Usefulness and Limitations of Multiple Ligation-Dependent Probe Amplification in Antithrombin Deficiency

Multiplex ligation-dependent probe amplification (MLPA) identifies genetic structural variants in in 5% of cases with antithrombin deficiency (ATD), the most severe congenital thrombophilia. Our aim was to unravel the utility and limitations of MLPA in a large cohort of unrelated patients with ATD (...

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Published in:International journal of molecular sciences 2023-03, Vol.24 (5), p.5023
Main Authors: Cifuentes, Rosa, Padilla, José, de la Morena-Barrio, María Eugenia, de la Morena-Barrio, Belén, Bravo-Pérez, Carlos, Garrido-Rodríguez, Pedro, Llamas, María, Miñano, Antonia, Vicente, Vicente, Lozano, María Luisa, Corral, Javier
Format: Article
Language:English
Subjects:
Algorithms
Anticoagulants
Antigens
Antithrombin
Antithrombins
Defects
DNA sequencing
Exons
Gene deletion
Genetic disorders
Genetic engineering
Genomes
Humans
Introns
Investigations
Methods
Multiplex Polymerase Chain Reaction - methods
Mutation
Nucleotide sequencing
Nucleotides
Patients
Plasma
Probes
Thrombophilia
Thrombophilia - genetics
Thrombosis
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Summary:Multiplex ligation-dependent probe amplification (MLPA) identifies genetic structural variants in in 5% of cases with antithrombin deficiency (ATD), the most severe congenital thrombophilia. Our aim was to unravel the utility and limitations of MLPA in a large cohort of unrelated patients with ATD (N = 341). MLPA identified 22 structural variants (SVs) causing ATD (6.5%). MLPA did not detect SVs affecting introns (four cases), and the diagnosis was inaccurate in two cases according to long-range PCR or nanopore sequencing. MLPA was used to detect possible hidden SVs in 61 cases with type I deficiency with single nucleotide variations (SNVs) or small insertion/deletion (INDEL). One case had a false deletion of exon 7, as the 29-bp deletion affected an MLPA probe. We evaluated 32 variants affecting MLPA probes: 27 SNVs and 5 small INDELs. In three cases, MLPA gave false-positive results, all diagnosed as deletions of the affected exon: a small INDEL complex, and two SNVs affecting MLPA probes. Our study confirms the utility of MLPA to detect SVs in ATD, but also shows some limitations in detecting intronic SVs. MLPA renders imprecise and false-positive results for genetic defects which affect MLPA probes. Our results encourage the validation of MLPA results.
ISSN:1422-0067
1661-6596
1422-0067
DOI:10.3390/ijms24055023