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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
› Author Affiliations 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



Publication History

Received: 01 March 2023

Accepted: 15 May 2023

Accepted Manuscript online:
07 June 2023

Article published online:
10 July 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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  • References

  • 1 Bolton-Maggs PH, Pasi KJ. Haemophilias A and B. Lancet 2003; 361 (9371): 1801-1809
    CrossrefPubMedGoogle Scholar
  • 2 White II GC, Rosendaal F, Aledort LM, Lusher JM, Rothschild C, Ingerslev J. Factor VIII and Factor IX Subcommittee. Definitions in hemophilia. Recommendation of the scientific subcommittee on factor VIII and factor IX of the scientific and standardization committee of the International Society on Thrombosis and Haemostasis. Thromb Haemost 2001; 85 (03) 560
    Article in Thieme ConnectPubMedGoogle Scholar
  • 3 Bagnall RD, Giannelli F, Green PM. Int22h-related inversions causing hemophilia A: a novel insight into their origin and a new more discriminant PCR test for their detection. J Thromb Haemost 2006; 4 (03) 591-598
    CrossrefPubMedGoogle Scholar
  • 4 Bagnall RD, Giannelli F, Green PM. Polymorphism and hemophilia A causing inversions in distal Xq28: a complex picture. J Thromb Haemost 2005; 3 (11) 2598-2599
    CrossrefPubMedGoogle Scholar
  • 5 Rossetti LC, Radic CP, Abelleyro MM, Larripa IB, De Brasi CD. Eighteen years of molecular genotyping the hemophilia inversion hotspot: from southern blot to inverse shifting-PCR. Int J Mol Sci 2011; 12 (10) 7271-7285
    CrossrefPubMedGoogle Scholar
  • 6 Mühle C, Zenker M, Chuzhanova N, Schneider H. Recurrent inversion with concomitant deletion and insertion events in the coagulation factor VIII gene suggests a new mechanism for X-chromosomal rearrangements causing hemophilia A. Hum Mutat 2007; 28 (10) 1045
    CrossrefPubMedGoogle Scholar
  • 7 Fujita J, Miyawaki Y, Suzuki A. et al. A possible mechanism for Inv22-related F8 large deletions in severe hemophilia A patients with high responding factor VIII inhibitors. J Thromb Haemost 2012; 10 (10) 2099-2107
    CrossrefPubMedGoogle Scholar
  • 8 Chen C, Xie X, Wu X. et al. Complex recombination with deletion in the F8 and duplication in the TMLHE mediated by int22h copies during early embryogenesis. Thromb Haemost 2017; 117 (08) 1478-1485
    Article in Thieme ConnectPubMedGoogle Scholar
  • 9 Antonarakis SE, Rossiter JP, Young M. et al. Factor VIII gene inversions in severe hemophilia A: results of an international consortium study. Blood 1995; 86 (06) 2206-2212
    CrossrefPubMedGoogle Scholar
  • 10 Bagnall RD, Waseem N, Green PM, Giannelli F. Recurrent inversion breaking intron 1 of the factor VIII gene is a frequent cause of severe hemophilia A. Blood 2002; 99 (01) 168-174
    CrossrefPubMedGoogle Scholar
  • 11 Abelleyro MM, Radic CP, Marchione VD. et al. Molecular insights into the mechanism of nonrecurrent F8 structural variants: full breakpoint characterization and bioinformatics of DNA elements implicated in the upmost severe phenotype in hemophilia A. Hum Mutat 2020; 41 (04) 825-836
    CrossrefPubMedGoogle Scholar
  • 12 Lannoy N, Hermans C. Principles of genetic variations and molecular diseases: applications in hemophilia A. Crit Rev Oncol Hematol 2016; 104: 1-8
    CrossrefPubMedGoogle Scholar
  • 13 Jourdy Y, Chatron N, Fretigny M, Dericquebourg A, Sanlaville D, Vinciguerra C. Comprehensive analysis of F8 large deletions: characterization of full breakpoint junctions and description of a possible DNA breakage hotspot in intron 6. J Thromb Haemost 2022; 20 (10) 2293-2305
    CrossrefPubMedGoogle Scholar
  • 14 Payne AB, Miller CH, Kelly FM, Michael Soucie J, Craig Hooper W. The CDC Hemophilia A Mutation Project (CHAMP) mutation list: a new online resource. Hum Mutat 2013; 34 (02) E2382-E2391
    CrossrefPubMedGoogle Scholar
  • 15 McVey JH, Rallapalli PM, Kemball-Cook G. et al. The European Association for Haemophilia and Allied Disorders (EAHAD) coagulation factor variant databases: important resources for haemostasis clinicians and researchers. Haemophilia 2020; 26 (02) 306-313
    CrossrefPubMedGoogle Scholar
  • 16 Kemball-Cook G, Tuddenham EG, Wacey AI. The factor VIII structure and mutation resource site: HAMSTeRS version 4. Nucleic Acids Res 1998; 26 (01) 216-219
    CrossrefPubMedGoogle Scholar
  • 17 Liu Q, Nozari G, Sommer SS. Single-tube polymerase chain reaction for rapid diagnosis of the inversion hotspot of mutation in hemophilia A. Blood 1998; 92 (04) 1458-1459
    CrossrefPubMedGoogle Scholar
  • 18 Rossetti LC, Radic CP, Larripa IB, De Brasi CD. Genotyping the hemophilia inversion hotspot by use of inverse PCR. Clin Chem 2005; 51 (07) 1154-1158
    CrossrefPubMedGoogle Scholar
  • 19 Rossetti LC, Radic CP, Larripa IB, De Brasi CD. Developing a new generation of tests for genotyping hemophilia-causative rearrangements involving int22h and int1h hotspots in the factor VIII gene. J Thromb Haemost 2008; 6 (05) 830-836
    CrossrefPubMedGoogle Scholar
  • 20 Santacroce R, Acquila M, Belvini D. et al; AICE-Genetics Study Group. Identification of 217 unreported mutations in the F8 gene in a group of 1,410 unselected Italian patients with hemophilia A. J Hum Genet 2008; 53 (03) 275-284
    CrossrefPubMedGoogle Scholar
  • 21 Citron M, Godmilow L, Ganguly T, Ganguly A. High throughput mutation screening of the factor VIII gene (F8C) in hemophilia A: 37 novel mutations and genotype-phenotype correlation. Hum Mutat 2002; 20 (04) 267-274
    CrossrefPubMedGoogle Scholar
  • 22 Chen J, Li Q, Lin S. et al. The spectrum of FVIII gene variants detected by next generation sequencing in 236 Chinese non-inversion hemophilia A pedigrees. Thromb Res 2021; 202: 8-13
    CrossrefPubMedGoogle Scholar
  • 23 Inaba H, Shinozawa K, Amano K, Fukutake K. Identification of deep intronic individual variants in patients with hemophilia A by next-generation sequencing of the whole factor VIII gene. Res Pract Thromb Haemost 2017; 1 (02) 264-274
    CrossrefPubMedGoogle Scholar
  • 24 Pezeshkpoor B, Zimmer N, Marquardt N. et al. Deep intronic ‘mutations’ cause hemophilia A: application of next generation sequencing in patients without detectable mutation in F8 cDNA. J Thromb Haemost 2013; 11 (09) 1679-1687
    CrossrefPubMedGoogle Scholar
  • 25 Rost S, Löffler S, Pavlova A, Müller CR, Oldenburg J. Detection of large duplications within the factor VIII gene by MLPA. J Thromb Haemost 2008; 6 (11) 1996-1999
    CrossrefPubMedGoogle Scholar
  • 26 Lu Y, Xin Y, Dai J. et al. Spectrum and origin of mutations in sporadic cases of haemophilia A in China. Haemophilia 2018; 24 (02) 291-298
    CrossrefPubMedGoogle Scholar
  • 27 Repessé Y, Slaoui M, Ferrandiz D. et al. Factor VIII (FVIII) gene mutations in 120 patients with hemophilia A: detection of 26 novel mutations and correlation with FVIII inhibitor development. J Thromb Haemost 2007; 5 (07) 1469-1476
    CrossrefPubMedGoogle Scholar
  • 28 Guo Z, Yang L, Qin X, Liu X, Zhang Y. Spectrum of molecular defects in 216 Chinese families with hemophilia A: identification of noninversion mutation hot spots and 42 novel mutations. Clin Appl Thromb Hemost 2018; 24 (01) 70-78
    CrossrefPubMedGoogle Scholar
  • 29 Johnsen JM, Fletcher SN, Huston H. et al. Novel approach to genetic analysis and results in 3000 hemophilia patients enrolled in the My Life, Our Future initiative. Blood Adv 2017; 1 (13) 824-834
    CrossrefPubMedGoogle Scholar
  • 30 Liang Q, Gu W, Chen P. et al. A more universal approach to Comprehensive Analysis of Thalassemia Alleles (CATSA). J Mol Diagn 2021; 23 (09) 1195-1204
    CrossrefPubMedGoogle Scholar
  • 31 Liu Y, Chen M, Liu J. et al. Comprehensive analysis of congenital adrenal hyperplasia using long-read sequencing. Clin Chem 2022; 68 (07) 927-939
    CrossrefPubMedGoogle Scholar
  • 32 Li S, Han X, Xu Y. et al. Comprehensive analysis of spinal muscular atrophy: SMN1 copy number, intragenic mutation, and 2 + 0 carrier analysis by third-generation sequencing. J Mol Diagn 2022; 24 (09) 1009-1020
    CrossrefPubMedGoogle Scholar
  • 33 Liang Q, Liu Y, Liu Y. et al. Comprehensive analysis of fragile X syndrome: full characterization of the FMR1 locus by long-read sequencing. Clin Chem 2022; 68 (12) 1529-1540
    CrossrefPubMedGoogle Scholar
  • 34 Zimmermann MA, Oldenburg J, Müller CR, Rost S. Unusual genomic rearrangements in introns 1 and 22 of the F8 gene. Hamostaseologie 2011; 31 (Suppl. 01) S69-S73
    Article in Thieme ConnectPubMedGoogle Scholar
  • 35 Jiang F, Mao AP, Liu YY. et al. Detection of rare thalassemia mutations using long-read single-molecule real-time sequencing. Gene 2022; 825: 146438
    CrossrefPubMedGoogle Scholar