Point mutation screening for 16 exons of the dystrophin gene by multiplex single-strand conformation polymorphism analysis

Abstract

We have developed a rapid and nonradioactive method to screen for point mutations using the Pharmacia PhastSystem. In an SSCP analysis, we applied the two multiplex exon PCR kits, commonly used for the detection of deletions in Duchenne and Becker muscular dystrophy patients. The different exon bands in the multiplex SSCP pattern could be identified by running well-characterised deletion patients in this system. Two common polymorphisms were easily identifiable and are helpful in the haplotype analysis in families. Screening of 70 patients in which no gross rearrangement was detectable with the multiplex PCR and Southern blot, resulted in the identification of 6 patients with a band shift after SSCP analysis. Of these 6 band shifts, 5 were the result of a frame shift or termination mutation. The other band shift was found to be a rare polymorphism unlikely to be the cause of the patient's phenotype. Application of this technique enabled us to improve diagnosis in the families involved and will allow us to extend the search for point mutations in the remaining exons of the dystrophin gene.

Point mutation and polymorphism in Duchenne/Becker Muscular Dystrophy (D/BMD) patients

“…SSCA (Orita et al, 1989;Glavac and Dean, 1993) and HA (Glavac and Dean, 1995) are mutation detection techniques that rely on detecting changes in the physical properties of DNA caused by the presence of sequence changes. These methods have shown an efficiency of 7-27% depending on the conditions and exons analysed (Soto and Sukumar, 1992;White et al, 1992;Kneppers et al, 1995;Eranslan et al, 1999). Prior et al (1995) screened around 80% of the dystrophin coding sequences for small mutations by using HA in 158 patients and identified mutations in 29 of them.…”

“…About 30-35% cases of D/BMD are assumed to result from microdeletions, microinsertions or substitutions of one or more nucleotide(s). Several independent studies have reported small mutations in the dystrophin gene (Kiliman et al, 1992;Nigro et al, 1992Nigro et al, , 1994Roberts et al, 1992;Tuffery et al, 1993Tuffery et al, , 1995Tuffery et al, , 1996Kneppers et al, 1995;Sitnik et al, 1997;Eranslan et al, 1999;Wibawa et al, 2000). These are randomly distributed throughout the dystrophin gene (http://www.dmd.nl).…”

CBFA2, frequently rearranged in leukemia, is not responsible for a familial leukemia syndrome

“…We also found no evidence of mutation in the IFNAR1 or CRFB4 genes, when studied by SSCP analysis. As the sensitivity of SSCP ranges from 70 to 98% 36,[46][47][48] and is dependent on nucleotide sequence, fragment size and electrophoretic conditions, it is possible that a mutation in IFNAR1 or CRFB4 could have been missed using this technique. Efforts are currently underway to analyze the other known candidate genes as well as to characterize and analyze novel genes which map to this locus.…”

Identification of point mutations in Turkish DMD/BMD families using multiplex-single stranded conformation analysis (SSCA)

“…Single or multiplex PCR products corresponding to the various exons of the dystrophin gene including exon-intron boundaries were analysed on long non-denaturing gels by SSCA technique and resulted in the identification of point mutations within this gene. [11][12][13] In the framework of this study, we have screened 18 deletion-prone exons of the dystrophin gene by using a modified non-isotopic multiplex SSCA. We have identified five different pathogenic mutations, two of which are novel, and six polymorphisms in 56 nondeletion DMD/BMD patients.…”