ABSTRACT
Genetic composition plays critical roles in the pathogenesis of autism spectrum disorder (ASD). Especially, inherited and de novo intronic variants are often seen in patients with ASD. However, the biological significance of intronic variants is difficult to address. Here, among a Chinese ASD cohort, we identified a recurrent inherited intronic variant in the CHD7 gene, which is specifically enriched in East Asian populations. CHD7 has been implicated in numerous developmental disorders including CHARGE syndrome and ASD. To investigate whether the ASD-associated CHD7 intronic variant affects neural development, we established human embryonic stem cells carrying this variant using CRISPR/Cas9 methods and found that the level of CHD7 mRNA significantly decreased compared to control. Upon differentiation towards the forebrain neuronal lineage, we found that neural cells carrying the CHD7 intronic variant exhibited developmental delay and maturity defects. Importantly, we found that TBR1, a gene also implicated in ASD, was significantly increased in neurons carrying the CHD7 intronic variant, suggesting the intrinsic relevance among ASD genes. Furthermore, the morphological defects found in neurons carrying CHD7 intronic mutations were rescued by knocking down TBR1, indicating that TBR1 may be responsible for the defects in CHD7-related disorders. Finally, the CHD7 intronic variant generated three abnormal forms of transcripts through alternative splicing, which all exhibited loss-of-function in functional assays. Our study provides crucial evidence supporting the notion that the intronic variant of CHD7 is potentially an autism susceptibility site, shedding new light on identifying the functions of intronic variants in genetic studies of autism.
ABSTRACT
Objective This study aimed to assess the diagnostic value of anti-mutated citrullinated vimentin (MCV) antibodies in rheumatoid arthritis (RA) and its correlation with disease progression, extra-articular manifestations and overlap syndrome. Methods Retrospective Studies. Clinical data of 837 patients in PekingUnionMedicalCollegeHospitalfrom June to August 2017 were collected, including the result of anti-MCV, anti-cyclic citrullinated peptide (anti-CCP) antibodies, rheumatoid factor (RF), erythrocyte sedimentation rate (ESR) and High-sensitivity-C-reactive protein (CRP). According to the 1987 American College of Rheumatology classification criteria for rheumatoid arthritis, there were 323 patients diagnosed with RA, including 59 males and 264 females with the average age of 51 years. According to whether the RA patients have overlap syndrome with other autoimmune disease (AID) or have extra-articular manifestations, 258 cases were categorized into RA group, including 47 males and 211 females with the average age of 50 years; 14 cases were categorized into the group of overlap syndrome, including 1 male and 13 females with the average age of 36 years;51 cases were categorized into the group of extra-articular manifestations, including 11 males and 40 females with the average age of 59 years.According to 2010 rheumatoid arthritis classification criteria for destruction in joints, the radiographic changes were divided into 4 stages. There were 203 casesenrolled in our study, 88 caseswere fitted into early stage group (stage I)including 21 males and 67 females with the average age of 48 years; 115 caseswere fitted into progressive stage group, which compromisedstageⅡ (interim stage), stage Ⅲ (severe stage) and stage Ⅳ(final stage) cases, including 19 males and 96 females with the average age of 53 years. Mann-Whitney U test, x2 test, Receiver operating characteristic (ROC) curves and Spearmancorrelation coefficientwere used in Statistical analysis. Results Ⅰ Amongdiagnosed RA patients, 199 (61.6％) cases were positive for anti-MCV, anti-CCP and RFsimultaneously, 42 (13％) cases were positive for anti-MCV, which was higher than anti-CCP positive (1 cases, 0.3％) or RF positive (7cases, 2.2％). The difference was statistically significant(P<0.001, P<0.001). ⅡROC was calculated and MCV=35.95 U/ml was used as best-fit cut-off value. The AUC for anti-MCV was 0.867, while the sensitivity was 80.5％and specificity was 80.9％.ⅢThe detection levels of anti-MCV (682.8 (106.4-1000.0)), anti-CCP (407 (4.0-1536.0)) and RF (82.8 (21.1-244.9)) in the group of progressive stage were higher than those in the group of early stage (114.5 (28.5-1000.0), 62.5 (5.0-1020.7), 50.1 (6.7-127.1)), which showed a significant difference(P<0.05, P<0.05, P<0.05). The anti-MCV, anti-CCP and RF were positively related to the degree of joint destruction (r=0.229, P<0.05;r=0.187, P<0.05;r=0.167, P<0.05);anti-MCV and anti-CCP were positively related to extra-articular manifestation (r=0.152, P<0.05;r=0.136, P<0.05). Conclusion Anti-MCV antibodies are more sensitive in patients with RA, and have complementary diagnostic value for anti-CCP and RF-negative patients; high levels of anti-MCV and anti-CCP in RA patients are associated with RA progression and extra-articular involvement.
ABSTRACT
Objective To explore the influence of prozone effect on anti-nuclear antibodies (ANA) testing by indirect immunofluorescence assay (IIFA).Methods The samples with high titer of ANA (≥1∶1 000) were selected from 880fresh serum samples, and were subsequently diluted in 1∶100, 1∶1 000and 1∶10 000ratio.Prozone effect was defined as fluorescence intensity from 1∶1 000dilution was stronger than that from1∶100dilution.The samples with prozone effect were determined manually or by Sprinter XL and EUROPattern.The samples with prozone effect were further characterized by combinations of fluorescence patterns, fluorescence intensities and autoantibody specificities.Results A total of 880samples were tested.Importantly, 34samples displayed prozone effect (3.86％in total and 29.57％in samples with ANA≥1∶1 000).Interestingly, prozone effect was identified by manual detection as well as by Sprinter XL with similar fluorescence patterns and fluorescence intensities.Notably, EUROPattern can only select the central area for identification.Among all samples with prozone effect, 74.42％samples exhibited fluorescence intensities of≥1∶10 000.Speckled pattern was the most prevalent fluorescence patterns in samples with prozone effect (46.51％).In addition, anti-RNP antibodies (62.79％) were the most popular autoantibodies in samples with prozone effect, followed by anti-dsDNA antibodies (51.16％) and anti-SSA antibodies (51.16％).Conclusion Prozone effect was present in ANA testing, especially in samples with high titers, resulting in underestimating the titers.The study highlighted that special attention should be paid to the prozone effect in clinical practice.
ABSTRACT
Objective To explore the effects of miR-21 antisense oligonucleotides(ASO)and cisplatin combined on the growth of subcutaneous xenotransplanted osteosarcomas in nude mice.Methods Xenotransplanted osteosarcoma models were established in nude mice by injection of human osteosarcoma cells MG-63.Inoculated mice were randomly divided into four groups(blank control group,cisplatin group,anti-miR-21 group,anti-miR-21+cisplatin group).The rats were treated by injec-tion of anti-miR-21 into the tumors in anti-miR-21 group,by injection of cisplatin through tail veins in cisplatin group or by in-jection both of them in anti-miR-21+ cisplatin group.The volume and weight of tumors were measured and the inhibition rates of tumors were calculated on a daily basis.Meanwhile,MG-63 cells were divided into the same four groups as mentioned be-fore.The cell proliferation was detected by MTT assay and the cell apoptosis by Annexin V analysis.Results The inhibition of tumor growth(volume and weight)was obvious in the anti-miR-21+cisplatin group compared to the control groups.In vitro cell growth was significantly inhibited and cell apoptosis increased obviously in the anti-miR-21 + cisplatin group compared to the control groups(P <0.05).Conclusion MiR-21 ASO combined with cisplatin can inhibit cell proliferation,promote cell apopto-sis in vitro ,and obviously suppress tumor growth in vivo.They may be used to treat osteosarcoma.
ABSTRACT
Objective To investigate the prevalence and clinical significance of different subtypes (IgG,IgM and IgA) of anticardiolipin antibodies (aCL) and anti-β2-glycoprotein Ⅰ antibodies (aβ 2GP1),as well as lupus anticoagulant (LA) in systemic lupus erythematosus (SLE).Methods IgG/IgM/IgA,IgG,IgM,IgA aCL and anti-β2GP1 were tested by enzyme-linked immunosorbent assay (ELISA) in 100 patients with SLE (42 patients were diagnosed as secondary antiphospholipid syndrome),44 healthy controls and 32 patients with other connective tissue diseases excluding SLE and antiphospholipid syndrome (APS).Meanwhile,LA was tested by modified Dilute Russell's viper venom time (dRVVT).The correlation between antiphospholipid antibodies and clinical manifestation was analyzed by Spearman correlation analysis.The postiverate of antiphospholipid antibodies in SLE patients,health controls and patients with other connective tissue diseases were compared by chi square test.The concentrations of antiphospholipid antibodies in different groups were compared using independent sample Kruskal Wallis test.The diagnostic efficacy of antiphospholipid antibodies in SLE patients was analyzed by crosstable using clinical diagnosis of APS as gold standard.P ＜ 0.05 was considered statistically significant.Results The prevalence of IgG aCL (x2 =15.031,P ＜ 0.001),IgA/G/M (x2 =11.678,P =0.003) and IgA (x2 =6.17,P =0.036) antiβ2GP1 were significantly higher in patients with SLE than in the other two groups.IgA/G/M (r =0.207,P=0.039),IgG (r=0.230,P=0.021) and IgA (r=0.217,P=0.030) aCL,IgA/G/M (r=0.218,P=0.029) and IgA (r =0.255,P =0.01) anti-β2GP1,as well as LA (r =0.233,P =0.02) were associated with thrombotic events.IgA/G/M anti-β2GP1 (r =0.22,P =0.029) and LA (r =0.254,P =0.011) were associated with pathological pregnancy.23.1％ (6/26) aCL positive SLE patients were IgM and/or IgA aCL positive.53.6％ (15/28) anti-β2GP1 positive SLE patients were IgM and/or IgA antiβ2GP1 positive.In SLE patients,the specificity and sensitivity of IgA/G/M aCL for APS were 98.3％ and 26.2％,respectively.The specificity and sensitivity of IgA/G/M anti-β2GP1 were 84.5％ and 40.5％,respectively.The specificity of at least two isotypes positive for APS (both aCL and anti-β2GP1 were 98.3％),was higher than IgG aCL (94.8％) or anti-β2GP1 (93.1％).The sensitivity of at least one isotype of aCL (47.6％) or anti-β2GP1 (42.9％) positive for APS were higher than IgG aCL (40.5％)and anti-β2GP1 (21.4％).Conclusions IgG and IgM aCL together would be better than IgA/G/M aCL for APS screening.IgA/G/M anti-β2GP1 would be better for APS screen due to higher sensitivity and strong association with thromboembolic events and pathologic pregnance.IgA aCL or anti-β2GP1 was associated with thromboembolic events.IgA aCL or anti-β2GP1 would be useful for APS diagnosis in IgG and IgM aCL or anti-β2GPl negative patients.
ABSTRACT
Copy number variation (CNV) is one kind of structural variations,which exists extensively in human genome.In addition,it is the crucial source of inter-individual gene differences.Recently,with the successful development of gene array and gene sequencing,molecular methods for CNV testing are more and more standardizing and automating.Therefore,ongoing attentions have been attached to the effects and mechanisms of CNV in complicated diseases like autoimmune diseases.Molecular methods for CNV testing can be divided into two categories:target screening and the whole genome scanning platforms.Currently,the studies about CNV and autoimmune diseases mainly focused on systemic lupus erythematosus and rheumatoid arthritis.Considering the limitations of measurement methods and testing costs,it is still in the infant.
ABSTRACT
The present study examined the role of Wnt/β-catenin signaling pathway in the degeneration of nucleus pulposus cells and the protective effect of DKK1 on nucleus pulposus cells. The model of nucleus pulposus cell degeneration was induced by intra-disc injection of TNF-α, and the expression of β-catenin protein was detected by Western blotting. The cultured rabbit nucleus pulposus cells were divided into 4 groups. In group A, the cells were cultured with normal medium and served as control group. In group B, the cells were cultured with TNF-α and acted as degeneration group. In group C, the cells were cultured with TNF-α and transfected with Adv-eGFP and was used as fluorescence control group. In group D, the cells were cultured with TNF-α and transfected with Adv-hDKK1-eGFP, serving as intervention group. The expression of type II collagen, proteoglycan, β-catenin, and MMP-13 in each group was detected by immunocytochemistry and RT-PCR. The result showed that TNF-α increased the expression of β-catenin and MMP-13, and significantly inhibited the synthesis of type II collagen and proteoglycan, which resulted in the degeneration of nucleus pulposus cells. This effect could be obviously reversed by DKK1. We are led to concluded that TNF-α could activate the Wnt/β-catenin signaling pathway, and increase the expression of MMP-13, thereby resulting in disc degeneration. Specifically blocking Wnt/β-catenin signaling pathway by DKK-1 could protect the normal metabolism of intervertebral disc tissue. The Wnt pathway plays an important role in the progression of the intervertebral disc degeneration.