Community Based Evaluation of Prevalence of Inhibitors in Patients with Severe Hemophilia A in India and Their Correlation with Environmental and Genetic Factors
Authors: David Sachin , Mathews Vikram , Apte Shashikant , Jayandharan Giridhara R , Nair Sukesh C , Daniel Dolly , Kumar Suresh , Alex Ansu Abu , Ganesan Saravanan , Subramanian Kannan , Ahmed Rayaz , Abraham Aby , Viswabandya Auro , George Biju , Srivastava Alok 
Published: October 2019

Abstract
Abstract 3380
The reported prevalence of inhibitors in patients with severe hemophilia A varies from 7 to 19% and there is a perception that the prevalence is lower in developing countries. A number of environmental and genetic factors have been reported to be associated with the risk of inhibitor development. Most of these data are from developed countries and a predominantly Caucasian population. We undertook a large multi-center, community based study in India to evaluate the prevalence of inhibitors among patients with severe hemophilia A and to study predisposing environmental and genetic factors in this cohort.
From 2009, we organized multiple community based camps in India and collected samples from cases after getting a written informed consent. Samples from a total of 470 patients were collected of which 312 fulfilled the inclusion criteria (diagnosis of severe hemophilia A confirmed in a central laboratory and at least 5 life time exposures to factor replacement). A detailed questionnaire was administered to all subjects to obtain demographic, clinical and treatment details. FVIII:C assay and Nijmegen modified Bethesda assay for inhibitors was done on all patients. Genomic DNA was used for PCR-RFLP's (IL4Rα Ile50Val; Arg551Gln, IL4 –590C/T, IL5 746T>C, CTLA4 49G/A), allele specific PCR's for identifying cytokine polymorphisms (TNF α −308G/A, TGF β 1 codon 10T/C; codon 25C/G, IL6 –174C/G, IL10 –592A/C; −819C/T; 1082A/G, IFNγ +874T/A) was done using cytokine genotyping SSP kits, VNTR's in IL5Rα 3'UTR by gene scan. HLA Class II typing was done using standard kit (AllSet+ SSP PCR kits). The immunophenotype of the Treg population was analyzed by flowcytometry using co-expression of the CD4+, CD25+ markers.
A total of 60/312 (19.2%) cases had evidence of inhibitors. Of these 22 (36.6%) had high titre inhibitors (≥5 BU). Table I compares the demographic and laboratory variables among cases that had inhibitors versus those that did not. In this study we could not find a correlation with environmental factors such as exposure to factor replacement in the first year of life, total factor replacement, surgery, chronic infections and additional medical illness or to number of joints affected and bleeding into other organs. While all patients in this study had FVIII levels <1%, patients with inhibitors had a significantly lower FVIII level than those that did not (Table 1). Cases with inhibitors were significantly more likely to have siblings with inhibitors (Table 1). 7 (11.5%) of cases with inhibitors had a discordant affected sibling who did not have inhibitors.
Of the evaluated immune-regulatory gene polymorphisms a significant protective association was noted with the heterozygous IL4–590 C/T allele and development of inhibitors (RR= 0.22; 95%CI 0.108–0.442: P=0.000). This protective effect was retained in the high titre subset when low titre inhibitor positive cases were excluded from the analysis. There was a significant increased risk of inhibitor formation among cases that were HLA DRB1–13 positive cases (RR=2.04; 95%CI 1.06 – 3.911; P=0.033) and this significance was retained in the high titre subset. There was also a trend to decreased risk of inhibitor formation among those that were HLA-DRB1–07 positive and this achieved statistical significance among the inhibitor cases that were high titre positive (RR=0.24; 95% CI0.055–1.05: P=0.047). There was no significant association with T-reg levels and inhibitors.
In conclusion the prevalence of inhibitors in patients with severe hemophilia A is similar to that reported in developed countries when evaluated in patients with at least 5 exposures. FVIII: C levels are significantly lower in cases with inhibitors. In this series there was no apparent association with any of the environmental parameters that were evaluated while there was a significant association with IL4–590 C/T polymorphism, HLA-DRB1–13 and a trend with HLA-DRB1–07. This along with the increased incidence of inhibitors in siblings with hemophilia suggests that inherited parameters may have a more significant pre-disposing effect than environmental factors in inhibitor formation in this population.

Disclosures:
No relevant conflicts of interest to declare.