Development of a Quantitative Multiplex PCR to Detect Three Common Alpha Thalassemia Deletions

Abstract

Alpha thalassemia is an autosomal recessive genetic disorder with a high prevalence in the Middle East. The severe form of alpha-thalassemia is incompatible with life and can cause significant obstetric complications in the mother. Therefore, it is important to determine the genotype in parents who have a chance of having a fetus with one of the severe forms of this disease. A total of 112 samples that were previously analyzed for common alpha thalassemia mutations in Iran were used in this study. A new multiplex PCR including quantitative polymerase chain reaction to amplify the homologous regions of the alpha-globin gene cluster and fluorescent gap PCR was designed to identify −α3.7, −α4.2, --MED deletions. The ROC curve was used to determine the optimum cutoff points. Statistical analysis showed that there is a significant difference between the peak height ratios for different genotypes. The peak corresponding to the 297 bp fragment resulting from the amplification of the allele with MED-I deletion was detected in all the samples with this deletion. Different cutoffs for a range of sensitivities and specificities were determined by the ROC curve. The suggested method can identify three common large deletions in the alpha-globin gene cluster. A study with a larger sample size can provide more accurate information about the sensitivity and specificity of this test.

Keywords:

• Alpha thalassemia
• multiplex PCR
• diagnosis
• deletion mutation

Acknowledgments

We are grateful to all the staff of the Molecular Medicine Department of Pasteur Institute of Iran and the Medical Genetics Department of Shiraz University of Medical Sciences.

Author contributions

Z. Hajimohammadi: performed the experiments, analyzed the data, and wrote the manuscript; S. Alimohammadi-Bidhendi: collaborated in the experiments; F. Bagheri Amiri: performed statistical analyses; M. Karimipoor; studied concepts, analysis, and interpretation of results. E. Davoudi-Dehaghani and M. Entezam: designed the study, analyzed and interpreted the results, and revised the paper.

Disclosure statement

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.

Additional information

Funding

This work was supported by the Pasteur Institute of Iran [grant number 66001856], and the Shiraz University of Medical Sciences [grant number 23732].