High-throughput sequencing in identifying somatic hypermutation in immunoglobulin heavy chain variable regions with complex clonal backgrounds
10.3760/cma.j.cn121090-20241016-00394
- VernacularTitle:采用高通量测序技术鉴别免疫球蛋白重链可变区体细胞高突变复杂克隆背景的研究
- Author:
Mengge GAO
1
;
Rong WEI
1
;
Yang LIU
1
;
Xiaojun HUANG
1
;
Shenmiao YANG
1
;
Xiaosu ZHAO
1
Author Information
1. 北京大学人民医院，北京大学血液病研究所，国家血液系统疾病临床医学研究中心，造血干细胞移植治疗血液病北京市重点实验室，北京 100044
- Publication Type:Journal Article
- Keywords:
Immunoglobulin heavy chain variable gene;
Somatic hypermutation;
Sanger sequencing;
Next generation sequencing
- From:
Chinese Journal of Hematology
2025;46(9):815-819
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To compare the performance of next-generation sequencing (NGS) and Sanger sequencing in investigating somatic hypermutation (SHM) status of immunoglobulin heavy chain variable region (IGHV) genes. It specifically focuses on identifying key factors contributing to discrepancies between the two methods, particularly under complex clonal backgrounds, to inform optimized strategies for clinical application.Methods:This retrospective analysis included 53 samples, comprising 43 identified as non-monoclonal and 10 as monoclonal using Sanger sequencing. All samples were further analyzed using NGS to assess IGHV SHM. The two methods were used for systematic comparison. For discordant cases, in-depth attribution analysis was conducted, considering factors, including clonal abundance quantification, differences in primer design, and interpretation criteria.Results:Among the 53 patients who underwent both Sanger and NGS testing, 36 were male and 17 were female, with a median age of 64 years (range: 33–88). Diagnoses included chronic lymphocytic leukemia (CLL) in 35 (66.0% ), diffuse large B-cell lymphoma in 9 (17.0% ), follicular lymphoma in 3 (5.7% ), mantle cell lymphoma in 3 (5.7% ), and other types in 3 (5.7% ) cases. In the 43 cases with non-monoclonal profiles using Sanger sequencing, NGS revealed 23 cases as biclonal or polyclonal, 17 as monoclonal, and 3 with no detectable clonality. The primary discrepancies between the two methods involved variations in clonality assessment, IGHV gene rearrangement types, and mutation rates. Among the 10 cases identified as monoclonal using Sanger sequencing, NGS detected biclonality and markedly different IGHV rearrangement types in 2 and 4 cases, respectively. Minor differences were observed in SHM percentage between the two methods; however, these did not substantially affect the overall determination of mutational status.Conclusion:Compared with Sanger sequencing, NGS exhibits superior performance in assessing IGHV SHM status under complex clonal conditions. It provides greater sensitivity and accuracy in detecting subclonal components and quantifying clonal proportions, thereby providing a more precise molecular basis for diagnosing and prognostically assessing lymphoid malignancies, including CLL.
