Identification of breakpoint regions and single nucleotide variations of RHD hybrid alleles by long-read sequencing.

IF 1.6 4区医学 Q3 HEMATOLOGY

Vox Sanguinis Pub Date : 2026-08-01 Epub Date: 2026-05-25 DOI:10.1111/vox.70292

Ti-Cheng Chang, Jing Yu, Bensheng Ju, Teha Kim, Melanie Loyd, Granger Ridout, Geoffrey Neale, Jane S Hankins, Mitchell J Weiss, Gorka Ochoa, Sunitha Vege, Stella T Chou, John Easton, Yan Zheng

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引用次数: 0

Abstract

Background and objectives: RHD*03N.01 (RHD*DIIIa-CEVS(4-7)-D) and RHD*01N.06(RHD*D-CEVS(4-7)-D) are hybrid alleles that encode a partial C antigen, resulting in increased risk of C alloimmunization in individuals without a conventional C antigen. As detection of the hybrid alleles remains challenging, new markers can aid accurate identification, ensuring provision of C-negative blood to individuals at risk.

Materials and methods: To identify breakpoint regions and distinct intronic single nucleotide variations (SNVs) of the hybrid alleles, Pacific Bioscience (PacBio) long-read sequencing was performed in two patients with RHD*DIIIa-CEVS(4-7)-D and one patient with RHD*D-CEVS(4-7)-D; two patients without hybrid alleles served as controls. Association of the identified SNVs with RHD*DIIIa-CEVS(4-7)-D was subsequently evaluated in 910 patients with sickle cell disease.

Results: The 5' breakpoint region of RHD*DIIIa-CEVS(4-7)-D was found in intron 3 at chr1:25293827-25293837 (RHD c.486+3036-3046) and chr1:25293907-25293980 (RHD c. 486+3116-3189) for the two patients. The 5' breakpoint region of RHD*D-CEVS(4-7)-D was at chr1:25294633-25295072 (RHD c.486+3842-4281). Both hybrid alleles had identical 3' breakpoint region in intron 7 at chr1:25310252-25310377 (RHD c.1073+3523-3648). Three intronic SNVs were found in RHD*DIIIa-CEVS(4-7)-D: chr1:25293603 (RHD c.486+2812) G>A, chr1:25293891 (RHD c.486+3100) A>G in intron 3; chr1:25310934 (RHD c.1073+4205) T>C in intron 7. In 910 patients, the SNVs demonstrated high sensitivities (94.83%, 94.83%, 96.55%) and specificities (99.88%, 99.88%, 99.88%) in detecting RHD*DIIIa-CEVS(4-7)-D. One SNV, chr1:25294894 (RHD c.486+4103) T>C in intron 3, was identified in RHD*D-CEVS(4-7)-D, but not confirmed due to lack of additional carriers.

Conclusion: Three intronic SNVs can be used to accurately identify RHD*DIIIa-CEVS(4-7)-D.

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RHD杂交等位基因断点区和单核苷酸变异的长读测序鉴定。

背景和目标：RHD*03N.01（RHD* diia - cevs (4-7)-D）和RHD*01N。06（RHD*D-CEVS(4-7)-D）是编码部分C抗原的杂交等位基因，导致没有常规C抗原的个体发生C异体免疫的风险增加。由于杂交等位基因的检测仍然具有挑战性，新的标记可以帮助准确识别，确保为有风险的个体提供c阴性血液。材料和方法：为了鉴定杂交等位基因的断点区域和不同的内含子单核苷酸变异（snv），对2例RHD* diia - cevs (4-7)-D患者和1例RHD*D-CEVS(4-7)-D患者进行了Pacific Bioscience （PacBio）长读测序；两名没有杂交等位基因的患者作为对照。随后在910例镰状细胞病患者中评估了鉴定的snv与RHD* diia - cevs (4-7)-D的相关性。结果：2例患者在内含子3 chr1:25293827-25293837 （RHD c.486+3036-3046）和chr1:25293907-25293980 （RHD c.486+ 316 -3189）处发现RHD* diia - cevs (4-7)-D的5′断点区域。RHD*D-CEVS(4-7)-D的5′断点位于chr1:25294633-25295072 （RHD c.486+3842-4281）。两个杂交等位基因在内含子7的chr1:25310252-25310377位点上具有相同的3′断点区（RHD c.1073+3523-3648）。在RHD* diia - cevs (4-7)-D中发现3个内含子snv: chr1:25293603 (RHD c.486+2812) G>A, chr1:25293891 (RHD c.486+3100) A>G；（RHD C .1073+4205） T>C in内含子7。在910例患者中，snv检测RHD* diia - cevs (4-7)-D的敏感性（94.83%,94.83%,96.55%）和特异性（99.88%,99.88%,99.88%）均较高。在RHD*D-CEVS(4-7)-D中发现了一个SNV chr1:25294894 (RHD C .486+4103) T>C，但由于缺乏其他载体而未得到证实。结论：3个内含子snv可准确鉴定RHD* diia - cevs (4-7)-D。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Vox Sanguinis 医学-血液学

CiteScore

4.40

自引率

11.10%

发文量

156

审稿时长

6-12 weeks

期刊介绍： Vox Sanguinis reports on important, novel developments in transfusion medicine. Original papers, reviews and international fora are published on all aspects of blood transfusion and tissue transplantation, comprising five main sections: 1) Transfusion - Transmitted Disease and its Prevention: Identification and epidemiology of infectious agents transmissible by blood; Bacterial contamination of blood components; Donor recruitment and selection methods; Pathogen inactivation. 2) Blood Component Collection and Production: Blood collection methods and devices (including apheresis); Plasma fractionation techniques and plasma derivatives; Preparation of labile blood components; Inventory management; Hematopoietic progenitor cell collection and storage; Collection and storage of tissues; Quality management and good manufacturing practice; Automation and information technology. 3) Transfusion Medicine and New Therapies: Transfusion thresholds and audits; Haemovigilance; Clinical trials regarding appropriate haemotherapy; Non-infectious adverse affects of transfusion; Therapeutic apheresis; Support of transplant patients; Gene therapy and immunotherapy. 4) Immunohaematology and Immunogenetics: Autoimmunity in haematology; Alloimmunity of blood; Pre-transfusion testing; Immunodiagnostics; Immunobiology; Complement in immunohaematology; Blood typing reagents; Genetic markers of blood cells and serum proteins: polymorphisms and function; Genetic markers and disease; Parentage testing and forensic immunohaematology. 5) Cellular Therapy: Cell-based therapies; Stem cell sources; Stem cell processing and storage; Stem cell products; Stem cell plasticity; Regenerative medicine with cells; Cellular immunotherapy; Molecular therapy; Gene therapy.