A canine model of Imerslund-Grasbeck syndrome: Genetic mapping, mutation identification and functional analysis

Imerslund-Grasbeck syndrome (I-GS, OMIM #261100) is an autosomal recessive disease characterized by congenital selective intestinal malabsorption of cobalamin (vitamin B12) and protemuria. Some human patients are known to carry mutations in the cubilin gene (CUBN), which encodes a multiligand receptor expressed on both enterocytes and kidney proximal tubule cells. A Giant Schnauzer (GS) kindred studied in our lab is a well-established animal model for human I-GS, and has contributed significantly to the research on I-GS and cubilin. Previous experiments demonstrated abnormality of cubilin in the affected dogs, but linkage analysis excluded the CUBN as the disease-causing gene.; We performed a whole genome scan linkage analysis and linked the canine I-GS to a marker on dog chromosome 8, which is orthologous to human chromosome 14q. Type I markers were developed in the GS kindred to refine the mapping. Haplotype analysis narrowed the candidates region to be between markers EML1 and SIVA, a 5Mb interval predicted from the human genome. KNS2, a marker located in the interval, was in complete linkage disequilibrium with I-GS, with a multipoint LOD score of 15.4.; One gene in the interval, AMN (amnionless), was a compelling candidate because it was demonstrated as the second I-GS gene in a recent human study. We cloned the cDNA of canine AMN based on human, rat and mouse AMN sequences. RT-PCR and genomic PCR disclosed an in-frame deletion of 33 bp in exon 10 of AMN, which was predicted to abolish the hypothetical transmembrane domain of AMN. The deletion segregated with the disease in the GS kindred and was absent in 112 unrelated normal dogs.; We also studied an Australian Shepherd (AS) kindred with similar clinical features to the GS kindred. Linkage analysis of this small pedigree to marker KNS2 gave a LOD score of 1.7, marginally suggesting linkage of the disease to AMN. Mutation screening in AMN identified a G > A transition in the start codon, which was predicted to abrogate translation initiation. The mutation segregated with the disease and was not seen in 112 chromosomes of unrelated normal dogs.; A recent study showed that cubilin and AMN form a tight complex, cubam, which is crucial for endocytosis of certain ligands, such as intrinsic factor-Cobalamin (IF-Cbl). Our RT-PCR in multiple canine tissues demonstrated that both genes have high expression levels in ileum and kidney, although they have different expression profiles in some other tissues. In an IF-Cbl pull-down assay, three AMN isoforms were present in the kidney homogenates of a normal dog but absent in the affected dogs of both kindreds. In a heterologous cell transfection system, wildtype canine AMN, but not the 33bp-deletion mutant, assists the membrane expression of cubilin. We therefore demonstrated in vivo that the fundamental cause of I-GS is the failure to express functional cubam, and canine I-GS is an orthologue of the human disease. The two pedigrees harboring different AMN mutations provide us a unique opportunity to study the functions of AMN and cubilin directly in tissues of both affected and normal dogs, which is nearly impossible in the human study of I-GS patients.