Screening of genetic alterations related to non-syndromic hearing loss using MassARRAY iPLEX® technology

BMC Medical Genetics

Research Article

Arthur Menino Castilho¹  Edi Lúcia Sartorato²  Priscila Zonzini Ramos²  Nathalia Zocal Pereira dos Santos²  Sueli Matilde Silva-Costa²  Fábio Tadeu Arrojo Martins²  Maria Carolina CCosta CMelo Svidnicki² 

[1] ENT Department, Faculty of Medical Sciences, University of Campinas (UNICAMP), Campinas, São Paulo, Brazil;Human Molecular Genetics Laboratory, Molecular Biology and Genetic Engineering Center (CBMEG), University of Campinas (UNICAMP), Campinas, São Paulo, Brazil;

关键词: Hearing loss;    Genetic testing;    MassARRAY;   

DOI  :  10.1186/s12881-015-0232-8

 received in 2014-10-08, accepted in 2015-09-15,  发布年份 2015

来源: Springer

【 摘 要 】

BackgroundRecent advances in molecular genetics have enabled to determine the genetic causes of non-syndromic hearing loss, and more than 100 genes have been related to the phenotype. Due to this extraordinary genetic heterogeneity, a large percentage of patients remain without any molecular diagnosis. This condition imply the need for new methodological strategies in order to detect a greater number of mutations in multiple genes. In this work, we optimized and tested a panel of 86 mutations in 17 different genes screened using a high-throughput genotyping technology to determine the molecular etiology of hearing loss.MethodsThe technology used in this work was the MassARRAY iPLEX® platform. This technology uses silicon chips and DNA amplification products for accurate genotyping by mass spectrometry of previous reported mutations. The generated results were validated using conventional techniques, as direct sequencing, multiplex PCR and RFLP-PCR.ResultsAn initial genotyping of control subjects, showed failures in 20 % of the selected alterations. To optimize these results, the failed tests were re-designed and new primers were synthesized. Then, the specificity and sensitivity of the panel demonstrated values above 97 %. Additionally, a group of 180 individuals with NSHL without a molecular diagnosis was screened to test the diagnostic value of our panel, and mutations were identified in 30 % of the cases. In 20 % of the individuals, it was possible to explain the etiology of the HL. Mutations in GJB2 gene were the most prevalent, followed by other mutations in in SLC26A4, CDH23, MT-RNR1, MYO15A, and OTOF genes.ConclusionsThe MassARRAY technology has the potential for high-throughput identification of genetic variations. However, we demonstrated that optimization is required to increase the genotyping success and accuracy. The developed panel proved to be efficient and cost-effective, being suitable for applications involving the molecular diagnosis of hearing loss.

【 授权许可】

CC BY   
© Svidnicki et al. 2015