Screening for minor changes in the distal part of the human dystrophin gene in Greek DMD/BMD patients

Abstract

The distal part of the human dystrophin gene is characterised by particular features and seems to play an important functional role. Additionally in recent years several data have implicated minor mutations in this gene region in some patients with mental retardation (MR). In order to screen for pathogenic mutations at the distal part of the human dystrophin gene we have used single-strand conformation analysis of products amplified by polymerase chain reaction (PCR-SSCA) in 35 unrelated male Greek DMD/BMD patients with no detectable deletions. Seven patients also had severe mental retardation. Direct sequencing of samples demonstrating a shift of SSCA mobility revealed six different and pathogenic minor changes, five in DMD and one in a BMD patient. Four of the mutations were found in DMD patients with severe MR. Three of these mutations were localised in exon 66, which presents an interesting similarity with part of the 3' end of the genome of eastern equine encephalomyelitis virus (EEEV). The present data from Greek DMD/BMD patients give further information about the phenotypic effects consequent on mutations in exons at the distal part of the human dystrophin gene.

DNA sequence analysis for structure/function and mutation studies in Becker muscular dystrophy

“…The only mutation in our study that was previously reported (19) is the DGA10716-10717 (Table 3). While the mutations are the same, the findings regarding the effect of mutation on mRNA and protein were very different.…”

“…Eighteen deletion-negative BMD patients tested by means of multiplex PCR were included in this study (4,18). We identified seven causative mutations, of which six were novel (Table 3) (19). Two were novel missense mutations in the aminoterminal actin-binding domain of the dystrophin protein (R82P, L172H) ( Fig.…”

“…We found that the GA deletion caused a corresponding 2 bp deletion, a subsequent frame-shift and 5% level of the protein with the expected 20 kDa reduction in its molecualr weight. On the contrary, Kekou et al studied illegitimate mRNA transcript from the lymphoblasts, and found that the same mutation led to an abnormal splicing with the removal of 4 exons (19). No protein data were provided.…”

Screening Human Genes for Small Alterations Performing an Enzymatic Cleavage Mismatched Analysis (ECMA) Protocol

“…PCR primers and conditions for the exons containing the 11 known mutations (Table 1) are as previously described [14][15][16][17][18]. In order to maximize product yield DNA was amplified by using 50 ng of genomic DNA and performing 35 cycles of PCR in a final reaction volume of 50 ll.…”

Long mutant dystrophins and variable phenotypes: evasion of nonsense-mediated decay?

“…Each class of exception has been convincingly explained by a distinct mechanism: (1) disruption of the "muscle-type" promoter and/or first exon results in phenotypic rescue by alternative promoter use in all tissues except the heart, and the consequence is cardiac rather than skeletal myopathy (Muntoni et al 1995), (2) frameshifting deletions early in the gene can allow in-frame translational re-initiation after abortive translation of the shortened cognate ORF, yielding an N-terminally truncated but otherwise full-length protein (Winnard et al 1995), (3) ORF-terminating mutations within the gene are sometimes associated with alternative splicing of exons bearing a point mutation (or adjacent to a rearrangement); this can restore the ORF in a proportion Fig. 1 A The 3' mutations of the dystrophin gene discussed in this paper (Barbieri et al 1996;Crawford et al 2000;Gardner et al 1995;Kekou et al 1999;Lasa et al 1997;Lenk et al 1993;McCabe et al 1989;Prior et al 1995;Roberts et al 1992). Dystrophin ORFs of the respective patients, drawn to scale against a schematic diagram of the corresponding regions of the message (labelled with exon numbers) and protein (labelled with known syntrophin and dystrobrevin interacting regions).…”