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.     

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.     

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.     

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.     

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.     

Diagnosis by sequencing: correction of misdiagnosis from FSHD2 to LGMD2A by whole-exome analysis

We studied and validated facioscapulohumeral muscular dystrophy (FSHD) samples from patients without a D4Z4 contraction (FSHD2 or 'phenotypic FSHD'). For this, we developed non-radioactive protocols to test D4Z4 allele constitution and DNA methylation, and applied these to samples from the Coriell Institute Cell Repository. The D4Z4 sizing showed two related subjects to have classic chromosome 4 contraction-dependent FSHD1. A third sample (GM17726) did not have a short chromosome 4 fragment, and had been assigned as non-4q FSHD (FSHD2). We tested D4Z4 haplotype and methylation for this individual but found both to be inconsistent with this diagnosis. Using exome sequencing, we identified two known pathogenic mutations in CAPN3 (Arg490Gln and Thr184Argfs(*)36), indicating a case of LGMD2A rather than FSHD. Our study shows how a wrong diagnosis can easily be corrected by whole-exome sequencing by constraining the variant analysis to candidate genes after the data have been generated. This new way of 'diagnosis by sequencing' is likely to become common place in genetic diagnostic laboratories. We also publish a digoxigenin-labeled Southern protocol to test D4Z4 methylation. Our data supports hypomethylation as a good epigenetic predictor for FSHD2. The non-radioactive protocol will help to make this assay more accessible to clinical diagnostic laboratories and the wider FSHD research community.     

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。     

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.     

Double SMCHD1 variants in FSHD2: the synergistic effect of two SMCHD1 variants on D4Z4 hypomethylation and disease penetrance in FSHD2

Facioscapulohumeral muscular dystrophy (FSHD) predominantly affects the muscles in the face, trunk and upper extremities and is marked by large clinical variability in disease onset and progression. FSHD is associated with partial chromatin relaxation of the D4Z4 repeat array on chromosome 4 and the somatic expression of the D4Z4 encoded DUX4 gene. The most common form, FSHD1, is caused by a contraction of the D4Z4 repeat array on chromosome 4 to a size of 1–10 units. FSHD2, the less common form of FSHD, is most often caused by heterozygous variants in the chromatin modifier SMCHD1, which is involved in the maintenance of D4Z4 methylation. We identified three families in which the proband carries two potentially damaging SMCHD1 variants. We investigated whether these variants were located in cis or in trans and determined their functional consequences by detailed clinical information and D4Z4 methylation studies. In the first family, both variants in trans were shown to act synergistically on D4Z4 hypomethylation and disease penetrance, in the second family both SMCHD1 function-affecting variants were located in cis while in the third family one of the two variants did not affect function. This study demonstrates that having two SMCHD1 missense variants that affect function is compatible with life in males and females, which is remarkable considering its role in X inactivation in mice. The study also highlights the variability in SMCHD1 variants underlying FSHD2 and the predictive value of D4Z4 methylation analysis in determining the functional consequences of SMCHD1 variants of unknown significance.     

DNA polymorphism and epigenetic marks modulate the affinity of a scaffold/matrix attachment region to the nuclear matrix

Mechanisms that regulate attachment of the scaffold/matrix attachment regions (S/MARs) to the nuclear matrix remain largely unknown. We have studied the effect of simple sequence length polymorphism (SSLP), DNA methylation and chromatin organization in an S/MAR implicated in facioscapulohumeral dystrophy (FSHD), a hereditary disease linked to a partial deletion of the D4Z4 repeat array on chromosome 4q. This FSHD-related nuclear matrix attachment region (FR-MAR) loses its efficiency in myoblasts from FSHD patients. Three criteria were found to be important for high-affinity interaction between the FR-MAR and the nuclear matrix: the presence of a specific SSLP haplotype in chromosomal DNA, the methylation of one specific CpG within the FR-MAR and the absence of histone H3 acetylated on lysine 9 in the relevant chromatin fragment.     

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.      

汉族人群4q亚端粒区等位片段4qA和4qB的结构特征

目的研究中国人4q35亚端粒区等位片段4qA和4qB的结构特征,探讨其与面肩肱型肌营养不良症（facioscapulohumeral muscular dystrophy,FSHD）的内在联系。方法研究对象包括80名无血缘关系的健康成年人。低熔点胶包埋法抽提基因组DNA。同一样品分别进行EcoRⅠ酶切、EcoRⅠ/BlnⅠ双酶切和HindⅢ酶切,脉冲电场电泳分离,p13E-11、4qA和4qB探针Southern印迹,计算4qA/4qB的频率、基因型频率及各型EcoRⅠ片段的长度,SPSS13.0统计软件分析数据。结果4q亚端粒区4qA的频率（46.9%）与4qB（53.1%）基本相等（χ2=1.250,P〉0.05）,4qA/4qB杂合型频率明显高于纯合型频率（P〈0.05）,4qA型和4qB型EcoRⅠ片段长度分别为（115.8±11.9）kb和（98.3±8.6）kb,两者差异有统计学意义（t=23.04,P〈0.001）。8.8%（7/80）的个体检测到易位构型。2名个体出现4qB型短EcoRⅠ片段。结论正常中国人4q亚端粒区4qA和4qB的频率基本相等,杂合型频率明显高于纯合型频率。4qB型D4Z4缺失不致病。4qA和4qB型EcoRⅠ片段具有动态性变化特征。     

Analysis of the tandem repeat locus D4Z4 associated with facioscapulohumeral muscular dystropothhy

The sequence of the tandem repeat sequence (D4Z4) associatedwith facioscapulohumeral muscular dystrophy (FSHD) has beendetermined: each copy of the 3.3 kb repeat contains two homeoboxesand two previously described repetitive sequences, LSau anda GC-rich low copy repeat designated hhspm3. By Southern blotting,FISH and isolation of cDNA and genomic clones we show that thereare repeat sequences similar to D4Z4 at other locations in thehuman genome. Southern blot analysis of primate genomic DNAindicates that the copy number of D4Z4-like repeats has increasedmarkedly within the last 25 million years. Two cDNA clones wereisolated and found to contain stop codons and frameshifts withinthe homeodomains. An STS was produced to the cDNAs and analysisof a somatic cell hybrid panel suggests they map to chromosome14. No cDNA clones mapping to the chromosome 4q35 D4Z4 repeatshave been Identified, although the possiblilty that they encodea protein cannot be ruled out. Although D4Z4 may not encodea protein, there is an association between deletions withinthis locus and FSHD. The D4Z4 repeats contain LSau repeats andare adjacent to 68 bp Sau3A repeats. Both of these sequencesare associated with heterochromatic regions of DNA, regionsknown to be involved in the phenomenon of position effect variegation.We postulate that deletion of D4Z4 sequences could produce aposition effect.     

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.     

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.