Mosaicism in Human Health and Disease.

Mosaicism refers to the occurrence of two or more genomes in an individual derived from a single zygote. Germline mosaicism is a mutation that is limited to the gonads and can be transmitted to offspring. Somatic mosaicism is a postzygotic mutation that occurs in the soma, and it may occur at any developmental stage or in adult tissues. Mosaic variation may be classified in six ways: (a) germline or somatic origin, (b) class of DNA mutation (ranging in scale from single base pairs to multiple chromosomes), (c) developmental context, (d) body location(s), (e) functional consequence (including deleterious, neutral, or advantageous), and (f) additional sources of mosaicism, including mitochondrial heteroplasmy, exogenous DNA sources such as vectors, and epigenetic changes such as imprinting and X-chromosome inactivation. Technological advances, including single-cell and other next-generation sequencing, have facilitated improved sensitivity and specificity to detect mosaicism in a variety of biological contexts.

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.

The FSHD jigsaw: are we placing the tiles in the right position?

PURPOSE OF REVIEW: Facioscapulohumeral muscular dystrophy (FSHD) is one of the most common myopathies, involving over 870,000 people worldwide and over 20 FSHD national registries. Our purpose was to summarize the main objectives of the scientific community on this topic and the moving trajectories of research from the past to the present. RECENT FINDINGS: To date, research is mainly oriented toward deciphering the molecular and pathogenetic basis of the disease by investigating DUX4-mediated muscle alterations. Accordingly, FSHD drug development has been escalating in the last years in an attempt to silence DUX4 or to block its downstream effectors. Breakthroughs in the field include the awareness that new biomarkers and outcome measures are required for tracking disease progression and patient stratification. The need to develop personalized therapeutic strategies is also crucial according to the phenotypic variability observed in FSHD subjects. SUMMARY: We analysed 121 literature reports published between 2021 and 2023 to assess the most recent advances in FSHD clinical and molecular research.

De novo variants and recombination at 4q35: Hints for preimplantation genetic testing in facioscapulohumeral muscular dystrophy.

Facioscapulohumeral muscular dystrophy (FSHD) has been associated with the deletion of an integral number of 3.3 kb units of the polymorphic D4Z4 repeat array at 4q35. The prenatal identification of this defect can be carried out on chorionic villi or amniocytes, whereas preimplantation genetic testing for monogenic disorders (PGT-M) requires molecular markers linked to the D4Z4 allele of reduced size. In this context the reliability of this association is crucial. To test the informativeness of the nearby polymorphic markers we investigated recombination at 4q35 using the polymorphic markers D4S1523, D4S163 and D4S139 positioned at 0.55, 0.5 and 0.21 Mb proximal to the D4Z4 array respectively. We determined the probability of recombination events to occur in the D4Z4-D4S1523 interval considering 86 subjects belonging to 12 FSHD families and found a recombination frequency of 14% between D4Z4 and D4S1523. Our study also revealed the occurrence of de novo variants and germline mosaicism. These findings highlight the recombinogenic nature of the 4q subtelomere and indicate that caution should be taken when interpreting PGT-M results. It is advisable that a woman who underwent a PGT-M cycle undertakes a prenatal DNA analysis to confirm the size of the D4Z4 alleles carried by the fetus.

Comparison of quantitative muscle ultrasound and whole-body muscle MRI in facioscapulohumeral muscular dystrophy type 1 patients.

INTRODUCTION: Muscle ultrasound is a fast, non-invasive and cost-effective examination that can identify structural muscular changes by assessing muscle thickness and echointensity (EI) with a quantitative analysis (QMUS). To assess applicability and repeatability of QMUS, we evaluated patients with genetically confirmed facioscapulohumeral muscular dystrophy type 1 (FSHD1), comparing their muscle ultrasound characteristics with healthy controls and with those detected by MRI. We also evaluated relationships between QMUS and demographic and clinical characteristics. MATERIALS AND METHODS: Thirteen patients were included in the study. Clinical assessment included MRC sum score, FSHD score and The Comprehensive Clinical Evaluation Form (CCEF). QMUS was performed with a linear transducer scanning bilaterally pectoralis major, deltoid, rectus femoris, tibialis anterior and semimembranosus muscles in patients and healthy subjects. For each muscle, we acquired three images, which were analysed calculating muscle EI by computer-assisted grey-scale analysis. QMUS analysis was compared with semiquantitative 1.5 T muscle MRI scale. RESULTS: All muscles in FSHD patients showed a significant increased echogenicity compared to the homologous muscles in healthy subjects. Older subjects and patients with higher FSHD score presented increased muscle EI. Tibialis anterior MRC showed a significant inverse correlation with EI. Higher median EI was found in muscles with more severe MRI fat replacement. CONCLUSIONS: QMUS allows quantitative evaluation of muscle echogenicity, displaying a tight correlation with muscular alterations, clinical and MRI data. Although a confirmation on larger sample is needed, our research suggests a possible future application of QMUS in diagnosis and management of muscular disorders.

Increased resistance towards fatigability in patients with facioscapulohumeral muscular dystrophy.

PURPOSE: In facioscapulohumeral muscular dystrophy (FSHD) fatigue is a major complaint. We aimed to investigate whether during isometric sustained elbow flexions, performance fatigability indexes differ in patients with FSHD with respect to healthy controls. METHODS: Seventeen patients with FSHD and seventeen healthy controls performed two isometric flexions of the dominant biceps brachii at 20% of their maximal voluntary contraction (MVC) for 2 min and then at 60% MVC until exhaustion. Muscle weakness was characterized as a percentage of predicted values. Maximal voluntary strength, endurance time and performance fatigability indices (mean frequency of the power spectrum (MNF), muscle fiber conduction velocity (CV) and fractal dimension (FD)), extracted from the surface electromyogram signal (sEMG) were compared between the two groups. RESULTS: In patients with FSHD, maximal voluntary strength was 68.7% of predicted value (p < 0.01). Compared to healthy controls, FSHD patients showed reduced MVC (p < 0.001; r = 0.62) and lower levels of performance fatigability, characterized by reduced rate of changes in MNF (p < 0.01; r = 0.56), CV (p < 0.05; 0.37) and FD (p < 0.001; r = 0.51) and increased endurance time (p < 0.001; r = 0.63), during the isometric contraction at 60% MVC. CONCLUSION: A decreased reduction in the slopes of all the considered sEMG parameters during sustained isometric elbow flexions suggests that patients with FSHD experience lower levels of performance fatigability compared to healthy controls.

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.

Phenotype may predict the clinical course of facioscapolohumeral muscular dystrophy.

INTRODUCTION: The correct phenotypic classification of patients with facioscapulohumeral muscular dystrophy (FSHD) is crucial for directing genetic diagnosis and for the definition of outcome measures in clinical trials. METHODS: Our objective was to ascertain the utility of the Comprehensive Clinical Evaluation Form (CCEF), the clinical classification proposed by the Italian Clinical Network for FSHD, in an independent FSHD patient population from the UK FSHD Patient Registry. We subdivided the patients into group 1, classic FSHD phenotype/category A of CCEF, and group 2, facial sparing phenotypes/category B1 of CCEF. RESULTS: Among 642 patients with FSHD1, 68.1% reported facial and shoulder weakness, whereas 24.1% reported shoulder weakness without facial impairment. The phenotype in group 2 was milder, with a higher mean age at onset (P < 0.0001) and less severe motor disability. DISCUSSION: Patients with different FSHD phenotypes may have different disease courses. Muscle Nerve 59:711-713, 2019.

Large-scale population analysis challenges the current criteria for the molecular diagnosis of fascioscapulohumeral muscular dystrophy.

Facioscapulohumeral muscular dystrophy (FSHD) is a common hereditary myopathy causally linked to reduced numbers (≤8) of 3.3 kilobase D4Z4 tandem repeats at 4q35. However, because individuals carrying D4Z4-reduced alleles and no FSHD and patients with FSHD and no short allele have been observed, additional markers have been proposed to support an FSHD molecular diagnosis. In particular a reduction in the number of D4Z4 elements combined with the 4A(159/161/168)PAS haplotype (which provides the possibility of expressing DUX4) is currently used as the genetic signature uniquely associated with FSHD. Here, we analyzed these DNA elements in more than 800 Italian and Brazilian samples of normal individuals unrelated to any FSHD patients. We find that 3% of healthy subjects carry alleles with a reduced number (4-8) of D4Z4 repeats on chromosome 4q and that one-third of these alleles, 1.3%, occur in combination with the 4A161PAS haplotype. We also systematically characterized the 4q35 haplotype in 253 unrelated FSHD patients. We find that only 127 of them (50.1%) carry alleles with 1-8 D4Z4 repeats associated with 4A161PAS, whereas the remaining FSHD probands carry different haplotypes or alleles with a greater number of D4Z4 repeats. The present study shows that the current genetic signature of FSHD is a common polymorphism and that only half of FSHD probands carry this molecular signature. Our results suggest that the genetic basis of FSHD, which is remarkably heterogeneous, should be revisited, because this has important implications for genetic counseling and prenatal diagnosis of at-risk families.

Altered Tnnt3 characterizes selective weakness of fast fibers in mice overexpressing FSHD region gene 1 (FRG1).

Facioscapulohumeral muscular dystrophy (FSHD), a common hereditary myopathy, is characterized by atrophy and weakness of selective muscle groups. FSHD is considered an autosomal dominant disease with incomplete penetrance and unpredictable variability of clinical expression within families. Mice overexpressing FRG1 (FSHD region gene 1), a candidate gene for this disease, develop a progressive myopathy with features of the human disorder. Here, we show that in FRG1-overexpressing mice, fast muscles, which are the most affected by the dystrophic process, display anomalous fast skeletal troponin T (fTnT) isoform, resulting from the aberrant splicing of the Tnnt3 mRNA that precedes the appearance of dystrophic signs. We determine that muscles of FRG1 mice develop less strength due to impaired contractile properties of fast-twitch fibers associated with an anomalous MyHC-actin ratio and a reduced sensitivity to Ca(2+). We demonstrate that the decrease of Ca(2+) sensitivity of fast-twitch fibers depends on the anomalous troponin complex and can be rescued by the substitution with the wild-type proteins. Finally, we find that the presence of aberrant splicing isoforms of TNNT3 characterizes dystrophic muscles in FSHD patients. Collectively, our results suggest that anomalous TNNT3 profile correlates with the muscle impairment in both humans and mice. On the basis of these results, we propose that aberrant fTnT represents a biological marker of muscle phenotype severity and disease progression.

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.

An integrated approach in a case of facioscapulohumeral dystrophy.

BACKGROUND: Muscle fatigue, weakness and atrophy are basilar clinical features that accompany facioscapulohumeral dystrophy (FSHD) the third most common muscular dystrophy.No therapy is available for FSHD. CASE PRESENTATION: We describe the effects of 6mo exercise therapy and nutritional supplementation in a 43-year-old woman severely affected by FSHD. CONCLUSION: A mixed exercise program combined with nutritional supplementation can be safely used with beneficial effects in selected patients with FSHD.

Facioscapulohumeral muscular dystrophy: new insights from compound heterozygotes and implication for prenatal genetic counselling.

Background Facioscapulohumeral muscular dystrophy (FSHD) is considered an autosomal dominant disease with a prevalence of 1 in 20 000. Almost all patients with FSHD carry deletions of integral copies of tandem 3.3 kb repeats (D4Z4) located on chromosome 4q35. However, FSHD families have been reported in which individuals carrying a D4Z4-reduced allele remain asymptomatic. Recently, it has been proposed that the D4Z4-reduced allele is pathogenic only in association with the permissive haplotype, 4APAS. Methods and results Through the Italian National Registry for FSHD (INRF), genotype-phenotype correlations were extensively studied in 11 non-consanguineous families in which two D4Z4-reduced alleles segregate. Overall, 68 subjects carrying D4Z4-reduced alleles were examined, including 15 compound heterozygotes. It was found that in four families the only FSHD-affected subject was the compound heterozygote for the D4Z4-reduced allele, and 52.6% of subjects carrying a single D4Z4-reduced 4A161PAS haplotype were non-penetrant carriers; moreover, the population frequency of the 4A161PAS haplotype associated with a D4Z4-reduced allele was found to be as high as 1.2%. Conclusions This study reveals a high frequency of compound heterozygotes in the Italian population and the presence of D4Z4-reduced alleles with the 4A161PAS pathogenic haplotype in the majority of non-penetrant subjects in FSHD families with compound heterozygosity. These data suggest that carriers of FSHD-sized alleles with 4A161PAS haplotype are more common in the general population than expected on the basis of FSHD prevalence. These findings challenge the notion that FSHD is a fully penetrant autosomal dominant disorder uniquely associated with the 4A161PAS haplotype, with relevant repercussions for genetic counselling and prenatal diagnosis.

Factors predicting disease progression in C9ORF72 ALS patients.

OBJECTIVE: To unveil clinical features, comorbidities, disease progression and prognostic factors in a population-based cohort of ALS patients carrying C9ORF72 expansion (C9 + ALS). METHODS: This is a retrospective observational study on ALS patients residing in Emilia Romagna and Piedmont-Valle D'Aosta regions whose data are available through population based registers. We analysed patients who underwent genetic testing, focusing on C9 + ALS subgroup. RESULTS: Among 2204 genotyped patients of the two registers, 150 were C9 + ALS. In comparison with patients without mutation, a higher proportion of family history (12.85 vs 68%, p < 0.001) and frontotemporal dementia (3.93% vs 10.67%, p < 0.001) was detected in C9 + ALS. C9 + ALS presented a faster disease progression as measured by monthly decline in ALS Functional Rating Scale-Revised (1.86 ± 3.30 vs 1.45 ± 2.35, p < 0.01) and in forced vital capacity (5.90 ± 5.24 vs 2.97 ± 3.47, p < 0.01), a shorter diagnostic delay (8.93 ± 6.74 vs 12.68 ± 12.86 months, p < 0.01) and earlier onset (58.91 ± 9.02 vs 65.04 ± 11.55 years, p < 0.01). Consistently, they reached death or tracheostomy earlier than other patients (31 vs 37 months, HR = 1.52, 95% C.I. 1.27-1.82, p < 0.001). With respect to other genotyped patients, C9 + ALS patients did not present a significantly higher prevalence of concomitant diseases. Independent prognostic factors of survival of C9 + ALS included sex, age, progression rate, presence of frontotemporal dementia and thyroid disorders, with the latter being associated with prolonged ALS survival (43 vs 29 months, HR = 0.42, 95% C.I. 0.24-0.74, p = 0.003). CONCLUSION: Even in the context of a more aggressive disease, C9 + ALS had a longer survival in presence of thyroid disorders. This finding may suggest protective pathogenic pathways in C9 + ALS to be explored, looking for therapeutic strategies to slow disease course.

Using Cluster Analysis to Overcome the Limits of Traditional Phenotype-Genotype Correlations: The Example of RYR1-Related Myopathies.

Thanks to advances in gene sequencing, RYR1-related myopathy (RYR1-RM) is now known to manifest itself in vastly heterogeneous forms, whose clinical interpretation is, therefore, highly challenging. We set out to develop a novel unsupervised cluster analysis method in a large patient population. The objective was to analyze the main RYR1-related characteristics to identify distinctive features of RYR1-RM and, thus, offer more precise genotype-phenotype correlations in a group of potentially life-threatening disorders. We studied 600 patients presenting with a suspicion of inherited myopathy, who were investigated using next-generation sequencing. Among them, 73 index cases harbored variants in RYR1. In an attempt to group genetic variants and fully exploit information derived from genetic, morphological, and clinical datasets, we performed unsupervised cluster analysis in 64 probands carrying monoallelic variants. Most of the 73 patients with positive molecular diagnoses were clinically asymptomatic or pauci-symptomatic. Multimodal integration of clinical and histological data, performed using a non-metric multi-dimensional scaling analysis with k-means clustering, grouped the 64 patients into 4 clusters with distinctive patterns of clinical and morphological findings. In addressing the need for more specific genotype-phenotype correlations, we found clustering to overcome the limits of the "single-dimension" paradigm traditionally used to describe genotype-phenotype relationships.

The MeCP2/YY1 interaction regulates ANT1 expression at 4q35: novel hints for Rett syndrome pathogenesis.

Rett syndrome is a severe neurodevelopmental disorder mainly caused by mutations in the transcriptional regulator MeCP2. Although there is no effective therapy for Rett syndrome, the recently discovered disease reversibility in mice suggests that there are therapeutic possibilities. Identification of MeCP2 targets or modifiers of the phenotype can facilitate the design of curative strategies. To identify possible novel MeCP2 interactors, we exploited a bioinformatic approach and selected Ying Yang 1 (YY1) as an interesting candidate. We demonstrate that MeCP2 interacts in vitro and in vivo with YY1, a ubiquitous zinc-finger epigenetic factor regulating the expression of several genes. We show that MeCP2 cooperates with YY1 in repressing the ANT1 gene encoding a mitochondrial adenine nucleotide translocase. Importantly, ANT1 mRNA levels are increased in human and mouse cell lines devoid of MeCP2, in Rett patient fibroblasts and in the brain of Mecp2-null mice. We further demonstrate that ANT1 protein levels are upregulated in Mecp2-null mice. Finally, the identified MeCP2-YY1 interaction, together with the well-known involvement of YY1 in the regulation of D4Z4-associated genes at 4q35, led us to discover the anomalous depression of FRG2, a subtelomeric gene of unknown function, in Rett fibroblasts. Collectively, our data indicate that mutations in MeCP2 might cause the aberrant overexpression of genes located at a specific locus, thus providing new candidates for the pathogenesis of Rett syndrome. As both ANT1 mutations and overexpression have been associated with human diseases, we consider it highly relevant to address the consequences of ANT1 deregulation in Rett syndrome.

Facioscapulohumeral muscular dystrophy in mice overexpressing FRG1.

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant neuromuscular disorder that is not due to a classical mutation within a protein-coding gene. Instead, almost all FSHD patients carry deletions of an integral number of tandem 3.3-kilobase repeat units, termed D4Z4, located on chromosome 4q35 (ref. 3). D4Z4 contains a transcriptional silencer whose deletion leads to inappropriate overexpression in FSHD skeletal muscle of 4q35 genes located upstream of D4Z4 (ref. 4). To identify the gene responsible for FSHD pathogenesis, we generated transgenic mice selectively overexpressing in skeletal muscle the 4q35 genes FRG1, FRG2 or ANT1. We find that FRG1 transgenic mice develop a muscular dystrophy with features characteristic of the human disease; by contrast, FRG2 and ANT1 transgenic mice seem normal. FRG1 is a nuclear protein and several lines of evidence suggest it is involved in pre-messenger RNA splicing. We find that in muscle of FRG1 transgenic mice and FSHD patients, specific pre-mRNAs undergo aberrant alternative splicing. Collectively, our results suggest that FSHD results from inappropriate overexpression of FRG1 in skeletal muscle, which leads to abnormal alternative splicing of specific pre-mRNAs.

Effects of creatine and exercise on skeletal muscle of FRG1-transgenic mice.

BACKGROUND: The FRG1-transgenic mouse displays muscle dysfunction and atrophy reminiscent of fascioscapulohumeral muscular dystrophy (FSHD) and could provide a model to determine potential therapeutic interventions. METHODS: To determine if FRG1 mice benefit from treatments that improve muscle mass and function, mice were treated with creatine alone (Cr) or in combination with treadmill exercise (CrEX). RESULTS: The CrEx treatment increased quadriceps weight, mitochondrial content (cytochome c oxidase (COX) activity, COX subunit one and four protein), and induced greater improvements in grip strength and rotarod fall speed. While Cr increased COX subunits one and four protein, no effect on muscle mass or performance was found. Since Cr resulted in no functional improvements, the benefits of CrEx may be mediated by exercise; however, the potential synergistic action of the combined treatment cannot be excluded. CONCLUSION: Treatment with CrEx attenuates atrophy and muscle dysfunction associated with FRG1 overexpression. These data suggest exercise and creatine supplementation may benefit individuals with FSHD.

RNA interference improves myopathic phenotypes in mice over-expressing FSHD region gene 1 (FRG1).

Muscular dystrophies, and other diseases of muscle, arise from recessive and dominant gene mutations. Gene replacement strategies may be beneficial for the former, while gene silencing approaches may provide treatment for the latter. In the last two decades, muscle-directed gene therapies were primarily focused on treating recessive disorders. This disparity at least partly arose because feasible mechanisms to silence dominant disease genes lagged behind gene replacement strategies. With the discovery of RNA interference (RNAi) and its subsequent development as a promising new gene silencing tool, the landscape has changed. In this study, our objective was to demonstrate proof-of-principle for RNAi therapy of a dominant myopathy in vivo. We tested the potential of adeno-associated viral (AAV)-delivered therapeutic microRNAs, targeting the human Facioscapulohumeral muscular dystrophy (FSHD) region gene 1 (FRG1), to correct myopathic features in mice expressing toxic levels of human FRG1 (FRG1(-high) mice). We found that FRG1 gene silencing improved muscle mass, strength, and histopathological abnormalities associated with muscular dystrophy in FRG1(-high) mice, thereby demonstrating therapeutic promise for treatment of dominantly inherited myopathies using RNAi. This approach potentially applies to as many as 29 different gene mutations responsible for myopathies inherited as dominant disorders.

Counseling and prenatal diagnosis in facioscapulohumeral muscular dystrophy: A retrospective study on a 13-year multidisciplinary approach.

Background and Aims: This is the first national population-based report about prenatal diagnosis for families with a history of facioscapulohumeral muscular dystrophy (FSHD), a complex hereditary disease. The incomplete disease penetrance and the phenotypic heterogeneity observed in carriers of D4Z4 alleles of reduced size, the FSHD molecular hallmark, make the estimate of genetic risk problematic. Methods: We considered all requests of preconception counseling and prenatal diagnosis received between January 2008 and December 2020 by the genetic counseling service associated with the Italian National Registry for FSHD (INRF). A multidisciplinary team managed the clinical and molecular data of each family. Results: Between 2008 and 2020, 60 couples required preconception counseling (PC) for FSHD. In 52 couples was observed at least one partner carried a D4Z4 reduced allele (DRA). Out of these 52 couples, 47 had a follow-up visit routine yearly. Out of these 47, 26 (55.3%) couples had children: eight asked for prenatal diagnosis (PND), two had assisted reproduction by heterologous in vitro fertilization (IVF), and 16 did not require further assistance. Regarding PND, 50 prenatal analyses were performed for 36 couples. The test resulted positive in 27 pregnancies, 12 (44.4%) were terminated, and 15 (55.6%) were carried to term. Conclusion: The different choices made by the couples show the importance of an integrated approach to support genetic counseling for FSHD. These results remark the relevance of the clinical and molecular investigation of the extended family, preferably before conception.