Autosomal dominant in cis D4Z4 repeat array duplication alleles in facioscapulohumeral dystrophy.

Facioscapulohumeral dystrophy (FSHD) has a unique genetic aetiology resulting in partial chromatin relaxation of the D4Z4 macrosatellite repeat array on 4qter. This D4Z4 chromatin relaxation facilitates inappropriate expression of the transcription factor DUX4 in skeletal muscle. DUX4 is encoded by a retrogene that is embedded within the distal region of the D4Z4 repeat array. In the European population, the D4Z4 repeat array is usually organized in a single array that ranges between 8 and 100 units. D4Z4 chromatin relaxation and DUX4 derepression in FSHD is most often caused by repeat array contraction to 1-10 units (FSHD1) or by a digenic mechanism requiring pathogenic variants in a D4Z4 chromatin repressor like SMCHD1, combined with a repeat array between 8 and 20 units (FSHD2). With a prevalence of 1.5% in the European population, in cis duplications of the D4Z4 repeat array, where two adjacent D4Z4 arrays are interrupted by a spacer sequence, are relatively common but their relationship to FSHD is not well understood. In cis duplication alleles were shown to be pathogenic in FSHD2 patients; however, there is inconsistent evidence for the necessity of an SMCHD1 mutation for disease development. To explore the pathogenic nature of these alleles we compared in cis duplication alleles in FSHD patients with or without pathogenic SMCHD1 variant. For both groups we showed duplication-allele-specific DUX4 expression. We studied these alleles in detail using pulsed-field gel electrophoresis-based Southern blotting and molecular combing, emphasizing the challenges in the characterization of these rearrangements. Nanopore sequencing was instrumental to study the composition and methylation of the duplicated D4Z4 repeat arrays and to identify the breakpoints and the spacer sequence between the arrays. By comparing the composition of the D4Z4 repeat array of in cis duplication alleles in both groups, we found that specific combinations of proximal and distal repeat array sizes determine their pathogenicity. Supported by our algorithm to predict pathogenicity, diagnostic laboratories should now be furnished to accurately interpret these in cis D4Z4 repeat array duplications, alleles that can easily be missed in routine settings.

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.

Epigenetic profiling of the D4Z4 locus: Optimization of the protocol for studying DNA methylation at single CpG site level.

The alteration of epigenetic modifications, including DNA methylation, can contribute to the etiopathogenesis and progression of many diseases. Among them, facioscapulohumeral dystrophy (FSHD) is a muscular disorder characterized by the loss of repressive epigenetic features affecting the D4Z4 locus (4q35). As a consequence, these alterations are responsible for DNA hypomethylation and a transcriptional-active chromatin conformation change that, in turn, lead to the aberrant expression of DUX4 in muscle cells. In the present study, methylation levels of 29 CpG sites of the DR1 region (within each repeat unit of the D4Z4 macrosatellite) were assessed on 335 subjects by employing primers designed for enhancing the performance of the assay. First, the DR1 original primers were optimized by adding M13 oligonucleotide tails. Moreover, the DR1 reverse primer was replaced with a degenerate one. As a result, the protocol optimization allowed a better sequencing resolution and a more accurate evaluation of DR1 methylation levels. Moreover, the assessment of the repeatability of measurements proved the reliability and robustness of the assay. The optimized protocol emerges as an excellent method to detect methylation levels compatible with FSHD.

Simultaneous measurement of the size and methylation of chromosome 4qA-D4Z4 repeats in facioscapulohumeral muscular dystrophy by long-read sequencing.

BACKGROUND: Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant muscular disorder characterized by asymmetric muscle wasting and weakness. FSHD can be subdivided into two types: FSHD1, caused by contraction of the D4Z4 repeat on chromosome 4q35, and FSHD2, caused by mild contraction of the D4Z4 repeat plus aberrant hypomethylation mediated by genetic variants in SMCHD1, DNMT3B, or LRIF1. Genetic diagnosis of FSHD is challenging because of the complex procedures required. METHODS: We applied Nanopore CRISPR/Cas9-targeted resequencing for the diagnosis of FSHD by simultaneous detection of D4Z4 repeat length and methylation status at nucleotide level in genetically-confirmed and suspected patients. RESULTS: We found significant hypomethylation of contracted 4q-D4Z4 repeats in FSHD1, and both 4q- and 10q-D4Z4 repeats in FSHD2. We also found that the hypomethylation in the contracted D4Z4 in FSHD1 is moderately correlated with patient phenotypes. CONCLUSIONS: Our method contributes to the development for the diagnosis of FSHD using Nanopore long-read sequencing. This finding might give insight into the mechanisms by which repeat contraction causes disease pathogenesis.

Single-molecule optical mapping enables quantitative measurement of D4Z4 repeats in facioscapulohumeral muscular dystrophy (FSHD).

PURPOSE: Facioscapulohumeral muscular dystrophy (FSHD) is a common adult muscular dystrophy. Over 95% of FSHD cases are associated with contraction of the D4Z4 tandem repeat (~3.3 kb per unit) at 4q35 with a specific genomic configuration (haplotype) called 4qA. Molecular diagnosis of FSHD typically requires pulsed-field gel electrophoresis with Southern blotting. We aim to develop novel genomic and computational methods for characterising D4Z4 repeat numbers in FSHD. METHODS: We leveraged a single-molecule optical mapping platform that maps locations of restriction enzyme sites on high molecular weight (>150 kb) DNA molecules. We developed bioinformatics methods to address several challenges, including the differentiation of 4qA with 4qB alleles, the differentiation of 4q35 and 10q26 segmental duplications, the quantification of repeat numbers with different enzymes that may or may not have recognition sites within D4Z4 repeats. We evaluated the method on 25 human subjects (13 patients, 3 individual control subjects, 9 control subjects from 3 families) labelled by the Nb.BssSI and/or Nt.BspQI enzymes. RESULTS: We demonstrated that the method gave a direct quantitative measurement of repeat numbers on D4Z4 repeats with 4qA allelic configuration and the levels of postzygotic mosaicism. Our method had high concordance with Southern blots from several cohorts on two platforms (Bionano Saphyr and Bionano Irys), but with improved quantification of repeat numbers. CONCLUSION: While the study is limited by small sample size, our results demonstrated that single-molecule optical mapping is a viable approach for more refined analysis on genotype-phenotype relationships in FSHD, especially when postzygotic mosaicism is present.

SMCHD1 mutation spectrum for facioscapulohumeral muscular dystrophy type 2 (FSHD2) and Bosma arhinia microphthalmia syndrome (BAMS) reveals disease-specific localisation of variants in the ATPase domain.

BACKGROUND: Variants in the Structural Maintenance of Chromosomes flexible Hinge Domain-containing protein 1 (SMCHD1) can cause facioscapulohumeral muscular dystrophy type 2 (FSHD2) and the unrelated Bosma arhinia microphthalmia syndrome (BAMS). In FSHD2, pathogenic variants are found anywhere in SMCHD1 while in BAMS, pathogenic variants are restricted to the extended ATPase domain. Irrespective of the phenotypic outcome, both FSHD2-associated and BAMS-associated SMCHD1 variants result in quantifiable local DNA hypomethylation. We compared FSHD2, BAMS and non-pathogenic SMCHD1 variants to derive genotype-phenotype relationships. METHODS: Examination of SMCHD1 variants and methylation of the SMCHD1-sensitive FSHD locus DUX4 in 187 FSHD2 families, 41 patients with BAMS and in control individuals. Analysis of variants in a three-dimensional model of the ATPase domain of SMCHD1. RESULTS: DUX4 methylation analysis is essential to establish pathogenicity of SMCHD1 variants. Although the FSHD2 mutation spectrum includes all types of variants covering the entire SMCHD1 locus, missense variants are significantly enriched in the extended ATPase domain. Identification of recurrent variants suggests disease-specific residues for FSHD2 and in BAMS, consistent with a largely disease-specific localisation of variants in SMCHD1. CONCLUSIONS: The localisation of missense variants within the ATPase domain of SMCHD1 may contribute to the differences in phenotypic outcome.

Interpretation of the Epigenetic Signature of Facioscapulohumeral Muscular Dystrophy in Light of Genotype-Phenotype Studies.

Facioscapulohumeral muscular dystrophy (FSHD) is characterized by incomplete penetrance and intra-familial clinical variability. The disease has been associated with the genetic and epigenetic features of the D4Z4 repetitive elements at 4q35. Recently, D4Z4 hypomethylation has been proposed as a reliable marker in the FSHD diagnosis. We exploited the Italian Registry for FSHD, in which FSHD families are classified using the Clinical Comprehensive Evaluation Form (CCEF). A total of 122 index cases showing a classical FSHD phenotype (CCEF, category A) and 110 relatives were selected to test with the receiver operating characteristic (ROC) curve, the diagnostic and predictive value of D4Z4 methylation. Moreover, we performed DNA methylation analysis in selected large families with reduced penetrance characterized by the co-presence of subjects carriers of one D4Z4 reduced allele with no signs of disease or presenting the classic FSHD clinical phenotype. We observed a wide variability in the D4Z4 methylation levels among index cases revealing no association with clinical manifestation or disease severity. By extending the analysis to family members, we revealed the low predictive value of D4Z4 methylation in detecting the affected condition. In view of the variability in D4Z4 methylation profiles observed in our large cohort, we conclude that D4Z4 methylation does not mirror the clinical expression of FSHD. We recommend that measurement of this epigenetic mark must be interpreted with caution in clinical practice.

Protein kinase A activation inhibits DUX4 gene expression in myotubes from patients with facioscapulohumeral muscular dystrophy.

Facioscapulohumeral muscular dystrophy (FSHD) is among the most prevalent of the adult-onset muscular dystrophies. FSHD causes a loss of muscle mass and function, resulting in severe debilitation and reduction in quality of life. Currently, only the symptoms of FSHD can be treated, and such treatments have minimal benefit. The available options are not curative, and none of the treatments address the underlying cause of FSHD. The genetic, epigenetic, and molecular mechanisms triggering FSHD are now quite well-understood, and it has been shown that expression of the transcriptional regulator double homeobox 4 (DUX4) is necessary for disease onset and is largely thought to be the causative factor in FSHD. Therefore, we sought to identify compounds suppressing DUX4 expression in a phenotypic screen using FSHD patient-derived muscle cells, a zinc finger and SCAN domain-containing 4 (ZSCAN4)-based reporter gene assay for measuring DUX4 activity, and ∼3,000 small molecules. This effort identified molecules that reduce DUX4 gene expression and hence DUX4 activity. Among those, ß2-adrenergic receptor agonists and phosphodiesterase inhibitors, both leading to increased cellular cAMP, effectively decreased DUX4 expression by >75% in cells from individuals with FSHD. Of note, we found that cAMP production reduces DUX4 expression through a protein kinase A-dependent mode of action in FSHD patient myotubes. These findings increase our understanding of how DUX4 expression is regulated in FSHD and point to potential areas of therapeutic intervention.

Facioscapulohumeral muscular dystrophy (FSHD) molecular diagnosis: from traditional technology to the NGS era.

Facioscapulohumeral muscular dystrophy (FSHD) is a genetic neuromuscular disorder which mainly affects the muscles of the face, shoulder, and upper arms. FSHD is generally associated with the contraction of D4Z4 macrosatellite repeats on 4q35 chromosome or mutations in SMCHD1, which are responsible of the toxic expression of DUX4 in muscle tissue. Despite the recent application of NGS techniques in the clinical practice, the molecular diagnosis of FSHD is still performed with dated techniques such as Southern blotting. The diagnosis of FSHD requires therefore specific skills on both modern and less modern analytical protocols. Considering that clinical and molecular diagnosis of FSHD is challenging, it is not surprising that only few laboratories offer a comprehensive characterization of FSHD, which requires the education of professionals on traditional techniques even in the era of NGS. In conclusion, the study of FSHD provides an excellent example of using classical and modern molecular technologies which are equally necessary for the analysis of DNA repetitive traits associated with specific disorders.

Clinical and genetic features of patients with facial-sparing facioscapulohumeral muscular dystrophy.

BACKGROUND AND PURPOSE: Facial-sparing scapular myopathy (SHD) is the most common atypical form of facioscapulohumeral muscular dystrophy (FSHD), clinically defined as without apparent facial muscle weakness on neurological examination. The clinical profiles and genetic features of SHD are limited. METHODS: A cohort of 21 Chinese patients with SHD were confirmed by molecular genetic analysis based on pulsed-field gel electrophoresis. The clinical assessments and methylation analysis were noted. RESULTS: The patients had FSHD-related EcoRI fragments with 4qA haplotype ranging from 18 kb to 33 kb (mean 26.3 ± 4.6 kb). The mean onset age was 25.52 ± 8.3 years. Over half of the patients had scapular winging and asymmetry weakness consistent with FSHD, without facial symptoms during their visit. Their facial electromyogram results were almost normal or mild myogenic damage, as well as the myopathology and serum creatine kinase. A conflict was unexpectedly found in intergenerational DR1 methylation analysis. CONCLUSION: Facial-sparing scapular myopathy is characterized as mild myopathic symptoms and chronic progression of weakness. The diagnosis should be accurately confirmed through FSHD-sized fragment detection and 4qA/B variant determination. Although the next generations of SHD had more severe muscular symptoms, local hypomethylation within D4Z4 was not found as a modifier for clinical heterogeneity.

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.

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.

Genetic and epigenetic characteristics of FSHD-associated 4q and 10q D4Z4 that are distinct from non-4q/10q D4Z4 homologs.

Facioscapulohumeral dystrophy (FSHD) is one of the most prevalent muscular dystrophies. The majority of FSHD cases are linked to a decreased copy number of D4Z4 macrosatellite repeats on chromosome 4q (FSHD1). Less than 5% of FSHD cases have no repeat contraction (FSHD2), most of which are associated with mutations of SMCHD1. FSHD is associated with the transcriptional derepression of DUX4 encoded within the D4Z4 repeat, and SMCHD1 contributes to its regulation. We previously found that the loss of heterochromatin mark (i.e., histone H3 lysine 9 tri-methylation (H3K9me3)) at D4Z4 is a hallmark of both FSHD1 and FSHD2. However, whether this loss contributes to DUX4 expression was unknown. Furthermore, additional D4Z4 homologs exist on multiple chromosomes, but they are largely uncharacterized and their relationship to 4q/10q D4Z4 was undetermined. We found that the suppression of H3K9me3 results in displacement of SMCHD1 at D4Z4 and increases DUX4 expression in myoblasts. The DUX4 open reading frame (ORF) is disrupted in D4Z4 homologs and their heterochromatin is unchanged in FSHD. The results indicate the significance of D4Z4 heterochromatin in DUX4 gene regulation and reveal the genetic and epigenetic distinction between 4q/10q D4Z4 and the non-4q/10q homologs, highlighting the special role of the 4q/10q D4Z4 chromatin and the DUX4 ORF in FSHD.

Risk of functional impairment in Facioscapulohumeral muscular dystrophy.

INTRODUCTION: Facioscapulohumeral muscular dystrophy (FSHD) is one of the most prevalent muscular dystrophies. Nevertheless, little is known about the risk of developing functional impairment. Here we determine the 6-year risk of functional impairment in FSHD. METHODS: A retrospective cohort of 313 genetically confirmed, clinically affected FSHD participants in a United States registry between January 2002 and June 2011. Our main outcome was wheelchair (WC) use. RESULTS: The 6-year risk of WC use was 24.0% (95% confidence interval 18.6-29.3). The distribution of WC risk was bimodal, with a peak in the second decade associated with large D4Z4 contractions, followed by an age-related increase in risk. Other functional categories showed moderate risk. Prevalence of hearing aid use and difficulty pronouncing words was increased in large D4Z4 contractions. CONCLUSIONS: The 6-year risk of functional impairment in FSHD is moderate, and early WC use is associated with large D4Z4 contractions.

Large scale genotype-phenotype analyses indicate that novel prognostic tools are required for families with facioscapulohumeral muscular dystrophy.

Facioscapulohumeral muscular dystrophy has been genetically linked to reduced numbers (≤ 8) of D4Z4 repeats at 4q35 combined with 4A(159/161/168) DUX4 polyadenylation signal haplotype. However, we have recently reported that 1.3% of healthy individuals carry this molecular signature and 19% of subjects affected by facioscapulohumeral muscular dystrophy do not carry alleles with eight or fewer D4Z4 repeats. Therefore, prognosis for subjects carrying or at risk of carrying D4Z4 reduced alleles has become more complicated. To test for additional prognostic factors, we measured the degree of motor impairment in a large group of patients affected by facioscapulohumeral muscular dystrophy and their relatives who are carrying D4Z4 reduced alleles. The clinical expression of motor impairment was assessed in 530 subjects, 163 probands and 367 relatives, from 176 unrelated families according to a standardized clinical score. The associations between clinical severity and size of D4Z4 allele, degree of kinship, gender, age and 4q haplotype were evaluated. Overall, 32.2% of relatives did not display any muscle functional impairment. This phenotype was influenced by the degree of relation with proband, because 47.1% of second- through fifth-degree relatives were unaffected, whereas only 27.5% of first-degree family members did not show motor impairment. The estimated risk of developing motor impairment by age 50 for relatives carrying a D4Z4 reduced allele with 1-3 repeats or 4-8 repeats was 88.7% and 55%, respectively. Male relatives had a mean score significantly higher than females (5.4 versus 4.0, P = 0.003). No 4q haplotype was exclusively associated with the presence of disease. In 13% of families in which D4Z4 alleles with 4-8 repeats segregate, the diagnosis of facioscapulohumeral muscular dystrophy was reported only in one generation. In conclusion, this large-scale analysis provides further information that should be taken into account when counselling families in which a reduced allele with 4-8 D4Z4 repeats segregates. In addition, the reduced expression of disease observed in distant relatives suggests that a family's genetic background plays a role in the occurrence of facioscapulohumeral muscular dystrophy. These results indicate that the identification of new susceptibility factors for this disease will require an accurate classification of families.

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.

Whole exome sequencing highlights rare variants in CTCF, DNMT1, DNMT3A, EZH2 and SUV39H1 as associated with FSHD.

Introduction: Despite the progress made in the study of Facioscapulohumeral Dystrophy (FSHD), the wide heterogeneity of disease complicates its diagnosis and the genotype-phenotype correlation among patients and within families. In this context, the present work employed Whole Exome Sequencing (WES) to investigate known and unknown genetic contributors that may be involved in FSHD and may represent potential disease modifiers, even in presence of a D4Z4 Reduced Allele (DRA). Methods: A cohort of 126 patients with clinical signs of FSHD were included in the study, which were characterized by D4Z4 sizing, methylation analysis and WES. Specific protocols were employed for D4Z4 sizing and methylation analysis, whereas the Illumina® Next-Seq 550 system was utilized for WES. The study included both patients with a DRA compatible with FSHD diagnosis and patients with longer D4Z4 alleles. In case of patients harboring relevant variants from WES, the molecular analysis was extended to the family members. Results: The WES data analysis highlighted 20 relevant variants, among which 14 were located in known genetic modifiers (SMCHD1, DNMT3B and LRIF1) and 6 in candidate genes (CTCF, DNMT1, DNMT3A, EZH2 and SUV39H1). Most of them were found together with a permissive short (4-7 RU) or borderline/long DRA (8-20 RU), supporting the possibility that different genes can contribute to disease heterogeneity in presence of a FSHD permissive background. The segregation and methylation analysis among family members, together with clinical findings, provided a more comprehensive picture of patients. Discussion: Our results support FSHD pathomechanism being complex with a multigenic contribution by several known (SMCHD1, DNMT3B, LRIF1) and possibly other candidate genes (CTCF, DNMT1, DNMT3A, EZH2, SUV39H1) to disease penetrance and expressivity. Our results further emphasize the importance of extending the analysis of molecular findings within the proband's family, with the purpose of providing a broader framework for understanding single cases and allowing finer genotype-phenotype correlations in FSHD-affected families.

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.

Long-read Nanopore sequencing identified D4Z4 contractions in patients with facioscapulohumeral muscular dystrophy.

Facioscapulohumeral muscular dystrophy (FSHD) is a genetic muscle disorder caused by abnormal expression of the DUX4 protein, commonly resulting from a contraction of D4Z4 repeat units with the presence of a polyadenylation (polyA) signal. More than 10 units of the D4Z4 repeat, with a length of 3.3 kb per unit, are typically required to silence DUX4 expression. Consequently, molecular diagnosis of FSHD is challenging. We used Oxford Nanopore technology to perform whole-genome sequencing of seven unrelated patients with FSHD, their six unaffected parents, and 10 unaffected controls. All seven patients were successfully identified to harbor one to five D4Z4 repeat units and the polyA signal, whereas none of the 16 unaffected individuals met the molecular diagnostic criteria. Our newly developed method provides a straightforward and powerful molecular diagnostic tool for FSHD.

Orofacial Muscle Weakening in Facioscapulohumeral Muscular Dystrophy (FSHD) Patients.

BACKGROUND: Facioscapulohumeral muscular dystrophy is the third most commonly found type of muscular dystrophy. The aim of this study was to correlate the D4Z4 repeat array fragment size to the orofacial muscle weakening exhibited in a group of patients with a genetically supported diagnosis of FSHD. METHODS: Molecular genetic analysis was performed for 52 patients (27 female and 25 male) from a group that consisted of 36 patients with autosomal dominant pedigrees and 16 patients with either sporadic or unknown family status. The patients were tested with the southern blotting technique, using EcoRI/Avrll double digestion, and fragments were detected by a p13E-11 telomeric probe. Spearman's correlation was used to compare the fragment size with the degree of muscle weakening found in the forehead, periocular and perioral muscles. RESULTS: A positive non-significant correlation between the DNA fragment size and severity of muscle weakness was found for the forehead (r = 0.27; p = 0187), the periocular (r = 0.24; p = 0.232) and the left and right perioral (r = 0.29; p = 0.122), (r = 0.32; p = 0.085) muscles. CONCLUSIONS: Although FSHD patients exhibited a decrease in muscular activity related to the forehead, perioral, and periocular muscles the genotype-phenotype associations confirmed a weak to moderate non-significant correlation between repeat size and the severity of muscle weakness. Orofacial muscle weakening and its association with a D4Z4 contraction alone may not have the significance to serve as a prognostic biomarker, due to the weak to moderate association. Further studies with larger sample sizes are needed to determine the degree of genetic involvement in the facial growth in FSHD patients.