Michal Prochazka

An autosomal genomic scan for loci linked to type II diabetes mellitus and body-mass index in Pima Indians

Genetic factors influence the development of type II diabetes mellitus, but genetic loci for the most common forms of diabetes have not been identified. A genomic scan was conducted to identify loci linked to diabetes and body-mass index (BMI) in Pima Indians, a Native American population with a high prevalence of type II diabetes. Among 264 nuclear families containing 966 siblings, 516 autosomal markers with a median distance between adjacent markers of 6.4 cM were genotyped. Variance-components methods were used to test for linkage with an age-adjusted diabetes score and with BMI. In multipoint analyses, the strongest evidence for linkage with age-adjusted diabetes (LOD = 1.7) was on chromosome 11q, in the region that was also linked most strongly with BMI (LOD = 3.6). Bivariate linkage analyses strongly rejected both the null hypothesis of no linkage with either trait and the null hypothesis of no contribution of the locus to the covariation among the two traits. Sib-pair analyses suggest additional potential diabetes-susceptibility loci on chromosomes 1q and 7q.

An amino acid substitution in the human intestinal fatty acid binding protein is associated with increased fatty acid binding, increased fat oxidation, and insulin resistance.

The intestinal fatty acid binding protein locus (FABP2) was investigated as a possible genetic factor in determining insulin action in the Pima Indian population. A polymorphism at codon 54 of FABP2 was identified that results in an alanine-encoding allele (frequency 0.71) and a threonine-encoding allele (frequency 0.29). Pimas who were homozygous or heterozygous for the threonine-encoding allele were found to have a higher mean fasting plasma insulin concentration, a lower mean insulin-stimulated glucose uptake rate, a higher mean insulin response to oral glucose and a mixed meal, and a higher mean fat oxidation rate compared with Pimas who were homozygous for the alanine-encoding allele. Since the FABP2 threonine-encoding allele was found to be associated with insulin resistance and increased fat oxidation in vivo, we further analyzed the FABP2 gene products for potential functional differences. Titration microcalorimetry studies with purified recombinant protein showed that the threonine-containing protein had a twofold greater affinity for long-chain fatty acids than the alanine-containing protein. We conclude that the threonine-containing protein may increase absorption and/or processing of dietary fatty acids by the intestine and thereby increase fat oxidation, which has been shown to reduce insulin action.

An autosomal genomic scan for loci linked to prediabetic phenotypes in Pima Indians.

Type 2 diabetes mellitus is a common chronic disease that is thought to have a substantial genetic basis. Identification of the genes responsible has been hampered by the complex nature of the syndrome. Abnormalities in insulin secretion and insulin action predict the development of type 2 diabetes and are, themselves, highly heritable traits. Since fewer genes may contribute to these precursors of type 2 diabetes than to the overall syndrome, such genes may be easier to identify. We, therefore, undertook an autosomal genomic scan to identify loci linked to prediabetic traits in Pima Indians, a population with a high prevalence of type 2 diabetes. 363 nondiabetic Pima Indians were genotyped at 516 polymorphic microsatellite markers on all 22 autosomes. Linkage analyses were performed using three methods (single-marker, nonparametric multipoint [MAPMAKER/SIBS], and variance components multipoint). These analyses provided evidence for linkage at several chromosomal regions, including 3q21-24 linked to fasting plasma insulin concentration and in vivo insulin action, 4p15-q12 linked to fasting plasma insulin concentration, 9q21 linked to 2-h insulin concentration during oral glucose tolerance testing, and 22q12-13 linked to fasting plasma glucose concentration. These results suggest loci that may harbor genes contributing to type 2 diabetes in Pima Indians. None of the linkages exceeded a LOD score of 3.6 (a 5% probability of occurring in a genome-wide scan). These findings must, therefore, be considered tentative until extended in this population or replicated in others.

Autosomal genomic scan for loci linked to obesity and energy metabolism in Pima Indians

An autosomal genomic scan to search for linkage to obesity and energy metabolism was completed in Pima Indians, a population prone to obesity. Obesity was assessed by percent body fat (by hydrodensitometry) and fat distribution (the ratio of waist circumference to thigh circumference). Energy metabolism was measured in a respiratory chamber as 24-h metabolic rate, sleeping metabolic rate, and 24-h respiratory quotient (24RQ), an indicator of the ratio of carbohydrate oxidation to fat oxidation. Five hundred sixteen microsatellite markers with a median spacing of 6.4 cM were analyzed, in 362 siblings who had measurements of body composition and in 220 siblings who had measurements of energy metabolism. These comprised 451 sib pairs in 127 nuclear families, for linkage analysis to obesity, and 236 sib pairs in 82 nuclear families, for linkage analysis to energy metabolism. Pointwise and multipoint methods for regression of sib-pair differences in identity by descent, as well as a sibling-based variance-components method, were used to detect linkage. LOD scores >=2 were found at 11q21-q22, for percent body fat (LOD=2.1; P=.001), at 11q23-q24, for 24-h energy expenditure (LOD=2.0; P=.001), and at 1p31-p21 (LOD=2.0) and 20q11.2 (LOD=3.0; P=.0001), for 24RQ, by pointwise and multipoint analyses. With the variance-components method, the highest LOD score (LOD=2.3 P=.0006) was found at 18q21, for percent body fat, and at 1p31-p21 (LOD=2.8; P=.0003), for 24RQ. Possible candidate genes include LEPR (leptin receptor), at 1p31, and ASIP (agouti-signaling protein), at 20q11.2.

Human PON2 gene at 7q21.3: cloning, multiple mRNA forms, and missense polymorphisms in the coding sequence.

We report the cloning and characterization of human PON2, a paraoxonase-related gene-2 that is physically linked with PON1 and PON3 on 7q2l.3. PON2 is ubiquitously expressed and we identified several mRNA forms produced by alternative splicing, or by the use of a second transcription start site. We also describe two polymorphisms in the coding sequences that, in the protein deduced from the longest open reading frame, predict an alanine-to-glycine substitution at residue 147 and a serine-to-cysteine substitution at residue 310.

NOR/Lt Mice: MHC-Matched Diabetes-Resistant Control Strain for NOD Mice

NOR/Lt is an insulitis-resistant and diabetes-free strain produced from an isolated genetic contamination within an NOD/Lt pedigree line. The albino coat-color phenotype, strain-specific endogenous retroviral profile, and skin graft tests indicated an NOD/Lt × C57BL/KsJ outcross-backcross segregant as the source of the contaminating genome. Analysis of 53 polymorphic DNA, biochemical, and immunologic markers distinguishing NOD/Lt from C57BL/KsJ revealed that 4 chromosomes (chromosomes 2, 4, 11, and 12) in NOR/Lt contained C57BL/KsJ-derived genes. The remaining markers on 14 chromosomes, including the diabetogenic H-2g7 complex on chromosome 17, were of NOD origin. Although completely resistant to cyclophosphamide-induced diabetes, NOR/Lt mice exhibited the same peripheral T-lymphocyte accumulation characteristic of NOD/Lt. Similarly, NOR/Lt peritoneal macrophages exhibited depressed interleukin-1 secretion characteristic of NOD/Lt. In addition to their diabetes resistance, NOR/Lt mice were distinguished from NOD/Lt by exhibiting more robust suppressor T-lymphocyte function. Outcross of NOR/Lt with NOD/Lt to generate heterozygosity at those chromosomal segments, defined by C57BL/KsJ markers in NOR/Lt parentals, did not produce insulitis or diabetes in F1 females. However, these F1 females were sensitive to cyclophosphamide-induced diabetes. In summary, the NOR/Lt strain is an MHC-matched diabetes-resistant control strain for NOD/Lt. Moreover, NOR/Lt will help identify the location and function of a non-MHC gene or genes capable of conferring resistance against insulitis and diabetes.

Crosses of NOD Mice With the Related NON Strain: A Polygenic Model for IDDM

Chromosome locations of non-major histocompatibility complex (MHC) genes contributing to insulin-dependent diabetes mellitus (IDDM) in mice have been determined by outcrossing NOD mice to other inbred strains congenic for the NOD MHC haplotype (H2g7). At least nine non-MHC IDDM susceptibility genes (Idd) were previously identified at first backcross (BC1) after outcross of NOD to C57BL/10.H2g7 congenic mice (B10.H2g7). We investigated whether the same set of Idd loci segregated with IDDM susceptibility after outcross of NOD to NON.H2g7 congenic mice. Since the outcrosses to NON.H2g7 and B10.H2g7 were performed in the same vivarium, direct comparisons were made of the chromosomal locations and relative strengths of Idd alleles in diabetic progeny from the two different outcrosses. In comparison with the NOD x B10.H2g7 outcross, the NOD x NON.H2g7 outcross produced significantly higher IDDM frequencies in F1, F2, and BC1 generations. The high F2 diabetes frequency allowed evaluation of the effects of homozygous expression of both the susceptibility and the resistance allele at Idd loci. This analysis demonstrated that no single non-MHC Idd locus was essential for the onset of diabetes in this cross. After outcross to NON.H2g7, Idd4 (chromosome [Chr] 11), Idd5 (Chr 1), and Idd8 (Chr 14) did not segregate with IDDM in either the BC1 or the F2 generation. Diabetogenic NOD-derived alleles at Idd2 (Chr 9), Idd3 (Chr 3), and Idd10 (Chr 3) were segregating in the BC1. An NON-derived allele contributing to susceptibility on Chr 7 (Idd7) was also detected. Dominant traits, detectable only in the F2 cross, were encoded by Chr 4 (Idd9) and two newly mapped loci on Chr 13 (Idd14) and 5 (Idd15). A third dominant trait was encoded by Chr 6 (possibly Idd6), but here, in contrast to Idd9, Idd14, and Idd15, the NON allele was diabetogenic. Stepwise logistic regression analysis of the BC1 and F2 data confirmed that the ability to identify certainty of the non-MHC Idd loci was contingent on the extent of homozygosity for NOD background genes. This study shows that the diabetogenic phenotype can be achieved through the actions of variable combinations of MHC-unlinked genes and a diabetogenic MHC haplotype.

Linkage of Chromosomal Markers on 4q With a Putative Gene Determining Maximal Insulin Action in Pima Indians

Insulin action in vivo varies widely in nondiabetic Pima Indians. Not all of this variance is attributable to individual differences in obesity, physical fitness, sex, or age, and after correcting for these co-variates, measures of insulin action aggregate in families. Insulin action at maximally stimulating insulin concentrations has a trimodal frequency distribution, particularly among obese individuals. This is consistent with the hypothesis that a codominantly inherited autosomal gene, unrelated to obesity, determines MaxM in the population. Preliminary sib-pair linkage analyses indicated the possibility of linkage between MaxM and the GYPA/B locus (encoding the MNSs red cell surface antigens) on chromosome 4q. To confirm and extend these findings, 10 additional loci on 4q were typed in 123 siblings and many of their parents from 46 nuclear families. The results indicate significant (P < 0.001) linkage of the FABP2 and ANX5 loci on 4q with MaxM, and of FABP2 with fasting insulin concentration. No linkage was found between the 4q markers and obesity. Our findings indicate that a gene on 4q, near the FABP2 and ANX5 loci, contributes to in vivo insulin action in Pima Indians.

High-throughput SNP detection by using DNA pooling and denaturing high performance liquid chromatography (DHPLC)

Abstract. One of the critical steps in the positional cloning of a complex disease gene involves association analysis between a phenotype and a set of densely spaced diallelic markers, typically single nucleotide repeats (SNPs), covering the region of interest. However, the effort and cost of detecting sufficient numbers of SNPs across relatively large physical distances represents a significant rate-limiting step. We have explored DNA pooling, in conjunction with denaturing high performance liquid chromatography (DHPLC), as a possible strategy for augmenting the efficiency, economy, and throughput of SNP detection. DHPLC is traditionally used to detect variants in polymerase chain reaction products containing both allelic forms of a polymorphism (e.g., heterozygotes or a 1:1 mix of both alleles) via heteroduplex separation and thereby requires separate analyses of multiple individual test samples. We have adapted this technology to identify variants in pooled DNA. To evaluate the utility and sensitivity of this approach, we constructed DNA pools comprised of 20 previously genotyped individuals with a frequency representation of 0%–50% for the variant allele. Mutation detection was performed by using temperature-modulated heteroduplex formation/DHPLC and dye-terminator sequencing. Using DHPLC, we could consistently detect SNPs at lower than 5% frequency, corresponding to the detection of one variant allele in a pool of 20 alleles. In contrast, fluorescent sequencing detected variants in the same pools only if the frequency of the less common allele was at least 10%. We conclude that DNA pooling of samples for DHPLC analysis is an effective way to increase throughput efficiency of SNP detection.

Genetic Control of Diabetogenesis in NOD/Lt Mice: Development and Analysis of Congenic Stocks

Genetic outcross and backcross analysis of nonobese diabetic (NOD/Lt) mice with a related but diabetes-resistant strain, nonobese normal (NON/Lt), has demonstrated that susceptibility to insulin-dependent diabetes mellitus is controlled in a recessive fashion by multiple genetic loci, including one (Idd-1s) associated with H-2 on chromosome 17 and another (Idd-2s) associated with Thy-1b/Apoa-1b (formerly Alp-1) on chromosome 9. To analyze the separate pathogenic contributions of Idd-1s and Idd-2s, two distinct congenie stocks of NOD/Lt mice homozygous on chromosomes 17 and 9 for NON/Lt linkage markers for the respective resistance alleles (Idd-1s and Idd-2r) were developed. The recessive nature of Idd-1s was confirmed at the fifth backcross generation in that 83% of females and 29% of males homozygous for NOD H-2 haplotype developed diabetes, whereas no diabetes occurred in any of the mice homozygous or heterozygous for the NON haplotype. However, codominant and recessive MHC-associated susceptibility genes in this congenie stock were indicated by the finding that at least one copy of the NOD/Lt MHC was required for insulitis development. Virtually no insulitis was detected in the pancreases of mice homozygous for NON haplotype at 42 wk of age, whereas heavy generalized insulitis was present in 3 of 19 H-2 heterozygotes and in 7 of 7 diabetic and 3 of 5 nondiabetic mice homozygous for NOD haplotype. Further indication of the presence of MHC-associated codominant and recessive MHC-associated susceptibility genes was the observation that the NOD MHC haplotype correlated in a codominant fashion with a relative increase in the percentage of splenic T-lymphocytes bearing the Ly-2 surface marker. Severe insulitis and concomitant high diabetes incidences occurred in all gentoypic classes of congenie mice carrying Thy-1/Apoa-1 linkage markers for either NOD or NON alleles at Idd-2. Molecular analysis indicated that the NON-derived Idd-2r resistance allele had been replaced by recombination with Idd-2s from NOD. Restriction-fragment–length polymorphism analysis of two polymorphic markers proximal to Thy-1, low-density lipoprotein receptor Ldlr and Ets-1, a protooncogene, confirmed a recombinant chromosome 9, because homozygosity for NOD genomie fragments was found centromeric to an NON congenie segment of at least 20 centiMorgans spanning the Thy-1 and Mod-1 loci. Of the polymorphic markers analyzed on chromosome 9 with a panel of first-backcross diabetic segregante, the strongest linkage association was with Thy-1b. The ease with which the Idd-2s susceptibility locus can apparently be uncoupled from the Thy-1 linkage marker underscores the difficulties of assessing gene action when a marker gene and the gene of interest recombine at a relatively high frequency. In summary, diabetogenesis in NOD/Lt mice requires a complex interaction between both recessive and dominant genes on multiple chromosomes, with both recessive and dominant expression indicated depending on the trait being assessed.