Pyrosequencing for detection of mutations in the connexin 26 (GJB2) and mitochondrial 12S RNA (MTRNR1) genes associated with hereditary hearing loss

Alessandro Ferraris, Eric Rappaport, Rosa Santacroce, Eleanor Pollak, Ian Krantz, Stephen Toth, Frida Lysholm, Maurizio Margaglione, Gabriella Restagno, Bruno Dallapiccola, Saul Surrey, Paolo Fortina

Research output: Contribution to journalArticlepeer-review


Hereditary hearing loss (HHL) is one of the most common congenital disorders and is highly heterogeneous. Mutations in the connexin 26 (CX26) gene (GJB2) account for about 20% of all cases of childhood deafness, and approach 50% in documented recessive cases of non-syndromic hearing loss. In addition, a single mitochondrial DNA mutation, mt1555A>G, in the 12S rRNA gene (MTRNR1), is associated with familial cases of progressive deafness. Effective screening of populations for HHL necessitates rapid assessment of several of these potential mutation sites. Pyrosequencing links a DNA synthesis protocol for determining sequence to an enzyme cascade that generates light whenever pyrophosphate is released during primer strand elongation. We assessed the ability of Pyrosequencing to detect common mutations causing HHL. Detection of the most common CX26 mutations in individuals of Caucasian (35delG), Ashkenazi (167delT), and Asian (235delC, V37I) descent was confirmed by Pyrosequencing. A total of 41 different mutations in the CX26 gene and the mitochondrial mt1555A>G mutation were confirmed. Genotyping of up to six different adjacent mutations was achieved, including simultaneous detection of 35delG 35deIG and 167delT. Accurate and reproducible results were achieved taking advantage of assay flexibility and experimental conditions easily optimized for a high degree of standardization and cost-effectiveness. The standardized sample preparation steps, including target amplification by PCR and preparation of single-stranded template combined with automated sequence reaction and automated genotype scoring, positions this approach as a potentially high throughput platform for SNP/mutation genotyping in a clinical laboratory setting.

Original languageEnglish
Pages (from-to)312-320
Number of pages9
JournalHuman Mutation
Issue number4
Publication statusPublished - 2002


  • Connexin 26
  • CX26
  • Deafness
  • DFNB1
  • Genotyping
  • GJB2
  • Hereditary hearing loss
  • HHL
  • Mitochondrial 12S rRNA
  • mtRNA
  • MTRNR1
  • Mutation detection
  • Pyrosequencing

ASJC Scopus subject areas

  • Genetics
  • Genetics(clinical)


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