Novel TUBA4A variant causes congenital myopathy with focal myofibrillar disorganisation.

BACKGROUND: Congenital myopathies are a clinical, histopathological and genetic heterogeneous group of inherited muscle disorders that are defined on peculiar architectural abnormalities in the muscle fibres. Although there have been at least 33 different genetic causes of the disease, a significant percentage of congenital myopathies remain genetically unresolved. The present study aimed to report a novel TUBA4A variant in two unrelated Chinese patients with sporadic congenital myopathy. METHODS: A comprehensive strategy combining laser capture microdissection, proteomics and whole-exome sequencing was performed to identify the candidate genes. In addition, the available clinical data, myopathological changes, the findings of electrophysiological examinations and thigh muscle MRIs were also reviewed. A cellular model was established to assess the pathogenicity of the TUBA4A variant. RESULTS: We identified a recurrent novel heterozygous de novo c.679C>T (p.L227F) variant in the TUBA4A (NM_006000), encoding tubulin alpha-4A, in two unrelated patients with clinicopathologically diagnosed sporadic congenital myopathy. The prominent myopathological changes in both patients were muscle fibres with focal myofibrillar disorganisation and rimmed vacuoles. Immunofluorescence showed ubiquitin-positive TUBA4A protein aggregates in the muscle fibres with rimmed vacuoles. Overexpression of the L227F mutant TUBA4A resulted in cytoplasmic aggregates which colocalised with ubiquitin in cellular model. CONCLUSION: Our findings expanded the phenotypic and genetic manifestations of TUBA4A as well as tubulinopathies, and added a new type of congenital myopathy to be taken into consideration in the differential diagnosis.

Diagnostic Value of Salivary Real-Time Quaking-Induced Conversion in Parkinson's Disease and Multiple System Atrophy.

BACKGROUND: Aggregation of α-synuclein (oligomeric α-syn) has been considered as the pathological hallmark of Parkinson's disease (PD) and multiple system atrophy (MSA). Studies showed oligomeric α-syn/total α-syn ratio was increased in the saliva of patients with PD, suggesting that seeding activity of salivary oligomeric α-syn may be a novel biomarker for the diagnosis of PD and MSA. OBJECTIVE: This study aimed to evaluate the diagnostic value of salivary α-syn seeding activity in patients with PD and MSA. METHODS: A total of 75 patients with PD, 18 patients with MSA, and 36 nonneurodegenerative healthy control subjects underwent salivary α-syn real-time quaking-induced conversion (RT-QuIC) assay. RESULTS: Salivary α-syn RT-QuIC assay distinguished patients with PD with 76.0% sensitivity (95% confidence interval [CI], 66.1-85.9) and 94.4% specificity (95% CI, 86.6-100.0). RT-QuIC assay sensitivity reached 61.1% (95% CI, 36.2-86.1) in patients with MSA. No significant differences were observed in the diameter of salivary α-syn fibrils examined by electron microscopy and in thioflavin T fluorescence intensity of salivary α-syn fibrils detected by RT-QuIC assay between patients with PD and MSA. Notably, the lag phase of RT-QuIC assay from patients with PD was significantly shorter than that of patients with MSA, which might be clinically applicable to the discrimination between PD and MSA. CONCLUSIONS: Salivary α-syn seeding activity may serve as a novel biomarker for the clinical diagnosis of PD and MSA.© 2022 International Parkinson and Movement Disorder Society © 2022 International Parkinson and Movement Disorder Society.

Comparison of the second consensus statement with the movement disorder society criteria for multiple system atrophy: A single-center analysis.

INTRODUCTION: This study aimed at comparing the differences between the second consensus statement and Movement Disorder Society (MDS) criteria for Multiple System Atrophy (MSA) in a single Chinese cohort. METHODS: We retrospectively reviewed 73 patients with MSA over the past five years. They were categorized as patients with probable and possible MSA according to the second consensus statement in addition to clinically established and clinically probable MSA according to the MDS criteria. The core clinical, supportive clinical, and imaging features were analyzed and compared between the two MSA subtypes. RESULTS: A total of 40 patients with MSA-P and 33 patients with MSA-C were included in this study. Approximately 78.7% of the category of probable patients in the second consensus statement can be categorized as clinically established MSA in the MDS criteria and five patients with non-supporting features in the second consensus statement criteria can be diagnosed as clinically probable MSA in the MDS criteria. "Rapid progression" and "moderate to severe postural instability" within three years of motor onset dominated among the supportive features. Approximately 78.9% of patients possessed at least one imaging marker with predominant signal decrease of putamen on iron-sensitive sequences (38.0% of patients). Twenty-two patients could not be diagnosed as clinically established MSA mainly due to the lack of supportive or imaging features. CONCLUSIONS: A high degree of agreement was noticed between the two criteria sets. The supportive and imaging features played important role in the diagnosis of MSA and affected the diagnostic level in the current criteria.

Ribosomal binding site sequences and promoters for expressing glutamate decarboxylase and producing γ-aminobutyrate in Corynebacterium glutamicum.

Glutamate decarboxylase (GAD) converts L-glutamate (Glu) into γ-aminobutyric acid (GABA). Corynebacterium glutamicum that expresses exogenous GAD gene, gadB2 or gadB1, can synthesize GABA from its own produced Glu. To enhance GABA production in C. glutamicum, ribosomal binding site (RBS) sequence and promoter were searched and optimized for increasing the expression efficiency of gadB2. R4 exhibited the highest strength among RBS sequences tested, with 6 nt the optimal aligned spacing (AS) between RBS and start codon. This combination of RBS sequence and AS contributed to gadB2 expression, increased GAD activity by 156% and GABA production by 82% compared to normal strong RBS and AS combination. Then, a series of native promoters were selected for transcribing gadB2 under optimal RBS and AS combination. P dnaK , P dtsR , P odhI and P clgR expressed gadB2 and produced GABA as effectively as widely applied P tuf and P cspB promoters and more effectively than P sod promoter. However, each native promoter did not work as well as the synthetic strong promoter P tacM , which produced 20.2 ± 0.3 g/L GABA. Even with prolonged length and bicistronic architecture, the strength of P dnaK did not enhance. Finally, gadB2 and mutant gadB1 were co-expressed under the optimal promoter and RBS combination, thus converted Glu into GABA completely and improved GABA production to more than 25 g/L. This study provides useful promoters and RBS sequences for gene expression in C. glutamicum.