Discovery of the BMPR1A promoter and germline mutations that cause juvenile polyposis

Department of Surgery, Carver College of Medicine, University of Iowa Hospitals and Clinics, 200 Hawkins Drive, Iowa City, IA 52242-1086, USA.
Human Molecular Genetics (Impact Factor: 6.39). 12/2010; 19(23):4654-62. DOI: 10.1093/hmg/ddq396
Source: PubMed


Juvenile polyposis (JP) is an autosomal dominant hamartomatous polyposis syndrome where affected individuals are predisposed
to colorectal and upper gastrointestinal cancer. Forty-five percent of JP patients have mutations or deletions involving the
coding regions of SMAD4 and BMPR1A, but the genetic basis of other cases is unknown. We set out to identify the JP gene in a large kindred having 10 affected
members without SMAD4 or BMPR1A coding sequence mutations or deletions. We found a germline deletion segregating in all affected members, mapping 119 kb
upstream of the coding region of BMPR1A by multiplex ligation-dependent probe amplification and comparative genomic hybridization. To further understand the genomic
structure of BMPR1A, we performed 5′ RACE from lymphoblastoid cell lines and normal colon tissue, which revealed four non-coding (NC) exons and
two putative promoters. Further analysis of this deletion showed that it encompassed 12 433 bp, including one promoter and
NC exon. The activities of each promoter and deletion constructs were evaluated by luciferase assays, and the stronger promoter
sequence analyzed for changes in JP patients without SMAD4 or BMPR1A alterations. A total of 6 of 65 JP probands were found to have mutations affecting this promoter. All probands examined had
diminished BMPR1A protein by ELISA, and all promoter mutations but one led to significantly reduced luciferase activity relative
to the wild-type promoter reporter. We conclude that we have identified the promoter for BMPR1A, in which mutations may be responsible for as many as 10% of JP cases with unknown mutations.

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Available from: Fadi S Dahdaleh, Aug 26, 2014
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    • "Aretz et al. (28) also found four JP patients with deletion of these four MLPA probes, but they also had deletions involving all the coding exons as well. These data suggest that promoter alterations play a role in the genesis of JP, as recently reported for BMPR1A (29), and therefore, further evaluation of promoter A in JP patients will be of interest. Whether epigenetic inactivation of the normal copy of SMAD4 leads to polyp formation is another important question to be examined in JP patients. "
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    ABSTRACT: Inactivation of SMAD4 has been linked to several cancers and germline mutations cause juvenile polyposis (JP). We set out to identify the promoter(s) of SMAD4, evaluate their activity in cell lines and define possible transcription factor binding sites (TFBS). 5′-rapid amplification of cDNA ends (5′-RACE) and computational analyses were used to identify candidate promoters and corresponding TFBS and the activity of each was assessed by luciferase vectors in different cell lines. TFBS were disrupted by site-directed mutagenesis (SDM) to evaluate the effect on promoter activity. Four promoters were identified, two of which had significant activity in several cell lines, while two others had minimal activity. In silico analysis revealed multiple potentially important TFBS for each promoter. One promoter was deleted in the germline of two JP patients and SDM of several sites led to significant reduction in promoter activity. No mutations were found by sequencing this promoter in 65 JP probands. The predicted TFBS profiles for each of the four promoters shared few transcription factors in common, but were conserved across several species. The elucidation of these promoters and identification of TFBS has important implications for future studies in sporadic tumors from multiple sites, and in JP patients.
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    ABSTRACT: BMPR1A is a cell surface receptor in the bone morphogenetic protein (BMP) pathway. Mutations in BMPR1A predispose to juvenile polyposis (JP). Sp1 and related proteins are widely expressed regulators of gene transcription, including members of the BMP pathway. We set out to identify important transcription factor binding sites (TFBS) in the recently identified BMPR1A promoter and to assess for the role of Sp1 and associated proteins in its regulation. The BMPR1A promoter was cloned into a luciferase reporter vector. Deletion fragments of this promoter insert were then constructed, of varying lengths and opposing directions, and were used to transfect HEK-293 and CRL-1459 cells. In silico analysis was performed to screen for relevant TFBS. Site-directed mutagenesis (SDM) was then employed to individually disrupt these TFBS in the wild-type (WT) vector. SDM constructs were then assessed for activity. Light activity from the deletion constructs ranged between 3% and 129% of the WT promoter. ModelInspector identified eight potential binding sites for Sp1- and Sp1-associated proteins that mapped to areas of marked loss or gain of activity from the deletion constructs. SDM of these TFBS led to a drop in activity in five mutants, which included 3 Sp1 sites, an ETSF site, and NFκB site. By combining in silico analysis and experimental data, Sp1 was found to be a candidate factor that likely plays a role in the transcriptional regulation of BMPR1A. This study potentially provides further insight toward the molecular basis of JP, and suggests that Sp1 plays a role in BMP signaling.
    Full-text · Article · Jul 2011 · Journal of Surgical Research
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    ABSTRACT: Dahdaleh FS, Carr JC, Calva D, Howe JR. Juvenile polyposis and other intestinal polyposis syndromes with microdeletions of chromosome 10q22–23. Juvenile polyposis (JP) is an autosomal dominant hamartomatous polyposis syndrome that carries a significant risk for the development of colorectal cancer. Microdeletions of one of the two predisposing genes to JP, BMPR1A, have been associated with a severe form of JP called juvenile polyposis of infancy. Many of these deletions have also been found to contiguously include PTEN, which is the gene responsible for the development of Cowden syndrome. The advent of molecular techniques that localize genomic copy number variations and others that target specific genes such as multiplex-ligation probe analysis has allowed researchers to explore this area further for deletions. Here, we review the literature for microdeletions described on chromosome 10q22–23 in patients with JP and other intestinal polyposis syndromes.
    Full-text · Article · Aug 2011 · Clinical Genetics
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