four functional SNVs that are predicted to be involved in alternative splicing regulation in traits Q1 and Q2, respectively

Mouse embryonic stem cell–based functional assay to evaluate mutations in BRCA2

Individuals with mutations in breast cancer susceptibility genes BRCA1 and BRCA2 have up to an 80% risk of developing breast cancer by the age of 70. Sequencing-based genetic tests are now available to identify mutation carriers in an effort to reduce mortality through prevention and early diagnosis. However, lack of a suitable functional assay hinders the risk assessment of more than 1,900 BRCA1 and BRCA2 variants in the Breast Cancer Information Core database that do not clearly disrupt the gene product. We have established a simple, versatile and reliable assay to test for the functional significance of mutations in BRCA2 using mouse embryonic stem cells (ES cells) and bacterial artificial chromosomes and have used it to classify 17 sequence variants. The assay is based on the ability of human BRCA2 to complement the loss of endogenous Brca2 in mouse ES cells. This technique may also serve as a paradigm for functional analysis of mutations found in other genes linked to human diseases. © 2008 Nature Publishing Group.Link_to_subscribed_fulltex

Colocalisation of predicted exonic splicing enhancers in BRCA2 with reported sequence variants

Disruption of the breast cancer susceptibility gene BRCA2 is associated with increased risk of developing breast and ovarian cancer. Over 1800 sequence changes in BRCA2 have been reported, although for many the pathogenicity is unclear. Classifying these changes remains a challenge, as they may disrupt regulatory sequences as well as the primary protein coding sequence. Sequence changes located in the splice site consensus sequences often disrupt splicing, however sequence changes located within exons are also able to alter splicing patterns. Unfortunately, the presence of these exonic splicing enhancers (ESEs) and the functional effect of variants within ESEs it is currently difficult to predict. We have previously developed a method of predicting which sequence changes within exons are likely to affect splicing, using BRCA1 as an example. In this paper, we have predicted ESEs in BRCA2 using the web-based tool ESEfinder and incorporated the same series of filters (increased threshold, 125 nt limit and evolutionary conservation of the motif) in order to identify predicted ESEs that are more likely to be functional. Initially 1114 ESEs were predicted for BRCA2, however after all the filters were included, this figure was reduced to 31, 3% of the original number of predicted ESEs. Reported unclassified sequence variants in BRCA2 were found to colocalise to 55% (17/31) of these conserved ESEs, while polymorphisms colocalised to 0 of the conserved ESEs. In summary, we have identified a subset of unclassified sequence variants in BRCA2 that may adversely affect splicing and thereby contribute to BRCA2 disruption

Cowden syndrome: mucocutaneous lesions as precursors of internal malignancy

BACKGROUND: Cowden syndrome is an autosomal-dominant hereditary cancer syndrome with high variability and susceptibility. It is characterized by multiple hamartomas and neoplasms of ectodermal, endodermal and mesodermal origin affecting many organs and also by the increased risk of development of internal malignancies. CASE REPORT: A 62-year-old woman was referred to our Maxillofacial Unit with a hamartomatous mass of the left lateral tongue which had slowly grown and was obstructing normal speech and restricting oral intake. The patient had a known history of Cowden syndrome and underwent excision of the lesion under general anaesthetic. DISCUSSION: Orofacial mucocutaneous features are very common in multiple hamartoma and neoplasia syndrome with almost up to 90% of the patients being affected. These cutaneous and mucosal lesions, which are predominantly benign, often manifest prior to the development of the internal malignant tumours associated with the syndrome. CONCLUSIONS: The prompt identification of Cowden syndrome's plethoric signs and symptoms can lead to appropriate surveillance and multidisciplinary management. Oral manifestations are frequent, prominent and usually precede the establishment of malignant tumours of visceral organs; hence, the maxillofacial surgeon or general dentist may have a significant role in the recognition of the disease. Overall prognosis is dependent on prevention or early treatment of internal malignancies; consequently, early diagnosis together with frequent follow-up forms the cornerstone of management