PB0267 - Long-read nanopore sequencing used for comprehensive structural variant characterization in severe hemophilia patients.

Sunday, June 23, 2024

13:45 – 14:45 ICT

Room: Exhibition Hall

Presenting Author(s)

NB

Nina Borràs

PhD
Laboratori de Coagulopaties Congènites, Banc de Sang i Teixits, Barcelona, Spain
Barcelona, Catalonia, Spain

Co-Author(s)

LC

Laia Closa

PhD
Laboratori d’Histocompatibilitat i Immunogenètica, Banc de Sang i Teixits, Barcelona, Spain
Barcelona, Catalonia, Spain
Belén de la Morena-Barrio, PhD

Post-doc
Servicio de Hematología, Hospital General Universitario Morales Messeguer, Centro Regional de Hemodonación, Universidad de Murcia, Universidad de Murcia, IMIB-Pascual Parrilla, CIBERER-ISCIII
Murcia, Murcia, Spain
LR

Lorena Ramírez

Technician
Laboratori de Coagulopaties Congènites, Banc de Sang i Teixits, Barcelona, Spain
Barcelona, Catalonia, Spain
Javier Corral

Professor
Servicio de Hematología, Hospital General Universitario Morales Messeguer, Centro Regional de Hemodonación, Universidad de Murcia, Universidad de Murcia, IMIB-Pascual Parrilla, CIBERER-ISCIII
Murcia, Murcia, Spain
OB

Olga Benítez

Hematologist
Department of Hematology, Hospital Universitari Vall d'Hebron Vall d' Hebron Institut of Oncology (VHIO)
Barcelona, Catalonia, Spain
CM

Cristina Marzo Alonso

hematologist
HUAV
Lleida, Catalonia, Spain
RN

Ramiro Núñez Vázquez

Head of thrombosis and hemostasis section
Hospital Universitario Virgen del Rocío
Sevilla, Andalucia, Spain
IS

Inmaculada Soto Ortega

Head of thrombosis and hemostasis section
Hospital Universitario Central de Asturias
Oviedo, Asturias, Spain
FV

Francisco Vidal

Head of Unit, PhD
Laboratori de Coagulopaties Congènites, Banc de Sang i Teixits, Barcelona, Spain
Barcelona, Catalonia, Spain
IC

Irene Corrales

Main researcher, PhD
Laboratori de Coagulopaties Congènites, Banc de Sang i Teixits, Barcelona, Spain
Barcelona, Catalonia, Spain

Background: Structural variants (SV) in F8 or F9 accounts for approximately 1-5% of the variants causing hemophilia A or B, respectively. Our laboratory has identified 25 different SV by PCR/MLPA, 11 have been fully characterized by gene-walking methods, while the exact breakpoints and underlying molecular mechanisms of the remaining 14 SVs continue unresolved.

Aims: Explore long-read sequencing (LRS) based on nanopore to fully characterize SVs identified in patients with hemophilia.

Methods: LRS was performed on Oxford Nanopore Technologies (ONT) devices. Two enrichment methods for regions of interest were tested: 1) long range PCR and 2) real-time computational enrichment using the Adaptive Sampling tool implemented in the MinKNOW platform.

Results: This technology was used to analyze 6 different SVs (4 involved intragenic F8 regions and 1 whole F9). Of them, 3 previously characterized were used to validate LRS procedures (Table 1, Figure 1). The exact breakpoints could be identified in 5 of them. The size of SVs varied from 9,706 bp to 4 Mb. Repetitive regions (LTR, LINE, SINE) were identified in 7/10 breakpoints. At all breakpoints, microhomologies between 2 and 8 bp were identified. About P4, preliminary results revealed a complex rearrangement involving intron 22 inversion and the duplication of exons 23-25 in F8 most likely mediated by Alu-elements. Specific strategies are being developed to fully unravel the molecular mechanism.

Conclusion(s): Our results highlight the value of LRS as an efficient, fast, and cost-effective method for comprehensive characterization of F8 and F9. It is of special interest for cases with duplications/insertions or when the ends of the gene are compromised, given the impossibility of limiting the size of the SV. The application of this technology will allow the comprehensive characterization of unresolved SVs, expanding our understanding of the mechanisms involved in these mutations.

PI18/01492, PI21/00137, PI23/01672, RFVE, Fundació Privada Catalana de l’Hemofília, PMP21/00052