Discussion
Mutation analysis in hemophilia has become a standard procedure over the
years, giving confirmation of the suspected disease, making carrier
diagnosis possible and enabling identification of variants with
inhibitor risk. In this study encompassing data from 1681 children
included in the PedNet Registry with hemophilia A and B, we can report
90 causative new variants in F8 and F9. These variants
were not previously reported in the HGMD or CHAMPS, CHMBS and EAHAD
hemophilia variant databases. The new variants were frequently found in
exon 14 in hemophilia A and exon 8 in hemophilia B as expected, since
both are the largest exons in F8 and F9, respectively. No
‘hotspot’ was identified, and the new variants occurred in all variant
types following the general spectrum of variants types in hemophilia A
and B. This is in line with a report from Johnsen et al ., in
which 3000 hemophilia patients were investigated with NGS and 285 new
variants were found in all variant types and F8 or F9 loci
(Johnsen, et al 2017).
In hemophilia A, 73 of 78 and in hemophilia B, 17 of 19 new variants
were found to be causative. However, seven variants could not be proven
to be causative by in silico analysis: five variants in mild
hemophilia patients (three hemophilia A, two hemophilia B), one in a
severe hemophilia A patient having another causative mutation and one in
a severe hemophilia A patient without other explanations. In general,
probable disease-causing variants are identified in approximately 95%
of hemophilia A cases and in almost all patients with hemophilia B
(Swystun and James 2017). It is a known phenomenon that in silicoanalysis – even if combined – can be non-conclusive and should be seen
as only one of the steps in categorizing variants as described by the
ACMG. Another explanation could be misdiagnosis due to overlapping
phenotypes, e.g. von Willebrand disease causing low FVIII levels, as
well as deep intronic variants altering mRNA splice sites.
Since the data were retrieved from the PedNet hemophilia foundation with
31 centers over the last two decades, reporting of genotype may vary
from centers over time. External quality assessment can be used to
ensure good quality genetic reports, which have become more complex
during the last decades: the availability and range of methodologies for
genetic diagnosis for hemophilia increases, e.g. by implementing NGS,
the evaluation of genotyping accuracy, standardization of nucleotide and
protein variant descriptions using Human Genome Variation Society (HGVS)
nomenclature has to be used correctly, and variant pathogenicity
assessment strategies such as in silico analysis have become
essential(Gomez, et al 2019). To ensure high-quality reporting in
the Pednet Registry, all reports were re-evaluated and updated with HGVS
nomenclature and classification in 2018/2019 (den Dunnen 2017) by a
genetic laboratory technician and two MDs (Lund University, Malmö/Lund,
Sweden). A regular update of genetic reports is planned for the Pednet
Registry and all new reports to be included are re-evaluated
continuously.
While hemophilia genetic variant databases are very useful, it should be
noted that they have certain limitations too. Reporting in hemophilia
variant databases, such as EAHAD, CHAMPS and CHBMPS, does not require a
specific standard on genetic reports and usually no follow-up reports or
re-evaluations are carried out. Also, the update of these registries is
not performed continuously and by now, according to their website
information and personal communication, the CHAMPS/CHMBPS database had
the latest update in December 2014 and the EAHAD variant database in
2017. To be sure that a new variant is being presented, a literature
search has to be performed additionally. The HGMD bases the entries on
published variants; however, not every new variant is published in a
medical journal and HGMD contain large number of misclassified variants
similar to scientific literature. The definition of variant type and
effect differs between databases and publications and adaptations are
needed for comparisons. There is no requirement to classify variants by
the guidelines or to prove specific variants – such as missense or
splice site – as likely causative by prediction programs, even if most
new reports will follow these standards today. Most probably, some
variants in the databases reported are polymorphisms not causing
hemophilia, which was also suggested by another group ‘My Life, Our
Future initiative’, finding 11 earlier reported variants unlikely to
cause hemophilia (Johnsen, et al 2017).
In 19 variants, inhibitor development was reported: 18 variants in
hemophilia A patients (18/73; 24.7%), and one in a patient with severe
hemophilia B (1/17; 5.9%). Although the new variants only represent a
subgroup of our population-based registry, this follows the expected
rate of inhibitor formation for hemophilia A and B. The highest rate of
inhibitors was found in hemophilia A as expected: most inhibitors
occurred in deletions causing large structural change (100%), followed
by complex variants (50%), variants with nonsense effect (45.4%), in
variants with duplication (33%), variants with splice site effect
(27.2%) and variants with deletions causing frameshift (18.2%).
Inhibitors were rarely reported in variants with missense effect. This
is in line with earlier publications (Gouw, et al 2012).
Information on inhibitor formation in specific variants is important for
clinical practical issues. The use and start of prophylaxis can be
influenced if the variant is known to cause inhibitors, especially in
the light of new emerging non-factor therapies in hemophilia that could
avoid early exposure to FVIII (Mahlangu, et al 2018). In mild and
moderate hemophilia A, it is known today that these patients have a
relevant risk for the development of inhibitors during their lifetime
(Eckhardt, et al 2013). A known risk of inhibitor development
could lead to the recommendation of the use of DDAVP as a first-line
therapy, which enhances the internal release of FVIII in mild hemophilia
A patients and avoids the formation of inhibitors.
In conclusion, we report 90 new causative variants in hemophilia A and
B, with detailed information on severity, factor level and inhibitor
formation. This will lead to further guidance for genetic laboratories
and the treating physician and these findings can be implemented in
variant databases. The strength of our study is the uniform collection
of variants in a large cohort with regular re-evaluation of genetic
reports following international guidelines. The study highlights the
need to re-evaluate and update earlier genetic reports both locally, but
also in variant databases in the light of changed nomenclature, the use
of population database data e.g. gnomAD, in silico prediction and
new sequencing techniques, such as NGS or MLPA.