<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

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

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

Luciferase assays with <i>SHOX</i> cis-regulatory elements.

<p>(A) Schematic overview of <i>SHOX</i> cis-regulatory elements (not drawn to scale). CpG 1 and 2, which contain the two <i>SHOX</i> promoters, encompass the regions of exon 1 and 2, respectively (CpG1 chrX/Y:504,564-505,326; CpG2 chrX/Y:510,430-512,197). In addition, there are six known limb specific enhancer elements (CNE-5: chrX/Y: 318,357-318,906; CNE-3 chrX/Y:380,279-380,664, CNE-2: chrX/Y:436,610-437,229; CNE4: chrX/Y:634,085-634,740; CNE5: chrX:670,705-671,956; CNE9: chrX:754,746+755,567). (B) Luciferase assays for the <i>SHOX</i> enhancers. <i>SHOX</i> enhancers were cloned upstream of a firefly luciferase into the vector pGL3-Promoter and cotransfected with a <i>HOXA9</i> expression vector or the empty or mutant control vectors, respectively. Overexpression of HOXA9 or its mutants produced only low increases of comparable levels of luciferase activity arguing for a HOXA9 independent effect. (C-D) Deletion analysis of <i>SHOX</i> CpG Islands 1 and 2 to narrow down the site of regulatory <i>HOXA9</i> activity by luciferase assays. (C) <i>SHOX</i> CpG Islands (schematically drawn as green bars) were cloned upstream a firefly luciferase into the vector pGL3-Basic and cotransfected with a <i>HOXA9</i> overexpression vector or the empty or mutant control vectors, respectively. Upon <i>HOXA9</i> expression, luciferase activity increases for CpG1 (8 fold) and for CpG2 (70 fold) (left and middle panel). As a control, CpG luciferase vectors were also cotransfected with <i>HOXD9</i> expression vectors and the respective control vectors. HOXD9 was not able to evoke an increase of luciferase activity as seen for HOXA9 (right panel). (D) Subdivision of CpG2 (as indicated by green bars). Upon <i>HOXA9</i> overexpression, a stronger increase of luciferase activity was seen for CpG2 part 2 than for part 1. (E) Subdivision of CpG2 part 2. CpG2 part 2a was able to evoke stronger luciferase activity compared with CpG2 part 2b. This region is therefore considered to inherit the main sites that are important for the HOXA9 mediated regulatory activity.</p

<i>HOXA9</i> overexpression in U2OS cells increases <i>SHOX</i> expression.

<p>(A) qRT-PCR analysis of <i>HOXA9</i> expression levels after transient overexpression of <i>HOXA9</i> in U2OS cells. A strong increase is seen upon transfection with wild type constructs as well as mutant constructs. (B) qRT-PCR analysis of <i>SHOX</i> expression levels after overexpression of <i>HOXA9</i>. <i>HOXA9</i> wild type, but not its mutants, is able to increase <i>SHOX</i> expression. <i>HOXA9 Mut1 = K223E; HOXA9 Mut2 = K223E, N256del, R257P, R258G.</i> All mutations affect highly conserved amino acids within the homeodomain.</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

qRT-PCR of precipitated DNA of a chromatin immunoprecipitation (ChIP) experiment.

<p>ChIPs were performed from U2OS cells transfected with <i>HOXA9</i>-wt-Flag using an α-Flag-Antibody or mouse IgG as control, respectively. Samples of immunoprecipitated DNA were checked for an enrichment of the putative binding sites compared to randomly selected sequences residing in 0.8 to 2.5 kb distance. In total, four primer pairs were established, two of which reside within <i>SHOX</i> promoter 2 containing potential HOXA9 binding sites, and two of which reside outside that region. For better comparability, the amount of DNA that was amplified out of the control sample (IgG precipitation) was set to 1. The two PCR products amplifying the potential HOXA9 binding sites (ChIP HOXA9 Amp1 and Amp2) show a higher enrichment of immunoprecipitated DNA compared to the control regions (ChIP HOXA9 Contr 1 and 2). ChIP HOXA9 Contr 1 and 2 both are residing more than 2 kb from the promoter.</p

An Efficient and Comprehensive Strategy for Genetic Diagnostics of Polycystic Kidney Disease

<div><p>Renal cysts are clinically and genetically heterogeneous conditions. Autosomal dominant polycystic kidney disease (ADPKD) is the most frequent life-threatening genetic disease and mainly caused by mutations in <i>PKD1</i>. The presence of six <i>PKD1</i> pseudogenes and tremendous allelic heterogeneity make molecular genetic testing challenging requiring laborious locus-specific amplification. Increasing evidence suggests a major role for <i>PKD1</i> in early and severe cases of ADPKD and some patients with a recessive form. Furthermore it is becoming obvious that clinical manifestations can be mimicked by mutations in a number of other genes with the necessity for broader genetic testing. We established and validated a sequence capture based NGS testing approach for all genes known for cystic and polycystic kidney disease including <i>PKD1</i>. Thereby, we demonstrate that the applied standard mapping algorithm specifically aligns reads to the <i>PKD1</i> locus and overcomes the complication of unspecific capture of pseudogenes. Employing careful and experienced assessment of NGS data, the method is shown to be very specific and equally sensitive as established methods. An additional advantage over conventional Sanger sequencing is the detection of copy number variations (CNVs). Sophisticated bioinformatic read simulation increased the high analytical depth of the validation study and further demonstrated the strength of the approach. We further raise some awareness of limitations and pitfalls of common NGS workflows when applied in complex regions like <i>PKD1</i> demonstrating that quality of NGS needs more than high coverage of the target region. By this, we propose a time- and cost-efficient diagnostic strategy for comprehensive molecular genetic testing of polycystic kidney disease which is highly automatable and will be of particular value when therapeutic options for PKD emerge and genetic testing is needed for larger numbers of patients.</p></div

Mutations and variants identified in other genes for cystic and polycystic kidney disease.

<p>NGS data that demonstrate the power of the setup for parallel analysis of all genes known to date for cystic and polycystic kidney disease and related disorders in a single step.</p><p>* Classification from PKD mutation database (ADPKD Mutation Database (<a href="http://pkdb.mayo.edu/" target="_blank">http://pkdb.mayo.edu/</a>)</p><p>** classification taken from ARPKD/<i>PKHD1</i> database (<a href="http://www.humgen.rwth-aachen.de" target="_blank">http://www.humgen.rwth-aachen.de</a>); het—heterozygous; LH—likely hypomorphic; LP—likely pathogenic; HLP—highly likely pathogenic; DP—definitely pathogenic; PP—probably pathogenic; P—pathogenic.</p><p>Mutations and variants identified in other genes for cystic and polycystic kidney disease.</p

Detection level and distribution of <i>PKD1</i> variants in our cohort and by variant simulation.

<p><b>A</b> Percentage of alternative reads detected by our NGS approach for all <i>PKD1</i> variants (black dots) from our cohort is displayed. All except seven variants could be detected above the standard detection threshold (black/grey lines displaying exon boundaries). The variants highlighted with red dots lie in the duplicated region and required second-step analysis with a lower detection threshold (8% alternative reads, red dashed line) to be detected in these critical exons. One variant in exon 1 could not be detected due to insufficient coverage on the MiSeq system. The variant in exon 42 lies at position +28 and is hardly covered due to insufficient coverage of this intronic region (no further adjustment of the analysis). Underneath, the <i>PKD1</i> genomic locus with a coverage plot is shown. <b>B</b> Percentage of alternative reads detected by our bioinformatic workflow for <i>PKD1</i> variants (black dots) from the read simulation by Wgsim. An evenly distributed variant density which is even higher than in the real dataset could be achieved across all coding exons in the duplicated region. Only one variant (red dot) in exon 5 could be detected below the default detection threshold (black/grey lines displaying exon boundaries). The red dashed line marks the lower detection threshold applied in negative cases. Underneath, the <i>PKD1</i> genomic locus with its coverage by simulated reads is shown. The coloured lines reflect the positions of detected variants.</p