Search

FSHD forms are associated to D4Z4 DNA hypomethylation on 4qA allelic chromosomes. These epigenetic changes provide a transcriptionally permissive chromatin environment leading to the production of a pathogenic protein DUX4. We have shown that the epigenetic changes in FSHD, can be investigated using methylation assays specific for permissive chromosomes 4qA (PAS+). Here, we report the analysis of a large cohort of individuals consisting of 287 FSHD1, 53 FSHD2 and 165 control subjects. This analysis showed highly significant difference of methylation levels between affected and control subjects further supported by strong correlation with the number of D4Z4 repeats both in FSHD1 and FSHD2. The different haplotype distribution between affected and control groups as well as particular methylation levels of some haplotypes suggested that diagnosis by methylation analysis can be improved by developing haplotype specific assays. To this aim, we developed haplotype specific assays and additional assays that allow precise genotyping. Furthermore, we set up bisulfite methylation assays to discriminate FSHD1 from FSHD2. Finally, assessment of methylation of intra-D4Z4 region and another SMCHD1 target sequence allowed evaluation of the effect of SMCHD1 mutations. Our results can allow the development of an integrate platform of methylation and sequencing assays to accelerate and improve the diagnostic procedure of FSHD.

The broad aim of biomedical science in the postgenomic era is to link genomic and phenotype information to allow deeper understanding of the processes leading from genomic changes to altered phenotype and disease. The EuroPhenome project (http://www.EuroPhenome.org) is a comprehensive resource for raw and annotated high-throughput phenotyping data arising from projects such as EUMODIC. EUMODIC is gathering data from the EMPReSSslim pipeline (http://www.empress.har.mrc.ac.uk/) which is performed on inbred mouse strains and knock-out lines arising from the EUCOMM project. The EuroPhenome interface allows the user to access the data via the phenotype or genotype. It also allows the user to access the data in a variety of ways, including graphical display, statistical analysis and access to the raw data via web services. The raw phenotyping data captured in EuroPhenome is annotated by an annotation pipeline which automatically identifies statistically different mutants from the appropriate baseline and assigns ontology terms for that specific test. Mutant phenotypes can be quickly identified using two EuroPhenome tools: PhenoMap, a graphical representation of statistically relevant phenotypes, and mining for a mutant using ontology terms. To assist with data definition and cross-database comparisons, phenotype data is annotated using combinations of terms from biological ontologies. © The Author(s) 2009. Published by Oxford University Press.

Background Facioscapulohumeral muscular dystrophy is associated with an epigenetic defect on 4q subtelomere. This dystrophy is caused by contraction of the D4Z4 macrosatellite array on chromosome 4qter in FSHD1, or by functional impairment of SMCHD1, a chromatin modifier binding to D4Z4, in FSHD2. Both genetic defects lead to D4Z4 DNA hypomethylation associated with inappropriate expression in skeletal muscle of the D4Z4-encoded DUX4 transcription factor in the presence of a polymorphic polyadenylation signal (PAS) distal to the last D4Z4 unit (4qA). Currently, hypomethylation is not diagnostic per se because of the interference of non-pathogenic arrays and because it does not interrogate the presence of DUX4-PAS. Importantly, the methylation status of the DUX4-PAS critical region has not been thoroughly investigated. Methods We investigated, by bisulfite sequencing, the DNA methylation levels of the region distal to the D4Z4 array critical to FSHD development in PAS-positive alleles. Results Comparison of FSHD1, FSHD2 and control subjects showed highly significant differences of methylation levels in all CpGs tested. Noteworthy, one of these CpGs (CpG6) is able to discriminate the affected individuals with a sensitivity of 0.95 in a fully characterized samples, supporting the potential usefulness of this assay for FSHD diagnosis. Moreover, our study evidenced a relationship between PAS-specific methylation and severity of the disease. Conclusions These data point to CpGs distal to the D4Z4 array as a critical region that summarizes multiple factors affecting epigenetics of FSHD. Additionally, methylation analysis of this region allows the establishment of a rapid and sensitive tool for FSHD diagnosis

SummaryPhysical 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.

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.