Molecular analysis of 4q35 rearrangements in fascioscapulohumeral muscular dystrophy (FSHD): application to family studies for a correct genetic advice and a reliable prenatal diagnosis of the disease.

In the majority of facioscapulohumeral muscular dystrophy (FSHD) families (about 95%) the genetic defect has been identified as a deletion of a variable number of KpnI repeats in the 4q35 region, although no specific transcripts from this locus have been isolated so far. Molecular diagnosis is based on the detection by probe p13E-11 of EcoRI small fragments, in the range 10-28 kb, that are resistant to BlnI digestion. In family studies this probe is used with other 4q35 polymorphic markers to assign the haplotype associated with the disease. So far, we performed DNA analysis in 145 FSHD families and identified the 4q35 DNA rearrangement not only in affected individuals, but also in healthy subjects at risk of transmitting the disease, such as non-penetrant gene carriers and somatic mosaics. In addition we applied prenatal tests to 19 fetuses, using DNA extracted from chorionic villi samples (CVS) at 10-11 weeks of gestation. The FSHD status, as determined by the presence of BlnI-resistant small fragments associated with the at risk haplotype, was assessed in nine fetuses; in the remaining 10 cases the disease was excluded. Our results show that molecular analysis of 4q35 rearrangements is a reliable indirect method to perform diagnostic, predictive and prenatal tests in FSHD.

Progress in the molecular diagnosis of facioscapulohumeral muscular dystrophy and correlation between the number of KpnI repeats at the 4q35 locus and clinical phenotype.

Genotype analysis by using the p13E-11 probe and other 4q35 polymorphic markers was performed in 122 Italian facioscapulohumeral muscular dystrophy families and 230 normal controls. EcoRI-BlnI double digestion was routinely used to avoid the interference of small EcoRI fragments of 10qter origin that were found in 15% of the controls. An EcoRI fragment ranging between 10 and 28 kb that was resistant to BlnI digestion was detected in 114 of 122 families (93%) comprising 76 familial and 38 isolated cases. Among the unaffected individuals, 3 were somatic mosaics and 7, carrying an EcoRI fragment larger than 20 kb, could be rated as nonpenetrant gene carriers. In a cohort of 165 patients with facioscapulohumeral muscular dystrophy we found an inverse correlation between fragment size and clinical severity. A severe lower limb involvement was observed in 100% of patients with an EcoRI fragment size of 10 to 13 kb (1-2 KpnI repeats left), in 53% of patients with a fragment size of 16 to 20 kb (3-4 KpnI repeats left), and in 19% of patients with a fragment size larger than 21 kb (>4 KpnI repeats left). Our results confirm that the size of the fragment is a major factor in determining the facioscapulohumeral muscular dystrophy phenotype and that it has an impact on clinical prognosis and genetic counseling of the disease.

Sequence homology between 4qter and 10qter loci facilitates the instability of subtelomeric KpnI repeat units implicated in facioscapulohumeral muscular dystrophy.

Physical mapping and in situ hybridization experiments have shown that a duplicated locus with a structural organization similar to that of the 4q35 locus implicated in facioscapulohumeral muscular dystrophy is present in the subtelomeric portion of 10q. We performed sequence analysis of the p13E-11 probe and of the adjacent KpnI tandem-repeat unit derived from a 10qter cosmid clone and compared our results with those published, by other laboratories, for the 4q35 region. We found that the sequence homology range is 98%-100% and confirmed that the only difference that can be exploited for differentiation of the 10qter from the 4q35 alleles is the presence of an additional BlnI site within the 10qter KpnI repeat unit. In addition, we observed that the high degree of sequence homology does facilitate interchromosomal exchanges resulting in displacement of the whole set of BlnI-resistant or BlnI-sensitive KpnI repeats from one chromosome to the other. However, partial translocations escape detection if the latter simply relies on the hybridization pattern from double digestion with EcoRI/BlnI and with p13E-11 as a probe. We discovered that the restriction enzyme Tru9I cuts at both ends of the array of KpnI repeats of different chromosomal origins and allows the use of cloned KpnI sequences as a probe by eliminating other spurious fragments. This approach coupled with BlnI digestion permitted us to investigate the structural organization of BlnI-resistant and BlnI-sensitive units within translocated chromosomes of 4q35 and 10q26 origin. A priori, the possibility that partial translocations could play a role in the molecular mechanism of the disease cannot be excluded.

Direct detection of 4q35 rearrangements implicated in facioscapulohumeral muscular dystrophy (FSHD).

The p13E-11 probe has been shown to detect DNA rearrangements in sporadic and familial cases of FSHD. Its use, however, has been hampered by the fact that it detects at least two pairs of EcoRI alleles, one derived from the 4q35 region (D4F104S1), the other from 10q26 (D10F104S2). We have cloned p13E-11 EcoRI fragments from the 4q35 and 10q26 subtelomeric regions and shown the presence of several restriction site differences within the KpnI tandem repeat units. The two loci present a different distribution of restriction sites for the enzyme BlnI which allows differential cleavage of the KpnI units derived from 10q26, leaving intact the 4q35 pair of alleles. This method of differential restriction greatly facilitates the interpretation of Southern blots obtained from affected and unaffected subjects, with an important improvement in reliability for diagnosis and genetic counselling. In addition, this method can be used to investigate the molecular mechanism of the 4q35 rearrangement implicated in the disease and to ascertain whether the rearrangement is because of interchromosomal exchange between 4qter and 10qter KpnI repeats.

Physical mapping evidence for a duplicated region on chromosome 10qter showing high homology with the facioscapulohumeral muscular dystrophy locus on chromosome 4qter.

p13E-11, a probe (D4F104S1 locus) derived from chromosome 4q35, detects EcoRI-rearranged fragments less than 28 kb in both sporadic and familial cases of facioscapulohumeral muscular dystrophy (FSHD). These fragments are smaller than those observed in healthy individuals. The interpretation of Southern blots is complicated by the fact that p13E-11 reveals two pairs of polymorphic alleles, one 4q35-specific and the other unlinked to 4q35, that sometimes overlap each other. We cloned a non-4q35 13-kb fragment not related to the disease from a sporadic FSHD patient of Italian origin. Haplotype analysis and in situ hybridization experiments showed that this fragment was located on the 10qter region. Restriction mapping of the 10qter clone, when compared with the 4q35 fragment, indicates a similar arrangement of KpnI tandemly repeated units and flanking sequences. However 4q35 and 10q26 EcoRI clones can be distinguished by restriction analysis with SfiI and StyI. This observation could be exploited for future applications in the field of molecular diagnosis and genetic counseling. In addition the isolation of two 10q26 cosmid clones (D10S1484 and D10S1485) from a human genomic library and the construction of a detailed physical map, spanning about 40 kb, showed that the structural homology extended upstream of the EcoRI sites, suggesting that a duplicated FSHD locus resided in the subtelomeric region of the long arm of chromosome 10. We cannot exclude the involvement of the duplicated locus in the molecular mechanism of the disease and in the genetic heterogeneity of FSHD syndromes.

Chromosome 4q35 haplotypes and DNA rearrangements segregating in affected subjects of 19 Italian families with facioscapulohumeral muscular dystrophy (FSHD).

Four DNA markers on the distal long arm of chromosome 4 have been analyzed for their linkage to facioscapulohumeral muscular dystrophy locus (FSHD) in a series of 16 Italian families. We found that, in two families, the disease is not linked to the 4q35 markers, indicating the presence of genetic heterogeneity among Italian FSHD families. Linkage analysis in the remaining families supports the order cen-D4S171-D4S163-D4S139-D4S810-FSHD-qter, in agreement with the physical map from the literature. EcoRI digestion and hybridization with the distal marker p13E-11 (D4S810)1 detected DNA rearrangements in the affected members of both sporadic and familial cases of FSHD, with family-specific fragments ranging in size between 15 kb and 28 kb. In three sporadic FSHD cases, the appearance of a new "small" fragment not present in either parent was clearly associated with the development of FSHD disease. However, in the familial cases analyzed, we observed two recombinations between all four 4q35 markers and the disease locus in apparently normal subjects, leaving open the possibility of nonpenetrance of the FSHD mutation.