SMCHD1 is involved in de novo methylation of the DUX4-encoding D4Z4 macrosatellite.

The DNA methylation epigenetic signature is a key determinant during development. Rules governing its establishment and maintenance remain elusive especially at repetitive sequences, which account for the majority of methylated CGs. DNA methylation is altered in a number of diseases including those linked to mutations in factors that modify chromatin. Among them, SMCHD1 (Structural Maintenance of Chromosomes Hinge Domain Containing 1) has been of major interest following identification of germline mutations in Facio-Scapulo-Humeral Dystrophy (FSHD) and in an unrelated developmental disorder, Bosma Arhinia Microphthalmia Syndrome (BAMS). By investigating why germline SMCHD1 mutations lead to these two different diseases, we uncovered a role for this factor in de novo methylation at the pluripotent stage. SMCHD1 is required for the dynamic methylation of the D4Z4 macrosatellite upon reprogramming but seems dispensable for methylation maintenance. We find that FSHD and BAMS patient's cells carrying SMCHD1 mutations are both permissive for DUX4 expression, a transcription factor whose regulation has been proposed as the main trigger for FSHD. These findings open new questions as to what is the true aetiology for FSHD, the epigenetic events associated with the disease thus calling the current model into question and opening new perspectives for understanding repetitive DNA sequences regulation.

Deciphering the complexity of the 4q and 10q subtelomeres by molecular combing in healthy individuals and patients with facioscapulohumeral dystrophy.

BACKGROUND: Subtelomeres are variable regions between telomeres and chromosomal-specific regions. One of the most studied pathologies linked to subtelomeric imbalance is facioscapulohumeral dystrophy (FSHD). In most cases, this disease involves shortening of an array of D4Z4 macrosatellite elements at the 4q35 locus. The disease also segregates with a specific A-type haplotype containing a degenerated polyadenylation signal distal to the last repeat followed by a repetitive array of ß-satellite elements. This classification applies to most patients with FSHD. A subset of patients called FSHD2 escapes this definition and carries a mutation in the SMCHD1 gene. We also recently described patients carrying a complex rearrangement consisting of a cis-duplication of the distal 4q35 locus identified by molecular combing. METHODS: Using this high-resolution technology, we further investigated the organisation of the 4q35 region linked to the disease and the 10q26 locus presenting with 98% of homology in controls and patients. RESULTS: Our analyses reveal a broad variability in size of the different elements composing these loci highlighting the complexity of these subtelomeres and the difficulty for genomic assembly. Out of the 1029 DNA samples analysed in our centre in the last 7 years, we also identified 54 cases clinically diagnosed with FSHD carrying complex genotypes. This includes mosaic patients, patients with deletions of the proximal 4q region and 23 cases with an atypical chromosome 10 pattern, infrequently found in the control population and never reported before. CONCLUSION: Overall, this work underlines the complexity of these loci challenging the diagnosis and genetic counselling for this disease.

Dysregulation of 4q35- and muscle-specific genes in fetuses with a short D4Z4 array linked to facio-scapulo-humeral dystrophy.

Facio-scapulo-humeral dystrophy (FSHD) results from deletions in the subtelomeric macrosatellite D4Z4 array on the 4q35 region. Upregulation of the DUX4 retrogene from the last D4Z4 repeated unit is thought to underlie FSHD pathophysiology. However, no one knows what triggers muscle defect and when alteration arises. To gain further insights into the molecular mechanisms of the disease, we evaluated at the molecular level, the perturbation linked to the FSHD genotype with no a priori on disease onset, severity or penetrance and prior to any infiltration by fibrotic or adipose tissue in biopsies from fetuses carrying a short pathogenic D4Z4 array (n = 6) compared with fetuses with a non-pathogenic D4Z4 array (n = 21). By measuring expression of several muscle-specific markers and 4q35 genes including the DUX4 retrogene by an RT-PCR and western blotting, we observed a global dysregulation of genes involved in myogenesis including MYOD1 in samples with <11 D4Z4. The DUX4-fl pathogenic transcript was detected in FSHD biopsies but also in controls. Importantly, in FSHD fetuses, we mainly detected the non-spliced DUX4-fl isoform. In addition, several other genes clustered at the 4q35 locus are upregulated in FSHD fetuses. Our study is the first to examine fetuses carrying an FSHD-linked genotype and reveals an extensive dysregulation of several muscle-specific and 4q35 genes at early development stage at a distance from any muscle defect. Overall, our work suggests that even if FSHD is an adult-onset muscular dystrophy, the disease might also involve early molecular defects arising during myogenesis or early differentiation.

Genome wide SNP array identified multiple mechanisms of genetic changes in Waldenstrom macroglobulinemia.

SNP array (SNPa) was developed to detect copy number alteration (CNA) and loss of heterozygosity (LOH) without copy number changes, CN-LOH. We aimed to identify novel genomic aberrations using SNPa in 31 WM with paired samples. Methylation status and mutation were analyzed on target genes. A total of 61 genetic aberrations were observed, 58 CNA (33 gains, 25 losses) in 58% of patients and CN-LOH in 6% of patients. The CNA were widely distributed throughout the genome, including 12 recurrent regions and identified new cryptic clonal chromosomal lesions that were mapped. Gene set expression analysis demonstrated a relationship between either deletion 6q or gain of chromosome 4 and alteration of gene expression profiling. We then studied methylation status and sought for mutations in altered regions on target genes. We observed methylation of DLEU7 on chromosome 13 in all patients (n = 12) with WM, and mutations of CD79B/CD79A genes (17q region), a key component of the BCR pathway, in 15% of cases. Most importantly, higher frequency of ≥3 CNA was observed in symptomatic WM. In conclusion, this study expands the view of the genomic complexity of WM, especially in symptomatic WM, including a potentially new mechanism of gene dysfunction, acquired uniparental disomy/CN-LOH. Finally, we have identified new potential target genes in WM, such as DLEU7 and CD79A/B.

Production of Innervated Skeletal Muscle Fibers Using Human Induced Pluripotent Stem Cells.

Only a limited number of large-scale protocols describe the production of mature skeletal muscle fibers from human induced pluripotent stem cells (hiPSCs). Here we describe a novel procedure for simultaneous differentiation of hiPSC into muscle cells and motor neurons, that generates innervated and contractile multinucleated skeletal muscle fibers with sarcomeric organization. Our protocol permits the production of expandable skeletal muscle progenitor cells and mature skeletal muscle fibers that can be used for the exploration of skeletal muscle differentiation for basic research, disease modeling, and drug discovery.

Facioscapulohumeral dystrophy weakened sarcomeric contractility is mimicked in induced pluripotent stem cells-derived innervated muscle fibres.

BACKGROUND: Facioscapulohumeral dystrophy (FSHD) is a late-onset autosomal dominant form of muscular dystrophy involving specific groups of muscles with variable weakness that precedes inflammatory response, fat infiltration, and muscle atrophy. As there is currently no cure for this disease, understanding and modelling the typical muscle weakness in FSHD remains a major milestone towards deciphering the disease pathogenesis as it will pave the way to therapeutic strategies aimed at correcting the functional muscular defect in patients. METHODS: To gain further insights into the specificity of the muscle alteration in this disease, we derived induced pluripotent stem cells from patients affected with Types 1 and 2 FSHD but also from patients affected with Bosma arhinia and microphthalmia. We differentiated these cells into contractile innervated muscle fibres and analysed their transcriptome by RNA Seq in comparison with cells derived from healthy donors. To uncover biological pathways altered in the disease, we applied MOGAMUN, a multi-objective genetic algorithm that integrates multiplex complex networks of biological interactions (protein-protein interactions, co-expression, and biological pathways) and RNA Seq expression data to identify active modules. RESULTS: We identified 132 differentially expressed genes that are specific to FSHD cells (false discovery rate < 0.05). In FSHD, the vast majority of active modules retrieved with MOGAMUN converges towards a decreased expression of genes encoding proteins involved in sarcomere organization (P value 2.63e-12 ), actin cytoskeleton (P value 9.4e-5 ), myofibril (P value 2.19e-12 ), actin-myosin sliding, and calcium handling (with P values ranging from 7.9e-35 to 7.9e-21 ). Combined with in vivo validations and functional investigations, our data emphasize a reduction in fibre contraction (P value < 0.0001) indicating that the muscle weakness that is typical of FSHD clinical spectrum might be associated with dysfunction of calcium release (P value < 0.0001), actin-myosin interactions, motor activity, mechano-transduction, and dysfunctional sarcomere contractility. CONCLUSIONS: Identification of biomarkers of FSHD muscle remain critical for understanding the process leading to the pathology but also for the definition of readouts to be used for drug design, outcome measures, and monitoring of therapies. The different pathways identified through a system biology approach have been largely overlooked in the disease. Overall, our work opens new perspectives in the definition of biomarkers able to define the muscle alteration but also in the development of novel strategies to improve muscle function as it provides functional parameters for active molecule screening.

Mesenchymal stem cells derived from patients with premature aging syndromes display hallmarks of physiological aging.

Progeroid syndromes are rare genetic diseases with most of autosomal dominant transmission, the prevalence of which is less than 1/10,000,000. These syndromes caused by mutations in the <i>LMNA</i> gene encoding A-type lamins belong to a group of disorders called laminopathies. Lamins are implicated in the architecture and function of the nucleus and chromatin. Patients affected with progeroid laminopathies display accelerated aging of mesenchymal stem cells (MSCs)-derived tissues associated with nuclear morphological abnormalities. To identify pathways altered in progeroid patients' MSCs, we used induced pluripotent stem cells (hiPSCs) from patients affected with classical Hutchinson-Gilford progeria syndrome (HGPS, c.1824C&gt;T-p.G608G), HGPS-like syndrome (HGPS-L; c.1868C&gt;G-p.T623S) associated with farnesylated prelamin A accumulation, or atypical progeroid syndromes (APS; homozygous c.1583C&gt; T-p.T528M; heterozygous c.1762T&gt;C-p.C588R; compound heterozygous c.1583C&gt;T and c.1619T&gt;C-p.T528M and p.M540T) without progerin accumulation. By comparative analysis of the transcriptome and methylome of hiPSC-derived MSCs, we found that patient's MSCs display specific DNA methylation patterns and modulated transcription at early stages of differentiation. We further explored selected biological processes deregulated in the presence of <i>LMNA</i> variants and confirmed alterations of age-related pathways during MSC differentiation. In particular, we report the presence of an altered mitochondrial pattern; an increased response to double-strand DNA damage; and telomere erosion in HGPS, HGPS-L, and APS MSCs, suggesting converging pathways, independent of progerin accumulation, but a distinct DNA methylation profile in HGPS and HGPS-L compared with APS cells.

AKT Signaling Modifies the Balance between Cell Proliferation and Migration in Neural Crest Cells from Patients Affected with Bosma Arhinia and Microphthalmia Syndrome.

Over the recent years, the SMCHD1 (Structural Maintenance of Chromosome flexible Hinge Domain Containing 1) chromatin-associated factor has triggered increasing interest after the identification of variants in three rare and unrelated diseases, type 2 Facio Scapulo Humeral Dystrophy (FSHD2), Bosma Arhinia and Microphthalmia Syndrome (BAMS), and the more recently isolated hypogonadotrophic hypogonadism (IHH) combined pituitary hormone deficiency (CPHD) and septo-optic dysplasia (SOD). However, it remains unclear why certain mutations lead to a specific muscle defect in FSHD while other are associated with severe congenital anomalies. To gain further insights into the specificity of SMCHD1 variants and identify pathways associated with the BAMS phenotype and related neural crest defects, we derived induced pluripotent stem cells from patients carrying a mutation in this gene. We differentiated these cells in neural crest stem cells and analyzed their transcriptome by RNA-Seq. Besides classical differential expression analyses, we analyzed our data using MOGAMUN, an algorithm allowing the extraction of active modules by integrating differential expression data with biological networks. We found that in BAMS neural crest cells, all subnetworks that are associated with differentially expressed genes converge toward a predominant role for AKT signaling in the control of the cell proliferation-migration balance. Our findings provide further insights into the distinct mechanism by which defects in neural crest migration might contribute to the craniofacial anomalies in BAMS.

Multilineage Differentiation for Formation of Innervated Skeletal Muscle Fibers from Healthy and Diseased Human Pluripotent Stem Cells.

Induced pluripotent stem cells (iPSCs) obtained by reprogramming primary somatic cells have revolutionized the fields of cell biology and disease modeling. However, the number protocols for generating mature muscle fibers with sarcolemmal organization using iPSCs remain limited, and partly mimic the complexity of mature skeletal muscle. Methods: We used a novel combination of small molecules added in a precise sequence for the simultaneous codifferentiation of human iPSCs into skeletal muscle cells and motor neurons. Results: We show that the presence of both cell types reduces the production time for millimeter-long multinucleated muscle fibers with sarcolemmal organization. Muscle fiber contractions are visible in 19-21 days, and can be maintained over long period thanks to the production of innervated multinucleated mature skeletal muscle fibers with autonomous cell regeneration of PAX7-positive cells and extracellular matrix synthesis. The sequential addition of specific molecules recapitulates key steps of human peripheral neurogenesis and myogenesis. Furthermore, this organoid-like culture can be used for functional evaluation and drug screening. Conclusion: Our protocol, which is applicable to hiPSCs from healthy individuals, was validated in Duchenne Muscular Dystrophy, Myotonic Dystrophy, Facio-Scapulo-Humeral Dystrophy and type 2A Limb-Girdle Muscular Dystrophy, opening new paths for the exploration of muscle differentiation, disease modeling and drug discovery.

Analysis of the 4q35 chromatin organization reveals distinct long-range interactions in patients affected with Facio-Scapulo-Humeral Dystrophy.

Facio-Scapulo Humeral dystrophy (FSHD) is the third most common myopathy, affecting 1 amongst 10,000 individuals (FSHD1, OMIM #158900). This autosomal dominant pathology is associated in 95% of cases with genetic and epigenetic alterations in the subtelomeric region at the extremity of the long arm of chromosome 4 (q arm). A large proportion of the remaining 5% of cases carry a mutation in the SMCHD1 gene (FSHD2, OMIM #158901). Here, we explored the 3D organization of the 4q35 locus by three-dimensions DNA in situ fluorescent hybridization (3D-FISH) in primary fibroblasts isolated from patients and healthy donors. We found that D4Z4 contractions and/or SMCHD1 mutations impact the spatial organization of the 4q35 region and trigger changes in the expression of different genes. Changes in gene expression were corroborated in muscle biopsies suggesting that the modified chromatin landscape impelled a modulation in the level of expression of a number of genes across the 4q35 locus in FSHD. Using induced pluripotent stem cells (hIPSC), we further examined whether chromatin organization is inherited after reprogramming or acquired during differentiation and showed that folding of the 4q35 region is modified upon differentiation. These results together with previous findings highlight the role of the D4Z4 macrosatellite repeat in the topological organization of chromatin and further indicate that the D4Z4-dependent 3D structure induces transcriptional changes of 4q35 genes expression.

Methylation hotspots evidenced by deep sequencing in patients with facioscapulohumeral dystrophy and mosaicism.

OBJECTIVE: To investigate the distribution of cytosine-guanine dinucleotide (CpG) sites with a variable level of DNA methylation of the D4Z4 macrosatellite element in patients with facioscapulohumeral dystrophy (FSHD). METHODS: By adapting bisulfite modification to deep sequencing, we performed a comprehensive analysis of D4Z4 methylation across D4Z4 repeats and adjacent 4qA sequence in DNA from patients with FSHD1, FSHD2, or mosaicism and controls. RESULTS: Using hierarchical clustering, we identified clusters with different levels of methylation and separated, thereby the different groups of samples (controls, FSHD1, and FSHD2) based on their respective level of methylation. We further show that deep sequencing-based methylation analysis discriminates mosaic cases for which methylation changes have never been evaluated previously. CONCLUSIONS: Altogether, our approach offers a new high throughput tool for estimation of the D4Z4 methylation level in the different subcategories of patients having FSHD. This methodology allows for a comprehensive and discriminative analysis of different regions along the macrosatellite repeat and identification of focal regions or CpG sites differentially methylated in patients with FSHD1 and FSHD2 but also complex cases such as those presenting mosaicism.

BACH2 promotes indolent clinical presentation in Waldenström macroglobulinemia.

Approximately 30% of the patients who fulfil the criteria of Waldenström's macroglobulinemia (WM) are diagnosed while asymptomatic (indolent), and will not require immediate therapy. Conversely, patients with a disease-related event will be considered for therapy. The physiopathology of these 2 groups remains unclear, and the mechanisms of progression from indolent to symptomatic WM have yet to be fully understood. Seventeen patients diagnosed with WM were included in this study, 8 asymptomatic WM (A-WM) and 9 symptomatic WM (S-WM). A differential analysis was performed on a first series of 11 patients and identified 48 genes whose expression separated samples from A- to S-WM. This gene signature was then confirmed on a second independent validation set of 6 WM. Within this expression profile, BACH2, a B-cell transcription factor known to be a tumor suppressor gene, was found to be over-expressed in A-MW relatively to S-MW. We specifically over-expressed BACH2 in a WM-related cell line and observed a significant reduction of the clonogenic activity. To the best of our knowledge, we report for the first time a specific gene expression signature that differentiates A-WM and S-WM. Within this expression profile, BACH2 was identified as a candidate gene that may help to understand better the behavior of tumor cells in indolent WM.

Differential DNA methylation of the D4Z4 repeat in patients with FSHD and asymptomatic carriers.

OBJECTIVE: We investigated the link between DNA hypomethylation and clinical penetrance in facioscapulohumeral dystrophy (FSHD) because hypomethylation is moderate and heterogeneous in patients and could not thus far be correlated with disease presence or severity. METHODS: To investigate the link between clinical signs of FSHD and DNA methylation, we explored 95 cases (37 FSHD1, 29 asymptomatic individuals carrying a shortened D4Z4 array, 9 patients with FSHD2, and 20 controls) by implementing 2 approaches: methylated DNA immunoprecipitation and sodium bisulfite sequencing. RESULTS: Both methods revealed statistically significant differences between asymptomatic carriers or controls and individuals with clinical FSHD, especially in the proximal region of the repeat. Absence of clinical expression in asymptomatic carriers is associated with a level of methylation similar to controls. CONCLUSIONS: We provide a proof of concept that the targeted approaches that we describe could be applied systematically to patient samples in routine diagnosis and suggest that local hypomethylation within D4Z4 might serve as a modifier for clinical expression of FSHD phenotype. CLASSIFICATION OF EVIDENCE: This study provides Class III evidence that assays for hypomethylation within the D4Z4 region accurately distinguish patients with FSHD from individuals with D4Z4 contraction without FSHD.

Acacetin and chrysin, two polyphenolic compounds, alleviate telomeric position effect in human cells.

We took advantage of the ability of human telomeres to silence neighboring genes (telomere position effect or TPE) to design a high-throughput screening assay for drugs altering telomeres. We identified, for the first time, that two dietary flavones, acacetin and chrysin, are able to specifically alleviate TPE in human cells. We further investigated their influence on telomere integrity and showed that both drugs drastically deprotect telomeres against DNA damage response. However, telomere deprotection triggered by shelterin dysfunction does not affect TPE, indicating that acacetin and chrysin target several functions of telomeres. These results show that TPE-based screening assays represent valuable methods to discover new compounds targeting telomeres.Molecular Therapy-Nucleic Acids (2013) 2, e116; doi:10.1038/mtna.2013.42; published online 20 August 2013.

High-throughput genomic analysis in Waldenström's macroglobulinemia.

Single-nucleotide polymorphism array (SNPa) and array-based comparative genomic hybridization (aCGH) are among the most sensitive genomic high-throughput screening techniques used in the exploration of genetic abnormalities in Waldenström's macroglobulinemia (WM). SNP and aCGH allow the identification of copy number abnormalities (CNA) at the kilobase level thus identifying cryptic genetic abnormalities unseen by lower-resolution approaches such as conventional cytogenetic or fluorescence in situ hybridization (FISH). CNA were identified in nearly 80% of cases by aCGH that delineated in addition minimal altered regions. At gene level, remarkable findings affecting genes involved in the regulation of the NF-kB signaling pathways were identified, such as biallelic inactivation of TNFAIP3 and TRAF3. SNPa also allowed characterization of copy neutral losses such as uniparental disomies (UPD), which is an important and frequent mechanism of gene alteration in cancer cells. Herein, we summarize the current knowledge of WM genomic basis using these high-throughput techniques.