title: Multiplexed genotyping of beta globin mutations with MALDI-TOF mass spectrometry.
abstract: BACKGROUND
  Beta thalassemia represents a great heterogeneity as over 300 mutations have been identified and each population at-risk has its own spectrum of mutations. Molecular characterization with high accuracy, sensitivity and economics is required for population screening and genetic counseling.
  
  
  METHODS
  We used the MALDI-TOF mass spectrometry (MS) platform to develop novel multiplex assays for comprehensive detection of 27 mutations in beta-thalassemia patients. Six multiplex assays were designed to detect 13 common known ß-mutations, namely CD41/42, CD71/72, IVS1-5, IVS1-1, CD26, IVS2-654, CAP+1, CD19, -28, -29, IVS1-2, InCD (T-G) and CD17; and 14 rare ß-mutations, i.e. InCD (A-C), CD8/9, CD43, -86, CD15, Poly A, Poly T/C, IVS2-1, CD1, CD35/36, CD27/28, CD16, CD37, and 619bpDEL in 165 samples. We compared the efficiencies of genotyping by MS and Amplification Refractory Mutation System (ARMS). Discrepant results were confirmed by sequencing analysis.
  
  
  RESULTS
  A total of 88.7% (260/293 allele) of MS and ARMS results was in agreement. More than fifty percent of the discrepant result was due to the false interpretation of ARMS results. Failed CD19 assay by MS method might be due to the assay design. The MS method detected 5 rare ß-mutations (CD15, CD35/36, CD8/9, Poly A and Poly T/C) presented in 13 alleles, which were not included in the ARMS screening panel.
  
  
  CONCLUSIONS
  We revealed that the MS method is a sensitive, high-throughput, highly automated, flexible, and cost-effective alternative to conventional ß-thalassemia genotyping methods.
authors.affiliation: UKM Medical Molecular Biology Institute, Department of Haematology, Faculty of Medicine, Universiti Kebangsaan Malaysia, Malaysia.
authors.forename: Mee-Lee
authors.initials: ML
authors.lastname: Looi
authors.forename: Mageswary
authors.initials: M
authors.lastname: Sivalingam
authors.forename: Nor Diana
authors.initials: ND
authors.lastname: Husin
authors.forename: Fara Zela Mohd
authors.initials: FZ
authors.lastname: Radin
authors.forename: Raihana Mohamed
authors.initials: RM
authors.lastname: Isa
authors.forename: Syed Zulkifli Syed
authors.initials: SZ
authors.lastname: Zakaria
authors.forename: Noor Hamidah
authors.initials: NH
authors.lastname: Hussin
authors.forename: Hamidah
authors.initials: H
authors.lastname: Alias
authors.forename: Zarina Abd
authors.initials: ZA
authors.lastname: Latiff
authors.forename: Hishamshah
authors.initials: H
authors.lastname: Ibrahim
authors.forename: Rahman
authors.initials: R
authors.lastname: Jamal
mesh_terms: D004252:DNA Mutational Analysis / Q000379:methods*; D060005:Genotyping Techniques / Q000379:methods*; D006801:Humans; D009154:Mutation; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D055544:beta-Globins / Q000235:genetics*; D017086:beta-Thalassemia / Q000235:genetics
chemical_list: D055544:beta-Globins