Multiplexed genotyping of beta globin mutations with MALDI-TOF mass spectrometry

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. We used th...

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Bibliographic Details
Published in:Clinica chimica acta 2011-05, Vol.412 (11-12), p.999-1002
Main Authors: Looi, Mee-Lee, Sivalingam, Mageswary, Husin, Nor Diana, Radin, Fara Zela Mohd, Isa, Raihana Mohamed, Zakaria, Syed Zulkifli Syed, Hussin, Noor Hamidah, Alias, Hamidah, Latiff, Zarina Abd, Ibrahim, Hishamshah, Jamal, Rahman
Format: Article
Language:English
Subjects:
Beta globin gene mutation
Beta thalassemia
beta-Globins - genetics
beta-Thalassemia - genetics
DNA Mutational Analysis - methods
Genotyping Techniques - methods
Humans
MALDI-TOF
Mass spectrometry
Molecular testing
Multiplexed genotyping
Mutation
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization - methods
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Summary: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. 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. 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. We revealed that the MS method is a sensitive, high-throughput, highly automated, flexible, and cost-effective alternative to conventional ß-thalassemia genotyping methods.
ISSN:0009-8981
1873-3492
DOI:10.1016/j.cca.2011.02.006