Large deletions and small insertions and deletions in the factor VIII gene predict unfavorable immune tolerance induction outcome in people with severe hemophilia A and high-responding inhibitors.

INTRODUCTION: Hemophilia A is an inherited bleeding disorder caused by pathogenic variants in the factor VIII gene (F8), which leads to factor VIII (FVIII) deficiency. Immune tolerance induction (ITI) is a therapeutic approach to eradicate alloantibodies (inhibitors) against exogenous FVIII in people with inherited hemophilia A. Few studies have evaluated the role of F8 variants on ITI outcome. MATERIAL AND METHODS: We included people with severe hemophilia A (FVIII ˂ 1 international units/dL) and high-responding inhibitors (≥ 5 Bethesda units/mL lifelong) who underwent a first course of ITI. Socio-demographic, clinical and laboratory data were collected. ITI outcomes were defined as total, partial successes, and failure. Detection of intron 1 and 22 inversions was performed by polymerase-chain reaction, followed by F8 sequencing. RESULTS: We included 168 people with inherited hemophilia A and high-responding inhibitors, median age 6 years at ITI start. Intron 22 inversion was the most prevalent variant (53.6 %), followed by nonsense (16.1 %), small insertion/deletion (11.3 %), and large deletion (10.7 %). In comparison with intron 22 inversion, the odds of ITI failure were 15.5 times higher (odds ratio [OR] 15.50; 95 % confidence interval [95 % CI] 4.59-71.30) and 4.25 times higher (95 % CI, 1.53-12.3) among carriers of F8 large deletions and small insertions and deletions, respectively. CONCLUSION: F8 large deletions and small insertions/deletions predicted ITI failure after a first course of ITI in patients with severe hemophilia A and high-responding inhibitors. This is the first study to show F8 large deletions and small insertions/deletions as predictors of ITI failure.

A Scan of Pleiotropic Immune Mediated Disease Genes Identifies Novel Determinants of Baseline FVIII Inhibitor Status in Hemophilia-A.

Hemophilia-A (HA) is caused by heterogeneous loss-of-function factor (F)VIII gene (F8)-mutations and deficiencies in plasma-FVIII-activity that impair intrinsic-pathway-mediated coagulation-amplification. The standard-of-care for severe-HA-patients is regular infusions of therapeutic-FVIII-proteins (tFVIIIs) but ~30% develop neutralizing-tFVIII-antibodies called "FVIII-inhibitors (FEIs)" and become refractory. We used the PATH study and ImmunoChip to scan immune-mediated-disease (IMD)-genes for novel and/or replicated genomic-sequence-variations associated with baseline-FEI-status while accounting for non-independence of data due to genetic-relatedness and F8-mutational-heterogeneity. The baseline-FEI-status of 450 North American PATH subjects-206 with black-African-ancestry and 244 with white-European-ancestry-was the dependent variable. The F8-mutation-data and a genetic-relatedness matrix were incorporated into a binary linear-mixed model of genetic association with baseline-FEI-status. We adopted a gene-centric-association-strategy to scan, as candidates, pleiotropic-IMD-genes implicated in the development of either ³2 autoimmune-/autoinflammatory-disorders (AADs) or ³1 AAD and FEIs. Baseline-FEI-status was significantly associated with SNPs assigned to NOS2A (rs117382854; p=3.2E-6) and B3GNT2 (rs10176009; p=5.1E-6), which have functions in anti-microbial-/-tumoral-immunity. Among IMD-genes implicated in FEI-risk previously, we identified strong associations with CTLA4 assigned SNPs (p=2.2E-5). The F8-mutation-effect underlies ~15% of the total heritability for baseline-FEI-status. Additive genetic heritability and SNPs in IMD-genes account for >50% of the patient-specific variability in baseline-FEI-status. Race is a significant determinant independent of F8-mutation-effects and non-F8-genetics.

Mutation detection and inhibitor risk in Iranian patients with Hemophilia A: Six novel mutations.

This investigation facilitates a better understanding of inhibitor development, the critical treatment morbidity in HA patients. Furthermore, six novel mutations are reported, which would expand the mutation spectrum of the F8 gene.

Spectrum of Molecular Defects in 216 Chinese Families With Hemophilia A: Identification of Noninversion Mutation Hot Spots and 42 Novel Mutations.

Hemophilia A (HA) is an X-linked bleeding disorder caused by heterogeneous mutations in the factor VIII gene ( F8). Our aim is to identify the causative mutations in a large HA cohort from China. We studied 216 unrelated HA families. Molecular analyses of F8 were performed using a combination of molecular techniques, including polymerase chain reaction, direct sequencing, and multiplex ligation-dependent probe amplification. The deleterious consequences of the unreported missense mutations were evaluated using various bioinformatics approaches. Causative mutations in F8 were identified in 209 families, intron 22 inversion (Inv22) was identified in 89 severe families, and intron 1 inversion (Inv1) was positive in 5 severe families; 95 mutations were detected among 115 noninversion families, of which 42 were novel, including 29 null variations and 13 missense mutations for which causality was demonstrated via bioinformatics. Among the 53 previously reported mutations, more nonsense (5 of 9) and missense (10 of 26) mutation sites were found to occur at Arginine (Arg) sites and multiple small deletions/insertions (5 of 10) located within the poly-A runs of the B domain. The majority of these sequence variants frequently recurred in the database. The odds ratios for the likelihood of developing inhibitors significantly increased in the presence of nonsense mutation. Our F8 defect spectrum was heterogeneous. Small deletions/insertions in the poly-A runs of the B domain and nonsense and missense mutations at Arg sites were identified as mutation hot spots. Nonsense mutation increased the risk of developing inhibitors.

Literature review and clinical observation of acquired idiopathic hemophilia with a new missense mutation in the factor VIII gene (His2026Arg).

The article provides review of possible mechanisms of inhibitor coagulopathies, in particular of acquired hemophilia A. This pathology is an extremely rare disease occurring in 1-2 cases in 1 million per year. In the present study we provide data for two clinical cases of hemophilia A in women. These cases had different development mechanisms, although both women have a newly discovered missense mutation His2026Arg in the VIII factor gene. The matter of main interest is the description of the disease development in the patient with an acquired idiopathic hemophilia A with a possible disease occurrence due to an asymmetric X-chromosome inactivation (lyonization). In this particular case lyonization led to the late manifestation of the hemophilia A carrier's state and development of severe form of the inhibitor-associated acquired hemophilia A. We also discuss therapeutic approaches to these forms of the disease, considering there are no concise protocols for case management due to an extreme rarity of the pathology. Acquainting the clinical personnel working it the different areas of medicine with suchlike inhibitor coagulopathies has a major practical importance.

Current strategy for genetic analysis of haemophilia A families.

Carrier detection and prenatal diagnosis of haemophilia A, which was based in the last decade mainly on linkage polymorphism analysis, has been greatly facilitated by the recent discovery that two types of inversion disrupting the factor VIII gene are common mutations observed in 42-48% of severe haemophilia A cases. In this study DNA analysis was performed in 64 unrelated severe haemophilia A patients and 173 women belonging to their families, and in four women from a family with a deceased haemophilic relative whose DNA was unavailable (a total of 177 females from 65 unrelated families). Factor VIII gene inversions were found in 32 out of the 65 families (49%), 29 involving recombination with the distal A gene and three with the proximal A gene. Definitive information regarding carriership of haemophilia was provided to all 81 women belonging to the 32 inversion-positive families, among them one woman previously uninformative for any of the polymorphisms examined, five women who were informative only for extragenic polymorphisms, and four suspected carriers who were relatives of the deceased haemophiliac. In 33 inversion-negative families, 96 females were examined by analysis of the BclI restriction fragment length polymorphism (RFLP) in intron 18 and of the multiallelic dinucleotide repeats in introns 13 and 22, followed by analysis of other intragenic polymorphisms. This procedure yielded an informativity rate of almost 100%. Of the 96 females examined by linkage polymorphism analysis, 78 belonged to 25 families with more than one haemophiliac and 29 of them were obligate carriers. In 47 of the 49 suspected carriers linkage polymorphism analysis enabled definition of carriership based on intragenic polymorphisms. 18 of the 96 females belonged to eight families with sporadic haemophilia cases and only eight of the 18 suspected carriers could be diagnosed by exclusion. In nine pregnant women carrying factor VIII gene inversions, mRNA extracted from chorionic villus samples (CVS) was analysed for factor VIII gene inversion by reverse transcription/polymerase chain reaction (RT/PCR). This procedure enabled rapid prenatal diagnoses in six male fetuses. Taken together, our data indicate that a high rate of informativity and carrier definition is possible by the strategy of first screening for factor VIII gene inversions, and, if none are found, sequential use of highly informative intragenic polymorphisms, followed by less informative intragenic and extragenic polymorphisms.