Heterozygous Variants in KCNJ10 Cause Paroxysmal Kinesigenic Dyskinesia Via Haploinsufficiency.

OBJECTIVE: Most paroxysmal kinesigenic dyskinesia (PKD) cases are hereditary, yet approximately 60% of patients remain genetically undiagnosed. We undertook the present study to uncover the genetic basis for undiagnosed PKD patients. METHODS: Whole-exome sequencing was performed for 106 PRRT2-negative PKD probands. The functional impact of the genetic variants was investigated in HEK293T cells and Drosophila. RESULTS: Heterozygous variants in KCNJ10 were identified in 11 individuals from 8 unrelated families, which accounted for 7.5% (8/106) of the PRRT2-negative probands. Both co-segregation of the identified variants and the significantly higher frequency of rare KCNJ10 variants in PKD cases supported impacts from the detected KCNJ10 heterozygous variants on PKD pathogenesis. Moreover, a KCNJ10 mutation-carrying father from a typical EAST/SeSAME family was identified as a PKD patient. All patients manifested dystonia attacks triggered by sudden movement with a short episodic duration. Patch-clamp recordings in HEK293T cells revealed apparent reductions in K+ currents of the patient-derived variants, indicating a loss-of-function. In Drosophila, milder hyperexcitability phenotypes were observed in heterozygous Irk2 knock-in flies compared to homozygotes, supporting haploinsufficiency as the mechanism for the detected heterozygous variants. Electrophysiological recordings showed that excitatory neurons in Irk2 haploinsufficiency flies exhibited increased excitability, and glia-specific complementation with human Kir4.1 rescued the Irk2 mutant phenotypes. INTERPRETATION: Our study established haploinsufficiency resulting from heterozygous variants in KCNJ10 can be understood as a previously unrecognized genetic cause for PKD and provided evidence of glial involvement in the pathophysiology of PKD. ANN NEUROL 2024.

Prevalence and genetic characterization of Campylobacter from clinical poultry cases in China.

IMPORTANCE: Campylobacter is a major cause of campylobacteriosis worldwide, and poultry is the main reservoir for its transmission. Campylobacter was generally considered to be a harmless commensal organism in poultry without pathogenic properties. However, it was proposed that a Campylobacter-like organism may be the cause of vibrionic hepatitis, which poses a significant public health risk. The occurrence and epidemiology of Campylobacter in healthy poultry have been studied systematically, but little is known about the epidemiology of Campylobacter isolates from diseased poultry in China. Therefore, this study determined the prevalence and molecular characterization of Campylobacter from diseased chickens, ducks, and geese in Yangzhou Veterinary Hospital between December 2016 and September 2017, which was critical for improving the diagnosis and prevention of Campylobacter infections.

Transcriptomic profiling of lipopolysaccharide-challenged bovine mammary epithelial cells treated with forsythoside A.

Mastitis is usually caused by a variety of pathogenic bacteria that seriously impact the health and milk-production ability of dairy cows, with consequent, economically detrimental effects on the dairy industry. Forsythoside A (FTA), isolated from the fruit and leaves of Forsythia suspensa (Thunb.) Vahl (Oleaceae), has been reported to have significant antioxidant, anti-inflammatory, and antibacterial effects. However, it is not clear whether FTA exerts a protective effect against lipopolysaccharide (LPS)-induced bovine mastitis and its potential gene signature. In this study, high-throughput sequencing technology was performed to analyze the differences between the mRNA and enrichment pathway of bovine mammary epithelial cells of the control, LPS, and LPS + FTA groups. The results showed that there were 139 differentially expressed genes (DEGs) (p-value < 0.05, |log2FoldChange| > 1, FPKM > 1) in the LPS group compared with the control group, including 121 up-regulated genes and 18 down-regulated genes, which were mainly enriched in the cellular response to lipopolysaccharide, cytokine activity, protein binding, and IL-17 signaling pathway based on Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, respectively. Compared with the control group and LPS + FTA group, there were 349 DEGs, including 322 up-regulated genes and 27 down-regulated genes. They were mainly enriched in protein localization to organelles, centrosomes, binding, and the IL-17 signaling pathway, based on GO and KEGG analysis. Compared to the LPS group, the LPS + FTA group had 272 DEGs, including 259 up-regulated genes and 13 down-regulated genes, which were mainly enriched in RNA processing, IL-6 receptor binding, and the lysosome pathway, based on GO and KEGG analyses. It can be seen that LPS stimulation induced the expression of inflammation-related genes, IL-17 and IL-6, whereas FTA treatment promoted the expression of the spliceosome-, lysosome-, and oxidative stress-related genes HSP70, HSPA8, and PARP2. The study utilized RNA-sequencing analysis of FTA against LPS-challenged bovine mammary epithelial cells to explore key mRNA findings that may be strongly associated with inflammation and oxidative stress, and provides a theoretical reference for further elucidation of molecular mechanisms of bovine mastitis and therapeutic effects of FTA against bovine mastitis.

Loss of function of CMPK2 causes mitochondria deficiency and brain calcification.

Brain calcification is a critical aging-associated pathology and can cause multifaceted neurological symptoms. Cerebral phosphate homeostasis dysregulation, blood-brain barrier defects, and immune dysregulation have been implicated as major pathological processes in familial brain calcification (FBC). Here, we analyzed two brain calcification families and identified calcification co-segregated biallelic variants in the CMPK2 gene that disrupt mitochondrial functions. Transcriptome analysis of peripheral blood mononuclear cells (PBMCs) isolated from these patients showed impaired mitochondria-associated metabolism pathways. In situ hybridization and single-cell RNA sequencing revealed robust Cmpk2 expression in neurons and vascular endothelial cells (vECs), two cell types with high energy expenditure in the brain. The neurons in Cmpk2-knockout (KO) mice have fewer mitochondrial DNA copies, down-regulated mitochondrial proteins, reduced ATP production, and elevated intracellular inorganic phosphate (Pi) level, recapitulating the mitochondrial dysfunction observed in the PBMCs isolated from the FBC patients. Morphologically, the cristae architecture of the Cmpk2-KO murine neurons was also impaired. Notably, calcification developed in a progressive manner in the homozygous Cmpk2-KO mice thalamus region as well as in the Cmpk2-knock-in mice bearing the patient mutation, thus phenocopying the calcification pathology observed in the patients. Together, our study identifies biallelic variants of CMPK2 as novel genetic factors for FBC; and demonstrates how CMPK2 deficiency alters mitochondrial structures and functions, thereby highlighting the mitochondria dysregulation as a critical pathogenic mechanism underlying brain calcification.

Cloning, expression, and characterization of a glycosaminoglycan lyase from Vibrio sp. H240.

Glycosaminoglycan lyase is an effective tool for the functional studies of glycosaminoglycans and for the preparation of oligosaccharides. In this study, a new glycosaminoglycan lyase HCLaseV with a molecular weight of 90 kDa was cloned, expressed, and characterized from Vibrio sp. H240. The lyase belonged to the polysaccharide lyase (PL)- 8 family. HCLaseV showed enzyme activities toward chondroitin sulfate A, chondroitin sulfate B, chondroitin sulfate C, and hyaluronic acid. HCLaseV exhibited the highest activity against HA at pH 7.0 and 40 °C. HCLaseV was an endo-type enzyme whose degradation end-product was unsaturated disaccharides. Ca2+ inhibited the activity of HCLaseV to a certain extent, which was different from most of the enzymes in the PL-8 family. Mutagenesis studies showed that the Ca2+ inhibition might be related to the Asn244 residue. The sequence homology was evaluated by mutagenesis studies, and the catalytic residues in HCLaseV were presumed to be His278, Trp485, and Tyr287. These characteristics are helpful for further basic research and application.

Mutation Analysis of MYORG in a Chinese Cohort With Primary Familial Brain Calcification.

Primary familial brain calcification (PFBC) is a progressive neurological disorder manifesting as bilateral brain calcifications in CT scan with symptoms as parkinsonism, dystonia, ataxia, psychiatric symptoms, etc. Recently, pathogenic variants in MYORG have been linked to autosomal recessive PFBC. This study aims to elucidate the mutational and clinical spectrum of MYORG mutations in a large cohort of Chinese PFBC patients with possible autosomal recessive or absent family history. Mutational analyses of MYORG were performed by Sanger sequencing in a cohort of 245 PFBC patients including 21 subjects from 10 families compatible with a possibly autosomal-recessive trait and 224 apparently sporadic cases. In-depth phenotyping and neuroimaging features were investigated in all patients with novel MYORG variants. Two nonsense variants (c.442C > T, p. Q148*; c.972C > A, p. Y324*) and two missense variants (c.1969G>C, p. G657R; c.2033C > G, p. P678R) of MYORG were identified in four sporadic PFBC patients, respectively. These four novel variants were absent in gnomAD, and their amino acid were highly conserved, suggesting these variants have a pathogenic impact. Patients with MYORG variants tend to display a homogeneous clinical spectrum, showing extensive brain calcification and parkinsonism, dysarthria, ataxia, or vertigo. Our findings supported the pathogenic role of MYORG variants in PFBC and identified two pathogenic variants (c.442C > T, c.972C > A), one likely pathogenic variant (c.2033C > G), and one variant of uncertain significance (c.1969G>C), further expanding the genetic and phenotypic spectrum of PFBC-MYORG.

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.

Identification of SLC20A2 deletions in patients with primary familial brain calcification.

Primary familial brain calcification (PFBC) is a rare neurological disorder. Mutations in five genes (SLC20A2, PDGFRB, PDGFB, XPR1, and MYORG) have been linked to PFBC. Here, we used SYBR green-based real-time quantitative polymerase chain reaction (PCR) assay and denaturing high-performance liquid chromatography analysis to detect copy number variants (CNVs) in 20 unrelated patients with PFBC, negatively sequenced for the five known genes. We identified three deletions in SLC20A2, including a large de novo full gene deletion and two exonic deletions confined to exon 2 and exon 6, respectively. Subsequent linked-read whole-genome sequencing of the patient with the large deletion showed a 1.7 Mb heterozygous deletion which removed the entire coding regions of SLC20A2 as well as 21 other genes. In the family with a deletion of exon 6, a missense variant of uncertain significance (SLC20A2: p.E267Q) also co-segregated with the disease. Functional assay showed the deletion could result in significantly impaired phosphate transport, whereas the p.E267Q variant did not. Our results confirm that deletion in SLC20A2 is a causal mechanism for PFBC and highlight the importance of functional study for classifying a rare missense variant as (likely) pathogenic.

c.835-5T>G Variant in SMN1 Gene Causes Transcript Exclusion of Exon 7 and Spinal Muscular Atrophy.

Spinal muscular atrophy (SMA) is an autosomal recessive genetic disorder caused by survival motor neuron (SMN) protein deficiency leading the loss of motor neurons in the anterior horns of the spinal cord and brainstem. More than 95% of SMA patients are attributed to the homozygous deletion of survival motor neuron 1 (SMN1) gene, and approximately 5% are caused by compound heterozygous with a SMN1 deletion and a subtle mutation. Here, we identified a rare variant c.835-5T>G in intron 6 of SMN1 in a patient affected with type I SMA. We analyzed the functional consequences of this mutation on mRNA splicing in vitro. After transfecting pCI-SMN1, pCI-SMN2, and pCI-SMN1 c.835-5T>G minigenes into HEK293, Neuro-2a, and SHSY5Y cells, reverse transcription polymerase chain reaction (RT-PCR) was performed to compare the splicing effects of these minigenes. Finally, we found that this mutation resulted in the skipping of exon 7 in SMN1, which confirmed the genetic diagnosis of SMA.

Theoretical Study on the Effects of Hydrogen-Bonding and Molecule-Cation Interactions on the Sensitivity of HMX.

To assess the effects of weak interactions on the sensitivity of HMX, 11 complexes of HMX (where six of them are hydrogen-bonding complexes, and the other five are molecular-cation complexes) have been studied via quantum chemical treatment. The geometric and electronic structures were determined using DFT-B3LYP and MP2(full) methods with the 6-311++G(2df, 2p) and aug-cc-pVTZ basis sets. The changes of the bond dissociation energy (BDE) of the trigger bond (N-NO2 in HMX) and nitro group charge have been computed on the detailed consideration to access the sensitivity changes of HMX. The results indicate that upon complex forming, the BDE increases and the charge of nitro group turns more negative in complexes, suggesting that the strength of the N-NO2 trigger bond is enhanced and then the sensitivity of HMX is reduced. Atom-in-molecules analyses have also been carried out to understand the nature of intermolecular interactions and the strength of trigger bonds.

VOCs elimination and health risk reduction in e-waste dismantling workshop using integrated techniques of electrostatic precipitation with advanced oxidation technologies.

Volatile organic compounds (VOCs) emitted during the electronic waste dismantling process (EWDP) were treated at a pilot scale, using integrated electrostatic precipitation (EP)-advanced oxidation technologies (AOTs, subsequent photocatalysis (PC) and ozonation). Although no obvious alteration was seen in VOC concentration and composition, EP technology removed 47.2% of total suspended particles, greatly reducing the negative effect of particles on subsequent AOTs. After the AOT treatment, average removal efficiencies of 95.7%, 95.4%, 87.4%, and 97.5% were achieved for aromatic hydrocarbons, aliphatic hydrocarbons, halogenated hydrocarbons, as well as nitrogen- and oxygen-containing compounds, respectively, over 60-day treatment period. Furthermore, high elimination capacities were also seen using hybrid technique of PC with ozonation; this was due to the PC unit's high loading rates and excellent pre-treatment abilities, and the ozonation unit's high elimination capacity. In addition, the non-cancer and cancer risks, as well as the occupational exposure cancer risk, for workers exposed to emitted VOCs in workshop were reduced dramatically after the integrated technique treatment. Results demonstrated that the integrated technique led to highly efficient and stable VOC removal from EWDP emissions at a pilot scale. This study points to an efficient approach for atmospheric purification and improving human health in e-waste recycling regions.