A novel TBK1 loss-of-function variant associated with ALS and parkinsonism phenotypes.

Loss-of-function (LoF) variants in the TANK binding kinase 1 (TBK1) gene are implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. In this study, we present the first familial cases of ALS and parkinsonism associated with a novel TBK1 variant. We describe two siblings: one diagnosed with classical ALS and the other with a unique syndrome overlapping ALS and parkinsonism. Comprehensive clinical and imaging evaluations supported these diagnoses. Genetic analysis through whole-genome sequencing revealed a previously unknown heterozygous splice site variant in TBK1. Functional assessments demonstrated that this splice site variant leads to abnormal splicing and subsequent degradation of the mutated TBK1 allele by nonsense-mediated decay, confirming its pathogenic impact. Our findings suggest a broader involvement of TBK1 in neurodegenerative diseases and underscore the need for further research into TBK1's role, advocating for screening for TBK1 variants in similar familial cases.

Association study of GBA1 variants with MSA based on comprehensive sequence analysis -Pitfalls in short-read sequence analysis depending on the human reference genome.

Multiple system atrophy (MSA) is a neurodegenerative disorder characterized by various combinations of autonomic failure, parkinsonism, and cerebellar ataxia. To elucidate variants associated with MSA, we have been conducting short-read-based whole-genome sequence analysis. In the process of the association studies, we initially focused on GBA1, a previously proposed susceptibility gene for MSA, to evaluate whether GBA1 variants can be efficiently identified despite its extraordinarily high homology with its pseudogene, GBA1LP. To accomplish this, we conducted a short-read whole-genome sequence analysis with alignment to GRCh38 as well as Sanger sequence analysis and compared the results. We identified five variants with inconsistencies between the two pipelines, of which three variants (p.L483P, p.A495P-p.V499V, p.L483_M489delinsW) were the results of misalignment due to minor alleles in GBA1P1 registered in GRCh38. The miscalling events in these variants were resolved by alignment to GRCh37 as the reference genome, where the major alleles are registered. In addition, a structural variant was not properly identified either by short-read or by Sanger sequence analyses. Having accomplished correct variant calling, we identified three variants pathogenic for Gaucher disease (p.S310G, p.L483P, and p.L483_M489delinsW). Of these variants, the allele frequency of p.L483P (0.003) in the MSA cases was higher than that (0.0011) in controls. The meta-analysis incorporating a previous report demonstrated a significant association of p.L483P with MSA with an odds ratio of 2.92 (95% CI; 1.08 - 7.90, p = 0.0353).

Citrin-deficient patient-derived induced pluripotent stem cells as a pathological liver model for congenital urea cycle disorders.

Citrin deficiency is a congenital secondary urea cycle disorder lacking useful disease models for effective treatment development. In this study, human induced pluripotent stem cells (iPSCs) were generated from two patients with citrin deficiency and differentiated into hepatocyte-like cells (HLCs). Citrin-deficient HLCs produced albumin and liver-specific markers but completely lacked citrin protein and expressed argininosuccinate synthase only weakly. In addition, ammonia concentrations in a medium cultured with citrin-deficient HLCs were higher than with control HLCs. Sodium pyruvate administration significantly reduced ammonia concentrations in the medium of citrin-deficient HLCs and slightly reduced ammonia in HLCs differentiated from control iPSCs, though this change was not significant. Our results suggest that sodium pyruvate may be an efficient treatment for patients with citrin deficiency. Citrin-deficient iPSCs are a pathological liver model for congenital urea cycle disorders to clarify pathogenesis and develop novel therapies.

Whole-Exome Sequencing Revealed a Pathogenic Germline Variant in the Fumarate Hydratase Gene, Leading to the Diagnosis of Hereditary Leiomyomatosis and Renal Cell Cancer.

The diagnosis of hereditary skin tumors is difficult for "old" diagnostic tools such as immunohistochemistry. Whole-exome sequencing analysis as a "new" diagnostic tool enables us to make a final diagnosis in spite of unknown hereditary diseases in the past. Hereditary leiomyomatosis and renal cell cancer are autosomal dominant hereditary cancer syndromes characterized by uterine myomas, cutaneous leiomyomas, and aggressive renal cell cancer. The syndrome is associated with pathogenic germline variants in the fumarate hydratase gene. Herein, we demonstrate a pathogenic germline variant of the fumarate hydratase gene in a 60-year-old woman with multiple cutaneous leiomyomas, leading to the diagnosis of hereditary leiomyomatosis and renal cell cancer. Whole-exome sequencing analysis using genomic DNA extracted from peripheral blood leukocytes revealed one germline variant in the FH gene on chromosome 1 (c.290G>A, p.Gly97Asp). She received total hysterectomy due to uterine myoma, which strongly supported the diagnosis. No tumor was detected in her kidney by computed tomography and ultrasound examination. Genetic examination for the mutation of the fumarate hydratase gene is important in order to reach the correct diagnosis and to detect renal cancer at its early stage.

Neurological disorders caused by novel non-coding repeat expansions: clinical features and differential diagnosis.

Nucleotide repeat expansions in the human genome are a well-known cause of neurological disease. In the past decade, advances in DNA sequencing technologies have led to a better understanding of the role of non-coding DNA, that is, the DNA that is not transcribed into proteins. These techniques have also enabled the identification of pathogenic non-coding repeat expansions that cause neurological disorders. Mounting evidence shows that adult patients with familial or sporadic presentations of epilepsy, cognitive dysfunction, myopathy, neuropathy, ataxia, or movement disorders can be carriers of non-coding repeat expansions. The description of the clinical, epidemiological, and molecular features of these recently identified non-coding repeat expansion disorders should guide clinicians in the diagnosis and management of these patients, and help in the genetic counselling for patients and their families.

Paraneoplastic Cerebellar Degeneration Leading to an Early Diagnosis of Peritoneal Serous Papillary Carcinoma.

A 77-year-old female with a subacute progression of ataxia and serum anti-Yo antibodies was suspected to have paraneoplastic cerebellar degeneration (PCD). An examination of an underlying cancer showed no abnormality in the gynecological organs, but the findings did show a mass in the Douglas fossa. The mass was resected and diagnosed as stage IIB peritoneal serous papillary carcinoma (PSPC), a rare gynecologic cancer that is difficult to diagnose in the early stages. PCD was treated with intravenous immunoglobulin (IVIG). For an early diagnosis and treatment, PSPC should be included in the list of malignancies that cause PCD with anti-Yo antibodies.

Genetic and Functional Analyses of Patients with Marked Hypo-High-Density Lipoprotein Cholesterolemia.

AIM: This study aimed to analyze two cases of marked hypo-high-density lipoprotein (HDL) cholesterolemia to identify mutations in ATP-binding cassette transporter A1 (ABCA1) and elucidate the molecular mechanism by which these novel pathological mutations contribute to hypo-HDL cholesterolemia in Tangier disease. METHODS: Wild type and mutant expression plasmids containing a FLAG tag inserted at the C-terminus of the human ABCA1 gene were generated and transfected into HEK293T cells. ABCA1 protein expression and cholesterol efflux were evaluated via Western blotting and efflux assay. The difference in the rate of change in protein expression was evaluated when proteolytic and protein-producing systems were inhibited. RESULTS: In case 1, a 20-year-old woman presented with a chief complaint of gait disturbance. Her HDL-C level was only 6.2 mg/dL. Tangier disease was suspected because of muscle weakness, decreased nerve conduction velocity, and splenomegaly. Whole-exome analysis showed compound heterozygosity for a W484* nonsense mutation and S1343I missense mutation, which confirmed Tangier disease. Cholesterol efflux decreased by a mixture of W484* and S1343I mutations. The S1343I mutation decreased the protein production rate but increased the degradation rate, decreasing the protein levels. This patient also had Krabbe disease. The endogenous ABCA1 protein level of macrophage cell decreased by knocking down its internal galactocerebrosidase.Case 2, a 51-year-old woman who underwent tonsillectomy presented with peripheral neuropathy, corneal opacity, and HDL-C of 3.4 mg/dL. Whole-exome analysis revealed compound heterozygosity for R579* and R1572* nonsense mutations, which confirmed Tangier disease. CONCLUSION: Case 1 is a new ABCA1 mutation with complex pathogenicity, namely, a W484*/S1343I compound heterozygote with marked hypo-HDL cholesterolemia. Analyses of the compound heterozygous mutations indicated that decreases in ABCA1 protein levels and cholesterol efflux activity caused by the novel S1343I mutation combined with loss of W484* protein activity could lead to marked hypo-HDL cholesterolemia. Galactocerebrosidase dysfunction could also be a potential confounding factor for ABCA1 protein function.

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.

Adrenomyeloneuropathy with Later Development of Cerebral Form Caused by a Hemizygous Splice-site Variant in ABCD1.

Adrenomyeloneuropathy (AMN)/adrenoleukodystrophy (ALD) is an X-linked genetic disorder caused by pathogenic variants in ABCD1. We treated a 54-year-old man with slowly progressive spastic paraparesis with later development of the cerebral form. A pathogenic splice-site variant of ABCD1 (c.1489-1G>A, p.Val497Alafs*51) and elevated levels of very long-chain fatty acids were found, leading to the diagnosis of AMN. Detailed ABCD1 mRNA expression analyses revealed decreased levels of ABCD1 mRNA accompanied by deletion of the first 31 bp in exon 6. The altered mRNA transcriptional patterns associated with splice site variants are diverse and may provide important insights into ALD pathogenesis.

Late-onset Myoclonic Seizure in a 78-year-old Woman with Gaucher Disease.

We herein report a 78-year-old woman with Gaucher disease (GD) who was initially diagnosed with GD type 1, had been receiving long-term enzyme replacement therapy since 58 years old, and developed neurological manifestations in her 70s. The neurological manifestations included myoclonic seizures and progressive cognitive decline. Although it is rare for GD patients to first develop neurologic manifestations at such an advanced age, physicians engaged in long-term care for GD patients should be alert for this possibility.

CGG repeat expansion in LOC642361/NUTM2B-AS1 typically presents as oculopharyngodistal myopathy.

CGG repeat expansions in LOC642361/NUTM2B-AS1 have recently been identified as a cause of oculopharyngeal myopathy with leukoencephalopathy. However, since only three patients from a single family were reported, it remains unknown whether their clinicopathological features are typical for CGG repeat expansions in LOC642361/NUTM2B-AS1. Here, using repeat-primed-polymerase chain reaction and long-read sequencing, we identify 12 individuals from 3 unrelated families with CGG repeat expansions in LOC642361/NUTM2B-AS1, typically presenting with oculopharyngodistal myopathy. The CGG repeat expansions range from 161 to 669 repeat units. Most of the patients present with ptosis, restricted eye movements, dysphagia, dysarthria, and diffuse limb muscle weakness. Only one patient shows T2-weighted hyperintensity in the cerebellar white matter surrounding the deep cerebellar nuclei on brain magnetic resonance imaging. Muscle biopsies from three patients show a myopathic pattern and rimmed vacuoles. Analyses of muscle biopsies suggest that CGG repeat expansions in LOC642361/NUTM2B-AS1 may deleteriously affect aggrephagic capacity, suggesting that RNA toxicity and mitochondrial dysfunction may contribute to pathogenesis. Our study thus expands the phenotypic spectrum for the CGG repeat expansion of LOC642361/NUTM2B-AS1 and indicates that this genetic variant typically manifests as oculopharyngodistal myopathy with chronic myopathic changes with rimmed vacuoles and filamentous intranuclear inclusions in muscle fibers.

Roles of the cerebellum and basal ganglia in temporal integration: Insights from a synchronized tapping task.

OBJECTIVE: The aim of this study was to clarify the roles of the cerebellum and basal ganglia for temporal integration. METHODS: We studied 39 patients with spinocerebellar degeneration (SCD), comprising spinocerebellar atrophy 6 (SCA6), SCA31, Machado-Joseph disease (MJD, also called SCA3), and multiple system atrophy (MSA). Thirteen normal subjects participated as controls. Participants were instructed to tap on a button in synchrony with isochronous tones. We analyzed the inter-tap interval (ITI), synchronizing tapping error (STE), negative asynchrony, and proportion of delayed tapping as indicators of tapping performance. RESULTS: The ITI coefficient of variation was increased only in MSA patients. The standard variation of STE was larger in SCD patients than in normal subjects, especially for MSA. Negative asynchrony, which is a tendency to tap the button before the tones, was prominent in SCA6 and MSA patients, with possible basal ganglia involvement. SCA31 patients exhibited normal to supranormal performance in terms of the variability of STE, which was surprising. CONCLUSIONS: Cerebellar patients generally showed greater STE variability, except for SCA31. The pace of tapping was affected in patients with possible basal ganglia pathology. SIGNIFICANCE: Our results suggest that interaction between the cerebellum and the basal ganglia is essential for temporal processing. The cerebellum and basal ganglia and their interaction regulate synchronized tapping, resulting in distinct tapping pattern abnormalities among different SCD subtypes.

Efficient protocol for the differentiation of kidney podocytes from induced pluripotent stem cells, involving the inhibition of mTOR.

The mechanistic/mammalian target of rapamycin (mTOR) is involved in a wide range of cellular processes. However, the role of mTOR in podocytes remains unclear. In this study, we aimed to clarify the role of mTOR in podocyte differentiation from human induced pluripotent stem cells (hiPSCs) and to establish an efficient differentiation protocol for human podocytes. We generated podocytes from hiPSCs by modifying protocol. The expression of the podocyte-specific slit membrane components nephrin and podocin was measured using PCR, western blotting, flow cytometry, and immunostaining; and the role of mTOR was evaluated using inhibitors of the mTOR pathway. Nephrin and podocin were found to be expressed in cells differentiated from hiPSCs, and their expression was increased by mTOR inhibitor treatment. S6, a downstream component of the mTOR pathway, was also found to be involved in podocyte differentiation. we evaluated its permeability to albumin, urea, and electrolytes. The induced podocytes were permeable to the small molecules, but only poorly permeable to albumin. We have shown that the mTOR pathway is involved in podocyte differentiation. Our monolayer podocyte differential protocol, using an mTOR inhibitor, provides a novel in vitro model for studies of kidney physiology and pathology.

Dysbiosis of the gut microbiota as a susceptibility factor for Kawasaki disease.

Introduction: Gut microbial imbalance (dysbiosis) has been reported in patients with acute Kawasaki disease (KD). However, no studies have analyzed the gut microbiota while focusing on susceptibility to KD. This study aimed to evaluate whether dysbiosis elevates susceptibility to KD by assessing children with a history of KD. Methods: Fecal DNA was extracted from 26 children with a history of KD approximately 1 year prior (KD group, 12 boys; median age, 32.5 months; median time from onset, 11.5 months) and 57 age-matched healthy controls (HC group, 35 boys; median age, 36.0 months). 16S rRNA gene analysis was conducted with the Illumina Miseq instrument. Sequence reads were analyzed using QIIME2. Results: For alpha diversity, Faith's phylogenetic diversity was significantly higher in the KD group. Regarding beta diversity, the two groups formed significantly different clusters based on Bray-Curtis dissimilarity. Comparing microbial composition at the genus level, the KD and HC groups were significantly different in the abundance of two genera with abundance over 1% after Benjamini-Hochberg false discovery rate correction for multiple comparisons. Compared with the HC group, the KD group had higher relative abundance of Ruminococcus gnavus group and lower relative abundance of Blautia. Discussion and conclusion: Ruminococcus gnavus group reportedly includes pro-inflammatory bacteria. In contrast, Blautia suppresses inflammation via butyrate production. In the predictive functional analysis, the proportion of gut microbiota involved in several pathways was lower in the KD group. Therefore, dysbiosis characterized by distinct microbial diversity and decreased abundance of Blautia in parallel with increased abundance of Ruminococcus gnavus group might be a susceptibility factor for KD.

Abnormal Development of Microbiota May Be a Risk Factor for Febrile Urinary Tract Infection in Infancy.

Febrile urinary tract infection (fUTI) is common in infants, but specific risk factors for developing it remain unclear. As most fUTIs are caused by ascending infections of intestinal bacteria, dysbiosis-an imbalance in gut microbial communities-may increase fUTI risk. This study was conducted to test the hypothesis that abnormal development of gut microbiota during infancy increases the risk of developing fUTI. Stool samples were collected from 28 infants aged 3-11 months with first-onset fUTI (fUTI group) and 51 healthy infants of the same age (HC group). After bacterial DNA extraction, 16S rRNA expression was measured and the diversity of gut microbiota and constituent bacteria were compared between the two groups. The alpha diversity of gut microbiota (median Shannon index and Chao index) was significantly lower in the fUTI group (3.0 and 42.5) than in the HC group (3.7 and 97.0; p < 0.001). The beta diversity also formed different clusters between the two groups (p < 0.001), suggesting differences in their microbial composition. The linear discriminant analysis effect size showed that the fUTI group proportionally featured significantly more Escherichia-Shigella in the gut microbiota (9.5%) than the HC group (3.1%; p < 0.001). In summary, abnormal gut microbiota development during infancy may increase the risk of fUTI.

Abnormal saccade profiles in hereditary spinocerebellar degeneration reveal cerebellar contribution to visually guided saccades.

OBJECTIVE: To study how the pathophysiology underlying hereditary spinocerebellar degeneration (spinocerebellar ataxia; SCA) with pure cerebellar manifestation evolves with disease progression using saccade recordings. METHODS: We recorded visually- (VGS) and memory-guided saccade (MGS) task performance in a homogeneous population of 20 genetically proven SCA patients (12 SCA6 and eight SCA31 patients) and 19 normal controls. RESULTS: For VGS but not MGS, saccade latency and amplitude were increased and more variable than those in normal subjects, which correlated with cerebellar symptom severity assessed using the International Cooperative Ataxia Rating Scale (ICARS). Parameters with significant correlations with cerebellar symptoms showed an aggravation after disease stage progression (ICARS > 50). The saccade velocity profile exhibited shortened acceleration and prolonged deceleration, which also correlated with disease progression. The main sequence relationship between saccade amplitude and peak velocity as well as saccade inhibitory control were preserved. CONCLUSIONS: The cerebellum may be involved in initiating VGS, which was aggravated acutely during disease stage progression. Dysfunction associated with disease progression occurs mainly in the cerebellum and brainstem interaction but may also eventually involve cortical saccade processing. SIGNIFICANCE: Saccade recording can reveal cerebellar pathophysiology underlying SCA with disease progression.