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。     

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.     

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.     

Molecular combing reveals complex 4q35 rearrangements in Facioscapulohumeral dystrophy

Facioscapulohumeral dystrophy (FSHD), one of the most common hereditary neuromuscular disorders, is associated with a complex combination of genetic variations at the subtelomeric 4q35 locus. As molecular diagnosis relying on Southern blot (SB) might be challenging in some cases, molecular combing (MC) was recently developed as an additional technique for FSHD diagnosis and exploration of the genomic organization of the 4q35 and 10q26 regions. In complement to the usual SB, we applied MC in a large cohort of 586 individuals with clinical FSHD. In 332 subjects, the two 4q alleles were normal in size, allowing exclusion of FSHD1 while we confirmed FSHD1 in 230 patients. In 14 patients from 10 families, we identified a recurrent complex heterozygous rearrangement at 4q35 consisting of a duplication of the D4Z4 array and a 4qA haplotype, irresolvable by the SB technique. In five families, we further identified variations in the SMCHD1 gene. Impact of the different mutations was tested using a minigene assay and we analyzed DNA methylation after sodium bisulfite modification and NGS sequencing. We discuss the involvement of this rearrangement in FSHD since all mutations in SMCHD1 are not associated with D4Z4 hypomethylation and do not always segregate with the disease.     

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.     

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.     

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 a BlnI 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. This BglII-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 polymorphic BlnI site in the first repeat unit.     

Inter- and intrachromosomal sub-telomeric rearrangements on 4q35: implications for facioscapulohumeral muscular dystrophy (FSHD) aetiology and diagnosis

The autosomal dominant myopathy facioscapulohumeral muscular dystrophy (FSHD) is causally related to a short Eco RI fragment detected by probe p13E-11. This remnant fragment is the result of a deletion of an integral number of tandemly arrayed 3.3 kb repeat units (D4Z4) on 4q35. Despite intensive efforts, no transcribed sequences have been identified within this array. Previously, we have shown that these repeats on 4q35 have been exchanged for a similar highly homologous repeat locus on 10q26 in 20% of the population and that a short chromosome 10-like array on 4q35 also results in FSHD. Here, we describe the hybrid structure of some of these repeat arrays, reflecting additional sub-telomeric instability. In three healthy individuals carrying a 4-like repeat on chromosome 10 or vice versa, one repeat array was shown to consist of hybrid clusters of 4-derived and 10-derived repeat units. Moreover, employing pulsed field gel electrophoresis analysis, we identified two unrelated individuals carrying deletions of a chromosomal segment (p13E-11) proximal to the repeat locus. These deletions were not associated with FSHD. In one of these cases, however, an expansion of the deletion into the repeat array was observed in one of his children suffering from FSHD. These data provide additional evidence for instability of this sub-telomeric region and suggests that the length of the repeat, and not its intrinsic properties, is crucial to FSHD. Moreover, they are in agreement with the hypothesis that FSHD is caused by a position effect in which the repeat structure influences the expression of genes nearby. Therefore, the region deleted proximal to the repeat locus in healthy individuals can be instrumental to refine the critical region for FSHD1.      

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.     

Facioscapulohumeral muscular dystrophy: new insights from compound heterozygotes and implication for prenatal genetic counselling

Background Facioscapulohumeral muscular dystrophy (FSHD) is considered an autosomal dominant disease with a prevalence of 1 in 20?000. Almost all patients with FSHD carry deletions of integral copies of tandem 3.3 kb repeats (D4Z4) located on chromosome 4q35. However, FSHD families have been reported in which individuals carrying a D4Z4-reduced allele remain asymptomatic. Recently, it has been proposed that the D4Z4-reduced allele is pathogenic only in association with the permissive haplotype, 4APAS. Methods and results Through the Italian National Registry for FSHD (INRF), genotype-phenotype correlations were extensively studied in 11 non-consanguineous families in which two D4Z4-reduced alleles segregate. Overall, 68 subjects carrying D4Z4-reduced alleles were examined, including 15 compound heterozygotes. It was found that in four families the only FSHD-affected subject was the compound heterozygote for the D4Z4-reduced allele, and 52.6% of subjects carrying a single D4Z4-reduced 4A161PAS haplotype were non-penetrant carriers; moreover, the population frequency of the 4A161PAS haplotype associated with a D4Z4-reduced allele was found to be as high as 1.2%. Conclusions This study reveals a high frequency of compound heterozygotes in the Italian population and the presence of D4Z4-reduced alleles with the 4A161PAS pathogenic haplotype in the majority of non-penetrant subjects in FSHD families with compound heterozygosity. These data suggest that carriers of FSHD-sized alleles with 4A161PAS haplotype are more common in the general population than expected on the basis of FSHD prevalence. These findings challenge the notion that FSHD is a fully penetrant autosomal dominant disorder uniquely associated with the 4A161PAS haplotype, with relevant repercussions for genetic counselling and prenatal diagnosis.     

Evaluation of the facioscapulohumeral muscular dystrophy (FSHD1) phenotype in correlation to the concurrence of 4q35 and 10q26 fragments

Probe p13E-11 (locus D4F104S1) detects two highly homologous polymorphic loci on chromosomes 4q35 and 10q26. Previous reports in the literature have described a correlation of shortened 4q35-specific fragments and facioscapulohumeral muscular dystrophy (FSHD1). We have identified 30 FSHDI families (46 patients) carrying one short 4q35 and one short 10q26 fragment. The clinical data of these patients were compared with those of 47 families (131 patients) showing a single short 4q35 fragment, in order to evaluate a potentially modifying influence of shortened 10q26 fragments on the phenotype. According to our results, the polymorphic locus on 10q26 does not modify the FSHDI phenotype. The normal population (14%) and our FSHDI population (13%) did not significantly differ in the overall frequency of short polymorphic 10q26 fragments. The specificity of the p13E-11/EcoRI-BlnI test for FSHD1 was 100%.     

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.     

Diagnostic, predictive, and prenatal testing for facioscapulohumeral muscular dystrophy: diagnostic approach for sporadic and familial cases.

Facioscapulohumeral muscular dystrophy (FSHD) is one of the common inherited neuromuscular disorders. The major gene involved, FSHD1, has been localised to chromosome 4q35. This 4q35 locus, detected by pE13-11 (D4F104S1), shows a mutation frequency of about 10% of the incidence. New mutants are characterised by de novo deletions of tens to hundreds of kilobases of DNA. Although these deletion fragments are very useful as a molecular genetic tool, their use in diagnostic DNA testing is hampered by multiple factors, particularly in familial cases. In this report we describe a protocol that can be used for DNA testing in well defined familial cases or proven de novo cases, and in the differential diagnosis of muscular dystrophy patients clinically suspected of having FSHD. In addition, we describe a prenatal diagnosis performed for FSHD1.     

Correlation between fragment size at D4F104S1 and age at onset or at wheelchair use, with a possible generational effect, accounts for much phenotypic variation in 4q35-facioscapulohumeral muscular dystrophy (FSHD)

In facioscapulohumeral muscular dystrophy (FSHD), the wide rangeof clinical severity observed both within and between familieshas obscured past attempts to identify any phenotypic differencesbetween families from which phenotype-genotype correlation couldbe proposed, although it is noted that age at onset is youngestand severity greatest in isolated cases. From 14/16 large 4q35-linkedFSHD families, and 25/34 isolated cases exhibiting a de novoD4F1O4S1 DNA fragment, we find a significant correlation betweenproband age at onset and FSHD-associated D4F104S1 fragment size(r = 0.56; p <0.001), with the smallest fragments occurringin isolated cases. A similar correlation (r = 0.70; p <0.01)with fragment size is observed for age to loss of ambulationin 16 subjects using a wheelchair. We find also that age atonset appears younger with successive generations in the 4q35families. We propose that fragment size at D4F1O4S1, togetherwith a possible generational effect, accounts for a significantpart of the wide phenotypic variation in FSHD. Our results predicta more limited range for severity within families, and in onefamily with a 4q35-linked 38kb fragment support scapulohumeralpresentation without facial involvement as a late onset variantof FSHD. We propose that in FSHD, quantitative variation ina uniform mutation mechanism influences age at onset, but bydeletion rather than expansion of DNA.     

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.