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.