Evaluation of new generation sequencing in genetic diagnosis of multiple endocrine neoplasia type 1

Abstract

The multiple endocrine neoplasia type 1 (MEN1) is a genetic, autossomic and dominant disease and is correlated with the development of endocrine tumors affecting pituitary gland, parathyroid, endocrine pancreas or duodenum. It's mainly caused by a germinative mutation in tumor suppressor gene MEN1 (11q13). The tumorigenesis follow the Knudson's model (1971). Genetic diagnosis of families with MEN1 is essential to recognizes asymptomatic mutation carriers, and allows an earlier detection and treatment of tumors leading to a reduction of mortality and morbidity associated to MEN1. Furthermore, it can exclude family members that do not carry mutations from the periodical screening. The genetic diagnosis for MEN1 is held using Sanger sequencing. However, limitations of this technique make it less cost-effective, mostly, the less capacity of data generation that leads to the need of PCR products up to 600 bp to obtain a suitable read. Moreover, specific conditions of the MEN1 gene contributes to make this process more laborious and expensive, like the need to read all gene sequence (9kb) to make a correct analysis due to the absence of "hot spots". This way, the need of "fragmentation" to allow the sequencing can hide important information to disease development, mostly in introns. These limitations preclude the clinical application of genetic diagnosis of MEN1. Since 2005, new technologies are available; they are called New Generation Sequencing (NGS) and consist in a new tool that allow the same sequencing, but with a larger generation data capacity, making them more attractive and cost-effective. The NGS also gives a higher speed to the process of data acquiring and is able make do the complete read of gene, including promoters and introns. Therefore, make the results more informative, do not forgetting quality aspects. Among lot of options of NGS available, lighter platforms are recommended, for example, Ion PGM and Illumina MiSeq. A strong tendency has been shown in order to change the Sanger sequencing to NGS, including clinical application to genetic diagnosis of complex diseases and inherited cancer. However, has not previous studies evaluating NGS to MEN1 genetic diagnosis. Thus, arises the interest to evaluate NGS like a genetic diagnosis method for MEN1, comparing with Sanger sequencing. This study aims to validate the NGS method using as model the Sanger sequencing and evaluate quality aspects and cost-effective of NGS. For this, will be analyzed 50 index-cases with MEN1. In a preliminary study, using Ion PGM, were analyzed 4 index-cases with a previously known germinative mutation in MEN1 gene. Was obtained an average coverage >100x, with 100% of MEN1 gene with at least 10 reads, allowing an accurate evaluation of every germinative mutation. Were found also an average of 37 SNPs per patient. However, a lot of in/dels were generated, making difficult the results analysis. These in/dels were generated by a specific limitation of Ion PGM platform. This way, Illumina MiSeq platform was chose due to the results analysis be made onboard, do not generate false in/dels and the less probability of errors in G-C rich sequences, common in MEN1. The sample enrichment will be made using long range PCR that will amplify all genomic region of MEN1 gene. This reaction was standardized during the preliminary study using Ion PGM platform and will be applied with Illumina MiSeq. Thus, this project pretends to introduce this technique as a genetic diagnosis tool for MEN1. (AU)