Establishment and characterization of the Komeda diabetes-prone rat as a segregating inbred strain.

The Komeda diabetes-prone (KDP) rat is a spontaneous animal model of human autoimmune type 1 diabetes. By positional cloning of the non-MHC major susceptibility locus lddm/kdp1, we recently identified a nonsense mutation in Cblb and also found that lymphocytes of KDP rats infiltrate into various tissues, indicating autoimmunity. The maintenance and production of KDP rats has been a critical problem owing to the poor reproductive ability of diabetic animals. To solve the problem, we here established the KDP rat as a segregating inbred strain. We first identified animals that were heterozygous at the lddm/kdp1 region in a breeding colony of KDP rats. The heterozygous region spans at least from D11Yok1 to Cblb on rat chromosome 11. By mating between the heterozygous rats, we obtained homozygotes, heterozygotes and wild-types with the expected ratio of 1:2:1 and found that only the homozygotes developed diabetes, suggesting that these genotypes represent those of lddm/kdp1. We then tried to maintain KDP rats by mating between the heterozygotes, which resulted in a segregating inbred strain. Within 210 d of age, about 80% of lddm/kdp1 homozygotes developed diabetes with severe insulitis, while neither heterozygotes nor wild-types developed diabetes. The phenotypic characteristics of the homozygotes are the same as those of progeny of diabetic parents in the original KDP rats. The segregating inbred KDP rat strain described here would serve as a useful animal model for autoimmune diseases, including type 1 diabetes.     

Analysis of the rat Iddm14 diabetes susceptibility locus in multiple rat strains: identification of a susceptibility haplotype in the Tcrb-V locus

Iddm14 (formerly Iddm4) is a non-MHC-linked genetic locus associated with autoimmune diabetes. Its effects have been well-documented in BB-derived rats in which diabetes is either induced by immunologic perturbation or occurs spontaneously. The role of Iddm14 in non-BB rat strains is unknown. Our goal was to extend the analysis of Iddm14 in new diabetes-susceptible strains and to identify candidate genes in the rat Iddm14 diabetes susceptibility locus that are common to these multiple diabetic strains. To determine if Iddm14 is important in strains other than BB, we first genotyped a (LEW.1WR1 × WF)F2 cohort in which diabetes was induced by perturbation with polyinosinic:polycytidylic acid. We found that Iddm14 is a major determinant of diabetes susceptibility in LEW.1WR1 rats. We then used nucleotide sequencing to establish a strain distribution pattern of polymorphisms (insertions, deletions, and single nucleotide polymorphisms [SNPs]) that predicts susceptibility to diabetes in a panel of inbred and congenic rats. Using the positional information from the congenic strains and the new linkage data, we identified a susceptibility haplotype in the T-cell receptor Vβ chain (Tcrb-V) locus. This haplotype includes Tcrb-V13, which is identical in five susceptible strains but different in resistant WF and F344 rats. We conclude that Iddm14 is a powerful determinant of both spontaneous and induced autoimmune diabetes in multiple rat strains, and that Tcrb-V13 SNPs constitute a haplotype of gene elements that may be critical for autoimmune diabetes in rats.     

Genes of SHR rats protect spontaneously diabetic BB/OK rats from diabetes: lessons from congenic BB.SHR rat strains

Diabetes in BB rats share many common features with human type 1 diabetes. One of them is the complex and polygenic nature of disease. Analysis of cross hybrids of diabetic BB/OK rats and rats of different diabetes-resistant strains has demonstrated that beside the MHC genes, Iddm1 and the lymphopenia, Iddm2, additional non-MHC genes are involved in diabetes development. To study the importance of the non-MHC genes, Iddm4 and Iddm3, two congenic BB.SHR rat strains were generated by recombining a segment of the SHR chromosome 6 (Iddm4; termed BB.6S; 15cM) or chromosome 18 (Iddm3; termed BB.18S; 24cM) into the BB/OK background by serial backcrossing and marker-aided selection. The characterization of both congenic strains demonstrates a drastic reduction of diabetes frequency in comparison to the BB/OK strain (86% vs 14% and 34%). It is supposed that diabetes protective genes of SHR must be located on both chromosomal segments and that these suppress the action of the essential and most important genes of diabetes development in the BB/OK rat, Iddm1, and Iddm2.     

The mutation of the LEW.1AR1-<Emphasis Type="Italic">iddm</Emphasis> rat maps to the telomeric end of rat chromosome 1

The LEW.1AR1-iddm rat is an animal model of human type 1 diabetes mellitus (T1DM) with an autosomal recessive mode of inheritance. T1DM susceptibility loci could be localized on chromosome (RNO) 20 in the major histocompatibility complex region (Iddm1) and on RNO1 (Iddm8, Iddm9) in a BN backcross cohort. In this study the impact of the different susceptibility regions on diabetes development was investigated in a backcross population of the diabetes-resistant PAR strain. A cohort of 130 [(PAR × LEW.1AR1-iddm) × LEW.1AR1-iddm] N2 rats was monitored for blood glucose and analyzed by linkage analysis. Sixteen percent of the PAR backcross animals developed T1DM. Genetic analysis revealed significant linkage to T1DM in the MHC region on RNO20p12. In contrast to the linkage analysis of the BN backcross cohort, only one susceptibility locus for T1DM could be identified on RNO1. This susceptibility region on RNO1 mapped to the telomeric end corresponding to Iddm8. Eighty-nine percent of diabetic PAR backcross animals were homozygous for Iddm8. The Iddm9 diabetes susceptibility region showed no linkage to diabetes in the PAR backcross cohort. The data of this study provide evidence that the mutation leading to T1DM in the LEW.1AR1-iddm rat is located at the telomeric end of RNO1 corresponding to Iddm8. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. H. Weiss and T. Arndt contributed equally to this study.     

Loci of the immune system are implicated in diabetes frequency and age at onset of diabetes in BB rats

The spontaneously diabetic BB rat is a well-established animal model in diabetes research developing an insulin-dependent type-1 diabetes mellitus closely resembling human diabetes. By several crossing studies using BB/OK rats it has been demonstrated that beside the MHC class-II genes of the RT1^u haplotype, Iddm1, and the lymphopenia, Iddm2, at least two additional non-MHC genes located on chromosomes 6 (Iddm4) and 18 (Iddm3) are involved in diabetes development. In addition, there are at least three genes located on chromosomes 6 (Dm1), 8 (Dm2) and 10 (Dm3) influencing the age at onset of diabetes. Comparing the homologous regions between rat and human, it is shown that most diabetogenic genes lie on human chromosomes near genes involved in immune processes providing human geneticist with new candidate regions for the analysis of diabetogenic non-MHC genes in human type-1 diabetes.     

Two genetic loci regulate T cell-dependent islet inflammation and drive autoimmune diabetes pathogenesis

Insulin-dependent diabetes mellitus (IDDM) is a polygenic disease caused by progressive autoimmune infiltration (insulitis) of the pancreatic islets of Langerhan, culminating in the destruction of insulin-producing beta cells. Genome scans of families with diabetes suggest that multiple loci make incremental contributions to disease susceptibility. However, only the IDDM1 locus is well characterized, at a molecular and functional level, as alleleic variants of the major histocompatibility complex (MHC) class II HLA-DQB1, DRB1, and DPB1 genes that mediate antigen presentation to T cells. In the nonobese diabetic (NOD) mouse model, the Idd1 locus was shown to be the orthologous MHC gene I-Ab. Inheritance of susceptibility alleles at IDDM1/Idd1 is insufficient for disease development in humans and NOD mice. However, the identities and functions of the remaining diabetes loci (Idd2-Idd19 in NOD mice) are largely undefined. A crucial limitation in previous genetic linkage studies of this disease has been reliance on a single complex phenotype-diabetes that displays low penetrance and is of limited utility for high-resolution genetic mapping. Using the NOD model, we have identified an early step in diabetes pathogenesis that behaves as a highly penetrant trait. We report that NOD-derived alleles at both the Idd5 and Idd13 loci regulate a T lymphocyte-dependent progression from a benign to a destructive stage of insulitis. Human chromosomal regions orthologous to the Idd5 and -13 intervals are also linked to diabetes risk, suggesting that conserved genes encoded at these loci are central regulators of disease pathogenesis. These data are the first to reveal a role for individual non-MHC Idd loci in a specific, critical step in diabetes pathogenesis-T cell recruitment to islet lesions driving destructive inflammation. Importantly, identification of intermediate phenotypes in complex disease pathogenesis provides the tools required to progress toward gene identification at these loci.     

Comparative mapping of rat <Emphasis Type="Italic">Iddm4</Emphasis> to segments on HSA7 and MMU6

Iddm4 is one of several susceptibility genes that have been identified in the BB rat model of type 1 diabetes. The BB rat allele of this gene confers dominant predisposition to diabetes induction by immune perturbation in both the diabetes-prone and the diabetes-resistant substrains, whereas the Wistar Furth (WF) allele confers resistance. We have positioned the gene in a 2.8-cM region on rat Chromosome (Chr) 4, proximal to Lyp/Ian4l1. We have produced a radiation hybrid map of the Iddm4-region that includes a number of rat genes with their mouse and human orthologs. We present a comparative map of the rat Iddm4 region in rat, human, and mouse, assigning the gene to a 6.3-Mb segment between PTN and ZYX at 7q32 in the human genome, and to a 5.7-Mb segment between Ptn and Zyx in the mouse genome.     

Leprosy: a paradigm for the study of human genetic susceptibility to infectious diseases

Fifty years ago, the first identification of a non Mendelian genetic contribution to the development of a common infectious disease, i.e. the association between malaria and sickle-cell trait, was shown using a supervised approach which tests a limited number of candidate genes selected by hypothesis. Since then, the few genes that were convincingly associated with susceptibility to human infectious diseases were identified following the same strategy. The study of leprosy has contributed to modifying this way of thinking. In the absence of a satisfying experimental model and because of the impossibility to grow the causative agent in vitro, the candidate gene approach has turned out to be of limited interest. Conversely, positional cloning led to the identification of two major genes involved in the control of the disease, establishing for the first time the oligogenic nature of a human genetic contribution to an infectious disease. It is likely that these major results obtained in leprosy and the recent burst of genomic tools will make the genome-wide screening (functional or positional) the main strategy of dissection of the genetic susceptibility to many common infectious diseases.     

Identification and functional analysis of CBLB mutations in type 1 diabetes

Casitas B-lineage lymphoma b (Cblb) is a negative regulator of T-cell activation and dysfunction of Cblb in rats and mice results in autoimmunity. In particular, a nonsense mutation in Cblb has been identified in a rat model of autoimmune type 1 diabetes. To clarify the possible involvement of CBLB mutation in type 1 diabetes in humans, we performed mutation screening of CBLB and characterized functional properties of the mutations in Japanese subjects. Six missense mutations (A155V, F328L, N466D, K837R, T882A, and R968L) were identified in one diabetic subject each, excepting N466D. Of these mutations, F328L showed impaired suppression of T-cell activation and was a loss-of-function mutation. These data suggest that the F328L mutation is involved in the development of autoimmune diseases including type 1 diabetes, and also provide insight into the structure-function relationship of CBLB protein.     

Mouse models of type 1 and type 2 diabetes derived from the same closed colony: genetic susceptibility shared between two types of diabetes

Except for rare subtypes of diabetes, both type 1 and type 2 diabetes are multifactorial diseases in which genetic factors consisting of multiple susceptibility genes and environmental factors contribute to the disease development. Due to complex interaction among multiple susceptibility genes and between genetic and environmental factors, genetic analysis of multifactorial diseases is difficult in humans. Inbred animal models, in which the genetic background is homogeneous and environmental factors can be controlled, are therefore valuable in genetic dissection of multifactorial diseases. We are fortunate to have excellent animal models for both type 1 and type 2 diabetes--the nonobese diabetic (NOD) mouse and the Nagoya-Shibata-Yasuda (NSY) mouse, respectively. Congenic mapping of susceptibility genes for type 1 diabetes in the NOD mouse has revealed that susceptibility initially mapped as a single locus often consists of multiple components on the same chromosome, indicating the importance of congenic mapping in defining genes responsible for polygenic diseases. The NSY mouse is an inbred animal model of type 2 diabetes established from Jcl:ICR, from which the NOD mouse was also derived. We have recently mapped three major loci contributing to type 2 diabetes in the NSY mouse. Interestingly, support intervals where type 2 diabetes susceptibility genes were mapped in the NSY mouse overlapped the regions where type 1 diabetes susceptibility genes have been mapped in the NOD mouse. Although additional evidence is needed, it may be possible that some of the genes predisposing to diabetes are derived from a common ancestor contained in the original closed colony, contributing to type 1 diabetes in the NOD mouse and type 2 diabetes in the NSY mouse. Such genes, if they exist, will provide valuable information on etiological pathways common to both forms of diabetes, for the establishment of effective methods for prediction, prevention, and intervention in both type 1 and type 2 diabetes.     

A major linkage region on distal chromosome 4 confers susceptibility to mouse autoimmune gastritis

Although much is known about the pathology of human chronic atrophic (type A, autoimmune) gastritis, its cause is poorly understood. Mouse experimental autoimmune gastritis (EAG) is a CD4+ T cell-mediated organ-specific autoimmune disease of the stomach that is induced by neonatal thymectomy of BALB/c mice. It has many features similar to human autoimmune gastritis. To obtain a greater understanding of the genetic components predisposing to autoimmune gastritis, a linkage analysis study was performed on (BALB/cCrSlc x C57BL/6)F2 intercross mice using 126 microsatellite markers covering 95% of the autosomal genome. Two regions with linkage to EAG were identified on distal chromosome 4 and were designated Gasa1 and Gasa2. The Gasa1 gene maps within the same chromosomal segment as the type 1 diabetes and systemic lupus erythematosus susceptibility genes Idd11 and Nba1, respectively. Gasa2 is the more telomeric of the two genes and was mapped within the same chromosomal segment as the type 1 diabetes susceptibility gene Idd9. In addition, there was evidence of quantitative trait locus controlling autoantibody titer within the telomeric segment of chromosome 4. The clustering of genes conferring susceptibility to EAG with those conferring susceptibility to type 1 diabetes is consistent with the coinheritance of gastritis and diabetes within human families. This is the first linkage analysis study of autoimmune gastritis in any organism and as such makes an important and novel contribution to our understanding of the etiology of this disease.     

MHC class II proteins and disease: a structural perspective

MHC class II molecules on the surface of antigen-presenting cells display a range of peptides for recognition by the T-cell receptors of CD4+ T helper cells. Therefore, MHC class II molecules are central to effective adaptive immune responses, but conversely, genetic and epidemiological data have implicated these molecules in the pathogenesis of autoimmune diseases. Indeed, the strength of the associations between particular MHC class II alleles and disease render them the main genetic risk factors for autoimmune disorders such as type 1 diabetes. Here, we discuss the insights that the crystal structures of MHC class II molecules provide into the molecular mechanisms by which sequence polymorphisms might contribute to disease susceptibility.     

Genome-wide linkage analysis of chronic relapsing experimental autoimmune encephalomyelitis in the rat identifies a major susceptibility locus on chromosome 9

The immunization of inbred Dark Agouti (DA) rats with an emulsion containing homogenized spinal cord and CFA induces chronic relapsing experimental autoimmune encephalomyelitis (EAE), a disease with many similarities to multiple sclerosis. We report here the first genome-wide search for quantitative trait loci regulating EAE in the rat using this model. We identified one quantitative trait locus on chromosome 9, Eae4, in a [DA(RT1av1) x BN(RT1n)]F2 intercross showing linkage to disease susceptibility and expression of mRNA for the proinflammatory cytokine IFN-gamma in the spinal cord. Eae4 had a larger influence on disease incidence among rats that were homozygous for the RT1av1 MHC haplotype (RT1av1 rats) compared with RT1n/av1 rats, suggesting an interaction between Eae4 and the MHC. Homozygosity for the DA allele at markers in Eae4 and in the MHC was sufficient for EAE. Thus, Eae4 is a major genetic factor determining susceptibility to EAE in this cross of DA rats. In addition, there was support for linkage to phenotypes of EAE on chromosomes 1, 2, 5, 7, 8, 12, and 15. The chromosome 12 region has been shown previously to predispose DA rats to arthritis, and the chromosome 2 region is syntenic to Eae3 in mice. We conclude that Eae4 and probably the other identified genome regions harbor genes regulating susceptibility to neuroinflammatory disease. The identification and functional characterization of these genes may disclose critical events in the pathogenesis of multiple sclerosis; understanding these events could be essential for the development of new therapies against the disease.     

Two mechanisms for the non-MHC-linked resistance to spontaneous autoimmunity

Genetic susceptibility and resistance to most autoimmune disorders are associated with highly polymorphic genes of the MHC and with non-MHC-linked polygenic modifiers. It is known that non-MHC-linked polymorphisms can override or enhance the susceptibility to an autoimmune disease provided by pathogenic MHC genes, but the mechanisms remain elusive. In this study, we have followed the fate of two highly diabetogenic beta cell-specific T cell receptors (Kd and I-Ag7 restricted, respectively) in NOR/Lt mice, which are resistant to autoimmune diabetes despite expressing two copies of the diabetogenic MHC haplotype H-2g7. We show that at least two mechanisms of non-MHC-linked control of pathogenic T cells operate in these mice. One segregates as a recessive trait and is associated with a reduction in the peripheral frequency of diabetogenic CD8+ (but not CD4+) T cells. The other segregates as a dominant trait and is mediated by IL-4- and TGF-beta1-independent immune suppressive functions provided by lymphocytes that target diabetogenic CD4+ and CD8+ T cells, without causing their deletion, anergy, immune deviation, or ignorance. These results provide explanations as to how non-MHC-linked polymorphisms can override the susceptibility to an autoimmune disease provided by pathogenic MHC haplotypes, and demonstrate that protective non-MHC-linked genes may selectively target specific lymphoid cell types in cellularly complex autoimmune responses.     

Understanding autoimmune diabetes: insights from mouse models.

Type 1 or insulin-dependent diabetes is an autoimmune disease that causes the selective destruction of insulin-secreting beta cells in the pancreatic islets. Although this is a polygenic disease, with at least 20 genes implicated, the dominant susceptibility locus maps to the major histocompatibility complex (MHC), both in humans and in rodent models. However, in spite of progress on several fronts, the molecular pathology of autoimmune diabetes remains incompletely defined. Major areas of research include environmental trigger factors, the identification and role of beta-cell antigens in inducing and maintaining the autoimmune response, and the nature of the pathogenic and protective lymphocytes involved. In this review, we will focus on these areas to highlight recent advances in understanding the pathogenesis of autoimmune diabetes, drawing extensively on insights gained by studying the non-obese diabetic (NOD) mouse.     

Selective IgA deficiency in autoimmune diseases

Selective immunoglobulin A deficiency (IgAD) is the most common primary immunodeficiency in Caucasians. It has previously been suggested to be associated with a variety of concomitant autoimmune diseases. In this review, we present data on the prevalence of IgAD in patients with Graves disease (GD), systemic lupus erythematosus (SLE), type 1 diabetes (T1D), celiac disease (CD), myasthenia gravis (MG) and rheumatoid arthritis (RA) on the basis of both our own recent large-scale screening results and literature data. Genetic factors are important for the development of both IgAD and various autoimmune disorders, including GD, SLE, T1D, CD, MG and RA, and a strong association with the major histocompatibility complex (MHC) region has been reported. In addition, non-MHC genes, such as interferon-induced helicase 1 (IFIH1) and c-type lectin domain family 16, member A (CLEC16A), are also associated with the development of IgAD and some of the above diseases. This indicates a possible common genetic background. In this review, we present suggestive evidence for a shared genetic predisposition between these disorders.     

Major histocompatibility complex controls susceptibility and dominant inheritance,but not the severity of the disease in mouse models of rheumatoid arthritis

Collagen-induced arthritis (CIA) in DBA/1 and proteoglycan-induced arthritis (PGIA) in BALB/c mice are the most frequently used mouse models for studying clinical, immunological and genetic factors contributing to rheumatoid arthritis. DBA/1 ( H2(q)) mice are susceptible to CIA but resistant to PGIA, whereas BALB/c mice ( H2 (d)) are susceptible to PGIA and resistant to CIA. To gain insight into the mechanisms of how the major clinical (disease susceptibility, severity and onset of arthritis) and immunological traits (antigen-specific T- and B-cell responses) are influenced by the major histocompatibility complex (MHC), we have generated a unique intercross of BALB/c and DBA/1 parent strains, and the F1 and F2 hybrids were immunized for either CIA or PGIA. The major clinical and immunological traits were identified as either binary (qualitative) or quantitative traits on Chromosome 17 with a peak at MHC when the entire population was analyzed. In contrast, when only arthritic (susceptible) mice were selected and analyzed, the major clinical traits (severity and onset) 'lost' the linkage to MHC. Thus, MHC dictates disease susceptibility, but not the severity of arthritis. This was even more evident in the case of the H2(q) allele, which was clearly responsible for the dominant inheritance of arthritis in F2 hybrids (either CIA or PGIA). In conclusion, while certain MHC alleles strongly affect disease susceptibility and determine the mode of inheritance of a polygenic autoimmune disease, neither the type of inheritance (dominant vs recessive) nor other MHC components have evident effects upon the clinical symptoms of arthritis.     

Rat models of type 1 diabetes: genetics, environment, and autoimmunity

For many years, the vast amount of data gathered from analysis of nonobese diabetic (NOD) and congenic NOD mice has eclipsed interest in the rat for the study of type 1 diabetes. The study of rat models has continued, however, and recently there has been a reanimation of interest for several reasons. First, genetic analysis of the rat has accelerated. Ian4L1, cblb, and Iddm4 are now known to play major roles in rat autoimmunity. Second, rats are amenable to study the interactions of genetics and environment that may be critical for disease expression in humans. Environmental perturbants that predictably enhance the expression of rat autoimmune diabetes include viral infection, toll-like receptor ligation, and depletion of regulatory T cell populations. Finally, data generated in the rat have correctly predicted the outcome of several human diabetes prevention trials, notably the failure of nicotinamide and low dose parenteral and oral insulin therapies.     

A comprehensive analysis of shared loci between systemic lupus erythematosus (SLE) and sixteen autoimmune diseases reveals limited genetic overlap

In spite of the well-known clustering of multiple autoimmune disorders in families, analyses of specific shared genes and polymorphisms between systemic lupus erythematosus (SLE) and other autoimmune diseases (ADs) have been limited. Therefore, we comprehensively tested autoimmune variants for association with SLE, aiming to identify pleiotropic genetic associations between these diseases. We compiled a list of 446 non–Major Histocompatibility Complex (MHC) variants identified in genome-wide association studies (GWAS) of populations of European ancestry across 17 ADs. We then tested these variants in our combined Caucasian SLE cohorts of 1,500 cases and 5,706 controls. We tested a subset of these polymorphisms in an independent Caucasian replication cohort of 2,085 SLE cases and 2,854 controls, allowing the computation of a meta-analysis between all cohorts. We have uncovered novel shared SLE loci that passed multiple comparisons adjustment, including the VTCN1 (rs12046117, P = 2.02×10⁻⁰⁶) region. We observed that the loci shared among the most ADs include IL23R, OLIG3/TNFAIP3, and IL2RA. Given the lack of a universal autoimmune risk locus outside of the MHC and variable specificities for different diseases, our data suggests partial pleiotropy among ADs. Hierarchical clustering of ADs suggested that the most genetically related ADs appear to be type 1 diabetes with rheumatoid arthritis and Crohn''s disease with ulcerative colitis. These findings support a relatively distinct genetic susceptibility for SLE. For many of the shared GWAS autoimmune loci, we found no evidence for association with SLE, including IL23R. Also, several established SLE loci are apparently not associated with other ADs, including the ITGAM-ITGAX and TNFSF4 regions. This study represents the most comprehensive evaluation of shared autoimmune loci to date, supports a relatively distinct non–MHC genetic susceptibility for SLE, provides further evidence for previously and newly identified shared genes in SLE, and highlights the value of studies of potentially pleiotropic genes in autoimmune diseases.     

Multifactorial inheritance in type 1 diabetes

To date, twelve separate chromosome regions have been implicated in the development of human type 1 (insulin-dependent) diabetes mellitus. The major disease locus, IDDM1 in the major histocompatibility complex (MHC) on chromosome 6p21, accounts for about 35% of the observed familial clustering and its contribution to disease susceptibility is likely to involve polymorphic residues of class II molecules in T-cell-mediated autoimmunity. IDDM2 is encoded by a minisatellite locus embedded in the 5 regulatory region of the insulin gene. Familial clustering of disease can be explained by the sharing of alleles of at least 10 loci. IDDM1 and IDDM2 interact epistatically. For a multifactorial disease, such as type 1 diabetes, important information concerning the pathways and mechanisms involved can be gained from examining such interactions between loci, using methods that simultaneously take account of the joint effects of the various underlying genetic components.