Diagnosis of α-thalassemia using droplet digital PCR

Creators Amao Yuki

Creators Yamashiro Yasuhiro

Creators Hattori Yukio

Creators Ogata Shizuka

Creators Ohto Fukumi

Creators Mella Ferania

Creators Kimoto Masafumi

Creators Mori Kentaro

Creators Suehiro Yutaka

Creators Yamasaki Takahiro

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.

[ISSN]0513-1812

[NCID]AA00594272