The human SMAD9 (GCC) repeat links to natural selection and late-onset neurocognitive disorders.

INTRODUCTION: Whereas (GCC)-repeats are overrepresented in genic regions, and mutation hotspots, they are largely unexplored with regard to their link with natural selection. Across numerous primate species and tissues, SMAD9 (SMAD Family Member 9) reaches highest level of expression in the human brain. This gene contains a (GCC)-repeat in the interval between + 1 and + 60 of the transcription start site, which is in the high-ranking (GCC)-repeats with respect to length. METHODS: Here we sequenced this (GCC)-repeat in 396 Iranian individuals, consisting of late-onset neurocognitive disorder (NCD) (N = 181) and controls (N = 215). RESULTS: We detected two predominantly abundant alleles of 7 and 9 repeats, forming 96.2% of the allele pool. The (GCC)7/(GCC)9 ratio was in the reverse order in the NCD group versus controls (p = 0.005), resulting from excess of (GCC)7 in the NCD group (p = 0.003) and (GCC)9 in the controls (p = 0.01). Five genotypes, predominantly consisting of (GCC)7 and lacking (GCC)9 were detected in the NCD group only (p = 0.008). The patients harboring those genotypes received the diagnoses of Alzheimer's disease (AD) and vascular dementia (VD). Five genotypes consisting of (GCC)9 and lacking (GCC)7 were detected in the control group only (p = 0.002). The group-specific genotypes formed approximately 4% of the genotype pool in the human samples studied. CONCLUSION: We propose natural selection and a novel locus for late-onset AD and VD at the SMAD9 (GCC)-repeat in humans.

Reduction of miR-744 delivered by NSCLC cell-derived extracellular vesicles upregulates SUV39H1 to promote NSCLC progression via activation of the Smad9/BMP9 axis.

BACKGROUND: Non-small cell lung cancer (NSCLC) is a common type of lung cancer. Extracellular vehicles (EVs) are nano-sized particles containing proteins, lipids, and miRNAs secreted by various cells, which play important roles in the development of lung cancer by regulating a wide range of signaling pathways. This study focused on elucidating a potential mechanism by which EVs promote the development of NSCLC. METHODS: Expression levels of miR-744, SUV39H1, Smad9, and BMP4 in clinical tissue samples of NSCLC patients and cell lines were quantified by RT-qPCR and/or western blot analysis. The interaction between SUV39H1 and miR-744 was identified by dual-luciferase reporter assay. miR-744, SUV39H1, and BMP4 expression levels were modulated in A549 and H460 cells, in order to evaluate their effects on cell proliferation, colony formation and cell cycle. A NSCLC xenograft mouse model was used to verify the in vitro findings. NSCLC cell-derived EVs and normal bronchial epithelial cell-derived EVs were isolated and their roles in NSCLC development were evaluated in vivo and in vitro. RESULTS: miR-744 expression was lower in cancer cell-derived derived EVs than in normal lung epithelial cell-derived EVs. Reduced miR-744 expression in EVs upregulated SUV39H1 in NSCLC cells and further increased BMP4 levels to promote NSCLC development. BMP4 was upregulated in NSCLC cells upon suppression of Smad9 mediated by SUV39H1. Reduced miR-744 expression transferred from cancer cell-derived EVs into NSCLC cells enhanced cancer development. CONCLUSION: Overall, our findings unveiled a mechanism whereby miR-744 delivered by NSCLC-derived EVs upregulated SUV39H1, activating the Smad9/BMP9 axis and thus promoted cancer development.

Thymopentin treatment of murine premature ovarian failure via attenuation of immune cell activity and promotion of the BMP4/Smad9 signalling pathway.

Premature ovarian failure (POF) is a typical form of pathological aging with complex pathogenesis and no effective treatment. Meanwhile, recent studies have reported that a high-fat and high-sugar (HFHS) diet adversely affects ovarian function and ovum quality. Here, we investigated the therapeutic effect of thymopentin (TP-5) as a treatment for murine POF derived from HFHS and its target. Pathological examination and hormone assays confirmed that TP-5 significantly improved murine POF symptoms. And, TP-5 could reduce oxidative stress injury and blood lipids in the murine POF derived from HFHS. Flow cytometry and qPCR results suggested that TP-5 attenuated activation of CD3+ T cells and type I macrophages. RNA-Seq results indicated somedifferences in gene transcription between the TP-5 intervention group and the control group. KEGG analysis indicated that the expression of genes involved in the mTOR signaling pathway was the most significantly different between the two groups. Additionally, compared with the control groups, the expression levels of interleukin, NFκB, and TNF families of genes were significantly downregulated in the POF+TP-5 group, whereas expression of the TGFß/Smad9 genes was significantly upregulated. Finally, immunofluorescence staining and qPCR confirmed that TP-5 promoted the polarization of Mø2 cells in the ovary by activating the expression of the BMP4/Smad9 signalling pathway. Thus, our study confirmed that TP-5 has a significant therapeutic effect on POF by upregulating BMP4/Smad9 signalling pathway so as to promote the balance and polarization of immune cell and reducing the release of inflammatory factors and reduce lipid oxidative stress injury.

Histone arginine methylation by Prmt5 is required for lung branching morphogenesis through repression of BMP signaling.

Branching morphogenesis is essential for the successful development of a functional lung to accomplish its gas exchange function. Although many studies have highlighted requirements for the bone morphogenetic protein (BMP) signaling pathway during branching morphogenesis, little is known about how BMP signaling is regulated. Here, we report that the protein arginine methyltransferase 5 (Prmt5) and symmetric dimethylation at histone H4 arginine 3 (H4R3sme2) directly associate with chromatin of Bmp4 to suppress its transcription. Inactivation of Prmt5 in the lung epithelium results in halted branching morphogenesis, altered epithelial cell differentiation and neonatal lethality. These defects are accompanied by increased apoptosis and reduced proliferation of lung epithelium, as a consequence of elevated canonical BMP-Smad1/5/9 signaling. Inhibition of BMP signaling by Noggin rescues the lung branching defects of Prmt5 mutant in vitro Taken together, our results identify a novel mechanism through which Prmt5-mediated histone arginine methylation represses canonical BMP signaling to regulate lung branching morphogenesis.

Pulmonary Arterial Hypertension: A Current Perspective on Established and Emerging Molecular Genetic Defects.

Pulmonary arterial hypertension (PAH) is an often fatal disorder resulting from several causes including heterogeneous genetic defects. While mutations in the bone morphogenetic protein receptor type II (BMPR2) gene are the single most common causal factor for hereditary cases, pathogenic mutations have been observed in approximately 25% of idiopathic PAH patients without a prior family history of disease. Additional defects of the transforming growth factor beta pathway have been implicated in disease pathogenesis. Specifically, studies have confirmed activin A receptor type II-like 1 (ACVRL1), endoglin (ENG), and members of the SMAD family as contributing to PAH both with and without associated clinical phenotypes. Most recently, next-generation sequencing has identified novel, rare genetic variation implicated in the PAH disease spectrum. Of importance, several identified genetic factors converge on related pathways and provide significant insight into the development, maintenance, and pathogenetic transformation of the pulmonary vascular bed. Together, these analyses represent the largest comprehensive compilation of BMPR2 and associated genetic risk factors for PAH, comprising known and novel variation. Additionally, with the inclusion of an allelic series of locus-specific variation in BMPR2, these data provide a key resource in data interpretation and development of contemporary therapeutic and diagnostic tools.

Long noncoding RNA KCNMA1-AS1 promotes osteogenic differentiation of human bone marrow mesenchymal stem cells by activating the SMAD9 signaling pathway.

The human bone marrow mesenchymal stem cells (hBMSCs) undergo intense osteogenic differentiation, a crucial bone formation mechanism. Evidence from prior studies suggested an association between long noncoding RNAs (lncRNAs) and the osteogenic differentiation of hBMSCs. However, precise roles and molecular mechanisms are still largely unknown. In this work, we report for the first time that lncRNA KCNMA1 antisense RNA 1 (KCNMA1-AS1) plays a vital role in regulating hBMSCs' osteogenic differentiation. Here, it was observed that the KCNMA1-AS1 expression levels were significantly upregulated during osteogenic differentiation. In addition, KCNMA1-AS1 overexpression enhanced in vitro osteogenic differentiation of hBMSCs and in vivo bone formation, whereas knockdown of KCNMA1-AS1 resulted in the opposite result. Additionally, the interaction between KCNMA1-AS1 and mothers against decapentaplegic homolog 9 (SMAD9) was confirmed by an RNA pull-down experiment, mass spectrometry, and RIP assay. This interaction regulated the activation of the SMAD9 signaling pathway. Moreover, rescue assays demonstrated that the inhibitor of the SMAD9 signaling pathway reversed the stimulative effects on osteogenic differentiation of hBMSCs by KCNMA1-AS1 overexpression. Altogether, our results stipulate that KCNMA1-AS1 promotes osteogenic differentiation of hBMSCs via activating the SMAD9 signaling pathway and can serve as a biomarker and therapeutic target in treating bone defects.

SMAD9-MYCN positive feedback loop represents a unique dependency for MYCN-amplified neuroblastoma.

BACKGROUND: Neuroblastoma (NB) is the most common extracranial solid tumor occurring during childhood and high-risk NB patients have a poor prognosis. The amplified MYCN gene serves as an important determinant of a high risk of NB. METHODS: We performed an integrative screen using public NB tissue and cell line data, and identified that SMAD9 played an important role in high-risk NB. An investigation of the super-enhancers database (SEdb) and chromatin immunoprecipitation sequencing (ChIP-seq) dataset along with biological experiments of incorporating gene knockdown and CRISPR interference (CRISPRi) were performed to identify upstream regulatory mechanism of SMAD9. Gene knockdown and rescue, quantitative real-time PCR (Q-RT-PCR), cell titer Glo assays, colony formation assays, a subcutaneous xenograft model and immunohistochemistry were used to determine the functional role of SMAD9 in NB. An integrative analysis of ChIP-seq data with the validation of CRISPRi and dual-luciferase reporter assays and RNA sequencing (RNA-seq) data with Q-RT-PCR validation was conducted to analyze the downstream regulatory mechanism of SMAD9. RESULTS: High expression of SMAD9 was specifically induced by the transcription factors including MYCN, PHOX2B, GATA3 and HAND2 at the enhancer region. Genetic suppression of SMAD9 inhibited MYCN-amplified NB cell proliferation and tumorigenicity both in vitro and in vivo. Further studies revealed that SMAD9 bound to the MYCN promoter and transcriptionally regulate MYCN expression, with MYCN reciprocally binding to the SMAD9 enhancer and transactivating SMAD9, thus forming a positive feedback loop along with the MYCN-associated cancer cell cycle. CONCLUSION: This study delineates that SMAD9 forms a positive transcriptional feedback loop with MYCN and represents a unique tumor-dependency for MYCN-amplified neuroblastoma.

Pediatric Germline Predisposition to Myeloid Neoplasms.

PURPOSE OF REVIEW: Advances in the understanding of germline predisposition to pediatric cancers, particularly myeloid neoplasms, have increased rapidly over the last 20 years. Here, we highlight the most up-to-date knowledge regarding known pathogenic germline variants that contribute to the development of myeloid neoplasms in children. RECENT FINDINGS: This discussion enumerates the most notable myeloid neoplasm-causing germline mutations. These mutations may be organized based on their molecular underpinnings-transcriptional control, splicing and signal transduction control, and a group of heterogeneous bone marrow failure syndromes. We review recent findings related to the biochemical mechanisms that predispose to malignant transformation in each condition. Key genetic discoveries such as novel mutations, degrees of penetrance, principles of the two-hit hypothesis, and co-occurrence of multiple mutations are shared. Clinical pearls, such as information regarding epidemiology, natural history, or prognosis, are also discussed. Germline mutations predisposing to pediatric myeloid neoplasms are frequent, but underrecognized. They hold major clinical implications regarding prognosis, treatment strategies, and screening for other malignancies. Further research is warranted to better characterize each of these conditions, as well as identify additional novel germline pathogenic variants of interest.

Pharmacological Manipulation of Early Zebrafish Skeletal Development Shows an Important Role for Smad9 in Control of Skeletal Progenitor Populations.

Osteoporosis and other conditions associated with low bone density or quality are highly prevalent, are increasing as the population ages and with increased glucocorticoid use to treat conditions with elevated inflammation. There is an unmet need for therapeutics which can target skeletal precursors to induce osteoblast differentiation and osteogenesis. Genes associated with high bone mass represent interesting targets for manipulation, as they could offer ways to increase bone density. A damaging mutation in SMAD9 has recently been associated with high bone mass. Here we show that Smad9 labels groups of osteochondral precursor cells, which are not labelled by the other Regulatory Smads: Smad1 or Smad5. We show that Smad9+ cells are proliferative, and that the Smad9+ pocket expands following osteoblast ablation which induced osteoblast regeneration. We further show that treatment with retinoic acid, prednisolone, and dorsomorphin all alter Smad9 expression, consistent with the effects of these drugs on the skeletal system. Taken together these results demonstrate that Smad9+ cells represent an undifferentiated osteochondral precursor population, which can be manipulated by commonly used skeletal drugs. We conclude that Smad9 represents a target for future osteoanabolic therapies.

A Rare Mutation in SMAD9 Associated With High Bone Mass Identifies the SMAD-Dependent BMP Signaling Pathway as a Potential Anabolic Target for Osteoporosis.

Novel anabolic drug targets are needed to treat osteoporosis. Having established a large national cohort with unexplained high bone mass (HBM), we aimed to identify a novel monogenic cause of HBM and provide insight into a regulatory pathway potentially amenable to therapeutic intervention. We investigated a pedigree with unexplained HBM in whom previous sequencing had excluded known causes of monogenic HBM. Whole exome sequencing identified a rare (minor allele frequency 0.0023), highly evolutionarily conserved missense mutation in SMAD9 (c.65T>C, p.Leu22Pro) segregating with HBM in this autosomal dominant family. The same mutation was identified in another two unrelated individuals both with HBM. In silico protein modeling predicts the mutation severely disrupts the MH1 DNA-binding domain of SMAD9. Affected individuals have bone mineral density (BMD) Z-scores +3 to +5, mandible enlargement, a broad frame, torus palatinus/mandibularis, pes planus, increased shoe size, and a tendency to sink when swimming. Peripheral quantitative computed tomography (pQCT) measurement demonstrates increased trabecular volumetric BMD and increased cortical thickness conferring greater predicted bone strength; bone turnover markers are low/normal. Notably, fractures and nerve compression are not found. Both genome-wide and gene-based association testing involving estimated BMD measured at the heel in 362,924 white British subjects from the UK Biobank Study showed strong associations with SMAD9 (PGWAS = 6 × 10-16 ; PGENE = 8 × 10-17 ). Furthermore, we found Smad9 to be highly expressed in both murine cortical bone-derived osteocytes and skeletal elements of zebrafish larvae. Our findings support SMAD9 as a novel HBM gene and a potential novel osteoanabolic target for osteoporosis therapeutics. SMAD9 is thought to inhibit bone morphogenetic protein (BMP)-dependent target gene transcription to reduce osteoblast activity. Thus, we hypothesize SMAD9 c.65T>C is a loss-of-function mutation reducing BMP inhibition. Lowering SMAD9 as a potential novel anabolic mechanism for osteoporosis therapeutics warrants further investigation. © 2019 The Authors. Journal of Bone and Mineral Research published by American Society for Bone and Mineral Research.

A potential role for SMAD9 in goose follicular selection through regulation of mRNA levels of luteinizing hormone receptor.

The egg production of poultry depends on follicular development and selection. Nonetheless, the mechanism underlying the priority of selecting of hierarchical follicles is completely unknown. SMAD9 is one of the important transcription factors in the BMP/SMAD pathway and is involved in goose follicular initiation. To identify its potential role in determination of the goose follicle hierarchy, we used BMP type I receptor inhibitor LDN-193189 both in vivo and in vitro and found that SMAD9 mRNA expression decreased in the presence of LDN-193189. While the level of SMAD9 mRNA decreased after treatment with LDN-193189, we found that the egg production (7.08 eggs per bird per year) of the animals increased, estradiol (E2) levels significantly increased, but the levels of progesterone (P4) remained unchanged. We also detected a significant increase in luteinizing hormone receptor (LHR) mRNA expression, but no change in follicle-stimulating hormone receptor (FSHR) mRNA amounts. The in vitro experimental results indicated that SMAD9 knockdown by RNA interference noticeably reduced E2 and P4 biosynthesis and FSHR and LHR mRNA expression in goose granulosa cells. Chromatin immunoprecipitation assay of goose granulosa cells revealed that phospho-SMAD9 bound to the LHR promoter and possibly regulated its transcriptional activity. These findings revealed that SMAD9 is differentially expressed in goose follicles, and acts as a key player in the control over goose follicular selection.

Association of the gene polymorphisms of BMPR2, ACVRL1, SMAD9 and their interactions with the risk of essential hypertension in the Chinese Han population.

Objective: Genetic factors are involved in the occurrence, development, and progression of essential hypertension (EH). To study the association between single nucleotide polymorphisms (SNPs) of the rs6435156 and rs1048829 loci of the bone morphogenetic protein receptor type 2 (BMPR2) gene, the rs121909287 and rs121909284 loci of the activin receptor-like kinase 1 (ACVRL1) gene, and the rs397514716 and rs121918359 loci of the mothers against decapentaplegic homolog 9 (SMAD9) gene with the risk of EH in the Chinese Han population. Materials and methods: A total of 460 EH patients and 460 healthy controls were recruited for the study. Genomic DNA of white blood cells was extracted, and the genotypes were analyzed by Sanger sequencing after polymerase chain reaction amplification. Multi-factor dimensionality reduction (MDR) was used to analyze the effect of gene-environment interactions on EH risk. Results: The risk of EH increased in the BMPR2 gene rs6435156 locus dominant model (adjusted odds ratio [OR] = 1.572, 95% confidence interval [CI]: 1.385-1.765, P<0.001) and recessive model (adjusted OR = 1.926, 95% CI: 1.693-2.067, P<0.001). The risk of EH increased in the rs1048829 recessive model (adjusted OR = 1.444, 95% CI: 1.142-1.696, P=0.003). The risk of EH increased in the recessive model of the ACVRL1 gene rs121909287 locus (adjusted OR = 1.403, 95% CI: 1.101-1.660, P=0.008). The risk of EH increased in the SMAD9 gene rs397514716 locus dominant model (adjusted OR = 1.370, 95% CI: 1.183-1.559, P<0.001) and recessive model (adjusted OR = 1.803, 95% CI: 1.470-1.983, P<0.001). The CG haplotype of the rs6435156 and rs1048829 loci of the BMPR2 gene, the CC haplotype of the ACVRL1 gene rs121909287 and rs121909284 loci, and the CC haplotype of the rs397514716 and rs121918359 loci of the SMAD9 gene were factors that protect against EH, whereas the TT haplotype of the rs6435156 and rs1048829 loci in the BMPR2 gene was a risk factor for EH. MDR analysis showed that the BMPR2 gene rs6435156 locus TT genotype carriers, the SMAD9 gene rs397514716 locus TT genotype carriers, and alcohol drinkers had the highest EH risk (OR = 4.523, 95% CI: 2.235-6.871, P<0.001). Conclusion: The SNPs of the rs6435156 and rs1048829 locus in the BMPR2 gene, the rs121909287 loci in the ACVRL1 gene, and the rs397514716 locus in the SMAD9 gene were associated with a risk of EH in Han Chinese.

The effects of mouse ovarian granulosa cell function and related gene expression by suppressing BMP/Smad signaling pathway.

BMP I type receptor inhibitor can selectively inhibit BMP/Smad signaling pathways, mainly by inhibiting the BMP I type receptor activity to prevent phosphorylation of Smad1, Smad5 and Smad9. The aim of the present study was to explore the effects of mouse ovarian granulosa cell function and related gene expression by suppressing BMP/Smad signaling pathway with LDN-193189(A type of BMP I type receptor inhibitor). In this study, we cultivate the original generation of mouse ovarian granular cells then collect cells and cell culture medium after treatment. Cellular localization and expression of Smad9 and P-smad9 proteins was studied by immunofluorescence (IF) in the ovarian granulosa cells of mouse; Related genes mRNA and proteins expression was checked by QRT-PCR and Western blot; Detected the concentration of related hormones by using ELISA kit; finally, the growth of the cells was analyzed by plotting cell growth curve with CCK-8 assay. The results indicate that, suppression of BMP/Smad signaling pathway can inhibit the expression of LHR and FSHR, inhibit cell proliferation and decrease E2 secretion, the mechanism of action maybe reduce the expression of smad9, at the same time, we found that the feedback regulation of smad9 may affect the expression of FSHR and cell proliferation.

Identification of novel point mutations in splicing sites integrating whole-exome and RNA-seq data in myeloproliferative diseases.

Point mutations in intronic regions near mRNA splice junctions can affect the splicing process. To identify novel splicing variants from exome sequencing data, we developed a bioinformatics splice-site prediction procedure to analyze next-generation sequencing (NGS) data (SpliceFinder). SpliceFinder integrates two functional annotation tools for NGS, ANNOVAR and MutationTaster and two canonical splice site prediction programs for single mutation analysis, SSPNN and NetGene2. By SpliceFinder, we identified somatic mutations affecting RNA splicing in a colon cancer sample, in eight atypical chronic myeloid leukemia (aCML), and eight CML patients. A novel homozygous splicing mutation was found in APC (NM_000038.4:c.1312+5G>A) and six heterozygous in GNAQ (NM_002072.2:c.735+1C>T), ABCC 3 (NM_003786.3:c.1783-1G>A), KLHDC 1 (NM_172193.1:c.568-2A>G), HOOK 1 (NM_015888.4:c.1662-1G>A), SMAD 9 (NM_001127217.2:c.1004-1C>T), and DNAH 9 (NM_001372.3:c.10242+5G>A). Integrating whole-exome and RNA sequencing in aCML and CML, we assessed the phenotypic effect of mutations on mRNA splicing for GNAQ, ABCC 3, HOOK 1. In ABCC 3 and HOOK 1, RNA-Seq showed the presence of aberrant transcripts with activation of a cryptic splice site or intron retention, validated by the reverse transcription-polymerase chain reaction (RT-PCR) in the case of HOOK 1. In GNAQ, RNA-Seq showed 22% of wild-type transcript and 78% of mRNA skipping exon 5, resulting in a 4-6 frameshift fusion confirmed by RT-PCR. The pipeline can be useful to identify intronic variants affecting RNA sequence by complementing conventional exome analysis.

Genetics of pulmonary arterial hypertension.

Painstaking research led to the discovery of gene mutations responsible for heritable forms of pulmonary arterial hypertension (PAH). Mutations in the gene BMPR2, which codes for a cell surface receptor (BMPRII), cause the approximately 80% of heritable cases of PAH. Less commonly mutations in ALK1, CAV1, ENG, and SMAD9, and newly discovered mutations in KCNK3, may cause heritable PAH. Other family members of many patients diagnosed with idiopathic PAH may be diagnosed with PAH. Genetic counseling and testing should be offered to patients diagnosed with heritable or idiopathic PAH.