Next-generation sequencing in Charcot-Marie-Tooth: a proposal for improvement of ACMG guidelines for variant evaluation.

BACKGROUND: The application of massive parallel sequencing technologies in the molecular analysis of Charcot-Marie-Tooth (CMT) has enabled the rapid and cost-effective identification of numerous potentially significant variants for diagnostic purposes. The objective is to reduce the number of variants, focusing only on those with pathogenic significance. The 2015 American College of Medical Genetics and Genomics (ACMG) guidelines aid in achieving this goal, but it is now evident that a pathology or gene-specific review of these rules is essential to avoid misinterpretations that may result from blindly applying the criteria. This study demonstrates how revised ACMG criteria, combined with CMT-specific literature data and expertise, can alter the final classification of a variant. METHODS: We reviewed ACMG criteria based on current knowledge of CMT and provided suggestions for adapting them to the specificities of CMT. RESULTS: Of the 226 index patients analysed, a diagnostic yield of 20% was obtained. It is worth noting that the 9% of cases had their final diagnosis changed with the application of the revised criteria, often resulting in the loss of the pathogenic classification of a variant. CONCLUSIONS: The widespread availability of high-throughput sequencing technologies has enabled genetic testing even for laboratories without specific disease expertise. Disease-specific ACMG criteria can be a valuable tool to prevent the proliferation of variants of uncertain significance and the misinterpretation of variants.

Phenotypic spectrum of myelin protein zero-related neuropathies: a large cohort study from five mutation clusters across Italy.

BACKGROUND: We aimed to investigate the clinical features of a large cohort of patients with myelin protein zero (MPZ)-related neuropathy, focusing on the five main mutation clusters across Italy. METHODS: We retrospectively gathered a minimal data set of clinical information in a series of patients with these frequent mutations recruited among Italian Charcot-Marie-Tooth (CMT) registry centres, including disease onset/severity (CMTES-CMT Examination Score), motor/sensory symptoms and use of orthotics/aids. RESULTS: We collected data from 186 patients: 60 had the p.Ser78Leu variant ('classical' CMT1B; from Eastern Sicily), 42 the p.Pro70Ser (CMT2I; mainly from Lombardy), 38 the p.Thr124Met (CMT2J; from Veneto), 25 the p.Ser44Phe (CMT2I; from Sardinia) and 21 the p.Asp104ThrfsX13 (mild CMT1B; from Apulia) mutation. Disease severity (CMTES) was higher (p<0.001) in late-onset axonal forms (p.Thr124Met=9.2±6.6; p.Ser44Phe=7.8±5.7; p.Pro70Ser=7.6±4.8) compared with p.Ser78Leu (6.1±3.5) patients. Disease progression (ΔCMTES/year) was faster in the p.Pro70Ser cohort (0.8±1.0), followed by p.Ser44Phe (0.7±0.4), p.Thr124Met (0.4±0.5) and p.Ser78Leu (0.2±0.4) patients. Disease severity (CMTES=1.2±1.5), progression (ΔCMTES/year=0.1±0.4) and motor involvement were almost negligible in p.Asp104ThrfsX13 patients, who, however, frequently (78%, p<0.001) complained of neuropathic pain. In the other four clusters, walking difficulties were reported by 69-85% of patients, while orthotic and walking aids use ranged between 40-62% and 16-28%, respectively. CONCLUSIONS: This is the largest MPZ (and late-onset CMT2) cohort ever collected, reporting clinical features and disease progression of 186 patients from five different clusters across Italy. Our findings corroborate the importance of differentiating between 'classical' childhood-onset demyelinating, late-onset axonal and mild MPZ-related neuropathy, characterised by different pathomechanisms, in view of different therapeutic targets.

The neurological core features of the infantile-onset multisystem neurologic, endocrine, and pancreatic disease: A novel nonsense mutation in an Italian family.

AIM: Biallelic mutations in the PTRH2 gene have been associated with infantile multisystem neurological, endocrine, and pancreatic disease (IMNEPD), a rare autosomal recessive disorder of variable expressivity characterized by global developmental delay, intellectual disability or borderline IQ level, sensorineural hearing loss, ataxia, and pancreatic insufficiency. Various additional features may be included, such as peripheral neuropathy, facial dysmorphism, hypothyroidism, hepatic fibrosis, postnatal microcephaly, cerebellar atrophy, and epilepsy. Here, we report the first Italian family presenting only predominant neurological features. METHODS: Extensive neurological and neurophysiological evaluations have been conducted on the two affected brothers and their healthy mother since 1996. The diagnosis of peripheral neuropathy of probable hereditary origin was confirmed through a sural nerve biopsy. Exome sequencing was performed after the analysis of major neuropathy-associated genes yielded negative results. RESULTS: Whole-exome sequencing analysis identified the homozygous substitution c.256C>T (p.Gln86Ter) in the PTRH2 gene in the two siblings. According to American College of Medical Genetics and Genomics (ACMG) guidelines, the variant has been classified as pathogenic. At 48 years old, the proband's reevaluation confirmed a demyelinating sensorimotor polyneuropathy with bilateral sensorineural hearing loss that had been noted since he was 13. Additionally, drug-resistant epileptic seizures occurred when he was 32 years old. No hepatic or endocrinological signs developed. The younger affected brother, 47 years old, has an overlapping clinical presentation without epilepsy. INTERPRETATION: Our findings expand the clinical phenotype and further demonstrate the clinical heterogeneity related to PTRH2 variants. We thereby hope to better define IMNEPD and facilitate the identification and diagnosis of this novel disease entity.

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

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

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

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

Clinical spectrum and frequency of Charcot-Marie-Tooth disease in Italy: Data from the National CMT Registry.

BACKGROUND AND PURPOSE: Data are reported from the Italian CMT Registry. METHODS: The Italian CMT Registry is a dual registry where the patient registers and chooses a reference center where the attending clinician collects a minimal dataset of information and administers the Charcot-Marie-Tooth (CMT) Examination/Neuropathy Score. Entered data are encrypted. RESULTS: Overall, 1012 patients had registered (535 females) and 711 had received a genetic diagnosis. Demyelinating CMT (65.3%) was more common than axonal CMT2 (24.6%) and intermediate CMT (9.0%). The PMP22 duplication was the most frequent mutation (45.2%), followed by variants in GJB1 and MPZ (both ~10%) and MFN2 (3.3%) genes. A relatively high mutation rate in some "rare" genes (HSPB1 1.6%, NEFL 1.5%, SH3TC2 1.5%) and the presence of multiple mutation clusters across Italy was observed. CMT4A was the most disabling type, followed by CMT4C and CMT1E. Disease progression rate differed depending on the CMT subtype. Foot deformities and walking difficulties were the main features. Shoe inserts and orthotic aids were used by almost one-half of all patients. Scoliosis was present in 20% of patients, especially in CMT4C. Recessive forms had more frequently walking delay, walking support need and wheelchair use. Hip dysplasia occurred in early-onset CMT. CONCLUSIONS: The Italian CMT Registry has proven to be a powerful data source to collect information about epidemiology and genetic distribution, clinical features and disease progression of CMT in Italy and is a useful tool for recruiting patients in forthcoming clinical trials.

Skeletal muscle involvement in biallelic SORD mutations: case report and review of the literature.

Biallelic mutations in the sorbitol dehydrogenase (SORD) gene have been identified as a genetic cause of autosomal recessive axonal Charcot-Marie-Tooth disease 2 (CMT2) and distal hereditary motor neuropathy (dHMN). We herein review the main phenotypes associated with SORD mutations and report the case of a 16-year-old man who was referred to our outpatient clinic for a slowly worsening gait disorder with wasting and weakness of distal lower limbs musculature. Since creatine phosphokinase (CPK) values were persistently raised (1.5fold increased) and a Next-Generation Sequencing CMT-associated panel failed in identifying pathogenic variants, a muscle biopsy was performed with evidence of alterations suggestive of a protein surplus distal myopathy. Finally, Whole-Exome Sequencing (WES) identified two pathogenic SORD variants in the heterozygous state: c.458C > A (p.Ala153Asp) and c.757delG (p.Ala253Glnfs*27). This is an isolated report of compound heterozygosity for two SORD mutations associated with clinical and histological signs of skeletal muscle involvement, expanding the phenotypic expression of SORD mutations.

Respiratory involvement and sleep-related disorders in CMT1A: case report and review of the literature.

Sleep-disordered breathing has been reported in Charcot-Marie-Tooth disease (CMT) type 1A in association with diaphragmatic weakness and sleep apnea syndrome, mainly of the obstructive type (OSA). Improvement has been observed not only in sleep quality but also in neuropathy symptoms in CMT1A patients with OSA following the initiation of continuous positive airway pressure. We report the cases of two siblings affected by CMT1A associated with hemidiaphragm relaxatio necessitating nocturnal non-invasive ventilation (NIV). Two twins, now 42 years old, with a family history of CMT1A, received a genetic diagnosis of CMT1A at the age of 16. Over the years, they developed a slowly worsening gait disorder and a decline in fine motor hand movements, currently presenting with moderate disability (CMTES:13). At the age of 40, they both started complaining of daytime sleepiness, orthopnea, and exertional dyspnea. They received a diagnosis of relaxatio of the right hemidiaphragm associated with impairment of nocturnal ventilation and they both have benefited from nocturnal NIV. Disorders of breathing during sleep may be underestimated in CMT1A since routine investigations of sleep quality are rarely performed. Our two clinical cases and a literature review suggest the importance of inquiring about symptoms of excessive daytime sleepiness and respiratory disturbances in individuals with CMT1A, even in the absence of severe neuropathy. In the presence of compatible symptoms, a pneumological assessment, along with an overnight polysomnogram and lung function tests, should be performed. Recognizing sleep-related symptoms is essential for providing accurate treatment and improving the quality of life for patients with CMT1A.

The SPTLC1 p.S331 mutation bridges sensory neuropathy and motor neuron disease and has implications for treatment.

AIMS: SPTLC1-related disorder is a late onset sensory-autonomic neuropathy associated with perturbed sphingolipid homeostasis which can be improved by supplementation with the serine palmitoyl-CoA transferase (SPT) substrate, l-serine. Recently, a juvenile form of motor neuron disease has been linked to SPTLC1 variants. Variants affecting the p.S331 residue of SPTLC1 cause a distinct phenotype, whose pathogenic basis has not been established. This study aims to define the neuropathological and biochemical consequences of the SPTLC1 p.S331 variant, and test response to l-serine in this specific genotype. METHODS: We report clinical and neurophysiological characterisation of two unrelated children carrying distinct p.S331 SPTLC1 variants. The neuropathology was investigated by analysis of sural nerve and skin innervation. To clarify the biochemical consequences of the p.S331 variant, we performed sphingolipidomic profiling of serum and skin fibroblasts. We also tested the effect of l-serine supplementation in skin fibroblasts of patients with p.S331 mutations. RESULTS: In both patients, we recognised an early onset phenotype with prevalent progressive motor neuron disease. Neuropathology showed severe damage to the sensory and autonomic systems. Sphingolipidomic analysis showed the coexistence of neurotoxic deoxy-sphingolipids with an excess of canonical products of the SPT enzyme. l-serine supplementation in patient fibroblasts reduced production of toxic 1-deoxysphingolipids but further increased the overproduction of sphingolipids. CONCLUSIONS: Our findings suggest that p.S331 SPTLC1 variants lead to an overlap phenotype combining features of sensory and motor neuropathies, thus proposing a continuum in the spectrum of SPTLC1-related disorders. l-serine supplementation in these patients may be detrimental.

Clinical and genetic features of a cohort of patients with MFN2-related neuropathy.

Charcot-Marie-Tooth disease type 2A (CMT2A) is a rare inherited axonal neuropathy caused by mutations in MFN2 gene, which encodes Mitofusin 2, a transmembrane protein of the outer mitochondrial membrane. We performed a cross-sectional analysis on thirteen patients carrying mutations in MFN2, from ten families, describing their clinical and genetic characteristics. Evaluated patients presented a variable age of onset and a wide phenotypic spectrum, with most patients presenting a severe phenotype. A novel heterozygous missense variant was detected, p.K357E. It is located at a highly conserved position and predicted as pathogenic by in silico tools. At a clinical level, the p.K357E carrier shows a severe sensorimotor axonal neuropathy. In conclusion, our work expands the genetic spectrum of CMT2A, disclosing a novel mutation and its related clinical effect, and provides a detailed description of the clinical features of a cohort of patients with MFN2 mutations. Obtaining a precise genetic diagnosis in affected families is crucial both for family planning and prenatal diagnosis, and in a therapeutic perspective, as we are entering the era of personalized therapy for genetic diseases.

Genetic Workup for Charcot-Marie-Tooth Neuropathy: A Retrospective Single-Site Experience Covering 15 Years.

Charcot-Marie-Tooth (CMT) disease is the most commonly inherited neurological disorder. This study includes patients affected by CMT during regular follow-ups at the CMT clinic in Genova, a neuromuscular university center in the northwest of Italy, with the aim of describing the genetic distribution of CMT subtypes in our cohort and reporting a peculiar phenotype. Since 2004, 585 patients (447 index cases) have been evaluated at our center, 64.9% of whom have a demyelinating neuropathy and 35.1% of whom have an axonal neuropathy. A genetic diagnosis was achieved in 66% of all patients, with the following distribution: CMT1A (48%), HNPP (14%), CMT1X (13%), CMT2A (5%), and P0-related neuropathies (7%), accounting all together for 87% of all the molecularly defined neuropathies. Interestingly, we observe a peculiar phenotype with initial exclusive lower limb involvement as well as lower limb involvement that is maintained over time, which we have defined as a "strictly length-dependent" phenotype. Most patients with this clinical presentation shared variants in either HSPB1 or MPZ genes. The identification of distinctive phenotypes such as this one may help to address genetic diagnosis. In conclusion, we describe our diagnostic experiences as a multidisciplinary outpatient clinic, combining a gene-by-gene approach or targeted gene panels based on clinical presentation.

RFC1 expansions are a common cause of idiopathic sensory neuropathy.

After extensive evaluation, one-third of patients affected by polyneuropathy remain undiagnosed and are labelled as having chronic idiopathic axonal polyneuropathy, which refers to a sensory or sensory-motor, axonal, slowly progressive neuropathy of unknown origin. Since a sensory neuropathy/neuronopathy is identified in all patients with genetically confirmed RFC1 cerebellar ataxia, neuropathy, vestibular areflexia syndrome, we speculated that RFC1 expansions could underlie a fraction of idiopathic sensory neuropathies also diagnosed as chronic idiopathic axonal polyneuropathy. We retrospectively identified 225 patients diagnosed with chronic idiopathic axonal polyneuropathy (125 sensory neuropathy, 100 sensory-motor neuropathy) from our general neuropathy clinics in Italy and the UK. All patients underwent full neurological evaluation and a blood sample was collected for RFC1 testing. Biallelic RFC1 expansions were identified in 43 patients (34%) with sensory neuropathy and in none with sensory-motor neuropathy. Forty-two per cent of RFC1-positive patients had isolated sensory neuropathy or sensory neuropathy with chronic cough, while vestibular and/or cerebellar involvement, often subclinical, were identified at examination in 58%. Although the sensory ganglia are the primary pathological target of the disease, the sensory impairment was typically worse distally and symmetric, while gait and limb ataxia were absent in two-thirds of the cases. Sensory amplitudes were either globally absent (26%) or reduced in a length-dependent (30%) or non-length dependent pattern (44%). A quarter of RFC1-positive patients had previously received an alternative diagnosis, including Sjögren's syndrome, sensory chronic inflammatory demyelinating polyneuropathy and paraneoplastic neuropathy, while three cases had been treated with immune therapies.

Validation of a new hand function outcome measure in individuals with Charcot-Marie-Tooth disease.

The symptomatology of Charcot-Marie-Tooth (CMT) disease mainly involves the feet and the hands. To date, there is no consensus on how to evaluate hand function in CMT. The aim of this study is to correlate the data of the engineered glove Hand Test System (HTS) with specific tests and the CMT examination score (CMTES). We analyzed 45 patients with the diagnosis of CMT using HTS, which measures the hand dexterity by specific sequences performed at maximum velocity. We completed the evaluation with the CMTES, tripod pinch and hand grip strength tested by a dynamometer, thumb opposition test (TOT), and Sollerman Hand function test (SHFT), and we conducted a test-retest with 20 normal subjects. Finger tapping (FT) and index-medium-ring-little (IMRL) sequence showed a significant correlation with CMTES (FT: dominant hand (DH): P = .036; non-dominant hand (NDH): P = .033; IMRL: DH: P = .009; NDH: P = .046). TOT correlated with CMTES significantly in both hands (P < .0001). tripod pinch showed a statistically significant correlation with CMTES (DH: P = .002; NDH: P = .005). Correlation between the hand grip and CMTES was significant only in DH (DH: P = .002). SHFT had a significant correlation with the CMTES (DH: P = .002). Test-retest showed a good reliability. HTS parameters correlate with CMTES confirming that this tool is sensitive to the hand deficits. In conclusion, we can state that HTS is a good, simple to use, and objective instrument to evaluate the hand function of CMT patients, but more studies on responsiveness and sensitivity are needed.

The suspected SARS-Cov-2 infection in a Charcot-Marie-Tooth patient undergoing postsurgical rehabilitation: the value of telerehabilitation for evaluation and continuing treatment.

We report, to the best of our knowledge, the first case of a probable COVID-19 infection in a 28-year-old man with Charcot-Marie-Tooth disease. The diagnosis was established through a remote interaction with the patient after early discharge from outpatient therapy due to upcoming traveling restrictions. The COVID-19 disease appeared mild, without major respiratory problems, and no obvious neuromuscular deterioration was reported or observed. Telerehabilitation provided an opportunity to continue with hand rehabilitation after tendon transfer surgery, perform an ad-hoc online evaluation, and advise the patient how to prevent the spread of infection and cope with restrictions limiting outpatient visits. This experience seems valuable for further development of telerehabilitation in anticipation of future pandemics or adversarial events since it allows reaching out to patients unable to travel and overcomes the need for regular outpatient visits.

A novel mutation of Twinkle in Perrault syndrome: A not rare diagnosis?

Perrault syndrome is a rare disorder characterized by ovarian dysgenesis, bilateral sensorineural hearing loss and associated with mutations in six mitochondrial proteins. Additional neurological features were also described. Herein, we report on a 27-year-old woman with Perrault syndrome (PS), moderate ataxia and axonal sensory-motor peripheral neuropathy in whom we identified compound heterozygous mutations in the TWNK gene (p.Val507Ile and the novel p.Phe248Ser variant). Fewer than 30 patients with PS have been reported worldwide. Neurological involvement is more frequently associated with mutations in TWNK and indicates possible genotype-phenotype correlations. TWNK mutations should be searched in patients with sensory ataxia, early onset bilateral sensorineural hearing loss, and ovarian dysfunction in women.

Early onset demyelinating Charcot-Marie-Tooth disease caused by a novel in-frame isoleucine deletion in peripheral myelin protein 2.

Peripheral myelin protein 2 (PMP2) is a small protein located on the cytoplasmic side of compact myelin, involved in the lipids transport and in the myelination process. In the last years few families affected with demyelinating Charcot-Marie-Tooth neuropathy (CMT1), caused by PMP2 mutations, have been identified. In this study we describe the first case of a PMP2 in-frame deletion. PMP2 was analyzed by direct sequencing after exclusion of the most frequent CMT-associated genes by using a next generation sequencing (NGS) genes panel. Sanger sequencing was used for family's segregation analysis. Molecular modeling analysis was used to evaluate the mutation impact on the protein structure. A novel PMP2: p.I50del has been identified in a child with early onset CMT1 and in three affected family members. All family members show an early onset demyelinating neuropathy without other distinguish features. Molecular modeling analysis and in silico evaluations do not suggest a strong impact on the overall protein structure, but a most likely altered protein function. This study suggests the importance to add PMP2 in CMT NGS genes panels or, at most, to test it after major CMT1 genes exclusion, due to the lack of diagnostic-addressing additional features.

Diagnostic Value of Sural Nerve Biopsy: Retrospective Analysis of Clinical Cases From 1981 to 2017.

Nerve biopsy represents the conclusive step in the diagnostic work-up of peripheral neuropathies, and its diagnostic yield is still debated. The aim of this study is to consider the impact of nerve biopsy on reaching a useful diagnosis in different peripheral neuropathies and its changing over time. We retrospectively analyzed 1,179 sural nerve biopsies performed in the period 1981-2017 at Neurological Clinic of Policlinico San Martino (Genoa). We relied on medical records and collected both clinical and pathological data in a database. Biopsy provided univocal diagnoses in 53% of cases (with an increase over time), multiple diagnostic options in 14%, while diagnosis was undetermined in 33% (undetermined reports decreased during the years). In 57% of patients, the pre-biopsy suspicion was confirmed, while in 43% sural biopsy modified the clinical diagnosis. The highest yield was in axonal neuropathies (29% undetermined reports vs. 40% in demyelinating and 48% in mixed neuropathies). In 68% of patients with vasculitic neuropathy, this etiology was already suspected, whereas in 32% nerve biopsy modified the clinical diagnosis. During the years, the number of annually performed biopsies decreased significantly (p = 0.007), with an increase in the mean age of patients (p < 0.0001). The percentage of hereditary neuropathies had a significant decrease (p = 0.016), while the rate of vasculitic and chronic inflammatory neuropathies increased (p < 0.0001). This is the largest Italian study addressing the yield of sural nerve biopsy. During the years, we observed a progressive refinement of the indication of this procedure, which confirms its utility for interstitial neuropathies, particularly if non-systemic vasculitic neuropathy is suspected.

Expanding the spectrum of genes responsible for hereditary motor neuropathies.

BACKGROUND: Inherited peripheral neuropathies (IPNs) represent a broad group of genetically and clinically heterogeneous disorders, including axonal Charcot-Marie-Tooth type 2 (CMT2) and hereditary motor neuropathy (HMN). Approximately 60%-70% of cases with HMN/CMT2 still remain without a genetic diagnosis. Interestingly, mutations in HMN/CMT2 genes may also be responsible for motor neuron disorders or other neuromuscular diseases, suggesting a broad phenotypic spectrum of clinically and genetically related conditions. Thus, it is of paramount importance to identify novel causative variants in HMN/CMT2 patients to better predict clinical outcome and progression. METHODS: We designed a collaborative study for the identification of variants responsible for HMN/CMT2. We collected 15 HMN/CMT2 families with evidence for autosomal recessive inheritance, who had tested negative for mutations in 94 known IPN genes, who underwent whole-exome sequencing (WES) analyses. Candidate genes identified by WES were sequenced in an additional cohort of 167 familial or sporadic HMN/CMT2 patients using next-generation sequencing (NGS) panel analysis. RESULTS: Bioinformatic analyses led to the identification of novel or very rare variants in genes, which have not been previously associated with HMN/CMT2 (ARHGEF28, KBTBD13, AGRN and GNE); in genes previously associated with HMN/CMT2 but in combination with different clinical phenotypes (VRK1 and PNKP), and in the SIGMAR1 gene, which has been linked to HMN/CMT2 in only a few cases. These findings were further validated by Sanger sequencing, segregation analyses and functional studies. CONCLUSIONS: These results demonstrate the broad spectrum of clinical phenotypes that can be associated with a specific disease gene, as well as the complexity of the pathogenesis of neuromuscular disorders.