title: Screening of genetic alterations related to non-syndromic hearing loss using MassARRAY iPLEX® technology.
abstract: BACKGROUND
  Recent 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.
  
  
  METHODS
  The 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.
  
  
  RESULTS
  An 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.
  
  
  CONCLUSIONS
  The 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.
authors.affiliation: Human Molecular Genetics Laboratory, Molecular Biology and Genetic Engineering Center (CBMEG), University of Campinas (UNICAMP), Campinas, São Paulo, Brazil. mcarolinasvid@gmail.com.
authors.forename: Maria Carolina Costa Melo
authors.initials: MC
authors.lastname: Svidnicki
authors.affiliation: Human Molecular Genetics Laboratory, Molecular Biology and Genetic Engineering Center (CBMEG), University of Campinas (UNICAMP), Campinas, São Paulo, Brazil. silvacosta.sm@gmail.com.
authors.forename: Sueli Matilde
authors.initials: SM
authors.lastname: Silva-Costa
authors.affiliation: Human Molecular Genetics Laboratory, Molecular Biology and Genetic Engineering Center (CBMEG), University of Campinas (UNICAMP), Campinas, São Paulo, Brazil. priscila.zonzini@yahoo.com.br.
authors.forename: Priscila Zonzini
authors.initials: PZ
authors.lastname: Ramos
authors.affiliation: Human Molecular Genetics Laboratory, Molecular Biology and Genetic Engineering Center (CBMEG), University of Campinas (UNICAMP), Campinas, São Paulo, Brazil. nathalia.zps@gmail.com.
authors.forename: Nathalia Zocal Pereira
authors.initials: NZ
authors.lastname: dos Santos
authors.affiliation: Human Molecular Genetics Laboratory, Molecular Biology and Genetic Engineering Center (CBMEG), University of Campinas (UNICAMP), Campinas, São Paulo, Brazil. fabio.arrojo@gmail.com.
authors.forename: Fábio Tadeu Arrojo
authors.initials: FT
authors.lastname: Martins
authors.affiliation: ENT Department, Faculty of Medical Sciences, University of Campinas (UNICAMP), Campinas, São Paulo, Brazil. arthurcastilho@gmail.com.
authors.forename: Arthur Menino
authors.initials: AM
authors.lastname: Castilho
authors.affiliation: Human Molecular Genetics Laboratory, Molecular Biology and Genetic Engineering Center (CBMEG), University of Campinas (UNICAMP), Campinas, São Paulo, Brazil. sartor@unicamp.br.
authors.forename: Edi Lúcia
authors.initials: EL
authors.lastname: Sartorato
mesh_terms: D000070585:Cadherin Related Proteins; D015820:Cadherins / Q000235:genetics; D000072259:Connexin 26; D017630:Connexins / Q000235:genetics*; D004252:DNA Mutational Analysis / Q000379:methods; D005820:Genetic Testing / Q000379:methods; D060005:Genotyping Techniques / Q000191:economics / Q000379:methods; D034381:Hearing Loss / Q000235:genetics*; D059014:High-Throughput Nucleotide Sequencing / Q000191:economics / Q000379:methods*; D006801:Humans; D026901:Membrane Transport Proteins / Q000235:genetics; D009154:Mutation; D009218:Myosins / Q000235:genetics; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D000076988:Sulfate Transporters
chemical_list: C442480:CDH23 protein, human; D000070585:Cadherin Related Proteins; D015820:Cadherins; D017630:Connexins; C000607042:GJB2 protein, human; C485245:MYO15A protein, human; D026901:Membrane Transport Proteins; C085609:SLC26A4 protein, human; D000076988:Sulfate Transporters; D000072259:Connexin 26; D009218:Myosins