Genetic and clinical specificity of 26 symptomatic carriers for dystrophinopathies at pediatric age

Abstract

The molecular basis underlying the clinical variability in symptomatic Duchenne muscular dystrophy (DMD) carriers are still to be precised. We report 26 cases of early symptomatic DMD carriers followed in the French neuromuscular network. Clinical presentation, muscular histological analysis and type of gene mutation, as well as X-chromosome inactivation (XCI) patterns using DNA extracted from peripheral blood or muscle are detailed. The initial symptoms were significant weakness (88%) or exercise intolerance (27%). Clinical severity varied from a Duchenne-like progression to a very mild Becker-like phenotype. Cardiac dysfunction was present in 19% of the cases. Cognitive impairment was worthy of notice, as 27% of the carriers are concerned. The muscular analysis was always contributive, revealing muscular dystrophy (83%), mosaic in immunostaining (81%) and dystrophin abnormalities in western blot analysis (84%). In all, 73% had exonic deletions or duplications and 27% had point mutations. XCI pattern was biased in 62% of the cases. In conclusion, we report the largest series of manifesting DMD carriers at pediatric age and show that exercise intolerance and cognitive impairment may reveal symptomatic DMD carriers. The complete histological and immunohistological study of the muscle is the key of the diagnosis leading to the dystrophin gene analysis. Our study shows also that cognitive impairment in symptomatic DMD carriers is associated with mutations in the distal part of the DMD gene. XCI study does not fully explain the mechanisms as well as the wide spectrum of clinical phenotype, though a clear correlation between the severity of the phenotype and inactivation bias was observed.

Figure 1

Histological findings and immunostaining on a DMD carrier muscle biopsy (patient #3). (a) Dystrophic aspect with fiber size variation (Hematein-eosine staining, × 200). (b, c and d) Mosaic aspect of dystrophin staining with DYS1 (b, × 200), DYS2 (c, × 200) and DYS3 (d, × 100) antibodies, respectively, targettting dystrophin rod, C-terminal and N-terminal domains.

Figure 2

Dystrophin analysis by multiplex western blot revealing decreased amount of dystrophin compared with the control. (a) DYS2 antibody: normal molecular weight (MW) (carrier #2); (b) DYS1 antibody: abnormal MW with an additional band of 220 kD (carrier #8). Dysferlin, calpain, α- and γ-sarcoglycan protein bands are also displayed.

Figure 3

Schematic representation of the DMD gene mutations detected in the 26 patients. Inheritance type, XCI pattern and cognitive impairment are indicated. Carrier number (Table 1) is represented in blue in brackets; cognitive impairment in red; DMD gene deletions, duplication, triplication, respectively, in red, green and blue lines (Numbers associated with deletions and duplications correspond to deletions and duplications boundaries); skewed XCI is indicated by a star and de novo occurrence by a box.

Figure 4

X-inactivation patterns in DMD carriers. AR locus, Xq11-q12: automatic sequencer traces correspond to the PCR products of undigested DNA (a, c) and after HpaII digestion (b, d). (a, b) Random X-inactivation pattern (52:48) in patient #5. (c, d) Completely skewed X-inactivation pattern (100:0) in patient #15.