title: Optimization of simultaneous screening of the main mutations involved in non-syndromic deafness using the TaqMan® OpenArray™ Genotyping platform.
abstract: BACKGROUND
  Hearing loss is the most common sensory deficit in humans, affecting approximately 10% of the global population. In developed countries, one in every 500 individuals suffers from severe to profound bilateral sensorineural hearing loss. For those up to 5 years old, the proportion is higher, at 2.7 in 1000 individuals, and for adolescents the average is 3.5 in 1000. Among the causes of hearing loss, more than 50% are related to genetic factors. To date, nearly 150 loci and 64 genes have been associated with hearing loss. Mutations in the GJB2 gene, which encodes connexin 26, constitute the main genetic cause. So far, more than 300 variations have been described in this gene.As a response to the clinical and genetic heterogeneity of hearing loss and the importance of correct molecular diagnosis of individuals with hereditary hearing loss, this study worked in the optimization for a diagnostic protocol employing a high-throughput genotyping technology.
  
  
  METHODS
  For this work, was used the TaqMan® OpenArray™ Genotyping platform. This is a high performance, high-throughput technology based on real-time PCR, which enables the evaluation of up to 3072 SNPs (Single Nucleotide Polymorphisms), point mutations, small deletions, and insertions, using a single genotyping plate. For the study, were selected the layout allowing to analyze 32 alterations in 96 individuals simultaneously. In the end, the generated results were validated by conventional techniques, as direct sequencing, Multiplex PCR and RFLP-PCR.
  
  
  RESULTS
  A total of 376 individuals were analyzed, of which 94 were healthy controls, totaling 4 plates in duplicate. All 31 of the changes analyzed were present in the nuclear genes GJB2, GJB6, CRYL1, TMC1, SLC26A4, miR-96, and OTOF, and in the mitochondrial genes MT-RNR1 and MT-TS1. The reactions were subsequently validated by established techniques (direct sequencing, multiplex PCR, and RFLP-PCR) that had previously been used to perform molecular screening of hearing loss at the Human Genetics Laboratory of the Center for Molecular Biology and Genetic Engineering (CBMEG), at the State University of Campinas (UNICAMP). In total, 11,656 genotyping reactions were performed. Of these, only 351 reactions failed, representing approximately 3.01% of the total. The average accuracy of genotyping using the OpenArray™ plates was 96.99%.
  
  
  CONCLUSIONS
  The results demonstrated the accuracy, low cost, and good reproducibility of the technique, indicating that the TaqMan® OpenArray™ Genotyping Platform is a useful and reliable tool for application in molecular diagnostic testing of hearing loss.
authors.affiliation: Human Molecular Genetics Laboratory, Center for Molecular Biology and Genetic Engineering (CBMEG), State University of Campinas - UNICAMP, Campinas, SP, Brazil. fabio.arrojo@cbmeg.unicamp.br.
authors.forename: Fábio Tadeu Arrojo
authors.initials: FT
authors.lastname: Martins
authors.forename: Priscila Zonzini
authors.initials: PZ
authors.lastname: Ramos
authors.forename: Maria Carolina Costa Melo
authors.initials: MC
authors.lastname: Svidnicki
authors.forename: Arthur Menino
authors.initials: AM
authors.lastname: Castilho
authors.forename: Edi Lúcia
authors.initials: EL
authors.lastname: Sartorato
mesh_terms: D000072259:Connexin 26; D000075302:Connexin 30; D017630:Connexins / Q000235:genetics; D003459:Crystallins / Q000235:genetics; D017353:Gene Deletion; D005838:Genotype; D034381:Hearing Loss / Q000175:diagnosis* / Q000235:genetics; D006801:Humans; D026901:Membrane Transport Proteins / Q000235:genetics; D017354:Point Mutation; D020641:Polymorphism, Single Nucleotide; D011933:Reagent Kits, Diagnostic; D060888:Real-Time Polymerase Chain Reaction; D012984:Software; D000076988:Sulfate Transporters
chemical_list: C472003:CRYL1 protein, human; D000075302:Connexin 30; D017630:Connexins; D003459:Crystallins; C000607042:GJB2 protein, human; C491020:GJB6 protein, human; D026901:Membrane Transport Proteins; D011933:Reagent Kits, Diagnostic; C085609:SLC26A4 protein, human; D000076988:Sulfate Transporters; D000072259:Connexin 26