Facioscapulohumeral muscular dystrophy (FSHD) is a common form of muscular disorder with an autosomal dominant trait. FSHD is characterized by weakness and atrophy of facial, shoulder-girdle and humeral muscles, with occasional subsequent pelvic-girdle and lower limb involvement. More than 95% of patients with FSHD have a smaller (< 35 kb) EcoRI fragment on chromosome 4q35 detected by probe p13E-11 and are called 4q35-FSHD 123. This EcoRI fragment in normal individuals contains tandem repeated 3.3-kb KpnI units (D4Z4) varying from 11 to 150 in number, while 4q35-FSHD patients showed less than ten units 23. No responsible gene has been isolated within the FSHD gene region.

Probe p13E-11 cross-hybridizes with chromosome 10q26, which contains highly homologous 3.3-kb KpnI repeated units. Since the BlnI restriction enzyme site exists exclusively within each unit derived from 10q26, but not in D4Z4 (an unit from 4q35), double enzyme digestion using EcoRI and BlnI can discriminate as 4q35 (BlnI-resistant) and 10q26 (BlnI-sensitive) units 4. In a Dutch control population, subtelomeric translocations between chromosomes 4 and 10 were seen in about 20% of individuals 567. Furthermore, somatic mosaicism was found in 40% of de novo FSHD families and 46% of these individuals had BlnI-resistant units on chromosome 10 8. These findings imply that frequent recombination between the subtelomeric region of chromosomes 4 and 10 has some roles for deletion of the FSHD region. In this study, we examined the frequency of subtelomeric translocation among three different ethnic populations of Japanese, Korean and Chinese, and compared with Japanese 4q35-FSHD patients.

Blood samples were obtained with informed consent. Genomic DNA was extracted from peripheral blood lymphocytes using a standard technique.

The BglII/BlnI dosage test was accomplished using the probe p13E-11 as previously reported 9. We examined 153 Japanese, 124 Korean and 114 Chinese healthy individuals, and unrelated 56 Japanese 4q35-FSHD patients. All individuals were classified into five groups according to the number of chromosomes with BlnI-resistant 4q-type KpnI units; nullsomy (N: 0), monosomy (M: 1), disomy (D: 2), trisomy (T: 3) and quatrosomy (Q: 4) (Figure 1A). Less than 5% of individuals showed unexpected ratios of chromosome 4q;10q, and excluded from this study. The frequency of translocation in each group was estimated and statistical analysis was performed using the chi-square test. We also tested 30 Japanese healthy individuals using pulsed field gel electrophoresis (PFGE) to certify the results obtained by the dosage test. For PFGE, we used agarose embedded "plug" DNA samples 10.

The molecular size of EcoRI fragments on chromosomes 4 and 10 detected by probe p13E-11 varies markedly from 10 to 300 kb. Since fragments longer than 50 kb are difficult to detect by conventional Southern blot analysis, PFGE analysis using agarose embedded plug DNA is often necessary to identify all four fragments from chromosome 4 and 10. However, we cannot always obtain such high quality DNA samples. The BglII/BlnI dosage test used in this study is a useful and easy method to reveal translocation between chromosome 4 and 10, which characterizes the first KpnI repeat unit as a BlnI-resistant 4.0-kb (chromosome 4-type) or a BlnI-sensitive 1.8-kb (chromosome 10-type) fragment. It should be noted, however, the dosage test cannot detect all inter-chromosomal exchanges, i.e., if one had exchanged KpnI repeats at the distal portion following the standard repeats, this exchange will be missed and judged as standard. van Overveld et al. reported that approximately 4.3% (9 among 208) of individuals with standard first repeat showed a hybrid consisting of both BlnI-resistant and -sensitive repeats 7. Therefore, the dosage test may underestimate the exchange ratio, although the present results of the PFGE and dosage tests were identical in 30 Japanese individuals. In the present study, less than 5% of individuals showed unclassified ratios of chromosome 4q;10q. These individuals may have complicated chromosomal rearrangements, or a deletion of the probe p13E-11 region as previously described 9. The limitation of the densitometric analysis should be also considered. We are currently examining in detail on these individuals.

The subtelomeric exchange between chromosomes 4 and 10 was frequently observed in all four populations we examined, and their ratios were similar to the Dutch population previously reported 57. The inter-chromosomal exchange may contribute to the deletion ofKpnI repeats on chromosome 4q35, although there was no difference between healthy and diseased individuals. The ratio of '4 on 10' (trisomy and quatrosomy of chromosome 4) was higher than that of '10 on 4' (nullsomy and monosomy) in all populations we examined. Translocations of chromosome ends have been reported to cause several disorders, such as alpha-thalassemia mental retardation syndrome, Wolf-Hirschhorn syndrome and Miller-Dieker syndrome. Further studies will be needed to clarify the influence of the subtelomeric exchange to the deletion of the repeated units on FSHD.

The frequency of translocation was 27.5% (Japanese), 29.8% (Korean), 19.3% (Chinese), and 32.1% (Japanese 4q35-FSHD patients). The ratio of '4 on 10' (trisomy and quatrosomy of chromosome 4) was higher than that of '10 on 4' (nullsomy and monosomy of chromosome 4) in all populations. In our study, there was no difference between healthy and diseased groups. Further studies will be needed to clarify the influence of the subtelomeric exchange to the deletion of the repeated units on FSHD.

None declared.

TM participated in the design of the study and performed the statistical analysis. KG and GY carried out the molecular genetic studies including the dosage test and PFGE. JHL and CZ obtained control DNA samples of Korean and Chinese. YKH and KA conceived of the study, and participated in its design and coordination.

All authors read and approved the final manuscript.