Abstract
Background: Multiplex Ligation Probe Amplification (MLPA) is a widely used technique for the diagnosis of lysosomal storage diseases (LSDs). It analyses over 40 DNA sequences in a single reaction, identifying copy number variations and large deletions/insertions in genes. The diagnostic process in LSDs starts with analysis of the missing or reduced enzyme, followed by genetic investigation and, if possible, a search for accumulated substrates. However, while genetic analysis using Sanger sequencing is excellent at detecting small genetic variations such as single-nucleotide variants (SNVs) and small insertions or deletions, it cannot detect large deletions or insertions. Methods: In the present study, a total of 800 patients with clinical suspicion of Fabry, Gaucher, or Pompe diseases were investigated. An enzyme assay was carried out on each patient, followed by genetic analysis using PCR, Sanger sequencing, and MLPA. Results: Nine patients with deficient or absent enzyme activity had Sanger sequencing results that could not confirm the molecular genetic diagnosis because either no mutation (Fabry) or only one mutation (Gaucher and Pompe) was identified. Subsequent analysis by MLPA identified two males with a hemizygous deletion and two females with a heterozygous deletion for FD. For PD, one female and two males had a heterozygous deletion. For GD, one male had a homozygous deletion and one female had a heterozygous deletion. The remaining patients were analyzed by MLPA with negative results. Conclusions: The results obtained suggest that MLPA should be used in combination with classical sequencing methods to ensure a correct and timely diagnosis of LSDs.