SPTLC2 variants are associated with early-onset ALS and FTD due to aberrant sphingolipid synthesis.

OBJECTIVE: Amyotrophic lateral sclerosis (ALS) is a devastating, incurable neurodegenerative disease. A subset of ALS patients manifests with early-onset and complex clinical phenotypes. We aimed to elucidate the genetic basis of these cases to enhance our understanding of disease etiology and facilitate the development of targeted therapies. METHODS: Our research commenced with an in-depth genetic and biochemical investigation of two specific families, each with a member diagnosed with early-onset ALS (onset age of <40 years). This involved whole-exome sequencing, trio analysis, protein structure analysis, and sphingolipid measurements. Subsequently, we expanded our analysis to 62 probands with early-onset ALS and further included 440 patients with adult-onset ALS and 1163 healthy controls to assess the prevalence of identified genetic variants. RESULTS: We identified heterozygous variants in the serine palmitoyltransferase long chain base subunit 2 (SPTLC2) gene in patients with early-onset ALS. These variants, located in a region closely adjacent to ORMDL3, bear similarities to SPTLC1 variants previously implicated in early-onset ALS. Patients with ALS carrying these SPTLC2 variants displayed elevated plasma ceramide levels, indicative of increased serine palmitoyltransferase (SPT) activity leading to sphingolipid overproduction. INTERPRETATION: Our study revealed novel SPTLC2 variants in patients with early-onset ALS exhibiting frontotemporal dementia. The combination of genetic evidence and the observed elevation in plasma ceramide levels establishes a crucial link between dysregulated sphingolipid metabolism and ALS pathogenesis. These findings expand our understanding of ALS's genetic diversity and highlight the distinct roles of gene defects within SPT subunits in its development.

LONRF2 is a protein quality control ubiquitin ligase whose deficiency causes late-onset neurological deficits.

Protein misfolding is a major factor of neurodegenerative diseases. Post-mitotic neurons are highly susceptible to protein aggregates that are not diluted by mitosis. Therefore, post-mitotic cells may have a specific protein quality control system. Here, we show that LONRF2 is a bona fide protein quality control ubiquitin ligase induced in post-mitotic senescent cells. Under unperturbed conditions, LONRF2 is predominantly expressed in neurons. LONRF2 binds and ubiquitylates abnormally structured TDP-43 and hnRNP M1 and artificially misfolded proteins. Lonrf2-/- mice exhibit age-dependent TDP-43-mediated motor neuron (MN) degeneration and cerebellar ataxia. Mouse induced pluripotent stem cell-derived MNs lacking LONRF2 showed reduced survival, shortening of neurites and accumulation of pTDP-43 and G3BP1 after long-term culture. The shortening of neurites in MNs from patients with amyotrophic lateral sclerosis is rescued by ectopic expression of LONRF2. Our findings reveal that LONRF2 is a protein quality control ligase whose loss may contribute to MN degeneration and motor deficits.

Clinical features of a family with late-onset distal hereditary motor neuropathy harboring p.Pro39Leu variant of HSPB1.

BACKGROUND AND AIMS: Pathogenic variants of HSPB1, the gene encoding the small heat shock protein 27, have been reported to cause autosomal dominant distal hereditary motor neuropathy (dHMN) type II and autosomal dominant Charcot-Marie-Tooth (CMT) disease with minimal sensory involvement (CMT2F). This study aimed to describe the clinical features of patients in a family with late-onset dHMN carrying the Pro39Leu variant of HSPB1. METHODS: Whole-exome sequence analysis identified a heterozygous pathogenic variant (Pro39Leu) of HSPB1 in the proband. The presence of the HSPB1 Pro39Leu variant in two affected individuals was confirmed using direct nucleotide sequence analysis. RESULTS: Both patients exhibited distal muscle weakness with lower extremity predominance and no obvious sensory deficits, leading to a clinical diagnosis of late-onset dHMN. Nerve conduction studies (NCSs) revealed a subclinical complication of sensory disturbance in one of the patients. The clinical and electrophysiological findings of patients with the HSPB1 Pro39Leu variant in this study and previous reports are summarized. INTERPRETATION: This study suggests that the clinical spectrum of patients carrying HSPB1 Pro39Leu variants, especially the disease onset, might be broader than expected, and HSPB1 variants should be considered in patients diagnosed with late-onset dHMN. Furthermore, patients with dHMN may have concomitant sensory deficits that should be evaluated using NCSs.

High-dose ubiquinol supplementation in multiple-system atrophy: a multicentre, randomised, double-blinded, placebo-controlled phase 2 trial.

Background: Functionally impaired variants of COQ2, encoding an enzyme in biosynthesis of coenzyme Q10 (CoQ10), were found in familial multiple system atrophy (MSA) and V393A in COQ2 is associated with sporadic MSA. Furthermore, reduced levels of CoQ10 have been demonstrated in MSA patients. Methods: This study was a multicentre, randomised, double-blinded, placebo-controlled phase 2 trial. Patients with MSA were randomly assigned (1:1) to either ubiquinol (1500 mg/day) or placebo. The primary efficacy outcome was the change in the unified multiple system atrophy rating scale (UMSARS) part 2 at 48 weeks. Efficacy was assessed in all patients who completed at least one efficacy assessment (full analysis set). Safety analyses included patients who completed at least one dose of investigational drug. This trial is registered with UMIN-CTR (UMIN000031771), where the drug name of MSA-01 was used to designate ubiquinol. Findings: Between June 26, 2018, and May 27, 2019, 139 patients were enrolled and randomly assigned to the ubiquinol group (n = 69) or the placebo group (n = 70). A total of 131 patients were included in the full analysis set (63 in the ubiquinol group; 68 in the placebo group). This study met the primary efficacy outcome (least square mean difference in UMSARS part 2 score (-1.7 [95% CI, -3.2 to -0.2]; P = 0.023)). The ubiquinol group also showed better secondary efficacy outcomes (Barthel index, Scale for the Assessment and Rating of Ataxia, and time required to walk 10 m). Rates of adverse events potentially related to the investigational drug were comparable between ubiquinol (n = 15 [23.8%]) and placebo (n = 21 [30.9%]). Interpretation: High-dose ubiquinol was well-tolerated and led to a significantly smaller decline of UMSARS part 2 score compared with placebo. Funding: Japan Agency for Medical Research and Development.

Noncanonical splice-site variant in peripheral myelin protein 22 gene (PMP22) in a patient with hereditary neuropathy with liability to pressure palsies.

AIM: Hereditary neuropathy with liability to pressure palsies (HNPP) is a peripheral neuropathy with autosomal dominant inheritance. Diagnosis can be made from the characteristic abnormalities determined by nerve conduction studies (NCS), including subclinical deficits at physiological compression sites. Heterozygous deletion of the chromosome 17p11.2-p12 region including the peripheral myelin protein 22 gene (PMP22) is the cause in the majority of cases. However, the loss of function of PMP22 due to frameshift-causing insertion/deletion, missense, nonsense, or splice-site disrupting variants cause HNPP in some patients. We report a case of a patient diagnosed with HNPP on the basis of clinical features and the results of NCS. No deletions of PMP22 were detected by fluorescence in situ hybridization. METHODS: We performed direct nucleotide sequence analysis and identified a heterozygous variant, c.78 + 3G > T, in PMP22. Since this variant is located outside the canonical splice site at the exon 2-intron 2 junction, we investigated whether the variant causes aberrant splicing and leads to the skipping of exon 2 of PMP22 by in vitro minigene splicing assay. RESULTS: We demonstrated that the c.78 + 3G > T variant causes the skipping of exon 2 and leads to loss of function of the mutant allele. CONCLUSION: Searching for sequence variants located outside the canonical splice sites should also be considered even when deletion of PMP22 is not found in a patient with a clinical diagnosis suggesting HNPP.

Clinical and electrophysiological findings of facial palsy in a case of hereditary gelsolin amyloidosis.

Hereditary gelsolin amyloidosis (HGA) is an autosomal dominant systemic amyloidosis, characterized by cranial and sensory peripheral neuropathy, corneal lattice dystrophy, and cutis laxa. We report a case of HGA presenting with bilateral facial palsy. A 70-year-old Japanese man presented with slowly progressive bilateral facial palsy and facial twitching, which had started in his 40s. His mother also had the same symptoms due to an unknown cause but rest of the family did not. He showed incomplete facial palsy with no frontal muscle movement and partial movement of the orbicularis oris and orbicularis oculi muscles. The patient showed no synkinesis. Electroneurography revealed symmetric low compound motor action potential amplitude of the orbicularis oris muscle, and a nerve excitability test showed a symmetric increase in the response threshold. Despite the partial voluntary movement of the orbicularis oculi muscle, bilateral blink reflexes were absent. He also showed facial spasms after contraction of the orbicularis oris muscle. Genetic testing revealed a heterozygous c.640G>A mutation (p. Asp214Asn); therefore, the patient was diagnosed with HGA. HGA related facial palsy showed moderate bilateral, upper blanch-dominant axonal degeneration of the facial nerve without reinnervation, and trigeminal nerve neuropathy.

Valosin-containing protein Asp395Gly mutation in a patient with frontotemporal dementia: a case report.

BACKGROUND: Variants in the valosin-containing protein (VCP) gene were identified as one of the causes for inclusion body myopathy associated with Paget disease of the bone and frontotemporal dementia (FTD). Previously identified pathogenic variants in VCP are associated with frontotemporal lobar degeneration with TDP-43 inclusions (FTLD-TDP) pathologically, but p.Asp395Gly VCP was recently reported to cause familial FTD with tauopathy characterized by neurofibrillary tau tangles (NFT) and not FTLD-TDP. We describe the clinical and genetic findings of a patient with p.Asp395Gly valosin-containing protein (VCP), who was diagnosed with FTD without a family history and in the absence of muscle or bone disease comorbidity. CASE PRESENTATION: The patient was a 62-year-old man, who developed atypical depression at the age of 37 years. Subsequently, he presented with self-centered behavior at the age of 45 years. The self-centered behavior intensified from around the age of 50 years, which was accompanied by the development of executive dysfunction; therefore, he visited our hospital at 52 years of age. Magnetic resonance imaging revealed bilateral frontal lobe atrophy. Brain perfusion single-photon emission computed tomography revealed bilateral frontal lobe hypoperfusion. The patient fulfilled the diagnostic criteria for behavioral variant of FTD. Ten years after the diagnosis, computed tomography of the trunk and limbs, muscle biopsy, and bone scintigraphy revealed the absence of concomitant muscle and bone disease. The concentrations of cerebrospinal fluid (CSF) total tau and phosphorylated tau proteins were 389 pg/mL and 53.2 pg/mL (cut-off: 50 pg/mL), respectively. Genetic analyses were performed using the whole-exome and Sanger sequencing methods. We identified p.Asp395Gly VCP in this patient with pure FTD. CONCLUSIONS: p.Asp395Gly VCP was identified in a patient with likely sporadic FTD without concomitant muscle and bone disease. The CSF analysis suggested that our patient may have FTD due to NFT accumulation similar to the familial FTD patients with p.Asp395Gly VCP recently reported. Our findings suggest that a genetic search for the pathogenic variants of VCP should be considered not only for familial FTD, but also for patients with sporadic FTD, even in the absence of comorbid muscle or bone disease.

Loss-of-function variants in NEK1 are associated with an increased risk of sporadic ALS in the Japanese population.

Loss-of-function (LoF) variants in NEK1 have recently been reported to be associated with amyotrophic lateral sclerosis (ALS). In this study, we investigated the association of NEK1 LoF variants with an increased risk of sporadic ALS (SALS) and the clinical characteristics of patients with SALS carrying LoF variants in a Japanese case series. Whole-exome sequencing analysis was performed for a series of 446 SALS patients in whom pathogenic variants in familial ALS-causative genes have not been identified and 1163 healthy control subjects in our Japanese series. We evaluated LoF variants, defined as nonsense, splice-site disrupting single-nucleotide variants (SNVs), or short insertion/deletion (indel) variants predicted to cause frameshifts in NEK1. We identified seven NEK1 LoF variants in patients with SALS (1.57%), whereas only one was identified in control subjects (0.086%) (P = 0.00073, Fisher's exact test). This finding is consistent with those in recent reports from other regions in the world. In conclusion, we demonstrated that NEK1 LoF variants are also associated with an increased risk of SALS in the Japanese population.

Juvenile amyotrophic lateral sclerosis with complex phenotypes associated with novel SYNE1 mutations.

Mutations in SYNE1 have been originally described to cause a slowly progressive, pure cerebellar ataxia (spinocerebellar ataxia, autosomal-recessive 8; SCAR8). Notably, recent studies revealed that affected patients with SYNE1-associated ataxia can present with complex phenotypes rather than pure cerebellar ataxia, including motor neuron and brainstem dysfunctions. We herein report a Japanese patient diagnosed with juvenile amyotrophic lateral sclerosis (ALS) with a complex phenotype, who carried compound heterozygous pathogenic variants in SYNE1. Of the variants, one was a novel frameshift variant and the other was a nonsense variant previously reported as pathogenic for SCAR8. The patient showed an early age at onset with a relatively slow but progressive course of ALS, accompanied by cognitive decline. Our findings suggest that the clinical spectrum of patients carrying pathogenic SYNE1 variants is broader than expected, and SYNE1 variants should be considered in patients diagnosed with juvenile ALS, even without prominent cerebellar ataxia.

Splice-site mutations in KIF5A in the Japanese case series of amyotrophic lateral sclerosis.

Our objective was to investigate the frequency of KIF5A variants in amyotrophic lateral sclerosis (ALS) and the clinical characteristics of familial ALS (FALS) associated with variants in KIF5A. Whole-exome sequence analysis was performed for a Japanese series of 43 families with FALS and 444 patients with sporadic ALS (SALS), in whom causative variants had not been identified. We compared the frequencies of rare variants (MAF < 0.01) in KIF5A, including missense and loss of function (LoF) variants, between ALS and control subjects (n = 1163). Clinical characteristics of patients with FALS carrying pathogenic variants in KIF5A were also described. LoF variants were identified only in the probands of two families with FALS, both of which were 3' splice-site variants leading to exon skipping and an altered C-terminal domain, located in the mutational hotspot causing FALS, and were considered to be pathogenic for FALS. Rare missense variants in KIF5A were identified in five patients with SALS (1.13%) and 11 control subjects (0.95%, carrier frequency), which were not significantly different. Consequently, the pathogenic LoF variants in KIF5A accounted for 2.1% of all FALS families in this study. These patients suffered from ALS characteristically associated with the predominant involvement of upper motor neuron. In conclusion, we identified two pathogenic splice-site variants in KIF5A in the probands in two Japanese families with FALS, which altered the C-terminal region of KIF5A. Our findings broaden the phenotype spectrum of ALS associated with variants in KIF5A in the Japanese series.

Atypical Familial Amyotrophic Lateral Sclerosis with Slowly Progressing Lower Extremities-predominant Late-onset Muscular Weakness and Atrophy.

Objective Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disease characterized by the progressive loss of the upper and lower motor neurons that progresses to paralysis of almost all skeletal muscles of the extremities, bulbar, and respiratory system. Although most ALS cases are sporadic, about 10% are dominantly inherited. We herein report an atypical phenotype of familial ALS (fALS). To elucidate the phenotype-genotype correlation of this atypical phenotype of fALS, clinical and genetic investigations were performed. Methods and Patients Five sibling patients (three men, two women) from a Japanese family and one healthy sibling (a woman) were clinically interviewed and examined. Genetic analyses, including genome-wide linkage analyses and whole-exome sequencing, were performed using genomic DNA extracted from the peripheral blood samples of these siblings. Results The clinical features of fALS are characterized by slow progression (mean duration of the disease±standard deviation [SD]: 19.6±3.9 years) and lower extremities-predominant late-onset muscular weakness (mean onset of muscular weakness±SD: 52.8±2.6 years). Genetic analyses revealed novel heterozygous missense mutations of c.2668C>T, p.R890C in the PLEC gene and c.421G>C, p.V141L in the ST3GAL6 gene in all affected siblings. Conclusion A new atypical fALS family with a benign clinical course is herein reported. We identified two candidate gene mutations of PLEC and ST3GAL6 linked to this phenotype.

Prominent Spasticity and Hyperreflexia of the Legs in a Nepalese Patient with Friedreich Ataxia.

Friedreich ataxia (FRDA) is an autosomal recessive spinocerebellar ataxia caused by mutations of FXN. Hypotonus and hyporeflexia of the lower extremities are observed in most FRDA patients. Patients with hyperreflexia, called Friedreich ataxia with retained reflexes (FARR), have also been identified. We herein report the case of a 16-year-old Nepalese boy presenting with early-onset ataxia with prominent spasticity and hyperreflexia of the legs. Mutational analyses established the diagnosis of FRDA presenting as FARR. A haplotype analysis revealed that expanded alleles of the patient shared a common haplotype with Indian and European FRDA patients, suggesting that the mutation descended from a common founder.

Association of ATXN2 intermediate-length CAG repeats with amyotrophic lateral sclerosis correlates with the distributions of normal CAG repeat alleles among individual ethnic populations.

Intermediate-length CAG repeats in ATXN2 have been widely shown to be a risk factor for sporadic amyotrophic lateral sclerosis (SALS). To evaluate the association of ATXN2 intermediate-length CAG repeat alleles with an increased risk of SALS, we investigated distributions of CAG repeat alleles in 394 patients with SALS and 490 control individuals in the Japanese population. In the intermediate-length repeat units of 29 or more, we identified one SALS patient with 31 repeat units and two control individuals with 30 repeat units. Thus, no significant differences in the carrier frequency of intermediate-length CAG repeat alleles were detected between patients with SALS and control individuals. When we investigated the distribution of "large normal alleles" defined as ATXN2 CAG repeats ranging from 24 up to 33 in the Japanese population compared with those in other populations in previous studies, the frequency of large normal alleles was significantly higher in the European and North American series than in the Japanese series. Moreover, these frequencies in the Turkish, Chinese, Korean, and Brazilian (Latin American) series were also higher than that in the Japanese series. These results raise the possibility that the frequencies of large normal alleles in individual populations underlie the frequencies of ALS risk alleles in the corresponding populations.

Burden of rare variants in causative genes for amyotrophic lateral sclerosis (ALS) accelerates age at onset of ALS.

OBJECTIVES: To evaluate the burden of rare variants in the causative genes for amyotrophic lateral sclerosis (ALS) on the age at onset of ALS in a Japanese case series. METHODS: We conducted whole-exome sequencing analysis of 89 families with familial ALS (FALS) and 410 patients with sporadic ALS (SALS) to identify known pathogenic mutations or rare functionally predicted deleterious variants in the causative genes for ALS. Rare variants (minor allele frequency <1%) with scaled Combined Annotation-Dependent Depletion score >20 were defined as rare functionally predicted deleterious variants. The patients with ALS were classified on the basis of the number of pathogenic and/or rare functionally predicted deleterious variants, and the age at onset was compared among the classified groups. RESULTS: Whole-exome sequencing analysis revealed known pathogenic mutations or rare functionally predicted deleterious variants in causative genes for ALS in 56 families with FALS (62.9%) and 87 patients with SALS (21.2%). Such variants in multiple genes were identified in seven probands with FALS and eight patients with SALS. The ages at onset in the patients with ALS with multiple variants were significantly earlier than those in other patients with ALS. Even when the patients with known pathogenic mutations were excluded, a significantly earlier onset of the disease was still observed in patients with multiple rare functionally predicted deleterious variants. CONCLUSIONS: A substantial number of patients carried rare variants in multiple genes, and the burden of rare variants in the known causative genes for ALS affects the age at onset in the Japanese ALS series.

Frequency and characteristics of the TBK1 gene variants in Japanese patients with sporadic amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a devastating neurological disease, and the etiology of sporadic ALS is generally unknown. The TANK-binding kinase 1 (TBK1) gene was identified as an ALS gene contributing to a predisposition toward ALS. To reveal the frequency and characteristics of variants of the TBK1 gene in sporadic ALS patients in Japan, we analyzed the TBK1 gene by exome sequencing in a large Japanese cohort of 713 sporadic ALS patients and 800 controls. We identified known or potentially toxic rare variants of TBK1 gene in 9 patients (1.26%) with sporadic ALS, including 4 novel missense variants (p.V23I, p.H322R, p.R358C, and p.T478I) and 3 loss-of-function variants (p.R357X, p.P378_I379del, and p.T419_G420del). The odds ratio between sporadic ALS patients and controls was 10.2 (p = 0.008, 95% confidence interval = 1.67-62.47). These findings support the contribution of TBK1 to the etiology of sporadic ALS in Japanese patients.

Molecular epidemiological study of familial amyotrophic lateral sclerosis in Japanese population by whole-exome sequencing and identification of novel HNRNPA1 mutation.

To elucidate the genetic epidemiology of familial amyotrophic lateral sclerosis (FALS) in the Japanese population, we conducted whole-exome sequencing analysis of 30 FALS families in whom causative mutations have not been identified in previous studies. Consequently, whole-exome sequencing analysis revealed novel mutations in HNRNPA1, TBK1, and VCP. Taken together with our previous results of mutational analyses by direct nucleotide sequencing analysis, a microarray-based resequencing method, or repeat-primed PCR analysis, causative mutations were identified in 41 of the 68 families (60.3%) with SOD1 being the most frequent cause of FALS (39.7%). Of the mutations identified in this study, a novel c.862/1018C>G (p.P288A/340A) mutation in HNRNPA1 located in the nuclear localization signal domain of hnRNPA1, enhances the recruitment of mutant hnRNPA1 into stress granules, indicating that an altered nuclear localization signal activity plays an essential role in amyotrophic lateral sclerosis pathogenesis.

Partial duplication of DHH causes minifascicular neuropathy: A novel mutation detection of DHH.

Minifascicular neuropathy (MN) is an extremely rare developmental malformation in which peripheral nerves are composed of many small fascicles. Only one patient with MN with 46XY gonadal dysgenesis (GD) was found to carry a mutation affecting the start codon in desert hedgehog (DHH). We identified an identical novel rearrangement mutation of DHH in two consanguineous families with MN, confirming mutations in DHH cause MN with 46XY GD. The patients with the 46XY karyotype developed GD, whereas a patient with the 46XX karyotype did not. These findings further support that DHH has important roles in perineural formation and male gonadal differentiation.