Clinical Phenotype of Pediatric and Adult Patients With Spinal Muscular Atrophy With Four SMN2 Copies: Are They Really All Stable?

OBJECTIVE: The aim of this study was to provide an overview of the clinical phenotypes associated with 4 SMN2 copies. METHODS: Clinical phenotypes were analyzed in all the patients with 4 SMN2 copies as part of a nationwide effort including all the Italian pediatric and adult reference centers for spinal muscular atrophy (SMA). RESULTS: The cohort includes 169 patients (102 men and 67 women) with confirmed 4 SMN2 copies (mean age at last follow-up = 36.9 ± 19 years). Six of the 169 patients were presymptomatic, 8 were classified as type II, 145 as type III (38 type IIIA and 107 type IIIB), and 8 as type IV. The remaining 2 patients were asymptomatic adults identified because of a familial case. The cross-sectional functional data showed a reduction of scores with increasing age. Over 35% of the type III and 25% of the type IV lost ambulation (mean age = 26.8 years ± 16.3 SD). The risk of loss of ambulation was significantly associated with SMA type (p < 0.0001), with patients with IIIB and IV less likely to lose ambulation compared to type IIIA. There was an overall gender effect with a smaller number of women and a lower risk for women to lose ambulation. This was significant in the adult (p = 0.009) but not in the pediatric cohort (p = 0.43). INTERPRETATION: Our results expand the existing literature on natural history of 4 SMN2 copies confirming the variability of phenotypes in untreated patients, ranging from type II to type IV and an overall reduction of functional scores with increasing age. ANN NEUROL 2023;94:1126-1135.

Six-minute walk test as outcome measure of fatigability in adults with spinal muscular atrophy treated with nusinersen.

INTRODUCTION/AIMS: Fatigue (subjective perception) and fatigability (objective motor performance worsening) are relevant aspects of disability in individuals with spinal muscular atrophy (SMA). The effect of nusinersen on fatigability in SMA patients has been investigated with conflicting results. We aimed to evaluate this in adult with SMA3. METHODS: We conducted a multicenter retrospective cohort study, including adult ambulant patients with SMA3, data available on 6-minute walk test (6MWT) and Hammersmith Functional Motor Scale-Expanded (HFMSE) at baseline and at least at 6 months of treatment with nusinersen. We investigated fatigability, estimated as 10% or higher decrease in walked distance between the first and sixth minute of the 6MWT, at baseline and over the 14-month follow-up. RESULTS: Forty-eight patients (56% females) were included. The 6MWT improved after 6, 10, and 14 months of treatment (p < 0.05). Of the 27 patients who completed the entire follow-up, 37% improved (6MWT distance increase ≥30 m), 48.2% remained stable, and 14.8% worsened (6MWT distance decline ≥30 m). Fatigability was found at baseline in 26/38 (68%) patients and confirmed at subsequent time points (p < 0.05) without any significant change over the treatment period. There was no correlation between fatigability and SMN2 copy number, sex, age at disease onset, age at baseline, nor with 6MWT total distance and baseline HFMSE score. DISCUSSION: Fatigability was detected at baseline in approximately 2/3 of SMA3 walker patients, without any correlation with clinical features, included motor performance. No effect on fatigability was observed during the 14-month treatment period with nusinersen.

A Schematic Approach to Defining the Prevalence of COL VI Variants in Five Years of Next-Generation Sequencing.

OBJECTIVE: To define the prevalence of variants in collagen VI genes through a next-generation sequencing (NGS) approach in undiagnosed patients with suspected neuromuscular disease and to propose a diagnostic flowchart to assess the real pathogenicity of those variants. METHODS: In the past five years, we have collected clinical and molecular information on 512 patients with neuromuscular symptoms referred to our center. To pinpoint variants in COLVI genes and corroborate their real pathogenicity, we sketched a multistep flowchart, taking into consideration the bioinformatic weight of the gene variants, their correlation with clinical manifestations and possible effects on protein stability and expression. RESULTS: In Step I, we identified variants in COLVI-related genes in 48 patients, of which three were homozygous variants (Group 1). Then, we sorted variants according to their CADD score, clinical data and complementary studies (such as muscle and skin biopsy, study of expression of COLVI on fibroblast or muscle and muscle magnetic resonance). We finally assessed how potentially pathogenic variants (two biallelic and 12 monoallelic) destabilize COL6A1-A2-A3 subunits. Overall, 15 out of 512 patients were prioritized according to this pipeline. In seven of them, we confirmed reduced or absent immunocytochemical expression of collagen VI in cultured skin fibroblasts or in muscle tissue. CONCLUSIONS: In a real-world diagnostic scenario applied to heterogeneous neuromuscular conditions, a multistep integration of clinical and molecular data allowed the identification of about 3% of those patients harboring pathogenetic collagen VI variants.

Deep RNA profiling identified CLOCK and molecular clock genes as pathophysiological signatures in collagen VI myopathy.

Collagen VI myopathies are genetic disorders caused by mutations in collagen 6 A1, A2 and A3 genes, ranging from the severe Ullrich congenital muscular dystrophy to the milder Bethlem myopathy, which is recapitulated by collagen-VI-null (Col6a1(-/-)) mice. Abnormalities in mitochondria and autophagic pathway have been proposed as pathogenic causes of collagen VI myopathies, but the link between collagen VI defects and these metabolic circuits remains unknown. To unravel the expression profiling perturbation in muscles with collagen VI myopathies, we performed a deep RNA profiling in both Col6a1(-/-)mice and patients with collagen VI pathology. The interactome map identified common pathways suggesting a previously undetected connection between circadian genes and collagen VI pathology. Intriguingly, Bmal1(-/-)(also known as Arntl) mice, a well-characterized model displaying arrhythmic circadian rhythms, showed profound deregulation of the collagen VI pathway and of autophagy-related genes. The involvement of circadian rhythms in collagen VI myopathies is new and links autophagy and mitochondrial abnormalities. It also opens new avenues for therapies of hereditary myopathies to modulate the molecular clock or potential gene-environment interactions that might modify muscle damage pathogenesis.

Influence of treatments in multiple sclerosis disability: a cohort study.

BACKGROUND AND OBJECTIVE: A critical aspect of multiple sclerosis (MS) treatments is understanding the effect of disease-modifying drugs (DMDs) on the long-term risk of disability and whether the effect is related to disability at start of treatment. METHODS: We performed an observational study on 3060 MS patients. The effect of therapy on progression to Expanded Disability Status Scale (EDSS) 3.0 and 6.0 from onset was analysed in treated vs untreated (UTP) patients using Cox regression analysis adjusted for propensity score and immortal time bias. RESULTS: Compared to UTP, the risks of EDSS 3.0 were 94% and 73% lower in immunomodulant (IMTP-) and immunosuppressant (ISTP-) treated patients, respectively, while the risk of EDSS 6.0 was 86% lower in IMTP. The risk of EDSS 6.0 was, respectively, 91% and 75% lower in 1275 IMTP before and 114 after EDSS 3.0 than in 539 UTP; the risk was higher in IMTP starting therapy after EDSS 3.0 than before (HR = 4.42). CONCLUSIONS: DMDs delayed long-term disability in MS patients treated either in the early or, to a lesser extent, in the later phase of the disease. Thus, the window of therapeutic opportunity is relatively extended, assuming that early is better than late treatment, but late is better than never.

Integrating D4Z4 methylation analysis into clinical practice: improvement of FSHD molecular diagnosis through distinct thresholds for 4qA/4qA and 4qA/4qB patients.

BACKGROUND: Facioscapulohumeral dystrophy (FSHD) is a myopathy characterized by the loss of repressive epigenetic features affecting the D4Z4 locus (4q35). The assessment of DNA methylation at two regions (DUX4-PAS and DR1) of D4Z4 locus proved to be an effective method to detect epigenetic signatures compatible with FSHD. The present study aims at validating the employment of this method into clinical practice and improving the protocol by refining the classification thresholds of 4qA/4qA patients. To this purpose, 218 subjects with clinical suspicion of FSHD collected in 2022-2023 were analyzed. Each participant underwent in parallel the traditional FSHD molecular testing (D4Z4 sizing) and the proposed methylation assay. The results provided by both analyses were compared to evaluate the concordance and calculate the performance metrics of the methylation test. RESULTS: Among the 218 subjects, the 4q variant type distribution was 54% 4qA/4qA, 43% 4qA/4qB and 3% 4qB/4qB. The methylation analysis was performed only on carriers of at least one 4qA allele. After refining the classification threshold, the test reached the following performance metrics: sensitivity = 0.90, specificity = 1.00 and accuracy = 0.93. These results confirmed the effectiveness of the methylation assay in identifying patients with genetic signature compatible with FSHD1 and FSHD2 based on their DUX4-PAS and DR1 profile, respectively. The methylation data were also evaluated with respect to the clinical information. CONCLUSIONS: The study confirmed the ability of the method to accurately identify methylation profiles compatible with FSHD genetic signatures considering the 4q genotype. Moreover, the test allows the detection of hypomethylated profiles in asymptomatic patients, suggesting its potential application in identifying preclinical conditions in patients with positive family history and FSHD genetic signatures. Furthermore, the present work emphasizes the importance of interpreting methylation profiles considering the patients' clinical data.

Budget impact analysis: comparing the costs of nusinersen and risdiplam in the treatment of type 3 spinal muscular atrophy patients.

OBJECTIVES: The aim of the budget impact analysis (BIA) was to determine the economic impact of introducing risdiplam in the treatment of type 3 spinal muscular atrophy (SMA) patients in a rare diseases reference centre on a 3-year time horizon, as compared with nusinersen. METHODS: Public databases were used to estimate the target population. Two market scenarios were assessed over a 3-year time horizon: with nusinersen and with the introduction of risdiplam. Drug acquisition and administration costs were considered. BIA is calculated as the difference between scenarios with nusinersen - scenario with risdiplam. RESULTS: The introduction of risdiplam would generate a 3-year saving of €3411.50. There could be a saving in the administration of risdiplam in the treatment of patients under the age of 2 with a weight of 5 kg (€26 382.08). CONCLUSION: The BIA shows the overlapping costs of these therapies, although the oral administration of risdiplam could be a decisive factor for the therapeutical switch from nusinersen.

Respiratory function in a large cohort of treatment-naïve adult spinal muscular atrophy patients: a cross-sectional study.

Due to poor data in literature, we aimed to investigate the respiratory function in a large cohort of naïve Italian adult (≥18 years) SMA patients in a multi-centric cross-sectional study. The following respiratory parameters were considered: forced vital capacity (FVC), forced expiratory volume in one second (FEV1) and need for non-invasive ventilation (NIV). We included 145 treatment-naïve adult patients (SMA2=18, SMA3=125; SMA4=2), 58 females (40 %), with median age at evaluation of 37 years (range 18-72). Fifty-six (37 %) and 41 (31 %) patients had abnormal (<80 %) values of FVC and FEV1, respectively. Fourteen (14 %) patients needed NIV, started at median age of 21 (range 4-68). Motor function, measured by Hammersmith Functional Motor Scale Expanded and Revised Upper Limb Module as well as SMA2, loss of walking ability, surgery for scoliosis, use of NIV, and cough assisting device (CAD) were all significantly associated to lower FVC and FEV1 values, while no association with age at baseline, disease duration, gender or 6 min walking test was observed, except for a correlation between FVC and age in SMA3 walkers (p < 0.05). In conclusion, respiratory function in adult SMA patients is relatively frequently impaired, substantially stable, and significantly correlated with motor function and disease severity.

Prevalence of Spinal Muscular Atrophy in the Era of Disease-Modifying Therapies: An Italian Nationwide Survey.

OBJECTIVE: Spinal muscular atrophy (SMA) is a neurodegenerative disorder caused by mutations in the SMN1 gene. The aim of this study was to assess the prevalence of SMA and treatment prescription in Italy. METHODS: An online survey was distributed to 36 centers identified by the Italian government as referral centers for SMA. Data on the number of patients with SMA subdivided according to age, type, SMN2 copy number, and treatment were collected. RESULTS: One thousand two hundred fifty-five patients with SMA are currently followed in the Italian centers with an estimated prevalence of 2.12/100,000. Of the 1,255, 284 were type I, 470 type II, 467 type III, and 15 type IV with estimated prevalence of 0.48, 0.79, 0.79 and 0.02/100,000, respectively. Three patients with SMA 0 and 16 presymptomatic patients were also included. Approximately 85% were receiving one of the available treatments. The percentage of treated patients decreased with decreasing severity (SMA I: 95.77%, SMA II: 85.11%, SMA III: 79.01%). DISCUSSION: The results provide for the first time an estimate of the prevalence of SMA at the national level and the current distribution of patients treated with the available therapeutical options. These data provide a baseline to assess future changes in relation to the evolving therapeutical scenario.

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.

Clinical and molecular characterization of limb-girdle muscular dystrophy due to LAMA2 mutations.

INTRODUCTION: In this study we describe the clinical and molecular characteristics of limb-girdle muscular dystrophy (LGMD) due to LAMA2 mutations. METHODS: Five patients clinically diagnosed with LGMD and showing brain white matter hyperintensities on MRI were evaluated using laminin α2 genetic and protein testing. RESULTS: The patients had slowly progressive, mild muscular dystrophy with various degrees of CNS involvement. Epilepsy was observed in 2, and subtle symptoms of CNS involvement (mild deficit in executive functions and low IQ scores) were noted in 3 patients. Novel LAMA2 mutations were identified in all patients. The amount of laminin α2 protein in the muscle biopsies ranged from trace to about 50% compared with controls. CONCLUSIONS: This study represents the largest series of LGMD laminin α2-deficient patients and expands the clinical phenotype associated with LAMA2 mutations. The findings suggest that brain MRI could be included in the diagnostic work-up of patients with undiagnosed LGMD.

Polymorphism in exercise genes and respiratory function in late-onset Pompe disease.

Genetic polymorphisms influencing muscle structure and metabolism may affect the phenotype of metabolic myopathies. We here analyze the possible influence of a wide panel of "exercise genes" on the severity and progression of respiratory dysfunction in late-onset Pompe disease (LOPD). We stratified patients with comparable age and disease duration according to the severity of their respiratory phenotype, assessed by both upright FVC% and postural drop in FVC%. We included 43 patients with LOPD (25 males, age 50.8 ± 13.6 yr) with a 2-yr follow-up since the beginning of enzyme replacement therapy (ERT). Twenty-two patients showed a postural drop >25% T0, seven other patients developed it during the follow-up. We analyzed the relationship between the progression of respiratory dysfunction and genetic polymorphisms affecting muscle function and structure [angiotensin converting enzyme (ACE), α-actinin 3 (ACTN3), peroxisome proliferator-activated receptor α (PPR-α), angiotensin (AGT)], glycogen metabolism [glycogen synthase (GYS), glycogen synthase kinase-3 isoform ß (GSK3ß)], and autophagy [sirtuin 1 (SIRT1), autophagy-related gene 7 (ATG7)]. Individuals carrying two copies of the ACE D-allele shared a 24-fold increase in the risk of severe respiratory dysfunction and progression during the 2-yr follow-up. ACTN3-XX polymorphism was also associated with worse respiratory outcome. The study of exercise genes is of particular interest in respiratory muscles, due to their peculiar features, that is, continuous, low-intensity contraction and prominent recruitment of type I fibers. In line with previous observations on skeletal muscles, ACE-DD and ACTN3-XX genotypes were associated with indirect evidence of more severe respiratory phenotypes. On the contrary, polymorphisms related to autophagy and glycogen metabolism did not seem to influence respiratory muscles.NEW & NOTEWORTHY Previous reports evaluated the role of exercise genes in influencing skeletal muscle phenotype and response to ERT in LOPD. Here, we investigate the role of polymorphisms in several exercise gene, focusing on respiratory muscles. ACE-DD and ACTN3-XX polymorphisms, possibly influencing muscle properties and fiber composition, were associated with more severe respiratory phenotypes.

Muscle imaging analogies in a cohort of patients with different clinical phenotypes caused by LMNA gene mutations.

Laminopathies are a heterogeneous group of LMNA-gene-mutation-related clinical disorders associated with alterations of cardiac and skeletal muscle and peripheral nerves, metabolic defects, and premature aging. Leg muscle imaging investigations were performed in a cohort of patients with LMNA gene alterations who were suffering from Emery-Dreifuss muscular dystrophy, limb-girdle muscular dystrophy type 1B, isolated cardiac disorders or a phenotype of cardiac disorders, and lipodystrophy, including one individual with peripheral neuropathy. Leg muscle imaging revealed varying degrees of alteration in the soleus and medial head of gastrocnemius in each subject. This study demonstrates that LMNA-gene-mutated patients devoid of any clinically detectable skeletal muscle involvement have the same pattern of leg muscle involvement as patients with overt skeletal muscle compromise. This finding suggests the presence of a continuum of skeletal muscle involvement among phenotypes of LMNA-gene-mutation-related skeletalmyopathy and cardiomyopathy.

Large genotype-phenotype study in carriers of D4Z4 borderline alleles provides guidance for facioscapulohumeral muscular dystrophy diagnosis.

Facioscapulohumeral muscular dystrophy (FSHD) is a myopathy with prevalence of 1 in 20,000. Almost all patients affected by FSHD carry deletions of an integral number of tandem 3.3 kilobase repeats, termed D4Z4, located on chromosome 4q35. Assessment of size of D4Z4 alleles is commonly used for FSHD diagnosis. However, the extended molecular testing has expanded the spectrum of clinical phenotypes. In particular, D4Z4 alleles with 9-10 repeat have been found in healthy individuals, in subjects with FSHD or affected by other myopathies. These findings weakened the strict relationship between observed phenotypes and their underlying genotypes, complicating the interpretation of molecular findings for diagnosis and genetic counseling. In light of the wide clinical variability detected in carriers of D4Z4 alleles with 9-10 repeats, we applied a standardized methodology, the Comprehensive Clinical Evaluation Form (CCEF), to describe and characterize the phenotype of 244 individuals carrying D4Z4 alleles with 9-10 repeats (134 index cases and 110 relatives). The study shows that 54.5% of index cases display a classical FSHD phenotype with typical facial and scapular muscle weakness, whereas 20.1% present incomplete phenotype with facial weakness or scapular girdle weakness, 6.7% display minor signs such as winged scapula or hyperCKemia, without functional motor impairment, and 18.7% of index cases show more complex phenotypes with atypical clinical features. Family studies revealed that 70.9% of relatives carrying 9-10 D4Z4 reduced alleles has no motor impairment, whereas a few relatives (10.0%) display a classical FSHD phenotype. Importantly all relatives of index cases with no FSHD phenotype were healthy carriers. These data establish the low penetrance of D4Z4 alleles with 9-10 repeats. We recommend the use of CCEF for the standardized clinical assessment integrated by family studies and further molecular investigation for appropriate diagnosis and genetic counseling. Especially in presence of atypical phenotypes and/or sporadic cases with all healthy relatives is not possible to perform conclusive diagnosis of FSHD, but all these cases need further studies for a proper diagnosis, to search novel causative genetic defects or investigate environmental factors or co-morbidities that may trigger the pathogenic process. These evidences are also fundamental for the stratification of patients eligible for clinical trials. Our work reinforces the value of large genotype-phenotype studies to define criteria for clinical practice and genetic counseling in rare diseases.

Assessing the Role of Anti rh-GAA in Modulating Response to ERT in a Late-Onset Pompe Disease Cohort from the Italian GSDII Study Group.

INTRODUCTION: Patients with late-onset Pompe disease (LOPD) receiving enzyme replacement therapy (ERT) may develop IgG antibodies against alglucosidase alpha (anti-rhGAA) in the first 3 months of treatment. The exact role of these antibodies in modulating efficacy of ERT in this group of patients is still not fully understood. To assess whether anti rh-GAA antibodies interfere with ERT efficacy, we studied a large Italian cohort of LOPD patients. METHODS: We analyzed clinical findings and performed serial measurements of IgG anti rh-GAA antibody titers from 64 LOPD patients treated with ERT. The first examination (T0) was completed on average at 17.56 months after starting ERT, while the follow-up (T1) was collected on average at 38.5 months. Differences in T0-T1 delta of the six-minute walking test (6MWT), MRC sum score (MRC), gait, stairs and chair performance (GSGC) and forced vital capacity (FVC) were considered and then related to the antibody titers. RESULTS: Almost 22% of the patients never developed antibodies against GAA, while 78.1% had a positive titer (31.2% patients developed a low titer, 43.8% a medium titer and 3.1% a high titer). No statistical significance was found in relating the T0-T1 delta differences and antibody titers, except for MRC sum score values in a subgroup of patients treated < 36 months, in which those with a null antibody titer showed a greater clinical improvement than patients with a positive titer. CONCLUSION: Our results confirm that in a large cohort of LOPD patients, anti rh-GAA antibody generation did not significantly affect either clinical outcome or ERT efficacy. However, in the first 36 months of treatment, a possible interference of low-medium antibody titers with the clinical status could be present. Therefore, a careful and regular evaluation of antibody titers, especially in cases with evidence of clinical decline despite ERT, should be performed.

Muscle MRI findings in patients with an apparently exclusive cardiac phenotype due to a novel LMNA gene mutation.

The case of a family in which several members displayed conduction defects inherited as a dominant trait is reported. The proband was a young woman with a 1st degree atrio-ventricular block and high serum creatine kinase. Several members of the family featured cardiologic symptoms. All adult family members were clinically evaluated and blood tests including serum creatine-kinase levels, standard and Holter ECG, echocardiogram and muscle MRI were performed. LMNA gene analysis was carried out and a novel missense mutation consisting in substitution of exon 4 c.799 T/C, p.Tyr267His was revealed. The mutation was present in seven family members, five of whom displayed cardiac defects alone with no involvement of the skeletal muscle. In all mutated individuals muscle MRI featured a pattern of skeletal muscle involvement similar to that observed in autosomal dominant Emery Dreifuss muscular dystrophy, suggesting that even patients bearing a LMNA gene mutation associated to an apparently selective cardiac phenotype may present subclinical skeletal muscle involvement.

Late-onset polyglucosan body myopathy in five patients with a homozygous mutation in GYG1.

Five Sardinian patients presented in their 5th or 6th decade with progressive limb girdle muscle weakness but their muscle biopsies showed vacuolar myopathy. The more or less abundant subsarcolemmal and intermyofibrillar vacuoles showed intense, partially α-amylase resistant, PAS-positive deposits consistent with polyglucosan. The recent description of late-onset polyglucosan myopathy has prompted us to find new genetic defects in the gene (GYG1) encoding glycogenin-1, the crucial primer enzyme of glycogen synthesis in muscle. We found a single homozygous intronic mutation harbored by five patients, who, except for two siblings, appear to be unrelated but all five live in central or south Sardinian villages.

A novel mutation in the central rod domain of lamin A/C producing a phenotype resembling the Emery-Dreifuss muscular dystrophy phenotype.

Lamins are the principal components of the nuclear lamina, a network constituting the major structural framework of the nuclear envelope. Alterations in lamin A/C have been associated with a heterogeneous series of human disorders known as laminopathies. We report the finding of a novel deletion in the central rod domain of lamin A/C exon 3 gene in four members of the same family. This genetic alteration was likely responsible for the relatively homogeneous clinical phenotype observed in our three patients, represented by a prominent cardiac conduction-system disease necessitating permanent pacemaker implantation, and limited skeletal involvement manifested by spinal rigidity and contractures. The findings from these cases further expand the clinical spectrum associated with mutations in the LMNA gene.