Noncoding variants alter GATA2 expression in rhombomere 4 motor neurons and cause dominant hereditary congenital facial paresis.

Hereditary congenital facial paresis type 1 (HCFP1) is an autosomal dominant disorder of absent or limited facial movement that maps to chromosome 3q21-q22 and is hypothesized to result from facial branchial motor neuron (FBMN) maldevelopment. In the present study, we report that HCFP1 results from heterozygous duplications within a neuron-specific GATA2 regulatory region that includes two enhancers and one silencer, and from noncoding single-nucleotide variants (SNVs) within the silencer. Some SNVs impair binding of NR2F1 to the silencer in vitro and in vivo and attenuate in vivo enhancer reporter expression in FBMNs. Gata2 and its effector Gata3 are essential for inner-ear efferent neuron (IEE) but not FBMN development. A humanized HCFP1 mouse model extends Gata2 expression, favors the formation of IEEs over FBMNs and is rescued by conditional loss of Gata3. These findings highlight the importance of temporal gene regulation in development and of noncoding variation in rare mendelian disease.

Chromosome 10q-linked FSHD identifies DUX4 as principal disease gene.

BACKGROUND: Facioscapulohumeral dystrophy (FSHD) is an inherited muscular dystrophy clinically characterised by muscle weakness starting with the facial and upper extremity muscles. A disease model has been developed that postulates that failure in somatic repression of the transcription factor DUX4 embedded in the D4Z4 repeat on chromosome 4q causes FSHD. However, due to the position of the D4Z4 repeat close to the telomere and the complex genetic and epigenetic aetiology of FSHD, there is ongoing debate about the transcriptional deregulation of closely linked genes and their involvement in FSHD. METHOD: Detailed genetic characterisation and gene expression analysis of patients with clinically confirmed FSHD and control individuals. RESULTS: Identification of two FSHD families in which the disease is caused by repeat contraction and DUX4 expression from chromosome 10 due to a de novo D4Z4 repeat exchange between chromosomes 4 and 10. We show that the genetic lesion causal to FSHD in these families is physically separated from other candidate genes on chromosome 4. We demonstrate that muscle cell cultures from affected family members exhibit the characteristic molecular features of FSHD, including DUX4 and DUX4 target gene expression, without showing evidence for transcriptional deregulation of other chromosome 4-specific candidate genes. CONCLUSION: This study shows that in rare situations, FSHD can occur on chromosome 10 due to an interchromosomal rearrangement with the FSHD locus on chromosome 4q. These findings provide further evidence that DUX4 derepression is the dominant disease pathway for FSHD. Hence, therapeutic strategies should focus on DUX4 as the primary target.

"Be an ambassador for change that you would like to see": a call to action to all stakeholders for co-creation in healthcare and medical research to improve quality of life of people with a neuromuscular disease.

BACKGROUND: Patient and public involvement for co-creation is increasingly recognized as a valuable strategy to develop healthcare research targeting patients' real needs. However, its practical implementation is not as advanced and unanimously accepted as it could be, due to cultural differences and complexities of managing healthcare programs and clinical studies, especially in the rare disease field. MAIN BODY: The European Neuromuscular Centre, a European foundation of patient organizations, involved its key stakeholders in a special workshop to investigate the position of the neuromuscular patient community with respect to healthcare and medical research to identify and address gaps and bottlenecks. The workshop took place in Milan (Italy) on January 19-20, 2018, involving 45 participants who were mainly representatives of the patient community, but also included experts from clinical centers, industry and regulatory bodies. In order to provide practical examples and constructive suggestions, specific topics were identified upfront. The first set of issues concerned the quality of life at specific phases of a patient's life, such as at the time of diagnosis or during pediatric to adult transition, and patient involvement in medical research on activities in daily living including patient reported outcome measures. The second set of issues concerned the involvement of patients in the management of clinical research tools, such as registries and biobanks, and their participation in study design or marketing authorization processes. Introductory presentations were followed by parallel working group sessions, to gain constructive contributions from all participants. The concept of shared decision making was used to ensure, in discussions, a partnership-based identification of the wishes and needs of all stakeholders involved, and the "ladder of participation" tool served as a model to evaluate the actual and the desired level of patients' involvement in all topics addressed. A general consensus on the outcome of the meeting was collected during the final plenary session. This paper reports the outcome of the workshop and the specific suggestions derived from the analysis of the first set of topics, related to quality of life. The outcomes of the second set of topics are reported elsewhere and are only briefly summarized herein for the sake of completeness. CONCLUSIONS: The neuromuscular community proved to be very active and engaged at different levels in the healthcare initiatives of interest. The workshop participants critically discussed several topics, providing practical examples where different stakeholders could play a role in making a change and bridging gaps. Overall, they indicated the need for education of all stakeholders for better communication, where everyone should become an ambassador to promote real change. Support should also come from institutions and healthcare bodies both at structural and economic level.

"The impact of European Neuromuscular Centre (ENMC) workshops on the neuromuscular field; 25 years on ".

Since 1992, the European Neuromuscular Centre facilitated workshops to bring experts in the field of neuromuscular disorders together. After organising more than 235 workshops, it is time to evaluate what impact these 25 years of ENMC workshops have had on the neuromuscular research field and on people affected by a neuromuscular condition. To measure this, workshop topics were retrospectively evaluated and bibliometric analyses on the citation scores of ENMC-derived publications were performed. In addition, a personalized survey was used to investigate the actual achievement and implementation of workshop deliverables. The evaluation of 25 years' workshop topics revealed a strong representation of muscular dystrophies, congenital and mitochondrial myopathies. The publications derived from ENMC workshops scored "high impact" as illustrated by the Mean Normalized Citation Score of 1.24. Also 16% of the ENMC papers belong to the top 10% best cited articles in the neuromuscular field. The main outcome of the personalised survey was that 90% of all workshop deliverables were started and either ongoing or completed. Of these deliverables, 78% were implemented in the field; bringing state-of-the-art knowledge and new collaborations to researchers and clinicians, improving designs of clinical trials and innovating tools to make accurate diagnoses.

The Position of Neuromuscular Patients in Shared Decision Making. Report from the 235th ENMC Workshop: Milan, Italy, January 19-20, 2018.

In the era of patient-centered medicine, shared decision-making (SDM) - in which healthcare professionals and patients exchange information and preferences and jointly reach a decision - has emerged as the gold standard model for the provision of formal healthcare. Indeed, in many geographical settings, patients are frequently invited to participate in choices concerning the design and delivery of their medical management. From a clinical perspective, benefits of this type of patient involvement encompass, for example, enhanced treatment satisfaction, improved medical compliance, better health outcomes, and maintained or promoted quality of life. Yet, although the theory and enactment of SDM in healthcare are well-described in the literature [1-3], comparatively less attention has been devoted to contextualizing questions relating to if, when, and how to include patients in decisions within medical research. In this context, patient involvement would be expected to be potentially relevant for and applicable to a wide range of activities and processes, from the identification of research priorities and development of grant applications, to the design of patient information and consent procedures, formulation of interventions, identification and recruitment of study sample populations, feasibility of a clinical trial, identification, selection, and specification of endpoints and outcomes in clinical trials and observational studies, data collection and analysis, and dissemination of results. To this end, 45 clinicians, healthcare professionals, researchers, patients, caregivers, and representatives from regulatory authorities and pharmaceutical companies from 15 different countries met to discuss the level of involvement of patients with neuromuscular diseases, specifically in the following settings of medical research for neuromuscular diseases: i) registries and biobanks; ii) clinical trials; and iii) regulatory processes. In this report, we present summaries of the talks that were given during the workshop, as well as discussion outcomes from the three topic areas listed above.

Ophthalmological findings in facioscapulohumeral dystrophy.

Ophthalmological abnormalities in facioscapulohumeral dystrophy may lead to treatable vision loss, facilitate diagnostics, could help unravelling the pathophysiology and serve as biomarkers. In this study, we provide a detailed description of the ophthalmological findings in a well-defined cohort of patients with facioscapulohumeral dystrophy using state of the art retina imaging techniques. Thirty-three genetically confirmed patients (aged 7-80 years) and 24 unrelated healthy controls (aged 6-68 years) underwent clinical ophthalmological examination, fundus photography, optical coherence tomography/angiography, genotyping and neurological examination. All patients had normal corrected visual acuity and normal intraocular pressure. In 27 of the 33 patients, weakness of the orbicularis oculi was observed. Central retinal pathology, only seen in patients and not in healthy controls, included twisting (tortuosity) of the retinal arteries in 25 of the 33 patients and retinal pigment epithelium defects in 4 of the 33 patients. Asymmetrical foveal hypoplasia was present in three patients, and exudative abnormalities were observed in one patient. There was a correlation between the severity of retinal tortuosity and the D4Z4 repeat array size (R 2 = 0.44, P < 0.005). Follow-up examination in a subgroup of six patients did not show any changes after 2 years. To conclude, retinal abnormalities were frequent but almost always subclinical in patients with facioscapulohumeral dystrophy and consisted primarily of arterial tortuosity and foveal abnormalities. Retinal tortuosity was seen in the retinal arterioles and correlated with the D4Z4 repeat array size, thereby providing clinical evidence for an underlying genetic linkage between the retina and facioscapulohumeral dystrophy.

Variants affecting diverse domains of MEPE are associated with two distinct bone disorders, a craniofacial bone defect and otosclerosis.

PURPOSE: To characterize new molecular factors implicated in a hereditary congenital facial paresis (HCFP) family and otosclerosis. METHODS: We performed exome sequencing in a four-generation family presenting nonprogressive HCFP and mixed hearing loss (HL). MEPE was analyzed using either Sanger sequencing or molecular inversion probes combined with massive parallel sequencing in 89 otosclerosis families, 1604 unrelated affected subjects, and 1538 unscreened controls. RESULTS: Exome sequencing in the HCFP family led to the identification of a rare segregating heterozygous frameshift variant p.(Gln425Lysfs*38) in MEPE. As the HL phenotype in this family resembled otosclerosis, we performed variant burden and variance components analyses in a large otosclerosis cohort and demonstrated that nonsense and frameshift MEPE variants were significantly enriched in affected subjects (p = 0.0006-0.0060). CONCLUSION: MEPE exerts its function in bone homeostasis by two domains, an RGD and an acidic serine aspartate-rich MEPE-associated (ASARM) motif inhibiting respectively bone resorption and mineralization. All variants associated with otosclerosis are predicted to result in nonsense mediated decay or an ASARM-and-RGD-truncated MEPE. The HCFP variant is predicted to produce an ASARM-truncated MEPE with an intact RGD motif. This difference in effect on the protein corresponds with the presumed pathophysiology of both diseases, and provides a plausible molecular explanation for the distinct phenotypic outcome.

Early onset as a marker for disease severity in facioscapulohumeral muscular dystrophy.

OBJECTIVE: To assess the relation between age at onset and disease severity in facioscapulohumeral muscular dystrophy (FSHD). METHODS: In this prospective cross-sectional study, we matched adult patients with FSHD with an early disease onset with 2 sex-matched FSHD control groups with a classic onset; the first group was age matched, and the second group was disease duration matched. Genetic characteristics, muscle performance, respiratory functioning, hearing loss, vision loss, epilepsy, educational level, and work status were compared with the 2 control groups. RESULTS: Twenty-eight patients with early-onset FSHD were age (n = 28) or duration (n = 27) matched with classic-onset patients. Patients with early-onset FSHD had more severe muscle weakness (mean FSHD clinical score 11 vs 5 in the age-matched and 9 in the duration-matched group, p < 0.05) and a higher frequency of wheelchair dependency (57%, 0%, and 30%, respectively, p < 0.05). In addition, systemic features were more frequent in early-onset FSHD, most important, hearing loss, decreased respiratory function and spinal deformities. There was no difference in work status. Genetically, the shortest D4Z4 repeat arrays (2-3 units) were found exclusively in the early-onset group, and the largest repeat arrays (8-9 units) were found only in the classic-onset groups. De novo mutations were more frequent in early-onset patients (46% vs 4%). CONCLUSIONS: Patients with early-onset FSHD more often have severe muscle weakness and systemic features. The disease severity is greater than in patients with classic-onset FSHD who are matched for disease duration, suggesting that the progression is faster in early-onset patients.

Experiences with bariatric surgery in patients with facioscapulohumeral dystrophy and myotonic dystrophy type 1: A qualitative study.

Overweight and obesity are common in patients with facioscapulohumeral dystrophy (FSHD) and myotonic dystrophy type 1 (DM1). Lifestyle change is often challenging for patients with neuromuscular diseases, especially to increase physical activity. When lifestyle changes have not been effective, bariatric surgery is a treatment option. However, very little is known about the benefits and risks in patients with neuromuscular disorders. This study therefore aims to obtain insight into the patients' perspectives and experiences, the outcome, effects and risks of bariatric surgery in these disorders. We performed a qualitative study, consisting of 14 in-depth interviews with six patients (three FSHD and three DM1; five women, one man; aged range 31-47 years), four relatives, three bariatric surgeons and one general practitioner. The study used a qualitative descriptive method. Four themes were formulated: (1) overweight as burden; (2) bariatric surgery as last option; (3) not your standard patient; and (4) a different life, a different me. This study shows that bariatric surgery has beneficial physical and mental effects for most patients with FSHD and DM1, and does not influence the muscular disease course. Bariatric surgery is feasible in patients with FSHD and DM1, but specific precautions and a suitable follow-up including tailored dietary and training advices are required.

Phenotype-genotype relations in facioscapulohumeral muscular dystrophy type 1.

To determine how much of the clinical variability in facioscapulohumeral muscular dystrophy type 1 (FSHD1) can be explained by the D4Z4 repeat array size, D4Z4 methylation and familial factors, we included 152 carriers of an FSHD1 allele (23 single cases, 129 familial cases from 37 families) and performed state-of-the-art genetic testing, extensive clinical evaluation and quantitative muscle MRI. Familial factors accounted for 50% of the variance in disease severity (FSHD clinical score). The explained variance by the D4Z4 repeat array size for disease severity was limited (approximately 10%), and varied per body region (facial muscles, upper and lower extremities approximately 30%, 15% and 3%, respectively). Unaffected gene carriers had longer repeat array sizes compared to symptomatic individuals (7.3 vs 6.0 units, P = 0.000) and slightly higher Delta1 methylation levels (D4Z4 methylation corrected for repeat size, 0.96 vs -2.46, P = 0.048). The D4Z4 repeat array size and D4Z4 methylation contribute to variability in disease severity and penetrance, but other disease modifying factors must be involved as well. The larger effect of the D4Z4 repeat array on facial muscle involvement suggests that these muscles are more sensitive to the influence of the FSHD1 locus itself, whereas leg muscle involvement seems highly dependent on modifying factors.

A family-based study into penetrance in facioscapulohumeral muscular dystrophy type 1.

OBJECTIVE: An observational cross-sectional study was conducted in a national facioscapulohumeral muscular dystrophy (FSHD) expertise center to estimate the penetrance of FSHD1 and to evaluate phenotype-genotype correlations. METHODS: Ten FSHD1 probands carrying 4-9 D4Z4 unit alleles and 140 relatives were examined. All 150 participants were genetically characterized, including D4Z4 methylation levels in the mutation carriers. Mutation carriers were classified as (1) symptomatic: with symptoms of muscle weakness on history and muscle FSHD signs on examination; (2) asymptomatic: without symptoms of muscle weakness but with muscle FSHD signs on examination; and (3) nonpenetrant: without symptoms of muscle weakness on history and without muscle FSHD signs on examination. We assessed the relationship between age-corrected clinical severity score and repeat size, sex, and D4Z4 methylation levels. RESULTS: The maximum likelihood estimates of symptomatic and those of symptomatic plus asymptomatic FSHD showed that penetrance depends on repeat size and increases until late adulthood. We observed many asymptomatic carriers with subtle facial weakness with or without mild shoulder girdle weakness (25% [17/69]). Nonpenetrance was observed less frequently than in recent population studies (17% [12/69]), and most asymptomatic patients reported some shoulder pain. D4Z4 methylation tended to be lower in moderately to severely affected mutation carriers with 7 or 9 repeats. DISCUSSION: This family-based study detected a lower overall nonpenetrance than previously observed, probably due to many asymptomatic mutation carriers identified by careful examination of facial and shoulder muscles. The recognition of asymptomatic mutation carriers is essential for selection of participants for future trials, and the likelihood estimates are helpful in counseling.

A 22-year follow-up reveals a variable disease severity in early-onset facioscapulohumeral dystrophy.

AIM: To assess the long-term natural course of early-onset facioscapulohumeral dystrophy (FSHD), which is important for patient management and trial-readiness, and is currently lacking. METHODS: We had the unique opportunity to evaluate 10 patients with early-onset FSHD after 22 years follow-up. Patients underwent a semi-structured interview, physical examination and additional genotyping. RESULTS: Nine initial study participants (median age 37 years) were included, one patient died shortly after first publication. At first examination, one patient was wheelchair dependent, one patient walked aided, and eight patients walked unaided. After 22 years, four patients were wheelchair dependent, three walked aided, and two walked unaided. Systemic features, including hearing loss (56%), intellectual disability (44%), and a decreased respiratory function (56%), were frequent. Patients participated socially and economically with most patients living in a regular house (n = 6) and/or having a paid job (n = 4). DISCUSSION: Patients with early-onset FSHD generally had a severe phenotype compared to classical onset FSHD. However, after 22 years of follow up they showed a wide variation in severity and, despite these physical limitations, participated socially and economically. These observations are important for patient management and should be taken into account in clinical trials.

Early onset facioscapulohumeral dystrophy - a systematic review using individual patient data.

Infantile or early onset is estimated to occur in around 10% of all facioscapulohumeral dystrophy (FSHD) patients. Although small series of early onset FSHD patients have been reported, comprehensive data on the clinical phenotype is missing. We performed a systematic literature search on the clinical features of early onset FSHD comprising a total of 43 articles with individual data on 227 patients. Additional data from four cohorts was provided by the authors. Mean age at reporting was 18.8 years, and 40% of patients were wheelchair-dependent at that age. Half of the patients had systemic features, including hearing loss (40%), retinal abnormalities (37%) and developmental delay (8%). We found an inverse correlation between repeat size and disease severity, similar to adult-onset FSHD. De novo FSHD1 mutations were more prevalent than in adult-onset FSHD. Compared to adult FSHD, our findings indicate that early onset FSHD is overall characterized by a more severe muscle phenotype and a higher prevalence of systemic features. However, similar as in adults, a significant clinical heterogeneity was observed. Based on this, we consider early onset FSHD to be on the severe end of the FSHD disease spectrum. We found natural history studies and treatment studies to be very scarce in early onset FSHD, therefore longitudinal studies are needed to improve prognostication, clinical management and trial-readiness.

Adding quantitative muscle MRI to the FSHD clinical trial toolbox.

OBJECTIVE: To add quantitative muscle MRI to the clinical trial toolbox for facioscapulohumeral muscular dystrophy (FSHD) by correlating it to clinical outcome measures in a large cohort of genetically and clinically well-characterized patients with FSHD comprising the entire clinical spectrum. METHODS: Quantitative MRI scans of leg muscles of 140 patients with FSHD1 and FSHD2 were assessed for fatty infiltration and TIRM hyperintensities and were correlated to multiple clinical outcome measures. RESULTS: The mean fat fraction of the total leg musculature correlated highly with the motor function measure, FSHD clinical score, Ricci score, and 6-minute walking test (correlation coefficients -0.845, 0.835, 0.791, -0.701, respectively). Fat fraction per muscle group correlated well with corresponding muscle strength (correlation coefficients up to -0.82). The hamstring muscles, adductor muscles, rectus femoris, and gastrocnemius medialis were affected most frequently, also in early stage disease and in patients without leg muscle weakness. Muscle involvement was asymmetric in 20% of all muscle pairs and fatty infiltration within muscles showed a decrease from distal to proximal of 3.9%. TIRM hyperintense areas, suggesting inflammation, were found in 3.5% of all muscles, with and without fatty infiltration. CONCLUSIONS: We show a strong correlation between quantitative muscle MRI and clinical outcome measures. Muscle MRI is able to detect muscle pathology before clinical involvement of the leg muscles. This indicates that quantitative leg muscle MRI is a promising biomarker that captures disease severity and motor functioning and can thus be included in the FSHD trial toolbox.

What's in a name? The clinical features of facioscapulohumeral muscular dystrophy.

Facioscapulohumeral muscular dystrophy (FSHD) is an inherited and progressive muscle disorder. Although its name suggests otherwise, it comprises weakness of the facial, shoulder and upper arm muscles, and also of the trunk and leg muscles. Its severity and disease course vary greatly and mild or early FSHD can be difficult to recognise. Knowledge of its subtle signs and symptoms can lead directly to the correct diagnosis without diagnostic delay and without needing multiple diagnostic procedures. We give an overview of the signs and symptoms of FSHD in severe as well as in mild cases, to facilitate correct and instant recognition of this relatively common muscle disorder.

De novo mutations in PLXND1 and REV3L cause Möbius syndrome.

Möbius syndrome (MBS) is a neurological disorder that is characterized by paralysis of the facial nerves and variable other congenital anomalies. The aetiology of this syndrome has been enigmatic since the initial descriptions by von Graefe in 1880 and by Möbius in 1888, and it has been debated for decades whether MBS has a genetic or a non-genetic aetiology. Here, we report de novo mutations affecting two genes, PLXND1 and REV3L in MBS patients. PLXND1 and REV3L represent totally unrelated pathways involved in hindbrain development: neural migration and DNA translesion synthesis, essential for the replication of endogenously damaged DNA, respectively. Interestingly, analysis of Plxnd1 and Rev3l mutant mice shows that disruption of these separate pathways converge at the facial branchiomotor nucleus, affecting either motoneuron migration or proliferation. The finding that PLXND1 and REV3L mutations are responsible for a proportion of MBS patients suggests that de novo mutations in other genes might account for other MBS patients.

Inter-individual differences in CpG methylation at D4Z4 correlate with clinical variability in FSHD1 and FSHD2.

Facioscapulohumeral muscular dystrophy (FSHD: MIM#158900) is a common myopathy with marked but largely unexplained clinical inter- and intra-familial variability. It is caused by contractions of the D4Z4 repeat array on chromosome 4 to 1-10 units (FSHD1), or by mutations in the D4Z4-binding chromatin modifier SMCHD1 (FSHD2). Both situations lead to a partial opening of the D4Z4 chromatin structure and transcription of D4Z4-encoded polyadenylated DUX4 mRNA in muscle. We measured D4Z4 CpG methylation in control, FSHD1 and FSHD2 individuals and found a significant correlation with the D4Z4 repeat array size. After correction for repeat array size, we show that the variability in clinical severity in FSHD1 and FSHD2 individuals is dependent on individual differences in susceptibility to D4Z4 hypomethylation. In FSHD1, for individuals with D4Z4 repeat arrays of 1-6 units, the clinical severity mainly depends on the size of the D4Z4 repeat. However, in individuals with arrays of 7-10 units, the clinical severity also depends on other factors that regulate D4Z4 methylation because affected individuals, but not non-penetrant mutation carriers, have a greater reduction of D4Z4 CpG methylation than can be expected based on the size of the pathogenic D4Z4 repeat array. In FSHD2, this epigenetic susceptibility depends on the nature of the SMCHD1 mutation in combination with D4Z4 repeat array size with dominant negative mutations being more deleterious than haploinsufficiency mutations. Our study thus identifies an epigenetic basis for the striking variability in onset and disease progression that is considered a clinical hallmark of FSHD.