Biallelic COQ4 Variants in Hereditary Spastic Paraplegia: Clinical and Molecular Characterization.

BACKGROUND: Hereditary spastic paraplegias (HSP) are neurologic disorders characterized by progressive lower-extremity spasticity. Despite the identification of several HSP-related genes, many patients lack a genetic diagnosis. OBJECTIVES: The aims were to confirm the pathogenic role of biallelic COQ4 mutations in HSP and elucidate the clinical, genetic, and functional molecular features of COQ4-associated HSP. METHODS: Whole exome sequences of 310 index patients with HSP of unknown cause from three distinct populations were analyzed to identify potential HSP causal genes. Clinical data obtained from patients harboring candidate causal mutations were examined. Functional characterization of COQ4 variants was performed using bioinformatic tools, single-cell RNA sequencing, biochemical assays in cell lines, primary fibroblasts, induced pluripotent stem cell-derived pyramidal neurons, and zebrafish. RESULTS: Compound heterozygous variants in COQ4, which cosegregated with HSP in pedigrees, were identified in 7 patients from six unrelated families. Patients from four of the six families presented with pure HSP, whereas probands of the other two families exhibited complicated HSP with epilepsy or with cerebellar ataxia. In patient-derived fibroblasts and COQ4 knockout complementation lines, stable expression of these missense variants exerted loss-of-function effects, including mitochondrial reactive oxygen species accumulation, decreased mitochondrial membrane potential, and lower ubiquinone biosynthesis. Whereas differentiated pyramidal neurons expressed high COQ4 levels, coq4 knockdown zebrafish displayed severe motor dysfunction, reflecting motor neuron dysregulation. CONCLUSIONS: Our study confirms that loss-of-function, compound heterozygous, pathogenic COQ4 variants are causal for autosomal recessive pure and complicated HSP. Moreover, reduced COQ4 levels attributable to variants correspond with decreased ubiquinone biosynthesis, impaired mitochondrial function, and higher phenotypic disease severity. © 2023 International Parkinson and Movement Disorder Society.

The Osmolality and Hemolysis of High-Concentration Monoclonal Antibody Formulations.

PURPOSES: Highly concentrated monoclonal antibody (mAb) formulations for subcutaneous administration are becoming increasingly preferred within the biopharmaceutical industry for ease of use and improved patient compliance. A common phenomenon observed in the industry is that osmolality detected via freezing-point depression (FPD) in high-concentration mAb formulations is much higher than the theoretical concentrations, yet the occurrence of this phenomenon and its possible safety issues have been rarely reported. METHODS: The current study summarized theoretical osmolality of U.S. Food and Drug Administration approved high-concentration mAb formulations and evaluated effects of high osmolality on safety using hemolysis experiments for the first time. Two mAbs formulated at 150 mg/mL were used as models and configured into two isotonic solutions: a, a theoretically calculated molarity in the isotonic range (H) and b, an osmolality value measured via the FPD in the isotonic range (I). The H and I formulations of each mAb were individually subjected to hemolysis experiments, and the hemolysis rates of the two formulations of the same mAb were compared. Besides, the effect of mAb concentration on osmolality detected by FPD was explored as well. RESULTS: The results indicated that the hemolysis rates were similar between the H and I formulations of mAbs at the same sample addition volume, and the osmolality values increased approximately linearly with the increase in mAb concentration. CONCLUSIONS: High osmolality for high-concentration mAb formulations would not affect product safety and the excipients could be added at relatively high levels to maintain product stability, especially for labile products.

Alu Retrotransposition Event in SPAST Gene as a Novel Cause of Hereditary Spastic Paraplegia.

OBJECTIVES: To diagnose the molecular cause of hereditary spastic paraplegia (HSP) observed in a four-generation family with autosomal dominant inheritance. METHODS: Multiplex ligation-dependent probe amplification (MLPA), whole-exome sequencing (WES), and RNA sequencing (RNA-seq) of peripheral blood leukocytes were performed. Reverse transcription polymerase chain reaction (RT-PCR) and Sanger sequencing were used to characterize target regions of SPAST. RESULTS: A 121-bp AluYb9 insertion with a 30-bp poly-A tail flanked by 15-bp direct repeats on both sides was identified in the edge of intron 16 in SPAST that segregated with the disease phenotype. CONCLUSIONS: We identified an intronic AluYb9 insertion inducing splicing alteration in SPAST causing pure HSP phenotype that was not detected by routine WES analysis. Our findings suggest RNA-seq is a recommended implementation for undiagnosed cases by first-line diagnostic approaches. © 2023 International Parkinson and Movement Disorder Society.

New insights into metabolomics profile generation in fermented tea: the relevance of bacteria and metabolites in Fuzhuan brick tea.

BACKGROUND: The contribution of bacteria to fermented tea is not clear and the associated research is relatively limited. To reveal the role of microorganisms in fermented tea processing, the microbial community and metabolites of Fuzhuan brick tea (FBT), a Chinese traditional fermented tea, were revealed via high-throughput sequencing and liquid chromatography-mass spectrometry (LC-MS). RESULTS: In FBT, bacterial communities had a higher abundance and diversity, Lactococcus and Bacillus were the main bacteria, and Eurotium was the predominant fungus. The predictive metabolic function indicated the pathways of cellular growth, environmental information, genetics and material metabolism of bacterial communities were abundant, whereas the fungal community predictive metabolic function was almost saprotroph. Using LC-MS, 1143 and 536 metabolites were defined in positive and negative ion mode, respectively. There were essential correlations between bacterial populations and metabolites, such that Bacillus was correlated significantly with 44 metabolites (P < 0.05) and Enterococcus was significantly associated with 15 metabolites (P < 0.05). Some of the main active components were significantly correlated with the bacteria, such as Enterococcus, Lactococcus and Carnobacterium. CONCLUSION: Not only Eurotium, but also the bacteria were involved in the changes of metabolomics profile in fermented FBT. The present study assists in providing new insights into metabolomics profile generation in fermented tea. The present research lays a foundation for controlling the FBT fermentation by artificial inoculation to improve quality. © 2021 Society of Chemical Industry.

[Extracts of Poria cocos polysaccharides improves alcoholic liver disease in mice via CYP2E1 and NF-κB inflammatory pathways].

The present study investigated the effect of extract of Poria cocos polysaccharides(PCP) on cytochrome P450 2 E1(CYP2 E1) and nuclear factor κB(NF-κB) inflammatory signaling pathways in alcoholic liver disease(ALD) mice and explored its protective effect and mechanism. Sixty male C57 BL/6 N mice of SPF grade were randomly divided into a control group, a model group, a positive drug group(bifendate, 200 mg·kg~(-1)), and high-(200 mg·kg~(-1)) and low-dose(50 mg·kg~(-1)) PCP groups. Gao-binge mo-del was induced and the mice in each group were treated correspondingly. Liver morphological and pathological changes were observed and organ index was calculated. Serum levels of alanine aminotransferase(ALT) and aspartate aminotransferase(AST) were detected. Malondialdehyde(MDA) and superoxide dismutase(SOD) in liver tissues were detected by assay kits. The levels of interleukin-6(IL-6) and tumor necrosis factor-α(TNF-α) were detected by ELISA. The activation of macrophages was observed by immunofluorescence staining and protein expression of CYP2 E1, Toll-like receptor 4(TLR4), NF-κB p65, and phosphorylated NF-κB p65(p-NF-κB p65) were analyzed by Western blot. The ALD model was properly induced. Compared with the model group, the PCP groups significantly improved the pathological injury of liver tissues. Immunofluorescence staining revealed that compared with the model group, the groups with drug intervention showed decreased macrophages in liver tissues. Additionally, the PCP groups showed reduced ALT, AST, MDA, IL-6, and TNF-α(P<0.05), and potentiated activity of SOD(P<0.01). PCP extract has the protective effect against alcoholic liver injury in mice, and the underlying mechanism may be related to the regulation of the expression of CYP2 E1 and inhibition of TLR4/NF-κB inflammatory signaling pathway to reduce oxidative stress and inflammatory injury, thereby inhibiting the development of ALD.

Neurological and imaging phenotypes of adults with untreated phenylketonuria: new cases and literature review.

OBJECTIVES: Phenylketonuria (PKU) is the most prevalent congenital disease of amino acid metabolism. Neurological manifestations usually complicate PKU in untreated adult patients. This study describes neurological and imaging phenotypes of adult patients with untreated PKU. METHODS: We investigated a cohort of 320 unrelated adult patients with suspected genetic leukoencephalopathies using whole-exome sequencing (WES). We analyzed the phenotypic features of adult PKU patients in our cohort and summarized cases reported in the literature. RESULTS: We identified 10 patients in our cohort and 12 patients in the literature, who presented with neurological manifestations and were diagnosed with PKU in adulthood. Approximately 60% of these patients had onset of clinical features in adulthood. The most common neurological symptoms of patients presenting in adulthood were cognitive disturbance and spastic paralysis, followed by vision loss, cerebellar ataxia, weakness of limbs, and seizure. This differed from that of patients presenting with PKU features in childhood, who consistently had mental retardation with various neurological complications emerging during a broad age range. Imaging findings were similar between patients presenting with clinical features in childhood compared with adulthood, comprising symmetric periventricular white matter hyperintense on T2-weighted imaging and diffusion-weighted imaging predominantly in the parietal and occipital lobes. Also, normal brain imaging and diffuse leukoencephalopathies were observed in both patient groups. CONCLUSION: PKU with clinical features presenting in adulthood is an atypical subtype and should be considered during diagnosis of adults with neurological symptoms and leukoencephalopathy. DWI seems to be most helpful to distinguish patients with PKU. Additionally, we demonstrate that PKU constitutes a part (3.1%) of adult genetic leukoencephalopathies.

Chinese patients with hereditary spastic paraplegias (HSPs): a protocol for a hospital-based cohort study.

INTRODUCTION: Hereditary spastic paraplegias (HSPs) are uncommon but not rare neurodegenerative diseases. More than 100 pathogenic genes and loci related to spastic paraplegia symptoms have been reported. HSPs have the same core clinical features, including progressive spasticity in the lower limbs, though HSPs are heterogeneous (eg, clinical signs, MRI features, gene mutation). The age of onset varies greatly, from infant to adulthood. In addition, the slow and variable rates of disease progression in patients with HSP represent a substantial challenge for informative assessment of therapeutic efficacy. To address this, we are undertaking a prospective cohort study to investigate genetic-clinical characteristics, find surrogates for monitoring disease progress and identify clinical readouts for treatment. METHODS AND ANALYSIS: In this case-control cohort study, we will enrol 200 patients with HSP and 200 healthy individuals in parallel. Participants will be continuously assessed for 3 years at 12-month intervals. Six aspects, including clinical signs, genetic spectrum, cognitive competence, MRI features, potential biochemical indicators and nerve electrophysiological factors, will be assessed in detail. This study will observe clinical manifestations and disease severity based on different molecular mechanisms, including oxidative stress, cholesterol metabolism and microtubule dynamics, all of which have been proposed as potential treatment targets or modalities. The analysis will also assess disease progression in different types of HSPs and cellular pathways with a longitudinal study using t tests and χ2 tests. ETHICS AND DISSEMINATION: The study was granted ethics committee approval by the first affiliated hospital of Fujian Medical University (MRCTA, ECFAH of FMU (2019)194) in 2019. Findings will be disseminated via presentations and peer-reviewed publications. Dissemination will target different audiences, including national stakeholders, researchers from different disciplines and the general public. TRIAL REGISTRATION NUMBER: NCT04006418.

Novel Compound Missense and Intronic Splicing Mutation in ALDH18A1 Causes Autosomal Recessive Spastic Paraplegia.

Background: Hereditary spastic paraplegia (HSP) caused by mutations in ALDH18A1 have been reported as spastic paraplegia 9 (SPG9), with autosomal dominant and autosomal recessive transmission (SPG9A and SPG9B). SPG9 is rare and has shown phenotypic and genotypic heterogeneity in previous reports. Methods: This study screened ALDH18A1 mutations in autosomal recessive HSP patients using combined whole exome sequencing and RNA splicing analysis. We conducted in silico investigations, co-segregation analysis, and ELISA-based analysis of P5CS (Δ1-pyrroline-5-carboxylate synthetase; encoded by ALDH18A1) concentration to validate the pathogenicity of the detected ALDH18A1 variants. All previously reported bi-allelic ALDH18A1 mutations and cases were reviewed to summarize the genetic and clinical features of ALDH18A1-related HSP. Results: A novel missense mutation c.880T>C, p.S294P and an intronic splicing mutation c.-28-13A>G were both detected in ALDH18A1 in an autosomal recessive family presenting with a complicated form HSP. ELISA assays revealed significantly decreased P5CS concentration in the proband's plasma compared with that in the healthy controls. Moreover, review of previously reported recessive cases showed that SPG9B patients in our cohort presented with milder symptoms, i.e., later age at onset and without cognitive impairment. Conclusion: The present study expands the genetic and clinical spectrum of SPG9B caused by ALDH18A1 mutation. Our work defines new genetic variants to facilitate future diagnoses, in addition to demonstrating the highly informative value of splicing mutation prediction in the characterization of disease-related intronic variants.

Effects of allyl isothiocyanate on the expression, function, and its mechanism of ABCA1 and ABCG1 in pulmonary of COPD rats.

BACKGROUND AND AIMS: Allyl isothiocyanate(AITC) has been shown to play an important role in the improved symptoms of chronic obstructive pulmonary disease(COPD) and the inhibition of inflammation, but the role in COPD lipid metabolism disorder and the molecular mechanism remains unclear. We aimed to explore whether and how AITC affects COPD by regulating lipid metabolism and inflammatory response. METHODS: The COPD rat model was established by cigarette smoke exposure. Cigarette smoke extract stimulated 16HBE cells to induce a cell model. The effect of AITC treatment was detected by lung function test, H&E staining, Oil red O staining, immunohistochemistry, ELISA, CCK-8, HPLC, fluorescence efflux test, siRNA, RT-PCR, and Western blotting. Biological analysis was performed to analyze the results. Graphpad Prism 8.0 software was used for statistical analysis. RESULTS: AITC can improve lung function and pathological injury in COPD rats. The levels of IL-1 ß and TNF- α in the AITC treatment group were significantly lower than those in the model group(P < 0.05), and the lipid metabolism was also improved (P < 0.05). AITC reverses CSE-induced down-regulation of LXR α, ABCA1, and ABCG1 expression and function in a time-and concentration-dependent manner (P < 0.05). AITC regulates the cholesterol metabolism disorder induced by CSE in NR8383 cells and attenuates macrophage inflammation (P < 0.05). In addition, after silencing LXR α with siRNA, the effect of AITC was also inhibited. CONCLUSION: These results suggest that AITC improves COPD by promoting RCT process and reducing inflammatory response via activating LXR pathways.

Novel CAPN1 mutations extend the phenotypic heterogeneity in combined spastic paraplegia and ataxia.

OBJECTIVE: Recessive mutations in the CAPN1 gene have recently been identified in spastic paraplegia 76 (SPG76), a complex hereditary spastic paraplegia (HSP) that is combined with cerebellar ataxia, resulting in an ataxia-spasticity disease spectrum. This study aims to assess the influence of CAPN1 variants on the occurrence of SPG76 and identify factors potentially contributing to phenotypic heterogeneity. METHODS: We screened a cohort of 240 unrelated HSP families for variants in CAPN1 using high-throughput sequencing analysis. We described in detail the clinical and genetic features of the SPG76 patients in our cohort and summarized all reported cases. RESULTS: Six unreported CAPN1-associated families containing eight patients with or without cerebellar ataxia were found in our cohort of HSP cases. These patients carried three previously reported homozygous truncating mutations (p.V64Gfs* 103, c.759+1G>A, and p.R285* ), and three additional novel compound heterozygous missense mutations (p.R481Q, p.P498L, and p.R618W). Lower limbs spasticity, hyperreflexia, and Babinski signs developed in about 94% of patients, with ataxia developing in 63% of cases. In total, 33 pathogenic mutations were distributed along the three reported functional domains of calpain-1 protein, encoded by CAPN1, with no hotspot region. A comparison of gender distribution between the two groups indicated that female SPG76 patients were significantly more likely to present with complicated HSP than male patients (P = 0.015). INTERPRETATION: Our study supports the clinically heterogeneous inter- and intra-family variability of SPG76 patients, and demonstrates that gender and calpain-1 linker structure may contribute to clinical heterogeneity in SPG76 cases.

Chinese patients with adrenoleukodystrophy and Zellweger spectrum disorder presenting with hereditary spastic paraplegia.

INTRODUCTION: X-linked adrenoleukodystrophy (ALD) and Zellweger spectrum disorder (ZSD) are peroxisomal diseases characterized by accumulation of very long chain fatty acids (VLCFA) in plasma and tissues. Considering the wide variability of manifestation, patients of ALD and atypical ZSD are easily misdiagnosed as hereditary spastic paraplegia (HSP) on their clinical grounds. Here, we aimed to determine the frequency of peroxisome diseases and compare their phenotypic spectra with HSP. METHODS: We first applied targeted sequencing in 120 pedigrees with spastic paraplegia, and subsequently confirmed 74 HSP families. We then performed whole exome sequencing for the probands of the 46 remaining pedigrees lacking known HSP-causal genes. Detailed clinical, radiological features, and VLCFA analyses are presented. RESULTS: Seven ALD pedigrees with ABCD1 mutations and one ZSD family harboring bi-allelic mutations of PEX16 were identified. Clinically, in addition to spastic paraplegia, four ALD probands presented adrenocortical insufficiency, and the ZSD proband and her affected sister both developed thyroid problems. VLCFA analysis showed that ratios of C24/C22 and C26/C22 were specifically increased in ALD probands. Moreover, three ALD probands and the ZSD proband had abnormalities in brain or spinal imaging. CONCLUSIONS: Our study reports the first ZSD case in China that manifested spastic paraplegia, and emphasized the finding that peroxisomal diseases comprise a significant proportion (8/120) of spastic paraplegia entities. These findings extend our current understanding of the ALD and ZSD diseases.

Genetic and Clinical Profile of Chinese Patients with Autosomal Dominant Spastic Paraplegia.

BACKGROUND: Hereditary spastic paraplegia (HSP) refers to a group of neurodegenerative disorders characterized by bilateral weakness, spasticity, and hyperreflexia in the lower limbs. The autosomal dominant HSP (ADHSP) predominantly presents as the pure form, but the clinical profiles and causal genetic variants underlying ADHSP are complex, and many remain unknown. METHODS: A cohort of 15 Chinese HSP pedigrees (including 35 patients and their 22 relatives) were screened by multiplex ligation-dependent probe amplification (MLPA) or whole-exome sequencing (WES). Neurological assessments were also conducted. RESULTS: The main subtypes of HSP above detected in our cohort were SPG4, SPG3A, and SPG6. Fifteen HSP-inducing mutations were identified, among which six were novel mutations: SPAST c.1277T>C, c.1292G>C, c.1562T>C, and c.1693A>T, NIPA1 c.748A>C, and KIDINS220 c.4448C>G. As expected, the most common presentation of the ADHSP cases was the pure form, manifesting spasticity of lower limbs and hyperreflexia, as well as pyramidal signs. Differing substantially from previous reports for KIDINS220 variants, our study family exhibited autosomal dominant inheritance, and only presented with spastic paraplegia, with no signs of intellectual disability, nystagmus, or obesity. CONCLUSION: Our work reveals a non-classical spastic paraplegia, intellectual disability, nystagmus, and obesity phenotype for a KIDINS220 mutation, which broadens both the clinical and genetic spectrum for ADHSP. Beyond underscoring the utility of using both MLPA and WES in studies of HSP, our work deepens the scientific understanding of phenotypes for ADHSP and defines new genetic variants to facilitate future diagnoses.