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CC BY-NC-ND 4.0 · Thromb Haemost 2023; 123(12): 1151-1164
DOI: 10.1055/a-2107-0702
New Technologies, Diagnostic Tools and Drugs

Comprehensive Analysis of Hemophilia A (CAHEA): Towards Full Characterization of the F8 Gene Variants by Long-Read Sequencing

Yingdi Liu   *
1   Center for Medical Genetics and Hunan Key Laboratory of Medical Genetics, School of Life Sciences, Central South University, Changsha, Hunan, China
,
Dongzhi Li   *
2   Prenatal Diagnostic Center, Guangzhou Women and Children's Medical Center, Guangzhou, Guangdong, China
,
Dongyi Yu   *
3   Center for Medical Genetics and Prenatal Diagnosis, Shandong Provincial Maternal and Child Health Care Hospital, Shandong Medicine and Health Key Laboratory of Birth Defect Prevention and Genetic Medicine, Key Laboratory of Birth Regulation and Control Technology of National Health Commission of China, Jinan, Shandong, China
,
Qiaowei Liang   *
4   Department of Medical Genetics, Hunan Jiahui Genetics Hospital, Changsha, Hunan, China
,
Guilan Chen
2   Prenatal Diagnostic Center, Guangzhou Women and Children's Medical Center, Guangzhou, Guangdong, China
,
Fucheng Li
2   Prenatal Diagnostic Center, Guangzhou Women and Children's Medical Center, Guangzhou, Guangdong, China
,
Lu Gao
3   Center for Medical Genetics and Prenatal Diagnosis, Shandong Provincial Maternal and Child Health Care Hospital, Shandong Medicine and Health Key Laboratory of Birth Defect Prevention and Genetic Medicine, Key Laboratory of Birth Regulation and Control Technology of National Health Commission of China, Jinan, Shandong, China
,
Zhuo Li
1   Center for Medical Genetics and Hunan Key Laboratory of Medical Genetics, School of Life Sciences, Central South University, Changsha, Hunan, China
,
Tiantian Xie
5   Berry Genomics Corporation, Beijing, China
,
Le Wu
5   Berry Genomics Corporation, Beijing, China
,
Aiping Mao
5   Berry Genomics Corporation, Beijing, China
,
Lingqian Wu
1   Center for Medical Genetics and Hunan Key Laboratory of Medical Genetics, School of Life Sciences, Central South University, Changsha, Hunan, China
4   Department of Medical Genetics, Hunan Jiahui Genetics Hospital, Changsha, Hunan, China
,
Desheng Liang
1   Center for Medical Genetics and Hunan Key Laboratory of Medical Genetics, School of Life Sciences, Central South University, Changsha, Hunan, China
4   Department of Medical Genetics, Hunan Jiahui Genetics Hospital, Changsha, Hunan, China
› Institutsangaben Funding This work was supported by the National Key Research and Development Program of China (2022YFC2703700, 2022YFC2703400, 2021YFC1005302) and the National Natural Science Foundation of China (82271752).
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Abstract

Background Hemophilia A (HA) is the most frequently occurring X-linked bleeding disorder caused by heterogeneous variants in the F8 gene, one of the largest genes known. Conventional molecular analysis of F8 requires a combination of assays, usually including long-range polymerase chain reaction (LR-PCR) or inverse-PCR for inversions, Sanger sequencing or next-generation sequencing for single-nucleotide variants (SNVs) and indels, and multiplex ligation-dependent probe amplification for large deletions or duplications.

Materials and Methods This study aimed to develop a LR-PCR and long-read sequencing-based assay termed comprehensive analysis of hemophilia A (CAHEA) for full characterization of F8 variants. The performance of CAHEA was evaluated in 272 samples from 131 HA pedigrees with a wide spectrum of F8 variants by comparing to conventional molecular assays.

Results CAHEA identified F8 variants in all the 131 pedigrees, including 35 intron 22-related gene rearrangements, 3 intron 1 inversion (Inv1), 85 SNVs and indels, 1 large insertion, and 7 large deletions. The accuracy of CAHEA was also confirmed in another set of 14 HA pedigrees. Compared with the conventional methods combined altogether, CAHEA assay demonstrated 100% sensitivity and specificity for identifying various types of F8 variants and had the advantages of directly determining the break regions/points of large inversions, insertions, and deletions, which enabled analyzing the mechanisms of recombination at the junction sites and pathogenicity of the variants.

Conclusion CAHEA represents a comprehensive assay toward full characterization of F8 variants including intron 22 and intron 1 inversions, SNVs/indels, and large insertions and deletions, greatly improving the genetic screening and diagnosis for HA.

Keywords

hemophilia A - gene rearrangements - long-range PCR - long-read sequencing - genetic testing

Note

Dr. Qiaowei Liang's present address is Department of Human Genetics, Graduate School of Medicine, Yokohama City University, Yokohama, Japan.


* These authors contributed equally to this work.


Supplementary Material



Publikationsverlauf

Eingereicht: 01. März 2023

Angenommen: 15. Mai 2023

Accepted Manuscript online:
07. Juni 2023

Artikel online veröffentlicht:
10. Juli 2023

© 2023. The Author(s). This is an open access article published by Thieme under the terms of the Creative Commons Attribution-NonDerivative-NonCommercial License, permitting copying and reproduction so long as the original work is given appropriate credit. Contents may not be used for commercial purposes, or adapted, remixed, transformed or built upon. (https://creativecommons.org/licenses/by-nc-nd/4.0/)

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