Identification of a novel large deletion in a patient with severe factor V deficiency using an in‐house F5 MLPA assay

Abstract

Factor V (FV) deficiency is a rare autosomal recessive bleeding disorder caused by mutations in the F5 gene. FV-deficient patients in whom no mutation or only one mutation is found may harbour large gene rearrangements, which are not detected by conventional mutation screening strategies. The aim of this study was to develop and validate a multiplex ligation-dependent probe amplification (MLPA) assay for the detection of large deletions and duplications in the F5 gene. Twenty-two MLPA probes targeting 19 of the 25 exons and the upstream and downstream regions of the F5 gene were designed and tested in 10 normal controls, a patient with a known heterozygous deletion of F5 exons 1-7 (positive control) and 14 genetically unexplained FV-deficient patients. MLPA results were confirmed by digital PCR on a QuantStudio(™) 3D Digital PCR System. The F5-specific probes yielded a reproducible peak profile in normal controls, correctly detected the known deletion in the positive control and suggested the presence of a novel deletion of exons 9-10 in a patient with undetectable FV levels and only one identified mutation. Follow-up by chip-based digital PCR, long-range PCR and direct sequencing confirmed that this patient carried a heterozygous F5 deletion of 1823 bp extending from intron 8 to intron 10. Bioinformatics sequence analysis pinpointed repetitive elements that might have originated the deletion. In conclusion, we have developed and validated an MLPA assay for the detection of gross F5 gene rearrangements. This assay may represent a valuable tool for the molecular diagnosis of FV deficiency.

Characterization of an apparently synonymous F5 mutation causing aberrant splicing and factor V deficiency

“…Patients with plasma FV levels in the 0–5% range are expected to be homozygous or compound heterozygous for F5 mutations, but 10–20% of cases remain genetically unexplained . Failure to identify one or both mutations by direct sequencing of the coding region may signify the presence of gross gene rearrangements or deep‐intronic splicing mutations , which are not detected by conventional mutation screening strategies. Alternatively, it may be due to misclassification of exonic variants, which are typically assessed only on the basis of their predicted effects on translation, whereas they may just as well impair pre‐mRNA splicing, as recently shown for missense mutations in the FGB and F11 genes as well as for a synonymous mutation in the LMAN1 gene .…”

Whole-exome sequencing and digital PCR identified a novel compound heterozygous mutation in the NPHP1 gene in a case of Joubert syndrome and related disorders

“…In the present case, we performed chip-based digital PCR to confirm the NPHP1 deletion. This new PCR technique allows absolute quantification of copy numbers without reference to a standard curve, and produces precise results compared with conventional real-time quantitative PCR [18, 19]. We show that digital PCR is an alternative efficient method to confirm clinical relevant CNVs in genetic diagnosis.…”

Antisense-Mediated Down-Regulation of Factor V-Short Splicing in a Liver Cell Line Model

“…Genomic DNA isolated from HepG2 cells was standardized to 20 ng/µL and the MLPA reaction was performed following the manufacturer's protocol. FAM-labelled MLPA amplification products were mixed with GeneScan TM 600 LIZ ® dye Size Standard and Hi-Di TM formamide (Life Technologies, Bleiswijk, The Netherlands), denatured and separated by capillary electrophoresis on an ABI 3730 DNA Analyzer (Life Technologies), as previously described [35]. The results were analysed using Coffalyser.Net (MRC Holland).…”