The CGG repeat expansion in RILPL1 is associated with oculopharyngodistal myopathy type 4.

Recent studies indicate that CGG repeat expansions in LRP12, GIPC1, and NOTCH2NLC are associated with oculopharyngodistal myopathy (OPDM) types 1, 2, and 3, respectively. However, some clinicopathologically confirmed OPDM cases continue to have unknown genetic causes. Here, through a combination of long-read whole-genome sequencing (LRS), repeat-primed polymerase chain reaction (RP-PCR), and fluorescence amplicon length analysis PCR (AL-PCR), we found that a CGG repeat expansion in the 5' UTR of RILPL1 is associated with familial and simplex OPDM type 4 (OPDM4). The number of repeats ranged from 139 to 197. Methylation analysis indicates that the methylation levels in RILPL1 were unaltered in OPDM4 individuals. Analyses of muscle biopsies suggested that the expanded CGG repeat might be translated into a toxic poly-glycine protein that co-localizes with p62 in intranuclear inclusions. Moreover, analyses suggest that the toxic RNA gain-of-function effects also contributed to the pathogenesis of this disease. Intriguingly, all four types of OPDM have been found to be associated with the CGG repeat expansions located in 5' UTRs. This finding suggests that a common pathogenic mechanism, driven by the CGG repeat expansion, might underlie all cases of OPDM.

Non-coding CGG repeat expansion in LOC642361/NUTM2B-AS1 is associated with a phenotype of oculopharyngodistal myopathy.

BACKGROUND: Oculopharyngodistal myopathy (OPDM) is a rare adult-onset neuromuscular disease, associated with CGG repeat expansions in the 5' untranslated region of LRP12, GIPC1, NOTCH2NLC and RILPL1. However, the genetic cause of a proportion of pathoclinically confirmed cases remains unknown. METHODS: A total of 26 OPDM patients with unknown genetic cause(s) from 4 tertiary referral hospitals were included in this study. Clinical data and laboratory findings were collected. Muscle samples were observed by histological and immunofluorescent staining. Long-read sequencing was initially conducted in six patients with OPDM. Repeat-primed PCR was used to screen the CGG repeat expansions in LOC642361/NUTM2B-AS1 in all 26 patients. RESULTS: We identified CGG repeat expansion in the non-coding transcripts of LOC642361/NUTM2B-AS1 in another two unrelated Chinese cases with typical pathoclinical features of OPDM. The repeat expansion was more than 70 times in the patients but less than 40 times in the normal controls. Both patients showed no leucoencephalopathy but one showed mild cognitive impairment detected by Montreal Cognitive Assessment. Rimmed vacuoles and p62-positive intranuclear inclusions (INIs) were identified in muscle pathology, and colocalisation of CGG RNA foci with p62 was also found in the INIs of patient-derived fibroblasts. CONCLUSIONS: We identified another two unrelated cases with CGG repeat expansion in the long non-coding RNA of the LOC642361/NUTM2B-AS1 gene, presenting with a phenotype of OPDM. Our cases broadened the recognised phenotypic spectrum and pathogenesis in the disease associated with CGG repeat expansion in LOC642361/NUTM2B-AS1.

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.

CGG repeat expansion in NOTCH2NLC causes mitochondrial dysfunction and progressive neurodegeneration in Drosophila model.

Neuronal intranuclear inclusion disease (NIID) is a neuromuscular/neurodegenerative disease caused by the expansion of CGG repeats in the 5' untranslated region (UTR) of the NOTCH2NLC gene. These repeats can be translated into a polyglycine-containing protein, uN2CpolyG, which forms protein inclusions and is toxic in cell models, albeit through an unknown mechanism. Here, we established a transgenic Drosophila model expressing uN2CpolyG in multiple systems, which resulted in progressive neuronal cell loss, locomotor deficiency, and shortened lifespan. Interestingly, electron microscopy revealed mitochondrial swelling both in transgenic flies and in muscle biopsies of individuals with NIID. Immunofluorescence and immunoelectron microscopy showed colocalization of uN2CpolyG with mitochondria in cell and patient samples, while biochemical analysis revealed that uN2CpolyG interacted with a mitochondrial RNA binding protein, LRPPRC (leucine-rich pentatricopeptide repeat motif-containing protein). Furthermore, RNA sequencing (RNA-seq) analysis and functional assays showed down-regulated mitochondrial oxidative phosphorylation in uN2CpolyG-expressing flies and NIID muscle biopsies. Finally, idebenone treatment restored mitochondrial function and alleviated neurodegenerative phenotypes in transgenic flies. Overall, these results indicate that transgenic flies expressing uN2CpolyG recapitulate key features of NIID and that reversing mitochondrial dysfunction might provide a potential therapeutic approach for this disorder.

Pathologic changes in neuronal intranuclear inclusion disease are linked to aberrant FUS interaction under hyperosmotic stress.

CGG repeat expansion in NOTCH2NLC is the genetic cause of neuronal intranuclear inclusion disease (NIID). Previous studies indicated that the CGG repeats can be translated into polyglycine protein (N2CpolyG) which was toxic to neurons by forming intranuclear inclusions (IIs). However, little is known about the factors governing polyG IIs formation as well as its molecular pathogenesis. Considering that neurogenetic disorders usually involve interactions between genetic and environmental stresses, we investigated the effect of stress on the formation of IIs. Our results revealed that under hyperosmotic stress, N2CpolyG translocated from the cytoplasm to the nucleus and formed IIs in SH-SY5Y cells, recapitulating the pathological hallmark of NIID patients. Furthermore, N2CpolyG interacted/ co-localized with an RNA-binding protein FUS in the IIs of cellular model and NIID patient tissues, thereby disrupting stress granule formation in cytoplasm under hyperosmotic stress. Consequently, dysregulated expression of microRNAs was found both in NIID patients and cellular model, which could be restored by FUS overexpression in cultured cells. Overall, our findings indicate a mechanism of stress-induced pathological changes as well as neuronal damage, and a potential strategy for the treatment of NIID.

Expansion of GGC Repeat in GIPC1 Is Associated with Oculopharyngodistal Myopathy.

Oculopharyngodistal myopathy (OPDM) is an adult-onset inherited neuromuscular disorder characterized by progressive ptosis, external ophthalmoplegia, and weakness of the masseter, facial, pharyngeal, and distal limb muscles. The myopathological features are presence of rimmed vacuoles (RVs) in the muscle fibers and myopathic changes of differing severity. Inheritance is variable, with either putative autosomal-dominant or autosomal-recessive pattern. Here, using a comprehensive strategy combining whole-genome sequencing (WGS), long-read whole-genome sequencing (LRS), linkage analysis, repeat-primed polymerase chain reaction (RP-PCR), and fluorescence amplicon length analysis polymerase chain reaction (AL-PCR), we identified an abnormal GGC repeat expansion in the 5' UTR of GIPC1 in one out of four families and three sporadic case subjects from a Chinese OPDM cohort. Expanded GGC repeats were further confirmed as the cause of OPDM in an additional 2 out of 4 families and 6 out of 13 sporadic Chinese individuals with OPDM, as well as 7 out of 194 unrelated Japanese individuals with OPDM. Methylation, qRT-PCR, and western blot analysis indicated that GIPC1 mRNA levels were increased while protein levels were unaltered in OPDM-affected individuals. RNA sequencing indicated p53 signaling, vascular smooth muscle contraction, ubiquitin-mediated proteolysis, and ribosome pathways were involved in the pathogenic mechanisms of OPDM-affected individuals with GGC repeat expansion in GIPC1. This study provides further evidence that OPDM is associated with GGC repeat expansions in distinct genes and highly suggests that expanded GGC repeat units are essential in the pathogenesis of OPDM, regardless of the genes in which the expanded repeats are located.

Subclinical peripheral neuropathy is common in neuronal intranuclear inclusion disease with dominant encephalopathy.

BACKGROUND AND PURPOSE: Neuronal intranuclear inclusion disease (NIID) is associated with CGG repeat expansion in the NOTCH2NLC gene. Although pure or dominant peripheral neuropathy has been described as a subtype of NIID in a few patients, most NIID patients predominantly show involvements of the central nervous system (CNS). It is necessary to further explore whether these patients have subclinical peripheral neuropathy. METHODS: Twenty-eight NIID patients, clinically characterized by CNS-dominant involvements, were recruited from two tertiary hospitals. Standard nerve conduction studies were performed in all patients. Skin and sural nerve biopsies were performed in 28 and 15 patients, respectively. Repeat-primed polymerase chain reaction and amplicon length polymerase chain reaction were used to screen the CGG repeat expansion in NOTCH2NLC. RESULTS: All 28 patients can be diagnosed with NIID based on skin pathological and genetic changes. All patients predominantly showed CNS symptoms mainly characterized by episodic encephalopathy and cognitive impairments, but no clinical symptoms of peripheral neuropathy could be observed initially. Electrophysiological abnormalities were found in 96.4% (27/28) of these patients, indicating that subclinical peripheral neuropathy is common in NIID patients with CNS-dominant type. Electrophysiological and neuropathological studies revealed that demyelinating degeneration was the main pathological pattern in these patients, although mild axonal degeneration was also observed in some patients. No significant association between CGG repeat size and the change of nerve conduction velocity was found in these patients. CONCLUSIONS: This study demonstrated that most patients with CNS-dominant NIID had subclinical peripheral neuropathy. Electrophysiological examination should be the routinely diagnostic workflow for every NIID patient.

Genetic origin of sporadic cases and RNA toxicity in neuronal intranuclear inclusion disease.

BACKGROUND: GGC repeat expansion in NOTCH2NLC has been recently linked to neuronal intranuclear inclusion disease (NIID) via unknown disease mechanisms. Herein, we explore the genetic origin of the sporadic cases and toxic RNA gain-of-function mechanism in NIID. METHODS: Multiple genetic screenings were performed on NIID individuals and their available family members. Methylation status of blood DNA, NOTCH2NLC mRNA level from muscle biopsies and RNA foci from skin biopsies of NIID individuals or asymptomatic carriers were evaluated and compared. RESULTS: In two sporadic NIID families, we identified two clinically and pathologically asymptomatic fathers carrying large GGC repeat expansion, above 300 repeats, with offspring repeat numbers of 172 and 148, respectively. Further evaluation revealed that the GGC repeat numbers in the sperm from two asymptomatic fathers were only 63 and 98, respectively. The CpG island in NOTCH2NLC of the asymptomatic carriers was hypermethylated, and accordingly, the NOTCH2NLC mRNA levels were decreased in the asymptomatic fathers. GGC repeat expansion RNA formed RNA foci and sequestered RNA binding proteins into p62 positive intranuclear inclusions in NIID individuals but not in the control or asymptomatic carrier. CONCLUSION: Our study suggested the GGC repeat expansion in NOTCH2NLC might have a disease-causing number ranging from ~41 to ~300 repeats. The contraction of GGC repeat expansion in sperm could be a possible mechanism for the paternal-biased origin in some sporadic or recessive inherited NIID individuals. The toxic RNA gain-of-function mechanism was identified to be involved in the pathogenicity of this disease.

Cryptic exon activation caused by a novel deep-intronic splice-altering variant in Becker muscular dystrophy.

BACKGROUND: An accurate genetic diagnosis of Becker muscular dystrophy (BMD) can be sometimes challenging due to deep intronic DMD variants. Here, we report on the genetic diagnosis of a BMD patient with a novel deep-intronic splice-altering variant in DMD. METHODS: The index case was a 3.8-year-old boy who was suspected of having a diagnosis of BMD based on his clinical, muscle imaging, and pathological features. Routine genomic detection approaches did not detect any disease-causing variants in him. Muscle-derived DMD mRNA studies, followed by genomic Sanger sequencing and in silico bioinformatic analyses, were performed in the patient. RESULTS: DMD mRNA studies detected a cryptic exon-containing transcript and normally spliced DMD transcript in the patient. The cryptic exon-containing transcript encoded a frameshift and premature termination codon (NP_003997.1:p.[=,Asp2740Valfs*52]). Further genomic Sanger sequencing and bioinformatic analysis identified a novel deep-intronic splice-altering variant in DMD (c.8217 + 23338A > G). The novel variant strengthened a cryptic donor splice site and activated a cryptic acceptor splice site in the deep-intronic region of DMD intron 55, resulting in the activation of a new dystrophin cryptic exon found in the patient. CONCLUSION: Our case report expands the genetic spectrum of BMD and highlights the essential role of deep-intronic cryptic exon-activating variants in genetically unsolved BMD patients.

Oculopharyngodistal myopathy.

PURPOSE OF REVIEW: Oculopharyngodistal myopathy (OPDM) is a rare adolescent or adult-onset neuromuscular disease that is characterized by progressive ocular, facial, pharyngeal and distal limb muscle weakness. The rimmed vacuoles and intranuclear inclusions in myofibers constitute the pathological hallmark of OPDM. In this review, the latest findings related to the genetic, molecular and clinical features of OPDM, as well as the diagnosis and management are summarized. RECENT FINDINGS: Four gene mutations, CGG repeats in the 5'-untranslated region of LRP12 , GIPC1 , NOTCH2NLC and RILPL1 have been reported to be disease-causing genes in OPDM, namely OPDM1, OPDM2, OPDM3 and OPDM4, accordingly. So far, limited studies have suggested that CGG repeat expansion within the pathogenic range may play a key role in the pathogenesis of OPDM with the gain-of-function mechanism at the RNA and/or protein level, while repeat expansion over a threshold limit may cause hypermethylation, leading to the transcriptional silencing of the CGG repeats in the expanded allele, which results in the existence of mild phenotype or asymptomatic carriers. SUMMARY: Novel gene mutations, possible molecular mechanisms and the clinical features related to different causative genes are discussed in this review. More studies on the exact pathogenic mechanism are needed.

The GGC repeat expansion in NOTCH2NLC is associated with oculopharyngodistal myopathy type 3.

Oculopharyngodistal myopathy (OPDM) is an adult-onset neuromuscular disease characterized by progressive ocular, facial, pharyngeal and distal limb muscle involvement. Trinucleotide repeat expansions in LRP12 or GIPC1 were recently reported to be associated with OPDM. However, a significant portion of OPDM patients have unknown genetic causes. In this study, long-read whole-genome sequencing and repeat-primed PCR were performed and we identified GGC repeat expansions in the NOTCH2NLC gene in 16.7% (4/24) of a cohort of Chinese OPDM patients, designated as OPDM type 3 (OPDM3). Methylation analysis indicated that methylation levels of the NOTCH2NLC gene were unaltered in OPDM3 patients, but increased significantly in asymptomatic carriers. Quantitative real-time PCR analysis indicated that NOTCH2NLC mRNA levels were increased in muscle but not in blood of OPDM3 patients. Immunofluorescence on OPDM muscle samples and expressing mutant NOTCH2NLC with (GGC)69 repeat expansions in HEK293 cells indicated that mutant NOTCH2NLC-polyglycine protein might be a major component of intranuclear inclusions, and contribute to toxicity in cultured cells. In addition, two RNA-binding proteins, hnRNP A/B and MBNL1, were both co-localized with p62 in intranuclear inclusions in OPDM muscle samples. These results indicated that a toxic protein gain-of-function mechanism and RNA gain-of-function mechanism may both play a vital role in the pathogenic processes of OPDM3. This study extended the spectrum of NOTCH2NLC repeat expansion-related diseases to a predominant myopathy phenotype presenting as OPDM, and provided evidence for possible pathogenesis of these diseases.

Long-read sequencing identified repeat expansions in the 5'UTR of the NOTCH2NLC gene from Chinese patients with neuronal intranuclear inclusion disease.

BACKGROUND: Neuronal intranuclear inclusion disease (NIID) is a heterogenous neurodegenerative disorder named after its pathological features. It has long been considered a disease of genetic origin. Recently, the GGC repeated expansion in the 5'-untranslated region (5'UTR) of the NOTCH2NLC gene has been found in adult-onset NIID in Japanese individuals. This study was aimed to investigate the causative mutations of NIID in Chinese patients. METHODS: Fifteen patients with NIID were identified from five academic neurological centres. Biopsied skin samples were analysed by histological staining, immunostaining and electron microscopic observation. Whole-genome sequencing (WGS) and long-read sequencing (LRS) were initially performed in three patients with NIID. Repeat-primed PCR was conducted to confirm the genetic variations in the three patients and the other 12 cases. RESULTS: Our patients included 14 adult-onset patients and 1 juvenile-onset patient characterised by degeneration of multiple nervous systems. All patients were identified with intranuclear inclusions in the nuclei of fibroblasts, fat cells and ductal epithelial cells of sweat glands. The WGS failed to find any likely pathogenic variations for NIID. The LRS successfully identified that three patients with adult-onset NIID showed abnormalities of GGC expansion in 5'UTR of the NOTCH2NLC gene. The GGC repeated expansion was further confirmed by repeat-primed PCR in seven familial cases and eight sporadic cases. CONCLUSION: Our findings provided evidence that confirmed the GGC repeated expansion in the 5'UTR of the NOTCH2NLC gene is associated with the pathogenesis of NIID. Additionally, the GGC expansion was not only responsible for adult-onset patients, but also responsible for juvenile-onset patients.

Efficacy, immunogenicity and safety of the HPV-16/18 AS04-adjuvanted vaccine in healthy Chinese women aged 18-25 years: results from a randomized controlled trial.

This phase II/III, double-blind, randomized trial assessed the efficacy, immunogenicity and safety of the human papillomavirus (HPV)-16/18 AS04-adjuvanted vaccine in young Chinese women (ClinicalTrials.gov registration NCT00779766). Women aged 18-25 years from Jiangsu province were randomized (1:1) to receive HPV vaccine (n = 3,026) or Al(OH)3 control (n = 3,025) at months 0, 1 and 6. The primary objective was vaccine efficacy (VE) against HPV-16/18 associated 6-month persistent infection (PI) and/or cervical intraepithelial neoplasia (CIN) 1+. Secondary objectives were VE against virological and clinical endpoints associated with HPV-16/18 and with high-risk HPV types, immunogenicity and safety. Mean follow-up for the according-to-protocol cohort for efficacy (ATP-E) was ∼15 months after the third dose. In the ATP-E (vaccine = 2,889; control = 2,894), for initially HPV DNA negative and seronegative subjects, HPV-16/18 related VE (95% CI) was 94.2% (62.7, 99.9) against 6-month PI and/or CIN1+ and 93.8% (60.2, 99.9) against cytological abnormalities. VE against HPV-16/18 associated CIN1+ and CIN2+ was 100% (-50.4, 100) and 100% (-140.2, 100), respectively (no cases in the vaccine group and 4 CIN1+ and 3 CIN2+ cases in the control group). At Month 7, at least 99.7% of initially seronegative vaccine recipients had seroconverted for HPV-16/18; geometric mean antibody titres (95% CI) were 6,996 (6,212 to 7,880) EU/mL for anti-HPV-16 and 3,309 (2,942 to 3,723) EU/mL for anti-HPV-18. Safety outcomes between groups were generally similar. The HPV-16/18 AS04-adjuvanted vaccine is effective, immunogenic and has a clinically acceptable safety profile in young Chinese women. Prophylactic HPV vaccination has the potential to substantially reduce the burden of cervical cancer in China.

Interaction of depressive and anxiety symptoms on the mortality of patients with COPD: a preliminary study.

BACKGROUND: Depressive and anxiety symptoms increase the risk of death in patients with Chronic Obstructive Pulmonary Disease (COPD), but the combined effects of the two factors are unknown. PURPOSE: To assess the possible interactive effects of depressive and anxiety symptoms on the death of patients with COPD. METHODS: Prospective data for 7787 Chinese patients with COPD was analyzed and the deaths were recorded. Depressive and anxiety symptoms were evaluated using the Hospital Anxiety and Depression Scale. A product of depressive and anxiety symptoms was added to the logistic regression model to evaluate the multiplicative interaction, and relative excess risk of interaction (RERI), attributable proportion (AP) of interaction, and synergy index (S) were applied to evaluate the additive interaction of the two factors. Bootstrap was used to calculate 95% confidence intervals (CIs) of RERI, AP and S. RERI > 0, AP > 0, or S > 1 indicated biological interaction. RESULTS: After 4 years' follow-up, the cumulative mortality was 20.0%, and the percentages of deaths in patients with depressive and anxiety symptoms were 28.5% and 30.9%, respectively. When adjusting for variables such as age, sex, disease duration, marital status, income, education level, co-morbidity, smoke status, biomass smoke, 6MWD, MRC, BMI and FEV1. the RERI, AP, and S (with 95% CIs) resulted in depression and anxiety scores of 6.54 (1.23-13.79), 0.54 (0.18-0.83), and 2.64 (1.33-4.75) respectively. CONCLUSION: Interaction effects were found between depressive and anxiety symptoms on the death of patients with COPD. Patients with both depressive and anxiety symptoms have an increased risk of death from COPD.

Spike 1 trimer, a nanoparticle vaccine against porcine epidemic diarrhea virus induces protective immunity challenge in piglets.

Porcine epidemic diarrhea virus (PEDV) is considered the cause for porcine epidemic diarrhea (PED) outbreaks and hefty losses in pig farming. However, no effective commercial vaccines against PEDV mutant strains are available nowadays. Here, we constructed three native-like trimeric candidate nanovaccines, i.e., spike 1 trimer (S1-Trimer), collagenase equivalent domain trimer (COE-Trimer), and receptor-binding domain trimer (RBD-Trimer) for PEDV based on Trimer-Tag technology. And evaluated its physical properties and immune efficacy. The result showed that the candidate nanovaccines were safe for mice and pregnant sows, and no animal death or miscarriage occurred in our study. S1-Trimer showed stable physical properties, high cell uptake rate and receptor affinity. In the mouse, sow and piglet models, immunization of S1-Trimer induced high-level of humoral immunity containing PEDV-specific IgG and IgA. S1-Trimer-driven mucosal IgA responses and systemic IgG responses exhibited high titers of virus neutralizing antibodies (NAbs) in vitro. S1-Trimer induced Th1-biased cellular immune responses in mice. Moreover, the piglets from the S1-Trimer and inactivated vaccine groups displayed significantly fewer microscopic lesions in the intestinal tissue, with only one and two piglets showing mild diarrhea. The viral load in feces and intestines from the S1-Trimer and inactivated vaccine groups were significantly lower than those of the PBS group. For the first time, our data demonstrated the protective efficacy of Trimer-Tag-based nanovaccines used for PEDV. The S1-Trimer developed in this study was a competitive vaccine candidate, and Trimer-Tag may be an important platform for the rapid production of safe and effective subunit vaccines in the future.

Impact of risk factors, activities and psychological disorders on the health of patients with chronic obstructive pulmonary disease in China: a cross-sectional study.

BACKGROUND: Patients with chronic obstructive pulmonary disease (COPD) often have organ dysfunction and resulting poor quality of life; however, in China little information is available regarding factors that affect their health. Here, the relationship between risk factors, activities and psychological disorders and health of patients with COPD in rural areas of Xuzhou, China was assessed. METHODS: A cross-sectional study of 7597 COPD patients randomly selected by place of residence from 24,641 COPD patients who had been identified by screening of the 1.10 million health records of all residents of the target area was carried out to evaluate the relationships between risk factors, activities, psychological disorders and the ADO index (age, dyspnea, and airflow obstruction). The participants were assessed by spirometry and by administering a newly designed face-to-face questionnaire, which included items on general factors, risk factors, activities and psychological disorders. Correlations between the ADO index and the items addressed by the questionnaire were calculated. RESULTS: The mean score of the ADO index was 3.7 ± 1.6. The ADO indices of current smokers, ex-smokers, and non-smokers were 3.9 ± 2.1, 3.7 ± 1.9, and 3.2 ± 1.5, respectively (P < 0.001). The ADO indices of cooks and non-cooks were 4.0 ± 2.2 and 3.5 ± 1.7, respectively (P < 0.001). The correlation coefficient between self-assessment of health status and ADO index was 0.976 (P < 0.001). Only 5.7% of patients reported no limitation of their daily living activities. The correlation coefficient between daily living activities and ADO index was 0.981 (P < 0.001). Only 5.5% of patients reported no limitation of social activities. The correlation coefficient between social activities and ADO index was 0.989 (P < 0.001), between the assessed anxiety score and ADO index 0.972 (P < 0.001), and between the assessed depression score and ADO index 0.989 (P < 0.001). CONCLUSIONS: COPD severity was significantly correlated with behavior (especially regarding smoking and cooking with biofuel in confined spaces), physical strength, daily living activities, social activities, anxiety and depression. Comprehensive approaches are required for the prevention and treatment of COPD.

Supporting smoking cessation in chronic obstructive pulmonary disease with behavioral intervention: a randomized controlled trial.

BACKGROUND: Cigarette smoking is the major risk factor for chronic obstructive pulmonary disease (COPD). But a fewer smoking cessation measures were conducted in communities for smokers with COPD in China. The aim of our study was to assess the preventive effects of behavioral interventions for smoking cessation and potential impact factors in smokers with COPD in China. METHODS: In a randomised controlled smoking cessation trial 3562 patients with COPD who were current smoker were allocated to intervention group received behavioral intervention and control group received the usual care for two years. The primary efficacy endpoint was the complete and continuous abstinence from smoking from the beginning of month 24 to the end of month 30. Participants were followed up at month 48. RESULTS: Continuous smoking abstinence rates from month 24 to 30 were significantly higher in participants receiving behavioral intervention than in those receiving usual care (46.4% vs 3.4%, p < 0.001). Continuous abstinence rates from months 24 to 36 (45.8% vs 4.0%) and months 24 to 48 (44.3% vs 5.1%) were also higher in participants receiving behavioral intervention than in those control group. Family members or family physicians/nurses smoking were first identified to influence smoking cessation. CONCLUSIONS: Behavioral intervention doubled the smoking cessation rate in patients with COPD and was complied well by the general practitioners. The family members and family physicians/nurses smoking were the main risk factors for smoking cessation. TRIAL REGISTRATION: Chinese Clinical Trials Registration (ChiCTR-TRC-12001958).

Complex I deficiency in m.3243A>G fibroblasts is alleviated by reducing NADH accumulation.

Introduction: Mitochondrial disease is a spectrum of debilitating disorders caused by mutations in the mitochondrial DNA (mtDNA) or nuclear DNA that compromises the respiratory chain. Mitochondrial 3243A>G (m.3243 A>G) is the most common mutation showing great heterogeneity in phenotype. Previous studies have indicated that NADH: ubiquinone oxidoreductase (complex I) deficiency accompanied by a decreased nicotinamide adenine dinucleotide (NAD+)/reduced NAD+ (NADH) ratio may play a pivotal role in the pathogenesis of m.3243A>G mutation. Methods: To evaluate the potential effects of strategies targeting the imbalanced NAD+/NADH ratio in m.3243A>G mutation, we treated fibroblasts derived from patients with the m.3243 A>G mutation using nicotinamide riboside (NR) or mitochondria-targeted H2O-forming NADH oxidase (mitoLbNOX). Results: M.3243 A>G fibroblasts showed a significant reduction in complex I core subunit 6, complex I enzymatic activity, complex I-dependent oxygen consumption rate (OCR), and adenosine triphosphate (ATP) production compared to the controls. The NAD+/NADH ratio was also significantly reduced in m.3243 A>G fibroblasts, and, using fluorescence lifetime imaging microscopy, we also found that the NADH level was elevated in m.3243 A>G fibroblasts. After NR treatment, the NAD+/NADH ratio, complex I-dependent OCR, and ATP levels increased, whereas NADH levels remained unchanged. More excitingly, after treatment with mitoLbNOX, the NAD+/NADH ratio, complex I-independent OCR, and ATP levels increased more pronouncedly compared with the NR treatment group, accompanied by significantly reduced NADH levels. Discussion: The present study suggests that compared with repletion of NAD+ alone, the combination of this therapeutic modality with alleviation of NADH overload may amplify the treatment effect of restoring NAD+/NADH balance in m.3243A>G fibroblasts.

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.