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

Originally reported and newly determined structure of the <i>Sdccag8</i>^<i>SBT</i> allele.

<p><i>Top</i>) originally reported structure of the <i>Sdccag8</i>^<i>SBT</i> allele; <i>middle</i>) structure of the wild-type allele; <i>bottom</i>) the <i>Sdccag8</i>^<i>SBT</i> allele structure determined in this study. The <i>Sdccag8</i> locus is in pink and the <i>Akt3</i> locus, in yellow. Contrary to the originally reported structure of the <i>Sdccag8</i>^<i>SBT</i> allele, a deletion that encompasses both <i>Sdccag8</i> exons 13–18 and <i>Akt3</i> exons 2–13 was identified. IR/DR: Inverted repeat/direct repeat sequence, the SBT recognition site (280 bp); AD2 SA: Adenovirus 2 splice acceptor site; STOP: Stop signal; pA: poly-A sequence; Tyr Prom: Tyrosinase upstream regulatory sequences (2.1 kb from the BALB/c tyrosinase promoter and the first 65 bp of exon 1); Tyr ORF: C57BL/6-derived Tyr^s-J cDNA sequence; loxP: Cre recombinase target site; Hox9c SA: Splice acceptor; Fw; WT forward primer site; Rw: WT reverse primer site; Fm: mutant forward primer site; Rm: mutant reverse primer site. All numbers provide an exon number for the corresponding box below it.</p

Comparison of phenotypes seen in SDCCAG8 mouse models (<i>Sdccag8</i>^{<i>Gt(OST40418)Tigm</i>}, <i>Sdccag8</i>^{<i>tm1e(EUCOMM)Wtsi</i>}, and <i>Sdccag8</i>^<i>SBT</i>) and the <i>Akt3</i>^<i>-/-</i> mouse.

<p>Comparison of phenotypes seen in SDCCAG8 mouse models (<i>Sdccag8</i>^{<i>Gt(OST40418)Tigm</i>}, <i>Sdccag8</i>^{<i>tm1e(EUCOMM)Wtsi</i>}, and <i>Sdccag8</i>^<i>SBT</i>) and the <i>Akt3</i>^<i>-/-</i> mouse.</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

Loss of <i>Sdccag8</i> exons 13–18 and <i>Akt3</i> exons 2–13 in the <i>Sdccag8</i>^<i>SBT</i> allele.

<p><b>A)</b> PCR primers were designed to amplify each exon of <i>Akt3</i> and exons 13–18 of <i>Sdccag8</i> from mouse genomic DNA. PCR products from <i>Sdccag8</i>^<i>+/+</i>, <i>Sdccag8</i>^<i>+/SBT</i>, and <i>Sdccag8</i>^{<i>SBT/SBT</i>} mice were loaded onto an agarose gel. <b>B</b>) RT-qPCR with primers spanning exons 3–4 of <i>Akt3</i> shows absence of <i>Akt3</i> transcripts in <i>Sdccag8</i>^{<i>SBT/SBT</i>} animals.</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