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

Overview

Journal Thromb Haemost

Publisher Thieme

Specialties Cardiology & Vascular Diseases
Hematology

Date 2023 Jun 7

PMID 37285902

Authors

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

Affiliations

Soon will be listed here.

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.

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