OC 74.3 - Characterization and dissection of new structural variants in FXI deficiency by nanopore sequencing.

Wednesday, July 13, 2022

10:45 AM – 11:00 AM

Location: ExCel Center, ICC Capital Suite Room 8&9

Presentation Format: In-Person

Presenting Author(s)

Belen de la morena-Barrio, PhD

Postdoctoral fellow
Servicio de Hematología y Oncología Médica, Hospital Universitario Morales Meseguer, Centro Regional de Hemodonación, Universidad de Murcia, IMIB-Pascual Parrilla, CIBERER, Murcia, Spain
murcia, Murcia, Spain

Co-Author(s)

�P

Ángeles Palomo

MD, Servicio de Hematología y Hemoterapia
Centro Materno-Infantil del Hospital Regional Universitario Carlos de Haya, Málaga
Málaga, Andalucia, Spain
AR

Ana María Rubio

Servicio de Hematología y Oncología Médica
Hospital Universitario Morales Meseguer-Centro Regional de Hemodonación, Universidad de Murcia, IMIB, CIBERER Spain
Murcia, Murcia, Spain
JP

Jose Padilla

Technician
Servicio de Hematología y Oncología Médica, Hospital Universitario Morales Meseguer, Centro Regional de Hemodonación, Universidad de Murcia, IMIB-Pascual Parrilla, CIBERER, Murcia, Spain
Murcia, Murcia, Spain
LM

Laura Martin

PhD
Unitat de Genòmica de Malalties Complexes. IIB-Sant Pau. Barcelona.
Barcelona, Catalonia, Spain
RC

Rosa Cifuentes

Predoctoral fellow
Servicio de Hematología y Oncología Médica, Hospital Universitario Morales Meseguer, Centro Regional de Hemodonación, Universidad de Murcia, IMIB-Pascual Parrilla, CIBERER, Murcia, Spain
Murcia, Murcia, Spain
Carlos Bravo Perez, MD, PhD

MD, PhD
Servicio de Hematología y Oncología Médica, Hospital Universitario Morales Meseguer, Centro Regional de Hemodonación, Universidad de Murcia, IMIB-Pascual Parrilla, CIBERER, Murcia, Spain
Murcia, Murcia, Spain
AM

Antonia Miñano

Servicio de Hematología y Oncología Médica
Hospital Universitario Morales Meseguer-Centro Regional de Hemodonación, Universidad de Murcia, IMIB, CIBERER Spain
Murcia, Murcia, Spain
VV

Vicente Vicente

Researcher and Medical Doctor
Servicio de Hematología y Oncología Médica, Hospital Universitario Morales Meseguer, Centro Regional de Hemodonación, Universidad de Murcia, IMIB-Pascual Parrilla, CIBERER, Murcia, Spain
Murcia, Murcia, Spain
ML

María Luisa Lozano

Leader of Hematology Service in Murcia
Servicio de Hematología y Oncología Médica, Hospital Universitario Morales Meseguer, Centro Regional de Hemodonación, Universidad de Murcia, IMIB-Pascual Parrilla, CIBERER, Murcia, Spain
Murcia, Murcia, Spain
Fv

Francisco vidal

Doctor
Coagulopaties congènites. Banc de Sang i Teixits. Barcelona. Unitat de Diagnòstic i Teràpia Molecular. Vall d’Hebron Institut de Recerca, Universitat Autònoma de Barcelona (VHIR-UAB). Barcelona.
Barcelona, Catalonia, Spain
JC

Javier Corral

Senior researcher
Servicio de Hematología y Oncología Médica, Hospital Universitario Morales Meseguer, Centro Regional de Hemodonación, Universidad de Murcia, IMIB-Pascual Parrilla, CIBERER, Murcia, Spain
Murcia, Murcia, Spain
Maria Eugenia de la Morena-Barrio, PhD

Reseacher
Servicio de Hematología y Oncología Médica, Hospital Universitario Morales Meseguer, Centro Regional de Hemodonación, Universidad de Murcia, IMIB-Arrixaca, CIBERER, Murcia, Spain
Murcia, Murcia, Spain

Background: Molecular diagnosis of FXI deficiency, usually restricted to F11 sequencing, has identified up to 280 variants, mostly SNVs or Indels. Only 3 causative structural variants (SV) have been reported: two whole gene deletions and one partial deletion.

Aims: Identification and characterization of new SVs causing FXI deficiency.

Methods: We studied 272 patients with FXI deficiency from 105 families identified by prolonged aPTT and characterized by functional and immunological methods. F11 was sequenced and SVs were identified by MLPA. The characterization of SVs was done by long-read nanopore sequencing using real-time enrichment methods in a MinION device. Validation involved PCR and NGS/sanger sequencing.

Results: Two cases with CRM- deficiency and no F11 defects detected by sequencing, carried heterozygous SVs identified by MLPA. P1 had a duplication of exons 8&9 located in intron 4, that segregated in 5 family members (FXI:C=44.3%). P2, with severe deficiency (FXI:C=5%), had a complete deletion of F11 in one allele, and a deleterious variant of maternal origin in the other allele (p.Phe295Cys). His father and sister had normal FXI levels and no F11 defects. STR analysis confirmed paternity. Deep NGS of F11 (>2000 reads) in 2 tissues of the patient discarded a mosaicism.
Nanopore sequencing accurately defined the extension of the deletion (7 MB, 142 genes) and duplication (1648 bp); and characterized the breakpoints, identifying repetitive elements (RE) in both cases. (Figure 1 and Figure 2)

Conclusion(s): SVs affecting F11, probably formed by recombination of RE, are heterogeneous in type and extension and may be relatively frequent (2%) in FXI deficiency. We detected the first de novo deletion of F11, which probably occurred during the spermatogenesis. These data support the screening of SVs affecting F11 in FXI deficiency. MLPA is able to detect SVs, but nanopore sequencing is the best method to characterize at nucleotide level these SVs.
PI21/00137 (ISCIII&FEDER)