Pitfalls in the detection of gross gene rearrangements using MLPA in Fabry disease.

MLPA (Multiplex Ligation-dependent Probe Amplification) is a semiquantitative molecular technique developed to uncover gross gene rearrangements in several monogenic diseases, including the X-linked Fabry disease (FD) caused by mutations in the GLA gene. Heterozygosity of the X chromosome in females makes it important to combine routine sequencing analysis with at least one allelic dosage assay (i.e. MLPA). We identified two new gross GLA gene rearrangements, which were not properly detected by MLPA in male patients with FD. In Patient 1, routine sequencing did not amplify GLA exon 7. MLPA failed to confirm such deletion. An alternative allele dosage, based on the Quantitative Fluorescent Multiplex-PCR (QFM-PCR), confirmed the mutation in the proband and excluded it in the mother, revealing that the mutation was de novo in the proband. Patient 2 harboured a gross GLA gene deletion encompassing almost the entire exon 5, the entire intron 5 and part of exon 6. MLPA confirmed the deletion of exon 5 but missed the partial deletion of exon 6. We characterised the breakpoint (c.652_886del452) and carried out screening for possible heterozygosity among at risk female family members. MLPA can miss some gross GLA gene rearrangements making the combination with other allele dosage assays mandatory to confirm or exclude FD at the molecular level.