CTD Patients Research Articles

Novel mutations of the SRF gene in Chinese sporadic conotruncal heart defect patients

BackgroundConotruncal heart defects (CTDs) are a group of congenital heart malformations that cause anomalies of cardiac outflow tracts. In the past few decades, many genes related to CTDs have been reported. Serum response factor (SRF) is a ubiquitous nuclear protein that acts as transcription factor, and SRF was found to be a critical factor in heart development and to be strongly expressed in the myocardium of the developing mouse and chicken hearts. The targeted inactivation of SRF during heart development leads to embryonic lethality and myocardial defects in mice.MethodsTo illustrate the relationship between SRF and human heart defects, we screened SRF mutations in 527 CTD patients, a cross sectional study. DNA was extracted from peripheral leukocyte cells for target sequencing. The mutations of SRF were detected and validated by Sanger sequencing. The affection of the mutations on wild-type protein was analyzed by in silico softwares. Western blot and real time PCR were used to analyze the changes of the expression of the mutant mRNA and protein. In addition, we carried out dual luciferase reporter assay to explore the transcriptional activity of the mutant SRF.ResultsAmong the target sequencing results of 527 patients, two novel mutations (Mut1: c.821A > G p.G274D, the adenine(A) was mutated to guanine(G) at position 821 of the SRF gene coding sequences (CDS), lead to the Glycine(G) mutated to Asparticacid(D) at position 274 of the SRF protein amino acid sequences; Mut2: c.880G > T p.G294C, the guanine(G) was mutated to thymine (T) at position 880 of the SRF CDS, lead to the Glycine(G) mutated to Cysteine (C) at position 294 of the SRF protein amino acid sequences.) of SRF (NM_003131.4) were identified. Western blotting and real-time PCR showed that there were no obvious differences between the protein expression and mRNA transcription of mutants and wild-type SRF. A dual luciferase reporter assay showed that both SRF mutants (G274D and G294C) impaired SRF transcriptional activity at the SRF promoter and atrial natriuretic factor (ANF) promoter (p < 0.05), additionally, the mutants displayed reduced synergism with GATA4.ConclusionThese results suggest that SRF-p.G274D and SRF-p.G294C may have potential pathogenic effects.

Right ventricular dimension index by cardiac magnetic resonance for prognostication in connective tissue diseases and pulmonary hypertension.

Pulmonary hypertension (PH) in patients with CTD is a heterogeneous condition affected by left heart disease, chronic lung disease and thromboembolism as well as pulmonary vascular disease. Recent studies using cardiac magnetic resonance (CMR) have shown that right ventricular dysfunction is predictive for mortality in patients with PH, but limited to pulmonary arterial hypertension. This study aimed to analyse prognostic factors in PH-CTD. This retrospective analysis comprised 84 CTD patients, including SSc, who underwent both CMR and right heart catheterization from 2008 to 2018. Demographics, laboratory findings, and haemodynamic and morphological parameters were extracted. The prognostic value of each parameter was evaluated by multivariate analysis using covariables derived from propensity score to control confounding factors. Of 84 patients, 65 had right heart catheterization-confirmed PH (54 pulmonary arterial hypertension, 11 non-pulmonary arterial hypertension). Nine out of these PH patients died during a median follow-up period of 25 months. In 65 patients with PH, right ventricular end-diastolic dimension index (RVEDDI) evaluated by CMR was independently associated with mortality (hazard ratio 1.24; 95% CI: 1.08-1.46; P = 0.003). In a receiver operating characteristic analysis, RVEDDI highly predicted mortality, with area under the curve of 0.87. The 0.5-2-year follow-up data revealed that RVEDDI in both survivors and non-survivors did not significantly change over the clinical course, leading to the possibility that an early determination of RVEDDI could predict the prognosis. RVEDDI simply evaluated by CMR could serve as a significant predictor of mortality in PH-CTD. A further validation cohort study is needed to confirm its usability.

Clinical Value of Asymmetrical Dimethylarginine Detection in Patients with Connective Tissue Disease-Associated Pulmonary Arterial Hypertension.

Objective To evaluate the clinical value of serum asymmetrical dimethylarginine (ADMA) in patients with connective tissue disease- (CTD-) associated pulmonary arterial hypertension (PAH). Methods 88 patients with CTD were recruited between December 2017 and August 2018 in Jiangxi Provincial People's Hospital. Patients were further divided into two groups: CTD-without PAH (n = 45 cases) and CTD-with PAH (n = 43 cases), according to the pulmonary systolic blood pressure measured by echocardiography. 40 healthy controls were also included (n = 40 cases). The clinical data, including laboratory examinations, echocardiographic measurements, pulmonary function, and serum ADMA levels determined by enzyme-linked immunosorbent assay, (ELISA) were collected. The correlation between ADMA levels and the occurrence of PAH, pulmonary function, and other laboratory indexes in CTD patients were analyzed. Statistical analyses were performed by SPSS (version 23); P < 0.05 was considered statistically significant. Results The serum levels of ADMA in the CTD-PAH group were significantly higher than those of the CTD-without PAH group and healthy control group (P < 0.05); the serum ADMA levels were (0.706 ± 0.153 μmol/L), (1.015 ± 0.122 μmol/L), and (0.661 ± 0.113 μmol/L), respectively. There was no significant difference between the CTD-without PAH group and healthy control group (P < 0.05). Correlation analysis showed that serum ADMA levels were positively correlated with sPAP and NT-proBNP and negatively correlated with DLCO% (r = 0.802, 0.475, −0.585, P < 0.001). Multivariate analysis indicated that elevated serum ADMA levels increased the risk for the appearance of PAH in CTD patients (OR = 57.460, P < 0.001). Using the receiver operating characteristic (ROC) curve analysis, at the cutoff level of 0.810 μmol/L, ADMA showed good diagnostic efficacy as follows: sensitivity was 97.7%, specificity was 75.6%, and the area under the curve (AUC) was 0.947 (P < 0.001). Conclusion Increased ADMA levels are independently associated with the presence and severity of PAH in CTD patients. The levels of ADMA in the serum may contribute to be a noninvasive indicator for early diagnosis of CTD-with PAH patients.

Serum KL-6 levels reflect the severity of interstitial lung disease associated with connective tissue disease

BackgroundBiomarkers have been actively investigated to supplement functional and imaging modalities to predict the severity, therapeutic responsiveness, and progression of connective tissue disease-associated interstitial lung disease (CTD-ILD). This study aimed to evaluate Krebs von den Lungen 6 (KL-6) as a potential biomarker reflecting the severity of CTD-ILD as assessed through computed tomography (CT) and pulmonary function test (PFT) parameters.MethodsThis retrospective study included 549 Korean patients with rheumatoid arthritis, systemic sclerosis, inflammatory myositis, and other CTDs with or without concurrent ILD. Serum KL-6 concentration (U/mL) was measured using the latex-enhanced immunoturbidimetric assay method. CT and PFT results were collected within 1 year of serum collection. A semiquantitative grade of ILD extent was evaluated through CT scan (grade 1, 0–25%; grade 2, 26–50%; grade 3, 51–75%; grade 4, 76–100%).ResultsCTD-ILD patients (n = 165) had elevated serum KL-6 levels compared to CTD patients without ILD (n = 384) (p < 0.001), and those findings were preserved after adjusting for age, sex, and CTD type. The semiquantitative grade of ILD on CT scan was significantly proportional to the KL-6 level, and the optimal cut-off KL-6 value effectively differentiated each ILD grade. The percent diffusing capacity of the lung for carbon monoxide (DLCO) (p < 0.001) and forced vital capacity (FVC) (p < 0.001) parameters had a moderate, negative correlation with the KL-6 level.ConclusionSerum KL-6 levels were increased in CTD-ILD patients and had a positive correlation with CT grade and a negative correlation with FVC and DLCO. Serum KL-6 levels may reflect CTD-ILD severity.

A Quadruplet Regimen Comprising Carfilzomib, Cyclophosphamide, Lenalidomide, Dexamethasone (KCRD) Vs an Immunomodulatory Agent Containing Triplet (CTD/CRD) Induction Therapy Prior to Autologous Stem Cell Transplant: Results of the Myeloma XI Study

A Quadruplet Regimen Comprising Carfilzomib, Cyclophosphamide, Lenalidomide, Dexamethasone (KCRD) Vs an Immunomodulatory Agent Containing Triplet (CTD/CRD) Induction Therapy Prior to Autologous Stem Cell Transplant: Results of the Myeloma XI Study

A loss-of-function mutation p.T52S in RIPPLY3 is a potential predisposing genetic risk factor for Chinese Han conotruncal heart defect patients without the 22q11.2 deletion/duplication

BackgroundConotruncal heart defect (CTD) is a complex congenital heart disease with a complex and poorly understood etiology. The transcriptional corepressor RIPPLY3 plays a pivotal role in heart development as a negative regulator of the key cardiac transcription factor TBX1. A previous study showed that RIPPLY3 contribute to cardiac outflow tract development in mice, however, the relationship between RIPPLY3 and human cardiac malformation has not been reported.Methods615 unrelated CTD Chinese Han patients were enrolled, we excluded the 22q11.2 deletion/duplication using a modified multiplex ligation-dependent probe amplification method—CNVplex®, and investigated the variants of RIPPLY3 in 577 patients without the 22q11.2 deletion/duplication by target sequencing. Functional assays were performed to testify the potential pathogenicity of nonsynonymous variants found in these CTD patients.ResultsFour rare heterozygous nonsynonymous variants (p.P30L, p.T52S, p.D113N and p.V179D) were identified in four CTD patients, the variant NM_018962.2:c.155C>G (p.T52S) is referred as rs745539198, and the variant NM_018962.2:c.337G>A (p.D113N) is referred as rs747419773. However, variants p.P30L and p.V179D were not found in multiple online human gene variation databases. Western blot analysis and immunofluorescence showed that there were no significant difference between wild type RIPPLY3 and these four variants. Luciferase assays revealed that the p.T52S variant altered the inhibition of TBX1 transcriptional activity in vitro, and co-immunoprecipitation assays showed that the p.T52S variant reduced the physical interaction of RIPPLY3 with TBX1. In addition to the results from pathogenicity prediction tools and evolutionary protein conservation, the p.T52S variant was thought to be a potentially deleterious variant.ConclusionOur results provide evidence that deleterious variants in RIPPLY3 are potential molecular mechanisms involved in the pathogenesis of human CTD.

Identification of TBX2 and TBX3 variants in patients with conotruncal heart defects by target sequencing

BackgroundConotruncal heart defects (CTDs) are heterogeneous congenital heart malformations that result from outflow tract dysplasia; however, the genetic determinants underlying CTDs remain unclear. Increasing evidence demonstrates that dysfunctional TBX2 and TBX3 result in outflow tract malformations, implying that both of them are involved in CTD pathogenesis. We screened for TBX2 and TBX3 variants in a large cohort of CTD patients (n = 588) and population-matched healthy controls (n = 300) by target sequencing and genetically analyzed the expression and function of these variants.ResultsThe probably damaging variants p.R608W, p.T249I, and p.R616Q of TBX2 and p.A192T, p.M65L, and p.A562V of TBX3 were identified in CTD patients, but none in controls. All altered amino acids were highly conserved evolutionarily. Moreover, our data suggested that mRNA and protein expressions of TBX2 and TBX3 variants were altered compared with those of the wild-type. We screened PEA3 and MEF2C as novel downstream genes of TBX2 and TBX3, respectively. Functional analysis revealed that TBX2R608W and TBX2R616Q variant proteins further activated HAS2 promoter but failed to activate PEA3 promoter and that TBX3A192T and TBX3A562V variant proteins showed a reduced transcriptional activity over MEF2C promoter.ConclusionsOur results indicate that the R608W and R616Q variants of TBX2 as well as the A192T and A562V variants of TBX3 contribute to CTD etiology; this was the first association of variants of TBX2 and TBX3 to CTDs based on a large population.

Study on difference of peripheral serum SP-D, anti-MDA5, IL-6 and TNF-α in CTD-ILD patients

Objective: To investigate the clinical value of peripheral serum levels of Surfactant Protein D (SP-D), anti-melanoma differentiation-associated gene 5 (MDA5), Insulation (IL-6) and Tumor Necrosis Factor (TNF-α) in the prognosis of the degree of Connective Tissue Disease combined with Interstitial Lung Disease (CTD-ILD). Methods: 339 CTD-ILD patients, 184 CTD patients and 64 healthy volunteers in our hospital from January 2010 to December 2015 were enrolled into the study. The clinical symptoms, the test of High Resolution CT (HRCT) and lung function of all patients were detected and compared between the CTDILD and CTD patients. The serum levels of SP-D, anti-MDA5, IL-6, TNF-α were detected by ELISA among the three groups. Results: The mean serum levels of SP-D, anti-MDA5, IL-6, TNF-α of CTD-ILD patients and CTD patients were both higher than the control group (P<0.05); and the mean serum levels of SP-D, anti- MDA5, IL-6 of CTD-ILD patients was higher than CTD patients (P<0.05). The mean level of SP-D of CTD-ILD patients with pant was higher than the patients without pant (P<0.05); the mean level of anti MDA5 of CTD-ILD patients with rash was higher than the patients without rash (P<0.05); the mean level of TNF-α of CTD-ILD patients with atony or cough was higher than the patients without atony or cough (P<0.05). The serum levels of SP-D, anti-MDA5, IL-6, TNF-α in CTD-ILD patients were related to the degree of pulmonary ventilation disorder and pulmonary diffuse dysfunction. Conclusion: The detection of SP-D, anti-MDA5, IL-6, TNF-α of CTD-ILD patients could be helpful to diagnose the degree of connective tissue disease combined with interstitial lung disease.

Mutation Screening of Gata4 Gene in CTD Patients Within Chinese Han Population.

Conotruncal heart defect is a complex form of congenital heart disease and usually has a poor prognosis. Although previous studies have identified several missense variants in GATA4 gene that may cause CTD, it remains unclear whether they are involved in CTD pathogenesis because the study population was limited. The aim of the study was to investigate the mutations of GATA4 gene in isolated CTD Chinese Han patients and identify the pathomechanism of the missense mutations. In this report, the coding exons and exon-intron boundaries of the GATA4 gene were sequenced in 600 CTD patients and 300 controls. Functional significance of the novel GATA4 gene mutation (p.A167D) was analyzed using PolyPhen 2 and SIFT. And, the functional characteristics of the mutant GATA4 gene were assayed in contrast to its wild-type counterpart using a luciferase reporter assay system as well as Western blot. Eight heterozygous nonsynonymous variants (V380M, G64E, A167D, V267M, S377G, P163S, P407Q, A66T) were found in 22 patients, of which one (A167D) was reported here for the first time and five (G64E, A167D, S377G, P163S, A66T) were only found in CTD patients when compared with 300 controls. The PolyPhen 2 and SIFT programs predicted that the A167D substitution was expected to influence protein function. Subsequent functional analyses revealed that the transcriptional activity and Western blot of A167D mutant GATA4 protein were not altered. These variants may be involved in other mechanisms underlying CTD or may be unrelated to CTD occurrence.

Microparticle subpopulations are potential markers of disease progression and vascular dysfunction across a spectrum of connective tissue disease

ObjectiveMicroparticles (MPs) are membrane-bound vesicles derived from vascular and intravascular cells such as endothelial cells (EMPs) and platelets (PMPs). We investigated EMP and PMP numbers across a spectrum of autoimmune rheumatic diseases (AIRDs) with the aim of comparing the levels of, and relationship between, EMPs and PMPs. MethodsPatients with Systemic Lupus Erythematosus (SLE) (n=24), Systemic Sclerosis (SSc) (n=24), Primary Raynauds Phenomenon (RP) (n=17) and “other CTD” (n=15) (Primary Sjogrens Syndrome, UCTD or MCTD) as well as 15 healthy controls were recruited. EMPs and PMPs were quantified using flow cytometry. Associations between MP levels and objective functional vascular assessments were evaluated. ResultsSLE patients had significantly higher EMPs compared with healthy controls and SSc patients. Higher PMP levels were noted in SSc and primary RP when compared to healthy controls and ‘other CTD’ patients. A modest correlation was noted between EMP and PMP levels in healthy controls (Spearman r=0.6, p=0.017). This relationship appeared stronger in SLE (r=0.72, p<0.0001) and other CTD patients (r=0.75, p<0.0001). The association between EMPs and PMPs was notably less strong in SSc (r=0.45, p=0.014) and RP (r=0.37, p=0.15). A significantly lower EMP/PMP ratio was detected in SSc/RP patients in comparison to both healthy controls and SLE/other CTD patients. Higher EMP and PMP levels were associated with higher digital perfusion following cold challenge in SSc. In contrast, higher PMP (but not EMP) levels were associated with lower digital perfusion at both baseline and following cold challenge in primary RP. Higher PMP levels were associated with greater endothelial-independent dilation in patients with SLE. ConclusionMP populations differ across the spectrum of AIRDS, possibly reflecting differences in vascular cell injury and activation. MP levels are associated with functional assessments of vascular function and might have a role as novel vascular biomarkers in AIRDs. Significance and innovationsLevels of circulating endothelial and platelet microparticles differ between SSc/primary RP compared with SLE and other CTDs (UCTD, MCTD and Primary Sjogrens). MP release may occur within different vascular sites across these disease groups (macrovascular and microvascular).The association between circulating MP levels and objective assessment of macro- and microvascular dysfunction within these disease areas suggests that MPs might have a useful role as novel circulating biomarkers of vascular disease within the CTDs.

Evaluation of differences in carotid intima-media thickness in patients affected by systemic rheumatic diseases.

The objective of this study is to investigate whether rheumatic autoimmune diseases, systemic sclerosis (SSc) in particular, are associated with increased carotid intima-media thickness (C-IMT). A total of 108 clinical outpatients (93 females), mean age 51±14years suffering from CTD were consecutively enrolled. Patients were subdivided into the following two groups: (1) Systemic Sclerosis (SSc, 60 patients); (2) non-Systemic Sclerosis (NoSSc, 48 patients). No randomization was managed. All patients underwent structured clinical interview, physical examination, laboratory evaluation and two-dimensional echo-color Doppler of the carotid arteries to measure C-IMT and atherosclerotic plaques. Framingham risk score was also calculated. We also enrolled 108 healthy controls (HC), matched by sex and age. The primary outcome was to stratify cardiovascular risk of CTD patients. There were no significant differences between SSc and NoSSc patients regarding any of the demographics and traditional cardiovascular risk factors. Mean C-IMT was not significantly different between the whole CTD patients (0.86±0.13mm) and HC (0.83±0.13mm). C-IMT was significantly higher in SSc than in NoSSc group (0.91±0.1mm vs 0.80±0.14mm, p<0.001). Furthermore, C-IMT in SSc group was significantly higher than C-IMT in controls (0.91±0.1mm vs 0.83±0.13mm, p<0.001). C-IMT did correlate neither with disease activity nor with drug intake. SSc patients had a significant increase in C-IMT as compared to NoSSc patients and healthy controls.

SAT0320 Coexistence of Left Heart Diseases is Common among Patients with Connective Tissue Diseases-Associated Pulmonary Arterial Hypertension

BackgroundThe cause of connective tissue diseases-associated pulmonary hypertension (CTD-PH) includes left heart disease and interstitial lung disease (ILD) as well as pulmonary arterial hypertension (PAH). Careful differential diagnosis of them...

FRI0268 Detection of borderline pulmonary hypertension using exercise doppler echocardiography in patients with connective tissue diseases

BackgroundConnective tissue disease (CTD)-associated pulmonary arterial hypertension (PAH), particular in systemic sclerosis (SSc), has lower survival than idiopathic PAH.(1) Additionally, CTD often develops left ventricular (LV) dysfunction.(2) Exercise Doppler echocardiography...

Human Pentraxin 3 (PTX3) as a Novel Biomarker for the Diagnosis of Pulmonary Arterial Hypertension

BackgroundAlthough inflammation is an important feature of pulmonary arterial hypertension (PAH), the usefulness of local inflammatory markers as biomarkers for PAH is unknown. In this study, we tested whether plasma concentrations of human pentraxin 3 (PTX3), a local inflammatory marker, would be a useful biomarker for detecting PAH.MethodsPlasma PTX3 concentrations were evaluated in 50 PAH patients (27 with idiopathic PAH, 17 with PAH associated with connective tissue disease (CTD-PAH), and six with congenital heart disease), 100 age and sex-matched healthy controls, and 34 disease-matched CTD patients without PAH. Plasma concentrations of B-type natriuretic peptide (BNP) and C-reactive protein (CRP) were also determined.ResultsMean PTX3 levels were significantly higher in all PAH patients than in the healthy controls (4.40±0.37 vs. 1.94±0.09 ng/mL, respectively; P<0.001). Using a threshold level of 2.84 ng/mL, PTX3 yielded a sensitivity of 74.0% and a specificity of 84.0% for the detection of PAH. In CTD-PAH patients, mean PTX3 concentrations were significantly higher than in CTD patients without PAH (5.02±0.69 vs. 2.40±0.14 ng/mL, respectively; P<0.001). There was no significant correlation between plasma levels of PTX3 and BNP or CRP. Receiver operating characteristic (ROC) curves for screening PAH in patients with CTD revealed that PTX3 (area under the ROC curve 0.866) is superior to BNP. Using a PTX3 threshold of 2.85 ng/mL maximized true-positive and false-negative results (sensitivity 94.1%, specificity 73.5%).ConclusionPlasma concentrations of PTX3 may be a better biomarker of PAH than BNP, especially in patients with CTD.

冠動脈疾患患者における末梢動脈硬化の進展に関する研究 特に四肢・頚動脈動脈硬化病変合併の臨床像と冠疾患臨床背景との関連性について

Coexisted peripheral vascular disease (PVD: ankle/brachial pressure index A/PBI less than 0.75) or carotid artery disease (CTD: Brochenbrough's supraorbital Doppler flow analysis) were diagnosed by non-invasive testings, and correlated with clinical pictures and coronary backgrounds in 121 consecutive patients with confirmed coronary artery disease. PVD were found in 16.5%, CTD in 33.1% and both PVD and CTD in 9.9%. The mean age of PVD(+) patients was significantly higher than that of PVD(-) patients, furthermore CTD(+) patients displayed a significantly larger number of coronary risk factors than CTD(-) patients. Concerning the subjective symptoms, 20% of PVD(+) patients and 45% of CTD(+) patients were asymptomatic regarding PVD or CTD symptoms. However the degree of calcification of the aortic arch on the chest X-film (n = 104) significantly correlated with A/BPI. In patients with AMI, PVD patients showed significantly higher peak CK and CK-MB values than those in PVD(-) patients, which suggested large infarct size in coexistent PVD patients. With respect to the relationship between coronary risk factors, there was a statistically significant difference between PVD(+) patients and PVD(-) patients in terms of the obesity ratio in males as well as hypercholesterolemia ratio and obesity ratio in females. In CTD patients, significant differences between CTD(+) and CTD(-) patients were found with respect to the smoking ratio and obesity ratio in males as well as the smoking ratio in females. In whole study patients, A/BPI with lowered and A.I. (atherogenic index) increased significantly when patients possessed coronary risk factors equal or more than 4 items, which suggested the progression of PVD with increasing number of coronary risk factors.