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.

Distribution of the C9orf72 hexanucleotide repeat expansion in healthy subjects: a multicenter study promoted by the Italian IRCCS network of neuroscience and neurorehabilitation.

Introduction: High repeat expansion (HRE) alleles in C9orf72 have been linked to both amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD); ranges for intermediate allelic expansions have not been defined yet, and clinical interpretation of molecular data lacks a defined genotype-phenotype association. In this study, we provide results from a large multicenter epidemiological study reporting the distribution of C9orf72 repeats in healthy elderly from the Italian population. Methods: A total of 967 samples were collected from neurologically evaluated healthy individuals over 70 years of age in the 13 institutes participating in the RIN (IRCCS Network of Neuroscience and Neurorehabilitation) based in Italy. All samples were genotyped using the AmplideXPCR/CE C9orf72 Kit (Asuragen, Inc.), using standardized protocols that have been validated through blind proficiency testing. Results: All samples carried hexanucleotide G4C2 expansion alleles in the normal range. All samples were characterized by alleles with less than 25 repeats. In particular, 93.7% of samples showed a number of repeats ≤10, 99.9% ≤20 repeats, and 100% ≤25 repeats. Conclusion: This study describes the distribution of hexanucleotide G4C2 expansion alleles in an Italian healthy population, providing a definition of alleles associated with the neurological healthy phenotype. Moreover, this study provides an effective model of federation between institutes, highlighting the importance of sharing genomic data and standardizing analysis techniques, promoting translational research. Data derived from the study may improve genetic counseling and future studies on ALS/FTD.

Clinical epidemiology of amyotrophic lateral sclerosis in Liguria, Italy: a ten year follow up study.

OBJECTIVE: This article presents an updated analysis of the LIGALS register, a prospective study conducted over a ten-year period (2009-2018) in Liguria, Italy, aimed at evaluating the incidence, prevalence, clinical presentation, and management of amyotrophic lateral sclerosis (ALS). METHODS: We calculated the mean annual crude incidence rate of ALS, assessed the point prevalence of ALS on January 1, 2018, and analyzed demographic factors, clinical characteristics, and clinical management strategies. Data analysis included Cox regression analysis to identify predictors of survival. RESULTS: The mean annual crude incidence rate of ALS was 3.16/100,000 per year (CI 95%) while the point prevalence of ALS on January 1, 2018, was 9.31/100,000 population (CI 95%). Among the patients, 6.5% were familial ALS, while 93.5% were sporadic cases. Clinical management strategies, including percutaneous endoscopic gastrostomy (PEG) and noninvasive ventilation (NIV), were employed. The study observed a stable frequency of NIV initiation and PEG placement over time, with a growing trend toward earlier PEG positioning. The mean survival from symptom onset was 39 months, whereas from diagnosis, it was 26 months. Cox regression analysis identified several predictors of survival, including gender, age at onset and diagnosis, site of onset, diagnostic category, phenotype, and diagnostic delay. CONCLUSIONS: This comprehensive analysis provides valuable insights into the long-term trends in ALS epidemiology and clinical management in Liguria, Italy. It underscores the importance of continued research efforts in understanding and addressing the challenges posed by ALS, particularly in terms of early diagnosis and optimizing clinical interventions to improve patient outcomes.

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.

A case of Huntington disease-like 2 in a patient of African ancestry: the everlasting support of clinical examination in the molecular era.

Chorea, cognitive decline, and psychiatric symptoms are shared by Huntington's disease (HD) and similar conditions called HD phenocopies. We describe the first case reported in Italy of Huntington disease-like 2 (HDL2), clinically and radiologically indistinguishable from HD, showing the importance of considering African ancestry in the diagnostic process.

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.

An integrated approach to the evaluation of patients with asymptomatic or minimally symptomatic hyperCKemia.

INTRODUCTION/AIMS: Currently, there are no straightforward guidelines for the clinical and diagnostic management of hyperCKemia, a frequent and nonspecific presentation in muscle diseases. Therefore, we aimed to describe our diagnostic workflow for evaluating patients with this condition. METHODS: We selected 83 asymptomatic or minimally symptomatic patients with persistent hyperCKemia for participation in this Italian multicenter study. Patients with facial involvement and distal or congenital myopathies were excluded, as were patients with suspected inflammatory myopathies or predominant respiratory or cardiac involvement. All patients underwent a neurological examination and nerve conduction and electromyography studies. The first step of the investigation included a screening for Pompe disease. We then evaluated the patients for myotonic dystrophy type II-related CCTG expansion and excluded patients with copy number variations in the DMD gene. Subsequently, the undiagnosed patients were investigated using a target gene panel that included 20 genes associated with isolated hyperCKemia. RESULTS: Using this approach, we established a definitive diagnosis in one third of the patients. The detection rate was higher in patients with severe hyperCKemia and abnormal electromyographic findings. DISCUSSION: We have described our diagnostic workflow for isolated hyperCKemia, which is based on electrodiagnostic data, biochemical screening, and first-line genetic investigations, followed by successive targeted sequencing panels. Both clinical signs and electromyographic abnormalities are associated with increased diagnostic yields.

A novel mutation in COL3A1 associates to vascular Ehlers-Danlos syndrome with predominant musculoskeletal involvement.

BACKGROUND: Vascular Ehlers-Danlos syndrome (vEDS) is a heritable connective tissue disorder caused by defects in the type III collagen protein. It is generally considered the most severe form of Ehlers-Danlos syndrome (EDS) due to an increased risk of spontaneous artery or organ rupture. vEDS has an extremely heterogeneous presentation and muscle rupture is considered a minor diagnostic criterium. METHODS: A patient with a long history of inconclusive examinations and investigations was referred to our unit. The clinical picture was mainly characterized by muscle ruptures, whereas the cardiovascular involvement was limited to mitral regurgitation. We performed a panel analysis of genes associated with inheritable heart diseases using the TruSight Cardio kit (Illumina). A skin biopsy was then performed for functional studies to analyze the different forms of collagen molecules produced in vitro by cutaneous fibroblasts. RESULTS: The patient presented the novel variant c.3478A>G (p.Ile1160Val) in COL3A1 (NM_000090.3), whose pathogenicity was supported by biochemical analysis of type III collagen. CONCLUSION: In this report, we describe a case of vEDS with predominant and severe musculoskeletal involvement. Our findings provide insight into genetic variants and clinical expression of vEDS, broadening the clinical scenario of the syndrome.

The heterozygous deletion c.1509_1510delAG in exon 14 of FUS causes an aggressive childhood-onset ALS with cognitive impairment.

We report a case of childhood-onset ALS with a FUS gene mutation presenting cognitive impairment and a rapid clinical progression. The patient, an 11-year-old girl, presented with right distal upper limb weakness and mild intellectual disability at the Griffith Mental Development Scales. The disease rapidly worsened and the patient became tetraplegic and bed-ridden 2 years after symptom onset. A c.1509_1510delAG mutation in exon 14 of the FUS gene was detected, resulting in a predicted truncated protein, p.G504Wfs*12, lacking the nuclear localization signal. The levels of FUS mRNA in the proband were not significantly different compared to controls. Western immunoblot analysis showed that one antibody (500-526) detected in the proband ~50% of the amount of FUS protein compared to controls, while 3 other antibodies (2-27, 400-450 and FUS C-terminal), which recognize both wild type and the mutated FUS, detected 60% to 75% of the amount of the protein. These findings indicate that p.G504Wfs*12 FUS is more prone to undergo post-translational modification respect to wild type FUS.

The role of anterior prefrontal cortex in prospective memory: an exploratory FDG-PET study in early Alzheimer's disease.

From previous studies in healthy volunteers the prefrontal regions are deeply involved in prospective memory (PM), although little is known about the functional neural basis of PM in prodromal Alzheimer's disease (AD). To this end, we retrospectively recruited 18 patients with mild cognitive impairment caused by AD and 23 matched healthy control subjects who had undergone 18F-fluorodeoxyglucose positron emission tomography and the PM-specific paradigm test. Brain metabolism was correlated with the PM score in the 2 groups separately to find those brain areas correlated with PM performance, which were then used as a hub for an inter-regional metabolic connectivity analyses (inter-regional correlation analysis). Of note, in mild cognitive impairment caused by AD, but not in healthy control subjects, PM score positively correlated with metabolic levels in the right anterior prefrontal cortex (middle and inferior frontal gyri), which disclosed a loss of interhemispheric connectivity in the inter-regional correlation analysis. According to our findings, the functioning of the right anterior prefrontal cortex and its interhemispheric metabolic connectivity is crucial in early AD to sustain PM performance, which deteriorates along with progressive metabolic failure of the interconnected areas.

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.

Autosomal-dominant transthyretin (TTR)-related amyloidosis is not a frequent CMT2 neuropathy "in disguise".

Transthyretin (TTR)-related familial amyloid polyneuropathy (TTR-FAP) is a life-threatening autosomal dominant, systemic disease. First symptoms usually occur from the second to over sixth decade of life with a length-dependent axonal neuropathy with prominent involvement of the small fibers and multi-organ systemic failure.Early diagnosis is pivotal for effective therapeutic options, but it is hampered by the heterogeneity of the clinical spectrum which can lead to misdiagnosis with other neurological condition/disorder such as axonal sensory-motor neuropathy (CMT2) as described in literature.The aim of our study was to search for TTR mutations in a large cohort of selected undiagnosed axonal sensory-motor neuropathy patients to establish if misdiagnosis is frequent or rare in the Italian population.No TTR pathogenic variants were found in our cohort. In conclusion, our study shows that TTR testing not should be straightforward recommended in CMT2 patients but only when "red flags" TTR's features are present.

Spinocerebellar ataxia 17: full phenotype in a 41 CAG/CAA repeats carrier.

BACKGROUND: Spinocerebellar ataxia 17 (SCA17) is one of the most heterogeneous forms of autosomal dominant cerebellar ataxias with a large clinical spectrum which can mimic other movement disorders such as Huntington disease (HD), dystonia and parkinsonism. SCA17 is caused by an expansion of CAG/CAA repeat in the Tata binding protein (TBP) gene. Normal alleles contain 25 to 40 CAG/CAA repeats, alleles with 50 or greater CAG/CAA repeats are pathological with full penetrance. Alleles with 43 to 49 CAG/CAA repeats were also reported and their penetrance is estimated between 50 and 80%. Recently few symptomatic individuals having 41 and 42 repeats were reported but it is still unclear whether CAG/CAA repeats of 41 or 42 are low penetrance disease-causing alleles. Thus, phenotypic variability like the disease course in subject with SCA17 locus restricted expansions remains to be fully understood. CASE PRESENTATION: The patients was a 63-year-old woman who, at 54 years, showed personality changes and increased frequency of falls. At 55 years of age neuropsychological tests showed executive attention and visuospatial deficit. At the age of 59 the patient developed dysarthria and a progressive cognitive deficit. The neurological examination showed moderate gait ataxia, dysdiadochokinesia and dysmetria, dysphagia, dysarthria and abnormal saccadic pursuit, severe axial asynergy during postural changes, choreiform dyskinesias. Molecular analysis of the TBP gene demonstrated an allele with 41 repeat suggesting that 41 CAG/CCG TBP repeats could be an allele associated with the full clinical spectrum of SCA17. CONCLUSIONS: The described case with the other similar cases described in the literature suggests that 41 CAG/CAA trinucleotides should be considered as critical threshold in SCA17. We suggest that SCA17 diagnosis should be suspected in patients presenting with movement disorders associated with other neurodegenerative signs and symptoms.

Twenty years of molecular analyses in amyotrophic lateral sclerosis: genetic landscape of Italian patients.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease with a heterogeneous genetic background. Because mutation analysis by Sanger sequencing is costly and time-consuming, in recent years, next-generation sequencing (NGS) techniques have become of much interest. This study analyses the results of 20 years of molecular analyses in ALS patients in our laboratory using traditional methods and NGS. Almost 300 ALS patients underwent genetic analysis with Sanger sequencing of 7 genes or with an NGS panel of 23 genes. The C9orf72 expansion was tested by fragment size analysis. Sanger sequencing revealed mutations in 23.8% of familial and 3.8% of sporadic cases, whereas NGS detected potentially pathogenic variants in 45.5% of familial and 5.4% of sporadic cases and variants of unknown significance in 30.3% of patients. In 11.8% of patients, potentially causative mutations were found in 2 or more ALS genes. Compared to traditional methods, NGS is more effective in revealing possibly causal variants, but counseling patients becomes more complicated due to frequent variants of unknown significance and potentially oligogenic cases.

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.

Genetic Counseling Dilemmas for a Patient with Sporadic Amyotrophic Lateral Sclerosis, Frontotemporal Degeneration & Parkinson's Disease.

Amyotrophic lateral sclerosis (ALS), frontotemporal degeneration and Parkinson's disease may be different expressions of the same neurodegenerative disease. However, association between ALS and parkinsonism-dementia complex (ALS-PDC) has only rarely been reported apart from the cluster detected in Guam. We report a patient presenting with ALS-PDC in whom pathological mutations/expansions were investigated. No other family members were reported to have any symptoms of a neurological condition. Our case demonstrates that ALS-PDC can occur as a sporadic disorder, even though the coexistence of the three clinical features in one patient suggests a single underlying genetic cause. It is known that genetic testing should be preferentially offered to patients with ALS who have affected first or second-degree relatives. However, this case illustrates the importance of genetic counseling for family members of patients with sporadic ALC-PDC in order to provide education on the low recurrence risk. Here, we dicuss the ethical, psychological and practical consequences for patients and their relatives.

Two novel cases of compound heterozygous mutations in mitofusin2: Finding out the inheritance.

MFN2 is the major gene involved in the axonal form of Charcot-Marie-Tooth disease. It usually has an autosomal dominant pattern of inheritance, but a few cases of homozygous or compound heterozygous mutations have been described. These patients usually present an earlier onset, more severe phenotype and their inheritance pattern can span from autosomal recessive to semidominant. Here we report two unrelated patients carrying two compound heterozygous MFN2 mutations. Both present a pure axonal neuropathy without any additional features. The first patient presents a mild clinical phenotype with onset in the 2nd decade, while the second patient shows a severe, early onset phenotype with loss of independent ambulation. Only a careful clinical examination as well as neurophysiological and genetic studies allowed us to establish the role and the transmission pattern of the identified variants. We discuss practical consequences of this finding in genetic counseling.

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.