Zhimei Liu

Aims: To characterize the clinical, electrophysiological, and genetic spectrum of pediatric CMS and evaluate genotype‐informed outcomes using an integrated phenotype–electrophysiology–genomics approach. Methods: We retrospectively reviewed 36 pediatric CMS patients evaluated at a single center between 2015 and 2025. Clinical features, RNS, targeted NGS/WES variants, ventilator use, treatments, ACMG/AMP classifications, and MG‐ADL outcomes were analyzed. Results: Of 36 patients, 28 (77.8%) developed symptoms in the neonatal period or infancy. Biallelic variants involved 17 CMS genes; postsynaptic CMS was most common (55.6%, 20/36). COLQ and CHRNE were the most frequent genes (13.9%, 5/36 each), followed by CHAT (11.1%, 4/36). VUS were detected in 19 patients (52.8%, 19/36), including 8 with biallelic VUS supported by phenotype, neuromuscular transmission findings, treatment response, and follow‐up. RNS showed a ≥ 10% decrement in 16/21 tested patients (76.2%). CHAT ‐CMS was associated with higher ventilator use (3/4 vs. 6/32; p = 0.041) and early mortality (3/4 vs. 1/32; p = 0.002). Median MG‐ADL improved from 5 to 3 after genotype‐informed therapy. Conclusion: Pediatric CMS shows marked genetic heterogeneity and frequent VUS‐related uncertainty. Integrating phenotype, electrophysiology, and genomics supports diagnosis and mechanism‐guided therapy. CHAT ‐CMS is high risk for early respiratory failure and mortality.

Objective: Mitochondrial diseases are the most common inherited metabolic disorders, characterized by pronounced clinical and genetic heterogeneity that complicates molecular diagnosis. Although DNA‐based sequencing approaches have become standard in genetic testing, up to half of patients remain without a definitive diagnosis. We aimed to perform RNA sequencing (RNA‐seq) of patient‐derived skin fibroblasts to enhance the molecular diagnostic efficacy of mitochondrial disease in undiagnosed cases in China. Methods: We performed RNA‐seq on skin fibroblasts from 140 pediatric patients with suspected mitochondrial disease who remained genetically undiagnosed after whole exome sequencing (WES). Aberrant RNA expression and splicing were identified using the detection of RNA outliers pipeline (DROP). Based on WES findings, patients were stratified into a candidate group ( n = 28), in which RNA‐seq evaluated the pathogenicity of WES‐identified variants of uncertain significance and an unsolved group ( n = 112), in which RNA‐seq was used to pinpoint candidate genes. In six cases where RNA‐seq identified the aberrant RNA event but WES did not detect the causative variants, whole genome sequencing (WGS) was performed. Results: Integrative RNA‐seq, WES, and WGS analysis resulted in a genetic diagnosis in 25% of patients overall (20/28 [71%] in the candidate group; 15/112 [13%] in the unsolved group). Aberrant splicing explained most candidate‐group diagnoses, including variants misclassified by in silico predictors such as SpliceAI. 14% of protein‐truncating variants predicted to undergo nonsense‐mediated decay (NMD) escaped degradation, highlighting the functional limits of current predictions. The variants identified in the unsolved cohort included synonymous, missense, deep intronic, near‐splice‐site variants, and large deletions. The most frequent among them was a recurrent synonymous East Asian founder mutation in ECHS1, accounting for seven cases. Interestingly, across 233 pathogenic variants associated with aberrant RNA phenotypes compiled from this study and prior reports, half were noncoding and half were coding variants. Conclusion: RNA‐seq substantially enhances molecular diagnosis in mitochondrial disease by exposing cryptic splicing, regulatory, and NMD‐escape events invisible to DNA sequencing alone. These data advocate transcriptome analysis as an essential component of comprehensive genomic diagnostics in neurometabolic disease.

Granulocyte colony-stimulating factor (G-CSF) can effectively improve body’s immunity. This study explored G-CSF’s role in dopaminergic neurons apoptosis in Parkinson’s disease mice model. In this study, Oxidopamine, also known as 6-hydroxydopamine (6-OHDA) drugs were used to establish mouse models, and 2,3,5-Triphenyltetrazolium chloride (TTC) measurement of brain nerve cell apoptosis and mouse motor function scores methods were used to analyze and clarify whether G-CSF’s role in inflammatory process is through regulating monocyte chemoattractant protein 1 (MCP-1) expression and T cell immune function. Meanwhile, neutrophil infiltration in apoptotic and surrounding areas was also assessed. Nerve cell apoptosis was significantly reduced After a single dose of G-CSF treatment in the model 24 h after successful modeling, compared with PBS control group (P P α, and TGF-β were significantly decreased through therapeutic intervention, and inflammation was controlled (P < 0.05). The number of CD11b infiltration in the apoptotic area in the G-CSF group did not increase, suggesting that intervention did not reduce apoptosis of nerve cells. Bcl-2 expression in the treatment group was significantly higher than in the control group, and Bax expression was lower (P P <0.05). G-CSF can thus promote bone marrow hematopoiesis, thereby stimulating endogenous nerve cell proliferation, resisting nerve damage in Parkinson’s mice, and promoting neuron regeneration to help preserve neuronal function in mice and improve prognosis.

Professor ZHANG Shan-chen's clinical experience and academic thoughts in the field of acupuncture are summarized. Professor ZHANG stresses on theoretical exploration and has written Zhenjiu Jiayijing Shuxue Chongji, published a series of articles on textual research and expounded the nomenclature of acupoints. He believes that clinical practice should be guided by theory and the comprehensive syndrome differentiation be emphasized. Hence, a holistic idea should be cultivated, in which, the human body is considered as an organic whole and should be adaptive to the nature. Based on the theory above, the diagnosis can be determined and the effective treatment be received. He suggests selecting few acupoints, identifying the deficiency from the excess so as to determine the reinforcing or replenishing method and exerting appropriate needling manipulation. Additionally, the response should be enhanced on the identification of deqi after needle insertion. Moreover, a great consideration is laid on the clinical trial and application of moxibustion, which is complemented with acupuncture technique each other and mutually conductive to the clinical effect.

Purpose: To study the regulatory influence of microRNA-146a (miR-146a) on 1-methyl-4-phenyl-1,2,3, 6-tetrahydropyridine (MPTP)-induced neuroinflammation in mice with Parkinson's disease (PD). Methods: Forty specific pathogen-free (SPF) male C57BL/6 mice were divided into 2 groups: normal control and PD groups. The 2 groups were each divided into 2 subgroups: miR-146a inhibitor and inhibitor control groups. The mRNA and protein expressions of miR-146a, interleukin-1 receptor- associated kinase-1 (IRAK-1) and P65-NF-κB were determined by quantitative real-time polymerase chain reaction (qRT-PCR) and immunoblot assay, respectively. Levels of interleukin (IL)-1, IL-6 and TNF-α were assayed by enzyme-linked immunosorbent assay (ELISA). Results: The level of expression of miR-146a was significantly and time-dependently increased in PD mice, relative to control (p < 0.05). In PD group, mRNA and protein expressions of IRAK-1 were markedly higher in miR-146a inhibitor group than in inhibitor control (p < 0.05). Protein expression of P65-NF-κB was significantly upregulated in brains of PD mice, relative to normal mice (p < 0.05). Moreover, IL-1, IL-6 and TNF-α levels were significantly higher in brains of PD mice than in control. Conclusion: These results show the involvement of miR-146a in the etiology of PD, and that its regulation of neuroinflammation occurs via inhibition of IRAK1 gene expression. This finding may be useful in the development of new anti-PD drugs based on miR-146a.

Pyruvate, the end product of glycolysis, is a key metabolic molecule enabling mitochondrial adenosine triphosphate synthesis and takes part in multiple biosynthetic pathways within mitochondria. The mitochondrial pyruvate carrier (MPC) plays a vital role in transporting pyruvate from the cytosol into the organelle. In humans, MPC is a hetero‐oligomeric complex formed by the MPC1 and MPC2 paralogs that are both necessary to stabilize each other and form a functional MPC. MPC deficiency (OMIM#614741) due to pathogenic MPC1 variants is a rare autosomal recessive disease involving developmental delay, microcephaly, growth failure, and increased serum lactate and pyruvate. To date, two MPC1 variants in four cases have been reported, though only one with a detailed clinical description. Herein, we report three novel pathogenic MPC1 variants in six patients from three unrelated families, identified within European, Kuwaiti, and Chinese mitochondrial disease patient cohorts, one of whom presented as a Leigh‐like syndrome. Functional analysis in primary fibroblasts from the patients revealed decreased expression of MPC1 and MPC2. We rescued pyruvate‐driven oxygen consumption rate in patient's fibroblasts by reconstituting with wild‐type MPC1. Complementing homozygous MPC1 mutant cDNA with CRISPR‐deleted MPC1 C2C12 cells verified the mechanism of variants: unstable MPC complex or ablated pyruvate uptake activity. Furthermore, we showed that glutamine and beta‐hydroxybutyrate were alternative substrates to maintain mitochondrial respiration when cells lack pyruvate. In conclusion, we expand the clinical phenotypes and genotypes associated with MPC deficiency, with our studies revealing glutamine as a potential therapy for MPC deficiency.

Objective: Variant non-ketotic hyperglycinaemia (NKH) is a rare disorder characterized by variable clinical, biochemical, and imaging features. The variant form of NKH is rare and characterized by variable clinical, biochemical and imaging features.Subjects: Herein, we report a girl with variant NKH with two mutations in glutaredoxin 5 (GLRX5), which has been described in only three patients.Results: The clinical and biochemical phenotypes of the patient are also described. She suffered from developmental regression associated with spasticity, developmental delay, anemia and optic atrophy. The mitochondrial leukoencephalopathy was used to designate these disorders. An increased T2 signal from the medulla oblongata to the C6 spinal region was also observed on spinal cord MRI. Tandem mass analysis of a dried blood sample revealed elevated levels of glycine. The patient has two compound heterozygous mutations (c.151_153 del AAG and c.196C>T) in the GLRX5 gene. The c.196C>T mutation led to a stop codon (p.Q66Ter). Activities of mitochondrial respiratory chain (MRC) complexes II+III in the patient's fibroblasts were abnormal.Conclusions: We present the case of a girl with variant NKH who manifested spasticity and bilateral cavitating leukoencephalopathy. The patient had a deficiency of a respiratory chain enzyme, and this is the first report. Genetic testing is important for physicians to evaluate suspected variant NKH patients and to provide proper genetic counseling.

Background: Progressive cavitating leukoencephalopathy (PCL) is thought to result from mutations in nuclear genes affecting mitochondrial function and energy metabolism. To date, mutations in two subunits of complex I, NDUFS1 and NDUFV1, have been reported to be related to PCL.Methods: Patients underwent clinical examinations, brain MRI, skin biopsy and muscle biopsy. Whole-genome or whole-exome sequencing was performed on the index patients from two unrelated families with PCL. The effects of the mutations were examined through complementation of the NDUFV2 mutation by cDNA expression.Results: The common clinical features of the patients in this study were recurring episodes of acute or subacute developmental regression that appeared in the first years of life, followed by gradual remissions and prolonged periods of stability. MRI showed leukoencephalopathy with multiple cavities. Three novel NDUFV2 missense mutations were identified in these families. Complex I deficiency was confirmed in affected individuals’ fibroblasts and a muscle biopsy. Functional and structural analyses revealed that these mutations affect the structural stability and function of the NDUFV2 protein, indicating that defective NDUFV2 function is responsible for the phenotypes in these individuals.Conclusions: Here, we report the clinical presentations, neuroimaging and molecular and functional analyses of novel mutations in NDUFV2 in two sibling pairs of two Chinese families presenting with PCL. We hereby expand the knowledge on the clinical phenotypes associated with mutations in NDUFV2 and the genotypes causative for PCL.

Shiga toxin (Stx)-producing Escherichia coli (STEC) causes foodborne outbreaks of bloody diarrhea. There are two major types of immunologically distinct Stxs: Stx1a and Stx2a. Stx1a is more cytotoxic to Vero cells than Stx2a, but Stx2a has a lower 50% lethal dose (LD₅₀) in mice. Epidemiological data suggest that infections by STEC strains that produce only Stx2a progress more often to a life-threatening sequela of infection called hemolytic-uremic syndrome (HUS) than isolates that make Stx1a only or produce both Stx1a and Stx2a. In this study, we found that an E. coli O26:H11 strain that produces both Stx1a and Stx2a was virulent in streptomycin- and ciprofloxacin-treated mice and that mice were protected by administration of an anti-Stx2 antibody. However, we discovered that in the absence of ciprofloxacin, neutralization of Stx1a enhanced the virulence of the strain, a result that corroborated our previous finding that Stx1a reduces the toxicity of Stx2a by the oral route. We further found that intraperitoneal administration of the purified Stx1a B subunit delayed the mean time to death of mice intoxicated with Stx2a and reduced the cytotoxic effect of Stx2a on Vero cells. Taken together, our data suggest that Stx1a reduces both the pathogenicity of Stx2 in vivo and cytotoxicity in vitro.