Over the last decade biomedical research has seen tremendous advancements in the field of genetics that enables unlimited access to >60 vertebrate genomes—including the human and mouse genomes, two of the most widely studied species in biomedical research. These advancements are largely due to rapid development of high throughput sequencing technologies such as next-generation sequencing (NGS) technologies that allow for more affordable and efficient sequencing compared to traditional Sanger technology. The availability of the entire human genome sequence has accelerated our efforts to gain insight into the genetics underlying human disease. Such efforts include Genome-Wide Association Studies (GWAS) — a widely used approach that examines the association between common genetic variants and specific human disease traits. GWAS has led to the successful identification of a large number of SNPs that are linked with chronic diseases ranging from Crohn’s disease, systemic lupus erythermatosus (SLE), type I diabetes (T1D), and many other common western world diseases(reviewed by Visscher et al.1). On the other hand, genetic deficiencies that cause severe disease—such as primary immunodeficiency diseases associated with a poor survival— represent mostly rare mutations within the human population2. Such patients can be found in pediatric clinics and more often than not, the genetic deficiencies underlying disease remain elusive. The availability of NGS, however, offers exciting new opportunities in that it enables the identification of all genome-wide variants in individual patients for limited costs. Nonetheless, both approaches are faced with a significant challenge to identify the causal variants. First of all, most GWAS identify loci that contain more than one SNP but more importantly, SNP maps are incomplete and require in depth probing of the identified genetic region (reviewed by Visscher et al.1). Thus the approach is generally not limited to a single SNP, but rather uncovers multiple gene candidates for a single locus and researchers are often left with the critical question to identify variant causality. This is further complicated by the fact that GWAS is often used for the analysis of complex polygenic traits where gene variants need to exist in combination with one other to assert an effect. In the case of monogenic traits underlying severe disease phenotypes, linkage analysis is rarely an option, whereas whole genome sequencing likely results in the identification of numerous “unique” variants. The biological consequences of such variants would again need to be confirmed and candidate gene selections are guided by a priori knowledge of gene function. Thus the challenges have rather shifted from identifying genetic changes to understanding gene-function and identifying gene causality.
Providing insight into the functional genome is not just limited to understanding gene or protein function, but also includes gene regulation and complex interactions with other genes within the context of cellular or organismal function. The mammalian genome is believed to consist of ~22,000 annotated genes—most of which have been poorly described. In addition, there is almost an unlimited number of phenotypes to be probed, making this an even more daunting task. Nonetheless, experimental models, including fruit fly and mouse models, have been extremely valuable in revealing unique insight into gene function. Typically, forward and reverse genetic approaches have been applied in parallel to uncover gene function. Reverse genetics begins with the creation of a genetic change and ends with the identification of a phenotype. This approach is hypothesis-based and assumes a specific gene function up front. On the other hand, forward (or classical) genetics proceeds from phenotype to the identification of a causal genetic change (SNP or mutation). This approach has led to important discoveries in the field of immunology most notably the identification of TLR4 as the sole LPS receptor3— a discovery recently awarded with the Nobel Prize. Until a few years ago, identification of such genetic variants required positional cloning. This was once considered an arcane art, requiring significant effort, time and financial resources. However, the current availability of the genome sequence for most inbred mouse strains has eliminated the need for contig construction and trivialized the identification of informative markers for high-resolution mapping and/or the identification of existing variants within an associated chromosomal region. Moreover, low cost high-throughput DNA sequencing has accelerated the process of finding unique mutations either introduced spontaneously or by following treatment with mutagens. The current limitation for forward genetics is rather the restricted number of strong monogenic phenotypes, something also referred to as the “phenotype gap”4. To overcome this limitation, germline mutagenesis— in which random mutations are introduced in spermatogonial stem cells— has proven to be an effective approach to expand the number of phenotypes.
2. N-ethyl-N-nitrosourea mutagenesis
In mice, a widely used mutagen to create and expand the number of phenotypes is the alkylating agent N-ethyl-N-nitrosourea (ENU). ENU is a powerful mutagen that according to our latest estimates can introduce more than 3 base-pair changes per million base-pairs of genomic DNA5. ENU introduces point mutations in spermatogonial stem cells, predominantly affecting A/T base pairs (44% A/T→T/A transversions and 38% A/T→G/C transitions), whereas at the protein level, ENU primarily results in missense mutations (64% missense, 26% splicing errors and 10% nonsense mutations)6. With three bp changes per million bps and a total length of ~2,717 Mb for the mouse genome, one can calculate that each G1 male carries ~8,000 bp changes genome-wide. With the coding region being 1.3% of total genomic sequence and 76% of random bp changes creating a coding change, it follows that each G1 mouse carries about 80 coding changes genome-wide, according to our latest estimates. These exist in a heterozygous form and do not necessarily cause a phenotype. In our experience, the majority of ENU-induced mutations, behave as recessive traits or are codominant at best. The approach entails a weekly injection of ~90mg/kg ENU for 3 weeks that is followed by a brief period of sterility for up to 12 weeks. After the recovery period, each G0 male is bred to untreated, wild type C57BL/6 female mice to generate G1 offspring. These G1 animals are then either used for phenotypic screens or can be used to produce G2 mice, which in turn are backcrossed to the G1 male to generate G3 offspring. While screening the G1 population for phenotypes is limited to the identification of dominant mutations, screening of G3 mice allows for the discovery of recessive mutations. Although the total number of base-pair changes in G3 mice will be reduced—each mouse will carry ~11 coding changes in homozygous form—this has proven to be the more powerful approach to capture mice with phenotypes of interest and more importantly allows for the retrieval of lethal phenotypes.
The rate-limiting step in ENU mutagenesis has long been the identification of causative mutations. Until recently, identified mutant lines were outcrossed to genetically different inbred strains and often the analyses of hundreds if not thousands of meiosis were needed to obtain a small enough critical region that could be sequenced. However, the availability of NGS has significantly facilitated the process of variant identification. Currently, targeted exon-enrichment—i.e. targeting exonic sequence within a critical region using sequence capture probes—, whole-exome and whole-genome sequencing are all proven strategies to effectively uncover mutations. The coverage of (targeted) genomic DNA is often exceptional, particularly for the exon-enrichment approach, where generally high quality sequence (minimal depth >10) for more than >98 % of the targeted region can be obtained5. Nonetheless, causality of the identified mutations remains a critical aspect of this approach and low resolution mapping (generally < 30 meioses) and/or genetic confirmation are still integral parts of the ENU mutagenesis approach. In addition, the availability of NGS also provides further opportunities for the phenotypic probing of ENU germline mutants. Often, phenotypes identified in ENU germline mice are lost or significantly influenced by modifier loci located on outcross strains carrying a high degree of genetic variation. For example, identification of genes required for optimal NK cell function has been difficult because of the large variation in NK cell ligands/receptors existing on different mouse backgrounds (Hoebe, unpublished results). By being able to analyze and sequence large genomic regions, fine mapping is superfluous and the exploration of subtle phenotypes can be traced following an outcross to strains with minimal genetic variation between the outcross and parent ENU strain. Ultimately, the genetic diversity should be just enough to allow low-resolution linkage analysis—a prime example being the genetic diversity between C57BL/6J and C57BL/10J strains.
3. Unraveling lymphocyte immune function using ENU mutagenesis
As referred to above, a critical aspect of ENU mutagenesis is the (biological) field of interest to be probed. ENU mutagenesis has been used to define the genetic footprint of a wide variety of phenotypes, including visible, behavioral, developmental and immunological phenotypes7. Nonetheless, its success is depending on: 1) the use of reliable screening assays with limited biological variation, 2) targeting large genomic footprints, and 3) probing a biological phenotype that is poorly defined. Our laboratory has used ENU mutagenesis to identify genes with non-redundant function in lymphocyte development, priming or effector function. Among the biological screens we apply is an in vivo cytotoxicity assay in which we test the ability of G3 mice to induce an antigen-specific CD8+ T cell response following immunization with irradiated cells containing antigen. In parallel, we test the ability of Natural Killer (NK) cells to recognize and eliminate “missing self” target cells in vivo—a process involving complex balancing interactions between activating and inhibitory NK cell receptors. Such screens are not just limited to identifying the presence/absence of NK cells and/or CD8+ T cells in vivo but challenges the host response to undergo NK cell recognition/killing, antigen uptake/ processing and presentation by Dendritic Cells (DCs), ultimately causing T cell priming, expansion and T cell cytolytic effector function. The in vivo immune responses assess the capacity of ENU mice to induce sterile inflammatory responses mediated by self-molecules that activate either NK cells and/or Toll-like receptor-independent sensing pathways—the latter presumably activated by cell-death- or “danger-” associated molecular patterns (DAMPs). Importantly, the induction of type I or II IFNs are essential for the generation of antigen-specific T cell responses mediated via cell-death induced immune responses8. Whereas IFNs have been shown to promote the maturation of DCs and stimulate T cell priming, the underlying pathways inducing type I IFN following exposure of DCs to dying cells are less well defined. It is well established that host molecules such as DNA and/or RNA in apoptotic cells can cause sustained and systemic type I IFN production when they escape degradation in macrophages9-11. The pathways by which such nucleotide structures drive type I IFN production following administration of apoptotic cells remains still elusive to date. Thus, the in vivo cytotoxicity screen performed in our laboratory presents a large genetic footprint, not only comprising lymphocyte development but also targeting NK-, DC- and T cell biological function. As a result, we have identified a number of germline mutants that are either deficient in the IFN pathways, but also includes germline mutants that exhibit impaired lymphocyte survival, T cell activation and/or actin-polymerization. Here we will provide two examples how ENU germline mutants can provide new insight into gene function, immunological pathways and/or disease development.
4. Gimap5 and loss of immunological tolerance driving auto-immune diseases
Using N-ethyl-N-nitrosourea (ENU) germline mutagenesis, our laboratory previously identified Gimap5-deficient mice—designated sphinx—that exhibit reduced lymphocyte survival and develop severe colitis around 10-12 weeks of age12. Specifically, these mice lack NK or CD8+ T cell populations in peripheral lymphoid organs, whereas relatively normal thymocyte development occurs, including the CD4+ T cell, CD8+ T cell, and Foxp3+ regulatory T cell lineages. Coarse mapping and sequencing of the critical region revealed a single G→T point mutation in Gimap5 to be the causal mutation. This mutation resulted in a G38C amino acid substitution in the predicted GTP-binding domain of Gimap5, destabilizing Gimap5 protein expression12.
Gimap5 is part of the family of Gimap genes which are predominantly expressed in lymphocytes and regulate lymphocyte survival during development and homeostasis 13. Gimap proteins contain a GTP-binding AIG1 homology domain, first identified in disease-resistance genes in higher plants9,10. More recent crystallographic studies showed that the Gimap proteins resemble a nucleotide coordination and dimerization mode previously observed for dynamin GTPase—a component essential for the scission and fusion of cellular vesicular compartments such as endosomes at the cell surface or the Golgi apparatus in the cytosol14. Members of the Gimap family appear to be localized to different subcellular compartments with Gimap5 reported to localize in lysosomes based on studies in human, mouse and rat lymphocytes15. Overall, the function of these proteins and their role in disease development remain poorly defined.
Genetic aberrancies in Gimap5 have been strongly linked to reduced lymphocyte survival and homeostasis, but importantly have also been associated with autoimmune diseases. In humans, polyadenylation polymorphisms in GIMAP5—causing relative modest changes in GIMAP5 RNA expression—were associated with increased concentrations of IA2 auto-antibodies in type 1 diabetes (T1D) patients and an increased risk of systemic lupus erythematosus (SLE)16,17. Studies using biobreeding (BB) rats— carrying a mutation (lyp/lyp) in Gimap5— show marked lymphopenia and predisposition to the development of T1D18-22 and intestinal inflammation23. Together these observations suggest that, beyond lymphocyte survival, Gimap5 is essential for maintaining immunological tolerance.
Although in Gimap5sph/sph mice no auto-antibodies can be detected, males and females developed severe colitis around 8-12 weeks of age, which was dependent on the microbiome and is CD4+ T cell driven12. Interestingly, inflammatory bowel disease (IBD) such as Crohn’s disease, ulcerative colitis and indeterminate colitis24,25 manifest generally in adolescence or adulthood and they behave as complex, polygenic diseases often sharing common risk factors with other autoimmune diseases26,27. Previous work suggests that impaired lymphocyte survival and consequent lymphopenia may be linked to the loss of immunological tolerance. Specifically, CD4+ T cells in a lymphopenic environment can undergo thymic independent expansion in the periphery. This process—also referred to as lymphopenia-induced proliferation (LIP)—is accompanied by marked alterations in T cell phenotype and is linked to auto-immunity28-30. Most notably, CD4+ T cells more readily adopt an effector phenotype, including the ability to robustly produce cytokines and can drive the development of colitis31-33. Importantly, the absence of Treg cells is an important determinant of immune-mediated sequelae, including colitis that is induced by CD4+ T cells undergoing LIP. Interestingly, studies in our laboratory show that in Gimap5sph/sph mice, the onset of colitis is preceded by a progressive reduction in circulating CD4+ T cells with remaining CD4+ T cells exhibiting a lymphopenia-induced proliferation (LIP) phenotype (CD44high and CD62low) with a large number of cells in S phase(12 and Figure 1). Moreover, CD4+ T cells derived from Gimap5sph/sph spleen or mesenteric lymph nodes (MLNs) exhibit a higher capacity to produce cytokines, i.e. IFNγ and/or IL-17A following activation of the T cell receptor.
Given the important role of regulatory T cells in immune-mediated sequelae induced by CD4+ T cells undergoing LIP, our laboratory assessed whether the colitis was driven by abnormalities in regulatory T cell development or function. Although relatively normal numbers of Foxp3+ Treg cells are found in 3-week-old mice, a loss of Treg cell numbers is observed by 6 weeks of age particularly in the MLNs34. In addition, regulatory T cells in Gimap5sph/sph mice show a progressive loss of suppressive function. Specifically, whereas Treg cells from 4-week-old Gimap5sph/sph mice show a slight, but significant reduction in their ability to suppress CD8+ T cell proliferation in vitro, Treg cells isolated from 6-week-old Gimap5sph/sph mice are incapable of suppressing CD8+ T cell proliferation entirely, thus indicating that a progressive impairment in Treg cell survival and function may underlie the colitis development in Gimap5sph/sph mice. Indeed, colitis can be prevented entirely by injecting wildtype regulatory T cells in 4-week-old Gimap5sph/sph mice.
Interestingly, the T cell phenotypes in Gimap5sph/sph mice show striking similarities with those seen in mice deficient in the family of Forkheadbox group O (Foxo) transcription factors. The family of Foxo transcription factors contain 4 members of which three (Foxo1, Foxo3 and Foxo4) have overlapping patterns of expression and transcriptional activities35-37. They play an essential role in the quiescence and survival of CD4+ T cells. Foxo1 expression is critical for maintaining naïve T cell quiescence38-40. In addition, Foxo1, 3 expression has been reported to be essential for Treg cell development and function41,42. Specifically, Foxo transcription factors serve a role as coactivators downstream of the TGFβ signaling pathway by interacting with SMAD proteins43,44, and directly regulate the induction of a number of Treg cell associated genes, including Foxp3, CTLA-4 and CD2541,42. Indeed immunoblot analysis of CD4+ T (including Treg cells) from Gimap5sph/sph mice at various ages, revealed a progressive loss of full-length Foxo1, -3a and -4 proteins, with normal levels at 3 weeks of age, but a complete loss of Foxo-expression in CD4+ T cells from 6-10 week-old Gimap5sph/sph mice34. The regulation of Foxo3 and Foxo4 protein expression appears to occur at the post-transcriptional level, although the exact mechanism underlying the loss of Foxo-expression remains to be determined. The progressive nature suggest a strong association with the loss of Treg function in Gimap5sph/sph over time and link the loss of full-length Foxo expression in Gimap5sph/sph lymphocytes with the onset of lymphopenia, impaired lymphocyte proliferation and increased effector function and differentiation into Th17 cells (Figure 1). The detailed mechanistic insight into the loss of immunological tolerance occurring in Gimap5sph/sph mice may ultimately provide important leads as to how polyadenylation polymorphisms in GIMAP5 predispose human patients to T1D or SLE.
5. Mutations in hematopietic protein 1; an immunodeficiency resulting in loss of a broad range of immunological functions
Genetic aberrancies causing severe combined immunodeficiency (SCID) are generally rare and associated with a high morbidity and/or mortality. They often present significant challenges in terms of treatment due to the wide variety of immune cells that can be affected. Therefore, besides defining the genetic footprint underlying SCID, a critical challenge lies in obtaining a thorough understanding of the degree of the immunodeficiency presented by specific mutations in genes, including defining the types of immune cells affected and functional aberrancies observed. Our laboratory previously identified a germline mutant, designated Lampe2, which exhibited impaired NK as well as CD8+ T cell cytoloytic effector function as determined by the in vivo cytotoxicity assay described above (Figure 2a). The G1 pedigrees of these germline mutants were selected to establish a homozygote colony used for genetic analysis and further phenotypic characterization. The mutation behaved as strictly recessive, in that normal cytolytic effector functions were observed in heterozygote mutant mice. Further characterization of 6-week-old homozygote Lampe2 mutants revealed markedly reduced numbers of CD8+ T, CD4+ T and B cell populations, and a slight reduction in NK cells (Figure 2b). In contrast, an increase in the number of macrophages in the spleen was observed (Figure 2b). Notably, upon necropsy, the liver exhibited white patches at the periphery (Figure 2c) which upon histological analysis revealed large areas of necrosis and significant hematopoietic infiltrate and inflammation (Figure 2 d-e).
To identify the causative mutation in Lampe2 mice, we performed coarse mapping by crossing Lampe2 C57BL/6J homozygotes males with 129S1/SvImJ females. The resulting F1 offspring were intercrossed to generate a F2 and a total of 24 offspring (6 Lampe2 mutant- and 18 wildtype-phenotypes) were analyzed for both phenotype and genotype. Genotyping was performed using a genome wide custom-made 353-SNP map distinguishing C57BL/6J and 129S1/SvImJ genetic backgrounds. Coarse mapping revealed a single peak with a LOD score of ~5.86 for SNP rs13482738 located on the distal end of chromosome 15 (Figure 3A). The critical region was defined by proximal marker rs6285067 (at position 95.14 Mb) and the distal end of chromosome 15 (at 103.40 Mb) and consisted of ~8.26 Mb genomic DNA containing 242 annotated genes. Among the annotated genes, Hematopoietic protein 1 (Hem-1 aka NCK associated protein 1 like or Nckap1l) presented a clear candidate gene, in that a previously reported ENU germline mutant carrying a point mutation (referred to as the NBT.1 mutation) causing a premature stop and absence of protein expression, exhibits striking similar phenotypes compared to Lampe2 mice. Specifically, these mice exhibit lymphopenia with a reduced number of peripheral CD4+ T, CD8+ T and B cells, and on the other hand showed marked expansion of myeloid cells, including neutrophils and macrophages. Moreover, the liver phenotype in mice carrying the NBT.1 mutation bears high resemblance with the liver phenotype observed in Lampe2 mice—i.e. the occurrence of whitish liver margins and large areas of inflammation. Hem1 is a member of the Hem family of cytoplasmic adaptor molecules predominantly and is expressed exclusively in hematopoietic cells, including T and B cells, macrophages, DCs and granulocytes. Hem1 plays a critical role in the reorganization of actin cytoskeleton and as such, affects a wide variety of immune functions, including chemotaxis/migration, adhesion, formation of an immune synapse and phagocytosis. Sequencing of Hem1 exons, including 50 bps of proximal/distal intronic sequence, was performed by Sanger sequencing methodology using genomic DNA and revealed a single A→G point mutation in intron 8-9 located 6 nucleotides upstream of the exon 9 acceptor splice site (Figure 3b). The A→G intronic nucleotide change potentially presented a new acceptor splice site and indeed sequencing of Hem-1 mRNA isolated from spleen showed the inclusion of 7 intronic nucleotides in mRNA derived from Lampe2 mice (Figure 3c). At the protein level, the inclusion of intronic nucleotides is predicted to result in a frame-shift and alternative coding following residue 261, and a premature stop at amino acid 381 resulting in a largely truncated Hem-1 protein in Lampe2 mice (Figure 3d). Given the similarities of the Lampe2 and NBT.1 mutant phenotypes and the predicted severe impact of the Lampe2 mutation on Hem-1 protein expression, we concluded that the mutation in Hem-1 caused the observed phenotypes in Lampe2 mice (hereafter referred to as Hem1lampe2).
Hem1 is part of the Wiskott-Aldrich syndrome protein family Verprolin-homologous protein (WAVE) protein complex in hematopoietic cells regulating cell mobility and intracellular processes requiring rearrangement of the cytoskeleton following immuno-receptor activation, including B and T cell, chemokine and innate immune receptors such as Toll-like receptors. Specifically, receptor triggering causes activation of Rho family of Guanosine triphosphatases (GTPases) such as CDC42, RhoA and Rac ultimately resulting in the activation of downstream adaptor complexes involved in the regulating of actin (de)polymerization. For hematopoietic cells, the adaptor complexes Wiskott-Aldrich syndrome protein (WASP) and WAVE are particularly important for the control of actin polymerization45-48. The hematopoietic cell-specific WAVE complex consists of a pentameric subunit complex including, Sra-1 (Specifically Rac-associated protein-1), Hem1, Abi (Abelson interactor 1 or 2), WAVE, and HSPC300 (Hematopoietic stem/progenitor cell protein 300)49. Under non-stimulated conditions, the WAVE complex is inactive, but following immunoreceptor activation, GTP-bound Rac binds the pentameric complex presumably through Sra149. In addition, this complex requires binding of phophatidylinositol (3,4,5) triphosphate (PIP3) interaction and phosphorylation by kinases50, including Abl kinase and Mitogen-activated protein kinases51. Ultimately, this results in a conformational change revealing the WAVE-specific VCA (Verprolin-homology, Cofillin-homology, and acidic) region and allow interaction with the actin-regulatory complex (Arp2/3), ultimately converting monomeric actin (G-actin) into filamentous actin (F-actin). Interestingly, the absence of individual subunit components often causes the degradation of all components of the WAVE complex resulting in aberrant actin polymerization. The consequences of deregulated actin polymerization in hematopoietic cells are wide-ranging and affect broad immunological functions, including but not limited to: 1) leukocyte migration/chemotaxis, 2) loss of immune synapse formation affecting T and B cell receptor signaling (thereby affecting T cell function and development), 3) leukocyte adhesion, and 4) DC-specific phagocytosis and their ability to cross present/prime T cells. As such, mutations in the specific subunit components of the WAVE complex resulting in abnormal gene expression/function cause severe combined immunodeficiencies that stretch beyond lymphocyte populations also affecting granulocyte function and are predicted to correlate with high mortality/morbidity.
6. Implications for human PID
Assessing the immune system using ENU mutagenesis in mice has previously led to important breakthrough discoveries in understanding the genetics in human patients with PID. A prime example is the identification of the 3d allele—a missense allele of Unc93b1, a gene encoding an ER membrane protein with 12 membrane spanning motifs with a previously unknown function. 3d germline mutants were identified in a screen probing the response of macrophages derived from ENU germline mice to a variety of TLR-ligands. Homozygote 3d mutant mice were found to be unresponsive to ligands activating endosomal TLRs, but exhibited normal responses to TLRs expressed at the surface39. Interestingly, at the same time Casrouge et al. identified two unrelated human patients that presented recurrent infections with Herpes simplex virus-1 (HSV-1) resulting in encephalitis (HSE) and showed remarkable similarities between the phenotypes observed in the human patients and in 3d mutant mice. Specifically, both patients were unresponsive to endosomal TLR stimulation and showed a high viral susceptibility. Following the identification of the causative mutation in 3d mice as being a missense allele of Unc93b1, subsequent sequencing of the human patients indeed revealed aberrant mutations in UNC93B52. This example highlights the power of ENU mutagenesis and its unbiased approach, by uncovering the function of genes for which a biological function is otherwise difficult to predict.
With regard to the Gimap5 and Hem-1 germline mutations described in this chapter, both present examples of genetic mutations leading to severe combined immunodeficiencies. Although limited information is available with regard to genetic mutations causing a null phenotype in human GIMAP5 or HEM1, ample evidence exist that dysregulation of these genes plays an important role in human disease. A previous report suggests that SLE patients were shown to have a trend for lower GIMAP5 mRNA expression in peripheral blood mononuclear cells compared to healthy controls16. Moreover, a poly-adenylation mutation in the 3’ region of GIMAP5, resulting in minor changes in GIMAP5 mRNA expression in peripheral blood mononuclear cells, are associated with increased predisposition to SLE and T1D15,16,53. Thus far, the effect on GIMAP protein expression, specifically in lymphocytes of homozygote/heterozygote carriers for this mutation, remains elusive and warrants further research. Our studies using Gimap5-deficient mice point to an important role for Gimap5 in maintaining peripheral immunological tolerance that is intrinsically related to the loss of Gimap5 expression in CD4+ T cells. Thus, research efforts may be directed to a better understanding of GIMAP5 and FOXO protein expression specifically in CD4+ T cells in human patients with SLE or T1D that carry the polyadenylation mutation in GIMAP5.
Finally, perhaps due to its indispensable role in a wide variety of immune pathways, mutations in human HEM1 leading to dysregulated actin polymerization, have thus far not been reported. Nonetheless, over- or under-expression of HEM1 is associated with disease prognosis in leukemia54. Specifically, HEM1 overexpression in B-cell chronic lymphocytic leukemia (CLL) is associated with a poor outcome, whereas down-regulation of HEM1 expression in CLL cells rendered tumor cells more susceptible to fludarabine-mediated killing54. These findings may indicate the critical role for HEM1 in invasion and/or metastasis of tumor cells from hematopoietic origin.
7. Concluding remarks
A major challenge in the field of genomics is to obtain a comprehensive understanding of the functions of all annotated mammalian genes. Whereas identification and analysis of genome wide SNPs and/or unique nucleotide changes are drastically improved following the development of next generation sequencing technologies, understanding the consequences of such genetic variants remains a major challenge in virtually all biomedical fields. ENU mutagenesis provides one approach that is both powerful and unbiased, uncovering gene function by introducing the sort of genetic abnormalities that can be observed in human patients (e.g. primary immuno-deficiencies). Ultimately, utilization of both forward and reverse genetic approaches will be instrumental in closing the existing phenotype gap and will help us understand the association between identified genetic variants, the implications for protein and biological function, and human disease.
This research was funded by grants from the NIH, including NIH/NIAID RO1 Grant 00426912 and PHS Grant P30 DK078392 (Integrative Morphology Core of the Cincinnati Digestive Disease Research Core Center)