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BACKGROUND AND PURPOSE Distal hereditary motor neuropathies (dHMNs) are a heterogeneous group of disorders characterized by degeneration of the motor component of peripheral nerves. Currently, only 15% to 32.5% of patients with dHMN are characterized genetically. Additionally, the prevalence of these genetic disorders is not well known. Recently, biallelic mutations in the sorbitol dehydrogenase gene (SORD) have been identified as a cause of dHMN, with an estimated frequency in undiagnosed cases of up to 10%. METHODS In the present study, we included 163 patients belonging to 108 different families who were diagnosed with a dHMN and who underwent a thorough genetic screening that included next-generation sequencing and subsequent Sanger sequencing of SORD. RESULTS Most probands were sporadic cases (62.3%), and the most frequent age of onset of symptoms was 2 to 10 years (28.8%). A genetic diagnosis was achieved in 37/108 (34.2%) families and 78/163 (47.8%) of all patients. The most frequent cause of distal hereditary motor neuropathies were mutations in HSPB1 (10.4%), GARS1 (9.8%), BICD2 (8.0%), and DNAJB2 (6.7%) genes. In addition, 3.1% of patients were found to be carriers of biallelic mutations in SORD. Mutations in another seven genes were also identified, although they were much less frequent. Eight new pathogenic mutations were detected, and 17 patients without a definite genetic diagnosis carried variants of uncertain significance. The calculated minimum prevalence of dHMN was 2.3 per 100,000 individuals. CONCLUSIONS This study confirms the genetic heterogeneity of dHMN and that biallelic SORD mutations are a cause of dHMN in different populations.

14 páginas, 4 figuras, 3 tablas, Aims: We aim to present data obtained from three patients belonging to three unrelated families with an infantile onset demyelinating neuropathy associated to somatic and neurodevelopmental delay and to describe the underlying genetic changes. Methods: We performed whole-exome sequencing on genomic DNA from the patients and their parents and reviewed the clinical, muscle and nerve data, the serial neurophysiological studies, brain and muscle MRIs, as well as the respiratory chain complex activity in the muscle of the three index patients. Computer modelling was used to characterise the new missense variant detected. Results: All three patients had a short stature, delayed motor milestone acquisition, intellectual disability and cerebellar abnormalities associated with a severe demyelinating neuropathy, with distinct morphological features. Despite the proliferation of giant mitochondria, the mitochondrial respiratory chain complex activity in skeletal muscle was normal, except in one patient in whom there was a mild decrease in complex I enzyme activity. All three patients carried the same two compound heterozygous variants of the TRMT5 (tRNA Methyltransferase 5) gene, one known pathogenic frameshift mutation [c.312_315del (p.Ile105Serfs*4)] and a second rare missense change [c.665 T > C (p.Ile222Thr)]. TRMT5 is a nuclear-encoded protein involved in the post-transcriptional maturation of mitochondrial tRNA. Computer modelling of the human TRMT5 protein structure suggests that the rare p.Ile222Thr mutation could affect the stability of tRNA binding. Conclusions: Our study expands the phenotype of mitochondrial disorders caused by TRTM5 mutations and defines a new form of recessive demyelinating peripheral neuropathy., Fundación Isabel Gemio; Generalitat Valenciana, Grant/Award Number:PROMETEO/2018/13; Fondo Europeo deDesarrollo Regional (FEDER); Instituto deSalud Carlos III, Grant/Award Numbers:PI19/01178, PI18/01374, PI16/00403

Background Mutations in the FGD4 gene cause an autosomal recessive demyelinating peripheral neuropathy referred to as CMT4H, characterized by its onset in infancy or early-childhood and its slow progression. Methods The clinical and genetic status of two patients with CMT4H was studied, performing genetic testing with a panel of genes and analysing FGD4 mRNA expression by quantitative PCR. Results Two novel FGD4 variants (c.514delG and c.2211dupA) were identified in two mildly affected Spanish siblings with CMT4H, and with disease onset in late adolescence/adulthood (one of them remaining asymptomatic at 20). On examination, foot deformity was observed without weakness or sensory involvement, and in the muscles of the lower extremities magnetic resonance imaging showed no fat replacement. Further analysis of FGD4 expression in peripheral blood suggested that neither mutation affected splicing, nor did they affect the dosage of FGD4 mRNA (compared to a healthy control). It was predicted that each allele would produce a truncated protein, p.Ala172Glnfs*28 (c.514delG) and p.Ala738Serfs*5 (c.2211dupA), the latter containing all the functional domains of the native protein. Conclusions The conservation of functional domains in the proteins produced from the FGD4 gene of two patients with CMT4H, could explain both the milder phenotype and the later disease onset in these patients. These results expand the clinical and mutational spectrum of FGD4-related peripheral neuropathies.

26 páginas, 4 figuras, 3 tablas, Our clinical series comprises 124 patients with movement disorders (MDs) and/or ataxia with cerebellar atrophy (CA), many of them showing signs of neurodegeneration with brain iron accumulation (NBIA). Ten NBIA genes are accepted, although isolated cases compatible with abnormal brain iron deposits are known. The patients were evaluated using standardised clinical assessments of ataxia and MDs. First, NBIA genes were analysed by Sanger sequencing and 59 patients achieved a diagnosis, including the detection of the founder mutation PANK2 p.T528M in Romani people. Then, we used a custom panel MovDisord and/or exome sequencing; 29 cases were solved with a great genetic heterogeneity (34 different mutations in 23 genes). Three patients presented brain iron deposits with Fe-sensitive MRI sequences and mutations in FBXO7, GLB1, and KIF1A, suggesting an NBIA-like phenotype. Eleven patients showed very early-onset ataxia and CA with cortical hyperintensities caused by mutations in ITPR1, KIF1A, SPTBN2, PLA2G6, PMPCA, and PRDX3. The novel variants were investigated by structural modelling, luciferase analysis, transcript/minigenes studies, or immunofluorescence assays. Our findings expand the phenotypes and the genetics of MDs and ataxias with early-onset CA and cortical hyperintensities and highlight that the abnormal brain iron accumulation or early cerebellar gliosis may resembling an NBIA phenotype., This work was supported by the Instituto de Salud Carlos III (ISCIII)—Subdirección General de Evaluación y Fomento de la Investigación within the framework of the National R + D+I Plan co-funded with European Regional Development Funds (ERDF) [Grants PI18/00147 and PI21/00103 to CE]; the Fundació La Marató TV3 [Grants 20143130 and 20143131 to BPD and CE]; and by the Generalitat Valenciana [Grant PROMETEO/2018/135 to CE]. Part of the equipment employed in this work was funded by Generalitat Valenciana and co-financed with ERDF (OP ERDF of Comunitat Valenciana 2014–2020). PS had an FPU-PhD fellowship funded by the Spanish Ministry of Education, Culture and Sport [FPU15/00964]. IH has a PFIS-PhD fellowship [FI19/00072]. ASM has a contract funded by the Spanish Foundation Per Amor a l’Art (FPAA)

Dystonia; Parkinson's disease/Parkinsonism; Genetic linkage Malaltia de Parkinson/Parkinsonisme; Vinculació genètica; Distonia Enfermedad de Parkinson/Parkinsonismo; Enlace genético; Distonía This work was supported by the Health Institute Carlos III—General Subdirectorate for Research Evaluation and Promotion (PI16/01575, PI18/01898, PI18/00147, PI19/01576), the Spanish Ministry of Economy and Competitiveness (SAF2007-60700), the Ministry of Economy, Innovation, Science and Business of the Government of Andalucía (CVI-02526, CTS-7685), the Ministry of Health and Social Welfare of the Government of Andalucía (PI-0459-2018, PE-0210-2018, PE-0186-2019) and by the Valencian Government (PROMETEO/2018/135), within the framework of the National Research and Development Plan co-funded with European Regional Development Funds. Part of the equipment employed in this study was funded by the Valencian Government and co-financed with European Regional Development Funds (OP ERDF of Valencian Community 2014-2020). I. Hinarejos has a PFIS-PhD fellowship (FI19/00072), S. Jesús has a contract “Acción B Clínicos-Investigadores” (Action B Clinicians-Researchers) contract (B-0007-2019) funded by the Ministry of Health and Family of the Government of Andalucía, and D. Macías-García has a Río Hortega contract (CM18/00142) funded by the Health Institute Carlos III.

The hereditary motor neuropathies (HMNs) comprise a heterogeneous group of rare diseases accompanied by a length-dependent predominantly motor neuropathy with distal muscle weakness and atrophy that cause distal HMN or distal spinal muscular atrophy (SMA). There is an overlap with axonal forms of Charcot–Marie–Tooth (CMT) disease when a patient suffers from minor sensory abnormalities, and if additional signs are also present, the clinical spectrum broadens and disorders such as proximal SMA, amyotrophic lateral sclerosis (ALS), spastic paraplegia (SPG) and even, ataxia must be considered. As new cases are described, more HMN genes are reported. Sometimes, these are new genes, and in others, these are known genes associated with other pathologies. This chapter focuses on the growing list of HMNs genes classified according to the pathways in which they play a role, to highlight the complexity of genetic bases of this group of neuropathies, and therefore, the difficulties to achieve a conclusive diagnosis.

Altres ajuts: Fondo Europeo de Desarrollo Regional (FEDER); Generalitat Valenciana (PROMETEO/2018/135, PROMETEO/2019/075). IIB Background and purpose: MORC2 mutations have been described as a rare cause of axonal Charcot-Marie-Tooth disease (CMT2Z). The aim of this work was to determine the frequency and distribution of these mutations throughout Spain, to provide a comprehensive phenotypical description and, if possible, to establish a genotype-phenotype correlation. Methods: Retrospectively, data on patients diagnosed with CMT2Z in Spain were collected and clinical, electrophysiological and muscle imaging information were analysed. Results: Fifteen patients with CMT2Z were identified throughout Spain, seven of them belonging to a single kindred, whilst the rest were sporadic. The most common mutation was p.R252W, and four new mutations were identified. Eleven patients were categorized as having a scapuloperoneal phenotype, with asymmetric muscle weakness, early proximal upper limb involvement and frequent spontaneous muscular activity with distal sensory impairment and pes cavus, whilst two presented with a more classic length dependent sensory motor phenotype. This distinction was corroborated by the distribution of muscle fatty infiltration in muscle imaging. Two other patients were classified as having a neurodevelopmental phenotype consisting in congenital or early onset, delay in motor milestones, and global developmental delay in one of them. Nerve conduction studies revealed an unequivocally axonal neuropathy with frequent spontaneous activity, and serum creatine kinase levels were increased in 50% of the patients. Conclusions: MORC2 mutations are a rare cause of CMT in Spain, but in-depth phenotyping reveals a recognizable phenotypic spectrum that will be clinically relevant for future identification of this disease.

Mutations in MORC2 lead to an axonal form of Charcot-Marie-Tooth (CMT) neuropathy type 2Z. To date, 31 families have been described with mutations in MORC2, indicating that this gene is frequently involved in axonal CMT cases. While the genetic data clearly establish the causative role of MORC2 in CMT2Z, the impact of its mutations on neuronal biology and their phenotypic consequences in patients remains to be clarified. We show that the full-length form of MORC2 is highly expressed in both embryonic and adult human neural tissues and that Morc2 expression is dynamically regulated in both the developing and the maturing murine nervous system. To determine the effect of the most common MORC2 mutations, p.S87L and p.R252W, we used several in vitro cell culture paradigms. Both mutations induced transcriptional changes in patient-derived fibroblasts and when expressed in rodent sensory neurons. These changes were more pronounced and accompanied by abnormal axonal morphology, in neurons expressing the MORC2 p.S87L mutation, which is associated with a more severe clinical phenotype. These data provide insight into the neuronal specificity of the mutated MORC2-mediated phenotype and highlight the importance of neuronal cell models to study the pathophysiology of CMT2Z.

BACKGROUND AND PURPOSE Mutations in the early growth response 2 gene (EGR2) cause demyelinating, but also axonal, neuropathies differing in severity and age of onset. Except for one family, all reported cases have autosomal dominant inheritance and mutations are localized within the three zinc finger (ZNF) DNA-binding domain. The aim of this study was to provide a clinical and molecular analysis of a novel recessive mutation in EGR2. METHODS Clinical and electrophysiological assessments of three affected patients, from a consanguineous family, were performed. Genetic analyses of EGR2 were carried out by Sanger sequencing. Functional effects of clinical recessive mutations were assessed using a mammalian two-hybrid assay. RESULTS A novel missense mutation (c.791C>T; p.P264L) in the homozygous state was detected outside the ZNF domains of the EGR2 gene. Three affected siblings presented with distal demyelinating polyneuropathy with severe sensory loss, progressive thoracolumbar scoliosis and trigeminal neuralgia. Respiratory compromise and cranial nerve dysfunction were also found. Our data indicate that the p.P264L mutation prevents interaction of EGR2 transcription factor with NAB corepressors, suggesting that a disruption of the NAB-EGR2 protein interactions can result in dramatic neuropathy. CONCLUSION Mutations in, or next to, the R1 domain of EGR2 should be considered with extreme caution for genetic counseling, since these could cause a severe neuropathy with an autosomal recessive manner of transmission.

BackgroundMutations in the metalloendopeptidase (MME) gene were initially identified as a cause of autosomal recessive Charcot-Marie-Tooth disease type 2 (CMT2). Subsequently, variants in MME were linked to other late-onset autosomal dominant polyneuropathies. Thus, our goal was to define the phenotype and mode of inheritance of patients carrying changes in MME.MethodsWe screened 197 index cases with a hereditary neuropathy of the CMT type or distal hereditary motor neuropathy (dHMN) and 10 probands with familial amyotrophic lateral sclerosis (fALS) using a custom panel of 119 genes. In addition to the index case subjects, we also studied other clinically and/or genetically affected and unaffected family members.ResultsWe found 17 variants in MME in a total of 20 index cases, with biallelic MME mutations detected in 13 cases from nine families (three in homozygosis and six in compound heterozygosis) and heterozygous variants found in 11 families. All patients with biallelic variants had a similar phenotype, consistent with late-onset axonal neuropathy. Conversely, the phenotype of patients carrying heterozygous mutations was highly variable [CMT type 1 (CMT1), CMT2, dHMN and fALS] and mutations did not segregate with the disease.ConclusionMME mutations that segregate in an autosomal recessive pattern are associated with a late-onset CMT2 phenotype, yet we could not demonstrate that MME variants in heterozygosis cause neuropathy. Our data highlight the importance of establishing an accurate genetic diagnosis in patients carrying MME mutations, especially with a view to genetic counselling.

Background Pantothenate kinase-associated neurodegeneration is a progressive neurological disorder occurring in both childhood and adulthood. The objective of this study was to design and pilot-test a disease-specific clinical rating scale for the assessment of patients with pantothenate kinase-associated neurodegeneration. Methods In this international cross-sectional study, patients were examined at the referral centers following a standardized protocol. The motor examination was filmed, allowing 3 independent specialists in movement disorders to analyze 28 patients for interrater reliability assessment. The scale included 34 items (maximal score, 135) encompassing 6 subscales for cognition, behavior, disability, parkinsonism, dystonia, and other neurological signs. Results Forty-seven genetically confirmed patients (30 ± 17 years; range, 6-77 years) were examined with the scale (mean score, 62 ± 21; range, 20-106). Dystonia with prominent cranial involvement and atypical parkinsonian features were present in all patients. Other common signs were cognitive impairment, psychiatric features, and slow and hypometric saccades. Dystonia, parkinsonism, and other neurological features had a moderate to strong correlation with disability. The scale showed good internal consistency for the total scale (Cronbach's α = 0.87). On interrater analysis, weighted kappa values (0.30-0.93) showed substantial or excellent agreement in 85% of the items. The scale also discriminated a subgroup of homozygous c.1583C>T patients with lower scores, supporting construct validity for the scale. Conclusions The proposed scale seems to be a reliable and valid instrument for the assessment of pediatric and adult patients with pantothenate kinase-associated neurodegeneration. Additional validation studies with a larger sample size will be required to confirm the present results and to complete the scale validation testing. © 2017 International Parkinson and Movement Disorder Society

Pulmonary arterial hypertension is a progressive disease that causes the obstruction of precapillary pulmonary arteries and a sustained increase in pulmonary vascular resistance. The aim was to analyze functionally the variants found in the BMPR2 gene and to establish a genotype-phenotype correlation. mRNA expression studies were performed using pSPL3 vector, studies of subcellular localization were performed using pEGFP-N1 vector and luciferase assays were performed using pGL3-Basic vector. We have identified 30 variants in the BMPR2 gene in 27 of 55 patients. In 16 patients we detected pathogenic mutations. Minigene assays revealed that 6 variants (synonymous, missense) result in splicing defect. By immunofluorescence assay, we observed that 4 mutations affect the protein localization. Finally, 4 mutations located in the 5′UTR region showed a decreased transcriptional activity in luciferase assays. Genotype-phenotype correlation, revealed that patients with pathogenic mutations have a more severe phenotype (sPaP p = 0.042, 6MWT p = 0.041), a lower age at diagnosis (p = 0.040) and seemed to have worse response to phosphodiesterase-5-inhibitors (p = 0.010). Our study confirms that in vitro expression analysis is a suitable approach in order to investigate the phenotypic consequences of the nucleotide variants, especially in cases where the involved genes have a pattern of expression in tissues of difficult access.

Background and purpose Mutations in the BICD2 gene cause autosomal dominant lower extremity-predominant spinal muscular atrophy 2A (SMALED2A), a condition that is associated with a specific pattern of thigh and calf muscle involvement when studied by magnetic resonance imaging (MRI). Patients may present minor clinical sensory impairment, but objective sensory involvement has yet to be demonstrated. Methods We collected clinical data from 11 patients from five different families carrying mutations in BICD2. Genetic diagnosis was achieved using gene panel testing and skin biopsies were taken from two patients to study the epidermal nerve fiber density. Results In the studied patients, three new pathogenic mutations were detected as well as the already defined pathogenic p.Ser107Leu mutation. The most frequent clinical picture was characterized by lower-limb weakness in combination with foot deformities. One patient manifested clinical and electrophysiological sensory impairment, and the epidermal nerve fiber density study of another patient revealed the existence of a small-fiber neuropathy. Muscle MRI showed a common pattern of fat deposition including selective involvement of gluteus medius and minimus at the pelvic level, the anterior compartment of the thigh and the posterior compartment of the calf, with only mild or no involvement of the intrinsic foot muscles. Conclusions We report three new pathogenic mutations in the BICD2 gene. Muscle MRI confirms the existence of a selective pattern of thigh and leg muscle involvement in SMALED2A, providing additional information regarding pelvic and foot muscles. Moreover, our results raise the possibility of sensory involvement in the disease.

Wilson's disease (WD) is an autosomal recessive disorder caused by ATP7B mutations. Subjects with only one mutation may show clinical signs and individuals with biallelic changes may remain asymptomatic. We aimed to achieve a conclusive genetic diagnosis for 34 patients clinically diagnosed of WD. Genetic analysis comprised from analysis of exons to WES (whole exome sequencing), including promoter, introns, UTRs (untranslated regions), besides of study of large deletions/duplications by MLPA (multiplex ligation-dependent probe amplification). Biallelic ATP7B mutations were identified in 30 patients, so that four patients were analyzed using WES. Two affected siblings resulted to be compound heterozygous for mutations in CCDC115, which is involved in a form of congenital disorder of glycosylation. In sum, the majority of patients with a WD phenotype carry ATP7B mutations. However, if genetic diagnosis is not achieved, additional genes should be considered because other disorders may mimic WD.

Mutations in the HSJ1 ( Heat-Shock Protein J1 ) gene, also called DNAJB2 (DnaJ (Hsp40) homologue, subfamily B, member 2), have been recently described as a cause of hereditary neuropathies. The HSJ1 c.352+1G>A mutation in homozygote state has been reported as the causative mutation in a single family with autosomal recessive distal hereditary motor neuropathy (dHMN).1 Since then, two other families with different HSJ1 mutations have been described: one with a dHMN phenotype and the other with a Charcot-Marie-Tooth disease type 2 (CMT2) phenotype.2 We identified the HSJ1 c.352+1G>A mutation in 10 patients who underwent long-lasting follow-up. We describe their phenotype and clinical evolution. Ten patients from five different Spanish families had been diagnosed with dHMN or CMT2 at two tertiary referral centres in Spain between 1976 and 2014. They all underwent neurological examination and electrophysiological studies using standard techniques.3 Regular follow-up was performed in all cases and repeated electrophysiological studies were carried out in some patients. All the families carried the HSJ1 c.352+1G>A homozygote pathogenic sequence variation. Family 1 was diagnosed by exome sequencing. Families 2 and 4 were diagnosed using a gene panel for genetic testing of CMT and dHMN. Families 3 and 5 were identified by mutational screening of the HSJ1 c.352+1G>A change, carried out in 52 patients from our registry, with autosomal recessive dHMN or CMT2, who still had no molecular diagnosis. In all cases, mutation was confirmed by Sanger sequencing and a segregation analysis was performed whenever possible. All the patients and relatives included in the present study signed informed consent. The research protocols were approved by the respective institutional boards of the Ethics Committee …

INTRODUCTION: PLA2G6-associated neurodegeneration (PLAN) comprises a continuum of three phenotypes with overlapping clinical and radiologic features. METHODS: Observational clinical study in a cohort of infantile and childhood onset PLAN patients and genetic analysis of the PLA2G6 gene. We analysed chronological evolution in terms of age at onset and disease course through a 66-item questionnaire. We performed qualitative and quantitative assessment of MRI abnormalities and searched for clinical and radiological phenotype and genotype correlations. RESULTS: Sixteen PLAN patients (mean age: 10.2 years, range 3-33) were evaluated, with a median onset (years) of signs/symptoms as follows: neurological regression (1.5), oculomotor abnormalities (1.5), hypotonia (1.8), gait loss (2.2), pyramidal signs (3.0), axonal neuropathy (3.0), dysphagia (4.0), optic atrophy (4.0), psychiatric symptoms (4.0), seizures (5.9), joint contractures (6.0), dystonia (8.0), bladder dysfunction (13.0) and parkinsonism (15.0). MRI assessment identified cerebellar atrophy (19/19), brain iron deposition (10/19), clava hypertrophy (8/19) and T2/FLAIR hyperintensity of the cerebellar cortex (6/19). The mid-sagittal vermis relative diameter (MVRD) correlated with age at onset of clinical variants, meaning that the earlier the onset, the more severe the cerebellar atrophy. All patients harboured missense, nonsense and frameshift mutations in PLA2G6, including four novel variants. CONCLUSIONS: Cerebellar atrophy was a universal radiological sign in infantile and childhood onset PLAN, and correlated with the severity of the phenotype. Iron accumulation within the globus pallidum and substantia nigra was also a common and strikingly uniform feature regardless of the phenotype.

Charcot-Marie-Tooth disease (CMT) is a complex disorder with wide genetic heterogeneity. Here we present a new axonal Charcot-Marie-Tooth disease form, associated with the gene microrchidia family CW-type zinc finger 2 (MORC2). Whole-exome sequencing in a family with autosomal dominant segregation identified the novel MORC2 p.R190W change in four patients. Further mutational screening in our axonal Charcot-Marie-Tooth disease clinical series detected two additional sporadic cases, one patient who also carried the same MORC2 p.R190W mutation and another patient that harboured a MORC2 p.S25L mutation. Genetic and in silico studies strongly supported the pathogenicity of these sequence variants. The phenotype was variable and included patients with congenital or infantile onset, as well as others whose symptoms started in the second decade. The patients with early onset developed a spinal muscular atrophy-like picture, whereas in the later onset cases, the initial symptoms were cramps, distal weakness and sensory impairment. Weakness and atrophy progressed in a random and asymmetric fashion and involved limb girdle muscles, leading to a severe incapacity in adulthood. Sensory loss was always prominent and proportional to disease severity. Electrophysiological studies were consistent with an asymmetric axonal motor and sensory neuropathy, while fasciculations and myokymia were recorded rather frequently by needle electromyography. Sural nerve biopsy revealed pronounced multifocal depletion of myelinated fibres with some regenerative clusters and occasional small onion bulbs. Morc2 is expressed in both axons and Schwann cells of mouse peripheral nerve. Different roles in biological processes have been described for MORC2. As the silencing of Charcot-Marie-Tooth disease genes have been associated with DNA damage response, it is tempting to speculate that a deregulation of this pathway may be linked to the axonal degeneration observed in MORC2 neuropathy, thus adding a new pathogenic mechanism to the long list of causes of Charcot-Marie-Tooth disease.

Early-onset hereditary motor and sensory neuropathies are rare diseases representing a broad clinical and genetic spectrum. Without a notable familial history, the clinical diagnosis is complicated because acquired causes of peripheral neuropathy, such as inflammatory neuropathies, neuropathies with toxic causes, and nutritional deficiencies, must be considered. We examined the clinical, electrophysiological, and pathologic manifestations of a boy with an initial diagnosis of chronic inflammatory demyelinating polyneuropathy. The progression of the disease despite treatment led to a suspicion of hereditary motor and sensory neuropathy. Genetic testing revealed the presence of the MPZ p.D90E mutation in heterozygosis. To clarify the pathogenicity of this mutation and achieve a conclusive diagnosis, we investigated the MPZ p.D90E mutation through in silico and cellular approaches. This study broadens the clinical phenotype of hereditary motor and sensory neuropathy due to MPZ mutation and emphasises the difficulty of achieving an accurate genetic diagnosis in a sporadic patient to provide an appropriate pharmacologic treatment.

Neurodegeneration with brain iron accumulation (NBIA) is a group of inherited heterogeneous neurodegenerative rare disorders. These patients present with dystonia, spasticity, parkinsonism and neuropsychiatric disturbances, along with brain magnetic resonance imaging (MRI) evidence of iron accumulation. In sum, they are devastating disorders and to date, there is no specific treatment. Ten NBIA genes are accepted: PANK2, PLA2G6, C19orf12, COASY, FA2H, ATP13A2, WDR45, FTL, CP, and DCAF17; and nonetheless, a relevant percentage of patients remain without genetic diagnosis, suggesting that other novel NBIA genes remain to be discovered. Overlapping complex clinical pictures render an accurate differential diagnosis difficult. Little is known about the pathophysiology of NBIAs. The reported NBIA genes take part in a variety of pathways: CoA synthesis, lipid and iron metabolism, autophagy, and membrane remodeling. The next-generation sequencing revolution has achieved relevant advances in genetics of Mendelian diseases and provide new genes for NBIAs, which are investigated according to 2 main strategies: genes involved in disorders with similar phenotype and genes that play a role in a pathway of interest. To achieve an effective therapy for NBIA patients, a better understanding of the biological process underlying disease is crucial, moving toward a new age of precision medicine.

Histidyl-tRNA synthetase (HARS) ligates histidine to cognate tRNA molecules, which is required for protein translation. Mutations in HARS cause the dominant axonal peripheral neuropathy Charcot-Marie-Tooth disease type 2W (CMT2W); however, the precise molecular mechanism remains undefined. Here, we investigated three HARS missense mutations associated with CMT2W (p.Tyr330Cys, p.Ser356Asn, and p.Val155Gly). The three mutations localize to the HARS catalytic domain and failed to complement deletion of the yeast ortholog (HTS1). Enzyme kinetics, differential scanning fluorimetry (DSF), and analytical ultracentrifugation (AUC) were employed to assess the effect of these substitutions on primary aminoacylation function and overall dimeric structure. Notably, the p.Tyr330Cys, p.Ser356Asn, and p.Val155Gly HARS substitutions all led to reduced aminoacylation, providing a direct connection between CMT2W-linked HARS mutations and loss of canonical ARS function. While DSF assays revealed that only one of the variants (p.Val155Gly) was less thermally stable relative to wild-type, all three HARS mutants formed stable dimers, as measured by AUC. Our work represents the first biochemical analysis of CMT-associated HARS mutations and underscores how loss of the primary aminoacylation function can contribute to disease pathology.

6 Páges, 1 Figure, 1 Table. The online version of this article (https://doi.org/10.1007/s10048-017-0524-6) contains supplementary material, which is available to authorized users., In two siblings, who suffer from an early childhood-onset axonal polyneuropathy with exclusive involvement of motor fibers, the c.629T>C (p.F210S) mutation was identified in the X-linked AIFM1 gene, which encodes for the apoptosis-inducing factor (AIF). The mutation was predicted as deleterious, according to in silico analysis. A decreased expression of the AIF protein, altered cellular morphology, and a fragmented mitochondrial network were observed in the proband's fibroblasts. This new form of motor neuropathy expands the phenotypic spectrum of AIFM1 mutations and therefore, the AIFM1 gene should be considered in the diagnosis of hereditary motor neuropathies., This project was funded by the Instituto de Salud Carlos III (ISCIII)–FEDER (grant nos. PI12/00453 and PI15/00187). P.S. has a FPU grant funded by the Ministerio de Educación, Cultura y Deporte (grant no. FPU15/00964). A.S.M. has a grant funded by the Fundació per amor a l’art (FPAA). C.E. has a BMiguel Servet^ contract funded by the ISCIII and Centro de Investigación Príncipe Felipe (CIPF) (grant no. CPII14/00002). The Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER) is an initiative from the ISCIII.

PHARC (Polyneuropathy, Hearing loss, Ataxia, Retinitis pigmentosa and Cataracts) (MIM# 612674 ) is an autosomal recessive neurodegenerative disease caused by mutations in the ABHD12 gene. We evaluated two Spanish siblings affected with pes cavus, sensorimotor neuropathy, hearing loss, retinitis pigmentosa and juvenile cataracts in whom the genetic test of ABHD12 revealed a novel homozygous frameshift mutation, c.211_223del (p.Arg71Tyrfs*26). The earliest clinical manifestation in these patients was a demyelinating neuropathy manifested with a Charcot-Marie-Tooth phenotype over three decades. Progressive hearing loss, cataracts and retinitis pigmentosa appeared after the age of 30. We herein describe the complete clinical picture of these two patients, and focus particularly on neuropathy characteristics. This study supports the fact that although PHARC is rare, its phenotype is very characteristic and we should include its study in patients affected with demyelinating polyneuropathy, hearing loss and retinopathy.

Mutations in the GDAP1 gene can cause Charcot-Marie-Tooth disease. These mutations are quite rare in most Western countries but not so in certain regions of Spain or other Mediterranean countries. This cross-sectional retrospective multicenter study analyzed the clinical and genetic characteristics of patients with GDAP1 mutations across Spain. 99 patients were identified, which were distributed across most of Spain, but especially in the Northwest and Mediterranean regions. The most common genotypes were p.R120W (in 81% of patients with autosomal dominant inheritance) and p.Q163X (in 73% of autosomal recessive patients). Patients with recessively inherited mutations had a more severe phenotype, and certain clinical features, like dysphonia or respiratory dysfunction, were exclusively detected in this group. Dominantly inherited mutations had prominent clinical variability regarding severity, including 29% of patients who were asymptomatic. There were minor clinical differences between patients harboring specific mutations but not when grouped according to localization or type of mutation. This is the largest clinical series to date of patients with GDAP1 mutations, and it contributes to define the genetic distribution and genotype-phenotype correlation in this rare form of CMT.

Mutations in SH3TC2 trigger autosomal recessive demyelinating Charcot-Marie-Tooth type 4C (CMT4C) neuropathy. Sh3tc2 is specifically expressed in Schwann cells and is necessary for proper myelination of peripheral axons. In line with the early onset of neuropathy observed in patients with CMT4C, our analyses of the murine model of CMT4C revealed that the myelinating properties of Sh3tc2-deficient Schwann cells are affected at an early stage. This early phenotype is associated with changes in the canonical Nrg1/ErbB pathway involved in control of myelination. We demonstrated that Sh3tc2 interacts with ErbB2 and plays a role in the regulation of ErbB2 intracellular trafficking from the plasma membrane upon Nrg1 activation. Interestingly, both the loss of Sh3tc2 function in mice and the pathological mutations present in CMT4C patients affect ErbB2 internalization, potentially altering its downstream intracellular signaling pathways. Altogether, our results indicate that the molecular mechanism for the axonal size sensing is disturbed in Sh3tc2-deficient myelinating Schwann cells, thus providing a novel insight into the pathophysiology of CMT4C neuropathy.

BACKGROUND: Pantothenate kinase-associated neurodegeneration is a progressive neurological disorder occurring in both childhood and adulthood. The objective of this study was to design and pilot-test a disease-specific clinical rating scale for the assessment of patients with pantothenate kinase-associated neurodegeneration. METHODS: In this international cross-sectional study, patients were examined at the referral centers following a standardized protocol. The motor examination was filmed, allowing 3 independent specialists in movement disorders to analyze 28 patients for interrater reliability assessment. The scale included 34 items (maximal score, 135) encompassing 6 subscales for cognition, behavior, disability, parkinsonism, dystonia, and other neurological signs. RESULTS: Forty-seven genetically confirmed patients (30 ± 17 years; range, 6-77 years) were examined with the scale (mean score, 62 ± 21; range, 20-106). Dystonia with prominent cranial involvement and atypical parkinsonian features were present in all patients. Other common signs were cognitive impairment, psychiatric features, and slow and hypometric saccades. Dystonia, parkinsonism, and other neurological features had a moderate to strong correlation with disability. The scale showed good internal consistency for the total scale (Cronbach's a = 0.87). On interrater analysis, weighted kappa values (0.30-0.93) showed substantial or excellent agreement in 85% of the items. The scale also discriminated a subgroup of homozygous c.1583C>T patients with lower scores, supporting construct validity for the scale. CONCLUSIONS: The proposed scale seems to be a reliable and valid instrument for the assessment of pediatric and adult patients with pantothenate kinase-associated neurodegeneration. Additional validation studies with a larger sample size will be required to confirm the present results and to complete the scale validation testing. © 2017 International Parkinson and Movement Disorder Society.

Pulmonary Arterial Hypertension (PAH; OMIM 178600) is a rare disease defined by a sustained increase in mean pulmonary artery pressure ≥ 25 mmHg at rest with normal wedge pressure ≥15 mmHg and ≥30 mmHg on exercise. Much of what is known about the genetic basis of PAH is related to BMPR2 gene. The aim of this study was to characterize the subcellular localization of BMPR2 gene when carries several missense and nonsense mutations. After in silico analysis, variants were selected according to their pathogenic character. Subcellular localization were performed using pEGFP-N1 vector. HELA cells were fixed 24h post-transfection in 4% paraformaldehyde and images were visualized using a Leica TCS SP2 confocal system. To determine the effect of clinical mutations over the subcellular localization of the BMPR2 protein, we performed immunofluorescence assay. Ours results indicated that in Hela cells, wild-type BMPR2 localized at plasma membrane and at perinuclear area. However, the two nonsense mutations p.S52Sfs*2 and p.W298* and, the missense mutation p.C84F mislocalized from plasma membrane and showed an abnormal cytoplasmatic pattern. The p.Y218* mutation displayed slight modifications on the cellular localization of the BMPR2 protein. All of the remaining clinical mutations analyzed showed the same pattern as wild-type BMPR2. Patients with these pathogenic mutations showed an early age of diagnosis (p=0.039) and greater sPaP (p=0.042). Confirming the effect of the mutated protein in the localization will allow us to understand the role of BMPR2 in the development of the disease. Likewise, these mutations predispose individuals to a more severe phenotype and this group shown an early age at diagnosis.

22 p., figuras y bibliografía, Urocanase is an enzyme in the histidine pathway encoded by the UROC1 gene. We describe the first putative mutations, p.L70P and p.R450C, in the coding region of the UROC1 gene in a girl who suffers from urocanic aciduria presenting with mental retardation and intermittent ataxia. In silico predictions, protein expression studies and enzyme activity assays suggest that none of the mutations can produce a fully functional enzyme. The p.L70P substitution, which probably imply the disruption of an alpha-helix in the N-terminus, would alter its properties and therefore, its function. The p.R450C change would render impossible any interaction between urocanase and its substrate and would loss its enzyme activity. Consequently, these studies suggest that both mutations could alter the correct activity of urocanase, which would explain the clinical and biochemical findings described in the patient., This work was supported by grants from the Fondo de Investigación Sanitaria (PI051318 and PI070548). The CIBERER is an initiative of the Instituto de Salud Carlos III.

Charcot-Marie-Tooth disease is characterized by broad genetic heterogeneity with >50 known disease-associated genes. Mutations in some of these genes can cause a pure motor form of hereditary motor neuropathy, the genetics of which are poorly characterized. We designed a panel comprising 56 genes associated with Charcot-Marie-Tooth disease/hereditary motor neuropathy. We validated this diagnostic tool by first testing 11 patients with pathological mutations. A cohort of 33 affected subjects was selected for this study. The DNAJB2 c.352+1G>A mutation was detected in two cases; novel changes and/or variants with low frequency (50 known disease-associated genes. Mutations in some of these genes can cause a pure motor form of hereditary motor neuropathy, the genetics of which are poorly characterized. We designed a panel comprising 56 genes associated with Charcot-Marie-Tooth disease/hereditary motor neuropathy. We validated this diagnostic tool by first testing 11 patients with pathological mutations. A cohort of 33 affected subjects was selected for this study. The DNAJB2 c.352+1G>A mutation was detected in two cases; novel changes and/or variants with low frequency (A mutation was detected in two cases; novel changes and/or variants with low frequency (A mutation was also detected in three additional families. On haplotype analysis, all of the patients from these five families shared the same haplotype; therefore, the DNAJB2 c.352+1G>A mutation may be a founder event. Our gene panel allowed us to perform a very rapid and cost-effective screening of genes involved in Charcot-Marie-Tooth disease/hereditary motor neuropathy. Our diagnostic strategy was robust in terms of both coverage and read depth for all of the genes and patient samples. These findings demonstrate the difficulty in achieving a definitive molecular diagnosis because of the complexity of interpreting new variants and the genetic heterogeneity that is associated with these neuropathies.

Charcot-Marie-Tooth (CMT) disease type 4 (CMT4) is the name given to autosomal recessive forms of hereditary motor and sensory neuropathy (HMSN). When we began this study, three genes or loci associated with inherited peripheral neuropathies had already been identified in the European Gypsy population: HMSN-Lom (MIM 601455), HMSN-Russe (MIM 605285) and the congenital cataracts facial dysmorphism neuropathy syndrome (MIM 604168). We have carried out genetic analyses in a series of 20 Spanish Gypsy families diagnosed with a demyelinating CMT disease compatible with an autosomal recessive trait. We found the p.R148X mutation in the N-myc downstream-regulated gene 1 gene to be responsible for the HMSN-Lom in four families and also possible linkage to the HMSN-Russe locus in three others. We have also studied the CMT4C locus because of the clinical similarities and showed that in 10 families, the disease is caused by mutations located on the SH3 domain and tetratricopeptide repeats 2 (SH3TC2) gene: p.R1109X in 20 out of 21 chromosomes and p.C737_P738delinsX in only one chromosome. Moreover, the SH3TC2 p.R1109X mutation is associated with a conserved haplotype and, therefore, may be a private founder mutation for the Gypsy population. Estimation of the allelic age revealed that the SH3TC2 p.R1109X mutation may have arisen about 225 years ago, probably as the consequence of a bottleneck.

Pulmonary Arterial Hypertension (PAH; OMIM 178600, ORPHA 422) is an autosomal dominant disease characterized by pulmonary vascular resistance increase, vascular remodelling and right heart failure. Symptoms of PAH include fatigue, shortness of breath and syncope. However, the result of missense, nonsense, synonymous and 5´UTR region mutations in BMPR2 gene is unknown. The aims were to determine pathological changes in BMPR2 gene presumably involved in an erroneous processing of mRNA, check the subcellular localization of missense and nonsense variats and correlate genotype/fenotype. Variants were selected from an in silico analysis. mRNA expression studies were performed using pSPL3 vector to confirm splicing alterations and studies of subcellular localization were performed using pEGFP-N1 vector. Correlation genotype-phenotype was performed using Spss v.19 software. Nine out of 24 changes analyzed were observed to affect mRNA processing (p.S52Sfs*2, p.C84F, p.P138A, p.R211R, p.Y218*, p.V278V, p.W298*, p.P327P, p.K467R). These changes produced either exon skipping or removed the main donor or acceptor sites to create a new donor or acceptor splice-site. Four out of 18 variants analyzed altered the subcellular localization of BMPR2 protein (p.S52Sfs*2, p.C84F, p.Y218*, W298*). Patients with these pathogenic mutations had a lower age of diagnosis (p=0.047) and greater sPaP (p=0.042). Functional and molecular studies have allowed us to check the real implication of the mutations studied and correlate the role in the phisiopathological pathway of the disease. Likewise, these mutations predispose individuals to a more severe phenotype and this group of patients showed an early age at diagnosis.

Background and purpose A three-generation family affected by axonal Charcot−Marie−Tooth disease (CMT) was investigated with the aim of discovering genetic defects and to further characterize the phenotype. Methods The clinical, nerve conduction studies and muscle magnetic resonance images of the patients were reviewed. A whole exome sequencing was performed and the changes were investigated by genetic studies, in silico analysis and luciferase reporter assays. Results A novel c.1226G>A change (p.R409Q) in the EGR2 gene was identified. Patients presented with a typical, late-onset axonal CMT phenotype with variable severity that was confirmed in the ancillary tests. The in silico studies showed that the residue R409 is an evolutionary conserved amino acid. The p.R409Q mutation, which is predicted as probably damaging, would alter the conformation of the protein slightly and would cause a decrease of gene expression. Conclusions This is the first report of an EGR2 mutation presenting as an axonal CMT phenotype with variable severity. This study broadens the phenotype of the EGR2-related neuropathies and suggests that the genetic testing of patients suffering from axonal CMT should include the EGR2 gene.

Mutations in SH3TC2 trigger autosomal recessive demyelinating Charcot-Marie-Tooth type 4C (CMT4C) neuropathy. Sh3tc2 is specifically expressed in Schwann cells and is necessary for proper myelination of peripheral axons. In line with the early onset of neuropathy observed in patients with CMT4C, our analyses of the murine model of CMT4C revealed that the myelinating properties of Sh3tc2-deficient Schwann cells are affected at an early stage. This early phenotype is associated with changes in the canonical Nrg1/ErbB pathway involved in control of myelination. We demonstrated that Sh3tc2 interacts with ErbB2 and plays a role in the regulation of ErbB2 intracellular trafficking from the plasma membrane upon Nrg1 activation. Interestingly, both the loss of Sh3tc2 function in mice and the pathological mutations present in CMT4C patients affect ErbB2 internalization, potentially altering its downstream intracellular signaling pathways. Altogether, our results indicate that the molecular mechanism for the axonal size sensing is disturbed in Sh3tc2-deficient myelinating Schwann cells, thus providing a novel insight into the pathophysiology of CMT4C neuropathy.

Charcot-Marie-Tooth (CMT) disease, a hereditary motor and sensory neuropathy that comprises a complex group of more than 50 diseases, is the most common inherited neuropathy. CMT is generally divided into demyelinating forms, axonal forms and intermediate forms. CMT is also characterized by a wide genetic heterogeneity with 29 genes and more than 30 loci involved. The most common pattern of inheritance is autosomal dominant (AD), although autosomal recessive (AR) forms are more frequent in Mediterranean countries. In this chapter we give an overview of the associated genes, mechanisms and epidemiology of AR-CMT forms and their associated phenotypes.

Charcot-Marie-Tooth (CMT) disease, a hereditary motor and sensory neuropathy that comprises a complex group of more than 50 diseases, is the most common inherited neuropathy. CMT is generally divided into demyelinating forms, axonal forms and intermediate forms. CMT is also characterized by a wide genetic heterogeneity with 29 genes and more than 30 loci involved. The most common pattern of inheritance is autosomal dominant (AD), although autosomal recessive (AR) forms are more frequent in Mediterranean countries. In this chapter we give an overview of the associated genes, mechanisms and epidemiology of AR-CMT forms and their associated phenotypes.

6 páginas, 1 figura, 2 tablas, Congenital hypomyelinating neuropathy (CHN) is a severe inherited neuropathy with neonatal or early infancy onset, reduced nerve conduction velocity, and pathological evidence of hypomyelination. We describe a case of CHN that presented with neonatal hypotonia and a progressive downhill clinical course, developing cranial nerve dysfunction, and respiratory failure. The nerve conduction velocities were severely slowed and sural nerve biopsy revealed non-myelinated and poorly myelinated axons, with no typical onion bulbs. The mutational screening showed that our proband harbored a novel missense mutation, p.S121F, in the MPZ gene. In silico analyses and molecular modeling predicted that the replacement of a serine by a phenylalanine is a non-tolerated change and may affect the folding and the stability of the protein. Subcellular location studies were performed and revealed that the mutant protein loses its correct location on the cell membrane surface and is mainly expressed in the cytosol, reducing its adhesive properties. This case illustrates the clinical heterogeneity that exists in neuropathies associated with MPZ mutations and highlights that in patients with mild hypotonia in the first months that develop a very severe demyelinating neuropathy, the MPZ gene must be taken into account., This work was supported by the Instituto de Salud Carlos III (Grants number PI08/90857, PI08/0889, CP08/00053 and PS09/00095) co-funded with FEDER funds. C.E. has a “Miguel Servet” contract funded by the Instituto de Salud Carlos III. The CIBERNED and the CIBERER are initiatives of the Instituto de Salud Carlos III.

32 p., figuras y bibliografía, Mutations in SH3TC2 cause Charcot-Marie Tooth disease (CMT) type 4C, a demyelinating inherited neuropathy characterized by early onset and scoliosis. Here we demonstrate that SH3TC2 is expressed in several components of the endocytic pathway including early endosomes, late endosomes and clathrin-coated vesicles close to the trans-Golgi network, and in the plasma membrane. Myristoylation of SH3TC2 in glycine 2 is necessary but not sufficient for the proper location of the protein in the cell membranes. In addition to myristoylation, correct anchoring also needs the presence of SH3 and TPR domains. Mutations that cause a stop codon and produce premature truncations that remove most of the TPR domains are expressed as the wild type protein. In contrast, missense mutations in or around the region of the first TPR domain are not expressed in early endosomes, have reduced expression in plasma membrane and late endosomes, and are variably expressed in clathrin-coated vesicles. Our findings suggest that the endocytic and membrane trafficking pathway is involved in the pathogenesis of CMT4C disease. We postulate that missense mutations of SH3TC2 could impair communication between the Schwann cell and the axon causing an abnormal myelin formation., This work was supported by the Fondo de Investigación Sanitaria [grant numbers PI08/90857, PI08/0889, CP08/00053] and the Spanish Ministry Science and Innovation [grant number SAF2006-01047]. V. L. is a recipient of JAE predoctoral fellowship from the Spanish National Research Council (CSIC). M.I.G. has a “Ramón y Cajal” contract funded by the Ministry of Science and Innovation. C.E. has a “Miguel Servet” contract funded by the Fondo de Investigación Sanitaria. Both CIBERER and CIBERNED are initiatives from the Instituto de Salud Carlos III.

14 p., figuras y bibliografía, Ataxia with isolated vitamin E deficiency (AVED) is a rare autosomal recessive neurodegenerative disease due to mutations in the alpha-TTP (alpha-tocopherol transfer protein) gene. In AVED, the biochemical hallmark is the low plasmatic levels of vitamin E and in most of patients, vitamin E supplementation allows a stabilization of the neurological conditions. We have investigated the genetic cause of ataxia and reduced levels of vitamin E, and apolipoproteins A1 (apoA1) and B (apoB) in a 16-year-old patient. Results revealed that our propositus is a compound heterozygote for the c.227_229delinsATT/c.744delA mutations in the -TTP gene, each inherited from one of the two parents. His sister is also a compound heterozygote for both mutations and she presents a biochemical pattern similar to that of his brother. After receiving the vitamin E supplementation, plasmatic levels of vitamin E and apoA1 have been normalized in the propositus. The detected mutations would justify the undetectable levels of vitamin E, but would not explain the also decreased levels of the apolipoproteins, as neither that after treatment with vitamin E, the levels of apoB do not become normal. These findings suggest that other genes may play a role in producing this atypical biochemical profile., This work was supported by the Spanish Network on Cerebellar Ataxias of the Instituto de Salud Carlos III (grant G03/56). The CIBERER is an initiative of the Instituto de Salud Carlos III.