Hybridization analysis of D4Z4 repeat arrays linked to FSHD

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant disease involving shortening of D4Z4, an array of tandem 3.3-kb repeat units on chromosome 4. These arrays are in subtelomeric regions of 4q and 10q and have 1–100 units. FSHD is associated with an array of 1–10 units at 4q35. Unambiguous clinical diagnosis of FSHD depends on determining the array length at 4q35, usually with the array-adjacent p13E-11 probe after pulsed-field or linear gel electrophoresis. Complicating factors for molecular diagnosis of FSHD are the phenotypically neutral 10q D4Z4 arrays, cross-hybridizing sequences elsewhere in the genome, deletions including the genomic p13E-11 sequence and part of D4Z4, translocations between 4q and 10q D4Z4 arrays, and the extremely high G + C content of D4Z4 arrays (73%). In this study, we optimized conditions for molecular diagnosis of FSHD with a 1-kb D4Z4 subfragment probe after hybridization with p13E-11. We demonstrate that these hybridization conditions allow the identification of FSHD alleles with deletions of the genomic p13E-11 sequence and aid in determination of the nonpathogenic D4Z4 arrays at 10q. Furthermore, we show that the D4Z4-like sequences present elsewhere in the genome are not tandemly arranged, like those at 4q35 and 10q26.     

Facioscapulohumeral muscular dystrophy and DUX4: breaking the silence

Autosomal dominant facioscapulohumeral muscular dystrophy (FSHD) has an unusual pathogenic mechanism. FSHD is caused by deletion of a subset of D4Z4 macrosatellite repeat units in the subtelomere of chromosome 4q. Recent studies provide compelling evidence that a retrotransposed gene in the D4Z4 repeat, DUX4, is expressed in the human germline and then epigenetically silenced in somatic tissues. In FSHD, the combination of inefficient chromatin silencing of the D4Z4 repeat and polymorphisms on the FSHD-permissive alleles that stabilize the DUX4 mRNAs emanating from the repeat result in inappropriate DUX4 protein expression in muscle cells. FSHD is thereby the first example of a human disease caused by the inefficient repression of a retrogene in a macrosatellite repeat array.     

Contractions of D4Z4 on 4qB subtelomeres do not cause facioscapulohumeral muscular dystrophy

Facioscapulohumeral muscular dystrophy (FSHD) is associated with contractions of the D4Z4 repeat in the subtelomere of chromosome 4q. Two allelic variants of chromosome 4q (4qA and 4qB) exist in the region distal to D4Z4. Although both variants are almost equally frequent in the population, FSHD is associated exclusively with the 4qA allele. We identified three families with FSHD in which each proband carries two FSHD-sized alleles and is heterozygous for the 4qA/4qB polymorphism. Segregation analysis demonstrated that FSHD-sized 4qB alleles are not associated with disease, since these were present in unaffected family members. Thus, in addition to a contraction of D4Z4, additional cis-acting elements on 4qA may be required for the development of FSHD. Alternatively, 4qB subtelomeres may contain elements that prevent FSHD pathogenesis.     

Specific Loss of Histone H3 Lysine 9 Trimethylation and HP1γ/Cohesin Binding at D4Z4 Repeats Is Associated with Facioscapulohumeral Dystrophy (FSHD)

Facioscapulohumeral dystrophy (FSHD) is an autosomal dominant muscular dystrophy in which no mutation of pathogenic gene(s) has been identified. Instead, the disease is, in most cases, genetically linked to a contraction in the number of 3.3 kb D4Z4 repeats on chromosome 4q. How contraction of the 4qter D4Z4 repeats causes muscular dystrophy is not understood. In addition, a smaller group of FSHD cases are not associated with D4Z4 repeat contraction (termed “phenotypic” FSHD), and their etiology remains undefined. We carried out chromatin immunoprecipitation analysis using D4Z4–specific PCR primers to examine the D4Z4 chromatin structure in normal and patient cells as well as in small interfering RNA (siRNA)–treated cells. We found that SUV39H1–mediated H3K9 trimethylation at D4Z4 seen in normal cells is lost in FSHD. Furthermore, the loss of this histone modification occurs not only at the contracted 4q D4Z4 allele, but also at the genetically intact D4Z4 alleles on both chromosomes 4q and 10q, providing the first evidence that the genetic change (contraction) of one 4qD4Z4 allele spreads its effect to other genomic regions. Importantly, this epigenetic change was also observed in the phenotypic FSHD cases with no D4Z4 contraction, but not in other types of muscular dystrophies tested. We found that HP1γ and cohesin are co-recruited to D4Z4 in an H3K9me3–dependent and cell type–specific manner, which is disrupted in FSHD. The results indicate that cohesin plays an active role in HP1 recruitment and is involved in cell type–specific D4Z4 chromatin regulation. Taken together, we identified the loss of both histone H3K9 trimethylation and HP1γ/cohesin binding at D4Z4 to be a faithful marker for the FSHD phenotype. Based on these results, we propose a new model in which the epigenetic change initiated at 4q D4Z4 spreads its effect to other genomic regions, which compromises muscle-specific gene regulation leading to FSHD pathogenesis.     

Specific Loss of Histone H3 Lysine 9 Trimethylation and HP1γ/Cohesin Binding at D4Z4 Repeats Is Associated with Facioscapulohumeral Dystrophy (FSHD)

Facioscapulohumeral dystrophy (FSHD) is an autosomal dominant muscular dystrophy in which no mutation of pathogenic gene(s) has been identified. Instead, the disease is, in most cases, genetically linked to a contraction in the number of 3.3 kb D4Z4 repeats on chromosome 4q. How contraction of the 4qter D4Z4 repeats causes muscular dystrophy is not understood. In addition, a smaller group of FSHD cases are not associated with D4Z4 repeat contraction (termed “phenotypic” FSHD), and their etiology remains undefined. We carried out chromatin immunoprecipitation analysis using D4Z4–specific PCR primers to examine the D4Z4 chromatin structure in normal and patient cells as well as in small interfering RNA (siRNA)–treated cells. We found that SUV39H1–mediated H3K9 trimethylation at D4Z4 seen in normal cells is lost in FSHD. Furthermore, the loss of this histone modification occurs not only at the contracted 4q D4Z4 allele, but also at the genetically intact D4Z4 alleles on both chromosomes 4q and 10q, providing the first evidence that the genetic change (contraction) of one 4qD4Z4 allele spreads its effect to other genomic regions. Importantly, this epigenetic change was also observed in the phenotypic FSHD cases with no D4Z4 contraction, but not in other types of muscular dystrophies tested. We found that HP1γ and cohesin are co-recruited to D4Z4 in an H3K9me3–dependent and cell type–specific manner, which is disrupted in FSHD. The results indicate that cohesin plays an active role in HP1 recruitment and is involved in cell type–specific D4Z4 chromatin regulation. Taken together, we identified the loss of both histone H3K9 trimethylation and HP1γ/cohesin binding at D4Z4 to be a faithful marker for the FSHD phenotype. Based on these results, we propose a new model in which the epigenetic change initiated at 4q D4Z4 spreads its effect to other genomic regions, which compromises muscle-specific gene regulation leading to FSHD pathogenesis.     

Equal proportions of affected cells in muscle and blood of a mosaic carrier of facioscapulohumeral muscular dystrophy

Autosomal dominant facioscapulohumeral muscular dystrophy (FSHD) is associated with contractions of D4Z4 repeat on 4q35. It displays a remarkable inter- and intra-familial clinical variability ranging from severe phenotype to asymptomatic carriers. Mosaicism for the contracted FSHD-sized allele is a recurrent finding, but only DNA from lymphocytes had been studied. It is currently not known if mosaicism is unequally distributed between different tissues and if muscle is relatively spared for the presence of the disease allele in mosaic asymptomatic carriers of a disease allele. Here we compare DNA extracted from peripheral blood lymphocytes (PBL), fibroblasts and muscle from a mosaic asymptomatic female carrier and mother of a FSHD patient. PFGE analysis showed a complex allelic segregation: two independent mitotic rearrangement episodes occurred, resulting in mosaicism for a contracted D4Z4 repeat on 4q35 in the mother and mosaicism for an expanded D4Z4 repeat on 10q26 in the affected daughter. The results show that the proportion of mosaicism in PBL and muscle were comparable, while in fibroblasts there was some variation in the mosaicism, which might be caused by culturing artefacts. This finding supports the hypothesis that a mitotic contraction of D4Z4 is an early embryonic event and indicates that the degree of mosaicism in PBL is representative for that of muscle.     

The 4q subtelomere harboring the FSHD locus is specifically anchored with peripheral heterochromatin unlike most human telomeres

This paper investigates the nuclear localization of human telomeres and, specifically, the 4q35 subtelomere mutated in facioscapulohumeral dystrophy (FSHD). FSHD is a common muscular dystrophy that has been linked to contraction of D4Z4 tandem repeats, widely postulated to affect distant gene expression. Most human telomeres, such as 17q and 17p, avoid the nuclear periphery to reside within the internal, euchromatic compartment. In contrast, 4q35 localizes at the peripheral heterochromatin with 4p more internal, generating a reproducible chromosome orientation that we relate to gene expression profiles. Studies of hybrid and translocation cell lines indicate this localization is inherent to the distal tip of 4q. Investigation of heterozygous FSHD myoblasts demonstrated no significant displacement of the mutant allele from the nuclear periphery. However, consistent association of the pathogenic D4Z4 locus with the heterochromatic compartment supports a potential role in regulating the heterochromatic state and makes a telomere positioning effect more likely. Furthermore, D4Z4 repeats on other chromosomes also frequently organize with the heterochromatic compartment at the nuclear or nucleolar periphery, demonstrating a commonality among chromosomes harboring this subtelomere repeat family.     

Fazioskapulohumerale Muskeldystrophie

Facioscapulohumeral muscular dystrophy (FSHD, MIM 158900) is one of the most frequent muscular dystrophies with a prevalence of at least 1:20,000. The slowly progressive myopathy normally begins in the second decade of life with weakness in facial muscles, shoulder muscles and proximal arm muscles. Lower extremities are typically affected in the later disease course. In rare pediatric cases, symptoms start in the first decade, and the disease takes a severe course with extreme manifestation in the face. The genetics of FSHD are complex and the pathomechanism is not well understood as yet. A direct gene defect is not known. There is a close association to deletions of subtelomeric D4Z4 repeats on chromosome 4q. A positional effect of these D4Z4 repeats on centromeric genes is suspected, which seem to be overexpressed in the muscles of patients. Molecular diagnosis is based on the detection of shortened D4Z4 fragments in Southern blot analysis. Diagnosis is complicated by 10q repeats highly homologous to D4Z4 repeats, which are both interchangeable. Only shortened repeat fragments in a special 4q environment are pathogenic.     

The FSHD-Associated Repeat, D4Z4, Is a Member of a Dispersed Family of Homeobox-Containing Repeats, Subsets of Which Are Clustered on the Short Arms of the Acrocentric Chromosomes

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant neuromuscular disorder that maps to human chromosome 4q35. FSHD is tightly linked to a polymorphic 3.3-kb tandem repeat locus, D4Z4. D4Z4 is a complex repeat: it contains a novel homeobox sequence and two other repetitive sequence motifs. In most sporadic FSHD cases, a specific DNA rearrangement, deletion of copies of the repeat at D4Z4, is associated with development of the disease. However, no expressed sequences from D4Z4 have been identified. We have previously shown that there are other loci similar to D4Z4 within the genome. In this paper we describe the isolation of two YAC clones that map to chromosome 14 and that contain multiple copies of a D4Z4-like repeat. Isolation of cDNA clones that map to the acrocentric chromosomes and Southern blot analysis of somatic cell hybrids show that there are similar loci on all of the acrocentric chromosomes. D4Z4 is a member of a complex repeat family, and PCR analysis of somatic cell hybrids shows an organization into distinct subfamilies. The implications of this work in relation to the molecular mechanism of FSHD pathogenesis is discussed. We propose the name 3.3-kb repeat for this family of repetitive sequence elements.     

Facioscapulohumeral muscular dystrophy

Facioscapulohumeral muscular dystrophy (FSHD) is caused by a cascade of epigenetic events following contraction of the polymorphic macrosatellite repeat D4Z4 in the subtelomere of chromosome 4q. Currently, the central issue is whether immediate downstream effects are local (i.e., at chromosome 4q) or global (genome-wide) and there is evidence for both scenarios. Currently, there is no therapy for FSHD, mostly because of our lack of understanding of the primary pathogenic process in FSHD muscle. Clinical trials based on suppression of inflammatory reactions or increasing muscle mass by drugs or training have been disappointing. A recent, probably the first evidence-based pilot trial to revert epigenetic changes did also not provide grounds for a larger clinical study. Clearly, better disease models need to be developed to identify and test novel intervention strategies to eventually improve the quality of life for patients with FSHD.     

Mechanism and timing of mitotic rearrangements in the subtelomeric D4Z4 repeat involved in facioscapulohumeral muscular dystrophy

Autosomal dominant facioscapulohumeral muscular dystrophy (FSHD1A) is associated with contractions of the polymorphic D4Z4 repeat on chromosome 4qter. Almost half of new FSHD mutations occur postfertilization, resulting in somatic mosaicism for D4Z4. Detailed D4Z4 analysis of 11 mosaic individuals with FSHD revealed a mosaic mixture of a contracted FSHD-sized allele and the unchanged ancestral allele in 8 cases, which is suggestive of a mitotic gene conversion without crossover. However, in 3 cases, the D4Z4 rearrangement resulted in two different-sized D4Z4 repeats, indicative of a gene conversion with crossover. In all cases, DNA markers proximal and distal to D4Z4 showed no allelic exchanges, suggesting that all rearrangements were intrachromosomal. We propose that D4Z4 rearrangements occur via a synthesis-dependent strand annealing model that relatively frequently allows for crossovers. Furthermore, the distribution of different cell populations in mosaic patients with FSHD suggests that mosaicism here results from D4Z4 rearrangements occurring during the first few zygotic cell divisions after fertilization.     

Genomic analysis of human chromosome 10q and 4q telomeres suggests a common origin

The subtelomeric region of human chromosome 4q contains the locus for facioscapulohumeral muscular dystrophy (FSHD). The FSHD mutation is a deletion within an array of 3.3-kb tandem repeats (D4Z4). The disease mechanism is unknown but is postulated to involve position effect. A closely related 3.3-kb array on chromosome 10qter, in contrast, is not associated with a disease phenotype. We show here that the 4q homology on chromosome 10 is not confined to the 3.3-kb repeats but extends both proximally (42 kb) and distally to include the telomere. We have also identified the most distal expressed gene on 10q known so far, mapping only 96 kb from the 3.3-kb repeat array. A 4q variant has also been identified; there is 92%nucleotide identity between the two 4q forms, 4qA and 4qB. The 4qter and 10qter forms show homology to other chromosome ends, including 4p, 21q, and 22q, and these regions may represent a relatively common subtelomeric domain.     

Large-scale population analysis challenges the current criteria for the molecular diagnosis of fascioscapulohumeral muscular dystrophy

Facioscapulohumeral muscular dystrophy (FSHD) is a common hereditary myopathy causally linked to reduced numbers (≤8) of 3.3 kilobase D4Z4 tandem repeats at 4q35. However, because individuals carrying D4Z4-reduced alleles and no FSHD and patients with FSHD and no short allele have been observed, additional markers have been proposed to support an FSHD molecular diagnosis. In particular a reduction in the number of D4Z4 elements combined with the 4A(159/161/168)PAS haplotype (which provides the possibility of expressing DUX4) is currently used as the genetic signature uniquely associated with FSHD. Here, we analyzed these DNA elements in more than 800 Italian and Brazilian samples of normal individuals unrelated to any FSHD patients. We find that 3% of healthy subjects carry alleles with a reduced number (4–8) of D4Z4 repeats on chromosome 4q and that one-third of these alleles, 1.3%, occur in combination with the 4A161PAS haplotype. We also systematically characterized the 4q35 haplotype in 253 unrelated FSHD patients. We find that only 127 of them (50.1%) carry alleles with 1–8 D4Z4 repeats associated with 4A161PAS, whereas the remaining FSHD probands carry different haplotypes or alleles with a greater number of D4Z4 repeats. The present study shows that the current genetic signature of FSHD is a common polymorphism and that only half of FSHD probands carry this molecular signature. Our results suggest that the genetic basis of FSHD, which is remarkably heterogeneous, should be revisited, because this has important implications for genetic counseling and prenatal diagnosis of at-risk families.     

The FSHD2 Gene SMCHD1 Is a Modifier of Disease Severity in Families Affected by FSHD1

Facioscapulohumeral muscular dystrophy type 1 (FSHD1) is caused by contraction of the D4Z4 repeat array on chromosome 4 to a size of 1–10 units. The residual number of D4Z4 units inversely correlates with clinical severity, but significant clinical variability exists. Each unit contains a copy of the DUX4 retrogene. Repeat contractions are associated with changes in D4Z4 chromatin structure that increase the likelihood of DUX4 expression in skeletal muscle, but only when the repeat resides in a genetic background that contains a DUX4 polyadenylation signal. Mutations in the structural maintenance of chromosomes flexible hinge domain containing 1 (SMCHD1) gene, encoding a chromatin modifier of D4Z4, also result in the increased likelihood of DUX4 expression in individuals with a rare form of FSHD (FSHD2). Because SMCHD1 directly binds to D4Z4 and suppresses somatic expression of DUX4, we hypothesized that SMCHD1 may act as a genetic modifier in FSHD1. We describe three unrelated individuals with FSHD1 presenting an unusual high clinical severity based on their upper-sized FSHD1 repeat array of nine units. Each of these individuals also carries a mutation in the SMCHD1 gene. Familial carriers of the FSHD1 allele without the SMCHD1 mutation were only mildly affected, suggesting a modifier effect of the SMCHD1 mutation. Knocking down SMCHD1 in FSHD1 myotubes increased DUX4 expression, lending molecular support to a modifier role for SMCHD1 in FSHD1. We conclude that FSHD1 and FSHD2 share a common pathophysiological pathway in which the FSHD2 gene can act as modifier for disease severity in families affected by FSHD1.     

Worldwide Population Analysis of the 4q and 10q Subtelomeres Identifies Only Four Discrete Interchromosomal Sequence Transfers in Human Evolution

Subtelomeres are dynamic structures composed of blocks of homologous DNA sequences. These so-called duplicons are dispersed over many chromosome ends. We studied the human 4q and 10q subtelomeres, which contain the polymorphic macrosatellite repeat D4Z4 and which share high sequence similarity over a region of, on average, >200 kb. Sequence analysis of four polymorphic markers in the African, European, and Asian HAPMAP panels revealed 17 subtelomeric 4q and eight subtelomeric 10qter haplotypes. Haplotypes that are composed of a mixture of 4q and 10q sequences were detected at frequencies >10% in all three populations, seemingly supporting a mechanism of ongoing interchromosomal exchanges between these chromosomes. We constructed an evolutionary network of most haplotypes and identified the 4q haplotype ancestral to all 4q and 10q haplotypes. According to the network, all subtelomeres originate from only four discrete sequence-transfer events during human evolution, and haplotypes with mixtures of 4q- and 10q-specific sequences represent intermediate structures in the transition from 4q to 10q subtelomeres. Haplotype distribution studies on a large number of globally dispersed human DNA samples from the HGDP-CEPH panel supported our findings and show that all haplotypes were present before human migration out of Africa. D4Z4 repeat array contractions on the 4A161 haplotype cause Facioscapulohumeral muscular dystrophy (FSHD), whereas contractions on most other haplotypes are nonpathogenic. We propose that the limited occurrence of interchromosomal sequence transfers results in an accumulation of haplotype-specific polymorphisms that can explain the unique association of FSHD with D4Z4 contractions in a single 4q subtelomere.     

Improved characterization of FSHD mutations

Facioscapulohumeral muscular dystrophy (FSHD) is caused by the shortest alleles of the 3.3kb-tandem repeat array D4Z4 at 4q35. Molecular diagnosis of FSHD depends upon the separation of unusually large alleles by pulse-field electrophoresis after EcoRI and EcoRI/BlnI digestion. The exact number of alleles could not however be directly inferred from the size of DNA fragments owing to polymorphisms in the telomeric region of the locus. Knowing the exact repeat number of disease causing alleles may benefit genetic counselling, help to understand the mechanism of this singular disease and the population dynamics of subtelomeric sequences variations. We present here a partial digestion mapping method giving the exact number of repeats for disease causing alleles, and we suggest that most inaccuracies induced by common polymorphisms could be reduced by using EcoRV in place of EcoRI. After studying more than 300 DNA samples with both the standard method and this new method, we show that alleles size can be evaluated with a precision of less than one half repeat, and that the variations in length of the truncated repeat in the telomeric region of the D4Z4 locus can be evaluated. The results suggest that at least one intact chromosome 4 type repeat at 4q35 is needed to cause FSHD.