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Intellectual disability (ID) has an estimated prevalence of 1.5%-2%. Whole exome sequencing (WES) studies have identified a multitude of novel causative gene defects and have shown that sporadic ID cases result from de novo mutations in genes associated with ID. Here, we report on a 10-year-old girl, who has been regularly presented in our neuropediatric and genetic outpatient clinic. A median cleft palate and a heart defect were surgically corrected in infancy. Apart from ID, she has behavioral anomalies, muscular hypotonia, scoliosis, and hypermobile joints. The facial phenotype is characterized by arched eyebrows, mildly upslanting long palpebral fissures, prominent nasal tip, and large, protruding ears. Trio WES revealed a de novo missense variant in MEIS2 (c.998G>A; p.Arg333Lys). Haploinsufficiency of MEIS2 had been discussed as the most likely mechanism of the microdeletion 5q14-associated complex phenotype with ID, cleft palate, and heart defect. Recently, four studies including in total 17 individuals with intragenic MEIS2 variants were reported. Here we present the evolution of the clinical phenotype and compare with the data of known individuals., (© 2021 The Authors. American Journal of Medical Genetics Part A published by Wiley Periodicals LLC.)

Background: Spinal muscular atrophy (SMA) is the most common neurodegenerative disease in childhood. Since motor neuron injury is usually not reversible, early diagnosis and treatment are essential to prevent major disability. Our objective was to assess the impact of genetic newborn screening for SMA on outcome., Methods: We provided clinical data from 43 SMA patients, identified via polymerase chain reaction of the SMN1 gene from dried blood spots between January 2018 and January 2020 in Germany. Follow-up included neurophysiological examinations and standardized physiotherapeutic testing., Results: Detection of SMA with newborn screening was consistent with known incidence in Germany. Birth prevalence was 1:6910; 39.5% had 2 SMN2 copies, 23% had 3 SMN2 copies, 32.5% had 4 copies, and 4.5% had 5 copies of the SMN2 gene. Treatment with SMA-specific medication could be started at the age of 14-39 days in 21 patients. Pre-symptomatically treated patients remained throughout asymptomatic within the observation period. 47% of patients with 2 SMN2 copies showed early, presumably intrauterine onset of disease. These patients reached motor milestones with delay; none of them developed respiratory symptoms. Untreated children with 2 SMN2 copies died. Untreated children with 3 SMN2 copies developed proximal weakness in their first year. In patients with ≥ 4 SMN2 copies, a follow-up strategy of "watchful waiting" was applied despite the fact that one of them was treated from the age of 6 months. Two infant siblings with 4 SMN2 copies were identified with a missed diagnosis of SMA type 3., Conclusion: Identification of newborns with infantile SMA and prompt SMA-specific treatment substantially improves neurodevelopmental outcome, and we recommend implementation in the public newborn screening in countries where therapy is available. Electrophysiology is a relevant parameter to support the urgency of therapy. There has to be a short time interval between a positive screening result and referral to a therapy-ready specialized treatment center.

Objective: To examine the effect of intrathecal application of nusinersen on the respiratory function in terms of vital capacity in pediatric patients with spinal muscular atrophy (SMA). SMA is characterized by on-going muscular atrophy and weakness that lead to respiratory insufficiency. In recent years therapy with nusinersen has been shown to improve motor function in patients with SMA., Methods: We retrospectively analyzed data from 12 pediatric patients (aged 4-12 years) with SMA II or III (7 walkers, 5 sitters) treated with nusinersen. We examined forced vital capacity (FVC) at baseline (i.e., before treatment) and 180 and 300 days after initiation of treatment., Results: No significant difference in the ranks of FVC of patients with SMA at baseline and day 300 was found and, thus, stable FVCs are implied (n = 6; Z = - 0.105, p exact  = 1.000; Median baseline  = 96.0%, 95%-CI [86.5, 110.5]; Median day300  = 96.0%, 95%-CI [92.0, 109.5]; s. Table 1). This also applied to the comparison between baseline and day 180 (n = 7; Z = 0.00, p exact  = 1.00; Median baseline  = 93.0%, 95%-CI [85.0, 110.0]; Median day180  = 91.0%, 95%-CI [72.0, 118.0]) and day 180 and 300 (n = 9; Z = - 0.533, p exact  = .652; Median day180  = 95.0%, 95%-CI [72.0, 118.0]; Median day300  = 90.0%, 95%-CI [74.0, 105.0])., Conclusion: Nusinersen therapy alone may not improve lung function of pediatric patients with SMA type II or III., Competing Interests: Declaration of competing interest Dr. Heitschmidt, Dr. Pichlmaier, Dr. Eckerland, Dr. Steindor, Dr. Olivier and Dr. Hirtz report no disclosures. Dr. Kölbel is serving on a scientific advisory board or data safety monitoring board for Biogen and Avexis, and received travel and speaker honoraria from Biogen, Avexis and Sanofi-Aventis. Dr. Stehling received speaker honoraria from Biogen., (Copyright © 2021 European Paediatric Neurology Society. Published by Elsevier Ltd. All rights reserved.)

Introduction : Congenital myasthenic syndromes (CMS) refer to a heterogenic group of neuromuscular transmission disorders. CMS-subtypes are diverse regarding exercise intolerance and muscular weakness, varying from mild symptoms to life-limiting forms with neonatal onset. Long-term follow-up studies on disease progression and treatment-response in pediatric patients are rare. Patients and Methods : We analyzed retrospective clinical and medication data in a cohort of 32 CMS-patients including the application of a standardized, not yet validated test (CMS-ST) to examine muscular strength and endurance in 21 patients at the last follow-up. Findings obtained in our cohort were compared with long-term follow-up studies of (adult) CMS-cohorts from the literature by considering the underlying molecular mechanisms. Outcomes of CMS-ST were compared to results of normal clinical assessment. Results : Thirty-two pediatric patients with defects in eight different CMS-genes were followed by a median time of 12.8 years. Fifty-nine percentage of patients manifested with first symptoms as neonates, 35% as infants. While 53% of patients presented a reduced walking distance, 34% were wheelchair-bound. Even under adequate therapy with pyridostigmine (PS) and 3,4-diaminopyridine, CHAT -mutations led to the progression of muscular weakness partly in combination with persistent respiratory and bulbar symptoms. RAPSN, CHRND , and CHRNB1 patients with neonatal manifestation, early respiratory problems, and bulbar symptoms showed a good and maintained treatment response. CHAT and CHRNE patients required higher PS dosages, whereas RAPSN patients needed a lower mean dosage at the last follow-up. The benefits of short-term medication and long-term progression of symptoms were highly dependent on the specific genetic defect. CMS-ST was carried out in 17/21 patients, determined affected muscle groups including bulbar and ocular symptoms, some of which were not reported by the patients. Conclusions : Our findings and comparison with the literature- suggest a better treatment-response and less severe progression of symptoms present in patients suffering from mutations in CMS-genes directly associated with receptor deficiency, while patients with defects leading to synaptopathy and presynaptic defects tend to have worse outcomes. Assessment of affected muscular groups and clinical symptoms by CMS-ST may be a useful tool for optimal therapeutic management of the patients, especially for future clinical studies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Della Marina, Wibbeler, Abicht, Kölbel, Lochmüller, Roos and Schara.)

Background: Walker-Warburg syndrome (WWS) is a rare form of alpha-dystroglycanopathy characterized by muscular dystrophy and severe malformations of the CNS and eyes. Bi-allelic pathogenic variants in POMK are the cause of a broad spectrum of alpha-dystroglycanopathies. POMK encodes protein-O-mannose kinase, which is required for proper glycosylation and function of the dystroglycan complex and is crucial for extracellular matrix composition., Results: Here, we report on male monozygotic twins with severe CNS malformations (hydrocephalus, cortical malformation, hypoplastic cerebellum, and most prominently occipital meningocele), eye malformations and highly elevated creatine kinase, indicating the clinical diagnosis of a congenital muscular dystrophy (alpha-dystroglycanopathy). Both twins were found to harbor a homozygous nonsense mutation c.640C>T, p.214* in POMK, confirming the clinical diagnosis and supporting the concept that POMK mutations can be causative of WWS., Conclusion: Our combined data suggest a more important role for POMK in the pathogenesis of meningoencephalocele. Only eight different pathogenic POMK variants have been published so far, detected in eight families; only five showed the severe WWS phenotype, suggesting that POMK-associated WWS is an extremely rare disease. We expand the phenotypic and mutational spectrum of POMK-associated WWS and provide evidence of the broad phenotypic variability of POMK-associated disease.

Filamin C (encoded by the FLNC gene) is a large actin-cross-linking protein involved in shaping the actin cytoskeleton in response to signaling events both at the sarcolemma and at myofibrillar Z-discs of cross-striated muscle cells. Multiple mutations in FLNC are associated with myofibrillar myopathies of autosomal-dominant inheritance. Here, we describe for the first time a boy with congenital onset of generalized muscular hypotonia and muscular weakness, delayed motor development but no cardiac involvement associated with a homozygous FLNC mutation c.1325C>G (p.Pro442Arg). We performed ultramorphological, proteomic, and functional investigations as well as immunological studies of known marker proteins for dominant filaminopathies. We show that the mutant protein is expressed in similar quantities as the wild-type variant in control skeletal muscle fibers. The proteomic signature of quadriceps muscle is altered and ultrastructural perturbations are evident. Moreover, filaminopathy marker proteins are comparable both in our homozygous and a dominant control case (c.5161delG). Biochemical investigations demonstrate that the recombinant mutant protein is less stable and more prone to degradation by proteolytic enzymes than the wild-type variant. The unusual congenital presentation of the disease clearly demonstrates that homozygosity for mutations in FLNC severely aggravates the phenotype., (© 2020 The Authors. Human Mutation published by Wiley Periodicals LLC.)

Introduction: Small fiber neuropathies (SFN) are associated with a reduction in quality of life. In adults, epidermal nerve fiber density (END) analysis is recommended for the diagnosis of SFN. In children, END assessment is not often performed. We analyzed small nerve fiber innervation to elucidate the potential diagnostic role of skin biopsies in young patients with pain., Methods: Epidermal nerve fiber density and sudomotor neurite density (SND) were assessed in skin biopsies from 26 patients aged 7 to 20 years (15 female patients) with unexplained chronic pain. The results were compared with clinical data., Results: Epidermal nerve fiber density was abnormal in 50% and borderline in 35% of patients. An underlying medical condition was found in 42% of patients, including metabolic, autoimmune, and genetic disorders., Discussion: Reduction of epidermal nerve fibers can be associated with treatable conditions. Therefore, the analysis of END in children with pain may help to uncover a possible cause and guide potential treatment options., (© 2019 The Authors. Muscle & Nerve published by Wiley Periodicals, Inc.)

Although the value of newborn screening (NBS) for early detection and treatment opportunity in SMA patients is generally accepted, there is still an ongoing discussion about the best strategy in children with 4 and more copies of the SMN2 gene. This gene is known to be the most important but not the only disease modifier.In our SMA-NBS pilot project in Germany comprising 278,970 infants screened between January 2018 and November 2019 were 38 positive cases with a homozygous SMN1 deletion. 40% of them had 4 or more SMN2 copies. The incidence for homozygous SMN1 deletion was 1 : 7350, which is within the known range of SMA incidence in Germany.Of the 15 SMA children with 4 SMN2 copies, one child developed physical signs of SMA by the age of 8 months. Reanalysis of the SMN2 copy number by a different test method revealed 3 copies. Two children had affected siblings with SMA Type III, who were diagnosed only after detection of the index patient in the NBS. One had a positive family history with an affected aunt (onset of disease at the age of 3 years). Three families were lost to medical follow up; two because of socioeconomic reasons and one to avoid the psychological stress associated with the appointments.Decisions on how to handle patients with 4 SMN2 copies are discussed in the light of the experience gathered from our NBS pilot SMA program.

Background: As genome-wide approaches prove difficult with genetically heterogeneous orphan diseases, we developed a new approach to identify candidate genes. We applied this to Emery-Dreifuss muscular dystrophy (EDMD), characterised by early onset contractures, slowly progressive muscular wasting, and life-threatening heart conduction disturbances with wide intra- and inter-familial clinical variability. Roughly half of EDMD patients are linked to six genes encoding nuclear envelope proteins, but the disease mechanism remains unclear because the affected proteins function in both cell mechanics and genome regulation., Methods: A primer library was generated to test for mutations in 301 genes from four categories: (I) all known EDMD-linked genes; (II) genes mutated in related muscular dystrophies; (III) candidates generated by exome sequencing in five families; (IV) functional candidates - other muscle nuclear envelope proteins functioning in mechanical/genome processes affected in EDMD. This was used to sequence 56 unlinked patients with EDMD-like phenotype., Findings: Twenty-one patients could be clearly assigned: 18 with mutations in genes of similar muscular dystrophies; 3 with previously missed mutations in EDMD-linked genes. The other categories yielded novel candidate genes, most encoding nuclear envelope proteins with functions in gene regulation., Interpretation: Our multi-pronged approach identified new disease alleles and many new candidate EDMD genes. Their known functions strongly argue the EDMD pathomechanism is from altered gene regulation and mechanotransduction due to connectivity of candidates from the nuclear envelope to the plasma membrane. This approach highlights the value of testing for related diseases using primer libraries and may be applied for other genetically heterogeneous orphan diseases., Funding: The Wellcome Trust, Muscular Dystrophy UK, Medical Research Council, European Community's Seventh Framework Programme "Integrated European -omics research project for diagnosis and therapy in rare neuromuscular and neurodegenerative diseases (NEUROMICS)"., Competing Interests: Declaration of Competing Interest Dr. Meinke reports personal fees from Greenovation Biopharmaceuticals, outside the submitted work; Dr. Harris reports grants from Muscular Dystrophy-UK, during the conduct of the study; Dr. Koelbel reports personal fees from Santhera, personal fees from Sarepta, personal fees from Biogen, personal fees from Avexis, outside the submitted work; Dr Schara reports personal fees for consulting from Alexion, Santhera, PTC, Ipsen, Biogen, Sarepta, Dynacure, Novartis, Avexis and Sanofi; Dr. Muntoni reports personal fees from Pfizer, personal fees from Roche, personal fees from Biogen, personal fees from Avexis, personal fees from Sarepta, grants from Sarepta, outside the submitted work; Dr. Schoser reports Advisory boards: Amicus Therapeutics, Audentes Therapeutics, Nexien Biopharm, Lupin therapeutics, Sanofi Genzyme. Contracted research: Amicus therapeutics, Greenovation Biopharm, Sanofi Genzyme. Honoraria: Kedrion. Travel expenses: Sanofi Genzyme. Dr. Kerr, Dr. Czapiewski, Dr. de las Heras, Mr. Dixon, Dr. Straub, Dr. Wehnert and Dr. Schirmer have nothing to disclose., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: The protein O-mannosyltransferase 1, encoded by the POMT1 gene, is a key enzyme in the glycosylation of α-dystroglycan. POMT1-related disorders belong to the group of dystroglycanopathies characterized by a proximally pronounced muscular dystrophy with structural or functional involvement of the brain and/or the eyes. The phenotypic spectrum ranges from the severe Walker-Warburg syndrome (WWS) to milder forms of limb girdle muscular dystrophy (LGMD). The phenotypic severity of POMT1-related dystroglycanopathies depends on the residual enzyme activity. A genotype-phenotype correlation can be assumed., Results: The clinical, neuroradiological, and genetic findings of 35 patients with biallelic POMT1 mutations (15 WWS, 1 MEB (muscle-eye-brain disease), 19 LGMD) from 27 independent families are reported. The representative clinical course of an infant with WWS and the long-term course of a 32 years old patient with LGMD are described in more detail. Specific features of 15 patients with the homozygous founder mutation p.Ala200Pro are defined as a distinct and mildly affected LGMD subgroup. Ten previously reported and 8 novel POMT1 mutations were identified. Type and location of each of the POMT1 mutations are evaluated in detail and a list of all POMT1 mutations reported by now is provided. Patients with two mutations leading to premature protein termination had a WWS phenotype, while the presence of at least one missense mutation was associated with milder phenotypes. In the patient with MEB-like phenotype two missense mutations were observed within the catalytic active domain of the enzyme., Conclusions: Our large cohort confirms the importance of type and location of each POMT1 mutation for the individual clinical manifestation and thereby expands the knowledge on the genotype-phenotype correlation in POMT1-related dystroglycanopathies. This genotype-phenotype correlation is further supported by the observation of an intrafamiliar analogous clinical manifestation observed in all affected 13 siblings from 5 independent families. Our data confirm the progressive nature of the disease also in milder LGMD phenotypes, ultimately resulting in loss of ambulation at a variable age. Our data define two major clinical POMT1 phenotypes, which should prompt genetic testing including the POMT1 gene: patients with a severe WWS manifestation predominantly present with profound neonatal muscular hypotonia and a severe and progressive hydrocephalus with involvement of brainstem and/or cerebellum. The presence of an occipital encephalocele in a WWS patient might point to POMT1 as causative gene within the different genes associated with WWS. The milder LGMD phenotypes constantly show markedly elevated creatine kinase values in combination with microcephaly and cognitive impairment.

Female carriers of DMD gene mutations may be symptomatic and show variable skeletal as well as cardiac muscle symptoms. Skeletal muscle can exhibit morphological alterations. However, inflammatory, degenerative and fibrotic changes as seen in Duchenne boys have not been specifically analysed yet, so we addressed the question whether skeletal muscle of female carriers show such alterations. Thirteen carriers with an age range of 3 to 50 years were studied retrospectively. Five out of 13 women had clinically affected relatives. Clinically, most women showed mild muscle weakness, while the CK levels were increased in nine of them. Histomorphological analyses highlighted the typical mosaic pattern of dystrophin-positive and -negative fibres. Regenerating fibres were diffusely scattered and focally pronounced, while endo- and perimysial fibrosis was a variable but constant feature. Infiltration of CD206 + TGFß + macrophages and scattered T cells was noted in the endomysium. TGFb and CCL18, were significantly increased. However, gene expression of markers involved in Th1/Th2 immunity did not reach statistical significance compared to non-diseased controls. In summary, skeletal muscle of clinically manifest female DMD gene mutation carriers shows mild fibrosis and increased regeneration associated with endomysial CD206 + TGFβ + and STAT6 + macrophages, which are most likely involved in fibrotic remodelling., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Laminin-211 deficiency leads to the most common form of congenital muscular dystrophy in childhood, MDC1A. The clinical picture is characterized by severe muscle weakness, brain abnormalities and delayed motor milestones defining MDC1A as one of the most severe forms of congenital muscular diseases. Although the molecular genetic basis of this neurological disease is well-known and molecular studies of mouse muscle and human cultured muscle cells allowed first insights into the underlying pathophysiology, the definition of marker proteins in human vulnerable tissue such as skeletal muscle is still lacking. To systematically address this need, we analyzed the proteomic signature of laminin-211-deficient vastus muscle derived from four patients and identified 86 proteins (35 were increased and 51 decreased) as skeletal muscle markers and verified paradigmatic findings in a total of two further MDC1A muscle biopsies. Functions of proteins suggests fibrosis but also hints at altered synaptic transmission and accords with central nervous system alterations as part of the clinical spectrum of MDC1A. In addition, a profound mitochondrial vulnerability of the laminin-211-deficient muscle is indicated and also altered abundances of other proteins support the concept that metabolic alterations could be novel mechanisms that underline MDC1A and might constitute therapeutic targets. Intersection of our data with the proteomic signature of murine laminin-211-deficient gastrocnemius and diaphragm allowed the definition of nine common vulnerable proteins representing potential tissue markers.

The heterooctameric mitochondrial trifunctional protein (MTP), composed of four α- and β-subunits harbours three enzymes that each perform a different function in mitochondrial fatty acid β-oxidation. Pathogenic variants in the MTP genes (HADHA and HADHB) cause MTP deficiency, a rare autosomal recessive metabolic disorder characterized by phenotypic heterogeneity ranging from severe, early-onset, cardiac disease to milder, later-onset, myopathy and neuropathy. Since metabolic myopathies and neuropathies are a group of rare genetic disorders and their associated muscle symptoms may be subtle, the diagnosis is often delayed. Here we evaluated data of 161 patients with myopathy and 242 patients with neuropathy via next generation sequencing (NGS) and report the diagnostic yield in three patients of this cohort by the detection of disease-causing variants in the HADHA or HADHB gene. The mitigated phenotypes of this treatable disease were missed by the newborn screening, highlighting the importance of phenotype-based NGS analysis in patients with rare and clinically very variable disorders such as MTP deficiency., (Copyright © 2019. Published by Elsevier Ltd.)

Aims: To define the neurological and neuropathological alterations caused by SYNE1 mutations., Methods: We describe 5 patients (3 males, 2 females; age 3-24 years) from 3 families. The diagnostic work-up included three muscle biopsies and two nerve biopsies in three of the cases., Results: Three different phenotypes were discerned. Two patients showed progressive ataxia, mental retardation, neuropathy and radially deviated thumbs (spinocerebellar ataxia, SCAR, type 8 phenotype). Two patients had mild congenital myopathy with restrictive lung disease, clubfeet and thumb anomalies (myopathic arthrogryposis). One patient had congenital myopathy with dilated cardiomyopathy and adducted thumbs (Emery-Dreifuss Muscular Dystrophy, EDMD, type 4). Light microscopy of the three muscle biopsies revealed chronic non-necrotizing myopathy without rimmed vacuoles in all cases combined with neurogenic atrophy in one case. The two nerve biopsies showed predominantly axonal neuropathy with demyelinating features. Nuclear alterations, most notably lobulation and focal widening of the space between inner and outer leaflet of the nuclear envelope, were a prominent consistent feature of myonuclei and Schwann cell nuclei in each of the three muscle specimens and one nerve specimen that could be examined by electron microscopy., Conclusion: Thumb abnormalities and nuclear envelope alterations are characteristic for SYNE 1 mutations. Schwann cell nuclei are affected, indicating that such nuclear envelope changes in glial cells contribute to the neurodegenerative phenotype in human nesprinopathies., (Copyright © 2018 European Paediatric Neurology Society. Published by Elsevier Ltd. All rights reserved.)

Objective: Spinal muscular atrophy (SMA) is the most common neurodegenerative disease in childhood. The study was conducted to assess the impact of early detection of SMA by newborn screening (NBS) on the clinical course of the disease., Methods: Screening was performed in two federal states of Germany, Bavaria and North Rhine Westphalia, between January 2018 and February 2019. The incidence in the screening population was calculated as number of detected patients with a homozygous deletion in the SMN1-gene per number of screened patients. To get an idea about the incidence of newly diagnosed SMA in the year prior to screening a survey covering all neuropediatric centers in the state of Bavaria was conducted, identifying all SMA-cases in 2017 and 2018. Following positive NBS and confirmatory diagnostic test, treatment was advised according to the recommendations of the "American SMA NBS Multidisciplinary Working Group". Immediate treatment with Nusinersen was recommended in children with 2 and 3 SMN2 copies and a conservative strict follow-up strategy in children with ≥4 copies. All children underwent regular standardized neuropediatric examination, CHOP INTEND and HINE-2 testing as well as electrophysiological exams every 2-3 months., Results: 165,525 children were screened. 22 cases of SMA were identified, meaning an incidence rate of 1:7524. SMN2 copy number analysis showed 2 SMN2 copies in 45% of patients, 3 SMN2 copies in 19 % and 4 SMN2 copies in 36%. These findings are confirmed in the most recent statistical data-cut from 31st August 2019 (incidence 1:7089, 2 SMN2 copies in 44%, 3 in 15% and 4 in 38%). Comparison with up-to-date German data on SMA incidence and the Bavarian survey give evidence that NBS did not lead to a relevant increase in incidence. 10 patients with 2 or 3 SMN2 copies were treated with Nusinersen, starting between 15- 39 days after birth, in 7/10 patients before onset of symptoms. Presymptomatically treated patients (age at last examination: 1- 12 months, median 8 months) showed no muscle weakness by the age of one month to one year. One child with 4 SMN2 copies became symptomatic at the age of 8 months., Conclusions: Newborn screening, resulting in presymptomatic treatment, improves outcome in children with genetically proven SMA. Newborn screening for SMA should be introduced in all countries where therapy is available. An immediate therapy in cases with 4 SMN2 copies should be considered.

Background: Spinal muscular atrophy (SMA) is a neuromuscular disorder characterized by muscle weakness and muscle atrophy. Nusinersen acts as a splicing modifier and has recently been approved for intrathecal treatment of SMA., Objective: Prior to approval, nusinersen was provided to patients with SMA type 1 in Germany within an Expanded Access Program (EAP). In contrast to previous clinical trials, children of different age groups and different stages of the disease were treated with nusinersen., Methods: We conducted a prospective, longitudinal data collection of patients treated with nusinersen within the EAP in seven neuromuscular centers in Germany. Standardized assessments including CHOP-INTEND and HINE-2 motor milestones were performed at baseline and 60 and 180 days after start of treatment., Results: Data from 61 SMA type 1 patients (mean age 21.08 months, range 1-93) were available for analysis. After six months of treatment, 47 children (77.0%) improved by ≥4 points in CHOP INTEND score. Mean change in CHOP INTEND score was 9.0±8.0 points. Nineteen patients (31.1%) improved by ≥2 points in HINE-2 motor milestones. Regression analysis revealed age at onset of treatment as major determinant of change in CHOP INTEND from baseline., Conclusion: When analyzing a broad spectrum of SMA type 1 patients, many children showed an improvement of motor function after six months of treatment with nusinersen, which is generally not expected within the natural course of the disease. Long-term observation and follow-up of patients with later onset types of SMA are crucial to understand the clinical impact of treatment with nusinersen.

Competing Interests: Disclosure The authors report no conflicts of interest in this work.

Dystrophinopathies are X-linked muscle diseases caused by mutations in the large DMD gene. The most common mutations are detected by standard diagnostic techniques. However, some patients remain without detectable mutation, most likely due to changes in the non-coding sequence. We report on a boy with complete absence of dystrophin in muscle biopsy but no causative mutation according to standard diagnostics. To search for deep intronic variations (DIV) in the DMD gene we isolated mRNA from muscle tissue and amplified overlapping cDNA fragments using RT-PCR. One cDNA product revealed an augmented fragment size showing an insertion of 77 bp between the exons 7 and 8 by sequencing. We sequenced the flanking sequences of gDNA and found two hemizygous single nucleotide variants (c.650-39575 A>C and c.650-39498 A>G) surrounding the inserted fragment. Both variants create cryptic splice sites which initiate the formation of a pseudoexon that produces a frameshift in the DMD gene., (Copyright © 2017 Elsevier B.V. All rights reserved.)

[This corrects the article DOI: 10.1371/journal.pone.0173144.].

Background: Autosomal-recessive proximal spinal muscular atrophies (SMA) are disorders characterized by a ubiquitous deficiency of the survival of motor neuron protein that leads to a multisystemic disorder, which mostly affects alpha motor neurons. Disease progression is clinically associated with failure to thrive or weight loss, mainly caused by chewing and swallowing difficulties. Although pancreatic involvement has been described in animal models, systematic endocrinological evaluation of the energy metabolism in humans is lacking., Methods: In 43 patients with SMA type I-III (8 type I; 22 type II; 13 type III), aged 0.6-21.8 years, auxological parameters, pubertal stage, motor function (Motor Function Measurement 32 -MFM32) as well as levels of leptin, insulin glucose, hemoglobin A1c, Homeostasis Model Assessment index and an urinary steroid profile were determined., Results: Hyperleptinemia was found in 15/35 (43%) of our patients; 9/15 (60%) of the hyperleptinemic patients were underweight, whereas 1/15 (7%) was obese. Hyperleptinemia was associated with SMA type (p = 0.018). There was a significant association with decreased motor function (MFM32 total score in hyperleptinemia 28.5%, in normoleptinemia 54.7% p = 0.008, OR 0.969; 95%-CI: 0.946-0.992). In addition, a higher occurrence of hirsutism, premature pubarche and a higher variability of the urinary steroid pattern were found., Conclusion: Hyperleptinemia is highly prevalent in underweight children with SMA and is associated with disease severity and decreased motor function. Neuronal degradation of hypothalamic cells or an increase in fat content by muscle remodeling could be the cause of hyperleptinemia.

The isolation perfusion model, including transbronchial ventilation of human lung, offers the possibility to study pharmacological interactions under physiological conditions. In view of the increasing importance of targeted therapy of lung diseases, this model of perfusion might attract major interest, particularly, in lung cancer. Our study investigated physiological, histological, and immunohistochemical alterations of lung and tumor tissue during isolated perfusion of lung lobectomy specimens to explore potential limitations of this model. Right after resection, 16 human lung resection specimens for primary lung cancer were isolated, ventilated, and perfused under physiological conditions with a modified Krebs-Henseleit solution over a period of 10, 60, 90, 120, and 240 min. Perfusion pressure, pH, lung weight gain, and histological edema formation were measured continuously before and during perfusion. After perfusion, lung and tumor tissue was investigated by hematoxylin-and-eosin stained sections. Immunohistochemistry of NADH, PECAM-1, angiotensin-converting-enzyme and NF-kappabeta were performed to determine lung tissue viability and changes at the endothelial layer. We found that perfusion up to 120 min could be performed with completely stable physiological conditions. Beyond that time span, edema formation and weight gain of the resection specimen started and were followed by an increase in inspiratory pressure and pulmonary artery pressure. Perfusion of more than 4 h led to a significant edema formation in lung tissue accompanied by loss of viability and significant histological alterations. We conclude that isolated ventilation and perfusion of human lung resections within the setup chosen is reliable for pharmacological studies up to a period of 120 min. Thereafter, edema formation and endothelial damage develop and limit the interpretation and reliability of drug delivery studies.