Functional Characterization of Rare Variants in the SHOX2 Gene Identified in Sinus Node Dysfunction and Atrial Fibrillation

Sinus node dysfunction (SND) and atrial fibrillation (AF) often coexist; however, the molecular mechanisms linking both conditions remain elusive. Mutations in the homeobox-containing SHOX2 gene have been recently associated with early-onset and familial AF. Shox2 is a key regulator of sinus node development, and its deficiency leads to bradycardia, as demonstrated in animal models. To provide an extended SHOX2 gene analysis in patients with distinct arrhythmias, we investigated SHOX2 as a susceptibility gene for SND and AF by screening 98 SND patients and 450 individuals with AF. The functional relevance of the novel mutations was investigated in vivo and in vitro, together with the previously reported p.H283Q variant. A heterozygous missense mutation (p.P33R) was identified in the SND cohort and four heterozygous variants (p.G77D, p.L129=, p.L130F, p.A293=) in the AF cohort. Overexpression of the pathogenic predicted mutations in zebrafish revealed pericardial edema for p.G77D and the positive control p.H283Q, whereas the p.P33R and p.A293= variants showed no effect. In addition, a dominant-negative effect with reduced heart rates was detected for p.G77D and p.H283Q. In vitro reporter assays demonstrated for both missense variants p.P33R and p.G77D significantly impaired transactivation activity, similar to the described p.H283Q variant. Also, a reduced Bmp4 target gene expression was revealed in zebrafish hearts upon overexpression of the p.P33R mutant. This study associates additional rare variants in the SHOX2 gene implicated in the susceptibility to distinct arrhythmias and allows frequency estimations in the AF cohort (3/990). We also demonstrate for the first time a genetic link between SND and AF involving SHOX2. Moreover, our data highlight the importance of functional investigations of rare variants

Evidence That Non-Syndromic Familial Tall Stature Has an Oligogenic Origin Including Ciliary Genes

Human growth is a complex trait. A considerable number of gene defects have been shown to cause short stature, but there are only few examples of genetic causes of non-syndromic tall stature. Besides rare variants with large effects and common risk alleles with small effect size, oligogenic effects may contribute to this phenotype. Exome sequencing was carried out in a tall male (height 3.5 SDS) and his parents. Filtered damaging variants with high CADD scores were validated by Sanger sequencing in the trio and three other affected and one unaffected family members. Network analysis was carried out to assess links between the candidate genes, and the transcriptome of murine growth plate was analyzed by microarray as well as RNA Seq. Heterozygous gene variants in CEP104, CROCC, NEK1, TOM1L2, and TSTD2 predicted as damaging were found to be shared between the four tall family members. Three of the five genes (CEP104, CROCC, and NEK1) belong to the ciliary gene family. All genes are expressed in mouse growth plate. Pathway and network analyses indicated close functional connections. Together, these data expand the spectrum of genes with a role in linear growth and tall stature phenotypes

miR-16 and miR-125b are involved in barrier function dysregulation through the modulation of claudin-2 and cingulin expression in the jejunum in IBS with diarrhoea

Micro-RNAs (miRNAs) play a crucial role in controlling intestinal epithelial barrier function partly by modulating the expression of tight junction (TJ) proteins. We have previously shown differential messenger RNA (mRNA) expression correlated with ultrastructural abnormalities of the epithelial barrier in patients with diarrhoea-predominant IBS (IBS-D). However, the participation of miRNAs in these differential mRNA-associated findings remains to be established. Our aims were (1) to identify miRNAs differentially expressed in the small bowel mucosa of patients with IBS-D and (2) to explore putative target genes specifically involved in epithelial barrier function that are controlled by specific dysregulated IBS-D miRNAs. Healthy controls and patients meeting Rome III IBS-D criteria were studied. Intestinal tissue samples were analysed to identify potential candidates by: (a) miRNA-mRNA profiling; (b) miRNA-mRNA pairing analysis to assess the co-expression profile of miRNA-mRNA pairs; (c) pathway analysis and upstream regulator identification; (d) miRNA and target mRNA validation. Candidate miRNA-mRNA pairs were functionally assessed in intestinal epithelial cells. IBS-D samples showed distinct miRNA and mRNA profiles compared with healthy controls. TJ signalling was associated with the IBS-D transcriptional profile. Further validation of selected genes showed consistent upregulation in 75% of genes involved in epithelial barrier function. Bioinformatic analysis of putative miRNA binding sites identified hsa-miR-125b-5p and hsa-miR-16 as regulating expression of the TJ genes CGN (cingulin) and CLDN2 (claudin-2), respectively. Consistently, protein expression of CGN and CLDN2 was upregulated in IBS-D, while the respective targeting miRNAs were downregulated. In addition, bowel dysfunction, perceived stress and depression and number of mast cells correlated with the expression of hsa-miR-125b-5p and hsa-miR-16 and their respective target proteins. Modulation of the intestinal epithelial barrier function in IBS-D involves both transcriptional and post-transcriptional mechanisms. These molecular mechanisms include miRNAs as master regulators in controlling the expression of TJ proteins and are associated with major clinical symptoms

Mutational analysis of the PITX2 coding region revealed no common cause for transposition of the great arteries (dTGA)

BACKGROUND: PITX2 is a bicoid-related homeodomain transcription factor that plays an important role in asymmetric cardiogenesis. Loss of function experiments in mice cause severe heart malformations, including transposition of the great arteries (TGA). TGA accounts for 5–7% of all congenital heart diseases affecting 0.2 per 1000 live births, thereby representing the most frequent cyanotic heart defect diagnosed in the neonatal period. METHODS: To address whether altered PITX2 function could also contribute to the formation of dTGA in humans, we screened 96 patients with dTGA by means of dHPLC and direct sequencing for mutations within the PITX2 gene. RESULTS: Several SNPs could be detected, but no stop or frame shift mutation. In particular, we found seven intronic and UTR variants, two silent mutations and two polymorphisms within the coding region. CONCLUSION: As most sequence variants were also found in controls we conclude that mutations in PITX2 are not a common cause of dTGA

Alternative Splicing and Nonsense-Mediated RNA Decay Contribute to the Regulation of SHOX Expression

The human SHOX gene is composed of seven exons and encodes a paired-related homeodomain transcription factor. SHOX mutations or deletions have been associated with different short stature syndromes implying a role in growth and bone formation. During development, SHOX is expressed in a highly specific spatiotemporal expression pattern, the underlying regulatory mechanisms of which remain largely unknown. We have analysed SHOX expression in diverse embryonic, fetal and adult human tissues and detected expression in many tissues that were not known to express SHOX before, e.g. distinct brain regions. By using RT-PCR and comparing the results with RNA-Seq data, we have identified four novel exons (exon 2a, 7-1, 7-2 and 7-3) contributing to different SHOX isoforms, and also established an expression profile for the emerging new SHOX isoforms. Interestingly, we found the exon 7 variants to be exclusively expressed in fetal neural tissues, which could argue for a specific role of these variants during brain development. A bioinformatical analysis of the three novel 3′UTR exons yielded insights into the putative role of the different 3′UTRs as targets for miRNA binding. Functional analysis revealed that inclusion of exon 2a leads to nonsense-mediated RNA decay altering SHOX expression in a tissue and time specific manner. In conclusion, SHOX expression is regulated by different mechanisms and alternative splicing coupled with nonsense-mediated RNA decay constitutes a further component that can be used to fine tune the SHOX expression level

The Homeobox Transcription Factor HOXA9 Is a Regulator of <em>SHOX</em> in U2OS Cells and Chicken Micromass Cultures

<div><p>The homeobox gene <em>SHOX</em> encodes for a transcription factor that plays an important role during limb development. Mutations or deletions of <em>SHOX</em> in humans cause short stature in Turner, Langer and Leri-Weill syndrome as well as idiopathic short stature. During embryonic development, <em>SHOX</em> is expressed in a complex spatio-temporal pattern that requires the presence of specific regulatory mechanisms. Up to now, it was known that <em>SHOX</em> is regulated by two upstream promoters and several enhancers on either side of the gene, but no regulators have been identified that can activate or repress the transcription of <em>SHOX</em> by binding to these regulatory elements. We have now identified the homeodomain protein HOXA9 as a positive regulator of <em>SHOX</em> expression in U2OS cells. Using luciferase assays, chromatin immunoprecipitation and electrophoretic mobility shift assays, we could narrow down the HOXA9 binding site to two AT-rich sequences of 31 bp within the <em>SHOX</em> promoter 2. Virus-induced <em>Hoxa9</em> overexpression in a chicken micromass model validated the regulation of <em>Shox</em> by Hoxa9 (negative regulation). As <em>Hoxa9</em> and <em>Shox</em> are both expressed in overlapping regions of the developing limb buds, a regulatory relationship of Hoxa9 and Shox during the process of limb development can be assumed.</p> </div

<i>In situ</i> hybridizations for <i>Hoxa9</i> and <i>Shox</i> in chicken embryos (d3-d7).

<p>The whole body is imaged for d3 to d4 embryos. Emerging limb buds are marked by an asterisk, pharyngeal arches are pointed by an arrow (A–D, A’-D’). For d5–d7 embryos, the right wing bud is presented to provide a detailed view of expression in the limb bud only (E–G, E’-G’). <i>Hoxa9</i> is expressed very early during embryonic development: expression is seen in d3 embryos along the vertebral axis of the posterior part of the body. In limb buds, expression starts at d3.25 (B) and persists until d6 (C–F). <i>Hoxa9</i> is expressed uniformly in the mesenchyme of the limb buds (A–G). <i>Shox</i> is also expressed during early embryonic stages and is already visible in the pharyngeal arches of d3 embryos (A’). With the outgrowth of the limb buds at d3.25 (B’), expression is also seen in wing and leg buds. Until stage d4, expression is seen in the whole limb bud (C’-D’); in later stages, expression is restricted to the middle segments of the limb buds (E’-G’). By stage d7, expression also begins to appear along the digital rays of the autopod (G’). Expression in the pharyngeal arches persists during all developmental stages analyzed (A’-G’).</p

EMSA experiments to confine the exact binding sites of HOXA9 within the <i>SHOX</i> promoter 2.

<p>(A) Division of the <i>SHOX</i> promoter 2 sequence into three DNA oligos (green, blue, red) of similar lengths. Upon addition of purified GST-tagged HOXA9 protein, oligo 2 (blue) and oligo 3 (red) were able to bind HOXA9 (left panel). Further subdivision of oligo 2 and 3 into three overlapping oligos of 31 bp each revealed that only oligo 2b and 3b can bind to HOXA9, thus narrowing down the binding sites to two sequences of 31 bp each (middle and right panel). (B) Mutations of five nucleotides in oligo 2b or 3b, respectively, inhibited the binding of HOXA9. (C) EMSA experiments confirm the binding sites of cHoxa9 to the chicken <i>Shox</i> promoter. ChOligo 2b and 3b are homologous to the human oligos 2b and 3b that were used in the EMSA experiments in (A). Both chOligo 2b and 3b were able to bind cHoxa9 protein. Mutations of five nucleotides in chOligo 2b and 3b, respectively, largely inhibited the binding of cHoxa9. As a control, oligos were incubated without protein (w/o) or with GST alone, where no shift was observed.</p

Analysis of the effect of Hoxa9 overexpression in chMM cultures by qRT-PCR and <i>in situ</i> hybridization.

<p>(A) <i>In situ</i> hybridization on chMM cultures (d3–d9). (a–i) overview of cultures; (a’-i’) detailed view. Especially in d6 and d9 cultures, <i>Hoxa9</i> infected cultures (a–c’) exhibit a generally weaker <i>Shox</i> expression compared to the control cultures (d–f’ and g–I’). Scale bar = 1000 µM. (B) Left panel: qRT-PCR analysis of <i>Hoxa9</i> expression levels after virus-induced Hoxa9 overexpression. Infection with Hoxa9-RCAS leads to a strong increase of <i>Hoxa9</i> expression for all time points analyzed. Right panel: qRT-PCR analysis of <i>Shox</i> expression levels in the corresponding samples. For all time points analyzed, <i>Shox</i> expression is reduced in the cultures that have been infected with Hoxa9 virus.</p