Profibrotic VEGFR3-Dependent Lymphatic Vessel Growth in Autoimmune Valvular Carditis.

BACKGROUND: Rheumatic heart disease is the major cause of valvular heart disease in developing nations. Endothelial cells (ECs) are considered crucial contributors to rheumatic heart disease, but greater insight into their roles in disease progression is needed. METHODS: We used a Cdh5-driven EC lineage-tracing approach to identify and track ECs in the K/B.g7 model of autoimmune valvular carditis. Single-cell RNA sequencing was used to characterize the EC populations in control and inflamed mitral valves. Immunostaining and conventional histology were used to evaluate lineage tracing and validate single-cell RNA-sequencing findings. The effects of VEGFR3 (vascular endothelial growth factor receptor 3) and VEGF-C (vascular endothelial growth factor C) inhibitors were tested in vivo. The functional impact of mitral valve disease in the K/B.g7 mouse was evaluated using echocardiography. Finally, to translate our findings, we analyzed valves from human patients with rheumatic heart disease undergoing mitral valve replacements. RESULTS: Lineage tracing in K/B.g7 mice revealed new capillary lymphatic vessels arising from valve surface ECs during the progression of disease in K/B.g7 mice. Unsupervised clustering of mitral valve single-cell RNA-sequencing data revealed novel lymphatic valve ECs that express a transcriptional profile distinct from other valve EC populations including the recently identified PROX1 (Prospero homeobox protein 1)+ lymphatic valve ECs. During disease progression, these newly identified lymphatic valve ECs expand and upregulate a profibrotic transcriptional profile. Inhibiting VEGFR3 through multiple approaches prevented expansion of this mitral valve lymphatic network. Echocardiography demonstrated that K/B.g7 mice have left ventricular dysfunction and mitral valve stenosis. Valve lymphatic density increased with age in K/B.g7 mice and correlated with worsened ventricular dysfunction. Importantly, human rheumatic valves contained similar lymphatics in greater numbers than nonrheumatic controls. CONCLUSIONS: These studies reveal a novel mode of inflammation-associated, VEGFR3-dependent postnatal lymphangiogenesis in murine autoimmune valvular carditis, with similarities to human rheumatic heart disease.

Association of HLA class II DR/DQ alleles in children and adolescents with rheumatic heart disease from a tertiary care centre in North India.

INTRODUCTION: Rheumatic fever and RHD constitutes an important public health problem in India. The relatively low attack rate of RF, the high concordance rate for RF in monozygotic twins (19%) compared to dizygotic twins (2.5%), and the high familial incidence of RF suggest the involvement of host genetic factors in susceptibility to RF with consequential progression to RHD. OBJECTIVE: To study the association of HLA CLASS II DR/DQ alleles in children and adolescents with RHD from a tertiary care center in North India. METHODS: 30 RHD patients and 30 age and sex-matched controls were included in our study and blood samples for HLA typing were processed through LAB Type™ reverse SSO DNA typing method. The assignment of the HLA typing was based on a comparison with already published HLA gene sequences. RESULTS: The mean age of RHD patients and matched control groups were 12.97 ± 2.95 and 11.93 ± 3.23, respectively. In the cases and control group, males accounted for 63.3% and 50% of the patients respectively. A significant difference was found between the cases and controls for HLA DR∗ 15 (p-value 0.002), HLA DR∗ B4 (p-value 0.045), HLA DR∗ B5 (p-value 0.017), and HLA DQB1∗ 02 (p-value 0.005). CONCLUSION: Our study suggests that HLA class II haplotypes may provide insight into the molecular mechanism of RHD and be a useful tool in predicting the clinical outcome in RF patients, thereby affording new means of intervention or vaccine design. Larger studies are needed to address this in our population.

Profiling system-wide variations and similarities between Rheumatic Heart Disease and Acute Rheumatic Fever-A pilot analysis.

Rheumatic heart disease (RHD) continues to affect developing countries with low income due to the lack of resources and effective diagnostic techniques. Understanding the genetic basis common to both the diseases and that of progression from its prequel disease state, Acute Rheumatic Fever (ARF), would aid in developing predictive biomarkers and improving patient care. To gain system-wide molecular insights into possible causes for progression, in this pilot study, we collected blood transcriptomes from ARF (5) and RHD (5) patients. Using an integrated transcriptome and network analysis approach, we identified a subnetwork comprising the most significantly differentially expressed genes and most perturbed pathways in RHD compared to ARF. For example, the chemokine signaling pathway was seen to be upregulated, while tryptophan metabolism was found to be downregulated in RHD. The subnetworks of variation between the two conditions provide unbiased molecular-level insights into the host processes that may be linked with the progression of ARF to RHD, which has the potential to inform future diagnostics and therapeutic strategies. We also found a significantly raised neutrophil/lymphocyte ratio in both ARF and RHD cohorts. Activated neutrophils and inhibited Natural Killer cell gene signatures reflected the drivers of the inflammatory process typical to both disease conditions.

Rationale, Design, and the Baseline Characteristics of the RHDGen (The Genetics of Rheumatic Heart Disease) Network Study†.

BACKGROUND: The genetics of rheumatic heart disease (RHDGen) Network was developed to assist the discovery and validation of genetic variations and biomarkers of risk for rheumatic heart disease (RHD) in continental Africans, as a part of the global fight to control and eradicate rheumatic fever/RHD. Thus, we describe the rationale and design of the RHDGen study, comprising participants from 8 African countries. METHODS: RHDGen screened potential participants using echocardiography, thereafter enrolling RHD cases and ethnically-matched controls for whom case characteristics were documented. Biological samples were collected for conducting genetic analyses, including a discovery case-control genome-wide association study (GWAS) and a replication trio family study. Additional biological samples were also collected, and processed, for the measurement of biomarker analytes and the biomarker analyses are underway. RESULTS: Participants were enrolled into RHDGen between December 2012 and March 2018. For GWAS, 2548 RHD cases and 2261 controls (3301 women [69%]; mean age [SD], 37 [16.3] years) were available. RHD cases were predominantly Black (66%), Admixed (24%), and other ethnicities (10%). Among RHD cases, 34% were asymptomatic, 26% had prior valve surgery, and 23% had atrial fibrillation. The trio family replication arm included 116 RHD trio probands and 232 parents. CONCLUSIONS: RHDGen presents a rare opportunity to identify relevant patterns of genetic factors and biomarkers in Africans that may be associated with differential RHD risk. Furthermore, the RHDGen Network provides a platform for further work on fully elucidating the causes and mechanisms associated with RHD susceptibility and development.

New insight into the role of lipid metabolism-related proteins in rheumatic heart valve disease.

PURPOSE: The aim of this study was to determine the expression of lipid metabolism-related proteins in rheumatic heart valve disease (RHVD). METHODS: This retrospective study involved a total of 20 cases of moderate or severe rheumatic mitral valve stenosis and 4 cases of mitral regurgitation due to secondary causes from September 2018 to September 2021. The patients enrolled included 12 males and 12 females who underwent surgical excision of the mitral valve at the cardiac surgery department of Hainan General Hospital. The samples of mitral valve were collected during surgery treatment as the study group, and mitral valves collected from patients with ischemic heart disease were allocated into the control group. Hematoxylin-eosin (HE), oil red staining and immunohistochemical (IHC) staining were conducted to compare the expression of lipid metabolism-related proteins (ATP-binding cassette transporter A1 and acyl-coenzyme A: cholesterol acyltransferase-1), and real-time polymerase chain reaction (RT-PCR) was applied to compare the mRNA levels of ABCA1, ACAT1, and the inflammatory cytokines TNF-α, IL-10, and MCP-1. RESULTS: In general, the rheumatic mitral valve showed leaflet thickening along with border adhesions and visible yellow fats. Oil red O staining also revealed the abovementioned results as well as fat cells. Both ABCA1 and ACAT1 were expressed in the rheumatic mitral valve via IHC, whereas only ACAT1 showed a faint level of expression in the ischemic mitral valve with no expression of ABCA1. In addition, compared with the ischemic mitral valve, RT-PCT showed increased mRNA expression levels of ABCA1, ACAT1, and the inflammatory cytokines TNF-α, IL-10, and MCP-1 (P < 0.05). After dividing the RMs into two groups for RT-PCR, we found that the higher the expression of ABCA1 and ACAT1 was, the lower the relative expression of inflammatory factors. CONCLUSION: This study showed that adipose tissue, adipose cells, and lipid transport-related proteins were expressed strongly in the rheumatic mitral valve, suggesting that adipose tissue formation might be one of the important pathways in the pathology of rheumatic heart disease. In addition, adipose tissue and adipocytes were also involved in the inflammatory process. These data provide new insight into pathological mechanisms in rheumatic heart disease.

Gender differences in genotypic distribution of endothelin-1 gene and endothelin receptor A gene in pulmonary hypertension associated with rheumatic mitral valve disease.

INTRODUCTION: The female gender is a risk factor for idiopathic pulmonary arterial hypertension. However, it is unknown whether females with rheumatic mitral valve disease are more predisposed to develop pulmonary hypertension compared to males. AIM: We aimed to investigate whether there was a difference in genotypic distribution of endothelin-1 (ET-1) and endothelin receptor A (ETA) genes between female and male patients of pulmonary hypertension associated with rheumatic mitral valve disease (PH-MVD). METHODS: We compared prevalence of ET-1 gene (Lys198Asn) and ETA gene (His323His) polymorphisms according to gender in 123 PH-MVD subjects and 123 healthy controls. RESULTS: The presence of mutant Asn/Asn and either mutant Asn/Asn or heterozygous Lys/Asn genotypes of Lys198Asn polymorphism when compared to Lys/Lys in females showed significant association with higher risk (odds ratio [OR] 4.5; p =0.007 and OR 2.39; p =0.02, respectively). The presence of heterozygous C/T and either mutant T/T or heterozygous C/T genotypes of His323His polymorphism when compared to wild C/C genotype in females showed a significant association with higher risk (OR 1.96; p =0.047 and OR 2.26; p =0.01, respectively). No significant difference was seen in genotypic frequencies in males between PH-MVD subjects and controls. Logistic regression analysis showed that mutant genotype Asn/Asn (p =0.007) and heterozygous genotype Lys/Asn of Lys198Asn polymorphism (p =0.018) were independent predictors of development of PH in females. CONCLUSIONS: ET-1 and ETA gene polymorphisms were more prevalent in females than males in PH-MVD signifying that females with rheumatic heart disease may be more susceptible to develop PH.

A study on the association of IL-10 promoter polymorphisms with rheumatic heart disease in Turkish female patients.

OBJECTIVE: Rheumatic heart disease (RHD) is an inflammatory disease that develops after streptococcal infections. The pleiotropic effect of IL-10 plays a role in the regulation of immune system responses. However, impaired IL-10 expression or signaling can impair antigen clearance during acute bacterial infections, creating a favorable environment for persistent inflammation. More than 30 single nucleotide polymorphisms (SNPs) in the promoter region of the gene encoding IL-10, which has a highly polymorphic structure, and the relationship of these SNPs with increased and decreased cytokine expression have been reported. Therefore, it is assumed that these polymorphisms may be predictors of an individual's susceptibility to RHD. In this study, we aimed to evaluate the relationship between sensitivity of IL-10 variants (-1082, -819, -592) and severity of RHD in the Turkish population. METHODS: In this case-control study, IL-10 promoter gene variants of the study group consisting of 390 women were examined using the TaqMan 5' allelic discrimination test method. RESULTS: There was no statistically significant difference between study groups in terms of IL-10 (-1082, -819, -592) genotypes. In patients with mild and severe valve damage, there was no statistically significant difference in terms of IL-10 (-1082, -819, -592) genotype distributions and allele frequencies (p>0.05). CONCLUSION: Our findings suggest that IL-10 (-1082, -819, -592) variants are not associated with the pathogenesis and severity of the disease in women in the Turkish population. In the Turkish population, IL-10 (-1082, -819, -592) variants cannot be recommended as a suitable genetic marker for RHD.

Association of HLA-DRB1 Alleles with Rheumatic Fever and Rheumatic Heart Disease: A Meta-analysis.

BACKGROUND: Rheumatic fever (RF) and its sequel rheumatic heart disease (RHD) is an autoimmune disease caused by an abnormal host immune response to group A streptococcus (GAS) infection. The HLA class II molecules are entailed in immune-mediated infectious, inflammatory, and autoimmune diseases including RHD. However, HLA class II genes are reported to be associated with RF/RHD across different populations with a very little consistency. OBJECTIVE: The aim of the study is to investigate the association between HLA class II genes and RF/RHD by meta-analysis. METHODS: A comprehensive literature search was conducted to identify all relevant case-control studies published before December 31, 2019. The data were extracted using standardized form and pooled odds ratio (OR) with 95% confidence interval (CI) are calculated to assess the strength of the association between HLA class II genes and RF/RHD. RESULTS: Thirteen studies for HLA-DRB1 alleles (1065 patients and 1691 controls) and eight studies for HLA-DQB1 alleles (644 patients and 1088 controls) were finally included. The meta-analysis showed a significantly higher frequency of HLA-DRB1*07 allele (OR = 1.68, P < .0001) in RF/RHD patients when compared to controls, while the frequency of HLA-DRB1*15 allele (OR = 0.60, P = .03) was significantly lower in RF/RHD patients than in controls. However, there were no significant differences in the frequency of HLA-DQB1 alleles between RF/RHD patients and controls. CONCLUSIONS: The results of the meta-analysis suggest that the differential presentation of autoimmune peptides by HLA-DRB1*07 (susceptible) and HLA-DRB1*15 (protective) alleles with different affinities may play a crucial role in the pathogenesis of RF/RHD.

Effect of Sox9 on TGF-ß1-mediated atrial fibrosis.

Atrial fibrosis is a crucial mechanism responsible for atrial fibrillation (AF). Sex-determining region Y-box containing gene 9 (Sox9) plays a pivotal role in fibrosis of many organs such as the skin, kidney, and liver. However, there are few studies about the occurrence and maintenance of Sox9 in atrial fibrosis. In this study, we investigated the role of Sox9 in the fibrotic phenotype of human atrial tissues and rat atrial fibroblasts in vitro. In the human right atrial tissue, Masson's trichrome staining, immunofluorescence, real-time quantitative polymerase chain reaction, and western blot analysis were carried out to explore the relationship between Sox9 and atrial fibrosis at the morphological, functional, and molecular levels. In cultured atrial fibroblasts, Sox9 was overexpressed by adenovirus or depleted by siRNA, and then, recombinant human transforming growth factor (TGF)-ß1 was added. Immunofluorescence analysis, western blot analysis, Transwell assay, and scratch assay were used to analyze the cells. In patient atrial tissues, Sox9 was increased with worsened atrial fibrosis, and this increase was related to AF severity. In rat atrial fibroblasts, Sox9 was promoted by TGF-ß1, and the α-smooth muscle actin (α-SMA) protein level and the ability of cell migration were increased after Sox9 overexpression by adenovirus, while the α-SMA protein level and the cell migration ability were decreased after Sox9 depletion by siRNA. In conclusion, Sox9 is involved in the regulation of fibrosis in the atria and may be located downstream of TGF-ß1. Our findings may provide a new perspective to treat atrial fibrosis during AF.

Susceptibility of CTLA-4 -1661A/G polymorphism towards severity of rheumatic heart disease.

AIM: Genetic contribution in acute rheumatic fever (ARF)/rheumatic heart disease (RHD) has been suggested but not according to severity of the valve involvement. This study attempts to identify the relevance of CTLA-4 polymorphism with severity of the disease. METHODS: In a case-control design, 291 healthy controls and 83 patients were genotyped for association between RHD and single-nucleotide polymorphisms -1661A/G of CTLA-4. RESULTS: Segregation of patients on the basis of severity i.e., MVL (Mitral Valve Lesion) and CVL (Combined Valve Lesion) revealed that the frequency of CTLA-4 -1661G allele depleted as the disease progressed to CVL (p < 0.05). Patients in the age group of 31-45 years were significantly more susceptible (p < 0.046). Whereas, female patients were more susceptible than the male patients. CONCLUSION: Our study suggests the risk associated with decreased frequency of CTLA-4 -1661G allele in the CVL group and in females.

Coordination of PRKCA/PRKCA-AS1 interplay facilitates DNA methyltransferase 1 recruitment on DNA methylation to affect protein kinase C alpha transcription in mitral valve of rheumatic heart disease.

In the present study, mitral valve tissues from three mitral stenosis patients with RHD by valve replacement and two healthy donors were harvested and conducted DNA methylation signature on PRKCA by MeDIP-qPCR. The presence of hypomethylated CpG islands at promoter and 5' terminal of PRKCA was observed in RHD accompanied with highly expressed PRKCA and down-regulated antisense long non-coding RNA (lncRNA) PRKCA-AS1 compared to health control. Furthermore, the enrichments of DNMT1/3A/3B on PRKCA were detected by ChIP-qPCR assay in vivo and in human cardiomyocyte AC16 and RL-14 cells exposed to TNF-α in vitro, and both demonstrated that DNMT1 substantially contributed to DNA methylation. Additionally, PRKCA-AS1 was further determined to bind with promoter of PRKCA via 5' terminal and interact with DNMT1 via 3' terminal. Taken together, our results illuminated a novel regulatory mechanism of DNA methylation on regulating PRKCA transcription through lncRNA PRKCA-AS1, and shed light on the molecular pathogenesis of RHD occurrence.

Novel FCN2 Variants and Haplotypes are Associated with Rheumatic Heart Disease.

Ficolins are pattern recognition molecules that are involved in innate immune defense. Ficonin-2 (FCN2) has a specific affinity for lipoteichoic acid present in the cell wall of Streptococcus pyogenes, an etiological agent for rheumatic heart disease (RHD). We have estimated FCN2 serum levels and analyzed the functional variants of FCN2 in 400 RHD patients, 617 healthy controls, and 581 individuals belonged to various ethnic populations, who are inhabited in various geographical regions of India. Our study revealed that the FCN2 -986A and +6359T alleles were the risk factors for RHD susceptibility (p = 0.0007 for -986G>A; p = 0.0004 for +6359C>T). The haplotype AGGT (p = 0.0024) was observed to be a risk factor for RHD susceptibility, and the haplotype GGAC (p = 0.002) was found to confer protection against RHD. The level of serum FCN2 was significantly higher in controls (p < 0.0001) and in controls with GGAC haplotypes (p < 0.0001). The frequency of the risk alleles -986A and +6359T was found to be more prevalent in Northern and North-Western (Indo-European) India. The protective GGAC haplotype was found more prevalent in Eastern (Tibeto-Burman) and South-Western (Dravidian) India. Alleles -986A and +6359T were in positive correlation with the prevalence of RHD (regression coefficient = 1.84 and 1.94, respectively), whereas GGAC haplotype was in negative correlation with prevalence of RHD (regression coefficient = -1.71). In conclusion, we found that low level of serum ficolin-2 is significantly associated with RHD. Further, FCN2 -986A and +6359T alleles and AGGT haplotype are associated with increased susceptibility to RHD, while GGAC haplotype is associated with moderate protection against RHD.

Interference with the expression of S1PR1 or STAT3 attenuates valvular damage due to rheumatic heart disease.

Rheumatic heart disease (RHD) affects numerous individuals annually; however, its pathogenesis remains unclear. The sphingosine 1­phosphate receptor 1 (S1PR1) and signal transducer and activator of transcription 3 (STAT3) have recently been shown to be involved in valvular damage via the promotion of the differentiation of T helper 17 (Th17) cells during the development of RHD­induced valvular damage. The present study investigated whether altering the expression of S1PR1 or STAT3 attenuates valvular damage due to RHD. Inactivated group A streptococcus (GAS) was used to establish a rat model of RHD. Recombinant adeno­associated viral vectors carrying an S1PR1 overexpression sequence were used to overexpress S1PR1. STAT3 small interfering RNA (STAT3­siRNA) was used to inhibit STAT3 expression. Reverse transcription­quantitative PCR (RT­qPCR) was performed to detect the mRNA expression of S1PR1, STAT3, collagen type III α1 chain (Col3a1) and fibroblast­specific protein 1. Western blotting (WB) and immunohistochemistry were used to detect the levels of S1PR1, STAT3, phosphorylated (p­) STAT3, and retinoic acid­related orphan receptor Î³T (RORγt) proteins. Enzyme­linked immunosorbent assays (ELISAs) and immunohistochemistry were used to detect the levels of interleukin (IL)­6 and IL­17. Hematoxylin and eosin (H&E) staining and Sirius Red staining were performed to evaluate the degree of inflammation and fibrosis in the valvular tissues. S1PR1 expression was decreased in the valvular tissues of the rats with RHD. The levels of IL­6, IL­17 and p­STAT3 in the rats with RHD were increased. The degree of valvular inflammation and fibrosis in the rats with RHD was also increased. The overexpression of S1PR1 and the inhibition of STAT3 reduced the total p­STAT3 level, resulting in decreased levels of IL­6, IL­17 and RORγt, and a reduced degree of valvular inflammation and fibrosis. These results suggest that the expression of S1PR1 and STAT3 may be involved in valvular tissue damage due to RHD. Thus, strategies designed to interfere with the expression of S1PR1 or STAT3 may affect the expression of Th17 cell­related cytokines and may thus attenuate valvular damage due to RHD.

Association of Novel Locus With Rheumatic Heart Disease in Black African Individuals: Findings From the RHDGen Study.

Importance: Rheumatic heart disease (RHD), a sequela of rheumatic fever characterized by permanent heart valve damage, is the leading cause of cardiac surgery in Africa. However, its pathophysiologic characteristics and genetics are poorly understood. Understanding genetic susceptibility may aid in prevention, control, and interventions to eliminate RHD. Objective: To identify common genetic loci associated with RHD susceptibility in Black African individuals. Design, Setting, and Participants: This multicenter case-control genome-wide association study (GWAS), the Genetics of Rheumatic Heart Disease, examined more than 7 million genotyped and imputed single-nucleotide variations. The 4809 GWAS participants and 116 independent trio families were enrolled from 8 African countries between December 31, 2012, and March 31, 2018. All GWAS participants and trio probands were screened by use of echocardiography. Data analyses took place from May 15, 2017, until March 14, 2021. Main Outcomes and Measures: Genetic associations with RHD. Results: This study included 4809 African participants (2548 RHD cases and 2261 controls; 3301 women [69%]; mean [SD] age, 36.5 [16.3] years). The GWAS identified a single RHD risk locus, 11q24.1 (rs1219406 [odds ratio, 1.65; 95% CI, 1.48-1.82; P = 4.36 × 10-8]), which reached genome-wide significance in Black African individuals. Our meta-analysis of Black (n = 3179) and admixed (n = 1055) African individuals revealed several suggestive loci. The study also replicated a previously reported association in Pacific Islander individuals (rs11846409) at the immunoglobulin heavy chain locus, in the meta-analysis of Black and admixed African individuals (odds ratio, 1.16; 95% CI, 1.06-1.27; P = 1.19 × 10-3). The HLA (rs9272622) associations reported in Aboriginal Australian individuals could not be replicated. In support of the known polygenic architecture for RHD, overtransmission of a polygenic risk score from unaffected parents to affected probands was observed (polygenic transmission disequilibrium testing mean [SE], 0.27 [0.16] SDs; P = .04996), and the chip-based heritability was estimated to be high at 0.49 (SE = 0.12; P = 3.28 × 10-5) in Black African individuals. Conclusions and Relevance: This study revealed a novel candidate susceptibility locus exclusive to Black African individuals and an important heritable component to RHD susceptibility in African individuals.

The clinical value of ficolin-3 gene polymorphism in rheumatic heart disease. An Egyptian adolescents study.

OBJECTIVE: Ficolin-3 is one of the innate immunity molecules that was thought to play a pivotal role in Streptococcus pyogenes autoimmunity and its complications; rheumatic fever (RF) and rheumatic heart disease (RHD). We aimed to disclose if there is an association between ficolin-3 (FCN3) gene polymorphisms (rs4494157 and rs10794501) and RF with or without RHD for the first time in Egyptian adolescents. RESULTS: Serum ficolin-3 level was significantly elevated in patients suffering from RF with and without RHD in comparison with control. Regarding FCN3 gene (rs4494157) polymorphism, a significant correlation was found between the A allele and the susceptibility to RF with or without RHD (OR = 2.93, P = 0.0002 and OR = 2.23, P = 0.008 respectively). Besides, AA homozygous genotype showed a significant association with RHD risk (OR = 3.47, P = 0.026). Patients carrying the A allele (CA + AA) had significantly higher serum ficolin-3 than those carrying the CC genotype (P ˂ 0.0001). While the frequency of (rs10794501) polymorphism revealed no significant differences between the controls and RF patients with or without RHD (OR = 1.43, P = 0.261 and OR = 1.48, P = 0.208 respectively).

Cytokine gene functional polymorphisms and phenotypic expression as predictors of evolution from latent to clinical rheumatic heart disease.

INTRODUCTION: Inflammation associated with rheumatic heart disease (RHD) is influenced by gene polymorphisms and inflammatory cytokines. There are currently no immunologic and genetic markers to discriminate latent versus clinical patients, critical to predict disease evolution. Employing machine-learning, we searched for predictors that could discriminate latent versus clinical RHD, and eventually identify latent patients that may progress to clinical disease. METHODS: A total of 212 individuals were included, 77 with latent, 100 with clinical RHD, and 35 healthy controls. Circulating levels of 27 soluble factors were evaluated using Bio-Plex ProTM® Human Cytokine Standard 27-plex assay. Gene polymorphism analyses were performed using RT-PCR for the following genes: IL2, IL4, IL6, IL10, IL17A, TNF and IL23. RESULTS: Serum levels of all cytokines were higher in clinical as compared to latent RHD patients, and in those groups than in controls. IL-4, IL-8, IL-1RA, IL-9, CCL5 and PDGF emerged in the final multivariate model as predictive factors for clinical, compared with latent RHD. IL-4, IL-8 and IL1RA had the greater power to predict clinical RHD. In univariate analysis, polymorphisms in IL2 and IL4 were associated with clinical RHD and in the logistic analysis, IL6 (GG + CG), IL10 (CT + TT), IL2 (CA + AA) and IL4 (CC) genotypes were associated with RHD. CONCLUSION: Despite higher levels of all cytokines in clinical RHD patients, IL-4, IL-8 and IL-1RA were the best predictors of clinical disease. An association of polymorphisms in IL2, IL4, IL6 and IL10 genes and clinical RHD was observed. Gene polymorphism and phenotypic expression of IL-4 accurately discriminate latent versus clinical RHD, potentially instructing clinical management.

miRNA-1183-targeted regulation of Bcl-2 contributes to the pathogenesis of rheumatic heart disease.

To determine whether up-regulation of miR-1183 targeting the gene for anti-apoptotic factor, B-cell lymphoma 2 (BCL-2) contributes to apoptosis in patients with rheumatic heart disease (RHD). Peripheral blood samples were isolated for miR-1183 characterization. The function of miRNA-1183 in RHD using miRNA mimic on PBMCs and THP-1 cell models. The binding of miR-1183 and Bcl-2 gene was confirmed by luciferase activity test. We also measured expression levels of BCL-2 in heart valve tissue from patients with RHD using ELISA and immunohistochemistry. In silico analysis and reporter gene assays indicated that miR-1183 directly targets the mRNA encoding BCL-2. It is found that miR-1183 binds directly to the 3'UTR of the BCL-2 mRNA and down-regulates the mRNA and protein levels of BCL-2. Overexpression of miR-1183 in RHD patients and cell lines down-regulated BCL-2 expression and induced apoptosis. With the progression of the disease, the expression of BCL-2 in the heart valve tissue of patients with RHD decreased. MiRNA-1183 is up-regulated in RHD and induces cardiac myocyte apoptosis through direct targeting and suppression of BCL-2, both of which might play important roles in RHD pathogenesis. During the compensatory period of RHD, up-regulated miR-1183 destroyed the balance of apoptosis proteins (Bax and BAK) in Bcl-2 family, enhance the apoptosis cascade reaction and reduce the anti apoptosis effect. The significantly higher expression levels of miR-1183 appear to play distinct roles in RHD pathogenesis by regulation BCL-2, possibly affecting myocardial apoptosis and remodeling in the context of RHD.

The Human Leukocyte Antigen Locus and Rheumatic Heart Disease Susceptibility in South Asians and Europeans.

Rheumatic heart disease (RHD), an autoinflammatory heart disease, was recently declared a global health priority by the World Health Organization. Here we report a genome-wide association study (GWAS) of RHD susceptibility in 1,163 South Asians (672 cases; 491 controls) recruited in India and Fiji. We analysed directly obtained and imputed genotypes, and followed-up associated loci in 1,459 Europeans (150 cases; 1,309 controls) from the UK Biobank study. We identify a novel susceptibility signal in the class III region of the human leukocyte antigen (HLA) complex in the South Asian dataset that clearly replicates in the Europeans (rs201026476; combined odds ratio 1.81, 95% confidence intervals 1.51-2.18, P = 3.48×10-10). Importantly, this signal remains despite conditioning on the lead class I and class II variants (P = 0.00033). These findings suggest the class III region is a key determinant of RHD susceptibility offering important new insight into pathogenesis while partly explaining the inconsistency of earlier reports.

Genetic variants in rheumatic fever and rheumatic heart disease.

Genetic association studies in rheumatic heart disease (RHD) have the potential to contribute toward our understanding of the pathogenetic mechanism, and may shed light on controversies about RHD etiology. Furthermore, genetic association studies may uncover biomarkers that can be used to identify susceptible individuals, and contribute toward developing vaccine and novel therapeutic targets. Genetic predisposition to rheumatic fever and RHD has been hypothesized by findings from familial studies and observed associations between genes located in the human leukocyte antigens on chromosome 6p21.3 and elsewhere in the genome. We sought to summarize, from published Genetic association studies in RHD, evidence on genetic variants implicated in RHD susceptibility. Using HuGENet™ systematic review methods, we evaluated 66 studies reporting on 42 genes. Existing meta-analyses of candidate gene studies suggest that TGF-ß1 [rs1800469], and IL-1ß [rs2853550] single nucleotide polymorphisms (SNPs) contribute to susceptibility to RHD, whereas the TNF-α [rs1800629 and rs361525], TGF-ß1 [rs1800470 and rs4803457], IL-6 [rs1800795], IL-10 [rs1800896] were not associated with RHD. However, candidate gene studies in RF/RHD are relatively small, thus lacking statistical power to identify reliable and reproducible findings, emphasizing the need for large-scale multicenter studies with different populations.