Generation and Characterization of hiPS Lines from Three Patients Affected by Different Forms of HPDL-Related Neurological Disorders.

Hereditary spastic paraplegias are rare genetic disorders characterized by corticospinal tract impairment. Spastic paraplegia 83 (SPG83) is associated with biallelic mutations in the HPDL gene, leading to varied severities from neonatal to juvenile onset. The function of HPDL is unclear, though it is speculated to play a role in alternative coenzyme Q10 biosynthesis. Here, we report the generation of hiPS lines from primary skin fibroblasts derived from three SPG83 patients with different HPDL mutations, using episomal reprogramming. The patients' clinical characteristics are carefully listed. The hiPS lines were meticulously characterized, demonstrating typical pluripotent characteristics through immunofluorescence assays for stemness markers (OCT4, TRA1-60, NANOG, and SSEA4) and RT-PCR for endogenous gene expression. Genetic integrity and identity were confirmed via Sanger sequencing and short tandem repeat analysis. These hiPS cells displayed typical pluripotent characteristics and were able to differentiate into neocortical neurons via a dual SMAD inhibition protocol. In addition, HPDL mutant neurons assessed via long-term culturing were able to achieve effective maturation, similarly to their wild-type counterparts. The HPDL hiPS lines we generated will provide a valuable model for studying SPG83, offering insights into its molecular mechanisms and potential for developing targeted therapies.

CACNA1G Causes Dominantly Inherited Myoclonus-Ataxia with Intellectual Disability: A Case Report.

Spinocerebellar ataxias (SCAs) are characterized by substantial phenotypic variability. Among them, SCA42 is a rare non-expansion entity presenting with slowly progressive cerebellar syndrome but whose clinical spectrum may be also wider. A 53-year-old male presented with progressive myoclonus-ataxia and intellectual disability. Genetic screening revealed a novel c.3835G > A (p. Asp1279Asn) variant in the CACNA1G gene. SCA42 is a rare non-expansion SCA caused by mutations in CACNA1G on chromosome 17q21, encoding the Ca(V)3.1, a low-threshold voltage-gated T-type calcium channel. The novel variant we identified is potentially involved in channel activity. This case expands the knowledge regarding CACNA1G-associated phenotype and highlights the importance of genetic screening in myoclonus-ataxia disorders.

Charcot-Marie-Tooth type 2CC misdiagnosed as Chronic Inflammatory Demyelinating Polyradiculoneuropathy.

BACKGROUND AND AIMS: Charcot-Marie-Tooth (CMT) is a heterogeneous group of genetic neuropathies and is typically characterized by distal muscle weakness, sensory loss, pes cavus and areflexia. Herein we describe a case of CMT2CC presenting with proximal muscle weakness and equivocal electrophysiological features, that was misdiagnosed as chronic inflammatory demyelinating polyneuropathy (CIDP). CASE REPORT: A 30-year-old woman complained of proximal muscle weakness with difficulty climbing stairs. Neurological examination showed weakness in lower limb (LL) muscles, that was marked proximally and mild distally, and absence of deep tendon reflexes in the ankles. Nerve conduction studies (NCS) showed sensory-motor neuropathy with non-uniform NC velocity and a partial conduction block (CBs) in peroneal nerve and tibial nerves. Thus, a diagnosis of CIDP was entertained and the patient underwent ineffective treatment with intravenous immunoglobulins. At electrophysiological revaluation CB in peroneal nerve was undetectable as also distal CMAP had decreased whereas the CBs persisted in tibial nerves. Hypothesizing a hereditary neuropathy, we examined the proband's son, who presented mild weakness of distal and proximal muscles at lower limbs. Neurophysiological investigation showed findings consistent with an intermediate-axonal electrophysiological pattern. A targeted-NGS including 136 CMT genes showed the heterozygous frameshift mutation (c.3057dupG; p.K1020fs*43) in the NEFH gene, coding for the neurofilament heavy chain and causing CMT2CC. INTERPRETATION: Diagnosis of a genetic neuropathy may be challenging when clinical features are atypical and/or electrophysiological features are misleading. The most common misdiagnosis is CIDP. Our report suggests that also CMT2CC patients with proximal muscle weakness and equivocal electrophysiological features might be misdiagnosed as CIDP.

Pseudodominance in RFC1-Spectrum Disorder.

Cerebellar ataxia, neuropathy, vestibular areflexia syndrome (CANVAS) and disease spectrum is an autosomal recessive disorder associated with biallelic repeat expansion (RE) in the RFC1 gene. A high carrier frequency in the healthy population determines the possibility of having affected members in two consecutive generations. We describe pseudodominance in two families affected with RFC1 disorder (10 affected, 5 oligo/asymptomatic individuals). In Family A, after the 75-year-old index case was diagnosed with CANVAS, the 73-year-old wife decided to undergo screening for carrier testing. Although she did not report any symptoms, she resulted positive for the biallelic AAGGG RE thus leading to a diagnosis in the asymptomatic offspring as well and revealing a pseudodominant pattern of inheritance. In Family B pseudodominance was suspected after the identification of the RFC1 RE in the proband affected by sensitive neuropathy because of a positive family history for undetermined polyneuropathy in the mother. The post-mortem identification of the RFC1 RE in a sample specimen from the deceased mother, who had been under our care, allowed the solution of a "cold case". Our report suggests that pseudodominance is a confounding phenomenon to consider in RFC1-spectrum disorder and genetic counselling is instrumental in families with affected individuals.

Early Diagnosis of AP5Z1/SPG48 Spastic Paraplegia: Case Report and Review of the Literature.

Hereditary spastic paraplegias (HSPs) are a genetically heterogeneous group of neurodegenerative disorders clinically characterized by progressive lower limb spasticity with pyramidal weakness. Around a dozen potential molecular mechanisms are recognized. Childhood HSP is a significant diagnostic challenge in clinical practice. Mutations in AP5Z1, which are associated with spastic paraplegia type 48 (SPG48), are extremely rare and seldom described in children.We report the clinical, radiologic, and molecular studies performed in a child harboring novel biallelic mutations in AP5Z1.The child presented a neurodevelopmental disorder with slight lower limb pyramidal signs. Brain magnetic resonance imaging (MRI) showed minimal white matter changes in the frontal horns of the lateral ventricles and a normally shaped corpus callosum. Western blotting in cultured skin fibroblasts indicated reduced protein expression, which confirmed the genetic diagnosis and framed this as a case of protein reduction in a context of impaired autophagy.Our findings expand the spectrum of phenotypes associated with mutations in AP5Z1, highlighting their clinical and pathophysiologic overlap with lysosomal storage disorders. SPG48 should be considered in the differential diagnosis of neurodevelopmental disorders even when pyramidal signs are minimal and brain MRI not fully informative.

Generation of a human induced pluripotent stem cell line (FSMi001-A) from fibroblasts of a patient carrying heterozygous mutation in the REEP1 gene.

Hereditary spastic paraplegias (HSPs) a group of rare, clinically, and genetically heterogeneous disorders characterized by progressive degeneration of the corticospinal tract. Among these HSPs, SPG31 is due to autosomal dominant mutations in the receptor expression-enhancing protein 1 (REEP1) gene. Over 80 genes have been associated with HSPs, and the list is constantly growing as research progresses. This study is aimed to create a patient-derived human induced pluripotent stem cell (hiPSC) line with a specific nonsense mutation to better characterize the etiopathogenesis of the disease.

Spinocerebellar ataxia 27B: a frequent and slowly progressive autosomal-dominant cerebellar ataxia-experience from an Italian cohort.

BACKGROUND: Autosomal-dominant spinocerebellar ataxia (ADCA) due to intronic GAA repeat expansion in FGF14 (SCA27B) is a recent, relatively common form of late-onset ataxia. OBJECTIVE: Here, we aimed to: (1) investigate the relative frequency of SCA27B in different clinically defined disease subgroups with late-onset ataxia collected among 16 tertiary Italian centers; (2) characterize phenotype and diagnostic findings of patients with SCA27B; (3) compare the Italian cohort with other cohorts reported in recent studies. METHODS: We screened 396 clinically diagnosed late-onset cerebellar ataxias of unknown cause, subdivided in sporadic cerebellar ataxia, ADCA, and multisystem atrophy cerebellar type. We identified 72 new genetically defined subjects with SCA27B. Then, we analyzed the clinical, neurophysiological, and imaging features of 64 symptomatic cases. RESULTS: In our cohort, the prevalence of SCA27B was 13.4% (53/396) with as high as 38.5% (22/57) in ADCA. The median age of onset of SCA27B patients was 62 years. All symptomatic individuals showed evidence of impaired balance and gait; cerebellar ocular motor signs were also frequent. Episodic manifestations at onset occurred in 31% of patients. Extrapyramidal features (17%) and cognitive impairment (25%) were also reported. Brain magnetic resonance imaging showed cerebellar atrophy in most cases (78%). Pseudo-longitudinal assessments indicated slow progression of ataxia and minimal functional impairment. CONCLUSION: Patients with SCA27B in Italy present as an adult-onset, slowly progressive cerebellar ataxia with predominant axial involvement and frequent cerebellar ocular motor signs. The high consistency of clinical features in SCA27B cohorts in multiple populations paves the way toward large-scale, multicenter studies.

Primary Coenzyme Q10 Deficiency-Related Ataxias.

Cerebellar ataxia is a neurological syndrome characterized by the imbalance (e.g., truncal ataxia, gait ataxia) and incoordination of limbs while executing a task (dysmetria), caused by the dysfunction of the cerebellum or its connections. It is frequently associated with other signs of cerebellar dysfunction, including abnormal eye movements, dysmetria, kinetic tremor, dysarthria, and/or dysphagia. Among the so-termed mitochondrial ataxias, variants in genes encoding steps of the coenzyme Q10 biosynthetic pathway represent a common cause of autosomal recessive primary coenzyme Q10 deficiencies (PCoQD)s. PCoQD is a potentially treatable condition; therefore, a correct and timely diagnosis is essential. After a brief presentation of the illustrative case of an Italian woman with this condition (due to a novel homozygous nonsense mutation in COQ8A), this article will review ataxias due to PCoQD.

Hereditary spastic paraparesis type 18 (SPG18): new ERLIN2 variants in a series of Italian patients, shedding light upon genetic and phenotypic variability.

INTRODUCTION: Hereditary spastic paraparesis (HSP) is a group of central nervous system diseases primarily affecting the spinal upper motor neurons, with different inheritance patterns and phenotypes. SPG18 is a rare, early-onset, complicated HSP, first reported as linked to biallelic ERLIN2 mutations. Recent cases of late-onset, pure HSP with monoallelic ERLIN2 variants prompt inquiries into the zygosity of such genetic conditions. The observed relationship between phenotype and mode of inheritance suggests a potential dominant negative effect of mutated ERLIN2 protein, potentially resulting in a milder phenotype. This speculation suggests that a wider range of HSP genes could be linked to various inheritance patterns. PURPOSE AND BACKGROUND: With documented cases of HSP loci exhibiting both dominant and recessive patterns, this study emphasizes that the concept of zygosity is no longer a limiting factor in the establishment of molecular diagnoses for HSP. Recent cases have demonstrated phenoconversion in SPG18, from HSP to an amyotrophic lateral sclerosis (ALS)-like syndrome. METHODS AND RESULTS: This report highlights two cases out of five exhibiting HSP-ALS phenoconversion, discussing an observed prevalence in autosomal dominant SPG18. Additionally, the study emphasizes the relatively high incidence of the c.502G>A variant in monoallelic SPG18 cases. This mutation appears to be particularly common in cases of HSPALS phenoconversion, indicating its potential role as a hotspot for a distinctive SPG18 phenotype with an ALS-like syndrome. CONCLUSIONS: Clinicians need to be aware that patients with HSP may show ALS signs and symptoms. On the other hand, HSP panels must be included in genetic testing methods for instances of familial ALS.

NOTCH1-Related Leukoencephalopathy: A Novel Variant and Literature Review.

NOTCH1-related leukoencephalopathy is a new diagnostic entity linked to heterozygous gain-of-function variants in NOTCH1 that neuroradiologically show some overlap with the inflammatory microangiopathy Aicardi-Goutières syndrome (AGS). To report a 16-year-old boy harbouring a novel NOTCH1 mutation who presented neuroradiological features suggestive of enhanced type I interferon signalling. We describe five years of follow-up and review the current literature on NOTCH1-related leukoencephalopathy. Clinical evaluation, standardised scales (SPRS, SARA, CBCL, CDI-2:P, WISCH-IV and VABS-2) and neuroradiological studies were performed, as well as blood DNA analysis. For the literature review, a search was performed on Pubmed, Scopus and Web of Science up to December 2023 using the following text word search strategy: (NOTCH1) AND (leukoencephalopathy). Our patient presents clinical features consistent with other reported cases with NOTCH1 mutations but is among the minority of patients with an onset after infancy. During the five-year follow-up, we observed an increase in the severity of spasticity and ataxia. However, at the age of 16 years, our proband is still ambulatory. As for other reported patients, he manifests psychiatric features ranging from hyperactivity during childhood to anxiety and depression during adolescence. The neuroradiological picture remained essentially stable over five years. In addition to the typical findings of leukoencephalopathy with cysts and calcifications already described, we report the presence of T2-hyperintensity and T1-hypotensity of the transverse pontine fibres, enhancement in the periventricular white matter after gadolinium administration and decreased NAA and Cho peaks in the periventricular white matter on MRS. We identified a novel heterozygous variant in NOTCH1 (c.4788_4799dup), a frame insertion located in extracellular negative regulatory region (NRR)-domain as in previously published cases. Blood interferon signalling was not elevated compared to controls. This case provides further data on a new diagnostic entity, i.e., NOTCH1-related leukoencephalopathy. By describing a standardised five-year follow-up in one case and reviewing the other patients described to date, we outline recommendations relating to monitoring in this illness, emphasising the importance of psychiatric and gastroenterological surveillance alongside neurological and neuropsychological management. Studies are needed to better understand the factors influencing disease onset and severity, which are heterogeneous.

Hereditary spastic paraparesis type 46 (SPG46): new GBA2 variants in a large Italian case series and review of the literature.

Hereditary spastic paraparesis (HSP) is a group of central nervous system diseases primarily affecting the spinal upper motor neurons, with different inheritance patterns and phenotypes. SPG46 is a rare, early-onset and autosomal recessive HSP, linked to biallelic GBA2 mutations. About thirty families have been described worldwide, with different phenotypes like complicated HSP, recessive cerebellar ataxia or Marinesco-Sjögren Syndrome. Herein, we report five SPG46 patients harbouring five novel GBA2 mutations, the largest series described in Italy so far. Probands were enrolled in five different centres and underwent neurological examination, clinical cognitive assessment, column imaging for scoliosis assessment, ophthalmologic examination, brain imaging, GBA2 activity in peripheral blood cells and genetic testing. Their phenotype was consistent with HSP, with notable features like upper gaze palsy and movement disorders. We review demographic, genetic, biochemical and clinical information from all documented cases in the existing literature, focusing on the global distribution of cases, the features of the syndrome, its variable presentation, new potential identifying features and the significance of measuring GBA2 enzyme activity.

A Variant in TBCD Associated with Motoneuronopathy and Corpus Callosum Hypoplasia: A Case Report.

Mutations in the tubulin-specific chaperon D (TBCD) gene, involved in the assembly and disassembly of the α/ß-tubulin heterodimers, have been reported in early-onset progressive neurodevelopment regression, with epilepsy and mental retardation. We describe a rare homozygous variant in TBCD, namely c.881G>A/p.Arg294Gln, in a young woman with a phenotype dominated by distal motorneuronopathy and mild mental retardation, with neuroimaging evidence of corpus callosum hypoplasia. The peculiar phenotype is discussed in light of the molecular interpretation, enriching the literature data on tubulinopathies generated from TBCD mutations.

Power of NGS-based tests in HSP diagnosis: analysis of massively parallel sequencing in clinical practice.

Hereditary spastic paraplegia (HSP) refers to a group of heterogeneous neurological disorders mainly characterized by corticospinal degeneration (pure forms), but sometimes associated with additional neurological and extrapyramidal features (complex HSP). The advent of next-generation sequencing (NGS) has led to huge improvements in knowledge of HSP genetics and made it possible to clarify the genetic etiology of hundreds of "cold cases," accelerating the process of reaching a molecular diagnosis. The different NGS-based strategies currently employed as first-tier approaches most commonly involve the use of targeted resequencing panels and exome sequencing, whereas genome sequencing remains a second-tier approach because of its high costs. The question of which approach is the best is still widely debated, and many factors affect the choice. Here, we aim to analyze the diagnostic power of different NGS techniques applied in HSP, by reviewing 38 selected studies in which different strategies were applied in different-sized cohorts of patients with genetically uncharacterized HSP.

Converging Role for REEP1/SPG31 in Oxidative Stress.

Mutations in the receptor expression-enhancing protein 1 gene (REEP1) are associated with hereditary spastic paraplegia type 31 (SPG31), a neurological disorder characterized by length-dependent degeneration of upper motor neuron axons. Mitochondrial dysfunctions have been observed in patients harboring pathogenic variants in REEP1, suggesting a key role of bioenergetics in disease-related manifestations. Nevertheless, the regulation of mitochondrial function in SPG31 remains unclear. To elucidate the pathophysiology underlying REEP1 deficiency, we analyzed in vitro the impact of two different mutations on mitochondrial metabolism. Together with mitochondrial morphology abnormalities, loss-of-REEP1 expression highlighted a reduced ATP production with increased susceptibility to oxidative stress. Furthermore, to translate these findings from in vitro to preclinical models, we knocked down REEP1 in zebrafish. Zebrafish larvae showed a significant defect in motor axon outgrowth leading to motor impairment, mitochondrial dysfunction, and reactive oxygen species accumulation. Protective antioxidant agents such as resveratrol rescued free radical overproduction and ameliorated the SPG31 phenotype both in vitro and in vivo. Together, our findings offer new opportunities to counteract neurodegeneration in SPG31.

New cellular imaging-based method to distinguish the SPG4 subtype of hereditary spastic paraplegia.

BACKGROUND AND PURPOSE: Microtubule defects are a common feature in several neurodegenerative disorders, including hereditary spastic paraplegia. The most frequent form of hereditary spastic paraplegia is caused by mutations in the SPG4/SPAST gene, encoding the microtubule severing enzyme spastin. To date, there is no effective therapy available but spastin-enhancing therapeutic approaches are emerging; thus prognostic and predictive biomarkers are urgently required. METHODS: An automated, simple, fast and non-invasive cell imaging-based method was developed to quantify microtubule cytoskeleton organization changes in lymphoblastoid cells and peripheral blood mononuclear cells. RESULTS: It was observed that lymphoblastoid cells and peripheral blood mononuclear cells from individuals affected by SPG4-hereditary spastic paraplegia show a polarized microtubule cytoskeleton organization. In a pilot study on freshly isolated peripheral blood mononuclear cells, our method discriminates SPG4-hereditary spastic paraplegia from healthy donors and other hereditary spastic paraplegia subtypes. In addition, it is shown that our method can detect the effects of spastin protein level changes. CONCLUSIONS: These findings open the possibility of a rapid, non-invasive, inexpensive test useful to recognize SPG4-hereditary spastic paraplegia subtype and evaluate the effects of spastin-enhancing drug in non-neuronal cells.

The clinical and molecular spectrum of ZFYVE26-associated hereditary spastic paraplegia: SPG15.

In the field of hereditary spastic paraplegia (HSP), progress in molecular diagnostics needs to be translated into robust phenotyping studies to understand genetic and phenotypic heterogeneity and to support interventional trials. ZFYVE26-associated hereditary spastic paraplegia (HSP-ZFYVE26, SPG15) is a rare, early-onset complex HSP, characterized by progressive spasticity and a variety of other neurological symptoms. While prior reports, often in populations with high rates of consanguinity, have established a general phenotype, there is a lack of systematic investigations and a limited understanding of age-dependent manifestation of symptoms. Here we delineate the clinical, neuroimaging and molecular features of 44 individuals from 36 families, the largest cohort assembled to date. Median age at last follow-up was 23.8 years covering a wide age range (11-61 years). While symptom onset often occurred in early childhood [median: 24 months, interquartile range (IQR) = 24], a molecular diagnosis was reached at a median age of 18.8 years (IQR = 8), indicating significant diagnostic delay. We demonstrate that most patients present with motor and/or speech delay or learning disabilities. Importantly, these developmental symptoms preceded the onset of motor symptoms by several years. Progressive spasticity in the lower extremities, the hallmark feature of HSP-ZFYVE26, typically presents in adolescence and involves the distal lower limbs before progressing proximally. Spasticity in the upper extremities was seen in 64%. We found a high prevalence of extrapyramidal movement disorders including cerebellar ataxia (64%) and dystonia (11%). Parkinsonism (16%) was present in a subset and showed no sustained response to levodopa. Cognitive decline and neurogenic bladder dysfunction progressed over time in most patients. A systematic analysis of brain MRI features revealed a common diagnostic signature consisting of thinning of the anterior corpus callosum, signal changes of the anterior forceps and non-specific cortical and cerebellar atrophy. The molecular spectrum included 45 distinct variants, distributed across the protein structure without mutational hotspots. Spastic Paraplegia Rating Scale scores, SPATAX Disability Scores and the Four Stage Functional Mobility Score showed moderate strength in representing the proportion of variation between disease duration and motor dysfunction. Plasma neurofilament light chain levels were significantly elevated in all patients (Mann-Whitney U-test, P < 0.0001) and were correlated inversely with age (Spearman's rank correlation coefficient r = -0.65, P = 0.01). In summary, our systematic cross-sectional analysis of HSP-ZFYVE26 patients across a wide age-range, delineates core clinical, neuroimaging and molecular features and identifies markers of disease severity. These results raise awareness to this rare disease, facilitate an early diagnosis and create clinical trial readiness.