Lymphopenia in the BB rat model of type 1 diabetes is due to a mutation in a novel immune-associated nucleotide (Ian)-related gene

Abstract

The BB (BioBreeding) rat is one of the best models of spontaneous autoimmune diabetes and is used to study non-MHC loci contributing to Type 1 diabetes. Type 1 diabetes in the diabetes-prone BB (BBDP) rat is polygenic, dependent upon mutations at several loci. Iddm1, on chromosome 4, is responsible for a lymphopenia (lyp) phenotype and is essential to diabetes. In this study, we report the positional cloning of the Iddm1/lyp locus. We show that lymphopenia is due to a frameshift deletion in a novel member (Ian5) of the Immune-Associated Nucleotide (IAN)-related gene family, resulting in truncation of a significant portion of the protein. This mutation was absent in 37 other inbred rat strains that are nonlymphopenic and nondiabetic. The IAN gene family, lying within a tight cluster on rat chromosome 4, mouse chromosome 6, and human chromosome 7, is poorly characterized. Some members of the family have been shown to be expressed in mature T cells and switched on during thymic T-cell development, suggesting that Ian5 may be a key factor in T-cell development. The lymphopenia mutation may thus be useful not only to elucidate Type 1 diabetes, but also in the function of the Ian gene family as a whole.

Figure 1

The lymphopenia gene region and the Ian5 transcript. (A) Physical map of the rat lyp gene region with genetic markers integrated (top). Overlapping PAC clones are shown along with the locations of genetic markers used to narrow the lyp interval, clone-end STS assays, and the limits of the lyp interval itself as a red arrow at both top and bottom. Distances between markers may not be strictly to scale because they are estimated on STS content. The bottom part shows an expanded view of the lyp interval, showing the locations of known genes and the extent of the assembled sequence contigs of rat genomic DNA, along with the framework of mouse genomic DNA sequence. A 13-kb-long rat genomic sequence contig includes the rat Ian5 gene. Position coordinates shown are those from mouse sequence supercontig Mm6_WIFeb01_100. (B) The cluster of Ian-related genes. In human, this gene family is present on chromosome 7q36.1. In the mouse, it is located on proximal chromosome 6. We have also indicated the position of the human and mouse orthologs of the LR8/Clast1 (mouse accession no. AB031386) gene as location aids, although this gene is not in the IAN family. We have indicated various alternative names associated with each gene, and provisionally named previously unnamed members of the family as follows (these are indicated by underlines). For those genes without a common name, we have chosen to continue using the Ian gene nomenclature. To avoid confusion, in this work we will refer to the genes in this family by using the name of the mouse ortholog and a prefix, h, m, or r, to specify which species is indicated (e.g., hIan2 for the human ortholog of mouse Ian2, otherwise known as himap1). Genes given the same Ian designation in different species have been determined to be orthologs of each other. Genes with different designations do not show enough similarity to be deemed orthologs, with the exception of hIan7, which is orthologous to both mIan7 and mIan3. Genes in species without a clear ortholog in the other have been given unique Ian numbers (e.g., hIan12). Positions shown are within the respective contigs (accession nos. NT_007704.8‖Hs7_7861 for human and supercontig Mm6_WIFeb01_100 for mouse). (C) Diagram of the rat, mouse, and human Ian5 gene transcripts, with exon structure shown to scale. Beneath each transcript diagram is a diagram of the extent of the major ORF (the Ian5-coding region). Continued on the following page.

Figure 1

The lymphopenia gene region and the Ian5 transcript. (A) Physical map of the rat lyp gene region with genetic markers integrated (top). Overlapping PAC clones are shown along with the locations of genetic markers used to narrow the lyp interval, clone-end STS assays, and the limits of the lyp interval itself as a red arrow at both top and bottom. Distances between markers may not be strictly to scale because they are estimated on STS content. The bottom part shows an expanded view of the lyp interval, showing the locations of known genes and the extent of the assembled sequence contigs of rat genomic DNA, along with the framework of mouse genomic DNA sequence. A 13-kb-long rat genomic sequence contig includes the rat Ian5 gene. Position coordinates shown are those from mouse sequence supercontig Mm6_WIFeb01_100. (B) The cluster of Ian-related genes. In human, this gene family is present on chromosome 7q36.1. In the mouse, it is located on proximal chromosome 6. We have also indicated the position of the human and mouse orthologs of the LR8/Clast1 (mouse accession no. AB031386) gene as location aids, although this gene is not in the IAN family. We have indicated various alternative names associated with each gene, and provisionally named previously unnamed members of the family as follows (these are indicated by underlines). For those genes without a common name, we have chosen to continue using the Ian gene nomenclature. To avoid confusion, in this work we will refer to the genes in this family by using the name of the mouse ortholog and a prefix, h, m, or r, to specify which species is indicated (e.g., hIan2 for the human ortholog of mouse Ian2, otherwise known as himap1). Genes given the same Ian designation in different species have been determined to be orthologs of each other. Genes with different designations do not show enough similarity to be deemed orthologs, with the exception of hIan7, which is orthologous to both mIan7 and mIan3. Genes in species without a clear ortholog in the other have been given unique Ian numbers (e.g., hIan12). Positions shown are within the respective contigs (accession nos. NT_007704.8‖Hs7_7861 for human and supercontig Mm6_WIFeb01_100 for mouse). (C) Diagram of the rat, mouse, and human Ian5 gene transcripts, with exon structure shown to scale. Beneath each transcript diagram is a diagram of the extent of the major ORF (the Ian5-coding region). Continued on the following page.

Figure 1

The lymphopenia gene region and the Ian5 transcript. (A) Physical map of the rat lyp gene region with genetic markers integrated (top). Overlapping PAC clones are shown along with the locations of genetic markers used to narrow the lyp interval, clone-end STS assays, and the limits of the lyp interval itself as a red arrow at both top and bottom. Distances between markers may not be strictly to scale because they are estimated on STS content. The bottom part shows an expanded view of the lyp interval, showing the locations of known genes and the extent of the assembled sequence contigs of rat genomic DNA, along with the framework of mouse genomic DNA sequence. A 13-kb-long rat genomic sequence contig includes the rat Ian5 gene. Position coordinates shown are those from mouse sequence supercontig Mm6_WIFeb01_100. (B) The cluster of Ian-related genes. In human, this gene family is present on chromosome 7q36.1. In the mouse, it is located on proximal chromosome 6. We have also indicated the position of the human and mouse orthologs of the LR8/Clast1 (mouse accession no. AB031386) gene as location aids, although this gene is not in the IAN family. We have indicated various alternative names associated with each gene, and provisionally named previously unnamed members of the family as follows (these are indicated by underlines). For those genes without a common name, we have chosen to continue using the Ian gene nomenclature. To avoid confusion, in this work we will refer to the genes in this family by using the name of the mouse ortholog and a prefix, h, m, or r, to specify which species is indicated (e.g., hIan2 for the human ortholog of mouse Ian2, otherwise known as himap1). Genes given the same Ian designation in different species have been determined to be orthologs of each other. Genes with different designations do not show enough similarity to be deemed orthologs, with the exception of hIan7, which is orthologous to both mIan7 and mIan3. Genes in species without a clear ortholog in the other have been given unique Ian numbers (e.g., hIan12). Positions shown are within the respective contigs (accession nos. NT_007704.8‖Hs7_7861 for human and supercontig Mm6_WIFeb01_100 for mouse). (C) Diagram of the rat, mouse, and human Ian5 gene transcripts, with exon structure shown to scale. Beneath each transcript diagram is a diagram of the extent of the major ORF (the Ian5-coding region). Continued on the following page.

Figure 2

Sequence of the BB rat Immune Associated Nucleotide (Ian5) gene. A representative sequencing trace of DNA from BBDP/WorAp compared with wild-type BBDR/WorAp and F344 rats. The frameshift mutation at nucleotide position 478 in the DP rat DNA is indicated. DNA sequences were determined on an ABI PRISM 3700 DNA Analyzer (Applied Biosystems) and analyzed by the use of Phred, Phrap, Consed, and PolyPhred for sequence assembly and identification of sequence variants.

Figure 3

Sequence comparisons between BBDR wild-type (+/+), lyp/lyp rat, mouse, and human Immune Associated Nucleotide-5 (Ian5) and mouse Ian4 predicted amino acid sequences. The deletion of a nucleotide in the codon for amino acid 85 of the rIan5 (lyp) changes the predicted downstream amino acids to include 19 amino acids (italicized) before the premature STOP codon at amino acid position 104. Putative ATP/GTP-binding sites are boxed/shadowed and a hydrophobic putative transmembrane region underlined.

Figure 4

Expression of rat Ian5 in tissues from lyp/lyp, lyp/+, and +/+ DR BB rats. (a) Northern blot containing 3 μg of poly(A⁺) RNA from thymus, spleen, or kidney of each of +/+ or lyp/lyp rats probed with a 695-bp region of Ian5 showing a 1.4-kb transcript (Ian5). The blot was stripped and reprobed with a 1420-bp GAPDH probe (GAPDH). The images were quantitated using a PhosphorImager and software and normalized to GAPDH expression in each lane. Size markers are indicated. (B) Northern blot containing 3 μg of poly(A⁺) RNA from thymus, spleen, lymph node, and kidney from each of +/+, +/lyp, and lyp/lyp rats probed as in A. Size markers are indicated. (C) Methylene blue stain of the blot in B before probing, showing even loading of 18S ribosomal RNA in each lane.