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.

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.

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.

Case report: Episodic ataxia without ataxia?

Hereditary myopathies represent a clinically and genetically heterogeneous group of neuromuscular disorders, characterized by highly variable clinical presentations and frequently overlapping phenotypes with other neuromuscular disorders, likely influenced by genetic and environmental modifiers. Genetic testing is often challenging due to ambiguous clinical diagnosis. Here, we present the case of a family with clinical and Electromyography (EMG) features resembling a myotonia-like disorder in which Whole Exome Sequencing (WES) analysis revealed the co-segregation of two rare missense variants in UBR4 and HSPG2, genes previously associated with episodic ataxia 8 (EA8). A review of the literature highlighted a striking overlap between the clinical and the molecular features of our family and the previously described episodic ataxias (EAs), which raises concerns about the genotype-phenotype correlation, clinical variability, and the confounding overlap in these groups of disorders. This emphasizes the importance of thoroughly framing the patient's phenotype. The more clear-cut the diagnosis, the easier the identification of a genetic determinant, and the better the prognosis and the treatment of patients.

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.

Expanding the Molecular Spectrum of ANKRD11 Gene Defects in 33 Patients with a Clinical Presentation of KBG Syndrome.

KBG syndrome (KBGS) is a neurodevelopmental disorder caused by the Ankyrin Repeat Domain 11 (ANKRD11) haploinsufficiency. Here, we report the molecular investigations performed on a cohort of 33 individuals with KBGS clinical suspicion. By using a multi-testing genomic approach, including gene sequencing, Chromosome Microarray Analysis (CMA), and RT-qPCR gene expression assay, we searched for pathogenic alterations in ANKRD11. A molecular diagnosis was obtained in 22 out of 33 patients (67%). ANKRD11 sequencing disclosed pathogenic or likely pathogenic variants in 18 out of 33 patients. CMA identified one full and one terminal ANKRD11 pathogenic deletions, and one partial duplication and one intronic microdeletion, with both possibly being pathogenic. The pathogenic effect was established by RT-qPCR, which confirmed ANKRD11 haploinsufficiency only for the three deletions. Moreover, RT-qPCR applied to six molecularly unsolved KBGS patients identified gene downregulation in a clinically typical patient with previous negative tests, and further molecular investigations revealed a cryptic deletion involving the gene promoter. In conclusion, ANKRD11 pathogenic variants could also involve the regulatory regions of the gene. Moreover, the application of a multi-test approach along with the innovative use of RT-qPCR improved the diagnostic yield in KBGS suspected patients.

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.

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.

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.

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.

1993-2014: two decades of predictive testing for Huntington's disease at the Medical Genetics Unit of the University of Genoa.

BACKGROUND: Predictive testing for Huntington's disease has been available at the Medical Genetics Unit of the University of Genoa from 1987. In 1989, an integrated counseling protocol (geneticist, psychologist, and neurologist) was developed following International Guidelines. METHODS: This is a retrospective analysis of the clinical charts and motivation questionnaires of persons seeking predictive testing through direct DNA analysis from 1993 until 2014, with the aim to evaluate their individual characteristics, motivations, and the outcomes of the counseling protocol. RESULTS: A total of 299 persons (164 women, 135 men) applied for predictive testing. Most applicants' features and motivations were similar to those previously described, but surprisingly the percentage of completed protocols was higher among men, 68.5% versus 53.5% (P = 0.011). Likewise, persons over 25 years of age were more likely to take the test than younger applicants (18-25 years): 63.4% versus 48.1% (P = 0.043). In addition, relationship status, having children, and the gender of the affected parent showed different effects on the decision about testing in males and females. No catastrophic reactions were reported during the study period. CONCLUSIONS: We observed that factors influencing the decision-making process might differ between males and females, and that predictive testing appears a safe procedure if framed within an integrated counseling protocol.

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.

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.