Using PCR to Identify the Mutation of α-Thalassemia

Chia-Chia Yu


Thalassemia is a recessively trait inherited disease of the red blood cells. In thalassemia, the genetic defect results in reduced presence of the normal hemoglobin subunit chains. The thalassemias are classified according to which subunit chain of the hemoglobin molecule is affected. In α-thalassemia, the production of α subunit chain is deficient, whereas in β-thalassemia the production of β subunit chain is defective.

The estimated prevalence of the disease-causing mutation is 16% in people from Cyprus, 3-14% in Thailand, and 3-8% in populations from India, Pakistan, Bangladesh, and China. A lower prevalence has been reported from black people in Africa (0.9%) and northern Europe (0.1%). In Southeast Asia, compound heterozygotes and homozygotes may have anemia which is mild to severe (hemoglobin HB H disease) or lethal (Hb Bartís hydrops fetalis). In Taiwan, a high incidence of the deletion and point mutation has been reported. Unlike β-thalassemia, in which nondeletional mutations predominate, over 95% of recognized α-thalassemia involves deletion of 1 or both α-globin genes from chromosome 16p13.3 (Figure 1).

The α-thalassemia involve the genes HBA1 (Online Mendelian Inheritance in Man, OMIM, 141800) and HBA2 (Online Mendelian Inheritance in Man, OMIM, 141850). In this paper, the identification of mutation in HBA1 and HBA2 genes which lead to α-thalassemia was analyzed by PCR and using specific primers to these two genes. My genomic DNA was used as a suspect sample for thalassemia, whereas Dr. Rubinís genomic DNA was used as a wild-type control.


Figure 1-The location and structure of HBA2 and HBA1 genes on chromosome 16p13.3. (A) Schematic representation of HBA2 and HBA1genes on chromosome 16p13.3. (B) and (C) present schematic representations of the structure of HBA2 and HBA1genes, respectively. The alpha-2 (HBA2) and alpha-1 (HBA1) coding sequences are identical. These genes differ slightly in the 5' untranslated regions and the introns, and they differ significantly in the 3' untranslated regions. Two alpha chains plus two beta chains constitute HbA, which in normal adult life comprises about 97% of the total hemoglobin; alpha chains combine with delta chains to constitute HbA-2, which with HbF (fetal hemoglobin) makes up the remaining 3% of adult hemoglobin. Alpha thalassemias result from deletions of each of the alpha genes as well as deletions of both HBA2 and HBA1. (adapted from NCBI)

Figure 2-Gel analysis of PCR products. (A) My Genomic DNA. (B) Dr. Rubinís genomic DNA. ―: negative control. The letter above each well indicates which primer set was used for PCR.

Figure 3-The DNA sequence alignments of my genomic DNA (CYU) and Dr. Rubinís genomic DNA (Rubin) in reference with the DNA sequence published on the NCBI website (NCBI). (A) The DNA sequence alignments of HBA2 gene. (B) The DNA sequence alignments of HBA1 gene.

Figure 4-The chromatogram showed these two possibilities, G and A, at the 107th nucleotide of my HBA1 gene. This is a SNP (rs33939620) which has been reported on the NCBI website.

Since I was suspect of thalassemia, the objective of this study was to identify if there is any mutation in HBA1 and HBA2 genes in my genomic DNA. Using primers specific to the HBA1 and HBA2 genes, these two genes were amplified by PCR and then sequenced. Dr. Rubinís genomic DNA was used as a wild-type control.

Gel analysis of PCR products revealed that HBA2 and HBA1 genes were successfully amplified by PCR. The PCR products were approximately 600 bp (Fig 2). The analysis of DNA sequence alignments revealed that except two SNPs and several deletions in 5í UTR were found in my HBA2 and HBA1 genes, the rest of the DNA sequences of my two genes are identical as those normal DNA sequences published on the NCBI (Fig 3). The SNP: rs33939620 found in my HBA1 gene have been reported to cause α-thalassemia (Fig 4).

Further studies can focus on confirming that the SNP: rs33939620 is a real mutation, the influence of 5í UTR deletions on gene expression, and the expression of my HBA2 and HBA1 genes are different from the normal level. Other genes involved in β-thalassemia and δ-thalassemia havenít been examined in my case yet. Future research on identification if there is any mutation present in these genes is also necessary.

Please refer to the attached full paper for more details.

Full Paper


I would like to thank Dr. Rubin for providing genomic DNA and guidance of this project. Also, lots of thank to Leleesha, Joe, Liu Bo, and Jinsong Qiu for helping me to complete this project.

This document was last modified 05/09/2007.
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