Here, all three patients diagnosed with unrelated neuromuscular disorders had a 4qB variant smaller than 38
kb on chromosome 4, which would have caused substantial diagnostic confusion in the absence of the 4qA/4qB variant determination. Neuromuscular disorders that may be clinically and histopathologically similar to FSHD include the various limb-girdle and scapuloperoneal syndromes, which also may cause mild facial weakness. Ancillary investigations, such as the assessments of CK, EMG and muscle biopsy, are often insufficient for diagnosis, as was the case in the probands from families 1 and 2. However, subsequent DNA analysis of the nonpathogenic short 4qB variant excluded the diagnosis of FSHD, and western blot analysis further confirmed the diagnosis of LGMD2 in these patients. The proband from family 3 also presented some FSHD-like symptoms and signs based on clinical observation, such as prominent involvements of the facial muscles and mild shoulder girdle and pelvic girdle weakness. The facial-sparing scapular myopathy was described as the most common clinical variant of FSHD, and some atypical phenotypes including LGMD, distal myopathy, mild or partial forms have been described in several papers published before 2004.18, 19, 20
However, the identification of the 4qA and 4qB variants had not been performed in these special cases. Here, the D4Z4 contraction in the proband from family 3 was confirmed as linked to the 4qB variant and considered as nonpathogenic.
We had previously demonstrated the presence of an FSHD-sized 4qB variant on chromosome 4 in healthy individuals.21
In the present study, we provided further evidence of the nonpathogenic 4qB variant in other neuromuscular disorders. This may also explain the rare finding of FSHD-sized D4Z4 repeats that do not co-segregate with unrelated disorders.22
Therefore, the extension of the clinical spectrum of FSHD needs to be carefully evaluated. Although some rare cases with ‘double trouble' had been reported,11, 23 Not
I digestions followed by hybridizations with B31 need to conclusively identify the correct chromosome 4, and some complex genetic constitutions with exchanged D4Z4 alleles of chromosome 4 and chromosome 10 origins are presented below.
There are two uncommon factors that made the genetic confirmation of the fourth pedigree confusing. First, both 4qA- and 4qB-type FSHD-sized D4Z4 alleles were found in the same family. Second, complex genetic constitutions were observed in the same family. Before complementary allelotyping analysis and integrated chromosome assignments, the members of the family who carried 4qB-type FSHD-sized alleles had been misdiagnosed as asymptomatic cases. Through PFGE-based DNA analysis with probes 4qA, 4qB and B31, we confirmed that the 18-kb 4qA variant correlated with FSHD in the proband and that the one carried by her mother resided on chromosome 4, whereas the 24-kb 4qB variant, which is considered as nonpathogenic, in the healthy husband and the two daughters resided on chromosome 10. Moreover, we were able to confirm the complex D4Z4 rearrangements of two exchanged repeat arrays, one on chromosome 4 and one on chromosome 10, inherited from the maternal 10q → 4q repeat and from the paternal 4q → 10q repeat. Although the haplotypes composed of a mixture of 4q and 10q sequences were detected at frequencies >15% in the Chinese FSHD,12
such exchanged constitution cannot be identified by conventional DNA diagnosis and never been observed in the Chinese population. As reported, all 4q subtelomeres originate from only four discrete interchromosomal sequence transfers during human evolution, and haplotypes with mixtures of 4q- and 10q-specific sequences represent intermediate structures in the transition from 4q to 10q subtelomeres.10
We also found that in this family, a 4qB variant resided on chromosome 10, confirming the same detection in other populations,10
and further suggesting that the breakpoint of the D4Z4 repeat was distal to the 4qA and 4qB variants. The pathophysiological pathway from 4q35 deletion together with the 4qA variant to the clinical picture of FSHD is unclear.
In summary, this is the first study to confirm that D4Z4 contractions on the 4qB variant do not cause FSHD in the Chinese population. Furthermore, our results emphasize that the D4Z4 repeat length analysis alone is insufficient for the diagnosis of FSHD, especially when used as an exclusion criterion. This analysis should be accompanied by 4qA/4qB variant determination and integrated chromosome assignments, especially in patients with obscure and unclassified myopathies similar to atypical forms of FSHD.