Evaluation of new generation sequencing efficiency in the analysis of MEN1, CDKN2B/p15, CDKN2C/p18 CDKN1A/p21, CDKN1B/p27Kip1 and AIP genes in patients with multiple endocrine neoplasia type 1 (MEN1)

• Grant number: 13/19810-2
• Support Opportunities: Regular Research Grants
• Duration: January 01, 2014 - December 31, 2015
• Field of knowledge: Biological Sciences - Genetics - Human and Medical Genetics
• Principal Investigator: Delmar Muniz Lourenço Jr
• Grantee: Delmar Muniz Lourenço Jr
• Host Institution: Hospital das Clínicas da Faculdade de Medicina da USP (HCFMUSP). Secretaria da Saúde (São Paulo - Estado). São Paulo , SP, Brazil
• Associated researchers: Alexander Augusto de Lima Jorge ; Sueli Mieko Oba Shinjo ; Suely Kazue Nagahashi Marie

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 germinal mutation in tumor suppressor gene MEN1 (11q13). Other genes were recently related to MEN1 (CDKN2B/p15, CDKN2C/p18 CDKN1A/p21, CDKN1B/p27Kip1 and AIP). Genetic diagnosis of families with MEN1 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, including, initially, only MEN1 gene. However, limitations of this technique make it less cost-effective, mostly, the lower capacity of data generation that leads to the need of PCR products up to 600 bp to obtain a suitable read of sequencing. 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. Is added to this the need of extend the genetic study for the genes cited above in cases which the analysis of MEN1 gene results negative. Once that Sanger sequencing limitations makes difficult the analysis of these genes too, the incorporation of complete genetic diagnosis for MEN1 in clinical practice is impossible. The NGS (New Generation Sequencing) is a new tool for genetic sequencing that has a greater capacity and speed of data generation and more coverage of genetic reading, including promoters, introns and regulatory regions. 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. This study aims to validate the NGS method using as model the Sanger sequencing; evaluate quality aspects and cost-effective of NGS including MEN1 gene and the others genes recently related to syndrome; exclude, using MLPA, big germinal deletions in MEN1 gene not found by Sanger sequencing and 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, CDKN2B/p15, CDKN2C/p18 CDKN1A/p21, CDKN1B/p27Kip1 and AIP genes. This project aims to introduce the NGS as a genetic diagnosis tool for MEN1 in our center. (AU)

Publicações científicas

(6)

(Referências obtidas automaticamente do Web of Science e do SciELO, por meio da informação sobre o financiamento pela FAPESP e o número do processo correspondente, incluída na publicação pelos autores)

RODRIGUES, KARINE C.; TOLEDO, RODRIGO A.; COUTINHO, FLAVIA L.; NUNES, ADRIANA B.; MACIEL, RUI M. B.; HOFF, ANA O.; TAVARES, MARCOS C.; TOLEDO, SERGIO P. A.; LOURENCO, JR., DELMAR M.. Assessment of Depression, Anxiety, Quality of Life, and Coping in Long-Standing Multiple Endocrine Neoplasia Type 2 Patients. THYROID, v. 27, n. 5, p. 693-706, MAY 2017. (13/19810-2, 13/01476-9, 12/04698-0)

TOLEDO, SERGIO P. A.; LOURENCO, JR., DELMAR M.; SEKIYA, TOMOKO; LUCON, ANTONIO M.; BAENA, MARCOS E. S.; CASTRO, CLAUDIO C.; BORTOLOTTO, LUIZ A.; ZERBINI, MARIA C. N.; SIQUEIRA, SHEILA A. C.; TOLEDO, RODRIGO A.; et al. Penetrance and Clinical Features of Pheochromocytoma in a Six-Generation Family Carrying a Germline TMEM127 Mutation. JOURNAL OF CLINICAL ENDOCRINOLOGY & METABOLISM, v. 100, n. 2, p. E308-E318, FEB 2015. (13/19810-2, 13/01476-9)

CARVALHO, RAFAEL A.; URTREMARI, BETSAIDA; JORGE, ALEXANDER A. L.; SANTANA, LUCAS S.; QUEDAS, ELISANGELA P. S.; SEKIYA, TOMOKO; LONGUINI, VIVIANE C.; MONTENEGRO, FABIO L. M.; LERARIO, ANTONIO M.; TOLEDO, SERGIO P. A.; et al. Germline mutation landscape of multiple endocrine neoplasia type 1 using full gene next-generation sequencing. EUROPEAN JOURNAL OF ENDOCRINOLOGY, v. 179, n. 6, p. 391-407, DEC 2018. (15/25444-4, 13/19810-2, 16/07504-2, 16/07965-0, 13/15388-4)

DANTAS, NAIARA C. B.; SOARES, CARLOS E. L.; MARTINS, MANOEL R. A.; LOURENCO, JR., DELMAR M.; QUIDUTE, ANA R. P.. Giant Prolactinoma Causing Hydrocephalus and Intracranial Hypertension as First Manifestations of Multiple Endocrine Neoplasia Type 1. FRONTIERS IN ENDOCRINOLOGY, v. 10, AUG 28 2019. (13/19810-2, 16/07504-2)

BRESCIA, MARILIA D'ELBOUX GUIMARAES; RODRIGUES, KARINE CANDIDO; D'ALESSANDRO, ANDRE FERNANDES; ALVES FILHO, WELLINGTON; VAN DER PLAS, WILLEMIJN Y.; KRUIJFF, SCHELTO; ARAP, SERGIO SAMIR; TOLEDO, SERGIO PEREIRA DE ALMEIDA; MONTENEGRO, FABIO LUIZ DE MENEZES; LOURENCO, DELMAR MUNIZ. Impact of parathyroidectomy on quality of life in multiple endocrine neoplasia type 1. ENDOCRINE CONNECTIONS, v. 11, n. 6, p. 13-pg., 2022-06-01. (16/25594-9, 15/25444-4, 13/19810-2, 16/07504-2)

LOURENCO, DELMAR M., JR.; DE HERDER, WOUTER W.. Editorial: Early Genetic and Clinical Diagnosis in MEN1. FRONTIERS IN ENDOCRINOLOGY, v. 11, p. 4-pg., 2020-04-15. (15/25444-4, 16/07504-2, 13/19810-2)