title: Novel multiplex primer extension and denaturing  high-performance liquid chromatography for  genotyping of the deafness gene mutations.
abstract: OBJECTIVE
  To find a rapid and accurate genotyping method for specific non-syndromic hearing loss (NSHL)-causing gene mutations for disease diagnosis in different ethnic populations.
  
  
  METHODS
  We performed a novel multiplex primer extension (PE) reaction in combination with denaturing high-performance liquid chromatography (DHPLC) to simultaneously detect and genotype the 6 most common mutations in 180 patients with NSHL (GJB2-235delC, GJB2-299delAT, PDS-A2168G, PDS IVS7-2A>G, mtDNA-A1555G, and mtDNA-C1494T) in Chinese population. This method involved the amplification of the target sequence, followed by a purification step, a multiplex PE reaction, and DHPLC analysis performed on the Transgenomic Wave DNA fragment analysis system under fully-denaturing conditions.
  
  
  RESULTS
  In a blind analysis, this technique successfully and accurately genotyped 100% of the samples simultaneously characterized by direct sequencing.
  
  
  CONCLUSIONS
  Combination of PE and DHPLC is simple, rapid, accurate, and cost-effective for genotyping common disease-causing mutations, including substitutions, insertions, and deletions in NSHL, and may be successfully used in other genetic diseases.
authors.affiliation: Department of Otolaryngology-Head and Neck Surgery, Xiangya Hospital, Central South University, Changsha 410008, China.
authors.forename: Meichao
authors.initials: M
authors.lastname: Men
authors.forename: Jinjie
authors.initials: J
authors.lastname: Xue
authors.forename: Lu
authors.initials: L
authors.lastname: Jiang
authors.forename: Honghan
authors.initials: H
authors.lastname: Wang
authors.forename: Qian
authors.initials: Q
authors.lastname: Pan
authors.forename: Yong
authors.initials: Y
authors.lastname: Feng
mesh_terms: D044466:Asian People; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D000072259:Connexin 26; D017630:Connexins / Q000235:genetics*; D004272:DNA, Mitochondrial / Q000235:genetics; D005260:Female; D005820:Genetic Testing / Q000379:methods; D005838:Genotype; D006319:Hearing Loss, Sensorineural / Q000175:diagnosis / Q000235:genetics*; D006801:Humans; D008297:Male; D026901:Membrane Transport Proteins / Q000235:genetics*; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity; D000076988:Sulfate Transporters
chemical_list: D017630:Connexins; D004272:DNA, Mitochondrial; C000607042:GJB2 protein, human; D026901:Membrane Transport Proteins; C085609:SLC26A4 protein, human; D000076988:Sulfate Transporters; D000072259:Connexin 26