Introduction
Hemophilia A and B are X-linked recessive congenital bleeding disorders caused by pathogenic variants in, respectively, the F8 orF9 gene. Hemophilia A, caused by lack or dysfunction of the plasma protein factor VIII (FVIII), affects about 1/5000 males, while hemophilia B, caused by lack or dysfunction of factor IX (FIX), affects approximately 1/30,000 males (Mannucci and Tuddenham 2001). Depending on the residual clotting activity in plasma levels of FVIII or FIX, hemophilia is categorized as severe (< 1%), moderate (1–5%), or mild (6–40%). The cornerstone of hemophilia treatment is replacement therapy with FVIII/FIX concentrates and – recommended by the World Health Organization (WHO) – treatment with prophylaxis in moderate and severe hemophilia (Andersson, et al 2017, Manco-Johnson, et al 2007). The main complication of replacement therapy is the development of anti-FVIII/FIX antibodies (inhibitors), which are able to neutralize the clotting activity of therapeutic clotting factors. (Gouw, et al 2013).
Since the F8/F9 mutation type is the main determinant of disease severity, the analysis of the F8 or F9 gene variant in hemophilia patients and their families has become standard in hemophilia treatment centers in recent years. Knowledge of the variant allows genetic counseling and provides information on the risk of inhibitor development. Additionally, information on clotting assays discrepancies, and in mild hemophilia A the probability of a therapeutic response to DDAVP, can be retrieved (Goodeve and Peake 2003, Seary, et al2012). Sporadic cases, i.e. with no known family history of hemophilia, accounts for approximately 50% of all cases. If hemophilia is diagnosed for the first time in a patient, studies show that the new variants are found in around 70–80% of the mothers of these index cases (Ljung, et al 1991, Martensson, et al 2016).
Currently, direct gene sequencing either through Sanger or next-generation sequencing (NGS) methodologies is the predominant technique for the testing of single nucleotide variants (SNVs) and small insertions and deletions (Gomez and Chitlur 2013). Nowadays, copy number variant (CNV) analysis for large deletions and duplications is performed by NGS or complimentary technologies such as array comparative genomic hybridization (aCGH) and multiplex ligation-dependent probe amplification (MLPA). For the F8 intron 22 inversion, Southern blot, long-range PCR, and inverse PCR protocols are used, while for theF8 intron 1 inversion, a PCR-based method is the standard technique. The most common variant causing severe hemophilia A is intron 22 inversion in F8 affecting roughly 40% of the patients but today a broad spectrum of more than 2000 mutations causing hemophilia A and more than 1000 mutations causing hemophilia B are described in FVIII or FIX variant databases, such as the American CDC Hemophilia Mutation Project databases CHAMPS/CHBMPS (https://www.cdc.gov/ncbddd/hemophilia/champs.html)or the European EAHAD Coagulation Factor Variant Databases (http://www.eahad-db.org), to which F8 and F9 gene variants from all over the world are reported randomly (Li, et al 2013, McVey, et al 2020, Payne, et al 2013). The variant types in hemophilia cover a broad spectrum: in addition to the F8 gene specific inversion 22 and inversion 1, SNVs, deletions, duplications, and complex mutations are found causing missense, nonsense, frameshift, deletion/insertion/duplication in frame, splice site mutations and promotor variants. Usually, new variants are crosschecked with the above-named hemophilia variant databases, such as the European Coagulation Factor Variant Databases from EAHAD, the CDC-based CHAMPS/CHBMPS or Human Gene Mutation database (HGMD), which collects large number of published gene alterations. In these databases, additional information about the number of patients with each reported variant and clinical information on severity of the disease, factor levels and inhibitor development on every reported patient may be available (Li, et al 2013, Payne, et al 2013).
The clinical interpretation of a new or an unpublished genetic variant in the F8 or F9 genes as well as other genes should be based on guidelines published by American College of Medical Genetics and Genomics and the Association for Molecular Pathology (Richards 2015). International F8/F9 gene variant databases assist effective variant classification especially when possible to combine with phenotypic and pedigree information. Various in silicoprediction programs developed for missense or splice site variants may be helpful but the provided information should be interpreted by caution as vast majority of predicted pathogenic missense variant are not disease causing. Recently, a guideline specific for genetic analysis in bleeding disorders has been published (Gomez, et al 2019). When predicting the pathogenicity of a gene variant it is recommended to combine several prediction programs (Richards, et al 2015).
The PedNet Registry cohort contains prospective data on children < 18 years with hemophilia A or B born since 1 January 2000 who are followed up regularly in 31 hemophilia centers in 18 countries in Europe, Canada and Israel. More than 2100 patients were included in 2019 and the F8/F9 gene mutation is known in 85% of the cases (Fischer, et al 2014). The purpose of the Registry is to promote and facilitate research and development of care in this large unselected patient population. The aim of this paper is to report all new variants, not previously published or known in databases, of F8 andF9 found in the PedNet cohort and predict the pathogenicity.