Evaluation of a high throughput method for the detection of mutations associated with thrombosis and hereditary hemochromatosis in Brazilian blood donors

PLoS One. 2015 May 8;10(5):e0125460. doi: 10.1371/journal.pone.0125460. eCollection 2015.

Abstract

Background: The aim of this study was to evaluate the OpenArray platform for genetic testing of blood donors and to assess the genotype frequencies of nucleotide-polymorphisms (SNPs) associated with venous thrombosis (G1691A and G20210A), hyperhomocysteinemia (C677T, A1298C), and hereditary hemochromatosis (C282Y, H63D and S65C) in blood donors from Sao Paulo, Brazil.

Methods: We examined 400 blood donor samples collected from October to November 2011. The SNPs were detected using OpenArray technology. The blood samples were also examined using a real-time PCR-FRET system to compare the results and determine the accuracy of the OpenArray method.

Results: We observed 100% agreement in all assays tested, except HFE C282Y, which showed 99.75% agreement. The HFE C282Y assay was further confirmed through direct sequencing, and the results showed that OpenArray analysis was accurate. The calculated frequencies of each SNP were FV G1691A 98.8% (G/G), 1.2% (G/A); FII G2021A 99.5% (G/G), 0.5% (G/A); MTHFR C677T 45.5% (C/C), 44.8% (C/T), 9.8% (T/T); MTHFR A1298C 60.3% (A/A), 33.6% (A/C), 6.1% (C/C); HFE C282Y 96%(G/G), 4%(G/A), HFE H63D 78.1%(C/C), 20.3% (C/G), 1.6% (G/G); and HFE S65C 98.1% (A/A), 1.9% (A/T).

Conclusion: Taken together, these results describe the frequencies of SNPs associated with diseases and are important to enhance our current knowledge of the genetic profiles of Brazilian blood donors, although a larger study is needed for a more accurate determination of the frequency of the alleles. Furthermore, the OpenArray platform showed a high concordance rate with standard FRET RT-PCR.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adult
  • Alleles
  • Blood Donors*
  • Brazil
  • Factor V / genetics
  • Female
  • Gene Expression
  • Gene Frequency
  • Genetic Testing
  • Genotype
  • Hemochromatosis / diagnosis
  • Hemochromatosis / genetics*
  • Hemochromatosis Protein
  • High-Throughput Screening Assays
  • Histocompatibility Antigens Class I / genetics
  • Humans
  • Hyperhomocysteinemia / diagnosis
  • Hyperhomocysteinemia / genetics*
  • Male
  • Membrane Proteins / genetics
  • Methylenetetrahydrofolate Reductase (NADPH2) / genetics
  • Middle Aged
  • Oligonucleotide Array Sequence Analysis
  • Polymorphism, Single Nucleotide*
  • Prothrombin / genetics
  • Real-Time Polymerase Chain Reaction
  • Sensitivity and Specificity
  • Venous Thrombosis / diagnosis
  • Venous Thrombosis / genetics*

Substances

  • HFE protein, human
  • Hemochromatosis Protein
  • Histocompatibility Antigens Class I
  • Membrane Proteins
  • Factor V
  • Prothrombin
  • MTHFR protein, human
  • Methylenetetrahydrofolate Reductase (NADPH2)

Associated data

  • figshare/10.6084/m9.figshare.1359935

Grants and funding

ECS was supported through funding from Conselho Nacional de Desenvolvimento Científico e Tecnlógico (CNPq) grant number 302202/2011-0, and JV was supported through Coordenação de aperfeiçoamento pessoal de nível superior (CAPES). This work was also supported through the Fundação Pró-Sangue Hemocentro de São Paulo and the Fundação Faculdade de Medicina. VDTN, CAN, and NG received no specific funding for this work. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.