Hossein Fakhrai-Rad

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.

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.

A rat genetic linkage map including 67 new microsatellite markers.

Genetic heterogeneity in outbred populations and a major impact of environmental factors on the disease phenotypes complicate genetic studies of multifactorial disorders. For control of these effects, inbred animal disease models are used for the genetic dissection of disease pathways that operate in concert to cause susceptibility to several human common disorders. Quantitative trait loci (QTLs) that contribute to disease phenotypes can be identified by analysis of experimental crosses between a strain with the disease phenotype of interest and a control strain in carefully controlled environments (Lander and Schork 1994). Several pathophysiologically well characterized inbred rat strains are developed as models for multifactorial disorders such as diabetes mellitus, inflammatory diseases, hypertension, and obesity (Greenhouse et al. 1990). In recent years, genome mapping of the rat has progressed with an increasing number of mapped genes and genetic markers (Bihoreau et al. 1997; Brown et al. 1998; Jacob et al. 1995; Pravenec et al. 1996), which has allowed genetic localization of QTLs in several rat models (Aitman et al. 1997; Galli et al. 1996; Gauguier et al. 1996; Hilbert et al. 1991; Jacob et al. 1991; Kanemoto et al. 1998; Kovacs and Kloting 1998). QTL identification and positional cloning of disease susceptibility genes rely on dense and accurate genetic maps, especially when new approaches with advanced intercross animals are used for highresolution genetic mapping (Darvasi 1998). In the present report, we describe the construction of a rat genetic linkage map based on anchor markers mapped in several hundred F2 progeny. The rats were derived from an intercross between the GK rat, a model for type 2 diabetes (Goto et al. 1975), and the F344 rat. Approximately 1200 microsatellite markers were PCR amplified, and 543 of these were informative in the GK/F344 cross and could be amplified readily. Most microsatellites were identified by screening genomic libraries and the Genebank database for repeated sequences (Jacob et al. 1995). Additional markers were also obtained from the Rat Map Database (http:// www.ratmap.gu.se/). GK and F344 rats were bred as described previously (Galli et al. 1996). Two reciprocal crosses (GK male × F344 female and F344 male × GK female) produced two types of F 1 progeny. The two sets of F1 progeny were intercrossed separately, and a total of 374 F2 progeny were obtained in two separate cohorts. We selected 45 F2 progeny (Galli et al. 1996) for genotyping with 400 informative microsatellites. Subsequently, 190 randomly selected rats from the two cohorts were genotyped with 194 of the 400 markers, spaced at an average of 10 cM. Finally, the remaining 139 rats were genotyped with 92 of the 194 markers to further improve resolution in previously identified QTLs (Galli et al. 1996). Rats were genotyped as previously described (Jacob et al. 1991), with the exception that one primer in each pair was labeled with g P-ATP. The linkage map was assembled by employing MAPMAKER/ EXP (version 3.0) linkage analysis software (Lander et al. 1987). The 400 markers were sorted into 27 separate linkage groups with a LOD of 5.0 as inclusion criterion. All linkage groups were asigned to specific chromosomes and oriented according to the rat cytogenetic map (Szpirer et al. 1998). All chromosomes except Chromosomes (Chrs) 6, 13, 15, and 16 were represented by single linkage groups. The linking process was subsequently repeated with a LOD of 4.0. This allowed mapping of all markers into 22 linkage groups. Each chromosome was represented by a single linkage group with the exception of Chr 13. The two linkage groups on Chr 13 were, however, linked with a LOD of 3.0. The resulting map consists of 400 markers in 21 linkage groups corresponding to the 20 autosomal chromosomes and the X Chr. This map includes 242 markers ordered with LOD

High-resolution QTL Analysis Reveals Multiple Diabetes Susceptibility Loci Mapped to Intervals less than 800-kb in the Species Conserved Niddm1i of the GK Rat.

3.0 and defined as framework (anchor) loci. The remaining 158 markers were placed according to their most likely positions in relation to the anchor markers. Considerable effort was made to assure accuracy in the mapping process: all genotypes were read twice, and all ambiguities were checked and resolved. The generated KI rat genetic linkage map spans a total length of 1782 cM (Kosambi 1943) ith an average spacing of consecutive markers of 4.4 cM (Table 1). Furthermore, the map includes 67 new markers, that is, 16 gene-specific markers and 51 anonymous microsatellites (Table 2). The map is available at http://www.ki.se/cmm. The sex-averaged genetic length of the map (1782 cM) is in

High-Resolution Quantitative Trait Locus Analysis Reveals Multiple Diabetes Susceptibility Loci Mapped to Intervals 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.

Identification of rat susceptibility loci for adjuvant-oil-induced arthritis

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.