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Biomedical subjects

M Prochazka

Publications and source records attributed to M Prochazka.

At least 37 records · Page 2Linked to original sources
PubMed1998

Structure and expression of the human MTG8/ETO gene.

Translocations involving the putative proto-oncogene MTG8/ETO on 8q22 are frequently found in acute myeloid leukemia. To date, little is known of the genomic organization of this gene. Here, we report that the MTG8 gene consists of 13 exons distributed over 87 kb of genomic DNA. Two polymorphic microsatellite repeats are described, including one in intron 3 (three alleles; heterozygosity 0.34) and another in the 3'UTR (15 alleles; heterozygosity 0.89). Expression of MTG8 was detected in a variety of normal human tissues with the highest mRNA levels occurring in brain and heart. Previously, two mRNA forms produced by the alternative usage of the first exon have been reported. We now describe a novel, abundantly expressed, alternatively spliced transcript resulting from the inclusion of a 155-bp exon (designated 9a) that changes the reading frame and introduces a premature stop codon. Identical alternatively spliced mRNA variants were found to be produced by the highly conserved homologous gene (Cbfa2t1) in the mouse, suggesting an evolutionary significance.

Alternative Splicing↗
PubMed1998

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.

Adult↗
PubMed1998

Insulin downregulates pyruvate dehydrogenase kinase (PDK) mRNA: potential mechanism contributing to increased lipid oxidation in insulin-resistant subjects.

Oxidative metabolism of glucose is regulated by pyruvate dehydrogenase (PDH) that can be inhibited by isoforms of PDH kinase (PDK). Recently, increased PDK activity has been implicated in the pathogenesis of insulin resistance and non-insulin-dependent diabetes mellitus (NIDDM) in obese subjects. Using quantitative RT-PCR, we measured mRNA of PDK2 and PDK4 isoforms in skeletal muscle biopsies from nondiabetic Pima Indians, a population with a high prevalence of NIDDM associated with obesity. PDK2 and PDK4 mRNAs were positively correlated with fasting plasma insulin concentration, 2-h plasma insulin concentration in response to oral glucose, and percentage body fat, whereas both isoforms were negatively correlated with insulin-mediated glucose uptake rates. Measurements of PDK2 and PDK4 mRNA during the hyperinsulinemic-euglycemic clamp and of PDK2 in cell culture indicated that both transcripts decrease in response to insulin. Increased fatty acid (FA) oxidation has been traditionally viewed as the cause for increased PDK activity contributing to insulin resistance in obese subjects. In contrast, our data indicate that insufficient downregulation of PDK mRNA in insulin-resistant individuals could be a cause of increased PDK expression leading to impaired glucose oxidation followed by increased FA oxidation.

Adult↗
PubMed1998

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.

Chromosome Mapping↗
PubMed1998

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.

Analysis of Variance↗
PubMed1998

A common variant in PPP1R3 associated with insulin resistance and type 2 diabetes.

Selected candidate genes have been analyzed in the Pima Indians of Arizona based on evidence that insulin resistance and type 2 diabetes have significant genetic determinants. An amino acid substitution at codon 905 of the glycogen-targeting subunit of type 1 protein phosphatase that regulates skeletal muscle glycogenesis was recently reported to be associated with changes in insulin action in Danish subjects. In addition to the variant at 905, we report here a novel substitution at codon 883 and common variant of an "ATTTA" element in the 3'-untranslated region (UTR) of the corresponding gene (PPP1R3). The 3'-UTR variant resembled the mRNA-destabilizing AT(AU)-rich elements (AREs) and resulted in a 10-fold difference in reporter mRNA half-life, was correlated with PPP1R3 transcript and protein concentrations in vivo, and was associated with insulin resistance and type 2 diabetes in the Pimas. The variant is more common in Pimas (0.56) than in Caucasians (0.40). Because of its apparent effect on expression of PPP1R3, it may, in part, contribute to the higher prevalence of type 2 diabetes in this Native American population.

Adult↗
PubMed1996

Cloning and characterization of PDK4 on 7q21.3 encoding a fourth pyruvate dehydrogenase kinase isoenzyme in human.

Different isoenzymes of pyruvate dehydrogenase kinase (PDK) inhibit the mitochondrial pyruvate dehydrogenase complex by phosphorylation of the E1alpha subunit, thus contributing to the regulation of glucose metabolism. By positional cloning in the 7q21.3-q22.1 region linked with insulin resistance and non-insulin-dependent diabetes mellitus in the Pima Indians, we identified a gene encoding an additional human PDK isoform, as evidenced by its amino acid sequence identity (>65%) with other mammalian PDKs, and confirmed by biochemical analyses of the recombinant protein. We performed detailed comparative analyses of the gene, termed PDK4, in insulin-resistant and insulin-sensitive Pima Indians, and detected five DNA variants with comparable frequencies in both subject groups. Using quantitative reverse transcription polymerase chain reaction, we found that the variants identified in the promoter and 5'-untranslated region did not correlate with differences in mRNA level in skeletal muscle and adipose tissue. We conclude that alterations in PDK4 are unlikely to be the molecular basis underlying the observed linkage at 7q21.3-q22.1 in the Pima Indians. Information about the genomic organization and promoter sequences of PDK4 will be useful in studies of other members of this family of mitochondrial protein kinases that are important for the regulation of glucose metabolism.

Adipose Tissue↗
PubMed1996

Association of the glycogen synthase locus on 19q13 with NIDDM in Pima Indians.

Skeletal muscle glycogen synthase (encoded by GYS1 on chromosome 19q13.3) is the rate-limiting enzyme in insulin-mediated non-oxidative glucose disposal. Our previous studies have demonstrated an impairment of insulin-stimulated GYS1 activities in insulin-resistant Pima Indians, and associations of non-insulin-dependent diabetes mellitus (NIDDM) with the GYS1 locus were reported recently in Finnish and Japanese populations. We have performed linkage and association analyses of GYS1 and seven additional DNA markers on 19q with NIDDM, and with parameters of insulin action in the Pima Indians. We have found a significant association of NIDDM with GYS1 genotypes (p = 0.009), and with common GYS1 alleles (p = 0.022) in the Pima Indians. We have performed a detailed comparative analysis of the GYS1 gene, mRNA, and protein product in insulin-sensitive and insulin-resistant Pima Indians. No mutations in GYS1 coding sequences were detected; nor did we find alterations of GYS1 mRNA expression or of its basal enzymatic activity in insulin-resistant Pima Indians. These results contrasted with a 25% reduction of immunoreactive protein in insulin-resistant subjects as detected by Western blotting with an antibody specific for the C-terminal end of GYS1 (t-test p = 0.024; Wilcoxon's rank-sum test, p = 0.04). Because no mutations were detected in the DNA encoding this epitope, the difference in immunoreactivity may reflect post-translational modification(s) of the protein rather than a difference in the gene itself, or it could have occurred by chance. We conclude that our data do not indicate alterations in the GYS1 gene as the cause for the observed association, and that a different locus near GYS1 may be the contributing genetic element.

Adult↗
PubMed1996

A novel (TA)n polymorphism in the hexokinase II gene: application to noninsulin-dependent diabetes mellitus in the Pima Indians.

Hexokinase II, one member of a family of structurally similar enzymes that catalyze the phosphorylation of glucose in the 6-position, has been suggested to play a role in the pathophysiology of noninsulin-dependent diabetes mellitus (NIDDM). The gene for hexokinase II, HK2, has been previously mapped to human chromosome 2p13 by fluorescence in situ hybridization, and two-point linkage analysis has placed it near the locus for transforming growth factor alpha, TGFA. We now report the characterization of a (TA)n polymorphism in intron 12 of HK2. Using multipoint analysis of CEPH family genotypes, we have determined the most likely locus order to be cen-D2S169-[D2S286-HK2]-[D2S145-D2S291]-[+ ++D2S45-D2S101-TGFA]-tel. As HKII is a candidate gene that could contribute to the manifestation of insulin resistance and NIDDM, we genotyped 1152 Pima Indians, a Native American tribe that has the highest reported prevalence of NIDDM in the world. Although we did not detect any linkage or association of HK2 with insulin resistance or NIDDM in the Pima Indians, the polymorphism and detailed mapping of HK2 described in this report should prove useful in the assessment of the role of this gene in the predisposition to NIDDM in other populations.

Base Sequence↗
PubMed1996

Microsatellite hybrid capture technique for simultaneous isolation of various STR markers.

The microsatellite hybrid capture technique was designed to enrich simultaneously for various microsatellite repeats from a genomic clone. It is illustrated in this report that different repeat motifs, including polymorphic ones, can be efficiently isolated in a single experiment. In principle, this technique can be applied to any type of genomic clones to facilitate the isolation of informative markers for fine mapping of subchromosomal regions of interest, and for linkage and association studies of candidate genes for which no polymorphic markers are available yet.

Asian People↗
PubMed1995

Molecular and linkage analysis of type-1 protein phosphatase catalytic beta-subunit gene: lack of evidence for its major role in insulin resistance in Pima Indians.

Insulin resistance is believed to be a prediabetic condition that results from reduced rates of insulin-mediated glycogen synthesis in skeletal muscle. A decrease in activities of skeletal muscle glycogen synthase and of its regulatory enzyme type-1 protein phosphatase (PP 1) have been previously identified in insulin-resistant Pima Indians. Because the PP1 catalytic beta-subunit is presumed to be the major isoform in the glycogen-bound PP1 complex, we have selected the structural gene for this subunit (PPP1CB) as a candidate for a detailed genetic analysis. We have determined the exon-intron structure of PPP1CB, and have identified a polymorphic (CA)-repeat marker (D2S1237) at this gene. No sequence abnormalities were detected in PPP1CB by Southern blot analysis or by single-stranded conformational polymorphism analysis of all eight coding exons. Using sib-pair linkage analyses, no evidence for linkage was found between the D2S1237 marker at this locus and fasting insulin, insulin-stimulated glucose uptake in vivo, obesity, or non-insulin-dependent diabetes mellitus. Similarly, we have found no evidence for association of D2S1237 with any of these phenotypes. Based on our data we conclude that the structural gene for the PP1 catalytic beta-subunit does not appear to be a major genetic determinant responsible for the PP1 abnormalities characteristic of insulin resistance in Pima Indians.

Alternative Splicing↗
PubMed1995

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.

Adult↗
PubMed1995

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.

Animals↗
PubMed1995

Evidence for linkage between a region on chromosome 1p and the acute insulin response in Pima Indians.

A low acute insulin response (AIR) is a predictor of non-insulin-dependent diabetes mellitus (NIDDM) in insulin-resistant Pima Indians. We have initiated a search for regions of the genome linked with the AIR using sib-pair linkage analysis as a first step in identifying genes that are determinants of this phenotype. Eighteen short tandem-repeat polymorphisms from chromosome 1 were genotyped in over 900 Pima Indians and tested for linkage with NIDDM and in a subset of Pima Indians for linkage with AIR. The anonymous DNA marker D1S198 on chromosome 1p was linked with AIR (P = 0.000056) in 175 sib pairs from 60 families, all with normal glucose tolerance, but no linkage was observed between D1S198 and NIDDM (P = 0.44, 996 sib pairs). Additional markers genotyped on chromosome 1 did not show linkage with AIR or NIDDM. This study indicates that a locus on chromosome 1p may be a determinant of the phenotypic variation seen in the AIR.

Chromosomes, Human, Pair 1↗
PubMed1994

Use of recombinant congenic and congenic strains of NOD mice to identify a new insulin-dependent diabetes resistance gene.

Insulin-dependent diabetes mellitus (IDDM) in NOD/Lt mice represents a complex polygenic disease. NOR/Lt is a recombinant congenic strain (RCS) in which limited regions of the NOD/Lt genome have been replaced by genome from the C57BL/KsJ strain. NOR mice are insulitis resistant and diabetes free despite genetic identity with NOD at numerous chromosomal regions containing previously described insulin-dependent diabetes (Idd) genes, including the strongly diabetogenic H2g7 major histocompatibility complex (MHC) haplotype. The present study revealed BKs-derived genome on segments of chromosomes (Chr) 1, 2, 4, 5, 7, 11, 12, and 18, approximating 11.6% of the total NOR genome analyzed. (NOD x NOR)F2 segregation analysis was employed to identify chromosomal regions in NOR containing Idd resistance alleles. IDDM developed in 33% (10/30) of F1 females, and 29.3% (36/123) of F2 females aged to 1 yr. A previously unrecognized diabetes resistance locus (designated Idd13r) strongly protective in homozygous state was identified on NOR Chr 2 in linkage with the Il1 alpha structural gene. The existence of this locus was confirmed by construction of a NOD stock congenic for NOR-derived markers on Chr 2. Our analysis shows the utility of RCS and congenic stocks for the identification and isolation of non-MHC genes with strong antidiabetogenic functions.

Alleles↗
PubMed1994

Organization of the human annexin V (ANX5) gene.

We characterized the region of human chromosome 4q26-q28 that contains the gene encoding annexin V (placental anticoagulant protein I), a member of a family of calcium-dependent phospholipid binding proteins. A total of 14.5 kb, containing 9 introns, could be directly amplified from genomic DNA; the remainder was characterized from genomic clones in phage lambda and a yeast artificial chromosome. The gene was mapped with restriction enzymes BamHI, EcoRI, HindIII, SacI, StuI, and XbaI; the transcribed region spans 28 kb and contains 13 exons (44 tp 530 bp in size) and 12 introns (0.23 to 8.8 kb in size). Several putative transcription factor binding sites are present in the 5'-region, but the promoter has no recognizable TATA box. This study will facilitate further analysis of the functions of annexin V and its role in disease.

Animals↗