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Evolutionary conservation of a coding function for D4Z4, the tandem DNA repeat mutated in facioscapulohumeral muscular dystrophy.

ABSTRACT: Facioscapulohumeral muscular dystrophy (FSHD) is caused by deletions within the polymorphic DNA tandem array D4Z4. Each D4Z4 repeat unit has an open reading frame (ORF), termed "DUX4," containing two homeobox sequences. Because there has been no evidence of a transcript from the array, these deletions are thought to cause FSHD by a position effect on other genes. Here, we identify D4Z4 homologues in the genomes of rodents, Afrotheria (superorder of elephants and related species), and other species and show that the DUX4 ORF is conserved. Phylogenetic analysis suggests that primate and Afrotherian D4Z4 arrays are orthologous and originated from a retrotransposed copy of an intron-containing DUX gene, DUXC. Reverse-transcriptase polymerase chain reaction and RNA fluorescence and tissue in situ hybridization data indicate transcription of the mouse array. Together with the conservation of the DUX4 ORF for >100 million years, this strongly supports a coding function for D4Z4 and necessitates re-examination of current models of the FSHD disease mechanism.

SUBMITTER: Clapp J

PROVIDER: S-EPMC1950813 | biostudies-literature | 2007 Aug

REPOSITORIES: biostudies-literature

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Evolutionary conservation of a coding function for D4Z4, the tandem DNA repeat mutated in facioscapulohumeral muscular dystrophy.

Clapp Jannine J Mitchell Laura M LM Bolland Daniel J DJ Fantes Judy J Corcoran Anne E AE Scotting Paul J PJ Armour John A L JA Hewitt Jane E JE

American journal of human genetics 20070627 2

Facioscapulohumeral muscular dystrophy (FSHD) is caused by deletions within the polymorphic DNA tandem array D4Z4. Each D4Z4 repeat unit has an open reading frame (ORF), termed "DUX4," containing two homeobox sequences. Because there has been no evidence of a transcript from the array, these deletions are thought to cause FSHD by a position effect on other genes. Here, we identify D4Z4 homologues in the genomes of rodents, Afrotheria (superorder of elephants and related species), and other speci ...[more]

PMID: 17668377

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Project description:ImportanceFacioscapulohumeral muscular dystrophy (FSHD) is considered an autosomal dominant disorder, associated with the deletion of tandemly arrayed D4Z4 repetitive elements. The extensive use of molecular analysis of the D4Z4 locus for FSHD diagnosis has revealed wide clinical variability, suggesting that subgroups of patients exist among carriers of the D4Z4 reduced allele (DRA).ObjectiveTo investigate the clinical expression of FSHD in the genetic subgroup of carriers of a DRA with 7 to 8 repeat units (RUs).Design, setting, and participantsThis multicenter cross-sectional study included 422 carriers of DRA with 7 to 8 RUs (187 unrelated probands and 235 relatives) from a consecutive sample of 280 probands and 306 relatives from the Italian National Registry for FSHD collected between 2008 and 2016. Participants were evaluated by the Italian Clinical Network for FSHD, and all clinical and molecular data were collected in the Italian National Registry for FSHD database. Data analysis was conducted from January 2017 to June 2018.Main outcomes and measuresThe phenotypic classification of probands and relatives was obtained by applying the Comprehensive Clinical Evaluation Form which classifies patients in the 4 following categories: (1) participants presenting facial and scapular girdle muscle weakness typical of FSHD (category A, subcategories A1-A3), (2) participants with muscle weakness limited to scapular girdle or facial muscles (category B, subcategories B1 and B2), (3) asymptomatic or healthy participants (category C, subcategories C1 and C2), and (4) participants with myopathic phenotypes presenting clinical features not consistent with FSHD canonical phenotype (category D, subcategories D1 and D2).ResultsA total of 187 probands (mean [SD] age at last neurological examination, 53.5 [15.2] years; 103 [55.1%] men) and 235 relatives (mean [SD] age at last neurologic examination, 45.1 [17.0] years; 104 [44.7%] men) with a DRA with 7 to 8 RUs and a molecular diagnosis of FSHD were evaluated. Of 187 probands, 99 (52.9%; 95% CI, 45.7%-60.1%) displayed the classic FSHD phenotype, whereas 86 (47.1%; 95% CI, 39.8%-54.3%) presented incomplete or atypical phenotypes. Of 235 carrier relatives from 106 unrelated families, 124 (52.8%; 95% CI, 46.4%-59.7%) had no motor impairment, whereas a small number (38 [16.2%; 95% CI, 9.8%-23.1%]) displayed the classic FSHD phenotype, and 73 (31.0%; 95% CI, 24.7%-38.0%) presented with incomplete or atypical phenotypes. In 37 of 106 families (34.9%; 95% CI, 25.9%-44.8%), the proband was the only participant presenting with a myopathic phenotype, while only 20 families (18.9%; 95% CI, 11.9%-27.6%) had a member with autosomal dominant FSHD.Conclusions and relevanceThis study found large phenotypic variability associated with individuals carrying a DRA with 7 to 8 RUs, in contrast to the indication that a positive molecular test is the only determining aspect for FSHD diagnosis. These findings suggest that carriers of a DRA with 7 to 8 RUs constitute a genetic subgroup different from classic FSHD. Based on these results, it is recommended that clinicians use the Comprehensive Clinical Evaluation Form for clinical classification and, whenever possible, study the extended family to provide the most adequate clinical management and genetic counseling.

Project description:Both forms of facioscapulohumeral muscular dystrophy (FSHD) are associated with aberrant epigenetic regulation of the chromosome 4q35 D4Z4 macrosatellite. Chromatin changes due to large deletions of heterochromatin (FSHD1) or mutations in chromatin regulatory proteins (FSHD2) lead to relaxation of epigenetic repression and increased expression of the deleterious double homeobox 4 (DUX4) gene encoded within the distal D4Z4 repeat. However, many individuals with the genetic requirements for FSHD remain asymptomatic throughout their lives. Here we investigated family cohorts of FSHD1 individuals who were either affected (manifesting) or without any discernible weakness (nonmanifesting/asymptomatic) and their unaffected family members to determine if individual epigenetic status and stability of repression at the contracted 4q35 D4Z4 array in myocytes correlates with FSHD disease.Family cohorts were analyzed for DNA methylation on the distal pathogenic 4q35 D4Z4 repeat on permissive A-type subtelomeres. We found DNA hypomethylation in FSHD1-affected subjects, hypermethylation in healthy controls, and distinctly intermediate levels of methylation in nonmanifesting subjects. We next tested if these differences in DNA methylation had functional relevance by assaying DUX4-fl expression and the stability of epigenetic repression of DUX4-fl in myogenic cells. Treatment with drugs that alter epigenetic status revealed that healthy cells were refractory to treatment, maintaining stable repression of DUX4, while FSHD1-affected cells were highly responsive to treatment and thus epigenetically poised to express DUX4. Myocytes from nonmanifesting subjects had significantly higher levels of DNA methylation and were more resistant to DUX4 activation in response to epigenetic drug treatment than cells from FSHD1-affected first-degree relatives containing the same contraction, indicating that the epigenetic status of the contracted D4Z4 array is reflective of disease.The epigenetic status of the distal 4qA D4Z4 repeat correlates with FSHD disease; FSHD-affected subjects have hypomethylation, healthy unaffected subjects have hypermethylation, and nonmanifesting subjects have characteristically intermediate methylation. Thus, analysis of DNA methylation at the distal D4Z4 repeat could be used as a diagnostic indicator of developing clinical FSHD. In addition, the stability of epigenetic repression upstream of DUX4 expression is a key regulator of disease and a viable therapeutic target.

Project description:Background and objectivesThe objective of this study was to examine whether the regional methylation levels at the most distal D4Z4 repeat units (RU) in the 4qA-permissive haplotype were associated with disease severity and progression in facioscapulohumeral muscular dystrophy type 1 (FSHD1).MethodsThis 21-year, retrospective, observational cohort study was conducted at the Fujian Neuromedical Center (FNMC) in China. Methylation levels of the most distal D4Z4 RU, including 10 CpGs, were assessed in all participants by bisulfite sequencing. Patients with FSHD1 were stratified into 4 groups based on methylation percentage quartiles, including LM1 (low methylation), LM2 (low to intermediate methylation), LM3 (intermediate to high methylation), and highest methylation (HM) levels. Patients received evaluations of motor function focusing on lower extremity (LE) progression at baseline and in follow-ups. FSHD clinical score (CS), age-corrected clinical severity scale (ACSS), and modified Rankin scale were used to assess motor function.ResultsThe methylation levels of the 10 CpGs were significantly lower in all 823 patients with genetically confirmed FSHD1 than in 341 healthy controls (HCs). CpG6 methylation levels could distinguish the following: (1) patients with FSHD1 from HCs; (2) symptomatic from asymptomatic/unaffected patients; (3) patients with LE involvement from those without LE involvement, with AUCs (95% CI) of 0.9684 (0.9584-0.9785), 0.7417 (0.6903-0.7931), and 0.6386 (0.5816-0.6956), respectively. Lower CpG6 methylation levels were correlated with higher CS (r = -0.392), higher ACSS (r = -0.432), and earlier onset age of first-ever muscle weakness (r = 0.297). For the LM1, LM2, LM3, and HM groups, the respective proportions of LE involvement were 52.9%, 44.2%, 36.9%, and 23.4%; and onset ages of LE involvement were 20, 26.5, 25, and 26.5 years. Cox regression analysis-adjusted for sex, age at examination, D4Z4 RU, and 4qA/B haplotype-showed that the LM1, LM2, and LM3 groups (i.e., groups with lower methylation levels) had a higher risk of independent ambulation loss, with HRs (95% CI) of 3.523 (1.565-7.930), 3.356 (1.458-7.727), and 2.956 (1.245-7.020), respectively.Discussion4q35 distal D4Z4 hypomethylation is correlated with disease severity and progression to lower extremity involvement.

Dataset Information

Evolutionary conservation of a coding function for D4Z4, the tandem DNA repeat mutated in facioscapulohumeral muscular dystrophy.

Publications

Evolutionary conservation of a coding function for D4Z4, the tandem DNA repeat mutated in facioscapulohumeral muscular dystrophy.

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