Three loci on mouse chromosome 6 influence onset and final incidence of type I diabetes in NOD.C3H congenic strains.

The development of insulin-dependent diabetes mellitus in both human and mouse is dependent on the interaction between genetic and environmental factors. The analysis of newly created NOD.C3H congenic strains for spontaneous and cyclophosphamide-induced diabetes has allowed the definition of three controlling genetic loci on mouse chromosome 6. A NOD-derived susceptibility allele at the Idd6 locus strongly influences the onset of diabetes in spontaneous diabetes. A NOD-derived resistance allele at the Idd19 locus affects the final diabetes incidence observed in both models, while a novel locus, provisionally termed Idd20, appears to control Idd19 in an epistatic manner. Decreased diabetes incidence is observed in CY-induced diabetes when Idd20 is homozygous for the C3H allele, while heterozygosity is associated with an increase in diabetes incidence. The Idd20, Idd19, and Idd6 candidate regions fall respectively within genetically defined intervals of 4, 7, and 4.5 cM on mouse chromosome 6. From our YAC contig, Idd6 would appear to localize within a ca. 1.5-Mb region on distal chromosome 6.

Evidence for the presence of insulin-dependent diabetes-associated alleles on the distal part of mouse chromosome 6.

Type 1 diabetes (IDDM) is a complex disorder with multifactorial and polygenic etiology. A genome-wide screen performed in a BC1 cohort of a cross between the nonobese diabetic (NOD) mouse with the diabetes-resistant feral strain PWK detected a major locus contributing to diabetes development on the distal part of chromosome 6. Unlike the majority of other Idd loci identified in intraspecific crosses, susceptibility is associated with the presence of the PWK allele. Genetic linkage analysis of congenic lines segregating PWK chromosome 6 segments in a NOD background confirmed the presence of the Idd locus within this region. The genetic interval defined by analysis of congenic animals showed a peak of significant linkage (P = 0.0005) centered on an approximately 9-cM region lying between D6Mit11 and D6Mit25 genetic markers within distal mouse chromosome 6. [Genetic markers polymorphic between the NOD and PWK strains are available as a supplement at http://www.genome.org]

Genetic analysis of diabetes and insulitis in an interspecific cross of the nonobese diabetic mouse with Mus spretus.

The nonobese diabetic (NOD) mouse is a widely used model for genetic studies of insulin-dependent diabetes mellitus due to the similarities between the murine and human diseases. To aid in the localization and identification of diabetes-related susceptibility genes, we have constructed an interspecific backcross between NOD and Mus spretus (SEG/Pas) mice. Although no diabetic animals were observed in the first backcross generation of (SEG/Pas x NOD) x NOD (BC1), the incidence of insulitis (lymphocyte infiltration of the islets of Langerhans) exceeded 20% after injections of cyclophosphamide, a treatment that provokes an acute form of diabetes in NOD mice. Insulitis, a prediabetic condition, is a useful phenotype in studies of diabetes susceptibility. In the second backcross (BC2) generation, 8% of the animals became diabetic and 76% were found to have insulitis. Genetic mapping studies in the BC2 families confirmed the importance of the major histocompatibility complex region on the severity of insulitis and suggested that additional susceptibility loci were linked to markers on mouse chromosomes 3, 6, and 15. Mus spretus crosses have been an important tool in recent advances in murine genetics, and our results extend their usefulness to the study of a multifactorial disease.

Two isoforms of the kidney androgen-regulated protein are encoded by two alleles of a single gene in OF1 mice.

Two cDNA clones coding for two forms of the mouse kidney androgen-regulated protein (KAP) distinguished by their electrophoretic mobilities on SDS gel electrophoresis have been isolated from libraries prepared from strains of mice having one (BALB/c) or two (OF1) forms of the KAP protein. The corresponding mRNAs have identical sizes, as well as identical sequences in their 5' non-translated regions. The size difference observed between the two proteins is due to two point mutations in the coding region of the KAP mRNA, leading to two amino-acid changes one of which resulted in the substitution of a glycine for a glutamic acid. As shown by in vitro transcription/translation experiments, these two amino-acid differences are responsible for the shift in the apparent molecular weight of the protein on SDS gels. Both forms of the protein are more abundant in males than in females. In vitro translation of kidney RNAs isolated from six different strains and species of mice revealed the presence of other forms of the KAP protein, characterized by small variations of their molecular weights. Southern blot analysis data are consistent with the presence of only one kap gene in the mouse genome. A restriction fragment length polymorphism has been observed, which does not correlate with the protein polymorphism, indicating the presence of another allele in the OF1 mouse genome.

Development of an enzyme-linked immunosorbent assay with a monoclonal antibody prepared against alpha 1-antitrypsin for diagnostic screening of inflammatory disorders.

A monoclonal antibody, designated A2a18b8, of IgG1 class prepared against human alpha 1-antitrypsin, cross-reacts with alpha 1-antitrypsin in the serum of rat and baboon, but not with alpha 1-antitrypsin in serum of rabbit, pig, hamster, guinea pig, dog, or turtle. We used A2a18b8 in an enzyme-linked immunosorbent assay (ELISA) developed for human alpha 1-antitrypsin. Preliminary ELISA screening of 247 serum samples from patients with various inflammatory disorders indicated that the concentration of a specific epitope(s) on alpha 1-antitrypsin recognized by this monoclonal antibody was increased significantly in patients with active systemic lupus erythematosus, mixed connective tissue disease, and rheumatoid arthritis, but not in patients with sclerodermic disorders or Sjögren's syndrome. Evidently, A2a18b8 has diagnostic value in that it selectively recognizes a specific epitope(s) on alpha 1-antitrypsin that is (are) apparently exposed during selective inflammatory disorders.

Genetic variation in androgen regulation of ornithine decarboxylase gene expression in inbred strains of mice.

Previous studies have indicated that androgen regulation of certain gene products in murine kidney is genetically controlled. In the present work, the expression of renal ornithine decarboxylase (ODC) gene(s) was used as a biological marker to study androgen responsiveness of eight inbred strains of mice (A/J, C57BR/cdJ, 129/J, C57L/J, BALB/cJ, SM/J, RF/J, and C57BL/6J). Kidneys of untreated females from these strains did not have significantly different basal ODC activities or ODC mRNA concentrations. However, renal enzyme concentrations in intact male mice exhibited marked strain-dependent variation; three strains (RF/J, SM/J, and C57BR/cdJ) had 5- to 20-fold higher activities than the other five strains. Renal ODC mRNA content showed similar genetic variability in the male mice; animals with highest enzyme activity had higher mRNA levels than those with low activity. These results could not be explained by differences in either serum testosterone levels or renal nuclear androgen receptor content, suggesting that the animals were differentially sensitive to endogenous androgens. To evaluate further the androgen regulation of ODC gene expression, female mice were treated with testosterone-releasing implants for 5-7 days. The two strains (A/J and C57BL/6J) that had low enzyme activity in response to endogenous testosterone in male mice also showed blunted responses to exogenous androgen administration, as measured by the induction of ODC and its mRNA. The relative distribution of the two mRNA species coding for ODC (2.2 and 2.7 kb in size) exhibited strain-dependent variation that did not, however, correlate with the androgen responsiveness. Studies of the mRNA levels in reciprocal F1 hybrids of C57BR/cdJ and C57BL/6J mice suggested that androgen sensitivity of ODC gene expression, at least in these crosses, was inherited in an autosomal dominant manner.

Androgen binding capacity and 5 alpha-reductase activity in pubic skin fibroblasts from hirsute patients.

We have measured the total (cytosolic plus nuclear) androgen binding capacity of pubic skin fibroblasts from nine patients with hirsutism of various origin. Confluent intact cell monolayers were incubated with increasing concentrations (0.05-2 nM) of [3H]dihydrotestosterone ([3H]DHT) with or without a 200-fold excess of unlabeled DHT. The androgen binding capacities (mean +/- SD) were similar in normal men (411 +/- 171 fmol/mg DNA), women (310 +/- 103 fmol/mg DNA), and hirsute patients (313 +/- 141 fmol/mg DNA) regardless of the plasma androgen levels. In contrast, the 5 alpha-reductase level in pubic skin fibroblasts (mean +/- SD) was, as previously described, higher in hirsute women (3.3 +/- 2.6 fmol/micrograms DNA . h) than in normal women (1.1 +/- 0.6 fmol/microgram DNA . h; P less than 0.05). We conclude from these data that: 1) increased androgen binding capacity cannot be held responsible for hypersensitivity to androgens in hirsutism; 2) the androgen receptor is not regulated by androgens in human skin, as similar levels are observed in men, women, and hirsute patients; 3) this contrasts with 5 alpha-reductase activity and emphasizes the importance of this enzyme as an amplifier of androgen action in areas where it is stimulated by androgens, such as pubic skin.

Dihydrotestosterone stimulates 5 alpha-reductase activity in pubic skin fibroblasts.

In vivo, the 5 alpha-reduction of testosterone (T) to dihydrotestosterone (DHT) is androgen dependent in pubic skin but not in the skin of the external genitalia. The aim of the present study was to determine whether pubic skin fibroblasts (PSF) had retained this androgen dependency. PSF were prepared from explants of skin from normal subjects (four men, three women) and three patients with complete form of the testicular feminization syndrome. Culture medium containing 5% fetal calf serum and DHT was added 24 h after subculture (day 1) and renewed every other day. 5 alpha-Reductase was assayed on day 4 or day 8 by incubation of intact cell monolayers with [3H]T (2 nM), extraction of the medium, and chromatography of the metabolites; DNA was assayed in the cell pellets; 5 alpha-reductase was expressed as fmol/micrograms DNA . h. Controls were untreated plates from the same subcultures. DHT had no effect on cell DNA, whereas it resulted in a dose-dependent increase in 5 alpha-reductase activity. In seven PSF strains tested, DHT (10(-7) M) increased 5 alpha-reductase activity 2- to 4-fold over the control levels. This effect was abolished by the simultaneous addition of cyproterone acetate (2 X 10(-6) M) and was not observed in PSF from testicular feminization syndrome patients, suggesting that it was indeed mediated via the androgen receptor. T but not estradiol or cortisol also increased 5 alpha-reductase activity in PSF. The effect of androgens was suppressed by protein synthesis inhibitors. These data provide strong evidence that PSF respond to androgens via a receptor mediated mechanism, and that 5 alpha-reductase can be used as a marker of androgen action in pubic skin in vitro as well as in vivo.