Multiplex Ligation-dependent Probe Amplification Analysis Subsequent to Direct DNA Full Sequencing for IdentifyingATP7BMutations and Phenotype Correlations in Children with Wilson Disease

Abstract

BackgroundMutations in ATP7B cause Wilson disease (WD). However, direct DNA full sequencing cannot detect all mutations in patients with WD. Multiplex ligation-dependent probe amplification (MLPA) analysis is reportedly useful in increasing the diagnostic yield in other genetic disorders with large deletions or insertions. The aim of this study was to evaluate whether the detection rate of ATP7B mutations can be increased by using MLPA.MethodsWe enrolled 114 children with WD from 104 unrelated families based on biochemical tests and direct DNA full sequencing. The patients with one or zero mutant allele were investigated using MLPA. We analyzed phenotypic correlations.ResultsTotal allele frequency by full sequencing was 87.5%. Full sequencing revealed two mutant alleles in 80 of 104 unrelated children. One mutant allele was detected in 22 children, and no mutations were found in two children. Novel mutations including small deletions with frameshift mutations were identified by DNA sequencing. MLPA revealed no gross deletion or duplication in 24 children with one or zero mutant alleles. The number of detected mutations was not associated with hepatic manifestation, age of onset, Kayser-Fleischer ring, ceruloplasmin, and urinary Cu concentrations.ConclusionMLPA showed a limited role to increase the mutation detection rate in children who do not receive a definite genetic diagnosis of WD through DNA full sequencing. This finding suggests that large deletions or duplications might be extremely rare in WD. Further development is needed to improve the genetic diagnosis of WD.

Challenges in molecular diagnosis of Wilson disease: viewpoint from the clinical laboratory

“…Our results suggest that large deletions and duplications in the ATP7B gene may be relatively uncommon. This is in concordance with a smaller study from Korea that showed no deletions or duplications detected by MLPA in the ATP7B gene in 24 children with one or no pathogenic variants detected by sequencing 14…”

Copper and copper nanoparticles toxicity and their impact on basic functions in the body

“…Under normal conditions, the Ptype ATPase is responsible for transport of copper from liver to bile (49, 50). It should be written that various mutations occur in the patients suffering from Wilson disease so different patients can have different mutation resulting in P-type ATPase impairment (51,52). Regardless of mutation type in the ATP7B gene, the impaired P-type ATPase leads to copper accumulation in multiple organs with fatal consequences if untreated (53).…”