Contributions
Abstract: PB1980
Type: Publication Only
Background
Wilson's disease is autosomal recessive disorder caused by mutations in copper-transporting ATPase (ATP7B). The clinical variants of the disease include isolated neurological and hepatic forms, 15% of disease cases manifest with hemolysis in the absence of autoantibodies. Confirmation of homozygous or compound heterozygous carriage of ATP7B gene mutations is essential for Wilson's disease diagnostics. Direct sequencing of a complete ATP7B gene the most reliable method of detecting mutations is still quite expensive for screening. Here we present alternative approach to the detection of polymorphisms in the ATP7B gene.
Aims
To develop screening method effective for the detection of the most common ATP7B gene mutations
Methods
Allele-specific real-time polymerase chain reaction was optimized for the detection of common ATP7B mutations. The primers were chosen so that the last nucleotide in the forward primer chain corresponded to the normal or mutant nucleotide of the detected polymorphism. TaqMan probes were synthesized to anneal inside the amplicon behind the mutation point. PCRs on DNAs isolated from PBMC were run on Rotor-Gene instrument. Initial denaturation 300 sec. at 95oC was followed by 45 cycles of 63oC (50 sec.) and 95oC (15 sec.). The raw data was analyzed by instrument software
Results
Primers and TaqMan probes were designed to measure HIS1069GLN, ARG778LEU, 3400delC, ARG969GLN and GLY1267ARG polymorphisms in the ATP7B gene. Probes were labeled by non-overlapping fluorescent dyes (FAM, R & G, ROX and Cy5) to facilitate multiplex detection of 5 mutations (see fig.1 for details). About 500 mutations of ATP7B gene are described so far, however, only a few of them are found frequently. HIS1069GLN homozygous mutation is responsible for 38-49% cases of Wilson's disease in the European population. Incidence of remaining 4 mutations reported not to exceed 2-3% for each. 260 patients with hemolysis or liver pathology of unknown origin admitted to National Research Center for Hematology between 2010 and 2017 years were tested using the method described and 28 patients carrying ATP7B gene mutation were found. Thirteen were heterozygous for HIS1069GLN; one heterozygous for ARG778LEU and 14 were homozygous for HIS1069GLN.
Conclusion
The test system developed is effective for the detection of most common ATP7B gene mutations and may be beneficial for the differential diagnosis between unclear hemolytic syndromes, liver pathology of unknown origin and Wilson's disease of various clinical manifestations.
Session topic: 29. Enzymopathies, membranopathies and other anemias
Keyword(s): Anemia, mutation analysis, Real time ASO PCR
Abstract: PB1980
Type: Publication Only
Background
Wilson's disease is autosomal recessive disorder caused by mutations in copper-transporting ATPase (ATP7B). The clinical variants of the disease include isolated neurological and hepatic forms, 15% of disease cases manifest with hemolysis in the absence of autoantibodies. Confirmation of homozygous or compound heterozygous carriage of ATP7B gene mutations is essential for Wilson's disease diagnostics. Direct sequencing of a complete ATP7B gene the most reliable method of detecting mutations is still quite expensive for screening. Here we present alternative approach to the detection of polymorphisms in the ATP7B gene.
Aims
To develop screening method effective for the detection of the most common ATP7B gene mutations
Methods
Allele-specific real-time polymerase chain reaction was optimized for the detection of common ATP7B mutations. The primers were chosen so that the last nucleotide in the forward primer chain corresponded to the normal or mutant nucleotide of the detected polymorphism. TaqMan probes were synthesized to anneal inside the amplicon behind the mutation point. PCRs on DNAs isolated from PBMC were run on Rotor-Gene instrument. Initial denaturation 300 sec. at 95oC was followed by 45 cycles of 63oC (50 sec.) and 95oC (15 sec.). The raw data was analyzed by instrument software
Results
Primers and TaqMan probes were designed to measure HIS1069GLN, ARG778LEU, 3400delC, ARG969GLN and GLY1267ARG polymorphisms in the ATP7B gene. Probes were labeled by non-overlapping fluorescent dyes (FAM, R & G, ROX and Cy5) to facilitate multiplex detection of 5 mutations (see fig.1 for details). About 500 mutations of ATP7B gene are described so far, however, only a few of them are found frequently. HIS1069GLN homozygous mutation is responsible for 38-49% cases of Wilson's disease in the European population. Incidence of remaining 4 mutations reported not to exceed 2-3% for each. 260 patients with hemolysis or liver pathology of unknown origin admitted to National Research Center for Hematology between 2010 and 2017 years were tested using the method described and 28 patients carrying ATP7B gene mutation were found. Thirteen were heterozygous for HIS1069GLN; one heterozygous for ARG778LEU and 14 were homozygous for HIS1069GLN.
Conclusion
The test system developed is effective for the detection of most common ATP7B gene mutations and may be beneficial for the differential diagnosis between unclear hemolytic syndromes, liver pathology of unknown origin and Wilson's disease of various clinical manifestations.
Session topic: 29. Enzymopathies, membranopathies and other anemias
Keyword(s): Anemia, mutation analysis, Real time ASO PCR