Grant Morahan

The nature and identification of quantitative trait loci: a community’s view

This white paper by eighty members of the Complex Trait Consortium presents a communitys view on the approaches and statistical analyses that are needed for the identification of genetic loci that determine quantitative traits. Quantitative trait loci (QTLs) can be identified in several ways, but is there a definitive test of whether a candidate locus actually corresponds to a specific QTL?

Linkage disequilibrium of a type 1 diabetes susceptibility locus with a regulatory IL12B allele

Type 1 diabetes (T1D; or insulin-dependent diabetes mellitus, IDDM) is an autoimmune disease with both genetic and environmental components. In addition to the human leukocyte antigen (HLA) complex, the single major genetic contributor of susceptibility, an unknown number of other unidentified genes are required to mediate disease. Although many loci conferring susceptibility to T1D have been mapped, their identification has proven problematic due to the complex nature of this disease. Our strategy for finding T1D susceptibility genes has been to test for human homologues of loci implicated in diabetes-prone NOD (non-obese diabetic) mice, together with application of biologically relevant stratification methods. We report here a new susceptibility locus, IDDM18, located near the interleukin-12 (IL-12)p40 gene, IL12B. Significant bias in transmission of IL12B alleles was observed in affected sibpairs and was confirmed in an independent cohort of simplex families. A single base change in the 3′ UTR showed strong linkage disequilibrium with the T1D susceptibility locus. The IL12B 3′ UTR alleles showed different levels of expression in cell lines. Variation in IL-12p40 production may influence T-cell responses crucial for either mediating or protecting against this and other autoimmune diseases.

Association of IL12B promoter polymorphism with severity of atopic and non-atopic asthma in children

BACKGROUND Severe asthma is a frequent cause of hospital admission, especially among children. The main environmental triggers of airway inflammation in asthma are viruses and aeroallergens. These agents elicit reciprocal immune responses, characterised by production of T helper 1 and T helper 2 cytokines, respectively. There is no genetic explanation for how hyper-responsiveness to these disparate environmental stimuli develops among individuals with asthma. Our aim was to assess relation between an IL12B promoter polymorphism and asthma. METHODS We did a cohort study in which we initially genotyped 411 6-year olds for the IL12B promoter polymorphism. We then assessed the relation between this polymorphism and asthma severity. A further 85 asthmatic children in an additional sample of 433 children from the same cohort were then assessed to confirm these findings. We also examined in-vitro interleukin-12 responses in a subgroup of individuals. FINDINGS Heterozygosity for the IL12B promoter polymorphism was observed in 76% (16) of atopic and non-atopic individuals with severe asthma in the initial sample. By comparison, heterozygotes comprised only 31% (17) of the moderate asthma group, and 48% (20) of individuals with mild asthma were heterozygous, as were unaffected controls. These findings were confirmed in the second sample (overall p<0.0001). Our data suggest that IL12B promoter heterozygosity contributes to asthma severity rather than susceptibility per se. The severity-predisposing genotype was associated with reduced interleukin 12 p40 gene transcription and decreased interleukin 12 p70 secretion. INTERPRETATION Interleukin 12 plays a key part in antagonism of T helper 2 differentiation, and in induction of antiviral host defense. Genetically determined attenuation of interleukin-12 response capacity would, therefore, provide a plausible common immunological pathway to disease severity for the two major forms of asthma.

Clonal Deletion of Autospecific B Lymphocytes

Using mice transgenic for functional, rearranged immunoglobulin heavy and light chain genes, it can be demonstrated that B lymphocytes reactive with cell surface-bound class I MHC antigen can be controlled by clonal elimination. Even low-affinity cell-bound ligands can induce deletion. Deletion can occur in the pre-B to B cell transitional stage or after the B cells exist the bone marrow, depending on where the cells first encounter autoantigen. IgD appears to play no role in protecting cells from deletion. It is argued that defects in B-cell tolerance alone may be sufficient to lead to systemic autoimmunity.

Markers on Distal Chromosome 2q Linked to Insulin-Dependent Diabetes Mellitus

Insulin-dependent diabetes mellitus (IDDM) is a multigenic autoimmune disease. An IDDM susceptibility gene was mapped to chromosome 2q34. This gene may act early in diabetogenesis, because “preclinical” individuals also showed linkage. Human leukocyte antigen (HLA)-disparate, but not HLA-identical, sibs showed linkage, which was even stronger in families with affected females. The genes encoding insulin-like growth factor-binding proteins 2 and 5 were mapped to a 4-megabase pair interval near this locus. These results indicate the existence of a gene that acts at an early stage in IDDM development, screening for which may identify a specific subset of at-risk individuals.

T-Cell Tolerance in Transgenic Mice Expressing Major Histocompatibility Class I Molecules in Defined Tissues

A fundamental question in immunology is concerned with the mechanism by which tolerance to self components is imparted to developing lymphocytes. This issue is especially relevant to T lymphocytes because of their ability to recognize antigen only if it can associate with the class I or class II tnolecules encoded by the major histocompatibility complex (MHC) (Zinkemagel & Doherty 1979), and also because of the particular selection process which operates during T-cell maturation in the thymus. A large proportion of the immature thymic lymphocytes bear both of the T-cell surface antigens CD4 and CD8 and express the ap heterodimeric antigen T-cell receptor (TCR) (Scollay et al. 1988). According to a widely accepted current model, these immature cells are positively selected on thymic epithelial cells for their capacity to bind with varying degrees of affmity to MHC molecules (Sprent et al. 1988). Since these molecules themselves are capable of binding self peptides (Bjorkman et al. 1987, Babbitt et al. 1985), any T cell with some affinity for self peptides in association with MHC molecules will also be selected. The binding to epithelial cells is presumed to have a protective effect, failure to bind being associated with apoptosis of thymus lymphocytes. Selection of the T-cell repertoire in this way will ensure that the mature T lymphocytes will recognize antigen when associated with self-MHC molecules, but it will also allow the differentiation of cells with dangerously high

Immunological tolerance: new approaches using transgenic mice

Transgenic technology allows the introduction into the germline of an animal of a known gene coding for a normally foreign antigen, and by means of a specific promoter, the direction of its expression to specific tissues. The antigen is therefore synthesized by the animal as an authentic self molecule, at a particular stage in development, and in a particular site. In this review, J.F.A.P. Miller and colleagues discuss this radically new approach to the investigation of the mechanism of acquired immunological tolerance to self components.

Localization of Idd11 using NOD congenic mouse strains: elimination of Slc9a1 as a candidate gene.

Abstract Type 1 diabetes is a multigenic autoimmune disease, the genetic basis for which is perhaps best characterized in the nonobese diabetic (NOD) mouse model. We previously located a NOD diabetes susceptibility locus, designated Idd11, on mouse Chromosome (Chr) 4 by analyzing diabetic backcross mice produced after crossing NOD/Lt with the nondiabetic resistant strain C57BL/6 (B6) strain. In order to confirm Idd11 and further refine its location, three NOD congenic mouse strains with different B6 derived intervals within Chr 4 were generated. Two of the congenic strains had a significant decrease in the cumulative incidence of diabetes compared with NOD/Lt control mice. The third NOD congenic strain, containing a B6 interval surrounding the Slc9a1 locus, was not protected against diabetes. These results define a new distal boundary for Idd11 and eliminate the Slc9a1 gene as a candidate. The Idd11 locus has now been definitively mapped to a 13cM interval on mouse Chr 4.

C57BL/6 and C57BL/10 inbred mouse strains differ at multiple loci on chromosome 4

Inbred mouse strains are valuable tools in biological research. As members of a given strain are all identical, independent studies on such mice can be readily reproduced and compared. C57BL is one of the most commonly used of these strains, and two of its major substrains are C57BL/6 (B6) and C57BL/10 (B10). The separation of B6 and B10 as lines occurred prior to 1937, and they have since provided the genetic backgrounds for the derivation of many congenic strains (for example, those congenic for the H-2; Boyse 1977). Since their separation, they have been characterized as differing at only 3 loci of 161 tested (Festing 1992): the minor histocompatability locus H-9, the immunoglobulin heavy chain locus Igh-2 (chromosome 12), and the locus for delta-aminolevulinate dehydratase Lv (chromosome 4). The two substrains thus have a close genetic relationship and experimental results using either strain have been considered equivalent. However, in genetic studies of diabetes using a backcross of nonobese diabetic (NOD) mice to the B6 strain, we observed that on distal chromosome 4, B 10 alleles differed from those of B6 and were instead the same as the NOD type. These surprising results led us to examine other chromosome 4 loci. DNA from B6, B10, and NOD mice was analyzed by conventional Southern blotting techniques (Sambrook et al . 1989) or by analysis of microsatellite markers (Dietrich et al. 1992). Probe and enzyme combinations were used that had previously identified polymorphisms between NOD and B6 mice (Morahan and co-workers, submitted; McClive et al. 1993). Probes were used to detect restriction fragment length polymorphisms (RFLP) at the following loci: ClqC (Petry et al. 1992),

IL10 and IL12B polymorphisms each influence IL-12p70 secretion by dendritic cells in response to LPS

Dendritic cells (DC) are the main producers of the cytokine IL‐12p70, through which they play a direct role in the development of IFN‐γ‐secreting Th1 cells, costimulation of CTL differentiation and NK‐cell activation. In contrast, IL‐10, which is also produced by DC, negatively regulates IL‐12 production. IL‐12p70 production varies widely between individuals, and several polymorphisms in the gene encoding IL‐12p40 (IL12B) have been identified that influence susceptibility and severity of infectious, autoimmune and neoplastic disease. Here we show that polymorphisms not only of IL12B, but also in the IL10 promoter, influence IL‐12p70 secretion by monocyte‐derived DC in response to LPS. Although IL12B promoter homozygotes were prone to making more IL‐12p70, presence of the IL10 high genotype restricted IL‐12p70 production in these individuals. These observations provide a further genetic control of IL‐12p70 regulation and emphasize the complexity of production of this cytokine. They also suggest genotypes that might influence the outcome of DC immunotherapy.