Clinical and genetic interpretation of uncertain DMD missense variants: evidence from mRNA and protein studies.

BACKGROUND: Pathogenic missense variants in the dystrophin (DMD) gene are rarely reported in dystrophinopathies. Most DMD missense variants are of uncertain significance and their pathogenicity interpretation remains complicated. We aimed to investigate whether DMD missense variants would cause aberrant splicing and re-interpret their pathogenicity based on mRNA and protein studies. METHODS: Nine unrelated patients who had an elevated serum creatine kinase level with or without muscle weakness were enrolled. They underwent a detailed clinical, imaging, and pathological assessment. Routine genetic testing and muscle-derived mRNA and protein studies of dystrophin and sarcoglycan genes were performed in them. RESULTS: Three of the 9 patients presented with a Duchenne muscular dystrophy (DMD) phenotype and the remaining 6 patients had a suspected diagnosis of Becker muscular dystrophy (BMD) or sarcoglycanopathy based on their clinical and pathological characteristics. Routine genetic testing detected only 9 predicted DMD missense variants in them, of which 6 were novel and interpreted as uncertain significance. Muscle-derived mRNA studies of sarcoglycan genes didn't reveal any aberrant transcripts in them. Dystrophin mRNA studies confirmed that 3 predicted DMD missense variants (c.2380G > C, c.4977C > G, and c.5444A > G) were in fact splicing and frameshift variants due to aberrant splicing. The 9 DMD variants were re-interpreted as pathogenic or likely pathogenic based on mRNA and protein studies. Therefore, 3 patients with DMD splicing variants and 6 patients with confirmed DMD missense variants were diagnosed with DMD and BMD, respectively. CONCLUSION: Our study highlights the importance of muscle biopsy and aberrant splicing for clinical and genetic interpretation of uncertain DMD missense variants.

Associations of sleep-related variables with reverse dipping patterns of blood pressure in α-synucleinopathies.

INTRODUCTION: The reverse dipping blood pressure (BP) pattern is very common in α-synucleinopathies. We aimed to explore the associations of sleep-related variables with abnormal BP circadian rhythms in Parkinson's disease (PD) and multiple system atrophy (MSA). METHODS: A total of 126 patients, 76 with PD and 50 with MSA, were included. All participants underwent ambulatory BP monitoring and full-night polysomnography (PSG). We analyzed abnormal dipping patterns and sleep-related parameters, including moderate to severe obstructive sleep apnea (OSA), rapid eye movement behavior disorder (RBD), average oxygen saturation (SaO2%), lowest SaO2%, duration of SaO2% <90%, and apnea-hypopnea index (AHI). Binary logistic regression was performed to explore the associations between paraclinical variables, sleep-related variables, and reverse dipping patterns. RESULTS: Reverse dipping patterns were predominant in patients with PD (58.5 %) and MSA (68.0 %). Patients with MSA had higher AHI, RBD, and lower average SaO2% than those with PD. Taking both diseases together as a whole group of α-synucleinopathies, logistic regression analysis indicates the Hoehn-Yahr stage (odds ratio [OR] = 2.00 for reverse systolic and 2.34 for reverse diastolic dipping patterns), moderate to severe OSA (OR = 2.71 for reverse systolic and 2.53 for reverse diastolic dipping patterns), average SaO2% (OR = 1.35 for reverse systolic dipping patterns), and male sex (OR = 2.70 for reverse diastolic dipping patterns) were independently associated with reverse dipping patterns. CONCLUSIONS: Reverse dipping patterns were common in patients with PD and MSA. Hoehn-Yahr stage, moderate to severe OSA, average SaO2%, and male sex were associated with reverse dipping patterns in α-synucleinopathy.

Abnormal Ocular Movement in the Early Stage of Multiple-System Atrophy With Predominant Parkinsonism Distinct From Parkinson's Disease.

BACKGROUND AND PURPOSE: The eye-movement examination can be applied as a noninvasive method to identify multiple-system atrophy (MSA). Few studies have investigated eye movements during the early stage of MSA with predominant parkinsonism (MSA-P). We aimed to determine the characteristic oculomotor changes in the early stage of MSA-P. METHODS: We retrospectively selected 17 patients with MSA-P and 40 with Parkinson's disease (PD) with disease durations of less than 2 years, and 40 age-matched healthy controls (HCs). Oculomotor performance in the horizontal direction was measured in detail using videonystagmography. RESULTS: We found that the proportions of patients with MSA-P and PD exhibiting abnormal eye movements were 82.4% and 77.5%, respectively, which were significantly higher than that in the HCs (47.5%, p<0.05). Compared with HCs, patients with MSA-P presented significantly higher abnormal proportions of fixation and gaze-holding (17.6% vs. 0%), without-fixation (47.1% vs. 0%), prolonged latency in reflexive saccades (29.4% vs. 5.0%), memory-guided saccades (93.3% vs. 10.0%), and catch-up saccades in smooth-pursuit movement (SPM, 41.2% vs. 0) (all p<0.05). Compared with those with PD, patients with MSA-P presented a significantly higher proportion of catch-up saccades in SPM (41.2% vs. 2.5%, p<0.001). CONCLUSIONS: MSA-P presented the characteristic of catch-up saccades in SPM in the early stage, which may provide some value in differentiating MSA-P from PD.

A first-in-human study of Brozopentyl Sodium, following single and multiple ascending intravenous infusion in Chinese healthy volunteers.

BACKGROUND: Brozopentyl Sodium (BZP), a novel agent for ischemic stroke, has shown promising results in preclinical pharmacological studies, prompting the initiation of the first-in-human investigation. PURPOSE: This study aimed to assess the safety, tolerability, and pharmacokinetic (PK) characteristics of BZP in Chinese healthy volunteers. METHODS: The study consisted of two parts. Part I was a single-center, randomized, single-blinded, placebo-controlled, single-ascending study with six BZP dose cohorts (SAD: 25, 50, 100, 200, 300, and 400 mg). Part II was a single-center, randomized, single-blinded, placebo-controlled, multi-dose- and dose-elevated study with three BZP dose cohorts (MAD: 50, 100, and 200 mg). Doses were administered once daily on days 1 and 7 and twice daily on days 2-6. The PK properties of BZP and its bioactive metabolites, BNBP, were assessed. Safety and tolerability evaluations were also conducted. RESULTS: In the SAD study, BZP reached peak plasma concentrations (Tmax) at the end of administration, with median Tmax values ranging from 1 to 1.03 h, while BNBP reached Tmax between 1.25 to 1.38 h. The terminal half-lives (T1/2) were approximately 8 h for BZP and 15 h for BNBP. In the MAD study, steady-state plasma concentrations of BZP were reached by day 5. There was minimal accumulation of both BZP and BNBP after 7 days of administration. The area under the plasma concentration-time curve from 0 to time of the last measurable concentration (AUC0-t) and maximum plasma drug concentration (Cmax) showed dose-proportional increases for BZP but not for BNBP in both study parts. Single and multiple doses of BZP demonstrated a good safety profile and were well-tolerated. CONCLUSION: BZP displayed safety, good tolerability and predictable PK characteristics following both single and multiple ascending intravenous administrations. These findings provide a basis for further clinical development of BZP for ischemic stroke patients.

Clinicopathological and circulating cell-free DNA profile in myositis associated with anti-mitochondrial antibody.

OBJECTIVE: Anti-mitochondrial antibodies (AMAs) are associated with idiopathic inflammatory myopathies (IIMs). We aimed to summarize the clinicopathological characteristics, assess circulating cell-free mitochondrial DNA (ccf-mtDNA), and circulating cell-free nuclear DNA (ccf-nDNA) in AMA-associated IIMs. METHODS: Medical records of 37 IIMs patients with AMAs were reviewed. Circulating cell-free mtDNA and ccf-nDNA levels in sera from IIMs patients with AMAs (n = 21), disease controls (n = 66) and healthy controls (HCs) (n = 23) were measured and compared. Twenty-eight immune-mediated necrotizing myopathy (IMNM) patients, 23 dermatomyositis (DM) patients, and 15 anti-synthetase syndrome (ASS) patients were enrolled as disease controls. Correlations between variables were analyzed. RESULTS: Limb weakness was observed in 75.7% and neck weakness in 56.8% of patients. Cardiac involvement occurred in 51.4% of patients. Muscle pathology revealed 81.1% of IMNM, 5.4% polymyositis, and 13.5% nonspecific myositis. Microinfarction was observed in 8.1% of patients. Serum ccf-mtDNA levels in AMA-associated IIMs were significantly higher than those in HCs (p < 0.001), but no significant differences between AMA-associated IIMs and IMNM, DM, or ASS. Serum ccf-nDNA levels in AMA-associated IIMs were significantly higher than those in HCs (p = 0.02), and significantly lower than those in DM (p = 0.02). Serum ccf-nDNA levels correlated negatively with MMT8 total scores (rs = -0.458, p = 0.037) and positively with mRS scores (rs = 0.486, p = 0.025). Serum ccf-nDNA levels were significantly higher in the non-remission group (p < 0.01). INTERPRETATION: AMA-associated IIMs exhibit distinct clinicopathological features. Serum ccf-nDNA may serve as a potential marker for disease severity and prognosis in AMA-associated IIMs.

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.

Clinical, muscle imaging, and genetic characteristics of dystrophinopathies with deep-intronic DMD variants.

BACKGROUND: Phenotypic heterogeneity within or between families with a same deep-intronic splice-altering variant in the DMD gene has never been systematically analyzed. This study aimed to determine the phenotypic and genetic characteristics of patients with deep-intronic DMD variants. METHODS: Of 1338 male patients with a suspected dystrophinopathy, 38 were confirmed to have atypical pathogenic DMD variants via our comprehensive genetic testing approach. Of the 38 patients, 30 patients from 22 unrelated families with deep-intronic DMD variants underwent a detailed clinical and imaging assessment. RESULTS: Nineteen different deep-intronic DMD variants were identified in the 30 patients, including 15 with Duchenne muscular dystrophy (DMD), 14 with Becker muscular dystrophy (BMD), and one with X-linked dilated cardiomyopathy. Of the 19 variants, 15 were single-nucleotide variants, 2 were structural variants (SVs), and 2 were pure-intronic large-scale SVs causing aberrant inclusion of other protein-coding genes sequences into the mature DMD transcripts. The trefoil with single fruit sign was observed in 18 patients and the concentric fatty infiltration pattern was observed in 2 patients. Remarkable phenotypic heterogeneity was observed not only in skeletal but also cardiac muscle involvement in 2 families harboring a same deep-intronic variant. Different skeletal muscle involvement between families with a same variant was observed in 4 families. High inter-individual phenotypic heterogeneity was observed within two BMD families and one DMD family. CONCLUSIONS: Our study first highlights the variable phenotypic expressivity of deep-intronic DMD variants and demonstrates a new class of deep-intronic DMD variants, i.e., pure-intronic SVs involving other protein-coding genes.

Quantitative assessment of oculomotor function by videonystagmography in multiple system atrophy.

OBJECTIVE: To quantitatively assess oculomotor impairments in multiple system atrophy (MSA) and to explore their correlation with clinical characteristics. METHODS: We recruited 45 patients with MSA, including 21 with dominant ataxia (MSA-C), 24 with dominant parkinsonism (MSA-P), and 40 age-matched healthy controls. Detailed oculomotor performance in the horizontal direction was measured using videonystagmography (VNG). RESULTS: We found that the proportion of abnormal eye movements in patients with MSA was 93.3% (37.7%, 51.1%, 73.3%, 71.1%, and 37.8% on fixation and gaze-holding, without fixation, saccade, smooth pursuit, and optokinetic nystagmus tests, respectively). Patients with MSA-C showed significantly lower gains in smooth pursuit test and optokinetic nystagmus test, and a higher incidence of hypermetria in the saccade test than patients with MSA-P (all P < 0.05). No oculomotor deficits were correlated with age, age of onset, sex, disease duration, or Unified Multiple System Atrophy Rating Scale (USMARS) (all r < 0.25, P > 0.1). CONCLUSIONS: An extremely high incidence of oculomotor impairments could be observed using VNG in both the MSA-C and MSA-P subtypes, although there were some differences between them. SIGNIFICANCE: A comprehensive oculomotor examination could serve as a valuable tool in the diagnostic workup of patients with MSA.

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.

Profiling of Differentially Expressed MicroRNAs in Saliva of Parkinson's Disease Patients.

Objective: This study aims to identify differentially expressed salivary miRNAs and validate the diagnostic potential for idiopathic Parkinson's disease (PD). Also, the disease specificity of candidate miRNAs was evaluated between PD, multiple system atrophy (MSA), and essential tremor (ET). Methods: We collected salivary samples from 50 PD, 20 ET, and 20 MSA patients, as well as 30 healthy controls (HCs). In the discovery phase, salivary miRNA microarray analysis was performed. In-silico analysis was used to investigate the target genes of differentially expressed miRNAs and clustered pathways. In validation phase, RT-qPCR was performed with samples from 30 PD patients and 30 HCs. Subsequently, we investigated candidate miRNAs in all recruited subjects. Receiver operating characteristic curve and Spearman correlation analysis was performed to determine diagnostic usefulness. Results: We identified 43 miRNAs that were differentially expressed between 5 PD patients and 5 HCs by miRNA microarray analysis. Computational analysis revealed the target genes were clustered in the pathways associated with ubiquitin protein ligase activity. The result of RT-qPCR showed that the miR-29a-3p and miR-29c-3p were found to be significantly downregulated (p = 0.004, p = 0.027), whereas the miR-6756-5p was significantly upregulated in 30 PD patients compared with 30 HCs (p = 0.032). The miR-29a-3p expression level in PD patients was significantly lower than ET patients (p = 0.035), but higher than MSA patients (p < 0.0001). The diagnostic efficacy reached a little higher when the combination of miR-29a-3p and miR-29c-3p. Conclusion: The miRNA combination of salivary miR-29a-3p and miR-29c-3p has potential to be a diagnostic biomarker for idiopathic PD.

NREM Sleep EEG Characteristics Correlate to the Mild Cognitive Impairment in Patients with Parkinsonism.

Early identification and diagnosis of mild cognitive impairment (MCI) in patients with parkinsonism (PDS) are critical. The aim of this study was to identify biomarkers of MCI in PDS using conventional electroencephalogram (EEG) power spectral analysis and detrended fluctuation analysis (DFA). In this retrospective study, patients with PDS who underwent an overnight polysomnography (PSG) study in our hospital from 2019 to 2020 were enrolled. Patients with PDS assessed by clinical examination and questionnaires were divided into two groups: the PDS with normal cognitive function (PDS-NC) group and the PDS with MCI (PDS-MCI) group. Sleep EEG signals were extracted and purified from the PSG and subjected to a conventional power spectral analysis, as well as detrended fluctuation analysis (DFA) during wakefulness, nonrapid eye movement (NREM) sleep, and rapid eye movement (REM) sleep. Forty patients with PDS were enrolled, including 25 with PDS-NC and 15 with PDS-MCI. Results revealed that compared with PDS-NC patients, patients with PDS-MCI had a reduced fast ratio ((alpha + beta)/(delta + theta)) and increased DFA during NREM sleep. DFA during NREM was diagnostic of PDS-MCI, with an area under the receiver operating characteristic curve of 0.753 (95% CI: 0.592-0.914) (p < 0.05). Mild cognitive dysfunction was positively correlated with NREM-DFA (r = 0.426, p = 0.007) and negatively correlated with an NREM-fast ratio (r = -0.524, p = 0.001). This suggested that altered EEG activity during NREM sleep is associated with MCI in patients with PDS. NREM sleep EEG characteristics of the power spectral analysis and DFA correlate to MCI. Slowing of EEG activity during NREM sleep may reflect contribution to the decline in NREM physiological function and is therefore a marker in patients with PDS-MCI.

Parkin is the most common causative gene in a cohort of mainland Chinese patients with sporadic early-onset Parkinson's disease.

INTRODUCTION: Genetic mutations associated with early-onset Parkinson's disease (EOPD) vary widely among different ethnicities. We detected the genes associated with EOPD in a Chinese cohort using next-generation sequencing (NGS) combined with multiplex ligation-dependent probe amplification (MLPA) and analyzed the phenotypic characteristics of the mutation carriers. METHODS: Cohort of 23 sporadic EOPD patients (onset age ≤ 45 years) were recruited. Genetic causes were identified by a targeted NGS panel containing 136 known extrapyramidal disease-causative genes. Multiplications or deletions of PD-causing genes were detected using the MLPA method. Demographic and clinical data were obtained, analyzed, and compared between patients with and those without Parkin gene variants. RESULTS: We identified 14 pathogenic or likely pathogenic variants (12 in Parkin, 1 in LRRK2, and 1 in VPS13C) in 10 patients (43.5%) and 8 rare variants of uncertain significance in 9 patients (39.1%). Parkin (34.8%) was the most common causative gene among our patients cohort, and exon deletion (62.5%) was the main type of variant. Patients with Parkin mutations had a younger age of onset, longer delay in diagnosis, slower disease progression, higher frequency of hyperreflexia, fatigue, and less hyposmia compared to patients without Parkin mutations. CONCLUSION: Our results revealed a higher prevalence of Parkin mutations in Chinese sporadic EOPD patients, and notably, exon deletion was the most common type of mutation. EOPD patients with Parkin mutations showed unique clinical characteristics.