Definitive molecular diagnosis of facioscapulohumeral dystrophy

To establish the usefulness of a molecular diagnostic protocol for the autosomal dominant disease facioscapulohumeral dystrophy (FSHD).

The genetic defect underlying the majority of cases is a deletion on chromosome 4q35 that is not associated with the coding sequence of any known gene. Molecular diagnosis of FSHD involves the visualization of this deletion as a "small" EcoRI restriction fragment. However, molecular diagnostics are complicated because of the homology of the telomeric regions of chromosomes 4q and 10q; the homologous 10q26 EcoRI fragments are also detected, and can fall into the size range considered to be diagnostic for FSHD. It is therefore important to distinguish the 4q35 and 10q26 EcoRI fragments, taking advantage of the presence of additional restriction sites (BlnI) in the alleles of chromosome 10q origin.

Paired digests of genomic DNA (EcoRI only and EcoRI/BlnI double digest), followed by pulsed field gel electrophoresis (PFGE), were used to establish the molecular diagnosis of FSHD in 82 unrelated index cases (46 familial, 24 proven sporadic with de novo mutations, and 12 with uncertain family history).

In all cases fulfilling FSHD diagnostic criteria, a 4q35 EcoRI allele size of < or = 38 kb was present. The smallest 4q35 EcoRI allele in 205 normal control subjects was 41 kb. EcoRI alleles < or = 38 kb of chromosome 10q26 origin were present in 11.2% of this control group. In problematic cases, it was possible to resolve the diagnostic question.

The combination of double digestion with EcoRI and BlnI followed by PFGE is the most reliable molecular protocol for distinguishing patients with FSHD.