Congenital factor V (FV) deficiency is a rare autosomal recessive bleeding disorder caused by pathogenic variants in F5 gene and characterized by heterogeneous clinical manifestations. The aim of this study was to define the mutational spectrum of F5 in Russian patients with congenital FV deficiency. We analyzed 16 unrelated patients with different disease severity and 9 relatives from five families. All functionally relevant regions of F5 were examined by Sanger sequencing. Multiplex ligation-dependent probe amplification (MLPA) was used to detect large deletions and duplications. Whole-genome sequencing and functional cDNA analysis were performed in selected cases. This study represents the first description of the F5 mutational spectrum in a Russian cohort. We identified 12 novel variants and demonstrated the functional effect of two previously unreported variants located outside canonical splice-site dinucleotides, leading to aberrant splicing. Notably, the proportion of variants undetectable by routine diagnostic approaches was higher than that reported in other populations. No clear genotype-phenotype correlation was observed. Despite the limited sample size, our findings expand current knowledge of the molecular basis of congenital FV deficiency and may improve genetic diagnostics in Russia.

Введение. Гемофилия А – наследственное заболевание, обусловленное дефицитом фактора VIII свертывания крови (FVIII). Основным принципом лечения гемофилии А является проведение специфической заместительной терапии концентратами дефицитного фактора свертывания крови с поддержанием активности, достаточной для предотвращения развития кровотечений. При проведении профилактической терапии общепризнаны целевые значения минимальной активности FVIII 3–5%. Остаточная активность FVIII зависит от множества факторов, в том числе от строения молекулы и фармакокинетических свойств применяемого препарата. Цель исследования: индивидуализированный фармакокинетический подбор режимов профилактики для поддержания минимальной остаточной активности FVIII ≥ 5% у пациентов с гемофилией А. Результаты. Проведено одноцентровое неинтервенционное проспективное исследование с анализом фармакокинетики у 11 пациентов с гемофилией А, находившихся на профилактике рекомбинантным препаратом FVIII – симоктокогом альфа. Результаты сравнивали с историческими данными лечения этих пациентов другими препаратами FVIII. При индивидуально подобранном режиме профилактики симоктокогом альфа у всех пациентов остаточная активность FVIII перед следующей инфузией препарата сохранялась на значимо более высоком уровне по сравнению с терапией другими непролонгированными концентратами препаратов FVIII (6,3% и 1,6%, соответственно), что является ключевым показателем, позволяющим предотвратить развитие кровотечений. Заключение. Продемонстрировано, что на протяжении 12 нед применения симоктокога альфа у 63,6% пациентов не было зарегистрировано ни одного кровотечения, тогда как в этой же группе пациентов средняя годовая частота кровотечений ранее составляла 3,5. Introduction. Hemophilia A is an inherited disorder caused by a deficiency of blood clotting factor VIII (FVIII). The primary treatment for hemophilia A is specific replacement therapy with concentrates of the deficient blood coagulation factor while maintaining activity sufficient to prevent bleeding. For prophylactic treatment, a generally accepted target for minimum FVIII activity is 3–5%. The residual activity of FVIII depends on various factors, including the molecule’s structure and the drug’s pharmacokinetic properties. Aim: individualized pharmacokinetic selection of prophylaxis regimens to maintain minimal residual FVIII activity ≥ 5% in patients with hemophilia A. Results. A single-center non-interventional prospective study with pharmacokinetics analysis included 11 patients with hemophilia A who were receiving prophylaxis with a recombinant FVIII drug, simoctocog alfa. The results were compared with historical data from treating these patients with other FVIII agents. With an individually selected prophylaxis regimen with simoctocog alfa, the residual FVIII activity prior to the next drug infusion remained at a significantly higher level in all patients compared with therapy with other non-extended-release FVIII drug concentrates (6.3% and 1.6%, respectively), which is a key indicator to prevent bleeding. Conclusion. During 12 weeks of treatment with simoctocog alfa, 63.6% of patients experienced no bleeding, compared with a previous average annual bleeding rate of 3.5 in the same group of patients.

Hemophilia A (HA) is one of the most widespread, X-linked, inherited bleeding disorders, which results from defects in the F8 gene. Nowadays, more than 3500 different pathogenic variants leading to HA have been described. Mutation analysis in HA is essential for accurate genetic counseling of patients and their relatives. We analyzed patients from 273 unrelated families with different forms of HA. The analysis consisted of testing for intron inversion (inv22 and inv1), and then sequencing all functionally important F8 gene fragments. We identified 101 different pathogenic variants in 267 patients, among which 35 variants had never been previously reported in international databases. We found inv22 in 136 cases and inv1 in 12 patients. Large deletions (1–8 exons) were found in 5 patients, and we identified a large insertion in 1 patient. The remaining 113 patients carried point variants involving either single nucleotide or several consecutive nucleotides. We report herein the largest genetic analysis of HA patients issued in Russia.

Book Citations: Authors, Title, HemaSphere, 2023;7(S3):pages. The individual abstract DOIs can be found at https://journals.lww.com/hemasphere/pages/default.aspx. Disclaimer: Articles published in the journal HemaSphere exclusively reflect the opinions of the authors. The authors are responsible for all content in their abstracts including accuracy of the facts, statements, citing resources, etc. 3158 characteristic for gene nor for pseudogene in patient A, and has untouched sequence (i.e. not carrying vWF gene nucleotides) in patient B. Therefore, it was not an exchange of complementary parts of gene and pseudogene, and we cannot suggest any mechanism of conversion in this case. Summary/Conclusion: Gene conversion took place in two cases out of 64, making its frequency in our sample to be 3,13%. The conversed gene allele in the heterozygous state caused mild form von Willebrand disease for patient A and carried only polymorphisms in patient B case. We assumed that gene conversion would be symmetrical, with gene and pseudogene swapping parts. However, it was not confirmed. HemaSphere | 2023;7(S3) EHA2023 Hybrid Congress Copyright Information: (Online) ISSN: 2572-9241 © 2023 the Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the European Hematology Association. This is an open access Abstract Book distributed under the Attribution-NonCommercial-NoDerivs (CC BY-NC-ND) which allows third parties to download the articles and share them with others as long as they credit the author and the Abstract Book, but they cannot change the content in any way or use them commercially. Abstract Book Citations: Authors, Title, HemaSphere, 2023;7(S3):pages. The individual abstract DOIs can be found at https://journals.lww.com/hemasphere/pages/default.aspx.Book Citations: Authors, Title, HemaSphere, 2023;7(S3):pages. The individual abstract DOIs can be found at https://journals.lww.com/hemasphere/pages/default.aspx. Disclaimer: Articles published in the journal HemaSphere exclusively reflect the opinions of the authors. The authors are responsible for all content in their abstracts including accuracy of the facts, statements, citing resources, etc. 3159

Introduction. Von Willebrand disease (vWD) is caused by von Willebrand factor (vWF) dysfunction resulting from pathogenic variants in the vWF gene coding the vWF protein. vWD type 2N is of particular interest, as it is characterized by almost normal vWF antigen level (Ag:vWF) and vWF loss of ability to bind FVIII and protect it from premature clearance, which leads to a low FVIII coagulation activity (FVIII:C). Therefore, the same phenotype occurs in patients with 2N type of vWD and hemophilia A. Aim — to identify patients with 2N type vWD using molecular genetic methods. Methods. Data from the medical histories of vWD patients were used. The major parameter in consideration was FVIII:C to vWF:Ag ratio, which is expected to be below 0.7 in type 2N of vWD. Pathogenic variants in exons and exon-intron junctions of the vWF gene were identified by Sanger sequencing. Due to recessive inheritance of type 2N, verification of the 2N vWD diagnosis required the identification of two pathogenic variants. Results. Three patients were considered as suffering from type 2N of vWD according to hemostasis parameters (FVIII:C/vWF:Ag < 0.7). One patient with a preliminary hemophilia A diagnosis was included after sequencing of the F8 gene, which showed no alterations, so 2N type of vWD was suspected. In all cases, sequencing of the relevant functional regions of the vWF gene led to verification of vWD type 2N. One woman (patient # 4) had a homozygous pathogenic variant p.Arg854Gln (c.2561 G>A) associated with type 2N vWD. One woman (patient # 3) was a compound heterozygote for the pathogenic variant p.Arg816Trp (c.2446 C>T) associated with type 2N and a newly described insertion c.2098_2099insG, that leads to a frameshift. The woman with FVIII:C/vWF:Ag < 0.7 (patient # 1) and the patient # 2 with preliminary hemophilia А diagnosis were both compound heterozygotes for the same combination of pathogenic variants — c.2435delC and p.Thr791Met (c.2372 C>T). Pathogenic variant p.Thr791Met is associated with type 2N, while the deletion c.2435delC should lead to allele disabling. Conclusion. Molecular methods allow more precise differentiation of type 2N from other types of vWD and hemophilia A.

Background:Congenital hemophilia A (HA) is an inherited recessive X‐linked bleeding disorder caused by a wide spectrum of mutations in the F8 gene and affected 1 in 5000 males. Its high degree of mutational heterogeneity complicates the carrier and prenatal diagnoses for genetic counseling but offers a wealth of data on the relationship between structure and function of FVIII.Aims:The aim of this study was to describe mutation spectrum in Russian patients with HA and to improve significantly genetic carriage and prenatal diagnostics of this disease in our country. Study results are especially important for families with sporadic form of HA (i.e. in families with the first reported patient), because in these cases only genetic analysis can give a reliable carriage status for proband's mothers.Methods:We conducted molecular genetic analysis for 239 unrelated patients with different forms of HA, recruited from 1990 to 2019 from different regions of Russia. Genomic DNA was isolated from peripheral blood using phenol–chloroform extraction. All patients with severe HA were examined for intron 22 (LD‐PCR method, modified from Liu et al., 1998 and Bagnall et al, 2006) and intron 1 (Bagnall et al., 2002) inversions. For patients without inversions we sequenced all functionally significant F8 gene fragments by Sanger.Results:We found inversion 22 in 130 unrelated patients with severe hemophilia A (68%, 17 patients with inhibitor). In 11 patients we found inversion 1 (5.8%, 1 patient with inhibitor), 3 patients had abnormal inversion 1 with additional deletion or duplication of nearby regions. In other 98 patients we detected 79 different mutations (12 patients with inhibitor), 40 of which had never been reported before in the international databases. Among them prevailed missense mutations (37), there were also nonsense (15), frameshift (13, 3 of them were indels), splicing mutations (9), large deletions (4) and inframe deletion (1). In three patients we found two mutations simultaneously. Five patients were women suffered from HA. One of them carried two mutations in F8 gene, others had only one mutation (one carried a missense mutation, while others had inversion 22), but we found for them evidence for an asymmetric inactivation of X‐chromosome using HUMARA test.Summary/Conclusion:All mutations detected in this study were distributed uniformly along the FVIII gene. As expected, significant part of missense and nonsense mutations occurred in CpG hot spot dinucleotides (21 out 52). We found two other mutation hot spots located in oligoA tracts of exon 14: p.Asn1460Lysfs∗1 and p.Ile1213Phefs∗5 detected in four and six unrelated patients, respectively. During our work we revealed a new mutation p.His634Arg found in 7 patients with mild HA (F8 = 10–30%) originated from Sverdlovsk region of Russia. We constructed four‐loci haplotypes for those patients and for 10 other patients with HA from the same region carrying other different mutations. All patients with this mutation shared the same unique haplotype, while all other patients had different haplotypes, which indicates founder effect for p.His634Arg substitution. For two large deletions (del Ex6 and del Ex6–12) we determined breakpoints, which were in both cases located within or nearby Alu‐repeats. In our cases HA in women mostly developed as a result of an asymmetric inactivation of X‐chromosome. For one woman with this inactivation we detected missense mutation led to a late manifestation of the disease (at 59 years old), which was at first diagnosed as acquired HA.

Introduction. Von Willebrand disease (vWD) — one of the most common coagulopathies — is characterised by a rather complicated inheritance pattern, which can be either dominant or recessive depending on the disease type. Aim. To compare clinical, coagulological and molecular genetic data obtained when examining patients with various types of vWD.Materials and methods. The vWF gene exons were sequenced in 16 patients suffering from VWD using the Sanger method.Results. In total, 12 various mutations were identified, one of which (Pro2527His) has not been previously observed in the world population. The c.2435delC microdeletion being a major mutation in many European countries was found to be the most common. This microdeletion was observed in 9 patients, 6 of whom had the most severe recessive form of the disease — type 3 (3 homozygotes). In two patients, this disorder was accompanied by the missense mutation Thr791Met, which allowed the authors to diagnose a rather rare recessive variant of vWD — 2N. In general, the data obtained by molecular genetic analysis correlated with the differential diagnosis of the vWD type, which is based on the clinical picture of the disease and coagulological properties. In only one case, the Arg1374Cys mutation characteristic of type 2 VWD (A/M) was observed in a patient with the alleged type 1 vWD. Most of the mutations were found in exons 18 (mainly c.2435delC deletion) and 28 which makes them the most perspective exons for the mutation search.Conclusion. The search for mutations in the vWF gene should start from exons 18 and 28. The obtained information provides a basis for developing an economical algorithm aimed at searching for mutations in the vWF gene in our counrtry vWD patients.