Efficient Detection of Mutations in Wilson Disease by Manifold Sequencing

Mutational analysis of ATP7B gene in Egyptian children with Wilson disease: 12 novel mutations

“…The 21 exons of the WD gene were amplified in a thermal cycler (Biometra T3 Thermocycler, Göttingen, Germany) as described elsewhere (Waldenström et al 1996). Direct sequencing of the amplified polymerase chain reaction (PCR) products was performed by cycle sequencing using fluorescent dye terminators in an automatic sequencer (Applied Biosystems, Darmstadt, Germany).…”

Determination of the frequencies of ten allelic variants of the Wilson disease gene (ATP7B), in pooled DNA samples

“…The forward 5'-biotinylated primer for exon 14 was 5'-AGA CTG TGA TGT TTG ACA AGA CTG G-3' and the reverse primer was 5'-CCA AGT CCA CGT ACC TCT TTA-3'. The sequences of the primers for amplifying the exons containing eight SNPs in the ATP7B gene are given in Waldenström et al 8 The DNA fragments containing the disease mutations or SNPs in the ATP7B gene were amplified from 200-400 ng of DNA using 1.25 units of Taq DNA polymerase, 200 µM dNTPs, the biotinylated primer at 0.2 µM concentration and the other primer at 1 µM concentration in 100 µl of DNA polymerase buffer (50 mM KCl, 10 mM Tris-HCl pH 8.3, 1.5 mM MgCl 2 , 12.5 mg/ml bovine serum albumin, PE Biosystems, Foster City, CA, USA). The temperature was varied during 35 cycles of 94°C for 30 s, an annealing temperature between 55°C and 65°C depending on the T m of the primers, for 30 s and 72°C for 45 s. (Table 1).…”

“…8 The frequencies of these sequence variants were determined rapidly and accurately using a different pooling strategy. An equal amount of DNA from 2500 individuals out of the 2640 analysed was combined into a 'super pool', which was subjected to quantitative analysis by the minisequencing method for each of the eight SNP sites.…”

“…The two most common mutations in Swedish patients are a C-A transversion in exon 14 (C3207A) and a C-T transition in exon 13 (C2930T), accounting for 33% and 11% of the Swedish disease alleles, respectively. 8 The prevalence of Wilson disease in Sweden has been estimated to be 1 in 250 000, whereas its prevalence has been estimated to be 1 in 30 000 in other populations. 9 In the present study we determined the frequency of the two major disease-causing alleles in the ATP7B gene in a large Swedish population sample to investigate if the 10-fold lower prevalence of Wilson disease could be due to patients being missed in the clinic.…”

“…The ATP7B gene also contains several singlenucleotide polymorphisms (SNPs) that are more common in the population than the disease-causing mutations. 8,10 To determine the Swedish allele frequencies of eight of the common SNPs in coding regions of the ATP7B gene in the same sample material, we devised a different pooling strategy. A large pool, consisting of 2500 Swedish DNA samples, was analysed quantitatively to determine the proportion of the two sequence variants of each SNP, which reflects directly the allele frequencies at these sites in the Swedish population.…”