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.