Dataset Information

An etiologic regulatory mutation in IRF6 with loss- and gain-of-function effects.

ABSTRACT: DNA variation in Interferon Regulatory Factor 6 (IRF6) causes Van der Woude syndrome (VWS), the most common syndromic form of cleft lip and palate (CLP). However, an etiologic variant in IRF6 has been found in only 70% of VWS families. To test whether DNA variants in regulatory elements cause VWS, we sequenced three conserved elements near IRF6 in 70 VWS families that lack an etiologic mutation within IRF6 exons. A rare mutation (350dupA) was found in a conserved IRF6 enhancer element (MCS9.7) in a Brazilian family. The 350dupA mutation abrogated the binding of p63 and E47 transcription factors to cis-overlapping motifs, and significantly disrupted enhancer activity in human cell cultures. Moreover, using a transgenic assay in mice, the 350dupA mutation disrupted the activation of MCS9.7 enhancer element and led to failure of lacZ expression in all head and neck pharyngeal arches. Interestingly, disruption of the p63 Motif1 and/or E47 binding sites by nucleotide substitution did not fully recapitulate the effect of the 350dupA mutation. Rather, we recognized that the 350dupA created a CAAAGT motif, a binding site for Lef1 protein. We showed that Lef1 binds to the mutated site and that overexpression of Lef1/?-Catenin chimeric protein repressed MCS9.7-350dupA enhancer activity. In conclusion, our data strongly suggest that 350dupA variant is an etiologic mutation in VWS patients and disrupts enhancer activity by a loss- and gain-of-function mechanism, and thus support the rationale for additional screening for regulatory mutations in patients with CLP.

SUBMITTER: Fakhouri WD

PROVIDER: S-EPMC3990169 | biostudies-literature | 2014 May

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

An etiologic regulatory mutation in IRF6 with loss- and gain-of-function effects.

Fakhouri Walid D WD Rahimov Fedik F Attanasio Catia C Kouwenhoven Evelyn N EN Ferreira De Lima Renata L RL Felix Temis Maria TM Nitschke Larissa L Huver David D Barrons Julie J Kousa Youssef A YA Leslie Elizabeth E Pennacchio Len A LA Van Bokhoven Hans H Visel Axel A Zhou Huiqing H Murray Jeffrey C JC Schutte Brian C BC

Human molecular genetics 20140116 10

DNA variation in Interferon Regulatory Factor 6 (IRF6) causes Van der Woude syndrome (VWS), the most common syndromic form of cleft lip and palate (CLP). However, an etiologic variant in IRF6 has been found in only 70% of VWS families. To test whether DNA variants in regulatory elements cause VWS, we sequenced three conserved elements near IRF6 in 70 VWS families that lack an etiologic mutation within IRF6 exons. A rare mutation (350dupA) was found in a conserved IRF6 enhancer element (MCS9.7) i ...[more]

PMID: 24442519

Similar Datasets

Project description:Comparison between transcriptomes of dash mutants vs WT plants and ectopic expression of 35S::DASH in hairy roots versus empty vector Loss of function: 3 WT plants A17 at 8 days after pollination and 3 dash mutant plants at 8 days afer pollination. Gain of function transformants: 3 transformed plants containing empty vector and 4 transformed plants containing 35S::DASH Total RNA was extracted using a modified cetyltrimethylammonium bromide method (Verdier et al., 2008). 5ug of total RNA from each sample was purified (RNeasy MinElute Cleanup kit; Qiagen) according to the manufacturer?s instructions. RNA was quantified and evaluated for purity using an ND-1000 Nanodrop Spectrophotometer (NanoDrop Technologies) and a Bioanalyzer 2100 (Agilent). The Affymetrix M. truncatula GeneChip Array (Affymetrix) was used for expression analysis during seed development. RNA from three (for dash mutant analysis) and four (for DASH ectopic expression analysis) independent biological replicates were analysed for each time point. Probe synthesis/labelling was carried out from 500 ng of RNA using the GeneChip 3?IVT express kit, according to the manufacturer?s instructions (Affymetrix). Array hybridization, scanning, and data normalization were performed as described by Benedito et al., (2008). Each file from the hybridized Affymetrix array was exported from GeneChip Operating Software version 1.4 (Affymetrix) and imported into Robust Multiarray Average Express (Irizarry et al., 2003) for global normalization. Presence/absence call for each probe set to remove background noise was obtained using dCHIP (Li and Wong, 2001). To identify probe sets differentially expressed in dash mutant vs WT control, the R package Anapuce (J. Aubert, UMR 518 AgroParisTech/INRA) was used. For each probe set, a paired t-test was performed on the log2 expression data from three arrays (3 independent biological repeats for 8 dap data), assuming that the variance of the log expression was the same for all transcripts per genotype. Spots with extreme specific variance, too low or too high, were excluded from the statistical analysis (details on the procedure given in Gagnot et al., 2008). P-values were adjusted by the Benjamini-Hochberg method (Benjamini and Hochberg, 1995), which controls the family-wise error rate.

Project description:ObjectiveSH3BP2 is a signaling adapter protein which regulates immune and skeletal systems. Gain-of-function mutations in SH3BP2 cause cherubism, characterized by jawbone destruction. This study was aimed to examine the role of SH3BP2 in inflammatory bone loss using a collagen-induced arthritis (CIA) model.MethodsCIA was induced in wild-type (Sh3bp2(+/+)) and heterozygous P416R SH3BP2 cherubism mutant knock-in (Sh3bp2(KI/+)) mice, an SH3BP2 gain-of-function model. Severity of the arthritis was determined by assessing the paw swelling and histological analyses of the joints. Micro-CT analysis was used to determine the levels of bone loss. Inflammation and osteoclastogenesis in the joints were evaluated by quantitating the gene expression of inflammatory cytokines and osteoclast markers. Furthermore, involvement of the T- and B-cell responses was determined by draining lymph node cell culture and measurement of the serum anti-mouse type II collagen antibody levels, respectively. Finally, roles of the SH3BP2 mutation in macrophage activation and osteoclastogenesis were determined by evaluating the TNF-? production levels and osteoclast formation in bone marrow-derived M-CSF-dependent macrophage (BMM) cultures.ResultsSh3bp2(KI/+) mice exhibited more severe inflammation and bone loss, accompanying an increased number of osteoclasts. The mRNA levels for TNF-? and osteoclast marker genes were higher in the joints of Sh3bp2(KI/+) mice. Lymph node cell culture showed that lymphocyte proliferation and IFN-? and IL-17 production were comparable between Sh3bp2(+/+) and Sh3bp2(KI/+) cells. Serum anti-type II collagen antibody levels were comparable between Sh3bp2(+/+) and Sh3bp2(KI/+) mice. In vitro experiments showed that TNF-? production in Sh3bp2(KI/+) BMMs is elevated compared with Sh3bp2(+/+) BMMs and that RANKL-induced osteoclastogenesis is enhanced in Sh3bp2(KI/+) BMMs associated with increased NFATc1 nuclear localization.ConclusionGain-of-function of SH3BP2 augments inflammation and bone loss in the CIA model through increased macrophage activation and osteoclast formation. Therefore, modulation of the SH3BP2 expression may have therapeutic potential for the treatment of rheumatoid arthritis.

Dataset Information

An etiologic regulatory mutation in IRF6 with loss- and gain-of-function effects.

Publications

An etiologic regulatory mutation in IRF6 with loss- and gain-of-function effects.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets