Diagnosis of α-thalassemia using droplet digital PCR

Most α-thalassemia occurs due to a large deletion in the α-globin gene. Common α-thalassemia with known mutations, such as Southeast Asian (SEA) type is readily diagnosed by Gap-PCR. However, there are many unknown mutations in α-thalassemia that need dosage or copy number assessment of the α-globin gene for diagnosis. Unlike β-thalassemia, a quantitative or real-time PCR approach for gene dosage study is not available for α-thalassemia. In real-time PCR, the gene needs to be amplified by two-fold at each cycle for dosage studies, which does not seem to be the case for the α-globin gene. The droplet digital PCR is not affected by amplification efficiency, and accurate quantification or copy number determination (copy/μg DNA) can be obtained. Here, we evaluated droplet digital PCR for detecting α-globin gene deletions. We analyzed DNA from 292 blood samples, including 62 normal samples, 35 heterozygous -α^{3.7}, 19 homozygous -α^{3.7}, 83 SEA type, 17 Filipino (FIL) type, 23 hemoglobin H disease (-α^{3.7}/SEA or FIL), 4α^{CS } /SEA and 49 non-SEA/FIL type α-thalassemia. Results of all α-thalassemia cases conformed to predicted values of copy numbers, except for six non-SEA/FIL α-thalassemias that had no deletion of the α-globin gene, but had deletions in a region containing multispecies conserved sequence R2 (DNase I hypersensitive sites 40) that is located 40 kb upstream of the ζ-globin gene and associates with the regulation of the α-globin gene. The α^{CS } allele or α^{T} has no deletion, and is diagnosed by sequencing. Thus, we show that digital droplet PCR gives accurate copy number of the α-globin gene, and is a reliable tool for determination of α-thalassemia.

Droplet Digital PCR (ddPCR)

α-thalassemia

quntitative PCR

copy number variation