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. 2019 May 23;14(1):114.
doi: 10.1186/s13023-019-1070-0.

Molecular diagnosis of hereditary spherocytosis by multi-gene target sequencing in Korea: matching with osmotic fragility test and presence of spherocyte

Hyoung Soo Choi  1 , Qute Choi  2 , Jung-Ah Kim  3 , Kyong Ok Im  4 , Si Nae Park  4 , Yoomi Park  5 , Hee Young Shin  4   6 , Hyoung Jin Kang  4   6 , Hoon Kook  7 , Seon Young Kim  8 , Soo-Jeong Kim  9 , Inho Kim  10 , Ji Yoon Kim  11 , Hawk Kim  12 , Kyung Duk Park  4   6 , Kyung Bae Park  13 , Meerim Park  14 , Sang Kyu Park  15 , Eun Sil Park  16 , Jeong-A Park  17 , Jun Eun Park  18 , Ji Kyoung Park  19 , Hee Jo Baek  7 , Jeong Ho Seo  20 , Ye Jee Shim  21 , Hyo Seop Ahn  6 , Keon Hee Yoo  22 , Hoi Soo Yoon  23 , Young-Woong Won  24 , Kun Soo Lee  11 , Kwang Chul Lee  25 , Mee Jeong Lee  26 , Sun Ah Lee  27 , Jun Ah Lee  28 , Jae Min Lee  29 , Jae Hee Lee  30 , Ji Won Lee  22 , Young Tak Lim  20 , Hyun Joo Jung  18 , Hee Won Chueh  31 , Eun Jin Choi  32 , Hye Lim Jung  33 , Ju Han Kim  34 , Dong Soon Lee  35 ; Hereditary Hemolytic Anemia Working Party of the Korean Society of Hematology
Affiliations

Molecular diagnosis of hereditary spherocytosis by multi-gene target sequencing in Korea: matching with osmotic fragility test and presence of spherocyte

Hyoung Soo Choi et al. Orphanet J Rare Dis. .

Abstract

Background: Current diagnostic tests for hereditary spherocytosis (HS) focus on the detection of hemolysis or indirectly assessing defects of membrane protein, whereas direct methods to detect protein defects are complicated and difficult to implement. In the present study, we investigated the patterns of genetic variation associated with HS among patients clinically diagnosed with HS.

Methods: Multi-gene targeted sequencing of 43 genes (17 RBC membrane protein-encoding genes, 20 RBC enzyme-encoding genes, and six additional genes for the differential diagnosis) was performed using the Illumina HiSeq platform.

Results: Among 59 patients with HS, 50 (84.7%) had one or more significant variants in a RBC membrane protein-encoding genes. A total of 54 significant variants including 46 novel mutations were detected in six RBC membrane protein-encoding genes, with the highest number of variants found in SPTB (n = 28), and followed by ANK1 (n = 19), SLC4A1 (n = 3), SPTA1 (n = 2), EPB41 (n = 1), and EPB42 (n = 1). Concurrent mutations of genes encoding RBC enzymes (ALDOB, GAPDH, and GSR) were detected in three patients. UGT1A1 mutations were present in 24 patients (40.7%). Positive rate of osmotic fragility test was 86.8% among patients harboring HS-related gene mutations.

Conclusions: This constitutes the first large-scaled genetic study of Korean patients with HS. We demonstrated that multi-gene target sequencing is sensitive and feasible that can be used as a powerful tool for diagnosing HS. Considering the discrepancies of clinical and molecular diagnoses of HS, our findings suggest that molecular genetic analysis is required for accurate diagnosis of HS.

Keywords: Hereditary spherocytosis; Molecular diagnosis; RBC membrane disorder.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Standard operating procedure for the diagnosis of hereditary hemolytic anemia (HHA) by HHA Working Party of Korean Society of Hematology [5]
Fig. 2
Fig. 2
Characteristics of significant variants for RBC membrane protein-encoding genes; SPTB, ANK1, SLC4A1, SPTA1, EPB41, EPB42. Abbreviations: SPTB, spectrin, beta; ANK1, ankyrin 1; SLC4A1, solute carrier family 4, member 1; SPTA1, spectrin, alpha 1; EPB41, erythrocyte membrane protein band 4.1; EPB42, erythrocyte membrane protein band 4.2
Fig. 3
Fig. 3
Number of patients with RBC membrane protein-encoding gene mutations. Abbreviations: SPTB, spectrin, beta; SPTA1, spectrin, alpha 1; EPB41, erythrocyte membrane protein band 4.1; EPB42, erythrocyte membrane protein band 4.2; ALDOB, aldolase B; ANK1, ankyrin 1; GSR, glutathione reductase; SLC4A1, solute carrier family 4, member 1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase
Fig. 4
Fig. 4
A diagram showing the number of patients with positive results of gene mutation, osmotic fragility test, and peripheral blood (PB) spherocytes in 58 of 59 patients with HS. One of 59 patients who had anemia and family history of HS showed negative result on all three tests
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