Genotypic and phenotypic characterization of the <i>Sdccag8^{Tn(sb-Tyr)2161B.CA1C2Ove}</i> mouse model

<div><p>Nephronophthisis-related ciliopathies (NPHP-RC) are a group of disorders that present with end-stage renal failure in childhood/adolescence, kidney cysts, retinal degeneration, and cerebellar hypoplasia. One disorder that shares clinical features with NPHP-RC is Bardet-Biedl Syndrome (BBS). Serologically defined colon cancer antigen 8 (<i>SDCCAG8</i>; also known as NPHP10 and BBS16) is an NPHP gene that is also associated with BBS. To better understand the patho-mechanisms of NPHP and BBS caused by loss of SDCCAG8 function, we characterized an SDCCAG8 mouse model (<i>Sdccag8</i>^{<i>Tn(sb-Tyr)2161B</i>.<i>CA1C2Ove</i>}) generated by Sleeping Beauty Transposon (SBT)-mediated insertion mutagenesis. Consistent with the previously reported, independent SDCCAG8 mouse models, our mutant mice display pre-axial polydactyly in their hind limbs. In addition, we report patterning defects in the secondary palate, brain abnormalities, as well as neonatal lethality associated with developmental defects in the lung in our mouse model. The neonatal lethality phenotype is genetic background dependent and rescued by introducing 129S6/SvEvTac background. Genetic modifier(s) responsible for this effect were mapped to a region between SNPs rs3714172 and rs3141832 on chromosome 11. While determining the precise genetic lesion in our mouse model, we found that SBT insertion resulted in a deletion of multiple exons from both <i>Sdccag8</i> and its neighboring gene <i>Akt3</i>. We ascribe the patterning defects in the limb and the secondary palate as well as lung abnormalities to loss of SDCCAG8, while the developmental defects in the brain are likely due to the loss of AKT3. This mouse model may be useful to study features not observed in other SDCCAG8 models but cautions are needed in interpreting data.</p></div

A region between rs3714172 and rs3141832 shows a significant association with survival.

<p>Comparison of allele distribution in mice that survive to P21 or later, analyzed using a Chi Square test with expected distribution.</p

Lack of <i>Sdccag8</i> expression in <i>Sdccag8</i>^<i>SBT</i> gene-trap mice.

<p><b>A)</b> Representative PCR genotyping results for <i>Sdccag8</i>^<i>+/+</i>, <i>Sdccag8</i>^<i>+/SBT</i>, <i>and Sdccag8</i>^{<i>SBT/SBT</i>} mice. Primer pairs Fw+Rw and Fm+Rm detect the presence of wild-type (WT) and mutant (Mut; SBT) alleles, respectively. <b>B)</b> qPCR results show the presence of <i>Sdccag8</i> mRNAs 5’ of the insertion site but their absence 3’ of the insertion. cDNAs from the brain, kidney, and lung were used for qPCR. Error bars represent standard errors. <b>C-E)</b> Immunoblot for SDCCAG8 shows loss of SDCCAG8 in the brain <b>(C)</b>, kidney <b>(D)</b>, and lung <b>(E)</b>. Arrowheads indicate full length SDCCAG8.</p

Developmental defects in the <i>Sdccag8</i>^{<i>SBT/SBT</i>} mutant lung.

<p><b>A</b>) <i>Sdccag8</i>^{<i>SBT/SBT</i>} mice (right) are cyanotic at P0 (before death). A wild-type (WT) littermate is shown on the left. <b>B</b>) H&E staining of lung sections from a WT (left) and a mutant littermate (right). Scale bar = 20 μm.</p

Neonatal <i>Sdccag8</i>^{<i>SBT/SBT</i>} mice have secondary palate anomalies, pre-axial polydactyly, and brain abnormalities but not cystic kidney.

<p><b>A)</b> Alcian blue and Alizarin red staining of the secondary palate. Arrowheads show alterations to the basisphenoid (BS), presphenoid (PS), and premaxilla (PM) regions of the palate. <b>B)</b> Alcian blue and Alizarin red staining of the forelimb. <b>C)</b> Alcian blue and Alizarin red staining of the hind limb. <b>D)</b> H&E staining of kidney sections. Scale bar = 200 μm <b>E)</b> H&E-stained brain sections. Anterior commissures are circled and the dashed line highlights the white matter and corpus callosum. Scale bar = 500 μm. In all panels, wild-type (at P0) is shown on the left and <i>Sdccag8</i>^{<i>SBT/SBT</i>} mutant littermates are on the right.</p