Multiplex ligation-dependent probe amplification (MLPA) identifies genetic structural variants in SERPINC1 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.
Keywords: antithrombin deficiency; genetic variants; multiplex ligation-dependent probe amplification; structural variants.