Trimetazidine Improves Mitochondrial Dysfunction in SOD1G93A Cellular Models of Amyotrophic Lateral Sclerosis through Autophagy Activation.

Amyotrophic Lateral Sclerosis (ALS) is considered the prototype of motor neuron disease, characterized by motor neuron loss and muscle waste. A well-established pathogenic hallmark of ALS is mitochondrial failure, leading to bioenergetic deficits. So far, pharmacological interventions for the disease have proven ineffective. Trimetazidine (TMZ) is described as a metabolic modulator acting on different cellular pathways. Its efficacy in enhancing muscular and cardiovascular performance has been widely described, although its molecular target remains elusive. We addressed the molecular mechanisms underlying TMZ action on neuronal experimental paradigms. To this aim, we treated murine SOD1G93A-model-derived primary cultures of cortical and spinal enriched motor neurons, as well as a murine motor-neuron-like cell line overexpressing SOD1G93A, with TMZ. We first characterized the bioenergetic profile of the cell cultures, demonstrating significant mitochondrial dysfunction that is reversed by acute TMZ treatments. We then investigated the effect of TMZ in promoting autophagy processes and its impact on mitochondrial morphology. Finally, we demonstrated the effectiveness of TMZ in terms of the mitochondrial functionality of ALS-rpatient-derived peripheral blood mononuclear cells (PBMCs). In summary, our results emphasize the concept that targeting mitochondrial dysfunction may represent an effective therapeutic strategy for ALS. The findings demonstrate that TMZ enhances mitochondrial performance in motor neuron cells by activating autophagy processes, particularly mitophagy. Although further investigations are needed to elucidate the precise molecular pathways involved, these results hold critical implications for the development of more effective and specific derivatives of TMZ for ALS treatment.

Comprehensive Cardiovascular Management of Myotonic Dystrophy Type 1 Patients: A Report from the Italian Neuro-Cardiology Network.

Myotonic dystrophy is a hereditary disorder with systemic involvement. The Italian Neuro-Cardiology Network-"Rete delle Neurocardiologie" (INCN-RNC) is a unique collaborative experience involving neurology units combined with cardio-arrhythmology units. The INCN facilitates the creation of integrated neuro-cardiac teams in Neuromuscular Disease Centers for the management of cardiovascular involvement in the treatment of myotonic dystrophy type 1 (MD1).

In Cis Effect of DMPK Expanded Alleles in Myotonic Dystrophy Type 1 Patients Carrying Variant Repeats at 5' and 3' Ends of the CTG Array.

Myotonic dystrophy type 1 (DM1) is an autosomal dominant multisystemic disease caused by a CTG repeat expansion in the 3'-untranslated region (UTR) of DMPK gene. DM1 alleles containing non-CTG variant repeats (VRs) have been described, with uncertain molecular and clinical consequences. The expanded trinucleotide array is flanked by two CpG islands, and the presence of VRs could confer an additional level of epigenetic variability. This study aims to investigate the association between VR-containing DMPK alleles, parental inheritance and methylation pattern of the DM1 locus. The DM1 mutation has been characterized in 20 patients using a combination of SR-PCR, TP-PCR, modified TP-PCR and LR-PCR. Non-CTG motifs have been confirmed by Sanger sequencing. The methylation pattern of the DM1 locus was determined by bisulfite pyrosequencing. We characterized 7 patients with VRs within the CTG tract at 5' end and 13 patients carrying non-CTG sequences at 3' end of the DM1 expansion. DMPK alleles with VRs at 5' end or 3' end were invariably unmethylated upstream of the CTG expansion. Interestingly, DM1 patients with VRs at the 3' end showed higher methylation levels in the downstream island of the CTG repeat tract, preferentially when the disease allele was maternally inherited. Our results suggest a potential correlation between VRs, parental origin of the mutation and methylation pattern of the DMPK expanded alleles. A differential CpG methylation status could play a role in the phenotypic variability of DM1 patients, representing a potentially useful diagnostic tool.

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.

NEFL-Related Charcot-Marie Tooth Disease due to P440L Mutation in Two Italian Families: Expanding the Phenotype and Defining Modulating Factors.

INTRODUCTION: Mutations in the neurofilament polypeptide light chain (NEFL) gene account for <1% of all forms of Charcot-Marie-Tooth (CMT) diseases and present with different phenotypes, including demyelinating, axonal and intermediate neuropathies, and with diverse pattern of transmission, with dominant and recessive inheritance being described. METHODS: Here, we present clinical and molecular data in two new unrelated Italian families, affected with CMT. RESULTS: We studied fifteen subjects (11 women, 4 men), age range 23-62 year. Onset of symptoms was mainly in childhood, with running/walking difficulties; some patients were pauci-asymptomatic; almost all shared variably distributed features of absent/reduced deep tendon reflexes, impaired gait, reduced sensation, and distal weakness in the legs. Skeletal deformities were seldom documented and were of mild degree. Additional features included sensorineural hearing loss in 3 patients, underactive bladder in 2 patients, and cardiac conduction abnormalities, requiring pacemaker implantation, in one child. Central nervous system (CNS) impairment was not documented in any subject. Neurophysiological investigation disclosed feature suggestive of demyelinating sensory-motor polyneuropathy in one family and resembling an intermediate form in the other. Multigene panel analysis of all known CMT genes revealed two heterozygous variants in NEFL: p.E488K and p.P440L. While the latter change segregated with the phenotype, the p.E488K variant appeared to act as a modifier factor being associated with axonal nerve damage. CONCLUSIONS: CMT related to P440L mutation in NEFL is associated with a mild, childhood-onset phenotype, showing prevalently sensory distal limbs involving and with motor impairment predominantly involving anterolateral leg muscles, in the absence of CNS involvement. Additional findings, never reported so far in patients with NEFL mutation, are cardiological and urinary dysfunctions. Our study expands the array of clinical features associated with NEFL-related CMT.

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.

Characterization of full-length CNBP expanded alleles in myotonic dystrophy type 2 patients by Cas9-mediated enrichment and nanopore sequencing.

Myotonic dystrophy type 2 (DM2) is caused by CCTG repeat expansions in the CNBP gene, comprising 75 to >11,000 units and featuring extensive mosaicism, making it challenging to sequence fully expanded alleles. To overcome these limitations, we used PCR-free Cas9-mediated nanopore sequencing to characterize CNBP repeat expansions at the single-nucleotide level in nine DM2 patients. The length of normal and expanded alleles can be assessed precisely using this strategy, agreeing with traditional methods, and revealing the degree of mosaicism. We also sequenced an entire ~50 kbp expansion, which has not been achieved previously for DM2 or any other repeat-expansion disorders. Our approach precisely counted the repeats and identified the repeat pattern for both short interrupted and uninterrupted alleles. Interestingly, in the expanded alleles, only two DM2 samples featured the expected pure CCTG repeat pattern, while the other seven presented also TCTG blocks at the 3' end, which have not been reported before in DM2 patients, but confirmed hereby with orthogonal methods. The demonstrated approach simultaneously determines repeat length, structure/motif, and the extent of somatic mosaicism, promising to improve the molecular diagnosis of DM2 and achieve more accurate genotype-phenotype correlations for the better stratification of DM2 patients in clinical trials.

A Clinical and Epidemiological Prevalence Study on Friedreich's Ataxia in Latium, Italy.

OBJECTIVE: The aim of this study was to estimate the Friedreich's ataxia (FRDA) prevalence in a highly populated region of Italy (previous studies in small geographic areas gave a largely variable prevalence) and to define the patients' molecular and clinical characteristics. METHODS: For the point-prevalence study, we considered patients belonging to families with a molecular diagnosis of FRDA and resident in Latium on 1 January 2019. The crude prevalence of FRDA, specific for age and sex, was calculated and standardized for age using the Italian population. Moreover, we investigated possible correlations among patients' genetic profile, symptoms, and age of onset. RESULTS: We identified 63 FRDA patients; the crude prevalence for total, males, and females were 1.07 (95% CI: 0.81-1.37), 0.81 (95% CI: 0.54-1.22), and 1.32 (95% CI: 0.97-1.79), per 100,000 inhabitants. We divided FRDA patients by three age-at-onset groups (early-EOFA 73%; late-LOFA 11.1%; very late-VLOFA 15.9%) and found significant differences in the scale for the assessment and rating of ataxia (SARA; p = 0.001), a biased distribution of the shorter allele (p = 0.001), an excess of scoliosis and cardiomyopathy (p = 0.001) in EOFA. To determine the contribution of patients' molecular and clinical characteristics to the annual rate of progression, we performed a multivariate regression analysis that gave an R2 value of 45.3%. CONCLUSIONS: We estimated the crude and standardized prevalence of FRDA in Latium. A clinical classification (EOFA, LOFA, VLOFA) gave significant correlations. This epidemiological estimate allows monitoring disease prevalence over time in cohort studies and/or for developing disease registry.

Clinical-Genetic Features Influencing Disability in Spastic Paraplegia Type 4: A Cross-sectional Study by the Italian DAISY Network.

Background and Objectives: Hereditary spastic paraplegias (HSPs) are a group of inherited rare neurologic disorders characterized by length-dependent degeneration of the corticospinal tracts and dorsal columns, whose prominent clinical feature is represented by spastic gait. Spastic paraplegia type 4 (SPG4, SPAST-HSP) is the most common form. We present both clinical and molecular findings of a large cohort of patients, with the aim of (1) defining the clinical spectrum of SPAST-HSP in Italy; (2) describing their molecular features; and (3) assessing genotype-phenotype correlations to identify features associated with worse disability. Methods: A cross-sectional retrospective study with molecular and clinical data collected in an anonymized database was performed. Results: A total of 723 Italian patients with SPAST-HSP (58% men) from 316 families, with a median age at onset of 35 years, were included. Penetrance was 97.8%, with men showing higher Spastic Paraplegia Rating Scale (SPRS) scores (19.67 ± 12.58 vs 16.15 ± 12.61, p = 0.009). In 26.6% of patients with SPAST-HSP, we observed a complicated phenotype, mainly including intellectual disability (8%), polyneuropathy (6.7%), and cognitive decline (6.5%). Late-onset cases seemed to progress more rapidly, and patients with a longer disease course displayed a more severe neurologic disability, with higher SPATAX (3.61 ± 1.46 vs 2.71 ± 1.20, p < 0.001) and SPRS scores (22.63 ± 11.81 vs 12.40 ± 8.83, p < 0.001). Overall, 186 different variants in the SPAST gene were recorded, of which 48 were novel. Patients with SPAST-HSP harboring missense variants displayed intellectual disability (14.5% vs 4.4%, p < 0.001) more frequently, whereas patients with truncating variants presented more commonly cognitive decline (9.7% vs 2.6%, p = 0.001), cerebral atrophy (11.2% vs 3.4%, p = 0.003), lower limb spasticity (61.5% vs 44.5%), urinary symptoms (50.0% vs 31.3%, p < 0.001), and sensorimotor polyneuropathy (11.1% vs 1.1%, p < 0.001). Increasing disease duration (DD) and abnormal motor evoked potentials (MEPs) were also associated with increased likelihood of worse disability (SPATAX score>3). Discussion: The SPAST-HSP phenotypic spectrum in Italian patients confirms a predominantly pure form of HSP with mild-to-moderate disability in 75% of cases, and slight prevalence of men, who appeared more severely affected. Early-onset cases with intellectual disability were more frequent among patients carrying missense SPAST variants, whereas patients with truncating variants showed a more complicated disease. Both longer DD and altered MEPs are associated with worse disability.

The actigraphic documentation of circadian sleep-wake rhythm dysregulation in myotonic dystrophy type 1.

STUDY OBJECTIVES: The present study aimed at identifying the sleep-wake rhythm in patients with myotonic dystrophy type 1 (DM1) compared to healthy controls. METHODS: Patients with genetic diagnosis of DM1 and healthy controls underwent a 7-day actigraphic recording and filled out a daily sleep diary to evaluate the sleep-wake rhythm. All participants underwent a physical and neurological examination to exclude conditions interfering with the sleep-wake cycle. Daytime activity, nocturnal sleep, and non-parametric circadian rhythm activity (NPCRA) were analysed. RESULTS: Twenty-nine patients affected by DM1 were included in the present study and were compared to 16 controls. Considering nocturnal actigraphic data, DM1 patients showed a longer time in bed, sleep period time, actual sleep time, and sleep latency compared to controls. Central phase measurement was significantly longer in DM1 patients than controls. At NPCRA analysis patients showed a lower degree of regularity in the activity-rest pattern compared to controls. Moreover, DM1 patients showed reduced motor activity during daytime and a lower synchronization of the rest-activity rhythm than controls. CONCLUSIONS: This study documented that patients with DM1 not only present the impairment of nocturnal sleep, but also show a dysregulation of the sleep-wake circadian rhythm; moreover, reduced amplitude of the circadian rhythmicity was also evident in comparison to controls, probably in relation to the reduced diurnal motor activity of patients. These findings add further evidence to the already documented sleep impairment and excessive daytime sleepiness in DM1 patients.

A 14-Year Italian Experience in DM2 Genetic Testing: Frequency and Distribution of Normal and Premutated CNBP Alleles.

Myotonic dystrophy type 2 (DM2) is a multisystemic disorder caused by a (CCTG) n in intron 1 of the CNBP gene. The CCTG repeat tract is part of a complex (TG) v (TCTG) w (CCTG) x (NCTG) y (CCTG) z motif generally interrupted in CNBP healthy range alleles. Here we report our 14-year experience of DM2 postnatal genetic testing in a total of 570 individuals. The DM2 locus has been analyzed by a combination of SR-PCR, TP-PCR, LR-PCR, and Sanger sequencing of CNBP alleles. DM2 molecular diagnosis has been confirmed in 187/570 samples analyzed (32.8%) and is mainly associated with the presence of myotonia in patients. This set of CNBP alleles showed unimodal distribution with 25 different alleles ranging from 108 to 168 bp, in accordance with previous studies on European populations. The most frequent CNBP alleles consisted of 138, 134, 140, and 136 bps with an overall locus heterozygosity of 90%. Sequencing of 103 unexpanded CNBP alleles in DM2-positive patients revealed that (CCTG)5(NCTG)3(CCTG)7 and (CCTG)6(NCTG)3(CCTG)7 are the most common interruption motifs. We also characterized five CNBP premutated alleles with (CCTG) n repetitions from n = 36 to n = 53. However, the molecular and clinical consequences in our cohort of samples are not unequivocal. Data that emerged from this study are representative of the Italian population and are useful tools for National and European centers offering DM2 genetic testing and counseling.

ATTRv in Lazio-Italy: A High-Prevalence Region in a Non-Endemic Country.

Hereditary transthyretin amyloidosis (ATTRv, v for variant) prevalence in Italy, a non-endemic region, has been established by ATTRv amyloidosis Italian Registry. However, values of prevalence were extremely heterogeneous, considering different regions. To properly establish the prevalence of the disease in the Lazio region, a survey was sent to university regional hospitals and to main regional hospitals, in order to collect all affected patients regularly followed. We identified 100 ATTRv patients and, considering a Lazio population of 5.8/million, we estimated a ATTRv prevalence of 17.2/million. The ATTRv amyloidosis Italian Registry reported a prevalence of 8.0/million in Lazio, while our survey showed a value of double this. Our survey documented a high-prevalence for a non-endemic country. The increased awareness of the disease among general practitioners and medical specialists is a fundamental step to reduce the diagnostic delay and start an effective treatment of this disease.

Assessment of self-reported and objective daytime sleepiness in adult-onset myotonic dystrophy type 1.

STUDY OBJECTIVES: Excessive daytime sleepiness (EDS) in myotonic dystrophy type 1 is mostly of central origin but it may coexist with sleep-related breathing disorders. However, there is no consensus on the sleep protocols to be used, assessments vary, and only a minority of patients are regularly tested or are on treatment for EDS. Our study presents data on self-reported and objective EDS in adult-onset myotonic dystrophy type 1. METHODS: Sixty-three patients with adult-onset DM1 were subjected to EDS-sleep assessments (polysomnography, Multiple Sleep Latency Test, Epworth Sleepiness Scale). Correlation coefficients were computed to assess the relationship between sleep and sleepiness test results, fatigue, and quality of life. RESULTS: 33% and 48% of patients had EDS based, respectively, on the Epworth Sleepiness Scale and the Multiple Sleep Latency Test, with a low concordance between these tests (k = 0.19). Thirteen patients (20%) displayed 2 or more sleep-onset rapid eye movement periods on Multiple Sleep Latency Test. Patients having EDS by Multiple Sleep Latency Test had a shorter disease duration (P < .05), higher total sleep time and sleep efficiency and lower wake after sleep onset on polysomnography. Patients with self-reported EDS reported significantly higher fatigue score compared with patients without EDS (P < .05). No other difference was found in demographic, clinical, and respiratory features. CONCLUSIONS: EDS test results are contradictory, making treatment options difficult. Combining quantitative tests and self-reported scales may facilitate physicians in planning EDS care with patients and families. CITATION: Sansone VA, Proserpio P, Mauro L, et al. Assessment of self-reported and objective daytime sleepiness in adult-onset myotonic dystrophy type 1. J Clin Sleep Med. 2021;17(12):2383-2391.

Movement disorders in primary central nervous system lymphoma: two unreported cases and a review of literature.

BACKGROUND: Recognition of secondary movement disorders (SMDs) is fundamental either to alleviate disabling disturbances or to treat potentially life-threatening conditions, such as brain tumors. Primary CNS lymphoma (PCNSL) is a rare form of CNS cancer that is often located in subcortical areas, accounting for both neuropsychiatric and motoric disorders. Nevertheless, an overview on PCNSL-related movement disorders (MDs) phenomenology has not been provided yet. OBJECTIVE: To outline the main features of PCNSL-related MDs. METHODS: A retrospective analysis was conducted on a cohort of patients with PCNSL presenting with MDs, including all existing cases identified by a systematic literature review (source: Medline; period: 1946-2020) and two unreported cases. Data on phenomenology, neuroimaging, histology, and clinical course were collected. RESULTS: A total cohort of fifteen subjects was defined, enrolling thirteen previously described patients extracted from eleven published studies, and our two unreported cases. A parkinsonian syndrome appearing at about 60 years of age, unresponsive to levodopa, associated to other neurological signs, resulted as the most common presentation of PCNSL-related MD. Chorea, dystonia, and dyskinesia occurred less frequently, with some degree of responsiveness to symptomatic treatments. Basal ganglia were involved in most cases and motoric disturbances often ameliorated after tumor mass reduction. CONCLUSIONS: This study identified those features of PCNSL-related MDs that could support an appropriate approach to such a rare condition. In fact, while the outcome remains still poor, the therapeutic scenario of PCNSL is changing; an early diagnosis together with an adequate management will be thus crucial for timely and successful interventions.

Cochlear Dysfunction Is a Frequent Feature of Facioscapulohumeral Muscular Dystrophy Type 1 (FSHD1).

INTRODUCTION: Facioscapulohumeral muscular dystrophy type 1 (FSHD) represents one of the most common forms of muscular hereditary diseases and it is characterized by a great clinical variability with the typical muscular symptoms and other clinical features, including hearing impairment. However, etiopathogenetic mechanisms of auditory dysfunction are still not completely understood and it has been suggested that it could be assigned to a cochlear alteration that is present even in those subjects with a normal pure tonal audiometry (PTA) examination. METHODS: We found out the cochlear function in 26 patients with molecular diagnosis of FSHD1 and in healthy controls. All patients underwent complete neurological and audiological examinations, including FSHD clinical score, pure-tone audiometry (PTA), and otoacoustic emissions (OAEs), in particular transient evoked otoacoustic emissions (TEOAEs) and distortion product evoked otoacoustic emissions (DPOAEs). RESULTS: All FSHD1 patients showed significantly reduced DPOAEs and TEOAEs, bilaterally and at all frequencies, even when considering only subjects with a normal PTA or a mild muscular involvement (FSHD score ≤ 2). No correlation between OAEs and FSHD clinical score was found. DISCUSSION: Cochlear echoes represent a sensitive tool in detecting subclinical cochlear dysfunction in FSHD1 even in subjects with normal hearing and/or subtle muscle involvement. Our study is focused on the importance of evaluating the cochlear alteration through OAEs and, in particular, by performing TEOAEs and DPOAEs sequentially, to evaluate more frequent specificities of cochlear dysfunction with a wider spectrum of analysis.

How to capture activities of daily living in myotonic dystrophy type 2?

Myotonic dystrophy type 2 (DM2) lacks validated patients´ reported outcomes (PROs). This represents a limit for monitoring disease progression and perceived efficacy of symptomatic treatments. Our aim was to investigate whether PROs for activities of daily living designed for other neuromuscular diseases could be used in DM2. Sixty-six DM2 patients completed the following PROs: DM1-Activ-c, Rasch-built Pompe-specific activity (R-PAct) scale, McGill-pain questionnaire, fatigue and daytime sleepiness scale and Beck depression inventory (BDI-II). Clinical data and motor outcome measures (6-minutes walking test - 6MWT, manual muscle testing, quick motor function test and myotonia behavior scale) were collected as well. Patients underwent one visit at baseline and one after 10 months. Ceiling/flooring effects, criterion validity and discriminant validity were calculated. DM1-activ-c and R-PAct showed acceptable ceiling effects despite being built for myotonic dystrophy type 1 and Pompe disease, respectively. The difficulty hierarchy of the single items was better preserved in R-PAct than in DM1-Activ-c. Both tests showed excellent criterion validity highly correlating with 6MWT, quick motor function test, myalgia and disease duration. They could partially discriminate patients with different disability grades. These results suggest that DM1-Activ-c, slightly better than R-PAct, might be adopted for monitoring activities of daily living also in DM2, at least until disease-specific PROs will be available.

Validation of Motor Outcome Measures in Myotonic Dystrophy Type 2.

Introduction: Myotonic dystrophy type 2 (DM2) lacks disease-specific, validated, motor outcome measures (OMs), and patients' reported outcomes (PROs). This represents a limit for the monitoring of disease progression and treatment response. Our aim was to identify the most appropriate OMs to be translated in clinical practice and clinical trials on DM2. This study has been registered on clinicaltrials.gov NCT03603171 (https://clinicaltrials.gov/ct2/show/NCT03603171). Methods: Sixty-six patients with genetically confirmed DM2 underwent a baseline and a follow-up visit after 1 year. The tested OMs included: hand opening time, pressure pain threshold (PPT), manual muscle testing (MMT), hand held dynamometry (HHD), scale for the assessment and rating of ataxia (SARA), quantitative motor function test (QMFT), gait stairs Gowers chair (GSGC), 30-s sit to stand test, functional index 2 (FI-2) and 6MWT. The PROs included DM1-Active-C, Rasch-built Pompe-specific activity scale (R-Pact), fatigue and daytime sleepiness (FDSS), brief pain inventory short form (BPI-sf), myotonia behavior scale (MBS), and the McGill pain questionnaire. Results: All patients completed the MBS and the results correlated well with the hand-opening time. The PPT showed a low reliability, no correlation with pain questionnaires, and did not differentiate patients with or without myalgia. Both muscle strength assessments, MMT and HHD, showed good construct validity. The QMFT showed an acceptable ceiling effect (14.5%), good convergent and differential validity and performed overall better than GSGC. The SARA score showed high flooring effect and is not useful in DM2. 6MWT proved a valid outcome measure in DM2. The 30-s sit to stand is a feasible test with good convergent validity, showing a flooring effect of 20% as it cannot be used in more severely affected patients. The FI-2 is time-consuming and has a high ceiling effect. At the 1-year visit the only assessments able to detect a worsening of DM2 were HHD, QMFT, and 6MWT, which are the most sensitive to change, and therefore clinically meaningful OMs in DM2. Conclusion: The clinical meaningful motor outcome measures that best depict the multifaceted phenotype of DM2 and its slow progression are MBS, MMT, or HHD (depending on the clinical setting), QMFT, and the 6MWT.

Design of a multiplex ligation-dependent probe amplification assay for SLC20A2: identification of two novel deletions in primary familial brain calcification.

Primary familial brain calcification (PFBC) is a rare disease characterized by brain calcifications that mainly affect the basal ganglia, thalamus, and cerebellum. Among the four autosomal-dominant genes known to be associated with the disease, SLC20A2 pathogenic variants are the most common, accounting for up to 40% of PFBC dominant cases; variants include both point mutations, small insertions/deletions and intragenic deletions. Over the last 7 years, we have collected a group of 50 clinically diagnosed PFBC patients, who were screened for single nucleotide changes and small insertions/deletions in SLC20A2 by Sanger sequencing. We found seven pathogenic/likely pathogenic variants: four were previously described by our group, and three are reported here (c.303delG, c.21delG, and c.1795-1G>A). We developed and validated a synthetic Multiplex Ligation-dependent Probe Amplification (MLPA) assay for SLC20A2 deletions, covering all ten coding exons and the 5' UTR (SLC20A2-MLPA). Using this method, we screened a group of 43 PFBC-patients negative for point mutations and small insertions/deletions, and identified two novel intragenic deletions encompassing exon 6 NC_000008.10:g.(42297172_42302163)_(423022281_42317413)del, and exons 7-11 including the 3'UTR NC_000008.10:g.(?_42275320)_(42297172_42302163)del. Overall, SLC20A2 deletions may be highly underestimated PFBC cases, and we suggest MLPA should be included in the routine molecular test for PFBC diagnosis.