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

Mee Lee Looi, Mageswary Sivalingam, Nor Diana Husin, Fara Zela Mohd Radin, Raihana Mohamed Isa, Syed Zulkifli Syed Zakaria, Noor Hamidah Hussin, Hamidah Alias, Zarina Abdul Latiff, Hishamshah Ibrahim, A. Rahman A. Jamal

Research output: Contribution to journalArticle

3 Citations (Scopus)

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. Conclusion: We revealed that the MS method is a sensitive, high-throughput, highly automated, flexible, and cost-effective alternative to conventional ß-thalassemia genotyping methods.

Original languageEnglish
Pages (from-to)999-1002
Number of pages4
JournalClinica Chimica Acta
Volume412
Issue number11-12
DOIs
Publication statusPublished - 12 May 2011

Fingerprint

beta-Globins
Matrix-Assisted Laser Desorption-Ionization Mass Spectrometry
Mass spectrometry
Mass Spectrometry
Refractory materials
Mutation
Amplification
Assays
Poly T
Screening
Poly C
beta-Thalassemia
Poly A
Alleles
Throughput
Thalassemia
Genetic Counseling
Population Genetics
Economics
Costs

Keywords

  • Beta globin gene mutation
  • Beta thalassemia
  • MALDI-TOF
  • Mass spectrometry
  • Molecular testing
  • Multiplexed genotyping

ASJC Scopus subject areas

  • Biochemistry
  • Clinical Biochemistry
  • Biochemistry, medical

Cite this

Multiplexed genotyping of beta globin mutations with MALDI-TOF mass spectrometry. / Looi, Mee Lee; Sivalingam, Mageswary; Husin, Nor Diana; Radin, Fara Zela Mohd; Isa, Raihana Mohamed; Syed Zakaria, Syed Zulkifli; Hussin, Noor Hamidah; Alias, Hamidah; Abdul Latiff, Zarina; Ibrahim, Hishamshah; A. Jamal, A. Rahman.

In: Clinica Chimica Acta, Vol. 412, No. 11-12, 12.05.2011, p. 999-1002.

Research output: Contribution to journalArticle

Looi, Mee Lee ; Sivalingam, Mageswary ; Husin, Nor Diana ; Radin, Fara Zela Mohd ; Isa, Raihana Mohamed ; Syed Zakaria, Syed Zulkifli ; Hussin, Noor Hamidah ; Alias, Hamidah ; Abdul Latiff, Zarina ; Ibrahim, Hishamshah ; A. Jamal, A. Rahman. / Multiplexed genotyping of beta globin mutations with MALDI-TOF mass spectrometry. In: Clinica Chimica Acta. 2011 ; Vol. 412, No. 11-12. pp. 999-1002.
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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. Conclusion: We revealed that the MS method is a sensitive, high-throughput, highly automated, flexible, and cost-effective alternative to conventional {\ss}-thalassemia genotyping methods.",
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AU - Sivalingam, Mageswary

AU - Husin, Nor Diana

AU - Radin, Fara Zela Mohd

AU - Isa, Raihana Mohamed

AU - Syed Zakaria, Syed Zulkifli

AU - Hussin, Noor Hamidah

AU - Alias, Hamidah

AU - Abdul Latiff, Zarina

AU - Ibrahim, Hishamshah

AU - A. Jamal, A. Rahman

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N2 - 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. Conclusion: We revealed that the MS method is a sensitive, high-throughput, highly automated, flexible, and cost-effective alternative to conventional ß-thalassemia genotyping methods.

AB - 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. Conclusion: We revealed that the MS method is a sensitive, high-throughput, highly automated, flexible, and cost-effective alternative to conventional ß-thalassemia genotyping methods.

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KW - Mass spectrometry

KW - Molecular testing

KW - Multiplexed genotyping

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