Molecular study of a large cohort of 109 haemophilia patients from Cuba using a gene panel with next generation sequencing‐based technology

Abstract

Introduction In several countries, molecular diagnosis of haemophilia A (HA) and B (HB) is hampered by a lack of resources for DNA analysis. The advent of next‐generation sequencing (NGS) has enabled gene analysis at a reasonable cost. Aim Describe a collaboration between Cuban and Spanish researchers to identify candidate variants and investigate the molecular epidemiology of 106 Cuban haemophilia patients using NGS. Patients/methods The molecular analysis protocol included well‐established LR‐PCR procedures to detect F8 inversions, NGS with a 30‐gene panel to sequence F8 and F9, and multiplex ligation‐dependent probe amplification to identify large structural variants. Results One‐hundred and thirty‐one candidate variants were identified along F8, F9, and VWF; 72 were unique and 28 (39%) had not been previously recorded. Putative variants were identified in 105/106 patients. Molecular characterization enabled confirmation and reclassification of: 90 HA (85%), 15 HB (14%), and one type 2N VWD (1%). Null variants leading to non‐production of FVIII or FIX were common in severe HA (64%), moderate HA (74%), and severe HB (60%), whereas missense variants were frequent in mild HA (57%) and moderate or mild HB (83%). Additional variants in VWF were identified in 16 patients. Conclusion This is the first description of the molecular epidemiology of HA and HB in Cuba. Variants identified in index cases will be of value for local implementation of familial studies and prenatal diagnosis using the molecular approaches available in Cuba. The results of this protocolled genetic study improved the accuracy of the clinical diagnosis and will facilitate management of these patients.

Clinical and molecular characteristics of hemophilia A affected individuals and carriers: A 24 years experience from three centers

“…PCR-based procedures with targeted analysis for intron 22 and intron 1 inversions, followed by sequencing analysis and multiplex ligation-dependent probe amplification (MLPA), are often employed to interrogate the F8 gene. The diagnostic yield in hemophilia A-affected individuals is consistently described as >95% across different cohorts, and it is later evident that a significant proportion of these affected individuals carry novel variants that are private to the family (Borras et al, 2022;Johnsen et al, 2022). The growth in knowledge of F8 molecular pathophysiology has been accompanied by the discovery of several wellestablished genotype-phenotype relationships: Common inversions in intron 22 and intron 1, as well as intragenic truncating variants (e.g., frameshift and nonsense variants), are known to be associated with severe hemophilia A (Konkle & Nakaya Fletcher, 1993).…”