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

Yingdi Liu; Dongzhi Li; Dongyi Yu; Qiaowei Liang; Guilan Chen; Fucheng Li; Lu Gao; Zhuo Li; Tiantian Xie; Le Wu; Aiping Mao; Lingqian Wu; Desheng Liang

doi:10.1055/a-2107-0702

Thrombosis and Haemostasis, Inhaltsverzeichnis

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^*

¹Center for Medical Genetics and Hunan Key Laboratory of Medical Genetics, School of Life Sciences, Central South University, Changsha, Hunan, China

,

Dongzhi Li^*

²Prenatal Diagnostic Center, Guangzhou Women and Children's Medical Center, Guangzhou, Guangdong, China

,

Dongyi Yu^*

³Center 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^*

⁴Department of Medical Genetics, Hunan Jiahui Genetics Hospital, Changsha, Hunan, China

,

Guilan Chen

²Prenatal Diagnostic Center, Guangzhou Women and Children's Medical Center, Guangzhou, Guangdong, China

,

Fucheng Li

²Prenatal Diagnostic Center, Guangzhou Women and Children's Medical Center, Guangzhou, Guangdong, China

,

Lu Gao

³Center 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

¹Center for Medical Genetics and Hunan Key Laboratory of Medical Genetics, School of Life Sciences, Central South University, Changsha, Hunan, China

,

Tiantian Xie

⁵Berry Genomics Corporation, Beijing, China

,

Le Wu

⁵Berry Genomics Corporation, Beijing, China

,

Aiping Mao

⁵Berry Genomics Corporation, Beijing, China

,

Lingqian Wu

¹Center for Medical Genetics and Hunan Key Laboratory of Medical Genetics, School of Life Sciences, Central South University, Changsha, Hunan, China

⁴Department of Medical Genetics, Hunan Jiahui Genetics Hospital, Changsha, Hunan, China

,

Desheng Liang

¹Center for Medical Genetics and Hunan Key Laboratory of Medical Genetics, School of Life Sciences, Central South University, Changsha, Hunan, China

⁴Department of Medical Genetics, Hunan Jiahui Genetics Hospital, Changsha, Hunan, China

› Institutsangaben

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

Volltext

Referenzen

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