Methylation of the FSHD syndrome-linked subtelomeric repeat in normal and FSHD cell cultures and tissues.

Facioscapulohumeral muscular dystrophy (FSHD) has an unusual molecular etiology. In a putatively heterochromatic subtelomeric region of each chromosome 4 homologue (4q35), unaffected individuals have 11 to about 95 tandem copies of a complex 3.3-kb repeat (D4Z4). Most FSHD patients have less than 10 copies at one allelic 4q35. This has been proposed to lead to the loss of heterochromatinization and, thereby, inappropriate gene expression by position effects, explaining the dominant nature of FSHD and the role of a decreased number of copies of D4Z4 at 4q35 but not at 10q26. Consistent with the proposed heterochromatinization of this repeat, by Southern blot analysis, we found that SmaI, MluI, SacII, and EagI sites in D4Z4 are highly methylated in normal and FSHD cell lines and somatic tissues, including skeletal muscle. Like repeated DNA sequences in the juxtacentromeric heterochromatin of chromosomes 1, 9, and 16, D4Z4 was hypomethylated at numerous CpGs in sperm and in cell lines from patients with an unrelated DNA methyltransferase deficiency syndrome (ICF; immunodeficiency, centromeric region instability, facial anomalies) in contrast to its hypermethylation in non-ICF postnatal somatic tissues. Our data on FSHD samples suggest that the disease-associated 4q35 D4Z4 repeats, which constitute a small percentage of the total D4Z4 repeats, are not generally hypomethylated relative to the other repeats of this sequence. However, in individuals not affected with FSHD, the hypermethylation of tandem, high-copy-number D4Z4 repeats might help stabilize heterochromatinization at allelic 4q35 regions just as hypermethylation elsewhere in the genome has been linked to chromatin compaction.     

Common epigenetic changes of D4Z4 in contraction‐dependent and contraction‐independent FSHD

Facioscapulohumeral muscular dystrophy (FSHD), caused by partial deletion of the D4Z4 macrosatellite repeat on chromosome 4q, has a complex genetic and epigenetic etiology. To develop FSHD, D4Z4 contraction needs to occur on a specific genetic background. Only contractions associated with the 4qA161 haplotype cause FSHD. In addition, contraction of the D4Z4 repeat in FSHD patients is associated with significant D4Z4 hypomethylation. To date, however, the methylation status of contracted repeats on nonpathogenic haplotypes has not been studied. We have performed a detailed methylation study of the D4Z4 repeat on chromosome 4q and on a highly homologous repeat on chromosome 10q. We show that patients with a D4Z4 deletion (FSHD1) have D4Z4‐restricted hypomethylation. Importantly, controls with a D4Z4 contraction on a nonpathogenic chromosome 4q haplotype or on chromosome 10q also demonstrate hypomethylation. In 15 FSHD families without D4Z4 contractions but with at least one 4qA161 haplotype (FSHD2), we observed D4Z4‐restricted hypomethylation on chromosomes 4q and 10q. This finding implies that a genetic defect resulting in D4Z4 hypomethylation underlies FSHD2. In conclusion, we describe two ways to develop FSHD: (1) contraction‐dependent or (2) contraction‐independent D4Z4 hypomethylation on the 4qA161 subtelomere. Hum Mutat 30:1–11, 2009. © 2009 Wiley‐Liss, Inc.     

The evolutionary distribution and structural organization of the homeobox-containing repeat D4Z4 indicates a functional role for the ancestral copy in the FSHD region [published erratum appears in Hum Mol Genet 1997 Mar;6(3):502]

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant neuromuscular disease that has been linked to deletions within a tandem array of 3.2 kb repeats adjacent to the telomere of 4q. These repeats are also present in other locations in the human genome, including the short arms of all the acrocentric chromosomes. Here, we examine two models for the role of this repeat in FSHD. First, because of the extensive similarity between the 3.2 kb repeats on 4q and those adjacent to rDNA on the acrocentric chromosomes, we investigated whether the FSHD region on 4q is involved in sub-nuclear localization, specifically to the nucleolus. The results likely exclude any involvement of nucleolar localization in the development of FSHD. Second, we investigated a model that suggests that a functional gene may be buried within the tandem array of 3.2 kb repeats. Toward this end, we evaluated the evolutionary conservation of the repeat and a double homeodomain sequence within the repeat in a variety of primate species. The genomic organization of the 3.2 kb repeat in humans, great apes and lower primates identified the FSHD-associated repeat on chromosome 4q as the likely ancestral copy. The sequence of the rhesus monkey double homeodomain reveals significant sequence identity with the human 4q sequence. These results strongly suggest a functional role for a component of the FSHD-associated repeat.      

Inflammatory facioscapulohumeral muscular dystrophy type 2 in 18p deletion syndrome

Facioscapulohumeral muscular dystrophy (FSHD) has been shown to be related to genetic and epigenetic derepression of DUX4 (mapping to chromosome 4), a gene located within a repeat array of D4Z4 sequences of polymorphic length. FSHD type 1 (FSHD1) is associated with pathogenic D4Z4 repeat array contraction, while FSHD type 2 (FSHD2) is associated with SMCHD1 variants (a chromatin modifier gene that maps to the short arm of chromosome 18). Both FSHD types require permissive polyadenylation signal (4qA) downstream of the D4Z4 array.     

Hemizygosity for SMCHD1 in Facioscapulohumeral Muscular Dystrophy Type 2: Consequences for 18p Deletion Syndrome

Facioscapulohumeral muscular dystrophy (FSHD) is most often associated with variegated expression in somatic cells of the normally repressed DUX4 gene within the D4Z4‐repeat array. The most common form, FSHD1, is caused by a D4Z4‐repeat array contraction to a size of 1–10 units (normal range 10–100 units). The less common form, FSHD2, is characterized by D4Z4 CpG hypomethylation and is most often caused by loss‐of‐function mutations in the structural maintenance of chromosomes hinge domain 1 (SMCHD1) gene on chromosome 18p. The chromatin modifier SMCHD1 is necessary to maintain a repressed D4Z4 chromatin state. Here, we describe two FSHD2 families with a 1.2‐Mb deletion encompassing the SMCHD1 gene. Numerical aberrations of chromosome 18 are relatively common and the majority of 18p deletion syndrome (18p‐) cases have, such as these FSHD2 families, only one copy of SMCHD1. Our finding therefore raises the possibility that 18p‐ cases are at risk of developing FSHD. To address this possibility, we combined genome‐wide array analysis data with D4Z4 CpG methylation and repeat array sizes in individuals with 18p‐ and conclude that approximately 1:8 18p‐ cases might be at risk of developing FSHD.     

A large patient study confirming that facioscapulohumeral muscular dystrophy (FSHD) disease expression is almost exclusively associated with an FSHD locus located on a 4qA-defined 4qter subtelomere

Facioscapulohumeral muscular dystrophy (FSHD), an autosomal dominant disorder, represents the third most common human muscular dystrophy. The FSHD disease locus, at chromosome 4q35, is associated with large contractions of the polymorphic repeat sequence array D4Z4. In addition to FSHD disease association with large D4Z4 deletions, a biased interaction with a specific 4qter subtelomeric sequence has been described in patients. Two distinct 4qter subtelomeres, defined as types 4qA and 4qB, have been identified and shown to be equally prevalent in the Caucasian population. In almost all 4q35-linked patients with FSHD, however, disease expression only occurs when large D4Z4 deletions are located on 4qA-defined 4qter subtelomeres. Conversely, large D4Z4 repeat contractions situated on 4qB-defined subtelomeres either are not disease-causing or exhibit a greatly reduced disease penetrance. This study was initiated to confirm this direct FSHD disease association data by measuring the frequency of type 4qA-defined and 4qB-defined subtelomeric sequences in a large cohort of 164 unrelated patients with FSHD from Turkey and the UK, all known to have large D4Z4 deletions. An almost complete association was found between large D4Z4 repeat array deletions located on 4qA-defined 4qter subtelomeres and disease expression in our large FSHD patient cohort. The observed failure of probes 4qA and 4qB to hybridise to two patient-derived DNA samples confirms the presence of an additional rare type of 4qter subtelomeric sequence in humans.     

Distinguishing the 4qA and 4qB variants is essential for the diagnosis of facioscapulohumeral muscular dystrophy in the Chinese population

Facioscapulohumeral muscular dystrophy (FSHD) is the third most common inherited muscular dystrophy with markedly clinical variability and complex genetic cause. Several reports pertaining to the Caucasian population have confirmed that there are 4qA and 4qB variants of the 4qter subtelomere, and FSHD is uniquely associated with the 4qA variant. However, few data relevant to the Chinese population have been published. In present paper, detailed clinical and genetic re-evaluations were performed in members of four special families who had been initially diagnosed as atypical or asymptomatic FSHD based only on the D4Z4 repeat length analysis. The FSHD-sized D4Z4 repeats in the probands from families 1, 2 and 3 were identified as 4qB variants. These patients were further confirmed as limb-girdle muscular dystrophy (LGMD2) or myotonic dystrophy (DM1) by molecular analyses. Specifically, we identified a 4qB variant on chromosome 10 in the healthy members of the fourth FSHD family with complex D4Z4 rearrangements of two exchanged repeat arrays. For the first time, we demonstrated in the Chinese population that D4Z4 contractions on the 4qB variant do not cause FSHD and 4qB variant on chromosome 10 might also represent intermediate structures in the transition from 4q to 10q. Furthermore, our results emphasize that D4Z4 repeat length analysis alone is not sufficient for the diagnosis of FSHD, especially when used as an exclusion criterion. This analysis should be accompanied by 4qA/4qB variant determination and integrated chromosome assignments, especially in patients with obscure and unclassified myopathies similar to atypical forms of FSHD.     

Monosomy of distal 4q does not cause facioscapulohumeral muscular dystrophy.

Facioscapulohumeral muscular dystrophy (FSHD) is a hereditary neuromuscular disorder transmitted in an autosomal dominant fashion. FSHD has been located by linkage analysis in the most distal part of chromosome 4q. The disease is associated with deletions within a 3.2 kb tandem repeat sequence, D4Z4. We have studied a family in which an abnormal chromosome 4 segregates through three generations in phenotypically normal subjects. This chromosome is the derivative of a (4;D or G) (q35;p12) translocation. Molecular analysis of the region 4q35 showed the absence of the segment ranging from the telomere to locus D4F104S1. Probe p13E-11 (D4F104S1), which detects polymorphic EcoRI fragments containing D4Z4, in Southern blot analysis showed only one allele in the carriers of the abnormal chromosome 4. Probe p13E-11 EcoRI fragments are contained in the subtelomeric region of 4q and their rearrangements associated with FSHD suggested that the gene responsible for the muscular dystrophy could be subject to a position effect variegation (PEV) because of its proximity to subtelomeric heterochromatin. The absence of the 4q telomeric region in our phenotypically normal cases indicates that haploinsufficiency of the region containing D4Z4 does not cause FSHD.     

Clinical and Genetic Analysis of Korean Patients with Facioscapulohumeral Muscular Dystrophy

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominantly inherited muscular disorder, which is characterized by weakness of facial, shoulder and hip girdle, humeral, and anterior distal leg muscles. The FSHD gene has been mapped to 4q35 and a deletion of integral copies of a 3.3-kb DNA repeat motif named D4Z4 was known to be the genetic background of the disorder. Although FSHD is the second most common muscular dystrophy in adulthood, there were few reports on the genetically confirmed patients in Korea. Recently, we experienced four Korean patients with clinical features resembling FSHD. In order to confirm the diagnosis, conventional Southern blot (SB) analysis by using double digestion with EcoRI and BlnI and hybridization with p13E-11 probe was performed in three patients and newly developed long polymerase chain reaction (PCR) method was used for one patient because genomic DNA was not enough for conventional SB for this patient. All patients were demonstrated to have shortened D4Z4 repeats that were consistent with FSHD. Therefore, we could confirm the diagnosis of FSHD in four Korean patients and appropriate genetic counseling was done for the patients and their families. It is of note that long-PCR method could be a good alternative for conventional SB when D4Z4 repeats were less than 5.     

The Facioscapulohumeral muscular dystrophy region on 4qter and the homologous locus on 10qter evolved independently under different evolutionary pressure

Background  

The homologous 4q and 10q subtelomeric regions include two distinctive polymorphic arrays of 3.3 kb repeats, named D4Z4. An additional BlnI restriction site on the 10q-type sequence allows to distinguish the chromosomal origin of the repeats. Reduction in the number of D4Z4 repeats below a threshold of 10 at the 4q locus is tightly linked to Facioscapulohumeral Muscular Dystrophy (FSHD), while similar contractions at 10q locus, are not pathogenic. Sequence variations due to the presence of BlnI-sensitive repeats (10q-type) on chromosome 4 or viceversa of BlnI-resistant repeats (4q-type) on chromosome 10 are observed in both alleles.     

面肩肱型肌营养不良症发病机制的研究进展

面肩肱型肌营养不良症,是一种常染色体显性遗传疾病,至今尚未找到其致病基因。大部分面肩肱型肌营养不良症患者和4q35区域3.3-kb的串联重复序列Z4D4的整倍缺失紧密连锁,几乎所有面肩肱型肌营养不良症患者,Southern杂交片段小于35 kb(少于11个D4Z4重复序列),而正常人群该片段为350 kb(11-150个D4Z4重复序列)。通过分子生物学研究与生物信息学分析,在4q35区域内,排除了FRG1、FRG2、ALP、ANT1、DUX4、YY I、HMGB2及Nu-c lolin等几个可能的候选基因;有关肩肱型肌营养不良症发病机制的位置变异效应假说,需要更多的证据支持;另一假说认为,面肩肱型肌营养不良症患者,D4Z4区域内类似沉默子的序列与转录抑制复合物相结合,由于D4Z4的缺失,该复合物不能形成并导致D4Z4上游基因的过表达,有关基因的过表达通过某种不明机制导致FSHD疾病的发生;D4Z4的缺失使4qter在细胞核内的定位异常,使许多基因的表达不正常,从而引起一系列的病理变化,并最终导致FSHD疾病的发生,也是FSHD发病的可能性机制之一。FSHD的发病相关基因和发病机制的研究有待深入。     

Towards the finer mapping of facioscapulohumeral muscular dystrophy at 4q35: Construction of a laser microdissection library

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal-dominant disorder which has been mapped to the 4q35 region. In order to saturate this distal 4q region with DNA markers, a laser-based chromosomal microdissection and microcloning procedure was used to construct a genomic library from the distal 20% of chromosome 4, derived from a single human metaphase spread. Of the 100 microclones analyzed from this library, 94 clones contained inserts sized from 80–800 bp, with an average size of 340 bp. Less than 20% of these clones hybridized to human repeat sequences. Seventy-two single-copy clones were further characterized by Southern blot hybridization against a DNA panel of somatic cell hybrids, containing various regions of chromosome 4. Forty-two clones mapped to chromosome 4, of which 8 clones mapped into the relevant 4q35 region. Twenty of these chromosome 4-specific clones were screened against “zoo-blots”; 11 clones, of which 3 mapped to 4q35, identified conserved sequences. This is the first report to describe the isolation of potential expressed sequences derived from the FSHD region. These chromosome region-specific microclones will be useful in the construction of the physical map of the region, the positional cloning of potential disease-associated genes, and the identification of additional polymorphic markers from within the distal 4q region. © 1995 Wiley-Liss, Inc.     

Exclusion mapping of chromosomal regions which cross hybridise to FSHD1A associated markers in FSHD1B

Facioscapulohumeral muscular dystrophy (FHSD) is a genetically heterogeneous, autosomal dominant primary disease of muscle. The predominant form of FSHD, which has been designated FSHD1A, has been localised to the 4q34 region of human chromosome 4. The disease locus (loci) for the remaining FSHD families, which are not linked to chromosome 4 and have been designated FSHD1B, has not yet been identified.

The D4F104S1 marker which detects copies of a 3·2 kb tandem repeat (D4Z4) which contains several types of repetitive sequences, including Hox gene-like elements, has been shown to be closely linked to the chromosome 4 FHSD disease locus. The loss of an integral number of the 3·2 kb tandem repeats has been associated with FSHD1A. When hybridised to chromosomal spreads these sequences cross hybridise with heterochromatin on acrocentric chromosomes and specific areas of human chromosomes 1, 3, and 10. Potentially these specific regions of cross hybridisation may be linked to FSHD1B. To examine this possiblity we have carried out linkage studies in our largest FSHD1B family. In this paper we exclude these areas of specific cross hybridisation as disease loci for FSHD1B.

     

Fine mapping of the FSHD gene region orientates the rearranged fragment detected by the probe p13E-11

We have produced a fine restriction map around the locus D4F104S1(previously designated D4S810); a probe to this locus, pl3E-11,identifies a polymorphic EcoRI fragment containing 3.2kb tandemrepeats and detects DNA rearrangements associated with facioscapulohumeralmuscular dystrophy (FSHD). We developed an STS (D4F106S1) whichmaps 2kb proximal to D4F104S1, and used this to isolate a 470kbYAC (y25C2E) from the ICI YAC library and a 930kb YAC (y956A11)from the CEPH megabase library. Both YACs contain the loci D4S139,D4F35S1 and D4F104S1. A cosmid library was produced from YACy25C2E and two cosmid contigs constructed; a 115kb contig encompassingD4S139, and one of 135kb linking D4F35S1 and D4F104S1 and extendingdistal to the EcoRl fragment detected by pl3E-11. A fine restrictionmap of both these contigs has been generated, allowing the orientationof the EcoRl fragment rearranged in FSHD to be determined. YACy956A11 was used to confirm the integrity of y25C2E and themap of this region. 9B6A, a probe to the homeobox region ofthe tandem repeat D4Z4, identified a cross-hybridising sequenceproximal to D4F10451, however, p13E-11 does not detect thisadditional locus. CpG islands were identified between D4S139and D4F35S1 and within each copy of the tandem repeat. The probe9B6A detected each copy of the repeat motif, suggesting thereis homeobox present in every copy of the 3.2kb repeat