Comprehensive genetic analysis of facioscapulohumeral muscular dystrophy by Nanopore long-read whole-genome sequencing.

BACKGROUND: Facioscapulohumeral muscular dystrophy (FSHD) is a high-prevalence autosomal dominant neuromuscular disease characterized by significant clinical and genetic heterogeneity. Genetic diagnosis of FSHD remains a challenge because it cannot be detected by standard sequencing methods and requires a complex diagnosis workflow. METHODS: We developed a comprehensive genetic FSHD detection method based on Oxford Nanopore Technologies (ONT) whole-genome sequencing. Using a case-control design, we applied this procedure to 29 samples and compared the results with those from optical genome mapping (OGM), bisulfite sequencing (BSS), and whole-exome sequencing (WES). RESULTS: Using our ONT-based method, we identified 59 haplotypes (35 4qA and 24 4qB) among the 29 samples (including a mosaic sample), as well as the number of D4Z4 repeat units (RUs). The pathogenetic D4Z4 RU contraction identified by our ONT-based method showed 100% concordance with OGM results. The methylation levels of the most distal D4Z4 RU and the double homeobox 4 gene (DUX4) detected by ONT sequencing are highly consistent with the BSS results and showed excellent diagnostic efficiency. Additionally, our ONT-based method provided an independent methylation profile analysis of two permissive 4qA alleles, reflecting a more accurate scenario than traditional BSS. The ONT-based method detected 17 variations in three FSHD2-related genes from nine samples, showing 100% concordance with WES. CONCLUSIONS: Our ONT-based FSHD detection method is a comprehensive method for identifying pathogenetic D4Z4 RU contractions, methylation level alterations, allele-specific methylation of two 4qA haplotypes, and variations in FSHD2-related genes, which will all greatly improve genetic testing for FSHD.

Clinical Application of Optical Genome Mapping for Molecular Diagnosis of Facioscapulohumeral Muscular Dystrophy.

Background: Facioscapulohumeral muscular dystrophy (FSHD) is a common form of muscular dystrophy that mainly affects skeletal muscle. FSHD1 accounts for 95% of all FSHD cases and can be diagnosed based on the pathogenic contraction of the D4Z4-repeat array on chromosome 4q35. Genetic diagnosis of FSHD1 is challenging because of the large size and repetitive nature of the D4Z4 region. We evaluated the clinical applicability of optical genome mapping (OGM) for the genetic diagnosis of FSHD1. Methods: We included 25 individuals with clinically confirmed or suspected/probable FSHD and their families. Ultra-high-molecular-weight DNA from peripheral blood was labeled, stained, and imaged using a single-molecule OGM platform (Bionano Genomics Saphyr system). D4Z4 repeat size and haplotype information were analyzed using the manufacturer's dedicated pipeline. We also compared the workflow and test time between Southern blot analysis and OGM. Results: We obtained concordant OGM and Southern blot results with 10 samples from patients with clinically confirmed FSHD. The D4Z4 repeat size differed within 1 unit between the Southern blot analysis and OGM. Among nine patients with clinically suspected or probable FSHD, six patients were confirmed to have pathogenic contractions by OGM. In our cohort, one de novo mosaic FSHD1 patient was successfully diagnosed with OGM. Moreover, OGM has a more straightforward and less time-consuming workflow than Southern blot analysis. Conclusions: OGM enables accurate and reliable detection of pathogenic contraction of the D4Z4-repeat array and is a valuable tool for the genetic diagnosis of FSHD1.

Best practice guidelines on genetic diagnostics of facioscapulohumeral muscular dystrophy: Update of the 2012 guidelines.

The gold standard for facioscapulohumeral muscular dystrophy (FSHD) genetic diagnostic procedures was published in 2012. With the increasing complexity of the genetics of FSHD1 and 2, the increase of genetic testing centers, and the start of clinical trials for FSHD, it is crucial to provide an update on our knowledge of the genetic features of the FSHD loci and renew the international consensus on the molecular testing recommendations. To this end, members of the FSHD European Trial Network summarized the evidence presented during the 2022 ENMC meeting on Genetic diagnosis, clinical outcome measures, and biomarkers. The working group additionally invited genetic and clinical experts from the USA, India, Japan, Australia, South-Africa, and Brazil to provide a global perspective. Six virtual meetings were organized to reach consensus on the minimal requirements for genetic confirmation of FSHD1 and FSHD2. Here, we present the clinical and genetic features of FSHD, specific features of FSHD1 and FSHD2, pros and cons of established and new technologies (Southern blot in combination with either linear or pulsed-field gel electrophoresis, molecular combing, optical genome mapping, FSHD2 methylation analysis and FSHD2 genotyping), the possibilities and challenges of prenatal testing, including pre-implantation genetic testing, and the minimal requirements and recommendations for genetic confirmation of FSHD1 and FSHD2. This consensus is expected to contribute to current clinical management and trial-readiness for FSHD.

First-trimester noninvasive prenatal diagnosis of seven facioscapulohumeral muscular dystrophy type 1 families using SNP-based amplicon sequencing: An earlier, rapid and safer way.

The study is to explore the feasibility and value of SNP-based noninvasive prenatal diagnosis (NIPD) for facioscapulohumeral muscular dystrophy type 1 (FSHD1) in early pregnancy weeks. We prospectively collected seven FSHD1 families, with an average gestational age of 8+6. Among these seven couples, there were three affected FSHD1 mothers and four affected fathers. A multiplex-PCR panel comprising 402 amplicons was designed to selective enrich for highly heterozygous SNPs upstream of the DUX4 gene. Risk haplotype was constructed based on familial linkage analysis. Fetal genotypes were accurately inferred through relative haplotype dosage analysis using Bayes Factor. All tests were successfully completed in a single attempt, and no recombination events were detected. NIPD results were provided within a week, which is 4 weeks earlier than karyomapping and 7 weeks earlier than Bionano single-molecule optical mapping (BOM). Ultimately, five FSHD1 fetuses and two normal fetuses were successfully identified, with a 100% concordance rate with karyomapping and BOM. Therefore, SNP-based NIPD for FSHD1 was demonstrated to be feasible and accurate in early weeks of gestation, although the risk of recombination events cannot be completely eliminated. In the future, testing of more cases is still necessary to fully determine the clinical utility.

A longitudinal study of disease progression in facioscapulohumeral muscular dystrophy (FSHD).

INTRODUCTION/AIMS: In preparation for clinical trials, it is important to better understand how disease burden changes over time in facioscapulohumeral muscular dystrophy (FSHD) and to assess the capability of select metrics to detect these changes. This study aims to evaluate FSHD disease progression over 1 year and to examine the sensitivity of several outcome measures in detecting changes during this interval. METHODS: We conducted a 12-month prospective observational study of 41 participants with FSHD. Participants were evaluated at baseline, 6 months, and 12 months with serial strength testing (manual muscle testing or MMT and maximum voluntary isometric contraction testing or MVICT), functional testing (FSHD-Composite Outcome Measure or FSHD-COM, FSHD Clinical Severity Score or CSS, and FSHD Evaluation Score or FES), sleep and fatigue assessments, lean body mass measurements, respiratory testing, and the FSHD-Health Index patient-reported outcome. Changes in these outcome measures were assessed over the 12-month period. Associations between changes in outcome measures and both age and sex were also examined. RESULTS: In a 12-month period, FSHD participant function remained largely stable with a mild worsening of strength, measured by MMT and standardized MVICT scores, and a mild loss in lean body mass. DISCUSSION: The abilities and disease burden of adults with FSHD are largely static over a 12-month period with participants demonstrating a mild average reduction in some measures of strength. Selection of patients, outcome measures, and trial duration should be carefully considered during the design and implementation of future clinical studies involving FSHD patients.

Living with facioscapulohumeral muscular dystrophy during the first two COVID-19 outbreaks: a repeated patient survey in the Netherlands.

BACKGROUND: Patients with facioscapulohumeral dystrophy (FSHD) suffer from slowly progressive muscle weakness. Approximately 20% of FSHD patients end up wheelchair-dependent. FSHD patients benefit from physical activity to maintain their muscle strength as much as possible. The impact of the COVID-19 pandemic on the health of FSHD patients was unknown. OBJECTIVE: This study assessed changes in daily care received, perceived psychosocial stress, and worsening of FSHD complaints in 2020. Furthermore, we compared COVID-19 infection incidence and severity of symptoms between FSHD patients and non-FSHD housemates. METHODS: Three online survey rounds were sent out to all adult participants of the Dutch FSHD registry regarding daily care received, perceived psychosocial stress, COVID-19 infection rate, and COVID-19 symptoms severity. They also included COVID-19-related questions regarding the participants' housemates, which served as control group. RESULTS: Participation rate was 210 (61%), 186 (54%), and 205 (59%) for survey 1, 2, and 3, respectively. Care reduction was reported by 42.7%, 40%, and 28.8% of the participants in the respective surveys. Perceived psychosocial stress increased in 44%, 30%, and 40% of the participants. Compared to the 197 non-FSHD housemates, the 213 FSHD patients reported more possibly COVID-19-related symptoms (27% vs. 39%, p = 0.017) of mostly minimal severity (63%). No difference in (possible) COVID-19 infection incidence rates was found (2.0% vs. 2.8%, p = 0.527). CONCLUSIONS: The COVID-19 pandemic negatively impacted care received and increased perceived psychosocial stress in FSHD patients. However, COVID-19 infection incidence in FSHD patients was similar to their non-FSHD housemates.

Physical activity practiced at a young age is associated with a less severe subsequent clinical presentation in facioscapulohumeral muscular dystrophy.

BACKGROUND: In facioscapulohumeral muscular dystrophy (FSHD), it is not known whether physical activity (PA) practiced at young age is associated with the clinical presentation of disease. To assess this issue, we performed a retrospective cohort study concerning the previous practice of sports and, among them, those with medium-high cardiovascular commitment in clinically categorized carriers of a D4Z4 reduced allele (DRA). METHODS: People aged between 18 and 60 were recruited as being DRA carriers. Subcategory (classical phenotype, A; incomplete phenotype, B; asymptomatic carriers, C; complex phenotype, D) and FSHD score, which measures muscle functional impairment, were assessed for all participants. Information on PAs was retrieved by using an online survey dealing with the practice of sports at a young age. RESULTS: 368 participants were included in the study, average age 36.6 years (SD = 9.4), 47.6% male. The FSHD subcategory A was observed in 157 (42.7%) participants with average (± SD) FSHD score of 5.8 ± 3.0; the incomplete phenotype (category B) in 46 (12.5%) participants (average score 2.2 ± 1.7) and the D phenotype in 61 (16.6%, average score 6.5 ± 3.8). Asymptomatic carriers were 104 (subcategory C, 28.3%, score 0.0 ± 0.2). Time from symptoms onset was higher for patients with A (15.8 ± 11.1 years) and D phenotype (13.3 ± 11.9) than for patients with B phenotype (7.3 ± 9.0). The practice of sports was associated with lower FSHD score (-17%) in participants with A phenotype (MR = 0.83, 95% CI = 0.73-0.95, p = 0.007) and by 33% in participants with D phenotype (MR = 0.67, 95% CI = 0.51-0.89, p = 0.006). Conversely, no improvement was observed in participants with incomplete phenotype with mild severity (B). CONCLUSIONS: PAs at a young age are associated with a lower clinical score in the adult A and D FSHD subcategories. These results corroborate the need to consider PAs at the young age as a fundamental indicator for the correct clinical stratification of the disease and its possible evolution.

Face to Face: deciphering facial involvement in inclusion body myositis.

OBJECTIVE: The objective of this study is to evaluate the frequency and characteristics of facial involvement in inclusion body myositis (IBM) patients and to compare it to the one previously described in facioscapulohumeral dystrophy (FSHD) patients. METHODS: Thirty-two IBM patients were included and compared to 29 controls and 39 FSHD patients. All participants were recorded in a video as they performed a series of seven facial tasks. Five raters independently assessed facial weakness using both a qualitative evaluation and a semi-quantitative facial weakness score (FWS). RESULTS: IBM patients had higher FWS than controls (7.89 ± 7.56 vs 1.06 ± 0.88, p < 0.001). Twenty IBM patients (63%) had a facial weakness with a FWS above the maximum value for controls. All facial tasks were significantly more impaired in IBM patients compared to controls (p < 0.001), task 2 evaluating orbiculari oculi muscle weakness being the most affected. IBM patients with facial weakness reported more swallowing troubles than IBM patients without facial weakness (p = 0.03). FSHD patients displayed higher FWS than IBM patients (12.16 ± 8.37 vs 7.89 ± 7.56, p = 0.01) with more pronounced facial asymmetry (p = 0.01). FWS inter-rater ICC was 0.775. CONCLUSION: This study enabled us to estimate the frequency of facial impairment in IBM in more than half of patients, to detail its characteristics and to compare them with those of FSHD patients. The standardized, semi-quantitative FWS is an interesting diagnostic help in IBM as it appeared more sensitive than qualitative evaluation to detect mild facial weakness.

Characterization of D4Z4 alleles and assessment of de novo cases in Facioscapulohumeral dystrophy (FSHD) in a cohort of Italian families.

Facioscapulohumeral dystrophy (FSHD) is an autosomal dominant disease, although 10%-30% of cases are sporadic. However, this percentage may include truly de novo patients (carrying a reduced D4Z4 allele that is not present in either of the parents) and patients with apparently sporadic disease resulting from mosaicism, non-penetrance, or complex genetic situations in either patients or parents. In this study, we characterized the D4Z4 Reduced Alleles (DRA) and evaluated the frequency of truly de novo cases in FSHD1 in a cohort of DNA samples received consecutively for FSHD-diagnostic from 100 Italian families. The D4Z4 testing revealed that 60 families reported a DRA compatible with FSHD1 (1-10 RU). The DRA co-segregated with the disease in most cases. Five families with truly de novo cases were identified, suggesting that this condition may be slightly lower (8%) than previously reported. In addition, D4Z4 characterization in the investigated families showed 4% of mosaic cases and 2% with translocations. This study further highlighted the importance of performing family studies for clarifying apparently sporadic FSHD cases, with significant implications for genetic counseling, diagnosis, clinical management, and procreative choices for patients and families.

Gastrointestinal and genitourinary symptoms in facioscapulohumeral muscular dystrophy: Prevalence and impact.

INTRODUCTION/AIMS: Anecdotally, patients with facioscapulohumeral muscular dystrophy (FSHD) describe gastrointestinal (GI) and genitourinary (GU) symptoms. We explored the prevalence of GI and GU symptoms and their impact on quality of life (QOL) in people with FSHD compared to healthy household controls. METHODS: In this descriptive, cross-sectional study, we emailed a survey exploring GI and GU symptoms to all FSHD Society patient contacts (n = 3507). We invited those with FSHD and unaffected household controls to respond. Non-parametric statistics were used to compare symptom frequency and impact of symptoms between respondents with FSHD and household controls. Within the FSHD group, symptom frequency was assessed relative to measures of disease progression (need for ambulatory or respiratory support). RESULTS: Surveys from 701 respondents (652 with FSHD) ≥18 years old were included in analysis. Those with FSHD had symptoms affecting both GI and GU systems more frequently than controls using ordinal rating of symptom frequency. Within the FSHD group, more advanced disease was associated with increased symptom frequency. QOL was negatively impacted by the GI and GU symptoms. There was no difference between groups in use of medications to treat these symptoms. DISCUSSION: Recognition and treatment of GI and GU symptoms in people with FSHD, particularly those with more advanced disease, could improve QOL. Additional investigation is required to confirm these findings and understand the physiology.

Evaluation of Optical Genome Mapping in Clinical Genetic Testing of Facioscapulohumeral Muscular Dystrophy.

Facioscapulohumeral muscular dystrophy (FSHD) is the third most common hereditary muscular dystrophy, caused by the contraction of the D4Z4 repeats on the permissive 4qA haplotype on chromosome 4, resulting in the faulty expression of the DUX4 gene. Traditional diagnostics are based on Southern blotting, a time- and effort-intensive method that can be affected by single nucleotide variants (SNV) and copy number variants (CNV), as well as by the similarity of the D4Z4 repeats located on chromosome 10. We aimed to evaluate optical genome mapping (OGM) as an alternative molecular diagnostic method for the detection of FSHD. We first performed optical genome mapping with EnFocus™ FSHD analysis using DLE-1 labeling and the Saphyr instrument in patients with inconclusive diagnostic Southern blot results, negative FSHD2 results, and clinically evident FSHD. Second, we performed OGM in parallel with the classical Southern blot analysis for our prospectively collected new FSHD cases. Finally, panel exome sequencing was performed to confirm the presence of FSHD2. In two patients with diagnostically inconclusive Southern blot results, OGM was able to identify shortened D4Z4 repeats on the permissive 4qA alleles, consistent with the clinical presentation. The results of the prospectively collected patients tested in parallel using Southern blotting and OGM showed full concordance, indicating that OGM is a useful alternative to the classical Southern blotting method for detecting FSHD1. In a patient showing clinical FSHD but no shortened D4Z4 repeats in the 4qA allele using OGM or Southern blotting, a likely pathogenic variant in SMCHD1 was detected using exome sequencing, confirming FSHD2. OGM and panel exome sequencing can be used consecutively to detect FSHD2.

Optical Genome Mapping for the Molecular Diagnosis of Facioscapulohumeral Muscular Dystrophy: Advancement and Challenges.

Facioscapulohumeral muscular dystrophy (FSHD) is the second most common muscular dystrophy in adults, and it is associated with local D4Z4 chromatin relaxation, mostly via the contraction of the D4Z4 macrosatellite repeat array on chromosome 4q35. In this study, we aimed to investigate the use of Optical Genome Mapping (OGM) as a diagnostic tool for testing FSHD cases from the UK and India and to compare OGM performance with that of traditional techniques such as linear gel (LGE) and Pulsed-field gel electrophoresis (PFGE) Southern blotting (SB). A total of 6 confirmed and 19 suspected FSHD samples were processed with LGE and PFGE, respectively. The same samples were run using a Saphyr Genome-Imaging Instrument (1-color), and the data were analysed using custom EnFocus FSHD analysis. OGM was able to confirm the diagnosis of FSHD1 in all FSHD1 cases positive for SB (n = 17), and D4Z4 sizing highly correlated with PFGE-SB (p < 0.001). OGM correctly identified cases with mosaicism for the repeat array contraction (n = 2) and with a duplication of the D4Z4 repeat array. OGM is a promising new technology able to unravel structural variants in the genome and seems to be a valid tool for diagnosing FSHD1.

The Dutch registry for facioscapulohumeral muscular dystrophy: Cohort profile and longitudinal patient reported outcomes.

Facioscapulohumeral dystrophy (FSHD) is the second most prevalent inherited muscular disorder and currently lacks a pharmaceutical treatment. The Dutch FSHD Registry was initiated in 2015 as a result of an international collaboration on trial readiness. This paper presents the cohort profile and six years of follow-up data of the registered FSHD patients. At the time of self-registration and every six months thereafter, participants were invited to complete a digital survey of patient and disease characteristics and the Dutch versions of the Checklist Individual Strength (CIS20R), the Individualised Neuromuscular Quality of Life Questionnaire (INQoL), the Beck Depression Index - Primary Care and the McGill Pain Questionnaire. From March 2015 to March 2021, 373 participants completed at least one survey. At baseline, fatigue and muscle weakness were the most frequently reported symptoms (median CIS20R sumscore 77 [IQR 60-92], median INQoL Fatigue score 58 [IQR 42-68] and median INQoL weakness score 58 [IQR 42-68]). Pain was experienced most often in the head and shoulder region (193, 52%). Nineteen of the 23 (sub)sections of questionnaires showed no significant changes over time. We conclude that the Dutch FSHD Registry was successfully set up, enabling collection of longitudinal data and facilitating recruitment in several studies.

Muscle fibrosis as a prognostic biomarker in facioscapulohumeral muscular dystrophy: a retrospective cohort study.

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant epigenetic disorder with highly variable muscle involvement and disease progression. Ongoing clinical trials, aimed at counteracting muscle degeneration and disease progression in FSHD patients, increase the need for reliable biomarkers. Muscle magnetic resonance imaging (MRI) studies showed that the appearance of STIR-positive (STIR+) lesions in FSHD muscles represents an initial stage of muscle damage, preceding irreversible adipose changes. Our study aimed to investigate fibrosis, a parameter of muscle degeneration undetectable by MRI, in relation to disease activity and progression of FSHD muscles. We histologically evaluated collagen in FSHD1 patients' (STIR+ n = 27, STIR- n = 28) and healthy volunteers' (n = 12) muscles by picrosirius red staining. All patients (n = 55) performed muscle MRI before biopsy, 45 patients also after 1 year and 36 patients also after 2 years. Fat content (T1 signal) and oedema/inflammation (STIR signal) were evaluated at baseline and at 1- and 2-year MRI follow-up. STIR+ muscles showed significantly higher collagen compared to both STIR- (p = 0.001) and healthy muscles (p < 0.0001). STIR- muscles showed a higher collagen content compared to healthy muscles (p = 0.0194). FSHD muscles with a worsening in fatty infiltration during 1- (P = 0.007) and 2-year (P < 0.0001) MRI follow-up showed a collagen content of 3.6- and 3.7-fold higher compared to FSHD muscles with no sign of progression. Moreover, the fibrosis was significantly higher in STIR+ muscles who showed a worsening in fatty infiltration in a timeframe of 2 years compared to both STIR- (P = 0.0006) and STIR+ muscles with no sign of progression (P = 0.02). Fibrosis is a sign of muscle degeneration undetectable at MRI never deeply investigated in FSHD patients. Our data show that 23/27 of STIR+ and 12/28 STIR- muscles have a higher amount of collagen deposition compared to healthy muscles. Fibrosis is higher in FSHD muscles with a worsening in fatty infiltration thus suggesting that its evaluation with innovative non-invasive techniques could be a candidate prognostic biomarker for FSHD, to be used to stratify patients and to evaluate the efficacy of therapeutic treatments.

A Targeted Approach for Evaluating DUX4-Regulated Proteins as Potential Serum Biomarkers for Facioscapulohumeral Muscular Dystrophy Using Immunoassay Proteomics.

BACKGROUND: Facioscapulohumeral muscular dystrophy (FSHD) is a progressive myopathy caused by misexpression of the double homeobox 4 (DUX4) embryonic transcription factor in skeletal muscle. Identifying quantitative and minimally invasive FSHD biomarkers to report on DUX4 activity will significantly accelerate therapeutic development. OBJECTIVE: The goal of this study was to analyze secreted proteins known to be induced by DUX4 using the commercially available Olink Proteomics platform in order to identify potential blood-based molecular FSHD biomarkers. METHODS: We used high-throughput, multiplex immunoassays from Olink Proteomics to measure the levels of several known DUX4-induced genes in a cellular myoblast model of FSHD, in FSHD patient-derived myotube cell cultures, and in serum from individuals with FSHD. Levels of other proteins on the Olink Proteomics panels containing these DUX4 targets were also examined in secondary exploratory analysis. RESULTS: Placental alkaline phosphatase (ALPP) levels correlated with DUX4 expression in both cell-based FSHD systems but did not distinguish FSHD patient serum from unaffected controls. CONCLUSIONS: ALPP, as measured with the Olink Proteomics platform, is not a promising FSHD serum biomarker candidate but could be utilized to evaluate DUX4 activity in discovery research efforts.

The Facioscapulohumeral Muscular Dystrophy-Health Index: Development and evaluation of a disease-specific outcome measure.

INTRODUCTION/AIMS: As promising therapeutic interventions are tested among patients with facioscapulohumeral muscular dystrophy (FSHD), there is a clear need for valid and reliable outcome tools to track disease progression and therapeutic gain in clinical trials and for clinical monitoring. Our aim was to develop and validate the Facioscapulohumeral Muscular Dystrophy-Health Index (FSHD-HI) as a multifaceted patient-reported outcome measure (PRO) designed to measure disease burden in adults with FSHD. METHODS: Through initial interviews with 20 individuals and a national cross-sectional study with 328 individuals with FSHD, we identified the most prevalent and impactful symptoms in FSHD. The most relevant symptoms were included in the FSHD-HI. We used patient interviews, test-retest reliability evaluation, known groups validity testing, and factor analysis to evaluate and optimize the FSHD-HI. RESULTS: The FSHD-HI contains 14 subscales that measure FSHD disease burden from the patient's perspective. Fourteen adults with FSHD participated in semistructured beta interviews and found the FSHD-HI to be clear, usable, and relevant to them. Thirty-two adults with FSHD participated in test-retest reliability assessments, which demonstrated the high reliability of the FSHD-HI total score (intraclass correlation coefficient = 0.924). The final FSHD-HI and its subscales also demonstrated a high internal consistency (Cronbach α = 0.988). DISCUSSION: The FSHD-HI provides researchers and clinicians with a reliable and valid mechanism to measure multifaceted disease burden in patients with FSHD. The FSHD-HI may facilitate quantification of therapeutic effectiveness, as demonstrated by its use as a secondary and exploratory measure in several clinical trials.

Late-onset facioscapulohumeral muscular dystrophy type 1 in previously undiagnosed families: Presenting clinical features in an often-misdiagnosed disorder.

INTRODUCTION/AIMS: In our experience, patients with late-onset facioscapulohumeral muscular dystrophy type 1 (FSHD1) are frequently misdiagnosed, some for many years. The aim of this report is to document this clinical experience including the presenting symptoms and misdiagnoses and to discuss the challenges in diagnosing patients with late-onset FSHD1. METHODS: We performed a retrospective medical record review and recorded clinical data on patients with a genetically confirmed diagnosis of FSHD1, who began to have symptoms at 50 years of age or older, and either had no family history of FSHD1 or had a history of an undiagnosed weakness in a family member. RESULTS: Thirteen patients, 7 men and 6 women, met the study inclusion criteria. Age of onset ranged from 52 to 74 (mean, 59.8) years, age of diagnosis ranged from 54 to 80 (mean, 66.5) years, and duration of symptoms from onset to diagnosis was 1 to 15 (mean, 6.7) years. Prior diagnoses included lumbosacral polyradiculopathy in five (38%); statin-related myopathy in two (15%); and one each of polymyositis, inclusion-body myositis, distal myopathy, limb-girdle muscular dystrophy, unspecific myopathy, and unspecified scapular winging. For eight patients (62%), family history was suspected in deceased members or if by confirmed DNA test postdiagnosis. DISCUSSION: The diagnosis of late-onset FSHD1 is often delayed by many years with patients frequently receiving misdiagnoses. FSHD1 may not be considered in the differential diagnosis of late-onset weakness due to its rarity and because its clinical features are subtler, nonspecific, and mimic other neuromuscular disorders.

Association of 4qA-Specific Distal D4Z4 Hypomethylation With Disease Severity and Progression in Facioscapulohumeral Muscular Dystrophy.

BACKGROUND AND OBJECTIVES: The objective of this study was to examine whether the regional methylation levels at the most distal D4Z4 repeat units (RU) in the 4qA-permissive haplotype were associated with disease severity and progression in facioscapulohumeral muscular dystrophy type 1 (FSHD1). METHODS: This 21-year, retrospective, observational cohort study was conducted at the Fujian Neuromedical Center (FNMC) in China. Methylation levels of the most distal D4Z4 RU, including 10 CpGs, were assessed in all participants by bisulfite sequencing. Patients with FSHD1 were stratified into 4 groups based on methylation percentage quartiles, including LM1 (low methylation), LM2 (low to intermediate methylation), LM3 (intermediate to high methylation), and highest methylation (HM) levels. Patients received evaluations of motor function focusing on lower extremity (LE) progression at baseline and in follow-ups. FSHD clinical score (CS), age-corrected clinical severity scale (ACSS), and modified Rankin scale were used to assess motor function. RESULTS: The methylation levels of the 10 CpGs were significantly lower in all 823 patients with genetically confirmed FSHD1 than in 341 healthy controls (HCs). CpG6 methylation levels could distinguish the following: (1) patients with FSHD1 from HCs; (2) symptomatic from asymptomatic/unaffected patients; (3) patients with LE involvement from those without LE involvement, with AUCs (95% CI) of 0.9684 (0.9584-0.9785), 0.7417 (0.6903-0.7931), and 0.6386 (0.5816-0.6956), respectively. Lower CpG6 methylation levels were correlated with higher CS (r = -0.392), higher ACSS (r = -0.432), and earlier onset age of first-ever muscle weakness (r = 0.297). For the LM1, LM2, LM3, and HM groups, the respective proportions of LE involvement were 52.9%, 44.2%, 36.9%, and 23.4%; and onset ages of LE involvement were 20, 26.5, 25, and 26.5 years. Cox regression analysis-adjusted for sex, age at examination, D4Z4 RU, and 4qA/B haplotype-showed that the LM1, LM2, and LM3 groups (i.e., groups with lower methylation levels) had a higher risk of independent ambulation loss, with HRs (95% CI) of 3.523 (1.565-7.930), 3.356 (1.458-7.727), and 2.956 (1.245-7.020), respectively. DISCUSSION: 4q35 distal D4Z4 hypomethylation is correlated with disease severity and progression to lower extremity involvement.