Simulated haemodynamic parameters and different infusion set-up affect drug delivery during syringe pump change over: A bench-top study in a laboratory setting.

BACKGROUND: Infusion therapy is widely used in clinical settings, particularly in intensive care units. AIM: to explore the influence of simulated cardiac output on "bolus" or "backflow" events that can occur during syringe pump changeover, considering several factors that have been previously outlined in published research. Syringe infusion pumps are commonly used for precise continuous intravenous drug delivery. Syringe pump changeover can be a challenging procedure. METHODS: Bench-top study in a laboratory setting. An extracorporeal circuit was used to simulate a cardiac output of 5 l/min. The following variables were used: three levels of vertical position of the syringe pump (-50 cm, 0, +50 cm), three levels of Central Venous Pressure (-5, 10, and 15 mmHg), presence/absence of carrier infusion (5 ml/h), and presence/absence of a needle-free connector between the syringe and extension line. RESULTS: A total of 108 syringe pump changes were performed with different combinations of the investigated variables. The mean time for syringe pump changeover was equal to 9.48 ± 2.45 s and the overall fluid displacement was 8 ± 40 µL (microlitres) (range, -262-156 µL). The CVP level and vertical position of the pump always statistically affected the overall displacement during syringe pump changeover. When a second infusion with an equal velocity rate to that of a syringe pump infusion is present in the same lumen, the presence of a needle-free device reduces the overall volume of displacement. CONCLUSIONS: Syringe pump changeover can be a critical moment for patients when vasoactive drugs are administered. IMPLICATIONS FOR CLINICAL PRACTICE: In a simulated environment with a cardiac output of 5 L/min, the CVP level and vertical position of the syringe pump generated bolus or backflow events during the syringe pump changeover. The application of carrier infusion appeared to intensify these phenomena. Employing a neutral, needle-free system can potentially aid in reducing the development of boluses or backflows.

Parental diagnostic delay and developmental outcomes in congenital and childhood-onset myotonic dystrophy type 1.

AIM: To investigate the timing of type 1 myotonic dystrophy (DM1) diagnosis in parents of affected children and describe children's perinatal characteristics and developmental outcomes. METHOD: This was a descriptive case series of children with congenital myotonic dystrophy (CDM) and childhood-onset myotonic dystrophy (ChDM). Parental timing of DM1 diagnosis and the perinatal, motor, and cognitive outcomes of paediatric patients were recorded. RESULTS: A total of 139 children followed by 12 highly specialized tertiary care neuromuscular centres in Italy and one tertiary neuromuscular centre in the USA were included: 105 children with CDM and 34 children with ChDM (mean age 8 years 8 months and 12 years 2 months respectively; 49 males and 17 males respectively). Seventy (50%) parents were diagnosed with adult-onset DM1 after the affected child was diagnosed. Only 12 (17%) of the 69 parents known to be affected had prenatal testing. Of the 105 children with CDM, 98% had maternally inherited CDM, 36% were born preterm, 83% required a stay in the neonatal intensive care unit for more than 48 hours, 84% and 79% had ambulation and speech delay, and 84% had an IQ lower than 70. Of the 34 children with ChDM, 59% had paternally inherited ChDM, 91% were born at term, and 36% had an IQ lower than 70. INTERPRETATION: Delay in diagnosing DM1 affects family planning. The prenatal and perinatal outcomes of the affected offspring emphasize the need for proactive counselling as parents may be reluctant to conduct prenatal testing.

Association between Reported Sleep Disorders and Behavioral Issues in Children with Myotonic Dystrophy Type 1-Results from a Retrospective Analysis in Italy.

Background: Sleep disorders have been poorly described in congenital (CDM) and childhood (ChDM) myotonic dystrophy despite being highly burdensome. The aims of this study were to explore sleep disorders in a cohort of Italian CDM and ChDM and to assess their association with motor and respiratory function and disease-specific cognitive and behavioral assessments. Methods: This was an observational multicenter study. Reported sleep quality was assessed using the Pediatric Daytime Sleepiness Scale (PDSS) and Pediatric Sleep Questionnaire (PSQ). Sleep quality was correlated to motor function (6 min walk test, 6MWT and grip strength; pulmonary function (predicted Forced Vital Capacity%, FVC% pred.); executive function assessed by BRIEF-2; autism traits assessed by Autism Spectrum Screening Questionnaire (ASSQ) and Repetitive Behavior Scale-revised (RBS-R); Quality of life (PedsQL) and disease burden (Congenital Childhood Myotonic Dystrophy Health Index, CCMDHI). Results: Forty-six patients were included, 33 CDM and 13 ChDM, at a median age of 10.4 and 15.1 years. Daytime sleepiness and disrupted sleep were reported by 30% children, in both subgroups of CDM and ChDM. Daytime sleepiness correlated with autism traits in CDM (p < 0.05). Disrupted sleep correlated with poorer executive function (p = 0.04) and higher disease burden (p = 0.03). Conclusions: Sleep issues are a feature of both CDM and ChDM. They correlate with behavioral issues and impact on disease burden.

Allelic heterogeneity and abnormal vesicle recycling in PLAA-related neurodevelopmental disorders.

The human PLAA gene encodes Phospholipase-A2-Activating-Protein (PLAA) involved in trafficking of membrane proteins. Through its PUL domain (PLAP, Ufd3p, and Lub1p), PLAA interacts with p97/VCP modulating synaptic vesicles recycling. Although few families carrying biallelic PLAA variants were reported with progressive neurodegeneration, consequences of monoallelic PLAA variants have not been elucidated. Using exome or genome sequencing we identified PLAA de-novo missense variants, affecting conserved residues within the PUL domain, in children affected with neurodevelopmental disorders (NDDs), including psychomotor regression, intellectual disability (ID) and autism spectrum disorders (ASDs). Computational and in-vitro studies of the identified variants revealed abnormal chain arrangements at C-terminal and reduced PLAA-p97/VCP interaction, respectively. These findings expand both allelic and phenotypic heterogeneity associated to PLAA-related neurological disorders, highlighting perturbed vesicle recycling as a potential disease mechanism in NDDs due to genetic defects of PLAA.

Early Onset Inherited Peripheral Neuropathies: The Experience of a Specialized Referral Center for Genetic Diagnosis Achievement.

BACKGROUND: Charcot-Marie-Tooth disease (CMT) is a heterogeneous group of inherited peripheral neuropathies. Although the typical disease onset is reported in the second decade, earlier onsets are not uncommon. To date, few studies on pediatric populations have been conducted and the achievement of molecular diagnosis remains challenging. METHODS: During the last 24 years we recruited 223 patients with early-onset hereditary peripheral neuropathies (EOHPN), negative for PMP22 duplication, 72 of them referred by a specialized pediatric hospital. Genetic testing for CMT-associated genes has been carried out with a range of different techniques. RESULTS: Of the 223 EOHPN cases, 43% were classified as CMT1 (demyelinating), 49% as CMT2 (axonal), and 8% as CMTi (intermediate). Genetic diagnosis was reached in 51% of patients, but the diagnostic yield increased to 67% when focusing only on cases from the specialized pediatric neuromuscular centers. Excluding PMP22 rearrangements, no significant difference in diagnostic rate between demyelinating and axonal forms was identified. De novo mutations account for 38% of cases. CONCLUSIONS: This study describes an exhaustive picture of EOHPN in an Italian referral genetic center and analyzes the molecular diagnostic rate of a heterogeneous cohort compared with one referred by a specialized pediatric center. Our data identify MPZ, MFN2, GDAP1, and SH3TC2 genes as the most frequent players in EOHPN. Our study underlines the relevance of a specific neurological pediatric expertise to address the genetic testing and highlights its importance to clarify possible unexpected results when neuropathy is only a secondary clinical sign of a more complex phenotype.

Caveolin-3 and Caveolin-1 Interaction Decreases Channel Dysfunction Due to Caveolin-3 Mutations.

Caveolae constitute membrane microdomains where receptors and ion channels functionally interact. Caveolin-3 (cav-3) is the key structural component of muscular caveolae. Mutations in CAV3 lead to caveolinopathies, which result in both muscular dystrophies and cardiac diseases. In cardiomyocytes, cav-1 participates with cav-3 to form caveolae; skeletal myotubes and adult skeletal fibers do not express cav-1. In the heart, the absence of cardiac alterations in the majority of cases may depend on a conserved organization of caveolae thanks to the expression of cav-1. We decided to focus on three specific cav-3 mutations (Δ62-64YTT; T78K and W101C) found in heterozygosis in patients suffering from skeletal muscle disorders. We overexpressed both the WT and mutated cav-3 together with ion channels interacting with and modulated by cav-3. Patch-clamp analysis conducted in caveolin-free cells (MEF-KO), revealed that the T78K mutant is dominant negative, causing its intracellular retention together with cav-3 WT, and inducing a significant reduction in current densities of all three ion channels tested. The other cav-3 mutations did not cause significant alterations. Mathematical modelling of the effects of cav-3 T78K would impair repolarization to levels incompatible with life. For this reason, we decided to compare the effects of this mutation in other cell lines that endogenously express cav-1 (MEF-STO and CHO cells) and to modulate cav-1 expression with an shRNA approach. In these systems, the membrane localization of cav-3 T78K was rescued in the presence of cav-1, and the current densities of hHCN4, hKv1.5 and hKir2.1 were also rescued. These results constitute the first evidence of a compensatory role of cav-1 in the heart, justifying the reduced susceptibility of this organ to caveolinopathies.

Variants in the WDR44 WD40-repeat domain cause a spectrum of ciliopathy by impairing ciliogenesis initiation.

WDR44 prevents ciliogenesis initiation by regulating RAB11-dependent vesicle trafficking. Here, we describe male patients with missense and nonsense variants within the WD40 repeats (WDR) of WDR44, an X-linked gene product, who display ciliopathy-related developmental phenotypes that we can model in zebrafish. The patient phenotypic spectrum includes developmental delay/intellectual disability, hypotonia, distinct craniofacial features and variable presence of brain, renal, cardiac and musculoskeletal abnormalities. We demonstrate that WDR44 variants associated with more severe disease impair ciliogenesis initiation and ciliary signaling. Because WDR44 negatively regulates ciliogenesis, it was surprising that pathogenic missense variants showed reduced abundance, which we link to misfolding of WDR autonomous repeats and degradation by the proteasome. We discover that disease severity correlates with increased RAB11 binding, which we propose drives ciliogenesis initiation dysregulation. Finally, we discover interdomain interactions between the WDR and NH2-terminal region that contains the RAB11 binding domain (RBD) and show patient variants disrupt this association. This study provides new insights into WDR44 WDR structure and characterizes a new syndrome that could result from impaired ciliogenesis.

DAG1 haploinsufficiency is associated with sporadic and familial isolated or pauci-symptomatic hyperCKemia.

DAG1 encodes for dystroglycan, a key component of the dystrophin-glycoprotein complex (DGC) with a pivotal role in skeletal muscle function and maintenance. Biallelic loss-of-function DAG1 variants cause severe muscular dystrophy and muscle-eye-brain disease. A possible contribution of DAG1 deficiency to milder muscular phenotypes has been suggested. We investigated the genetic background of twelve subjects with persistent mild-to-severe hyperCKemia to dissect the role of DAG1 in this condition. Genetic testing was performed through exome sequencing (ES) or custom NGS panels including various genes involved in a spectrum of muscular disorders. Histopathological and Western blot analyses were performed on muscle biopsy samples obtained from three patients. We identified seven novel heterozygous truncating variants in DAG1 segregating with isolated or pauci-symptomatic hyperCKemia in all families. The variants were rare and predicted to lead to nonsense-mediated mRNA decay or the formation of a truncated transcript. In four cases, DAG1 variants were inherited from similarly affected parents. Histopathological analysis revealed a decreased expression of dystroglycan subunits and Western blot confirmed a significantly reduced expression of beta-dystroglycan in muscle samples. This study supports the pathogenic role of DAG1 haploinsufficiency in isolated or pauci-symptomatic hyperCKemia, with implications for clinical management and genetic counseling.

Gas Humidification During Helmet Continuous Positive Airway Pressure: A Crossover Randomized Physiologic Study in Healthy Subjects.

BACKGROUND: In the last decades, the use of the helmet interface to deliver noninvasive respiratory support has steadily increased. When delivering oxygen therapy, conditioning of inspired gas is mandatory, as the water content of medical gas is neglectable. To minimize the risk of airway damage, it is recommended to achieve an absolute humidity greater than 10 mg H2O/L. The primary aim of the study was to assess the performance of 3 different heated humidifiers to condition gas during helmet continuous positive airway pressure and to compare them with a setting without active humidification. METHODS: We performed a crossover randomized physiological study in healthy volunteers. Absolute humidity, relative humidity, and temperature were measured during 4 steps (no humidification and the 3 heated humidifiers, performed in a randomized order) and at 3 time points (after 1, 5, and 10 minutes). RESULTS: Eight subjects participated to the study. Without active humidification, absolute humidity was constantly below the recommended level. All humidifiers conditioned the inspired gases to an average absolute humidity greater than 10 mg H2O/L. Overall, the best performance, in terms of absolute humidity, was obtained with H900 (19.74 ± 0.50 mg H2O/L), followed by TurbH2O (-6.82 mg H2O/L vs H900; 95% confidence interval, -8.49 to -5.14; P < .05) and F&P 810 (-9.03 mg H2O/L vs H900; 95% confidence interval, -10.70 to -7.35; P < .05). CONCLUSIONS: Active humidification is required to achieve adequate conditioning of inspired gas during helmet continuous positive airway pressure. The choice between different humidifiers available on the market should be made according to the local needs and expertise.

Statin-Induced Necrotizing Autoimmune Myopathy: Case Report of a Patient under Chronic Treatment.

Introduction: 3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) inhibitors are widely used worldwide to treat dyslipidaemia and prevent cardiovascular events. Statins can cause a wide variety of muscle injuries ranging from myalgia to severe rhabdomyolysis. In most cases, these symptoms are mild and self-limiting and do not require specific treatment besides drug withdrawal. Statin-induced autoimmune necrotizing myopathy (SINAM) is a rare but potentially fatal complication, characterized by the subacute onset of progressive proximal muscle weakness and considerably high creatine phosphokinase (CK) levels in patients exposed to statins. The diagnosis is supported by the presence of antibodies HMGCR, which allows the differentiation from other forms of necrotizing autoimmune myopathies. Symptoms usually progress even after statin discontinuation and can determine severe muscle damage. Summary. We describe the case of a 77-year-old man who developed SINAM after 5 years of statin use. He suffered from muscle functional impairment mainly involving proximal lower limb muscles which progressed to the point that he almost became bedridden. Initial treatment with prednisone alone was not effective, and he required a combination therapy with steroids, methotrexate, and intravenous immunoglobulins. After 5 months of therapy and rehabilitation, he showed complete laboratory response and muscle strength recovery. Conclusion: Recognizing SINAM is paramount in order to promptly start treatment and avoid permanent muscle damage. Using a combination therapy from the beginning could contribute to a better outcome. Prompt statin cessation, categorization of the muscle disease by autoantibody testing, imaging, and histology, exclusion of malignancy, and anti-inflammatory therapy with corticosteroids, antimetabolites, immunoglobulins, and in some cases rituximab are currently accepted approaches to this entity.

Case report: Episodic ataxia without ataxia?

Hereditary myopathies represent a clinically and genetically heterogeneous group of neuromuscular disorders, characterized by highly variable clinical presentations and frequently overlapping phenotypes with other neuromuscular disorders, likely influenced by genetic and environmental modifiers. Genetic testing is often challenging due to ambiguous clinical diagnosis. Here, we present the case of a family with clinical and Electromyography (EMG) features resembling a myotonia-like disorder in which Whole Exome Sequencing (WES) analysis revealed the co-segregation of two rare missense variants in UBR4 and HSPG2, genes previously associated with episodic ataxia 8 (EA8). A review of the literature highlighted a striking overlap between the clinical and the molecular features of our family and the previously described episodic ataxias (EAs), which raises concerns about the genotype-phenotype correlation, clinical variability, and the confounding overlap in these groups of disorders. This emphasizes the importance of thoroughly framing the patient's phenotype. The more clear-cut the diagnosis, the easier the identification of a genetic determinant, and the better the prognosis and the treatment of patients.

Myopathologic trajectory in Duchenne muscular dystrophy (DMD) reveals lack of regeneration due to senescence in satellite cells.

Duchenne muscular dystrophy (DMD) is a devastating X-linked muscular disease, caused by mutations in the DMD gene encoding Dystrophin and affecting 1:5000 boys worldwide. Lack of Dystrophin leads to progressive muscle wasting and degeneration resulting in cardiorespiratory failure. Despite the absence of a definitive cure, innovative therapeutic avenues are emerging. Myopathologic studies are important to further understand the biological mechanisms of the disease and to identify histopathologic benchmarks for clinical evaluations. We conducted a myopathologic analysis on twenty-four muscle biopsies from DMD patients, with particular emphasis on regeneration, fibro-adipogenic progenitors and muscle stem cells behavior. We describe an increase in content of fibro-adipogenic progenitors, central orchestrators of fibrotic progression and lipid deposition, concurrently with a decline in muscle regenerative capacity. This regenerative impairment strongly correlates with compromised activation and expansion of muscle stem cells. Furthermore, our study uncovers an early acquisition of a senescence phenotype by DMD-afflicted muscle stem cells. Here we describe the myopathologic trajectory intrinsic to DMD and establish muscle stem cell senescence as a pivotal readout for future therapeutic interventions.

A novel de novo variant in POLR3B gene associated with a primary axonal involvement of the largest nerve fibers.

BACKGROUND AND AIMS: POLR3B gene encodes a subunit of RNA polymerase III (Pol III). Biallelic mutations in POLR3B are associated with leukodystrophies, but recently de novo heterozygous mutations have been described in early onset peripheral demyelinating neuropathies with or without central involvement. Here, we report the first Italian case carrying a de novo variant in POLR3B with a pure neuropathy phenotype and primary axonal involvement of the largest nerve fibers. METHODS: Nerve conduction studies, sympathetic skin response, dynamic sweat test, tactile and thermal quantitative sensory testing and brain magnetic resonance imaging were performed according to standard procedures. Histopathological examination was performed on skin and sural nerve biopsies. Molecular analysis of the proband and his relatives was performed with Next Generation Sequencing. The impact of the identified variant on the overall protein structure was evaluated through rotamers method. RESULTS: Since his early adolescence, the patient presented with signs of polyneuropathy with severe distal weakness, atrophy, and reduced sensation. Neurophysiological studies showed a sensory-motor axonal polyneuropathy, with confirmed small fiber involvement. In addition, skin biopsy and sural nerve biopsy showed predominant large fibers involvement. A trio's whole exome sequencing revealed a novel de novo variant p.(Arg1046Cys) in POLR3B, which was classified as Probably Pathogenic. Molecular modeling data confirmed a deleterious effect of the variant on protein structure. INTERPRETATION: Neurophysiological and morphological findings suggest a primary axonal involvement of the largest nerve fibers in POLR3B-related neuropathies. A partial loss of function mechanism is proposed for both neuropathy and leukodystrophy phenotypes.

An early onset benign myopathy with glycogen storage caused by a de novo 1.4 Mb-deletion of chromosome 14.

Early onset myopathies are a clinically and histologically heterogeneous monogenic diseases linked to approximately 90 genes. Molecular diagnosis is challenging, especially in patients with a mild phenotype. We describe a 26-year-old man with neonatal hypotonia, motor delay and seizures during infancy, and non-progressive, mild muscular weakness in adulthood. Serum Creatine kinase level was normal. Whole-body muscle MRI showed thin muscles, and brain MRI was unremarkable. A deltoid muscle biopsy showed glycogen storage. WGS revealed a de novo 1.4 Mb-deletion of chromosome 14, confirmed by Array-CGH. This microdeletion causes the loss of ten genes including RALGAPA1, encoding for RalA, a regulator of glucose transporter 4 (GLUT4) expression at the membrane of myofibers. GLUT4 was overexpressed in patient's muscle. Here we highlight the importance to search for chromosomal alterations in the diagnostic workup of early onset myopathies.

Early Muscle MRI Findings in a Pediatric Case of Emery-Dreifuss Muscular Dystrophy Type 1.

Emery-Dreifuss muscular dystrophy (EDMD) is a rare disease characterized by early contractures, progressive muscle weakness, and cardiac abnormalities. Different subtypes of EDMD have been described, with the two most common forms represented by the X-linked EDMD1, caused by mutations in the EMD gene encoding emerin, and the autosomal EDMD2, due to mutations in the LMNA gene encoding lamin A/C. A clear definition of the magnetic resonance imaging (MRI) pattern in the two forms, and especially in the rarer EDMD1, is still lacking, although a preferential involvement of the medial head of the gastrocnemius has been suggested in EDMD2. We report a 13-year-old boy with mild limb girdle muscle weakness, elbow and ankle contractures, with absence of emerin at muscle biopsy, carrying a hemizygous frameshift mutation on the EMD gene (c.153dupC/p.Ser52Glufs*9) of maternal inheritance. Minor cardiac rhythm abnormalities were detected at 24-hour Holter electrocardiogram and required ß-blocker therapy. MRI scan of the thighs showed a mild diffuse involvement, while tibialis anterior, extensor digitorum longus, peroneus longus, and medial gastrocnemius were the most affected muscles in the leg. We also provide a review of the muscular MRI data in EDMD patients and highlight the relative heterogeneity of the MRI patterns found in EDMDs, suggesting that muscle MRI should be studied in larger EDMD cohorts to better define disease patterns and to cover the wide disease spectrum.

Clinical and functional characterization of a long survivor congenital titinopathy patient with a novel metatranscript-only titin variant.

Congenital titinopathies are an emerging group of a potentially severe form of congenital myopathies caused by biallelic mutations in titin, encoding the largest existing human protein involved in the formation and stability of sarcomeres. In this study we describe a patient with a congenital myopathy characterized by multiple contractures, a rigid spine, non progressive muscular weakness, and a novel homozygous TTN pathogenic variant in a metatranscript-only exon: the c.36400A > T, p.Lys12134*. Muscle biopsies showed increased internalized nuclei, variability in fiber size, mild fibrosis, type 1 fiber predominance, and a slight increase in the number of satellite cells. RNA studies revealed the retention of intron 170 and 171 in the open reading frame, and immunoflourescence and western blot studies, a normal titin content. Single fiber functional studies showed a slight decrease in absolute maximal force and a cross-sectional area with no decreases in tension, suggesting that weakness is not sarcomere-based but due to hypotrophy. Passive properties of single fibers were not affected, but the observed increased calcium sensitivity of force generation might contribute to the contractural phenotype and rigid spine of the patient. Our findings provide evidence for a pathogenic, causative role of a metatranscript-only titin variant in a long survivor congenital titinopathy patient with distal arthrogryposis and rigid spine.

Thyroid-stimulating hormone receptor signaling restores skeletal muscle stem cell regeneration in rats with muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a severe and progressive myopathy leading to motor and cardiorespiratory impairment. We analyzed samples from patients with DMD and a preclinical rat model of severe DMD and determined that compromised repair capacity of muscle stem cells in DMD is associated with early and progressive muscle stem cell senescence. We also found that extraocular muscles (EOMs), which are spared by the disease in patients, contain muscle stem cells with long-lasting regenerative potential. Using single-cell transcriptomics analysis of muscles from a rat model of DMD, we identified the gene encoding thyroid-stimulating hormone receptor (Tshr) as highly expressed in EOM stem cells. Further, TSHR activity was involved in preventing senescence. Forskolin, which activates signaling downstream of TSHR, was found to reduce senescence of skeletal muscle stem cells, increase stem cell regenerative potential, and promote myogenesis, thereby improving muscle function in DMD rats. These findings indicate that stimulation of adenylyl cyclase leads to muscle repair in DMD, potentially providing a therapeutic approach for patients with the disease.

Long-term clinical and MRI follow-up in two POMT2-related limb girdle muscular dystrophy (LGMDR14) patients.

INTRODUCTION: POMT2-related limb girdle muscular dystrophy (LGMDR14) is a rare muscular dystrophy caused by mutations in the POMT2 gene. Thus far only 26 LGMDR14 subjects have been reported and no longitudinal natural history data are available. CASE REPORT: We describe two LGMDR14 patients followed for 20 years since infancy. Both patients presented a childhood-onset, slowly progressive pelvic girdle muscular weakness leading to loss of ambulation in the second decade in one patient, and cognitive impairment without detectable brain structural abnormalities. Glutei, paraspinal, and adductor muscles were the primarily involved muscles at MRI. DISCUSSION: This report provides natural history data on LGMDR14 subjects, with a focus on longitudinal muscle MRI. We also reviewed the LGMDR14 literature data, providing information on the LGMDR14 disease progression. Considering the high prevalence of cognitive impairment in LGMDR14 patients, a reliable application of functional outcome measures can be challenging, therefore a muscle MRI follow-up to assess disease evolution is recommended.

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.