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

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

Autosomal recessive limb-girdle and Miyoshi muscular dystrophies in the Netherlands: The clinical and molecular spectrum of 244 patients.

In this retrospective study, we conducted a clinico-genetic analysis of patients with autosomal recessive limb-girdle muscular dystrophy (LGMD) and Miyoshi muscular dystrophy (MMD). Patients were identified at the tertiary referral centre for DNA diagnosis in the Netherlands and included if they carried two mutations in CAPN3, DYSF, SGCG, SGCA, SGCB, SGCD, TRIM32, FKRP or ANO5 gene. DNA was screened by direct sequencing and multiplex ligand-dependent probe amplification (MLPA) analysis. A total of 244 patients was identified; 68 LGMDR1/LGMD2A patients with CAPN3 mutations (28%), 67 sarcoglycanopathy patients (LGMDR3-5/LGMD2C-E) (27%), 64 LGMDR12/LGMD2L and MMD3 patients with ANO5 mutations (26%), 25 LGMDR2/LGMD2B and MMD1 with DYSF mutations (10%), 21 LGMDR9/LGMD2I with FKRP mutations (9%) and one LGMDR8/LGMD2H patient with TRIM32 mutations (<1%). The estimated minimum prevalence of AR-LGMD and MMD in the Netherlands amounted to 14.4 × 10-6 . Thirty-three novel mutations were identified. A wide range in age of onset (0-72 years) and loss of ambulation (5-74 years) was found. Fifteen patients (6%) initially presented with asymptomatic hyperCKemia. Cardiac abnormalities were found in 35 patients (17%). Non-invasive ventilation was started in 34 patients (14%). Both cardiac and respiratory involvement occurs across all subtypes, stressing the need for screening in all included subtypes.

Social participation of adult patients with spinal muscular atrophy: Frequency, restrictions, satisfaction, and correlates.

INTRODUCTION: In this study we assessed social participation in 62 adults with spinal muscular atrophy (SMA) types 1c-4. METHODS: The outcome measure used was the Utrecht Scale of Evaluation Rehabilitation-Participation (USER-P) with Frequency, Restrictions, and Satisfaction scores, and a hierarchical regression analysis. RESULTS: Early-onset (types 1, 2, and 3a) and late-onset (types 3b and 4) SMA patients reported similar frequency and satisfaction scores. "Age," "motor skills," "pain," and "feelings of depression" correlated with frequency; "motor skills" and "feelings of depression" correlated with restrictions; and "level of education," "fatigue," and "feelings of depression" correlated with satisfaction. "Motor skills" and "feelings of depression" explained 33% of variance in frequency of participation. "Motor skills" explained 26% of variance of restrictions in participation. "Fatigue" and "feelings of depression" explained 50% of variance in satisfaction with participation. DISCUSSION: Motor skills, feelings of depression and fatigue are correlates of participation in daily life. This knowledge can be used to optimize care for SMA patients. Muscle Nerve 58:805-811, 2018.

Fatty acid oxidation flux predicts the clinical severity of VLCAD deficiency.

PURPOSE: Very-long-chain acyl-CoA dehydrogenase (VLCAD) deficiency (VLCADD) is an inherited disorder of mitochondrial long-chain fatty acid ß-oxidation (LC-FAO) and is included in many newborn screening (NBS) programs worldwide. Patients may present with hypoketotic hypoglycemia, cardiomyopathy, and/or myopathy, but clinical severity varies widely and the clinical outcome is unpredictable. We investigated predictive markers that may determine clinical severity. METHODS: We developed a clinical severity score (CSS), which was determined for 13 Dutch patients with VLCADD, all of whom were diagnosed before the introduction of VLCADD in NBS to prevent bias from early diagnosis. In cultured skin fibroblasts from these patients, we measured LC-FAO flux (the rate of oleate oxidation), VLCAD activity, and acylcarnitine profiles following palmitate loading. RESULTS: The strongest correlation (r = 0.93; P < 0.0001) was observed between LC-FAO flux and the CSS. VLCAD activity measurement and the C14/C16-to-acylcarnitine ratio correlated much less. A median LC-FAO flux of 6% of control values (range 5.6-6.8%) was associated with cardiomyopathy (P < 0.01), and 32.4% (range 5.6-50.5%) was associated with myopathy (P < 0.05). CONCLUSION: Our results demonstrate a very strong correlation between LC-FAO flux in fibroblasts and the clinical severity of VLCADD. LC-FAO flux measurements may thus predict whether patients are likely to develop symptoms.

Oscillometry: A substitute of spirometry in children with neuromuscular diseases?

INTRODUCTION: Spirometry plays an important role in the assessment of possible respiratory failure in children with neuromuscular diseases (NMDs). However, obtaining reliable spirometry results is a major challenge. We studied the relation between oscillometry and spirometry results. Oscillometry is an easy, noninvasive method to measure respiratory resistance R and reactance X. We hypothesized an increased R and reduced X in patients with more reduced lung function. METHODS: In this prospective single-center study, we included all children with NMDs able to perform spirometry. We consecutively measured R and X at 5, 11, and 19 Hz and (forced) vital capacity, peak expiratory flow. Spearman correlation coefficients and positive and negative predictive values were calculated. Regression curves were estimated. RESULTS: We included 148 patients, median age 13 years (interquartile range: 8-16). A negative correlation was found between R and spirometry outcomes (Spearman correlation coefficient [ρ]: -0.5 to -0.6, p < 0.001). A positive correlation was found between X (i.e., less negative outcomes) and spirometry outcomes (ρ: 0.4-0.6, p < 0.001). Highest correlation was found at lower frequencies. Regression analysis showed a nonlinear relation. Measurement of inspiratory and expiratory R and X did not provide added value. Positive predictive values of 80%-85% were found for z-scores of R measured at 5 Hz versus (F)VC ≤ 60%. CONCLUSION: We found a nonlinear relation between oscillometry and spirometry results with increased R and reduced X in patients with more restrictive lung function decline. Given the difficulties with performing spirometry, oscillometry may be a promising substitute.

Short-term effect and effect on rate of lung function decline after surgery for neuromuscular or syndromic scoliosis.

INTRODUCTION: Understanding the impact of scoliosis surgery on lung function is important for counseling patients about risks and benefits of surgery. We prospectively compared the trends in lung function test (LFT) results before and after scoliosis surgery in children with neuromuscular diseases or dysmorphic syndromes. We hypothesized a stabilization. METHODS: We prospectively included children with neuromuscular or syndromic scoliosis able to perform LFTs. We studied (forced) vital capacity ([F]VC), ratio of forced expiratory volume in 1 s (FEV1 ) and FVC, and peak expiratory flow (PEF). Preoperative LFT results were compared with results 3-4 months after surgery. The mean monthly change in LFT results up to 2 years after surgery was compared with the preoperative natural history using linear mixed-effects models. RESULTS: We included 43 patients. No significant change was observed in absolute values of (F)VC, FEV1 /FVC, and PEF before and after surgery. In 23 neuromuscular patients median standardized VC, FVC, and PEF decreased significantly after surgery from 43% to 33%, 42% to 31%, and 51% to 40%, respectively. In 20 syndromic patients, median FVC decreased from 68% to 65%. The monthly rate of change in FVC did not change significantly in both groups with a mean difference of 0.18% (95% CI: -0.27, -0.61) and -0.44% (95% CI: -1.05, 0.16). CONCLUSION: No stabilization of lung function 3-4 months after scoliosis surgery was observed in children with neuromuscular and syndromic scoliosis with restrictive lung function disease. The effect on the rate of lung function decline remains inconclusive.

Nerve ultrasound for diagnosing chronic inflammatory neuropathy: A multicenter validation study.

OBJECTIVE: To validate the diagnostic accuracy of a previously described short sonographic protocol to identify chronic inflammatory neuropathy (CIN), including chronic inflammatory demyelinating polyneuropathy (CIDP), Lewis Sumner syndrome, and multifocal motor neuropathy (MMN), and to determine the added value of nerve ultrasound to detect treatment-responsive patients compared to nerve conduction studies (NCS) in a prospective multicenter study. METHODS: We included 100 consecutive patients clinically suspected of CIN in 3 centers. The study protocol consisted of neurologic examination, laboratory tests, NCS, and nerve ultrasound. We validated a short sonographic protocol (median nerve at forearm, upper arm, and C5 nerve root) and determined its diagnostic accuracy using the European Federation of Neurological Societies/Peripheral Nerve Society criteria of CIDP/MMN (reference standard). In addition, to determine the added value of nerve ultrasound in detecting treatment-responsive patients, we used previously published diagnostic criteria based on clinical, NCS, and sonographic findings and treatment response (alternative reference standard). RESULTS: Sensitivity and specificity of the sonographic protocol for CIN according to the reference standard were 87.4% and 67.3%, respectively. Sensitivity and specificity of this protocol according to the alternative reference standard were 84.6% and 72.8%, respectively, and of NCS 76.1% and 93.4%. With addition of nerve ultrasound, 44 diagnoses of CIN were established compared to 33 diagnoses with NCS alone. CONCLUSIONS: A short sonographic protocol shows high diagnostic accuracy for detecting CIN. Nerve ultrasound is able to detect up to 25% more patients who respond to treatment. CLASSIFICATION OF EVIDENCE: This multicenter study provides Class IV evidence that nerve ultrasound improves diagnosis of CIN.

Effect of mechanical insufflation-exsufflation in children with neuromuscular weakness.

INTRODUCTION: Children with neuromuscular diseases develop cough impairment. Airway clearance techniques (ACTs) may help to prevent recurrent respiratory tract infections (RTIs). A commonly used ACT is mechanical insufflation-exsufflation (MI-E), but evidence for efficacy is limited. We hypothesize that MI-E has beneficial effect on RTI related hospital admission rate. METHODS: In this single-center retrospective study, we reviewed all children who used daily MI-E between 2005 till June 2019. Primary outcome studied was the number of RTIs requiring hospital admission. Patient satisfaction and burden experienced by MI-E use were explored by questionnaires using a Likert scale. The relative number of RTIs requiring admission and the number of admission days per eligible period before and after the introduction of MI-E were compared using the Friedman test and the Wilcoxon signed-rank test. RESULTS: Thirty-seven children were included. The median number of RTI related hospital admissions per 1000 eligible days after the introduction of MI-E was 0.9 (interquartile range [IQR] 0.0-3.1) compared to the 3 preceding years (median 3.7; IQR 1.4-5.9; P = .006). The median number of RTI related admission days per 1000 eligible days after the introduction of MI-E was significantly lower with a median of 2.7 (IQR 0.0-17.4) compared to the 3 preceding years (median 33.6; IQR 15.0-51.1; P = .001). Patient satisfaction was high with low burden, even in patients who discontinued treatment. CONCLUSION: A significantly lower number of RTIs requiring hospital admission and shorter admission duration after the introduction of MI-E was found, with high patient satisfaction and low burden.

Glycogen Storage Disease Type IV: A Rare Cause for Neuromuscular Disorders or Often Missed?

Advancements in genetic testing now allow early identification of previously unresolved neuromuscular phenotypes. To illustrate this, we here present diagnoses of glycogen storage disease IV (GSD IV) in two patients with hypotonia and delayed development of gross motor skills. Patient 1 was diagnosed with congenital myopathy based on a muscle biopsy at the age of 6 years. The genetic cause of his disorder (two compound heterozygous missense mutations in GBE1 (c.[760A>G] p.[Thr254Ala] and c.[1063C>T] p.[Arg355Cys])), however, was only identified at the age of 17, after panel sequencing of 314 genes associated with neuromuscular disorders. Thanks to the availability of next-generation sequencing, patient 2 was diagnosed before the age of 2 with two compound heterozygous mutations in GBE1 (c.[691+2T>C] (splice donor variant) and the same c.[760A>G] p.[Thr254Ala] mutation as patient 1). GSD IV is an autosomal recessive metabolic disorder with a broad and expanding clinical spectrum, which hampers targeted diagnostics. The current cases illustrate the value of novel genetic testing for rare genetic disorders with neuromuscular phenotypes, especially in case of clinical heterogeneity. We argue that genetic testing by gene panels or whole exome sequencing should be considered early in the diagnostic procedure of unresolved neuromuscular disorders.

Mapping the differences in care for 5,000 spinal muscular atrophy patients, a survey of 24 national registries in North America, Australasia and Europe.

Spinal muscular atrophy (SMA) is an autosomal recessive genetic disorder characterised by the degeneration of motor neurons and progressive muscle weakness. It is caused by homozygous deletions in the survival motor neuron gene on chromosome 5. SMA shows a wide range of clinical severity, with SMA type I patients often dying before 2 years of age, whereas type III patients experience less severe clinical manifestations and can have a normal life span. Here, we describe the design, setup and utilisation of the TREAT-NMD national SMA patient registries characterised by a small, but fully standardised set of registry items and by genetic confirmation in all patients. We analyse a selection of clinical items from the SMA registries in order to provide a snapshot of the clinical data stratified by SMA subtype, and compare these results with published recommendations on standards of care. Our study included 5,068 SMA patients in 25 countries. A total of 615 patients were ventilated, either invasively (178) or non-invasively (437), 439 received tube feeding and 455 had had scoliosis surgery. Some of these interventions were not available to patients in all countries, but differences were also noted among high-income countries with comparable wealth and health care systems. This study provides the basis for further research, such as quality of life in ventilated SMA patients, and will inform clinical trial planning.

Cholesterol-rich domains are involved in Bordetella pertussis phagocytosis and intracellular survival in neutrophils.

Bordetella pertussis-specific antibodies protect against whooping cough by facilitating host defense mechanisms such as phagocytosis. However, the mechanism involved in the phagocytosis of the bacteria under non-opsonic conditions is still poorly characterized. We report here that B. pertussis binding and internalization is cholesterol dependent. Furthermore, we found cholesterol to be implicated in B. pertussis survival upon interaction with human neutrophils. Pre-treatment of PMN with cholesterol sequestering drugs like nystatin or methyl-beta-cyclodextrin (MbetaCD) resulted in a drastic decrease of uptake of non-opsonized B. pertussis. Conversely, phagocytosis of opsonized bacteria was not affected by these drugs, showing that cholesterol depletion affects neither the viability of PMN nor the route of entry of opsonized B. pertussis. Additionally, intracellular survival rate of non-opsonized bacteria was significantly decreased in cholesterol-depleted PMN. Accordingly, confocal laser microscopy studies showed that non-opsonized B. pertussis co-localized with lysosomal markers only in cholesterol-depleted PMN but not in normal PMN. Our results indicate that B. pertussis docks to molecules that eventually prevent cellular bactericidal activity.