Sequence Variation and Expression of the Gimap Gene Family in the BB Rat

Positional cloning of lymphopenia (lyp) in the BB rat revealed a frameshift mutation in Gimap5, a member of at least seven related GTPase Immune Associated Protein genes located on rat chromosome 4q24. Our aim was to clone and sequence the cDNA of the BB diabetes prone (DP) and diabetes resistant (DR) alleles of all seven Gimap genes in the congenic DR.lyp rat line with 2 Mb of BB DP DNA introgressed onto the DR genetic background. All (100%) DR.lyp/lyp rats are lymphopenic and develop type 1 diabetes (T1D) by 84 days of age while DR.+/+ rats remain T1D and lyp resistant. Among the seven Gimap genes, the Gimap5 frameshift mutation, a mutant allele that produces no protein, had the greatest impact on lymphopenia in the DR.lyp/lyp rat. Gimap4 and Gimap1 each had one amino acid substitution of unlikely significance for lymphopenia. Quantitative RT-PCR analysis showed a reduction in expression of all seven Gimap genes in DR.lyp/lyp spleen and mesenteric lymph nodes when compared to DR.+/+. Only four; Gimap1, Gimap4, Gimap5, and Gimap9 were reduced in thymus. Our data substantiates the Gimap5 frameshift mutation as the primary defect with only limited contributions to lymphopenia from the remaining Gimap genes.


Introduction
Lymphopenia (lyp) is a prerequisite for spontaneous type 1 diabetes (T1D) in the BioBreeding (BB) diabetes prone (DP) rat [1]. Positional cloning of the lyp gene revealed a frame shift mutation in Gimap5 (previously known as Ian5 or Ian4L1). Gimap5 is a member of at least seven related GTPase Immune Associated Protein (Gimap) genes located within 150 Kilobases (Kb) on rat chromosome (RNO) 4 [2,3]. DR. lyp/lyp rats, where 2 Mb of DP DNA was introgressed onto the BB diabetes resistant (DR) genetic background, are lymphopenic and 100% develop spontaneous T1D by 84 days of age [4].
The positional cloning and subsequent identification of the Gimap5 gene on RNO4 were in part established through generation of the DR.lyp congenic rat line along with recombination events following our method of marker assisted breeding of DP with F344 rats [2,4,5]. Analysis of the lyp phenotype in the F344 DNA recombinant rats helped us define the critical lyp interval as a region of approximately 33 Kb between D4Rhw6 (76.83 Mb) and IIsnp3 (77. 16 Mb) containing Gimap1, Gimap5, and Gimap3 (formerly known as Ian2, Ian5, and Ian4, resp.) [2,4]. Gimap5 was identified as the lyp gene in the BBDP rat through a frameshift mutation and premature truncation of the Gimap5 protein [2,6] 2 Experimental Diabetes Research and can be rescued in a P1-derived artificial chromosome (PAC) transgenic rat [7]. However, potential contributions to lymphopenia and/or T1D from the other Gimap genes are still unknown. Similarly, how the mechanisms by which reduced Gimap5 transcript levels and the absence of the Gimap5 protein [2,7,8] contribute to lymphopenia and T1D are still being elucidated [9][10][11][12][13].
The predicted structures of the Gimap proteins show common sequences and motifs, such as GTP-binding domains in the N-terminal half, but with differing Cterminal ends [2,3]. Some C-terminal regions are consistent with transmembrane domains as in the case of Gimap1 and Gimap5, while others, as in Gimap9 and Gimap4, predict coiled coil domains [3,14]. Both GIMAP4 and GIMAP7 from human Jurkat cells [3] localize to the endoplasmic reticulum and Golgi apparatus while mouse Gimap3 from murine IL-3-dependent 32D myeloid precursor cells was expressed at the outer mitochondrial membrane [15]. Conflicting reports show that GIMAP5, from human primary T cells [10] and from GIMAP5 transfected 293T cells [16], localizes to the centrosome, Golgi apparatus, or endoplasmic reticulum (ER), whereas Gimap5, cloned from Rat2 fibroblasts, localizes to a distinct subcellular fraction that is neither mitochondrial nor ER [11]. Gimap proteins may therefore have similar function, but different subcellular locations. At this time, there is a paucity of information as to the expression of the Gimap genes in specific cell types.
The fact that the Gimap genes are located together in a tight cluster on RNO4 (and in conserved synteny with many other species), combined with their sequence similarities, suggests the possibility that the proteins carry out similar function. While there is sufficient evidence to support the frameshift mutation in Gimap5 as the cause of lymphopenia, we could not exclude that either Gimap1 or Gimap3 play a role, as they are located within the lymphopenia critical interval between D4Rhw6 and IIsnp3 as well as within the PAC used in the transgenic rescue of lymphopenia [7]. In addition, it is possible that the remaining Gimap family members outside the lymphopenia critical interval play a role in T1D development. In order to substantiate the frameshift mutation in Gimap5 and the subsequent protein null allele as the cause of lymphopenia as well as explore a possible contribution by other Gimap family members, we sequenced DR. +/+ and DR. lyp/lyp cDNA from rat thymus. In addition, we examined Gimap gene expression across multiple tissues and quantified mRNA expression of all annotated and putative Gimap genes in DR. +/+ and DR. lyp/lyp rat thymus, spleen, and mesenteric lymph node (MLN).

cDNA Cloning and
Sequencing. cDNA synthesis was performed using SuperScript II Reverse Transcriptase (Invitrogen, Carlsbad, Calif, USA) according to the manufacturer's recommendations. PCR products were amplified from thymus cDNA as follows: PCR products were generated by using either Herculase (Stratagene) or Roche Taq polymerase (Roche Diagnostics, Indianapolis, Ind, USA). Reactions with Herculase were 25 μL, consisting of 100 ng cDNA, 0.5 μL of Herculase polymerase, 2.5 μL of the supplied buffer, 0.5 μL of a mix of 10 mM each dNTP, and 2 μL each 5 μM primer. Amplification was carried out in a PTC-200 Peltier Thermal Cycler (Bio-Rad, Hercules, Calif, USA) with the following conditions: 95 • C for 3 minutes, 35 cycles of 95 • C for 30 seconds, 60 • C for 30 seconds, 72 • C for 6 minutes, and Table 1: Probes and primers used in qRT-PCR.

Primer name
Primer sequence 5 to 3 Probes and primers were designed to bind near the 3 end of the transcripts. f is forward primer, r is reverse primer, p is probe, 6FAM is 6-carboxyfluorescein, HEX is hexachlorofluorescein, and BHQ1 is black hole quencher 1.  Representative qRT-PCR products for each gene, from each tissue, were run on an agarose gel to check for primer pair binding specificity. Each assay was also optimized and validated with serial dilutions of RNA to produce a standard curve that was then translated into a reaction efficiency, or specificity, of each Gimap gene assay. Results from each assay were validated and normalized against cyclophilin. The standard curves, multiplexed with cyclophilin, showed the following reaction efficiencies: Gimap8: 90%±2 SD, Gimap7: 87% ± 4 SD, Gimap4: 92% ± 2 SD, Gimap6: 94% ± 3 SD, Gimap9: 98%±7 SD, Gimap1: 100%±11 SD, Gimap5: 88%± 6 SD, Lr8: 93% ± 4 SD, and cyclophilin: 90% ± 6 SD.
2.6. Statistical Analysis. Three DR. +/+ rats from different litters were used to determine the expression of Gimap genes in mesenteric lymph node, thymus, spleen, bone marrow, and kidney. To compare Gimap and Lr8 gene expression across multiple tissues, data was first normalized to cyclophilin then scaled and expressed as a percentage of DR. +/+ Gimap5 mesenteric lymph node (MLN), the highest expressing gene overall. For analysis of Gimap expression in DR. lyp/lyp , DR. lyp/+ , and DR. +/+ rat thymus, spleen, and mesenteric lymph node, 15 rats (5 rats per genotype) were Gimap8, Gimap9, Gimap4, Gimap6, and Gimap7 (in the order as they appear on the chromosome) are located outside the lymphopenia critical interval (Figure 1). Sequence analysis of thymus cDNA encoding Gimap4 showed three single nucleotide polymorphisms (SNPs) at positions 216, 510, and 618, relative to the ATG start site, and two nucleotides deleted at position 922-923 in DR. lyp/lyp rats as compared to DR. +/+ rats ( Table 2). The first three base pair substitutions resulted in synonymous amino acid changes in the hypothetical protein sequence, while the deletion resulted in a frameshift mutation in the last three predicted amino acid residues and eliminated the normal stop codon at position 311. 3 RACE from DR. lyp/lyp thymus cDNA showed that the reading frame continued for other 21 amino acids before generating a new stop codon (Table 2). This same frameshift mutation was also identified in F344/Rhw (nonlymphopenic) rats, which were used in the positional cloning of lymphopenia (Table 2). One nucleotide substitution was identified in Gimap7 at position 603, relative to the ATG start site, between DR. +/+ and DR. lyp/lyp that resulted in a synonymous amino acid change in the hypothetical protein sequence (Table 2). No SNPs were found in the coding sequences of Gimap6, Gimap8, and Gimap9.

cDNA Cloning and Sequencing of Gimap1 and Gimap3
in DR. +/+ and DR. lyp/lyp Rats. Gimap1 and Gimap3 are located inside the lymphopenia critical interval (Figure 1). Sequence analysis of thymic cDNA showed a single SNP in the coding sequence of Gimap1 at nucleotide position 752, relative to the ATG start site, that produced an amino   acid change in DR. lyp/lyp rats as compared to the DR. +/+ ( Table 2). The SNP produced a methionine (M) to threonine (T) substitution at amino acid 251, which is located near the C-terminus and is not in any of the predicted GTP binding domains. Sorting intolerant from tolerant (SIFT) analysis (http://blocks.fhcrc.org/sift/SIFT.html) predicted the T substitution to be tolerated at this position and did not predict to affect protein function.

MNWL Y S KT L G S I GS CC I DT L PWP FHS F FQR NL L A L PGE PG NP L E S S AT E S GKQS R S C L S A S PVME . E EGC EHS LQKNP TR
Gimap3 is not annotated in the rat genome sequence. Genomic sequencing of the putative ortholog of mouse Gimap3 from base pair positions 76,846,091 to 76,852,162 on RNO 4 (the orthologous DNA interval to mouse Gimap3) in DR. +/+ and DR. lyp/lyp rats revealed repetitive single or dinucleotide repeats throughout the region that likely resulted in early termination of the sequencing reactions. As such, no specific PCR products could be generated. Attempts were also made to amplify Gimap3 from DR. +/+ and DR. lyp/lyp rat thymus cDNA; however, again, no specific PCR products were obtained. Comparative analysis of the Brown Norway (BN/Hsdmcwi) database sequence, available at the University of California Santa Cruz (UCSC; Nov. To compare Gimap gene expression across multiple tissues, data was first normalized to cyclophilin then scaled and expressed as a percentage of DR. +/+ Gimap5 mesenteric lymph node (MLN), the highest expressing gene overall. Genes appear in the order at which they appear on rat chromosome 4. Tissues appear in the following order per gene: MLN (dots), thymus (white), spleen (hash marks), bone marrow (black), and kidney (stripes). Significance is represented as follows: * * * is P < .0001 and * * is P < .001.
blots of DR. +/+ and DR. lyp/lyp or from qRT-PCR of DR. +/+ rat thymus or spleen (data not shown). Therefore, Gimap3 is likely a pseudogene in rat.

Predicted Protein Alignment.
Alignment of the Gimap family predicted protein sequences in the DR. +/+ rat ( Figure 2) showed predicted GTP binding domains and conserved box characteristics for all of the Gimap proteins with the most divergent regions located near the Cterminal ends. Gimap1 and Gimap5 are predicted to contain transmembrane domains while Gimap4 and Gimap9 are predicted to contain coiled coil domains. Gimap8, Gimap7, and Gimap6 are predicted to have neither transmembrane nor coiled coil domains. Gimap8 was larger than the other Gimap proteins, containing 688 amino acids and three repeated GTP binding domains ( Figure 2).

Gimap Expression Pattern across Multiple Tissues.
The relative expression levels of the Gimap genes in mesenteric lymph node, thymus, spleen, bone marrow, and kidney were determined in the DR. +/+ rat ( Figure 3). All of the Gimap genes expressed more robustly in the mesenteric lymph nodes, thymus, and spleen as compared to bone marrow and kidney (P < .0001). In the mesenteric lymph node, Gimap4, Gimap5, and Gimap8 were expressed significantly higher than Gimap9 (P < .0001) while in kidney, Gimap4 and Gimap8 were expressed significantly higher than Gimap1 and Gimap9 (P < .001) (Figure 3). No significant expression differences were detected between any of the Gimap genes (Gimap8, 9, 4, 6, 7, 1, and 5) in thymus, spleen, and bone marrow. Overall, Lr8, a gene unassociated with the Gimap family but also within the 2 Mb of DP DNA in the congenic DR. lyp/lyp rat line, expressed predominantly in the spleen, an expression pattern unique relative to the Gimap family.

Gimap
Expression in DR. lyp/lyp and DR. +/+ Thymus, Spleen, and Mesenteric Lymph Node. In thymus, expression of Gimap4, Gimap9, Gimap1, and Gimap5 was significantly decreased in DR. lyp/lyp rats as compared to DR. +/+ (Figure 4). In contrast, Gimap7 expression in thymus was higher in DR. lyp/lyp as compared to DR. +/+ while Gimap8, Gimap6, and Lr8 showed no differential expression. Expression of all of the Gimap genes (8, 9, 4, 6, 7, 1, and 5) was reduced in DR. lyp/lyp rat spleen and mesenteric lymph node as compared to DR. +/+ (Figure 4). We observed the same expression pattern whether the data were normalized to cyclophilin or to total RNA (data not shown). Data from bone marrow and kidney is not shown due to the very low expression in these tissues. The low expression observed in these tissues is not due to RNA degradation, but rather to the low mRNA levels relative to cyclophilin levels.

Discussion
While the frameshift mutation in Gimap5 is likely necessary and sufficient for lymphopenia, the possibility remains that additional mutation(s) in the Gimap family may contribute to the development of lymphopenia, spontaneous T1D, or both in the DR. lyp/lyp rat. Aside from Gimap5, only Gimap1 and Gimap3 could potentially play a role in the development of lymphopenia, as they are the only remaining genes located within the 33 Mb interval critical for lymphopenia between D4Rhw6 and IIsnp3. The methionine to threonine base pair substitution at amino acid position 251 in Gimap1 is not located in any of the predicted GTP binding domains and SIFT analysis predicted that the mutation is not likely to alter normal Gimap1 protein function. Furthermore, the DR amino acid at position 251 is not conserved across species; human GIMAP1 has a valine at this same position [3]. Lastly, no Gimap3 transcript could be found and no open reading frame could be identified. We suspect that this region of the rat genome does not code for a protein, which is similar to the human GIMAP3 pseudogene [3] and unlike mouse Gimap3 [15]. Therefore, the sequence analysis of Gimap1 and Gimap3 supports the hypothesis that Gimap5 is the cause of lymphopenia in the DR. lyp/lyp rat. In addition, the sequence data from all of the remaining Gimap family members suggest that these genes are not likely to play a role in the onset of T1D diabetes. We cannot however exclude the possibility that there are mutations outside of the coding regions, such as in transcription factor binding sites or other regulatory sites, that play a role in the regulation of Gimap gene expression and/or the onset of T1D in the DR. lyp/lyp rat.
While we can exclude the involvement of the members of the Gimap family proximal to D4Rhw6 (Gimap8, Gimap9, Gimap4, Gimap6, and Gimap7) in the development of lymphopenia, we cannot exclude that they may play a role in the development of T1D. Coding sequence analysis of these family members revealed that only Gimap4 had genetic differences, specifically a two-base pair deletion, that would result in a nonsynonymous amino acid change between the nondiabetic DR. +/+ and the diabetes susceptible DR. lyp/lyp ( Table 2). Although the effect of this variation is unknown, we did discover this same deletion in the nonlymphopenic, diabetes resistant F344 rat. F344 DNA introgressed through this interval on the DR. lyp/lyp background protects from  Figure 4: Gimap gene expression in the DR. +/+ and DR. lyp/lyp rats. The mean ± standard deviation is shown for DR. +/+ and DR. lyp/lyp rat Gimap gene expression in thymus (n = 5), spleen (n = 5) and mesenteric lymph node (MLN) (n = 4) after normalization to cyclophilin. Black columns represent DR. +/+ and grey hatched columns represent DR. lyp/lyp . Data is expressed as a percentage of DR. +/+ . Significant differences are follows; * for P < .05, * * for P < .01, * * * for P < .001. Genes appear in the order at which they appear on rat chromosome 4. The average Gimap expression in DR. +/+ rat bone marrow and kidney s is shown in Figure 3.
onset of T1D [4] suggesting that the deletion mutation in Gimap4 is not deleterious. In addition, the predicted protein sequences of both human (AK001972) and mouse (NP 778155.2) Gimap4 show that the 23 C-terminal amino acids are similar to those of DR. lyp/lyp (data not shown). It is therefore unlikely that the Gimap4 two-base pair deletion mutation in the DR. lyp/lyp rat is functionally relevant to development of T1D or lymphopenia, rather it is likely an additional natural isoform [20].
All of the Gimap genes were predominantly expressed in organs of the immune system: mesenteric lymph node, thymus, and spleen, consistent with previous findings of a role of the Gimap gene family in lymphocyte development [21]. Interestingly, there was an overall reduction in expression of all seven Gimap genes in DR. lyp/lyp rat spleen and mesenteric lymph node and four (Gimap4, Gimap9, Gimap1, and Gimap5) of the seven genes in DR. lyp/lyp rat thymus. In contrast, Lr8, a gene unrelated to the Gimap family located