Giancarlo Deidda

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.

De Novo Facioscapulohumeral Muscular Dystrophy: Frequent Somatic Mosaicism, Sex-Dependent Phenotype, and the Role of Mitotic Transchromosomal Repeat Interaction between Chromosomes 4 and 10

Autosomal dominant facioscapulohumeral muscular dystrophy (FSHD) is caused by deletion of most copies of the 3.3-kb subtelomeric D4Z4 repeat array on chromosome 4q. The molecular mechanisms behind the deletion and the high proportion of new mutations have remained elusive. We surveyed 35 de novo FSHD families and found somatic mosaicism in 40% of cases, in either the patient or an asymptomatic parent. Mosaic males were typically affected; mosaic females were more often the unaffected parent of a nonmosaic de novo patient. A genotypic-severity score, composed of the residual repeat size and the degree of somatic mosaicism, yields a consistent relationship with severity and age at onset of disease. Mosaic females had a higher proportion of somatic mosaicism than did mosaic males. The repeat deletion is significantly enhanced by supernumerary homologous repeat arrays. In 10% of normal chromosomes, 4-type repeat arrays are present on chromosome 10. In mosaic individuals, 4-type repeats on chromosome 10 are almost five times more frequent. The reverse configuration, also 10% in normal chromosomes, was not found, indicating that mutations may arise from transchromosomal interaction, to which the increase in 4-type repeat clusters is a predisposing factor. The somatic mosaicism suggests a mainly mitotic origin; mitotic interchromosomal gene conversion or translocation between fully homologous 4-type repeat arrays may be a major mechanism for FSHD mutations.

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.

FRG1P is localised in the nucleolus, Cajal bodies, and speckles

T he highly conserved facioscapulohumeral muscular dystrophy (FSHD) region gene 1 (FRG1) was initially cloned as candidate gene of unknown function for FSHD. To explore the biological function of the FRG1 protein (FRG1P), we studied its cellular localisation in untransfected and FRG1 transfected cell lines. In interphase cells, FRG1P is localised in the dense structures of the nucleolus, in Cajal bodies, and in 60–80% of cells in nuclear speckles. A time course study revealed that FRG1P accumulates in nuclear speckles before its appearance in nucleoli, whereas the localisation in Cajal bodies remains unchanged, as does the localisation of NHPX protein. In accordance with the presence of FRG1P in these nuclear structures, transcription inhibition experiments showed an effect of RNA polymerases I and II on the localisation of FRG1P. Finally, by deletion of the predicted nuclear localisation signals of FRG1P, we demonstrated that both signals are necessary for this subnuclear localisation. FRG1P is an attractive candidate for the pathogenesis of FSHD because of its high evolutionary conservation; its transcriptional deregulation in FSHD; and its colocalisation with proteins that are defective in the neuromuscular disorders oculopharyngeal muscular dystrophy (OPMD) and spinal muscular atrophy (SMA). FRG1 (FSHD Region Gene 1, accession number L76159) was isolated as the first candidate gene for the autosomal dominant myopathy, facioscapulohumeral muscular dystrophy, in 1996. FSHD is primarily characterised by progressive weakness and atrophy of the facial, upper arm, and shoulder muscles. The FSHD locus on 4q35 contains a polymorphic repeat array consisting of 3.3 kb repeated elements (D4Z4). In control individuals, this array may vary between 11–100 units, whereas FSHD patients carry 1–10 units on one of their chromosomes 4, owing to the deletion of an integral number of D4Z4 units. It is becoming increasingly evident that FSHD is caused by a local chromatin alteration, in which contraction of the D4Z4 repeat array results in the transcriptional deregulation of genes located on 4q35 (position effect). Indeed, Gabellini et al recently claimed evidence for transcriptional upregulation of genes close to the repeat in FSHD muscle, including FRG1. In cultured cells, a transcriptional repressor complex binds to D4Z4, and partial loss of this complex leads to transcriptional upregulation of at least one 4qter gene, as observed in FSHD muscle. New expression data based on quantitative RT-PCR suggest that the expression of the 4q35 copy of FRG1 is in fact changed, although the nature of the deregulation is still controversial. FRG1P is highly conserved in vertebrates and nonvertebrates; the human protein is 97% identical to the murine protein and 46% identical to its orthologue in C elegans. Database searches have identified potential orthologues in—for example—Drosophila, tomato, Xenopus and Schizosaccharomyces pombe. As a result of multiple ectopic duplications, in humans FRG1 belongs to a multigene family, with copies on many different chromosomes. The ancestral copy is located on chromosome 4q35, and is demonstrably transcribed in all tissues tested. Additionally, this chromosome 4 copy of FRG1 is deregulated in FSHD muscle. 5 The FRG1 transcript is 1042 bp in length, and is distributed over nine exons encoding an open reading frame (ORF) of 258 amino acid residues. This ORF is coding for a 30 kDa protein, which does not show homology to known proteins. Computer algorithms predict an amino terminal nuclear localisation signal (NLS) and a carboxy terminal bipartite (BP) nuclear localisation signal in FRG1P. In fact, the amino terminal signal consists of two NLS motifs, each four residues in size. Moreover, an imperfect lipocalin motif is predicted, which is involved in transport of hydrophobic particles. Despite its impressive conservation during evolution, indicating a fundamental function, little is known about the role of FRG1P. To obtain more insight into the biological function of FRG1P and its putative involvement in FSHD pathogenesis, we used immunological and GFP based techniques to study its subcellular localisation during different cell stages in untransfected and transfected cell lines. These studies demonstrate that FRG1P colocalises with PABPN1 and SMN1, both involved in related neuromuscular disorders.

A new dosage test for subtelomeric 4;10 translocations improves conventional diagnosis of facioscapulohumeral muscular dystrophy (FSHD)

Facioscapulohumeral muscular dystrophy (FSHD) is caused by the size reduction of a polymorphic repeat array on 4q35. Probe p13E-11 recognises this chromosomal rearrangement and is generally used for diagnosis. However, diagnosis of FSHD is complicated by three factors. First, the probe cross hybridises to a highly homologous repeat array locus on chromosome 10q26. Second, although aBlnI polymorphism allows discrimination between the repeat units on chromosomes 4 and 10 and greatly facilitates FSHD diagnosis, the occurrence of translocations between chromosomes 4 and 10 further complicates accurate FSHD diagnosis. Third, the recent identification of deletions of p13E-11 in both control and FSHD populations is an additional complicating factor. Although pulsed field gel electrophoresis is very useful and sometimes necessary to detect these rearrangements, this technique is not operational in most FSHD diagnostic laboratories. Moreover, repeat arrays >200 kb are often difficult to detect and can falsely suggest a deletion of p13E-11. Therefore, we have developed an easy and reliable Southern blotting method to identify exchanges between 4 type and 10 type repeat arrays and deletions of p13E-11. ThisBglII-BlnI dosage test addresses all the above mentioned complicating factors and can be carried out in addition to the standard Southern blot analysis for FSHD diagnosis as performed in most laboratories. It will enhance the specificity and sensitivity of conventional FSHD diagnosis to the values obtained by PFGE based diagnosis of FSHD. Moreover, this study delimits the FSHD candidate gene region by mapping the 4;10 translocation breakpoint proximal to the polymorphicBlnI site in the first repeat unit.

Molecular analysis of 4q35 rearrangements in facioscapulohumeral 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.

Allele-specific DNA hypomethylation characterises FSHD1 and FSHD2

Background Facioscapulohumeral muscular dystrophy (FSHD) is associated with an epigenetic defect on 4qter. Two clinically indistinguishable forms of FSHD are known, FSHD1 and FSHD2. FSHD1 is caused by contraction of the highly polymorphic D4Z4 macrosatellite repeat array on chromosome 4q35. FSHD2 is caused by pathogenic mutations of the SMCHD1 gene. Both genetic defects lead to D4Z4 DNA hypomethylation. In the presence of a polymorphic polyadenylation signal (PAS), DNA hypomethylation leads to inappropriate expression of the D4Z4-encoded DUX4 transcription factor in skeletal muscle. Currently, hypomethylation is not diagnostic per se because of the interference of non-pathogenic arrays and the lack of information about the presence of DUX4-PAS. Methods We investigated, by bisulfite sequencing, the DNA methylation levels of the region distal to the D4Z4 array selectively in PAS-positive alleles. Results Comparison of FSHD1, FSHD2 and Control subjects showed a highly significant difference of methylation levels in all CpGs tested. Importantly, using a cohort of 112 samples, one of these CpGs (CpG6) is able to discriminate the affected individuals with a sensitivity of 0.95 supporting this assay potential for FSHD diagnosis. Moreover, our study showed a relationship between PAS-specific methylation and severity of the disease. Conclusions These data point to the CpGs distal to the D4Z4 array as a critical region reflecting multiple factors affecting the epigenetics of FSHD. Additionally, methylation analysis of this region allows the establishment of a rapid and sensitive tool for FSHD diagnosis.

Estrogens enhance myoblast differentiation in facioscapulohumeral muscular dystrophy by antagonizing DUX4 activity

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant neuromuscular disorder that is characterized by extreme variability in symptoms, with females being less severely affected than males and presenting a higher proportion of asymptomatic carriers. The sex-related factors involved in the disease are not known. Here, we have utilized myoblasts isolated from FSHD patients (FSHD myoblasts) to investigate the effect of estrogens on muscle properties. Our results demonstrated that estrogens counteract the differentiation impairment of FSHD myoblasts without affecting cell proliferation or survival. Estrogen effects are mediated by estrogen receptor &bgr; (ER&bgr;), which reduces chromatin occupancy and transcriptional activity of double homeobox 4 (DUX4), a protein whose aberrant expression has been implicated in FSHD pathogenesis. During myoblast differentiation, we observed that the levels and activity of DUX4 increased progressively and were associated with its enhanced recruitment in the nucleus. ER&bgr; interfered with this recruitment by relocalizing DUX4 in the cytoplasm. This work identifies estrogens as a potential disease modifier that underlie sex-related differences in FSHD by protecting against myoblast differentiation impairments in this disease.

Highly efficient, in vivo optimized, archaeal endonuclease for controlled RNA splicing in mammalian cells

ARCHAEA‐ExPRESs is an mRNA modification technology that makes use of components derived from the Archaeon Methanocaldococcus jannaschii, namely the tRNA splicing endonuclease (MJ‐EndA) and its natural substrate, the bulge‐helix‐bulge (BHB) structure (1). These components can perform both cis‐ and trans‐splicing in cellular and animal models and may provide a convenient way to modulate gene expression using components independent of cellular regulatory networks. To use MJ‐EndA in stable expression mammalian systems, we developed variants characterized by high efficiency and sustainable in vivo activity. The MJ‐EndA variants were created by the introduction of proper localization signals followed by mutagenesis and direct selection in mammalian cells. Of note, enzyme selection used an in vivo selection method based on puromycin resistance conferred to cells by BHB‐mediated intron splicing from an out‐of‐frame puromycin N‐acetyl transferase (PAC) gene. This approach yielded several endonuclease variants, the best of which showed 40‐fold higher activity compared to the parental enzyme and stable processing of 30% of the target mRNA. Notably, these variants showed complete compatibility with long‐term expression in mammalian cells, suggesting that they may be usefully applied in functional genomics and genetically modified animal models.—Putti, S., Calandra, P., Rossi, N., Scarabino, D., Deidda, G., Tocchini‐Valentini, G. P., Highly efficient, in vivo optimized, archaeal endonuclease for controlled RNA splicing in mammalian cells. FASEB J. 27, 3466–3477 (2013). www.fasebj.org