Alpha-Thalassemia Caused by αα (HBA1: c.95 + 1G > A) Mutation and its Combinations with Other Forms of Thalassemia or Hemoglobinopathy in Northern Thailand.

The αα mutation is typically disregarded during routine thalassemia testing because of its mild or absent pathology in heterozygous individuals as well as its interactions with modulators that can result in variable phenotypes, particularly in β-thalassemia (β-thal) and hemoglobin E (HbE) carriers. This study was performed to analyze the αα mutation in Northern Thailand using targeted next-generation sequencing (NGS). Hb analysis was performed by HPLC and/or CE methods. The α-thal-1 --, --, and -- type deletions were detected by real-time PCR with high-resolution melting analysis, and the α-thal-2 (-α and -α) deletion was detected by conventional gap-PCR. Thalassemia genotypes were further investigated using an NGS panel targeting the coding regions of the , , and genes. The αα mutation was misdiagnosed by routine thalassemia diagnostic methods. NGS results showed that 7 (3 Burmese and 4 Thai originations) of 1107 (0.63%) blood samples were heterozygous for the αα mutation, which appeared in 5 different genotypes. Clinical and hematological features varied with their combination forms. The targeted NGS analysis utilized in this study is a promising genetic testing method for the identification of uncommon globin gene mutations. It shows particular promise in thalassemia screening and genetic counseling.