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

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.

Regulatory Mutation Study in Cases with Unsolved Hypochromic Microcytic Anemia and α-Major Regulatory Element Haplotype Analysis in Iran.

α-Thalassemia (α-thal) is an inherited blood disorder with different clinical manifestations. Although genetic causes of anemia are identified routinely in the majority of α-thal cases, a pathogenic variant in a few cases remains undiagnosed. In this study, some reported regulatory mutations have been investigated in five unsolved α-thal carriers. α-Major regulatory element (α-MRE) haplotype analysis has also been performed in Iran for the first time. Four regions, including the HBA2 core promoter, the highly conserved sequence of hypersensitive-40 (HS-40), a region containing regulatory single nucleotide polymorphism (SNP) CR062116, and a region containing rs7203560, were screened for changes by Sanger sequencing in a total of five unsolved suspected α-thal carriers. The frequencies of α-MRE haplotypes B and C were also determined in control samples with normal hematological indices. No pathogenic variant was found in the investigated regions. Haplotype frequencies observed for B and C haplotypes fell into the range of frequencies observed in previous studies. The investigated genotypes in the control group were in the Hardy-Weinberg equilibrium. This study can provide evidence that there is no association between the B haplotype and microcytic hypochromic anemia. The cause of anemia remains a mystery in our unsolved cases, which demonstrates the need for further studies on the causes of hypochromic microcytic anemia in individuals with intact α- and ß-globin genes without iron deficiency.