Genes and Immunity (2007) 8, 416–421 & 2007 Nature Publishing Group All rights reserved 1466-4879/07 $30.00 www.nature.com/gene ORIGINAL ARTICLE Spontaneous mutations in the mouse Sharpin gene result in multiorgan inflammation, immune system dysregulation and dermatitis RE Seymour1, MG Hasham1, GA Cox1, LD Shultz1, H HogenEsch2, DC Roopenian1 and JP Sundberg1 1The Jackson Laboratory, Bar Harbor, ME, USA and 2Department of Veterinary Pathobiology, Purdue University, West Lafayette, IN, USA Homologues of the SHARPIN (SHANK-associated RH domain-interacting protein) gene have been identified in the human, rat and mouse genomes. SHARPIN and its homologues are expressed in many tissues. SHARPIN protein forms homodimers and associates with SHANK in the post-synaptic density of excitatory neurotransmitters in the brain. SHARPIN is hypothesized to have roles in the crosslinking of SHANK proteins and in enteric nervous system function. We demonstrate that two independently arising spontaneous mutations in the mouse Sharpin gene, cpdm and cpdmDem, cause a chronic proliferative dermatitis phenotype, which is characterized histologically by severe inflammation, eosinophilic dermatitis and defects in secondary lymphoid organ development. These are the first examples of disease-causing mutations in the Sharpin gene and demonstrate the importance of SHARPIN protein in normal immune development and control of inflammation. Genes and Immunity (2007) 8, 416–421; doi:10.1038/sj.gene.6364403; published online 31 May 2007 Keywords: Sharpin; lymphoid organ development; B cell; eosinophil Introduction between markers D15Mit156 and D15Mit2 using an intraspecific cross of C57BL/KaLawRij-cpdm/cpdm with The chronic proliferative dermatitis mutation (cpdm) BALB/cJ. An intersubspecific cross using CAST/EiJ was was first observed as a spontaneous genetic disease in created to increase polymorphisms in this region for C57BL/KaLawRij mice at the TNO Institute, Rijswijk, high-resolution mapping; however, the skin phenotype The Netherlands. Mutant mice have a shortened lifespan, in some mice was highly variable, likely due to the runted appearance and a characteristic progressive contribution of CAST modifier genes.4 To avoid these dermatitis with alopecia that first becomes clinically complications, we developed a congenic strain, C.CAST- apparent at 3–4 weeks of age. Males and females are (D15Mit156-D15Mit2)/Sun, moving the CAST critical affected equally. The mutant phenotype includes multi- interval for cpdm onto the BALB/cJ background to organ inflammation with eosinophilia, defective TH1 advance fine mapping. This congenic strain retained a cytokine production, splenomegaly and dermatitis high rate of polymorphism in the m.Chr.15 interval of with granulocytic infiltrates, ortho- and parakeratotic 1,2 interest compared to C57BL/KaLawRij-cpdm/cpdm but hyperplasia, and follicular dystrophy. Peyer’s patches lacked the purported unlinked CAST-derived modifier are absent in adult mice, and remaining secondary genes altering the cpdm phenotype. Using the original lymphoid organs lack B-cell follicles, follicular dendritic (CAST/EiJ þ / þÂC57BL/KaLawRij-cpdm/cpdm) F2 in- cells and germinal centers. The B- and T-cell areas of tercross and over 1100 mice from the new C.CAST- the spleen are poorly defined, and the marginal zone (D15Mit156-D15Mit2)/Sun  C57BL/KaLawRij-cpdm/cpdm is absent. B-cell isotype switching and immuno- F2 intercross, seven critical recombinants were identified, globulin production is also affected, with significantly and the cpdm genetic interval was narrowed to o0.2 cM diminished serum immunoglobulins (Ig) IgG, IgA 3 (372 kb). The cpdm interval contained 21 identified genes and IgE. according to the Ensembl (http://www.ensembl.org/ index.html) mouse genome database annotation (Sup- plementary Table 1). The interval is located in a region of Results conserved synteny with human Chromosome 8q24.3 We initially localized the cpdm gene to an approximately (h.Chr.8q24.3); comparison of the mouse and human 1.2 cM interval on mouse Chromosome 15 (m.Chr.15) genomic sequences demonstrates a complete conserva- tion of gene order and gene content between the two species. Correspondence: Dr RE Seymour, The Jackson Laboratory, An independent spontaneous mutation resulting in a 600 Main Street, Bar Harbor, ME 04609, USA. E-mail: [email protected] similar phenotype was identified in OcB-3/Dem recom- Received 20 February 2007; revised 1 May 2007; accepted 3 May binant congenic mice at The Netherlands Cancer 2007; published online 31 May 2007 Institute, Amsterdam. Spontaneous mutations in the mouse Sharpin gene RE Seymour et al 417 Allelism of this second mutation with cpdm was con- the 14-bp deletion disrupts the reading frame and results firmed by heterozygous matings (C57BL/KaLawRij þ / in an early stop codon beginning at position 552. To cpdm  Ocb-3/Dem þ /cpdm) that produced affected F1 ensure that this change represented a mutation from the offspring at expected Mendelian ratios. This second strain of origin, we obtained and sequenced genomic strain was given the gene symbol cpdmDem,4 and back- DNA from wild-type Ocb3/Dem mice (kind gift of Dr M crossed onto the BALB/cByJ inbred strain for 10 Lipoldova, Institute of Molecular Genetics, Academy of generations for colony maintenance purposes, creating Sciences, Czech Republic), and determined that these the CBy.OcB3 congenic strain. changes are not seen in the wild-type allele (Figure 2). To Genes in the critical region for cpdm were initially validate a relationship between the Ocb-3/Dem control screened by real-time quantitative PCR (QPCR) assay, DNA and the CBy.OcB3-cpdmDem/cpdmDem mice main- which suggested Sharpin as a strong candidate, the only tained at The Jackson Laboratory, we also sequenced the gene in the interval that was significantly downregulated nearby Slc39a4 gene amplified from the control DNA to in testes or spleen of both C57BL/KaLawRij-cpdm/cpdm look for the presence of 10 benign polymorphisms that and CBy.OcB3-cpdmDem/cpdmDem mice as compared to the we observed previously between CBy.OcB3-cpdmDem/ same tissues in their respective controls (data not cpdmDem mice and the other strains used (data not shown). We began to examine genes in the interval by shown). These polymorphisms were also confirmed in direct sequence analysis of cDNA, using sequence the Ocb-3/Dem control mouse DNA, verifying the close obtained from NCBI m34 via Ensembl for comparison. relationship to CBy.OcB3-cpdmDem/cpdmDem. Slc39a4, Gpaa1, Hsf1 and Sharpin were completed, and Western blot analysis of spleen tissue homogenates independent mutations were identified in exon 1 of from pre-clinical (o12-day old) mice with anti-SHARPIN Sharpin for each of the two cpdm alleles. Examination of antibody demonstrated an approximately 40 kDa the complete genomic sequence of Sharpin revealed that protein, present in control mice but absent in both the these changes were not found in control mice of the C57BL/KaLawRij-cpdm/cpdm and CBy.OcB3-cpdmDem/ parent strains nor in C57BL/10J or BALB/cByJ strains. In cpdmDem mutants (Figure 3). The size is consistent with the C57BL/KaLawRij-cpdm/cpdm mice, a single base pair the predicted 39.9 kDa protein sequence for the mouse, deletion was observed at the 30-end of exon 1 (Figure 1), and slightly smaller than the 45-kDa major band resulting in a shift of the open reading frame predicted demonstrated by Lim et al. (2001) in rat tissues, probably to cause an early stop codon beginning at position reflecting species differences. Minor bands (o37 and 624 (numbered sequence is based on Ensembl cDNA 440 kDa) similar to those described for the rat were also transcript ENSMUST00000023211). The CBy.OcB3- observed in adult wild-type mice (data not shown). The cpdmDem/cpdmDem mouse showed a C to A transition at larger bands appear to be non-specific, as they were position 434 near the 50-end of Exon 1, followed by a visible in adult cpdm/cpdm mice and controls, though 14-bp deletion of nucleotides 438-451 (Figure 2). The barely detectable in mice o2 weeks of age. The smaller transition does not result in an amino acid change, but bands were not found in young mutant mice, and barely Figure 3 Western blot using polyclonal rabbit anti-SHARPIN antibody demonstrates absence of the protein in mutant mice. (a) Anti-SHARPIN antibody. (b) Anti-BETA ACTIN antibody. Protein size marker is shown at top left; numbers below lanes indicate the Figure 1 Chromatogram showing the C57BL/KaLawRij-cpdm/ ratio of SHARPIN to BETA ACTIN loading control. Lanes 1 and 2, cpdm mutation. There is a single base pair deletion in exon 1 C57BL/KaLawRij þ / þ ; lane 3 CBy.OcB3/Dem þ / þ . Lanes 4 (box). (a) C57BL/KaLawRij þ / þ genomic sequence. (b) cpdm/ and 5, C57BL/KaLawRij-cpdm/cpdm; lane 6, CBy.OcB3-cpdmDem/ cpdm. cpdmDem. Figure 2 Composite image of chromatograms demonstrating the C to A transition (box) and the 14-basepair deletion in exon 1 of the CBy.OcB3-cpdmDem/cpdmDem Sharpin gene. (a) BALB/cByJ þ / þ .(b) OcB3/Dem þ / þ .(c) CBy.OcB3-cpdmDem/cpdmDem. Genes and Immunity Spontaneous mutations in the mouse Sharpin gene RE Seymour et al 418 detectable in adult mutants. To further confirm that Discussion mutations in Sharpin cause the cpdm phenotype, we used in vivo complementation with a bacterial artificial SHARPIN and its homologues are expressed in multiple chromosome (BAC) clone expressed in FVB/NJ trans- tissues in humans and mice (GNF SymAtlas, v.1.1.1, genic mice created for that purpose. The BAC clone http://www.wombat.gnf.org/SymAtlas/). Orthologues RP24-173I23 was selected, as it carries a C57BL/B6 (B6)- are also found in rats, dogs, chimpanzees (Mouse derived chromosome 15 segment including Sharpin Genome Informatics, http://www.informatics.jax.org/), (UCSC Genome browser http://www.genome.ucsc. and probably other species (Ensembl, http://www.