J Korean Med Sci.  2018 Jun;33(26):e177. 10.3346/jkms.2018.33.e177.

Multiplex Ligation-dependent Probe Amplification Analysis Subsequent to Direct DNA Full Sequencing for Identifying ATP7B Mutations and Phenotype Correlations in Children with Wilson Disease

• Shim JO
• Yang HR
• Moon JS
• Chang JY
• Ko JS
• Park SS
• Seo JK

• KMID: 2414030
• DOI: http://doi.org/10.3346/jkms.2018.33.e177

Abstract

BACKGROUND
Mutations 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.
METHODS
We 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.
RESULTS
Total 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.
CONCLUSION
MLPA 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.

Keyword

Wilson Disease; Mutation; Multiplex Ligation-dependent Probe Amplification; Sequence Analysis; Child

MeSH Terms

Age of Onset
Alleles
Ceruloplasmin
Child*
Diagnosis
DNA*
Frameshift Mutation
Gene Frequency
Hepatolenticular Degeneration*
Humans
Multiplex Polymerase Chain Reaction*
Phenotype*
Sequence Analysis
Sequence Analysis, DNA
Ceruloplasmin
DNA