title: Whole-exome sequencing to identify genetic risk variants underlying inhibitor development in severe hemophilia A patients.
abstract: The development of neutralizing antibodies (inhibitors) against coagulation factor VIII (FVIII) is the most problematic and costly complication of FVIII replacement therapy that affects up to 30% of previously untreated patients with severe hemophilia A. The development of inhibitors is a multifactorial complication involving environmental and genetic factors. Among the latter, F8 gene mutations, ethnicity, family history of inhibitors, and polymorphisms affecting genes involved in the immune response have been previously investigated. To identify novel genetic elements underling the risk of inhibitor development in patients with severe hemophilia A, we applied whole-exome sequencing (WES) and data analysis in a selected group of 26 Italian patients with (n = 17) and without (n = 9) inhibitors. WES revealed several rare, damaging variants in immunoregulatory genes as novel candidate mutations. A case-control association analysis using Cochran-Armitage and Fisher's exact statistical tests identified 1364 statistically significant variants. Hierarchical clustering of these genetic variants showed 2 distinct patterns of homozygous variants with a protective or harmful role in inhibitor development. When looking solely at coding variants, a total of 28 nonsynonymous variants were identified and replicated in 53 inhibitor-positive and 174 inhibitor-negative Italian severe hemophilia A patients using a TaqMan genotyping assay. The genotyping results revealed 10 variants showing estimated odds ratios in the same direction as in the discovery phase and confirmed the association of the rs3754689 missense variant (OR 0.58; 95% CI 0.36-0.94; P = .028) in a highly conserved haplotype region surrounding the LCT locus on chromosome 2q21 with inhibitor development.
authors.affiliation: Department of Pathophysiology and Transplantation, Università degli Studi di Milano, Milan, Italy; and.
authors.forename: Marcin M
authors.identifier: 0000-0002-8276-3866
authors.initials: MM
authors.lastname: Gorski
authors.affiliation: Angelo Bianchi Bonomi Hemophilia and Thrombosis Center, Fondazione Istituto di Ricovero e Cura a Carattere Scientifico Ca' Granda, Ospedale Maggiore Policlinico and Fondazione Luigi Villa, Milan, Italy.
authors.forename: Kevin
authors.initials: K
authors.lastname: Blighe
authors.affiliation: Angelo Bianchi Bonomi Hemophilia and Thrombosis Center, Fondazione Istituto di Ricovero e Cura a Carattere Scientifico Ca' Granda, Ospedale Maggiore Policlinico and Fondazione Luigi Villa, Milan, Italy.
authors.forename: Luca A
authors.initials: LA
authors.lastname: Lotta
authors.affiliation: Department of Pathophysiology and Transplantation, Università degli Studi di Milano, Milan, Italy; and.
authors.forename: Emanuela
authors.identifier: 0000-0002-4808-0498
authors.initials: E
authors.lastname: Pappalardo
authors.affiliation: Department of Pathophysiology and Transplantation, Università degli Studi di Milano, Milan, Italy; and.
authors.forename: Isabella
authors.identifier: 0000-0002-2699-0394
authors.initials: I
authors.lastname: Garagiola
authors.affiliation: Department of Pathophysiology and Transplantation, Università degli Studi di Milano, Milan, Italy; and.
authors.forename: Ilaria
authors.identifier: 0000-0002-5059-5212
authors.initials: I
authors.lastname: Mancini
authors.affiliation: Angelo Bianchi Bonomi Hemophilia and Thrombosis Center, Fondazione Istituto di Ricovero e Cura a Carattere Scientifico Ca' Granda, Ospedale Maggiore Policlinico and Fondazione Luigi Villa, Milan, Italy.
authors.forename: Maria Elisa
authors.identifier: 0000-0002-7113-4028
authors.initials: ME
authors.lastname: Mancuso
authors.affiliation: Angelo Bianchi Bonomi Hemophilia and Thrombosis Center, Fondazione Istituto di Ricovero e Cura a Carattere Scientifico Ca' Granda, Ospedale Maggiore Policlinico and Fondazione Luigi Villa, Milan, Italy.
authors.forename: Maria Rosaria
authors.initials: MR
authors.lastname: Fasulo
authors.affiliation: Angelo Bianchi Bonomi Hemophilia and Thrombosis Center, Fondazione Istituto di Ricovero e Cura a Carattere Scientifico Ca' Granda, Ospedale Maggiore Policlinico and Fondazione Luigi Villa, Milan, Italy.
authors.forename: Elena
authors.initials: E
authors.lastname: Santagostino
authors.affiliation: Department of Pathophysiology and Transplantation, Università degli Studi di Milano, Milan, Italy; and Angelo Bianchi Bonomi Hemophilia and Thrombosis Center, Fondazione Istituto di Ricovero e Cura a Carattere Scientifico Ca' Granda, Ospedale Maggiore Policlinico and Fondazione Luigi Villa, Milan, Italy.
authors.forename: Flora
authors.identifier: 0000-0001-7423-9864
authors.initials: F
authors.lastname: Peyvandi
mesh_terms: D057134:Antibodies, Neutralizing / Q000096:biosynthesis*; D019774:Blood Coagulation Factor Inhibitors / Q000096:biosynthesis*; D016022:Case-Control Studies; D004252:DNA Mutational Analysis / Q000379:methods; D020022:Genetic Predisposition to Disease; D055106:Genome-Wide Association Study; D006467:Hemophilia A / Q000235:genetics* / Q000276:immunology; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D009154:Mutation; D020641:Polymorphism, Single Nucleotide; D012307:Risk Factors; D012720:Severity of Illness Index
chemical_list: D057134:Antibodies, Neutralizing; D019774:Blood Coagulation Factor Inhibitors