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.

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.

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.

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.

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.

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.

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.

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.

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.

Role of MAPT in Pure Motor Neuron Disease: Report of a Recurrent Mutation in Italian Patients.

The aim of our study was to evaluate the role of mutations in the MAPT gene in patients with pure amyotrophic lateral sclerosis (ALS). A cohort of 120 ALS patients, both sporadic and familial, without cognitive impairment was analyzed by next-generation sequencing with a multiple-gene panel comprising 23 genes, including MAPT, known to be associated with ALS and frontotemporal dementia. The presence of the C9orf72 expansion was also investigated. Twelve patients had mutations in the SOD1, TARDBP, MATR3, and FUS genes, while 10 patients carried the C9orf72 expansion. One female patient was found to carry the D348G mutation in MAPT, previously reported in an Italian family with lower motor neuron disease. Our patient presented both upper and lower motor neuron signs, early development of dyspnea, resting and kinetic tremor, and a slow disease course (> 11 years). The present case further broadens the clinical phenotype associated with MAPT mutations and suggests that, although rarely, MAPT mutations can cause ALS and, therefore, should be analyzed in ALS patients, especially in those with early breathing difficulties and long-lasting disease.

Alternative Splicing in the Human PMP22 Gene: Implications in CMT1A Neuropathy.

CMT1A patients commonly share PMP22 genetic overloading but they show phenotypic heterogeneity and variability in PMP22 mRNA and protein expression. Moreover, PMP22 mRNA levels do not correlate with clinical outcome measures in these patients, suggesting their uselessness as a disease biomarker. Thus, in-depth analysis of PMP22 transcription and translation might help to define its pathogenic role in CMT1A. We focused on the alternative splicing of PMP22 gene to verify whether mRNA processing is altered in CMT1A. We identified three new PMP22 transcripts enriched in human sural nerve biopsies. One of them was an untranslated variant, whereas the other two originated from a PMP22 undescribed exon and encoded for a new putative protein localized in the endoplasmic reticulum. As splicing events in the PMP22 gene are differently regulated in tissues and during development, we analyzed the levels of PMP22 transcripts and their splicing pattern in human and experimental CMT1A. We found an altered PMP22 splicing ratio in the CMT1A rat. In addition, we showed a remarkable derangement in rat QKI expression, which is a critical regulator of splicing during myelination. Overall, our data suggest that an alteration of mRNA processing could be a pathogenic mechanism in CMT1A.

Molecular Chaperones in the Pathogenesis of Amyotrophic Lateral Sclerosis: The Role of HSPB1.

Genetic discoveries in amyotrophic lateral sclerosis (ALS) have a significant impact on deciphering molecular mechanisms of motor neuron degeneration but, despite recent advances, the etiology of most sporadic cases remains elusive. Several cellular mechanisms contribute to the motor neuron degeneration in ALS, including RNA metabolism, cellular interactions between neurons and nonneuronal cells, and seeding of misfolded protein with prion-like propagation. In this scenario, the importance of protein turnover and degradation in motor neuron homeostasis gained increased recognition. In this study, we evaluated the role of the candidate gene HSPB1, a molecular chaperone involved in several proteome-maintenance functions. In a cohort of 247 unrelated Italian ALS patients, we identified two variants (c.570G>C, p.Gln190His and c.610dupG, p.Ala204Glyfs* 6). Functional characterization of the p.Ala204Glyfs* 6 demonstrated that the mutant protein alters HSPB1 dynamic equilibrium, sequestering the wild-type protein in a stable dimer and resulting in a loss of chaperone-like activity. Our results underline the relevance of identifying rare but pathogenic variations in sporadic neurodegenerative diseases, suggesting a possible correlation between specific pathomechanisms linked to HSPB1 mutations and the associated neurological phenotype. Our study provides additional lines of evidence to support the involvement of HSPB1 in the pathogenesis of sporadic ALS.

Innovative quantitative testing of hand function in Charcot-Marie-Tooth neuropathy.

To describe a new test to quantitatively evaluate hand function in patients affected by Charcot-Marie-Tooth neuropathy (CMT). The sensor-engineered glove test (SEGT) was applied to CMT patients (N: 26) and compared with a cohort of healthy controls (HC, N: 26). CMT patients were further divided into subjects with clinically normal (group 1) or impaired hand (group 2) function. The SEGT parameters evaluated were touch duration, inter-tapping interval, and movement rate parameters of two different sequences: finger tapping (FT) and index-medium-ring-little (IMRL) performed at self-paced mode (SPM) and maximum velocity (MV). Hand function and strength were assessed by the 9-hole peg test (9HPT) and dynamometry. Disability of patients was measured by the CMT neuropathy score. CMT patients had significantly worst performances at SEGT than controls regarding the rate of execution of both FT (at MV) and IMRL sequences (at SPM and MV). The rate parameter at MV in IMRL sequence showed a significant trend of decreasing in its average between HC (n: 26, rate = 3.08 ± 0.52 Hz), group 1 (n: 9, rate = 2.64 ± 0.66 Hz) and group 2 (n: 17, rate = 2.19 ± 0.45 Hz) (p for trend <0.001). No correlations were found with either 9HPT, dynamometry, electrophysiology, and the CMT neuropathy score. The SEGT test is sensitive to show hand dysfunction in CMT patients, with and without clinically impaired hands.

Neuroimaging features in C9orf72 and TARDBP double mutation with FTD phenotype.

Increasing evidence has shown that morphological and functional neuroimaging may help to understand the pathophysiological mechanisms leading to behavioral disturbances in patients with genetic or sporadic frontotemporal dementia (FTD). The C9orf72 expansion was found in association with the N267S TARDBP mutation in two siblings with behavioral-variant FTD (bvFTD). In one of them with very mild dementia, MRI showed symmetric atrophy of temporal, inferolateral and orbital frontal cortex, while [18F]FDG-PET disclosed more extended hypometabolism in dorsolateral and inferolateral frontal cortex, anterior cingulate, and caudate nucleus. Hypometabolism in right lateral and orbital frontal cortex was confirmed also in comparison with a group of sporadic bvFTD patients. These findings appear as the neuroimaging hallmark of double C9orf72 and TARDBP gene mutation with a bvFTD phenotype.

Sural nerve biopsy and functional studies support the pathogenic role of a novel MPZ mutation.

Our patient is a 65-year-old woman presenting with bilateral pes cavus, pronounced distal muscle wasting, weakness and areflexia. Electrophysiological findings included diffuse unrecordable motor and sensory responses. While the CMT phenotype was evident, the lack of family history and the severe, but unspecific electrophysiological impairment, was a challenge for genetic diagnosis. A sural nerve biopsy was performed, showing a severe loss of myelinated fibers with residual axons surrounded by myelin outfoldings. Whereas myelin outfoldings are a pathological hallmark of autosomal recessive CMT4B1 and CMT4B2, due to mutations in myotubularin-related 2 (MTMR2) and 13 (MTMR13) genes respectively, they may also occur in nerve biopsies from CMT1B patients. By direct sequencing, a novel heterozygous transversion c.410G>T in MPZ gene was demonstrated, producing an amino acid change from glycine to valine in position 108 (p.G108V). In HeLa cells the fusion P0G108V-EGFP was normally trafficked to the cell membrane, but with decreased P0 adhesion function, compared with wild-type P0, thus supporting a pathogenic role of the new variant. In conclusion this case highlights the relevance, in selected cases, of sural nerve biopsy to orient the genetic/molecular tests, while in vitro analyses may strengthen the pathogenic role of novel mutations.

Complexities of Genetic Counseling for ALS: A Case of Two Siblings with Discordant Genetic Test Results.

Rapid advances in the genetics of amyotrophic lateral sclerosis (ALS) have dramatically changed the approach of clinicians and researchers to the motor neuron diseases. We report two siblings in whom the genetic study provided conflicting results, hence raising a number of issues which deserve to be considered by clinicians involved in genetic testing for ALS. The first patient died within 2 years of ALS onset, while her brother still manages to walk unaided, 7 years into onset. Genetic analyses, performed on the first patient as part of a research protocol, and as clinical genetic testing on the brother, provided different results. Results for Patient 1 were negative for all investigated genes, thus suggesting that her disease may be a phenocopy, while her brother carried an autosomal dominant TARDBP mutation (p.A382T). A multidisciplinary approach may help patients and clinicians face the emerging dilemmas in such a complex field. Sharing and updating of advances, not to mention uncertainties inherent to current knowledge, with patients and families may prove to be an effective way to support them and to make them aware of the present limits of our knowledge and of the blurred border between research and clinical practice.

Charcot-Marie-Tooth disease: frequency of genetic subtypes in a Southern Italy population.

The objective of this study is to assess the genetic distribution of Charcot-Marie-Tooth (CMT) disease in Campania, a region of Southern Italy. We analyzed a cohort of 197 index cases and reported the type and frequency of mutations for the whole CMT population and for each electrophysiological group (CMT1, CMT2, and hereditary neuropathy with susceptibility to pressure palsies [HNPP]) and for familial and isolated CMT cases. Genetic diagnosis was achieved in 148 patients (75.1%) with a higher success rate in HNPP and CMT1 than CMT2. Only four genes (PMP22, GJB1, MPZ, and GDAP1) accounted for 92% of all genetically confirmed CMT cases. In CMT1, PMP22 duplication was the most common mutation while the second gene in order of frequency was MPZ in familial and SH3TC2 in isolated cases. In CMT2, GJB1 was the most frequent mutated gene and GJB1 with GDAP1 accounted for almost 3/4 of genetically defined CMT2 patients. The first gene in order of frequency was GJB1 in familial and GDAP1 in isolated cases. In HNPP, the majority of patients harbored the PMP22 gene deletion. The novelty of our data is the relatively high frequency of SH3TC2 and GDAP1 mutations in demyelinating and axonal forms, respectively. These epidemiological data can help in panel design for our patients' population.

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.