Alternative titles; symbols
HGNC Approved Gene Symbol: SMAD9
Cytogenetic location: 13q13.3 Genomic coordinates (GRCh38): 13:36,844,831-36,920,854 (from NCBI)
| Location | Phenotype |
Phenotype MIM number |
Inheritance |
Phenotype mapping key |
|---|---|---|---|---|
| 13q13.3 | Pulmonary hypertension, primary, 2 | 615342 | Autosomal dominant | 3 |
The SMAD9 gene encodes a downstream modulator of the bone morphogenetic protein (BMP) signaling pathway, which is part of the TGF-beta (see TGFB1, 190180) superfamily that regulates growth, differentiation, apoptosis, and development. SMAD9 is closely related to SMAD1 (601595) and SMAD5 (603110), which are collectively known as the receptor SMADs. In canonical BMP signaling, receptor SMADs are phosphorylated and associate with SMAD4 (600993) to translocate to the nucleus (summary by Nishita et al., 1999, Drake et al., 2011).
Watanabe et al. (1997) identified SMAD9, which they called MADH6. They isolated human fetal brain cDNAs representing 2 alternatively spliced MADH6 transcripts, MADH6a and MADH6b. The MADH6a and MADH6b cDNAs encode deduced 467- and 430-amino acid proteins, respectively. Compared with full-length MADH6a, MADH6b lacks exon 3, leading to a deletion of 37 amino acids in a central, proline-rich domain. The authors reported 80% amino acid identity between MADH6 and MADH1 (601595) and 67% identity between MADH6 and MADH2 (601366). Northern blot analysis detected weak expression of a 6.6-kb MADH6 transcript in adult heart, brain, placenta, lung, skeletal muscle, prostate, testis, ovary, and small intestine. Somewhat stronger expression was detected in fetal brain, lung, and kidney, but expression was absent in fetal liver.
Using Xenopus Smad1 (601595) to probe a placenta cDNA library, Nishita et al. (1999) cloned 2 splice variants of SMAD9, which they called SMAD8 and SMAD8B. Full-length SMAD8 contains N- and C-terminal Mad homology domains (MH1 and MH2, respectively), and MH2 includes an SSxS phosphorylation motif. Compared with full-length SMAD8, SMAD8B lacks exon 6, resulting in a 47-amino acid truncation of the MH2 domain that removes the SSxS motif. Northern blot analysis detected variable expression of a 6.2-kb transcript in all tissues examined. RT-PCR showed that SMAD8B was more weakly expressed than SMAD8 in brain, but both were equally expressed in placenta, kidney, and heart.
Watanabe et al. (1997) and Nishita et al. (1999) reported that the SMAD9 gene contains 6 coding exons.
By radiation hybrid mapping and fluorescence in situ hybridization, Watanabe et al. (1997) localized the MADH9 gene to chromosome 13q12-q14.
Constitutively active ALK2 (ACVR1, 102576), a bone morphogenetic protein (BMP; see 112264) type I receptor, phosphorylates SMAD8 on the SSxS motif. Phosphorylated SMAD8 binds SMAD4 (600993), and the dimer translocates into the nucleus (Chen et al., 1997). By cotransfecting human embryonic kidney cells with epitope-tagged constructs, Nishita et al. (1999) showed that SMAD8 interacted with SMAD4, but not with other SMADs. SMAD8B interacted with both SMAD4 and SMAD8. Both SMAD8 and SMAD8B were predominantly cytoplasmic when expressed in COS-7 cells. Phosphorylation by constitutively active ALK2 resulted in nuclear translocation of SMAD8, but not SMAD8B, which lacks the phosphorylation motif. In mouse myoblasts, cotransfection of SMAD8 and constitutively active ALK2 activated transcription of a reporter gene, and activation was reduced by inclusion of SMAD8B. Nishita et al. (1999) concluded that SMAD8 and SMAD8B positively and negatively regulate BMP signaling, respectively.
Davis et al. (2008) demonstrated that induction of a contractile phenotype in human vascular smooth muscle cells by TGF-beta and BMPs is mediated by miR21 (611020). miR21 downregulates PDCD4 (608610), which in turn acts as a negative regulator of smooth muscle contractile genes. Surprisingly, TGF-beta and BMP signaling promoted a rapid increase in expression of mature miR21 through a posttranscriptional step, promoting the processing of primary transcripts of miR21 (pri-miR21) into precursor miR21 (pre-miR21) by the Drosha complex (608828). TGF-beta and BMP-specific SMAD signal transducers SMAD1 (601595), SMAD2 (601366), SMAD3 (603109), and SMAD5 (603110) are recruited to pri-miR21 in a complex with the RNA helicase p68 (DDX5; 180630), a component of the Drosha microprocessor complex. The shared cofactor SMAD4 (600993) is not required for this process. Davis et al. (2008) concluded that regulation of microRNA biogenesis by ligand-specific SMAD proteins is critical for control of the vascular smooth muscle cell phenotype and potentially for SMAD4-independent responses mediated by the TGF-beta and BMP signaling pathways. In a follow-up to the report of Davis et al. (2008), Drake et al. (2011) found that BMPR2 was essential for the SMAD-mediated miR processing. Loss of SMAD9 (603295) also affected miR processing in smooth muscle cells and in endothelial cells, but it did not affect canonical BMP signaling. Knockdown of individual receptor SMADs 1, 5, and 9 decreased levels of processed miR21 levels in both types of cells, suggesting that this miR processing pathway forms a complex. The findings indicated that SMAD9 is also essential for this miR pathway.
Primary Pulmonary Hypertension 2
In a patient with primary pulmonary hypertension-2 (PPH2; 615342), Shintani et al. (2009) identified a heterozygous truncating mutation in the SMAD9 gene (603295.0001). In vitro functional expression assays showed that the mutant SMAD9 protein could not be phosphorylated, did not interact with SMAD4, and was inefficient in activating the BMP responsive promoter-reporter, resulting in downregulation of downstream TGFB/BMP signaling.
In a woman with PPH2, Drake et al. (2011) identified a heterozygous truncating mutation in the SMAD9 gene (R294X; 603295.0003). Pulmonary artery endothelial cells derived from this patient showed no significant induction of miR21 when treated with BMP9. Mutant cells showed increased proliferation compared to controls, and overexpression of miR21 induced growth suppression. Overexpression of SMAD9 in SMAD9-mutant cells normalized the proliferation rate and normalized miR levels. Canonical BMP signaling remained relatively intact in SMAD9-mutant cells, with less than a 50% reduction in fold-change of downstream target genes. The findings suggested that aberrant miR processing may play an important role in the pathogenesis of PPH.
Associations Pending Confirmation
For discussion of a possible association between variation in the SMAD9 gene and hamartomatous polyposis and gastrointestinal ganglioneuromas, see 603295.0004.
Pulmonary artery hypertension (PAH; see 178600), a progressive, lethal condition that results in pathologic changes in the pulmonary arterial tree, eventually leading to right heart failure. Identification of mutations in the type II bone morphogenetic protein (Bmp) receptor BMPR2 (600799) in families with PAH has implicated Bmp-signaling in the pathogenesis of PAH. Huang et al. (2009) investigated Smad8, which is a divergent receptor-regulated Smad downstream of Bmp-signaling, by creating Smad8-null mice. Loss of Smad8 function in adult mice resulted in characteristic changes in distal pulmonary arteries including medial thickening and smooth muscle hyperplasia as observed in patients with PAH. Smad8-mutant pulmonary vasculature had upregulated activin (see 147290)/Tgf-beta (190180) signaling and pathologic remodeling with aberrant Prx1 (PRRX1; 167420) and tenascin C (TNC; 187380) expression. A subset of Smad8 mutants had pulmonary adenomas, possibly uncovering a function for Smad8 in normal growth control. Huang et al. (2009) hypothesized that Smad8 may play a role in both pulmonary hypertension and lung tumorigenesis.
Using mice with skeletal muscle-specific knockout or knockdown of BMP signaling molecules, Sartori et al. (2013) found that BMP signaling, acting via Smad1, Smad5, and Smad8 (Smad1/5/8) and Smad4, regulated muscle mass. Inhibition of BMP signaling caused muscle atrophy, abolished the hypertrophic phenotype of myostatin (MSTN; 601788)-deficient mice, and exacerbated the muscle-wasting effects of denervation and fasting. Bmp14 (GDF5; 601146) was required to prevent excessive muscle loss following denervation. The BMP-Smad1/5/8-Smad4 pathway negatively regulated Fbxo30 (609101), a ubiquitin ligase required for muscle loss. Inhibition of Fbxo30 protected denervated muscle from atrophy and blunted atrophy in Smad4-deficient muscle. Sartori et al. (2013) concluded that BMP signaling is the dominant pathway controlling muscle mass and that the hypertrophic phenotype caused by myostatin inhibition results from unrestrained BMP signaling.
In a patient with primary pulmonary hypertension-2 (PPH2; 615342), Shintani et al. (2009) identified a heterozygous 606C-A transversion in exon 2 of the SMAD9 gene, resulting in a cys202-to-ter (C202X) substitution. The truncated protein is predicted to lack 228 C-terminal residues, including the MH2 domain and the SXS phosphorylation site. The mutation was not identified in 150 controls. The boy was diagnosed with pulmonary hypertension at age 8 years. Family history revealed that the unaffected father also carried the mutation, indicating reduced penetrance. Two sibs of the proband had died of pulmonary disease at ages 13 years and less than 2 years, respectively. In vitro functional expression assays showed that the mutant SMAD9 protein could not be phosphorylated, did not interact with SMAD4 (600993), and was inefficient in activating the BMP responsive promoter-reporter.
In a 7-year-old Japanese girl with primary pulmonary hypertension-2 (PPH2; 615342), Nasim et al. (2011) identified a heterozygous c.127A-G transition in the SMAD9 gene, resulting in a lys43-to-glu (K43E) substitution at a highly conserved residue in the MH1 domain. SMAD9 was chosen for study because of its role in the BMP signaling pathway. The patient's parents were unavailable for study, but the variant was not found in several large control databases or in 340 Japanese control samples. Expression of the mutant protein in a reporter construct generated reduced basal activity and impaired responses to ligand stimulation compared to wildtype. There were no differences in expression of the mutant protein compared to wildtype. Nasim et al. (2011) suggested that the moderate effect of this variant indicates that it may be a susceptibility factor in the development of PPH.
In a 26-year-old woman with primary pulmonary hypertension (PPH2; 615342), Drake et al. (2011) identified a heterozygous arg294-to-ter (R294X) substitution in the SMAD9 gene. No further clinical information was provided.
This variant is classified as a variant of unknown significance because its contribution to hamartomatous polyposis and gastrointestinal ganglioneuromas (see 158350) has not been confirmed.
In a 38-year-old Caucasian man with hamartomatous polyposis syndrome (HPS), who was negative for mutation in known HPS-associated genes, Ngeow et al. (2015) performed whole-exome sequencing and identified a heterozygous germline val90-to-met (V90M) substitution. Functional analysis in transfected HEK293 cells showed lower PTEN (601728) mRNA and protein levels in patient lymphoblastoid cell lines compared to healthy controls, and reduced PTEN protein expression was also observed in polyp tissue from the patient. However, because SMAD9 was unable to directly bind the PTEN promoter, Ngeow et al. (2015) studied miR21 (611020), a direct downstream effector of the BMP-SMAD9 signaling pathway known to target PTEN directly and suppress PTEN expression. The authors demonstrated that miR21 expression increased significantly in BMP4 (112262)-treated mutant cells compared to wildtype, due to increased binding of mutant SMAD9 to primary miR21 (pri-miR21) and consequent increased processing of pri-miR21 into mature miR21. Ngeow et al. (2015) concluded that V90M is a gain-of-function mutation resulting in increased miR21 expression, which causes decreased PTEN mRNA and protein stability. The proband presented with persistent diarrhea and weight loss, and colonoscopy revealed diffuse 3- to 5-mm polyps throughout his colon. Histologic analysis showed that these were hamartomatous polyps admixed with either mature adipose or ganglioneuromatous proliferation. The proband had an affected brother who was diagnosed with polyposis in his 20s; their father had diffuse polyposis and died in his 40s, and a paternal aunt and uncle died from early-onset colorectal cancer in their 40s. DNA was unavailable from affected family members.
Chen, Y., Bhushan, A., Vale, W. Smad8 mediates the signaling of the ALK-2 receptor serine kinase. Proc. Nat. Acad. Sci. 94: 12938-12943, 1997. Note: Erratum: Proc. Nat. Acad. Sci. 95: 1968 only, 1998. [PubMed: 9371779] [Full Text: https://doi.org/10.1073/pnas.94.24.12938]
Davis, B. N., Hilyard, A. C., Lagna, G., Hata, A. SMAD proteins control DROSHA-mediated microRNA maturation. Nature 454: 56-61, 2008. [PubMed: 18548003] [Full Text: https://doi.org/10.1038/nature07086]
Drake, K. M., Zygmunt, D., Mavrakis, L., Harbor, P., Wang, L., Comhair, S. A., Erzurum, S. C., Aldred, M. A. Altered microRNA processing in heritable pulmonary arterial hypertension: an important role for Smad-8. Am. J. Resp. Crit. Care Med. 184: 1400-1408, 2011. [PubMed: 21920918] [Full Text: https://doi.org/10.1164/rccm.201106-1130OC]
Huang, Z., Wang, D., Ihida-Stansbury, K., Jones, P. L., Martin, J. F. Defective pulmonary vascular remodeling in Smad8 mutant mice. Hum. Molec. Genet. 18: 2791-2801, 2009. [PubMed: 19419974] [Full Text: https://doi.org/10.1093/hmg/ddp214]
Nasim, M. T., Ogo, T., Ahmed, M., Randall, R., Chowdhury, H. M., Snape, K. M., Bradshaw, T. Y., Southgate, L., Lee, G. J., Jackson, I., Lord, G. M., Gibbs, J. S. R., and 10 others. Molecular genetic characterization of SMAD signaling molecules in pulmonary arterial hypertension. Hum. Mutat. 32: 1385-1389, 2011. [PubMed: 21898662] [Full Text: https://doi.org/10.1002/humu.21605]
Ngeow, J., Yu, W., Yehia, L., Niazi, F., Chen, J., Tang, X., Heald, B., Lei, J., Romigh, T., Tucker-Kellogg, L., Lim, K. H., Song, H., Eng, C. Exome sequencing reveals germline SMAD9 mutation that reduces phosphatase and tensin homolog expression and is associated with hamartomatous polyposis and gastrointestinal ganglioneuromas. Gastroenterology 149: 886-889, 2015. [PubMed: 26122142] [Full Text: https://doi.org/10.1053/j.gastro.2015.06.027]
Nishita, M., Ueno, N., Shibuya, H. Smad8B, a Smad8 splice variant lacking the SSXS site that inhibits Smad8-mediated signalling. Genes Cells 4: 583-591, 1999. [PubMed: 10583507] [Full Text: https://doi.org/10.1046/j.1365-2443.1999.00285.x]
Sartori, R., Schirwis, E., Blaauw, B., Bortolanza, S., Zhao, J., Enzo, E., Stantzou, A., Mouisel, E., Toniolo, L., Ferry, A., Stricker, S., Goldberg, A. L., Dupont, S., Piccolo, S., Amthor, H., Sandri, M. BMP signaling controls muscle mass. Nature Genet. 45: 1309-1318, 2013. [PubMed: 24076600] [Full Text: https://doi.org/10.1038/ng.2772]
Shintani, M., Yagi, H., Nakayama, T., Saji, T., Matsuoka, R. A new nonsense mutation of SMAD8 associated with pulmonary arterial hypertension. J. Med. Genet. 46: 331-337, 2009. [PubMed: 19211612] [Full Text: https://doi.org/10.1136/jmg.2008.062703]
Watanabe, T. K., Suzuki, M., Omori, Y., Hishigaki, H., Horie, M., Kanemoto, N., Fujiwara, T., Nakamura, Y., Takahashi, E. Cloning and characterization of a novel member of the human Mad gene family (MADH6). Genomics 42: 446-451, 1997. [PubMed: 9205116] [Full Text: https://doi.org/10.1006/geno.1997.4753]