Supplementary Material for: Transcriptional Effects of E3 Ligase Nrdp1 on Hypertrophy in Neonatal Rat Cardiomyocytes by Microarray and Integrated Gene Network Analysis

<i>Objective:</i> Neuregulin receptor degradation protein-1 (Nrdp1) is a novel E3 ubiquitin ligase, and we have previously shown that overexpression of Nrdp1 increased cardiomyocyte injury. However, the role of Nrdp1 in myocardial hypertrophy is unclear. In the present study, we clarified the molecular mechanisms of angiotensin II (Ang II)-induced cardiomyocyte hypertrophy regulated by Nrdp1 based on genome-wide transcriptional analysis. <i>Methods:</i> Neonatal rat cardiomyocytes were infected with adenoviruses containing green fluorescent protein (Ad-GFP) or wild-type Nrdp1 (Ad-Nrdp1), and then treated with Ang II for 36 h. Detection of differentially expressed genes was achieved with an Affymetrix Rat Gene 2.0 Array and Cluster and Java TreeView software. <i>Results and Conclusion:</i> Microarray data analysis demonstrated that Nrdp1 overexpression affected the expression of 12,140 mRNA genes in Ang II-induced cardiomyocyte hypertrophy, including the upregulation of 12,044 and the downregulation of 96. Gene ontology and globe signal transduction network analysis showed that Nrdp1 affected the expression of many genes related to stimulus response, the cell receptor pathway, and cell growth. Pathway network analysis identified myocardial metabolism, DNA replication, and the cell cycle as the most important pathways targeted by Nrdp1. lncRNA-mRNA coexpression network analysis showed that two core lncRNAs, NONRATT057160 and NONRATT054243, were involved in cardiomyotrophy regulated by Nrdp1 in cardiomyocytes. Taken together, these data provide compelling clues for further exploration of the function of Nrdp1 in heart disease

Supplementary Material for: miR-499 Ameliorates Podocyte Injury by Targeting Calcineurin in Minimal Change Disease

<b><i>Background:</i></b> Podocyte injury is a hallmark of minimal change disease (MCD). Calcineurin inhibitors have been widely used in the current treatment of MCD, and miR-499 may target calcineurin. We aimed to study the function of miR-499 in MCD and test whether miR-499 delivery can improve MCD. <b><i>Methods:</i></b> An MCD mouse model was generated using puromycin aminonucleoside (PAN). MiR-499 was delivered using lentiviruses. Biochemical indicators including serum albumin, triglyceride, cholesterol, and 24-h urine protein were determined. Targets of miR-499 were confirmed using reporter gene activity assays. The ultrastructure of podocytes was analyzed using transmission electron microscopy. <b><i>Results:</i></b> MiR-499 significantly improved MCD-related symptoms and signs. Foot-process effacement was caused by PAN and partially reversed by miR-499. We identified that both CnAα and CnAβ were targets of miR-499, and were overexpressed in the presence of PAN. However, miR-499 reduced the expression of CnAα and CnAβ, leading to a decreased activity of calcineurin signaling in mouse podocytes in vitro and in vivo. In addition, miR-499 recovered PAN-induced reduction of cell viability. <b><i>Conclusions:</i></b> MiR-499 ameliorated podocyte injury by targeting CnAα and CnAβ in a PAN-induced MCD mouse model. Delivery of miR-499 can be a novel strategy for MCD treatment

Supplementary Material for: Aortic Regurgitation Requiring Unplanned Surgery following Transcatheter Closure of Ventricular Septal Defect in Children: Incidence and Risk Factors

Introduction: Our aim is to investigate the incidence and risk factors for aortic regurgitation (AR) requiring unplanned surgery after transcatheter closure of ventricular septal defect (VSD) in children. Methods: Medical records of 876 children with VSD who underwent transcatheter closure from July 2009 to September 2018 in our hospital were retrospectively reviewed. Groups with and without new-onset or increasing AR requiring unplanned surgery were compared. Univariate and multivariate analysis were used to identify the possible risk factors. Smoothing plot and threshold effect analysis were carried to find the relationship between possible factors and risk of new-onset or increasing AR. Results: A total of 29 children (3.3%) underwent unplanned surgery after transcatheter closure owing to new-onset or increasing AR, including 6 children with new-onset AR and 23 children with increasing AR. Multivariate regression analysis revealed that preoperative mild AR (OR: 60.39, 95%CI 11.53-316.30 ，P＜0.001), larger ratio between diameter to body surface area (OR: 1.25, 95%CI 1.01-1.55，P=0.039), intracristal VSD (OR: 34.09, 95%CI 4.07-285.65，P＜0.001, and shorter distance from the upper edge of defect to the aortic valve (or the sub-aortic rim) (OR: 0.12, 95%CI 0.05-0.27，P＜0.001) were risk factors for new-onset or increasing AR requiring unplanned surgery. And, low risk of AR after muscular ventricular septal defect transcatheter closure was found. An L-shaped non-linear relationship between the sub-aortic rim and risk of new-onset or increasing AR was observed, and the risk of new-onset or increasing AR increased with the sub-aortic rim up to the turning point (2 mm) (adjusted OR:0.00, 95 % CI 0.00-0.08; P=0.001). With a median time of 7.3 years’ follow-up, no new-onset or increasing AR has been found for children who initially didn’t have unplanned surgery. Conclusion: Preoperative mild AR, larger ratio between diameter to body surface area, intracristal VSD and shorter distance of the sub-aortic rim (especially less than 2 mm) could increase the risk of new-onset or increasing AR requiring unplanned surgery after transcatheter closure of VSD

Supplementary Material for: Uniparental Disomy of Chromosome 15 in Two Cases by Chromosome Microarray: A Lesson Worth Thinking

<p>Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are neurogenetic disorders caused by loss of function of the imprinted genes at 15q11q13. A 5-7 Mb paternal/maternal deletion of chromosomal region 15q11.2q13 is the major genetic cause of PWS/AS, but in a small group of patients, the PWS/AS phenotype can result from maternal/paternal uniparental disomy (UPD) of chromosome 15. Various mechanisms leading to UPD include gametic complementation, trisomy rescue, and compensatory UPD, which can be inferred from the pattern of uniparental heterodisomy (heteroUPD) or uniparental isodisomy (isoUPD). However, heteroUPD and isoUPD, especially mixed heteroUPD and isoUPD, are very rare in patients with PWS/AS. Here, we report 2 children with PWS/AS caused by mixed segmental heteroUPD 15 and isoUPD 15 which failed to be identified by chromosome microarray (CMA) but could be detected by other molecular genetic methods. The present report unravels the mechanism of mixed iso/heteroUPD 15 in PWS/AS and phenotype-genotype correlations. Moreover, our study suggests that CMA is prone to misdiagnosis for imprinting disorders such as PWS/AS, though it is considered a highly useful tool for copy number variations. As a result, other molecular detection methods, such as methylation analysis and STR marker analysis for UPD, should be supplementary used in this situation.</p

Supplementary Material for: Enhanced Prophylactic and Therapeutic Effects of Polylysine-Modified Ara h 2 DNA Vaccine in a Mouse Model of Peanut Allergy

<p><b><i>Background:</i></b> The prevalence of food allergy has been increasing, but treatment is very limited. DNA vaccination has been recognized as a promising method for the treatment of allergic diseases; however, poor immunogenicity has hindered its application. <b><i>Methods:</i></b> BALB/c mice were intradermally injected with plasmid DNA encoding the peanut protein Ara h 2 (pAra h 2) or pAra h 2 pretreated with poly-L- lysine (PLL) before or after sensitization with Ara h 2 protein. Ara h 2-specific antibodies were measured by ELISA. CD207+ dendritic cells (DCs) and Treg cells in draining lymph nodes were analyzed by flow cytometry after DNA immunization, and cytokine production in splenocytes was also analyzed. <b><i>Results:</i></b> In the prophylactic study, pretreatment with pAra h 2 or PLL-pAra h 2 resulted in lower levels of Ara h 2-specific IgG1, IgG2a, and IgE after sensitization with Ara h 2 protein, and mice in the PLL-pAra h 2 group had a significantly lower level of antibodies than those in the pAra h 2 group. In the treatment study, intradermal injection with pAra h 2 or PLL-pAra h 2 after Ara h 2 protein sensitization significantly decreased the level of Ara h 2-specific antibodies, and PLL- pAra h 2 had stronger effects than pAra h 2. There were increased numbers of CD207+ DCs and Treg cells in the mice receiving intradermal injection with PLL-pAra h 2, and splenocytes from PLL-pAra h 2-treated mice secreted increased levels of IFN-γ and IL-10. <b><i>Conclusions:</i></b> Modification of pAra h 2 with PLL improved its prophylactic and therapeutic effects in peanut-allergic mice.</p

Supplementary Material for: Lysophosphatidic Acid Induces Ligamentum Flavum Hypertrophy Through the LPAR1/Akt Pathway

<b><i>Background/Aims:</i></b> Hypertrophic ligamentum flavum (LF) is a major cause of lumbar spinal stenosis. Our previous work showed that high levels of lysophosphatidic acid (LPA) expression are positively correlated with LF hypertrophy. This study aimed to further unveil how LPA regulates LF hypertrophy <b><i>Methods:</i></b> We studied LPAR1 expression in human LF cells using PCR and western blotting. Cell viability cell cycle, apoptosis rate and molecular mechanisms were assayed in LPAR1 knockdown or overexpression LF cells. LF hypertrophy and the molecular mechanism was confirmed in human samples and in <i>in vivo</i> studies. <b><i>Results:</i></b> The expression of LPA and its receptor LPAR1 is significantly higher in tissues or cells harvested from hypertrophic LF compared to healthy controls. Moreover, LPA promoted LF cell proliferation by interacting with LPAR1. This conclusion is supported by the fact that depletion or overexpression of LPAR1 changed the effect of LPA on LF cell proliferation. LPA also inhibits apoptosis in LF cells through the receptor LPAR1. Importantly, we demonstrated that the LPA-LPAR1 interaction initiated Akt phosphorylation and determined cell proliferation and apoptosis. Our <i>in vitro</i> findings were supported by our <i>in vivo</i> evidence that lyophilized LPA significantly induced LF hypertrophy via the LPAR1-Akt signaling pathway. More importantly, targeted inhibition of LPAR1 by Ki16425 with a gel sponge implant effectively reduced LPA-associated LF hypertrophy. Taken together, these data indicate that LPA binds to the receptor LPAR1 to induce LF cell proliferation and inhibit apoptosis by activating AKT signaling cascades. Targeting this signaling cascade with Ki16425 is a potential therapeutic strategy for preventing LF hypertrophy. <b><i>Conclusion:</i></b> LPA-LPAR1-Akt activation is positively correlated with the proliferation and survival of LF cells. LPAR1 could be a target for new drugs and the development of new therapeutic methods for treating LF hypertrophy

Supplementary Material for: A Sequence Kernel Association Test for Dichotomous Traits in Family Samples under a Generalized Linear Mixed Model

<b><i>Objective:</i></b> The existing methods for identifying multiple rare variants underlying complex diseases in family samples are underpowered. Therefore, we aim to develop a new set-based method for an association study of dichotomous traits in family samples. <b><i>Methods:</i></b> We introduce a framework for testing the association of genetic variants with diseases in family samples based on a generalized linear mixed model. Our proposed method is based on a kernel machine regression and can be viewed as an extension of the sequence kernel association test (SKAT and famSKAT) for application to family data with dichotomous traits (F-SKAT). <b><i>Results:</i></b> Our simulation studies show that the original SKAT has inflated type I error rates when applied directly to family data. By contrast, our proposed F-SKAT has the correct type I error rate. Furthermore, in all of the considered scenarios, F-SKAT, which uses all family data, has higher power than both SKAT, which uses only unrelated individuals from the family data, and another method, which uses all family data. <b><i>Conclusion:</i></b> We propose a set-based association test that can be used to analyze family data with dichotomous phenotypes while handling genetic variants with the same or opposite directions of effects as well as any types of family relationships

Supplementary Material for: Narrowband Ultraviolet B Interferes with Gene Expression in the Peripheral Blood T Cells of Patients with Psoriasis

<b><i>Background:</i></b> Psoriasis pathogenesis and development are closely related to abnormal T cell activity. Narrowband ultraviolet B (NB-UVB) treatment markedly improves skin lesions in psoriasis. <b><i>Objectives:</i></b> To investigate differential gene expression in psoriasis and to understand the possible mechanisms of NB-UVB therapy for psoriasis. <b><i>Methods:</i></b> The mRNA expression profiles and differentially expressed genes from peripheral blood T cells of psoriatic patients before and after NB-UVB treatment were examined using RNA sequencing and validated by real-time reverse-transcription polymerase chain reaction. <b><i>Results:</i></b> A total of 129 genes were differentially expressed in the peripheral blood T cells of psoriatic patients: 83 genes were downregulated and 46 were upregulated in psoriatic patients compared to those of healthy subjects. These genes were enriched in intracellular membrane-bound organelles, membrane-bound organelles and the nucleus, and are involved in the cell cycle, apoptosis, inflammation and other processes. These changes are reversed in psoriatic patients with good clinical outcomes following NB-UVB treatment. <b><i>Conclusion:</i></b> NB-UVB treatment has beneficial effects on local psoriatic lesions, possibly due to its effect on peripheral blood T cell gene expression

Supplementary Material for: Rare-Variant Kernel Machine Test for Longitudinal Data from Population and Family Samples

<b><i>Objective:</i></b> The kernel machine (KM) test reportedly performs well in the set-based association test of rare variants. Many studies have been conducted to measure phenotypes at multiple time points, but the standard KM methodology has only been available for phenotypes at a single time point. In addition, family-based designs have been widely used in genetic association studies; therefore, the data analysis method used must appropriately handle familial relatedness. A rare-variant test does not currently exist for longitudinal data from family samples. Therefore, in this paper, we aim to introduce an association test for rare variants, which includes multiple longitudinal phenotype measurements for either population or family samples. <b><i>Methods:</i></b> This approach uses KM regression based on the linear mixed model framework and is applicable to longitudinal data from either population (L-KM) or family samples (LF-KM). <b><i>Results:</i></b> In our population-based simulation studies, L-KM has good control of Type I error rate and increased power in all the scenarios we considered compared with other competing methods. Conversely, in the family-based simulation studies, we found an inflated Type I error rate when L-KM was applied directly to the family samples, whereas LF-KM retained the desired Type I error rate and had the best power performance overall. Finally, we illustrate the utility of our proposed LF-KM approach by analyzing data from an association study between rare variants and blood pressure from the Genetic Analysis Workshop 18 (GAW18). <b><i>Conclusion:</i></b> We propose a method for rare-variant association testing in population and family samples using phenotypes measured at multiple time points for each subject. The proposed method has the best power performance compared to competing approaches in our simulation study