Holger Luthman

MHC2TA is associated with differential MHC molecule expression and susceptibility to rheumatoid arthritis, multiple sclerosis and myocardial infarction

Antigen presentation to T cells by MHC molecules is essential for adaptive immune responses. To determine the exact position of a gene affecting expression of MHC molecules, we finely mapped a previously defined rat quantitative trait locus regulating MHC class II on microglia in an advanced intercross line. We identified a small interval including the gene MHC class II transactivator (Mhc2ta) and, using a map over six inbred strains combined with gene sequencing and expression analysis, two conserved Mhc2ta haplotypes segregating with MHC class II levels. In humans, a –168A → G polymorphism in the type III promoter of the MHC class II transactivator (MHC2TA) was associated with increased susceptibility to rheumatoid arthritis, multiple sclerosis and myocardial infarction, as well as lower expression of MHC2TA after stimulation of leukocytes with interferon-γ. We conclude that polymorphisms in Mhc2ta and MHC2TA result in differential MHC molecule expression and are associated with susceptibility to common complex diseases with inflammatory components.

Epigenetic regulation of PPARGC1A in human type 2 diabetic islets and effect on insulin secretion.

Aims/hypothesisInsulin secretion in pancreatic islets is dependent upon mitochondrial function and production of ATP. The transcriptional coactivator peroxisome proliferator activated receptor gamma coactivator-1 alpha (protein PGC-1α; gene PPARGC1A) is a master regulator of mitochondrial genes and its expression is decreased and related to impaired oxidative phosphorylation in muscle from patients with type 2 diabetes. Whether it plays a similar role in human pancreatic islets is not known. We therefore investigated if PPARGC1A expression is altered in islets from patients with type 2 diabetes and whether this expression is influenced by genetic (PPARGC1A Gly482Ser polymorphism) and epigenetic (DNA methylation) factors. We also tested if experimental downregulation of PPARGC1A expression in human islets influenced insulin secretion.MethodsThe PPARGC1A Gly482Ser polymorphism was genotyped in human pancreatic islets from 48 non-diabetic and 12 type 2 diabetic multi-organ donors and related to PPARGC1A mRNA expression. DNA methylation of the PPARGC1A promoter was analysed in pancreatic islets from ten type 2 diabetic and nine control donors. Isolated human islets were transfected with PPARGC1A silencing RNA (siRNA).ResultsPPARGC1A mRNA expression was reduced by 90% (p < 0.005) and correlated with the reduction in insulin secretion in islets from patients with type 2 diabetes. After downregulation of PPARGC1A expression in human islets by siRNA, insulin secretion was reduced by 41% (p ≤ 0. 01). We were able to ascribe reduced PPARGC1A expression in islets to both genetic and epigenetic factors, i.e. a common PPARGC1A Gly482Ser polymorphism was associated with reduced PPARGC1A mRNA expression (p < 0.00005) and reduced insulin secretion (p < 0.05). In support of an epigenetic influence, the PPARGC1A gene promoter showed a twofold increase in DNA methylation in diabetic islets compared with non-diabetic islets (p < 0.04).Conclusions/interpretationWe have shown for the first time that PPARGC1A might be important in human islet insulin secretion and that expression of PPARGC1A in human islets can be regulated by both genetic and epigenetic factors.

Genetic analysis of non-insulin dependent diabetes mellitus in the GK rat

Non-insulin dependent diabetes mellitus (NIDDM) is a major public health problem, but its aetiology remains poorly understood. We have performed a comprehensive study of the genetic basis of diabetes in the Goto-Kakizaki (GK) rat, the most widely used animal model of non-obese NIDDM. The genetic dissection of NIDDM using this model has allowed us to map three independent loci involved in the disease. In addition, we identify a major factor affecting body weight, but not glucose tolerance, on chromosome 7 and map a further 10 regions that are suggestive for linkage. We conclude that NIDDM is polygenic and fasting hyperglycaemia and postprandial hyperglycaemia clearly have distinct genetic bases.

Overexpression of Alpha2A-Adrenergic Receptors Contributes to Type 2 Diabetes

Ratting Out a Diabetes Gene Inbred animals with inherited susceptibility to disease can be especially informative regarding pathogenetic mechanisms because they carry naturally occurring genetic variants of the same type that cause disease in humans. This principle is illustrated by Rosengren et al. (p. 217; published online 19 November), whose analysis of an inbred strain of rats prone to develop type 2 diabetes led to the discovery of a gene whose aberrant overexpression suppresses pancreatic insulin secretion in both rats and humans. The culprit gene, ADRA2A, encodes the alpha2A adrenergic receptor and is potentially a valuable lead for diabetes therapy because it can be targeted pharmacologically. Sequence variations in an adrenergic receptor gene cause reduced insulin secretion and contribute to type 2 diabetes. Several common genetic variations have been associated with type 2 diabetes, but the exact disease mechanisms are still poorly elucidated. Using congenic strains from the diabetic Goto-Kakizaki rat, we identified a 1.4-megabase genomic locus that was linked to impaired insulin granule docking at the plasma membrane and reduced β cell exocytosis. In this locus, Adra2a, encoding the alpha2A-adrenergic receptor [alpha(2A)AR], was significantly overexpressed. Alpha(2A)AR mediates adrenergic suppression of insulin secretion. Pharmacological receptor antagonism, silencing of receptor expression, or blockade of downstream effectors rescued insulin secretion in congenic islets. Furthermore, we identified a single-nucleotide polymorphism in the human ADRA2A gene for which risk allele carriers exhibited overexpression of alpha(2A)AR, reduced insulin secretion, and increased type 2 diabetes risk. Human pancreatic islets from risk allele carriers exhibited reduced granule docking and secreted less insulin in response to glucose; both effects were counteracted by pharmacological alpha(2A)AR antagonists.

Quantitative trait loci disposing for both experimental arthritis and encephalomyelitis in the DA rat; impact on severity of myelin oligodendrocyte glycoprotein‐induced experimental autoimmune encephalomyelitis and antibody isotype pattern

Quantitative trait loci (QTL) controlling inflammatory diseases with different organ specificity may hypothetically either be unique for one disease or shared among different diseases. We have investigated whether five non‐MHC QTL controlling susceptibility to experimental arthritis in the DA rat also influence myelin oligodendrocyte glycoprotein (MOG)‐induced experimental autoimmune encephalomyelitis (EAE) in an F2 intercross between inbred DA and PVG.RT1a rats. Two of the five chromosome regions affecting arthritis in the DA rat also regulate phenotypes of EAE. The DA allele at markers in Cia3(collagen‐induced arthritis QTL) on chromosome 4 is associated with more severe EAE and high levels of anti‐MOG antibodies of the IgG2c subclass. Since production of antibodies of the IgG2c subclass may be stimulated by Th1 cells, and there is previous evidence that such cells promote EAE, it is possible that both of the studied phenotypes are controlled by the same gene or genes regulating Th1/Th2 cell differentiation. Furthermore, we show that Oia2(oil‐induced arthritis QTL) on chromosome 4 regulates levels of anti‐MOG antibodies of the IgG1 subclass and of anti‐MOG IgE, but that this gene region does not affect clinical disease severity in our study. Since production of IgE and IgG1 may be stimulated by Th2 cells, this QTL may also control Th1/Th2 bias. We conclude that Cia3and Oia2regulate MOG‐induced EAE in rats. Furthermore, since both EAE and arthritis phenotypes co‐localize to these gene regions, they may harbor genes which are key regulators of pathogenic immune responses.

Steroid 21-hydroxylase (P450c21): a new allele and spread of mutations through the pseudogene

Lesions in the gene encoding the adrenal enzyme steroid 21-hydroxylase (P450c21) result in defective adrenal cortisol synthesis, often accompanied by aldosterone deficiency. The symptoms range from severe neonatal disease to inconspicuous symptoms in adulthood depending on the nature of the mutations. The 21-hydroxylase gene is present in close proximity to a highly homologous pseudogene, and both genes show variation in copy number between individuals. For complete DNA sequence characterization, we have applied selective polymerase chain reaction amplification and direct sequencing of all full-length steroid 21-hydroxylase genes present in individuals. Using healthy individuals with only one remaining steroid 21-hydroxylase allele as normal references, a new allele was found in two siblings, in whom clinical and laboratory findings demonstrated moderate enzyme deficiency. Full-length sequencing of this allele displayed an Arg 484 to Pro codon change in exon 10, in the same position as a previously identified GG to C mutation found in a patient with severe 21 -hydroxylase deficiency. Arg 484 is located within a stretch of amino acids that are highly conserved between mammalian 21-hydroxylases. The finding of the presently reported 21-hydroxylase allele indicates that the GG to C mutation from the severely affected patient has arisen by a two-step mechanism, consisting of a G to C transversion accompanied by an adjacent G deletion. When sequencing 26 pseudogenes, both these mutations, which are not present in the pseudogenes hitherto reported, were found at low frequency together with a number of other polymorphisms. Thus, also rare mutations can spread via the pseudogene and can therefore be expected to arise independently in unrelated individuals.

Impact of Diabetic Inheritance on Glucose Tolerance and Insulin Secretion in Spontaneously Diabetic GK-Wistar Rats

The impact of genetic factors and maternal diabetes on glucose tolerance and pancreatic β-cell function was studied in first generation (F1) offspring generated in crosses between the spontaneously diabetic Goto-Kakizaki (GK)-Wistar rat and normoglycemic control Wistar rats (W). The (GK × W) F1 hybrids were offspring of either male GK (mGK) and female Wistar (fW) (mGK × fW) or male Wistar (mW) and female GK (fGK) (mW × fGK) rats. Already at 8 days of age, blood glucose levels were elevated in GK (7.6 ± 0.5 vs. 4.8 ± 0.3 mM in W; P < 0.001) and in F1 rats (6.0 ± 0.3 in mGK × fW and 6.6 ± 0.4 mM in mW × fGK; both P < 0.01 vs. W). In 2-month-old male rats, glucose (2 g/kg, intraperitoneally) markedly increased blood glucose levels after 60 min in GK rats (18.1 ± 0.6 vs. 5.5 ± 0.3 mM in W; P < 0.001) and moderately increased levels in F1 rats (9.9 ± 0.9 in mGK × fW and 11.6 ± 1.0 mM in mW × fGK, both P < 0.01 vs. W). Similar patterns were obtained in female rats. Repeated backcrossing of F1 with W rats successively improved glucose tolerance. In perfused pancreases of male rats, the 20-min insulin response to 16.7 mM glucose was −7.44 ± 5.18 pmol in GK rats, 71.57 ± 12.25 pmol in W rats, 9.00 ± 0.89 pmol in mGK × fW rats, and 18.20 ± 3.97 pmol in mW × fGK rats. In female W rats, the glucose-induced insulin response was significantly lower than in males (P < 0.05). However, as in males, insulin responses to glucose were impaired in both GK and F1 female rats. Arginine-induced insulin release was similar in all groups. In mGK × fW, glucose-stimulated somatostatin release was 50% of that in W rats, whereas arginine-stimulated responses of glucagon and somatostatin were not different from W rats. Pancreatic contents of insulin and glucagon were similar in mGK × fW and W rats, whereas somatostatin content was lower in mGK × fW rats (P < 0.05). In conclusion, the diabetic state in GK and F1 rats was evident early in life. Hybrid rats were intermediate between W and GK rats with regard to glucose tolerance and glucose-stimulated insulin response in vitro, but had normal pancreatic insulin content. Results of repeated backcrossing of F1 rats with W rats showed that genes in >1 locus contribute to the diabetic state. Furthermore, the absence of significant differences between impact of maternal and paternal origin of the GK genes for glucose intolerance suggests that hyperglycemia in utero does not influence the severity of diabetes in the F1 offspring.

High-Resolution Quantitative Trait Locus Analysis Reveals Multiple Diabetes Susceptibility Loci Mapped to Intervals <800 kb in the Species-Conserved Niddm1i of the GK Rat

Niddm1i, a 16-Mb locus within the major diabetes QTL in the diabetic GK rat, causes impaired glucose tolerance in the congenic NIDDM1I strain. Niddm1i is homologous to both human and mouse regions linked with type 2 diabetes susceptibility. We employed multiple QTL analyses of congenic F2 progeny selected for one recombination event within Niddm1i combined with characterization of subcongenic strains. Fine mapping located one hyperglycemia locus within 700 kb (Niddm1i4, P = 5 × 10−6). Two adjacent loci were also detected, and the GK allele at Niddm1i2 (500 kb) showed a glucose-raising effect, whereas it had a glucose-lowering effect at Niddm1i3 (400 kb). Most proximally, Niddm1i1 (800 kb) affecting body weight was identified. Experimental data from subcongenics supported the four loci. Sorcs1, one of the two known diabetes susceptibility genes in the region, resides within Niddm1i3, while Tcf7l2 maps outside all four loci. Multiple-marker QTL analysis incorporating the effect of cosegregating QTL as cofactors together with genetically selected progeny can remarkably enhance resolution of QTL. The data demonstrate that the species-conserved Niddm1i is a composite of at least four QTL affecting type 2 diabetes susceptibility and that two adjacent QTL (Niddm1i2GK and Niddm1i3GK) act in opposite directions.

Contiguous localization of the genes encoding human insulin-like growth factor binding proteins 1(IGBP1) and 3(IGBP3) on chromosome 7

In extracellular fluids the insulin-like growth factors (IGFs) are bound to specific binding proteins (IGBPs). The genes for two members of this protein family have been mapped, the IGBP1 gene to human chromosomal region 7p14-p12 and the IGBP2 gene to region 2q33-q34. In this study, somatic cell hybrid analysis indicated that IGBP3 is also located on chromosome 7. Pulsed-field gel electrophoresis was used to demonstrate the close physical linkage between IGBP1 and IGBP3. Overlapping cosmid clones encompassing these genes were isolated, and restriction endonuclease mapping showed that the genes are arranged in a tail-to-tail fashion separated by 20 kb of DNA. Further characterization of the IGBP1 DNA sequence disclosed a duplication of the intron 3-exon 4 junction within the third intron. In addition, we report RFLPs for ApaLI and TaqI in the IGBP1 locus.

Differences in the Ratio of RNA Encoding Two Isoforms of the Insulin Receptor Between Control and NIDDM Patients: The RNA Variant Without Exon 11 Predominates in Both Groups

Two alternative forms of the insulin receptor with different affinities for insulin are expressed as a result of alternative splicing of RNA corresponding to exon 11 of the IR gene. The percentage of IR-RNA molecules without exon 11, encoding the high-affinity isoform, was determined by cDNA-mediated PCR amplification of RNA extracts from the quadriceps femoris muscle of healthy control subjects (n = 9) and NIDDM patients (n = 7). In both patients and control individuals, a majority of the IR-RNA molecules contained exon 11. In addition, the proportion of IR-RNA molecules without exon 11 was decreased in patients (21 ± 1%) compared with control subjects (31 ± 3%) (P = 0.018). Careful investigation of the kinetics of the PCR-based assay system, as well as the conditions for separation of the PCR products, allowed us to suggest a possible explanation of the discrepant results concerning the alternative splicing presented in previous reports. The diabetic subjects as a group had higher fasting insulin levels and lower insulin-mediated glucose uptake during a euglycemic-hyperinsulinemic clamp (P = 0.042). However, identification of the regulatory pathways leading to the splicing alteration in NIDDM patients requires further investigation.