Molecular Diagnosis of Hereditary Spherocytosis By Multi-Gene Target Sequencing in Korea

Background: Hereditary spherocytosis (HS) is the most common cause of hereditary hemolytic anemia. Spherocytes formed by defective membrane proteins are selectively captured in the spleen and destroyed, leading to hemolytic anemia. Current tests used to diagnose HS focus on the detection of hemolysis or indirectly assess protein defects. Direct methods to detect protein defects are complicated and difficult to implement. Recent next-generation sequencing (NGS) methods enable large-scale gene mutation analyses to be used for such diagnoses. In this study, we investigated the patterns of genetic variation associated with HS to determine the molecular mechanisms underlying the condition. Specifically, we analyzed mutations in red blood cell membrane protein-encoding genes in Korean HS patients using NGS.

Methods: In total, 60 patients with HS were enrolled in this study. Targeted sequencing of 43 genes (17 membrane protein-encoding genes, 20 enzyme-encoding genes, and 6 additional candidate genes) was performed using the Illumina HiSeq platform and variants were called according to a data-processing pipeline.

Results: Of the 60 patients, 50 (83%) had one or more significant variants in a membrane protein gene. A total of 54 significant variants (8 previously reported and 46 novel) were detected in 6 membrane protein-encoding genes, i.e., SPTB, ANK1, SPTA1, SLC4A1, EPB41, and EPB42. The most variants (28) were detected in SPTB. Four significant variants, all of which were previously reported, were detected in genes encoding enzymes (ALDOB, G6PD, GAPDH, and GSR). Additionally, 5 previously reported variants were detected in UGT1A1. These results suggest 35 primer sets that can be used to diagnose HS.

Conclusion: This was the first large-scaled genetic study of Korean HS patients. These results clarify the pattern of genetic variation associated with HS in Korean patients. They will enable easier, more rapid diagnosis of HS.