Limits...
Multiplex ARMS PCR to Detect 8 Common Mutations of ATP7B Gene in Patients With Wilson Disease.

Dastsooz H, Imanieh MH, Dehghani SM, Haghighat M, Moini M, Fardaei M - Hepat Mon (2013)

Bottom Line: Two sets of ARMS mutant and normal specific primer pairs were designed for genotyping of p.R778L, p.R969Q, p.H1069Q, and p.3400delC mutations as Set 1 and p.W779G, c.3061-1G > A, p.I1102T, and p.N1270S mutations as Set 2.Using these two sets, we identified H1069Q mutation in four patients, c.2335T > G mutation in three, c.3061-1G > A splice site mutation in five, c.3305T > C mutation in one, and c.3809A > G mutation in two patients.The Multiplex ARMS assay used in this study can be an efficient, reliable, and cost effective method as a primary screen for patients with Wilson disease.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Genetics, Shiraz University of Medical Sciences, Shiraz, IR Iran ; Department of Molecular Medicine, Shiraz University of Medical Sciences, Shiraz, IR Iran.

ABSTRACT

Background: Wilson disease is a rare disorder of copper metabolism due to mutation in ATP7B gene. Proper counseling of patients with Wilson disease, and their families necessitates finding mutation in ATP7B gene. Finding mutations in ATP7B gene with 21 exons, and more than 500 mutations is expensive and time-consuming.

Objectives: The aim of this study was to provide a simple multiplex amplification refractory mutation system PCR (M-ARMS-PCR) for screening eight common mutations in ATP7B gene.

Patients and methods: Two sets of ARMS mutant and normal specific primer pairs were designed for genotyping of p.R778L, p.R969Q, p.H1069Q, and p.3400delC mutations as Set 1 and p.W779G, c.3061-1G > A, p.I1102T, and p.N1270S mutations as Set 2. The Multiplex ARMS assay was then subsequently tested in 65 patients with Wilson disease with known and unknown ATP7B mutations.

Results: Using these two sets, we identified H1069Q mutation in four patients, c.2335T > G mutation in three, c.3061-1G > A splice site mutation in five, c.3305T > C mutation in one, and c.3809A > G mutation in two patients.

Conclusions: The Multiplex ARMS assay used in this study can be an efficient, reliable, and cost effective method as a primary screen for patients with Wilson disease.

No MeSH data available.


Related in: MedlinePlus

Chromatograms and gel Electrophoresis Images of the Mutation and the DNA Bands of Set 2 and 2A(A) Gel electrophoresis of Set 1, U: upper and L: lower controls are depicted for all lines. 3rd, 4th, and 6th wells show bands for the normal DNA. 100bp DNA ladder is depicted in right and left. Using Mix Set 2, Samples in 1st, 2nd, 5th, and 7th wells show the band for c.3061-1G > A, c.3305T > C, c.2335T > G, and c.3809A > G mutations, respectively. (B) Gel electrophoresis of the DNA bands of the hetero- and homozygote c.2335T > G, and c.3061-1G > A mutations. 1: the normal DNA with Set 2A. 2 and 3: heterozygotec.3061-1G > A mutation. 4 and 5: heterozygote c.2335T > G mutation; 6 and 7: homozygote c.3061-1G > A mutation. 8 and 9: homozygote c.2335T > G. 100bp DNA ladder is depicted in middle and left. (C) Chromatograms of main mutation detected in Set 1.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3753551&req=5

fig4700: Chromatograms and gel Electrophoresis Images of the Mutation and the DNA Bands of Set 2 and 2A(A) Gel electrophoresis of Set 1, U: upper and L: lower controls are depicted for all lines. 3rd, 4th, and 6th wells show bands for the normal DNA. 100bp DNA ladder is depicted in right and left. Using Mix Set 2, Samples in 1st, 2nd, 5th, and 7th wells show the band for c.3061-1G > A, c.3305T > C, c.2335T > G, and c.3809A > G mutations, respectively. (B) Gel electrophoresis of the DNA bands of the hetero- and homozygote c.2335T > G, and c.3061-1G > A mutations. 1: the normal DNA with Set 2A. 2 and 3: heterozygotec.3061-1G > A mutation. 4 and 5: heterozygote c.2335T > G mutation; 6 and 7: homozygote c.3061-1G > A mutation. 8 and 9: homozygote c.2335T > G. 100bp DNA ladder is depicted in middle and left. (C) Chromatograms of main mutation detected in Set 1.

Mentions: M-ARMS PCR of Set 2 was performed for 65 patients with Wilson disease. Similarly for Set 2, all samples showed both control bands, and bands from c.2335T > G mutation in three cases, c.3061-1G > A splice site mutation in four cases, c.3305T > C mutation in one case, and c.3809A > G mutation in two cases (examples of cases with these mutations are shown in Figure 2-A. first, 2nd, 5th, and 7th wells). Distribution of ATP7B mutations (n = 65) detected by Set 1 and 2 of multiplex ARMS, and sequencing technique are given in Table 3. No band other than the internal control was amplified in the DNA without these mutations (Figure 2-A. 3rd, 4th, and 6th wells). The assay was designated as a fail try if both PCR control bands were not amplified, and the M-ARMS assay was repeated. To confirm the efficiency of this method for genotyping, Set 2A was performed for c.2335T > G mutation in samples with a homozygote and a heterozygote (a sample from mother of this homozygous patient) condition. Set 2A was also performed for c.3061-1G > A mutation in patients with a homozygote, and a compound heterozygote condition. Using Set 2 of M-ARMS assay, patients with the homozygote c.2335T > G mutation (case 3), and c.3061-1G > A mutation (case 8) showed the control bands (153 bp, and 585 bp), and the related mutant bands (294 bp for c.2335T > G and 330 bp for c.3061-1G > A), and using Set 2A all bands except the bands for these mutations were amplified (Figure 2-B, 8th, and 9th wells for the case 3; and 6th and 7th wells for case 8).


Multiplex ARMS PCR to Detect 8 Common Mutations of ATP7B Gene in Patients With Wilson Disease.

Dastsooz H, Imanieh MH, Dehghani SM, Haghighat M, Moini M, Fardaei M - Hepat Mon (2013)

Chromatograms and gel Electrophoresis Images of the Mutation and the DNA Bands of Set 2 and 2A(A) Gel electrophoresis of Set 1, U: upper and L: lower controls are depicted for all lines. 3rd, 4th, and 6th wells show bands for the normal DNA. 100bp DNA ladder is depicted in right and left. Using Mix Set 2, Samples in 1st, 2nd, 5th, and 7th wells show the band for c.3061-1G > A, c.3305T > C, c.2335T > G, and c.3809A > G mutations, respectively. (B) Gel electrophoresis of the DNA bands of the hetero- and homozygote c.2335T > G, and c.3061-1G > A mutations. 1: the normal DNA with Set 2A. 2 and 3: heterozygotec.3061-1G > A mutation. 4 and 5: heterozygote c.2335T > G mutation; 6 and 7: homozygote c.3061-1G > A mutation. 8 and 9: homozygote c.2335T > G. 100bp DNA ladder is depicted in middle and left. (C) Chromatograms of main mutation detected in Set 1.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3753551&req=5

fig4700: Chromatograms and gel Electrophoresis Images of the Mutation and the DNA Bands of Set 2 and 2A(A) Gel electrophoresis of Set 1, U: upper and L: lower controls are depicted for all lines. 3rd, 4th, and 6th wells show bands for the normal DNA. 100bp DNA ladder is depicted in right and left. Using Mix Set 2, Samples in 1st, 2nd, 5th, and 7th wells show the band for c.3061-1G > A, c.3305T > C, c.2335T > G, and c.3809A > G mutations, respectively. (B) Gel electrophoresis of the DNA bands of the hetero- and homozygote c.2335T > G, and c.3061-1G > A mutations. 1: the normal DNA with Set 2A. 2 and 3: heterozygotec.3061-1G > A mutation. 4 and 5: heterozygote c.2335T > G mutation; 6 and 7: homozygote c.3061-1G > A mutation. 8 and 9: homozygote c.2335T > G. 100bp DNA ladder is depicted in middle and left. (C) Chromatograms of main mutation detected in Set 1.
Mentions: M-ARMS PCR of Set 2 was performed for 65 patients with Wilson disease. Similarly for Set 2, all samples showed both control bands, and bands from c.2335T > G mutation in three cases, c.3061-1G > A splice site mutation in four cases, c.3305T > C mutation in one case, and c.3809A > G mutation in two cases (examples of cases with these mutations are shown in Figure 2-A. first, 2nd, 5th, and 7th wells). Distribution of ATP7B mutations (n = 65) detected by Set 1 and 2 of multiplex ARMS, and sequencing technique are given in Table 3. No band other than the internal control was amplified in the DNA without these mutations (Figure 2-A. 3rd, 4th, and 6th wells). The assay was designated as a fail try if both PCR control bands were not amplified, and the M-ARMS assay was repeated. To confirm the efficiency of this method for genotyping, Set 2A was performed for c.2335T > G mutation in samples with a homozygote and a heterozygote (a sample from mother of this homozygous patient) condition. Set 2A was also performed for c.3061-1G > A mutation in patients with a homozygote, and a compound heterozygote condition. Using Set 2 of M-ARMS assay, patients with the homozygote c.2335T > G mutation (case 3), and c.3061-1G > A mutation (case 8) showed the control bands (153 bp, and 585 bp), and the related mutant bands (294 bp for c.2335T > G and 330 bp for c.3061-1G > A), and using Set 2A all bands except the bands for these mutations were amplified (Figure 2-B, 8th, and 9th wells for the case 3; and 6th and 7th wells for case 8).

Bottom Line: Two sets of ARMS mutant and normal specific primer pairs were designed for genotyping of p.R778L, p.R969Q, p.H1069Q, and p.3400delC mutations as Set 1 and p.W779G, c.3061-1G > A, p.I1102T, and p.N1270S mutations as Set 2.Using these two sets, we identified H1069Q mutation in four patients, c.2335T > G mutation in three, c.3061-1G > A splice site mutation in five, c.3305T > C mutation in one, and c.3809A > G mutation in two patients.The Multiplex ARMS assay used in this study can be an efficient, reliable, and cost effective method as a primary screen for patients with Wilson disease.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Genetics, Shiraz University of Medical Sciences, Shiraz, IR Iran ; Department of Molecular Medicine, Shiraz University of Medical Sciences, Shiraz, IR Iran.

ABSTRACT

Background: Wilson disease is a rare disorder of copper metabolism due to mutation in ATP7B gene. Proper counseling of patients with Wilson disease, and their families necessitates finding mutation in ATP7B gene. Finding mutations in ATP7B gene with 21 exons, and more than 500 mutations is expensive and time-consuming.

Objectives: The aim of this study was to provide a simple multiplex amplification refractory mutation system PCR (M-ARMS-PCR) for screening eight common mutations in ATP7B gene.

Patients and methods: Two sets of ARMS mutant and normal specific primer pairs were designed for genotyping of p.R778L, p.R969Q, p.H1069Q, and p.3400delC mutations as Set 1 and p.W779G, c.3061-1G > A, p.I1102T, and p.N1270S mutations as Set 2. The Multiplex ARMS assay was then subsequently tested in 65 patients with Wilson disease with known and unknown ATP7B mutations.

Results: Using these two sets, we identified H1069Q mutation in four patients, c.2335T > G mutation in three, c.3061-1G > A splice site mutation in five, c.3305T > C mutation in one, and c.3809A > G mutation in two patients.

Conclusions: The Multiplex ARMS assay used in this study can be an efficient, reliable, and cost effective method as a primary screen for patients with Wilson disease.

No MeSH data available.


Related in: MedlinePlus