Variants in the gene encoding aldose reductase (AKR1B1) and diabetic nephropathy in American Indians.

AIMS: The aldose reductase gene (AKR1B1) is a strong candidate for diabetic nephropathy, and the T allele at rs759853 and the Z-2 allele at an [AC]n microsatellite are associated with diabetic kidney disease in some populations. As AKR1B1 is located on 7q35, where we have previously reported linkage to diabetic nephropathy in Pima Indians, this study examined the association of AKR1B1 variants with diabetic nephropathy in this population. METHODS: AKR1B1 variants were identified by sequencing and genotyped using allelic discrimination and pyrosequencing. Genotype distributions were compared between 107 cases with diabetic end-stage renal disease and 108 control subjects with diabetes for > or = 10 years and no evidence of nephropathy, and between 141 individuals with nephropathy and 416 individuals without heavy proteinuria in a family study of 257 sibships. RESULTS: We identified 11 AKR1B1 single nucleotide polymorphisms (SNPs) and the [AC]n microsatellite polymorphism. Three SNPs were rare and two were in 100% genotypic concordance; thus, eight polymorphisms were genotyped. No variant was associated with diabetic kidney disease in the case-control or family-based study. For example, the T allele at rs759853 had an allele frequency of 0.165 in cases and 0.171 in control subjects (OR = 0.96, 95% CI, 0.57-1.59, P = 0.86); in the family study its frequency was 0.140 and 0.169 in affected and unaffected individuals, respectively (OR = 0.90, 95% CI, 0.53-1.54 P = 0.71). Corresponding values for the Z-2 allele at the [AC]n microsatellite were OR = 1.09 (95% CI 0.72-1.66, P = 0.67) and OR = 1.25 (95% CI 0.81-1.95, P = 0.31) in the case-control and family studies, respectively. CONCLUSIONS: Common AKR1B1 polymorphisms are unlikely to be major determinants of diabetic nephropathy in this population.

Variant screening of PRKAB2, a type 2 diabetes mellitus susceptibility candidate gene on 1q in Pima Indians.

The AMP-activated protein kinase (AMPK) is a key enzyme involved in the regulation of lipid and glucose metabolism. There are multiple isoforms of the three subunits of this enzymatic complex, each encoded by a different gene in humans. We have investigated the PRKAB2 gene encoding the beta2 subunit, which is located on chromosome 1q within a region linked with type 2 diabetes mellitus (T2DM) in the Pima Indians and four different Caucasian populations. The gene consists of eight exons spanning about 15 kb, and we detected nine variants in the introns and 3' UTR, including eight informative single nucleotide polymorphisms (SNPs) and one rare 4 bp insertion/deletion. In an analysis of representative markers in selected Pima Indians including 149 diabetic cases (onset age < 25 years) and 150 controls (at least 45 years old, with normal glucose tolerance), we found no evidence for association of this locus with T2DM. We conclude that variants in PRKAB2 are unlikely to contribute to the disease susceptibility in Pima Indians.

Polymorphism screening of the insulin receptor-related receptor gene (INSRR) on 1q in Pima Indians.

INSRR coding for the insulin receptor-related receptor (IRR) is located within the 1q21-q23 region linked with type-2 diabetes mellitus in Pima Indians and Caucasians. Although the ligand and biological function of this receptor are not yet known, its tyrosine kinase phosphorylates proteins involved in insulin signaling, and IRR may also play a role in the control of the insulin producing beta-cell mass. Therefore, defects in INSRR could contribute to susceptibility to type-2 diabetes. By screening the 22 exons, 5' and 3' flanking sequences, and most introns in 20 Pima Indians and one Caucasian control, we detected nine diallelic variants, including eight single nucleotide polymorphisms (SNPs), and a length polymorphism involving a 26-nt motif. In this study sample, four of the identified SNPs were rare, while the remaining five common variants located within 4.5 kb from the 3' end of the gene were in linkage disequilibrium. When analysed in selected diabetic and non-diabetic Pimas, none of the markers was associated with the disease. We conclude that INSRR has no detectable mutations contributing to diabetes in the Pima Indians. However, information on the novel markers may prove useful for association studies of this candidate gene in other populations.

Analysis of the lamin A/C gene as a candidate for type II diabetes susceptibility in Pima Indians.

AIMS/HYPOTHESIS: Lamin A/C (LMNA) is located within a region on chromosome 1q that has been linked with Type II (non-insulin-dependent) diabetes mellitus in Pima Indians. Rare mutations in exon 8 of LMNA underlie Dunnigan-Type familial partial lipodystrophy, a disease characterized by regional adipocyte degeneration and frequently accompanied by insulin resistance, glucose intolerance, and diabetes. A more common variant in exon 10 (3408C/T) has recently been associated with obesity in non-diabetic aboriginal Canadian subjects. Because obesity is a strongly predisposing factor for Type II diabetes, we hypothesized that the LMNA 3408C/T variant could be associated with diabetes and body mass index in Pima Indians. METHODS: To determine whether the LMNA 3408C/T variant contributes to Type II diabetes susceptibility, we genotyped the polymorphism in 1,338 Pimas using allelic discrimination technology. The locus was screened for additional variants in 20 diabetic Pima Indians and non-diabetic Pima Indians using denaturing high performance liquid chromatography and dideoxy sequencing. RESULTS: We found no evidence for association of 3408C/T with diabetes, body mass index, total cholesterol, HDL cholesterol, triglycerides, leptin concentrations, or indices of insulin sensitivity and secretion. Subsequent screening of the remaining LMNA exons and flanking sequences revealed only rare variants in intron 4 and the 3'UTR, showing no frequency differences between diabetic and non-diabetic Pima Indians. We reassessed the linkage with diabetes following adjustment for the LMNA 3408C/T variant; adjustment for the effects of LMNA did not substantially modify the evidence for linkage. CONCLUSION/INTERPRETATION: We conclude that the LMNA 3408C/T variant probably does not play a role in susceptibility to diabetes or obesity in Pima Indians.

Analysis of linkage disequilibrium between polymorphisms in the KCNJ9 gene with type 2 diabetes mellitus in Pima Indians.

The KCNJ9 gene encodes a G-protein-coupled inwardly rectifying potassium channel and is located within a region on human chromosome 1 that has been linked with type 2 diabetes mellitus in Pima Indians and Caucasians. To assess the potential contribution of genetic alterations within KCNJ9 to diabetes susceptibility in the Pimas, we have genotyped 11 single nucleotide polymorphisms (SNPs) in 50 Pimas with diabetes and 50 Pimas over the age of 45 without diabetes and in 51 sib pairs, discordant for the disease, who were characterized by decreased allele sharing at the chromosomal location of the maximum LOD score. We detected three SNP clusters exhibiting distinct linkage disequilibria. Polymorphisms in intron 2, exon 3, and the 3'-UTR were in statistically significant linkage disequilibrium with diabetes in the case-control group (P = 0.006), but not the sibling pairs (P = 0.097). A weak association with diabetes was also found in the original linkage set comprising 1150 Pimas (odds ratio = 0.64/P = 0.079 for a dominant model and OR = 0.67/P = 0.005 for a recessive model). However, no effect on linkage was detected following adjustment for one of the most strongly associated SNPs in the entire original linkage set. Our results indicate that variants in KCNJ9 are associated with diabetes in Pimas but do not account for the linkage of 1q with diabetes in this population.

Analysis of slc19a2, on 1q23.3 encoding a thiamine transporter as a candidate gene for type 2 diabetes mellitus in pima indians.

Mutations in the SLC19A2 gene cause thiamine-responsive megaloblastic anemia (TRMA) frequently combined with diabetes mellitus and deafness. Type 2 diabetes mellitus is heritable and a region on 1q21-q23 encompassing SLC19A2 was linked with the disease in Pima Indians and Caucasians. We therefore investigated this candidate gene in selected diabetic and nondiabetic Pimas and found no variants. We conclude that mutations in SLC19A2 do not contribute to type 2 diabetes in this population.

Analysis of PBX1 as a candidate gene for type 2 diabetes mellitus in Pima Indians.

The human proto-oncogene PBX1 codes for a homeodomain containing protein that modulates expression of several genes, including those contributing to regulation of insulin action and glucose metabolism. PBX1 is located on chromosome 1q22, a region linked with type 2 diabetes in Pima Indians, Caucasians, and an Old Order Amish population. We have investigated the PBX1 genomic sequence to identify polymorphisms that may contribute to diabetes susceptibility in the Pimas. PBX1 is composed of nine exons spanning approx. 117 kb and is located within 300 kb of microsatellite D1S1677, which marks the peak of linkage to diabetes susceptibility in the Pima Indians. We detected 16 single nucleotide polymorphisms in PBX1 including one causing a glycine to serine substitution at residue 21. Comparison of the frequencies of the polymorphisms between affected and unaffected Pima Indians did not detect any significant differences, indicating that mutations in PBX1 do not explain the linkage of 1q with type 2 diabetes in this population. The genomic structure of PBX1 provides a basis for similar systematic examinations of this candidate locus in other populations in relation to both type 2 diabetes and other metabolic disorders.

Subcutaneous abdominal adipocyte size, a predictor of type 2 diabetes, is linked to chromosome 1q21--q23 and is associated with a common polymorphism in LMNA in Pima Indians.

Large subcutaneous abdominal adipocyte size (s.c. abd. AS) is associated with insulin resistance and predicts type 2 diabetes in Pima Indians. Because type 2 diabetes is familial, we aimed to determine whether mean s.c. abd. AS is also familial and if so, to identify chromosomal regions linked to this measure. Body composition (hydrodensitometry) and mean s.c. abd. AS (fat biopsy) were measured in 295 Pima Indians (179 with normal, 80 with impaired, and 36 with diabetic glucose tolerance) representing 164 nuclear families. Mean s.c. abd. AS, adjusted for age, sex, and percentage body fat was a familial trait (heritability h(2) = 0.48, P < 0.0001). A genome-wide autosomal scan revealed suggestive evidence for linkage (LOD 1.73) of adjusted mean s.c. abd. AS to chromosome 1q21--q23, a region containing LMNA, the gene encoding for the nuclear envelope proteins lamin A/C. Rare mutations in LMNA were recently shown to underlie familial partial lipodystrophy (FPLD), a syndrome characterized by regional loss of adipose tissue, insulin resistance, and glucose intolerance. A common (allelic frequency 0.43) single nucleotide polymorphism (silent 1908C --> T substitution) in exon 10 of LMNA (GenBank X03444) was associated with reduced age-, sex- and percentage body fat-adjusted mean s.c. abd. AS [0.80 +/- 0.17 (CC), 0.76 +/- 0.15 (CT), 0.73 +/- 0.16 (TT) microg lipid/cell, P < 0.05 for CC vs TT]. These findings indicate that approximately half of the variance in mean s.c. abd. AS can be attributed to familial factors and that genetic variation in LMNA might not only underlie rare cases of FPLD, but may also contribute to variation in adipocyte size in the general population.

Genomic structure and expression of human KCNJ9 (Kir3.3/GIRK3).

The human KCNJ9 (Kir 3.3, GIRK3) is a member of the G-protein-activated inwardly rectifying potassium (GIRK) channel family. Here we describe the genomic organization of the KCNJ9 locus on chromosome 1q21-23 as a candidate gene for Type II diabetes mellitus in the Pima Indian population. The gene spans approximately 7.6 kb and contains one noncoding and two coding exons separated by approximately 2.2 and approximately 2.6 kb introns, respectively. We identified 14 single nucleotide polymorphisms (SNPs), including one that predicts a Val366Ala substitution, and an 8 base-pair (bp) insertion/deletion. Our expression studies revealed the presence of the transcript in various human tissues including pancreas, and two major insulin-responsive tissues: fat and skeletal muscle. The characterization of the KCNJ9 gene should facilitate further studies on the function of the KCNJ9 protein and allow evaluation of the potential role of the locus in Type II diabetes.

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

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.

Molecular characterization of the human PEA15 gene on 1q21-q22 and association with type 2 diabetes mellitus in Pima Indians.

The PEA15 gene encoding a protein kinase C substrate is widely expressed, and its overexpression may contribute to impairment of glucose uptake. PEA15 is located within a region on human 1q linked with type 2 diabetes in both Pima Indians and Caucasians. To assess the potential contribution of genetic alterations within this locus to disease susceptibility in the Pimas, we have investigated its genomic sequences. The PEA15 locus is composed of four exons spanning approximately 10.2kb of genomic DNA, flanked upstream by an potentially expressed Alu element, downstream by the H326 gene, and is located within 250kb of KCNJ9. We also sequenced over 2kb of the promoter region and identified various motifs analogous to known transcription factor binding sites. By analysis of 22 Pimas, including 13 diabetic subjects, we detected four single nucleotide polymorphisms (SNPs) in the non-coding regions of PEA15, including three frequent variants that were in allelic disequilibrium, and one variant found only in a single Pima. The three SNPs were not associated with type 2 diabetes mellitus in 50 affected and 50 control Pimas (p=0.12-0.17), and we conclude that mutations in this gene probably do not contribute significantly to disease susceptibility in this Native American tribe. However, knowledge of the genomic structure of PEA15 provides the basis for similar systematic examinations of this candidate locus in relation to type 2 diabetes and other metabolic disorders in other populations.

Genome-wide scan for CAG/CTG repeat expansions in Pimas with early onset of type 2 diabetes mellitus.

The expansion of polymorphic CAG/CTG repeats in specific genes causes several neurodegenerative disorders and in many instances the length of the disease-causing repeat correlates with the onset age and/or severity of symptoms. Type 2 diabetes mellitus has features in common with diseases resulting from trinucleotide repeat expansion, including a variable age of disease onset and penetrance. We have investigated whether CAG/CTG repeat expansion contributes to the genetic etiology of type 2 diabetes in the Pima Indians, a population with the highest reported prevalence of this disease. Using the Repeat Expansion Detection (RED) method, we determined the size range in nondiabetic Pimas to be between (CAG)20 and (CAG)130 (mean repeat length = 195 bp), which is significantly larger than the mean size reported in Caucasians (150 bp). We compared the distribution of CAG/CTG repeat lengths among 40 Pimas with an early onset of type 2 diabetes (<22 years) and 38 nondiabetic subjects (>55 years). A 240-bp CAG/CTG RED product was found more frequently in early onset diabetics relative to nondiabetic controls (26% vs 11%), whereas a 210-bp band was more prominent in unaffected subjects (29% vs 13%); however, these differences were not statistically significant. In one Pima kindred, we also identified large RED products (>/=360 bp) that displayed intergenerational instability among family members. However, these expansions were not associated with diabetes or any other clinical abnormalities in the carriers. We conclude that this unstable CAG/CTG repeat may represent a novel locus, consisting of large, but apparently nonpathogenic, unstable sequences.

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.

Polymorphism in the 3' untranslated region of MTG8 is associated with obesity in Pima Indian males.

Obesity has a genetic component and predisposes for the development of type 2 diabetes mellitus. One approach to identifying new candidate genes for obesity is to explore potential regulatory factors expressed in fat tissue that may play a role in adipocyte development or metabolic control. Because we found relatively abundant mRNA levels of the putative transcription factor MTG8 in human adipose tissue, a polymorphic microsatellite marker in the 3' untranslated region of this gene was genotyped in 281 Pima Indians, a population with one of the highest reported rates of obesity. We detected a male-specific association with age-adjusted percentage body fat (p = 0.0002), body mass index (p = 0.01), waist circumference (p = 0.008), and thigh circumference (p = 0.02). Comparative analysis of all 13 MTG8 exons in 30 Pimas did not reveal any genetic variants which could explain the association with obesity in males.

Uniparental disomy of the entire X chromosome in a female with Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a severe, progressive, X-linked muscle-wasting disorder with an incidence of approximately 1/3,500 male births. Females are also affected, in rare instances. The manifestation of mild to severe symptoms in female carriers of dystrophin mutations is often the result of the preferential inactivation of the X chromosome carrying the normal dystrophin gene. The severity of the symptoms is dependent on the proportion of cells that have inactivated the normal X chromosome. A skewed pattern of X inactivation is also responsible for the clinical manifestation of DMD in females carrying X;autosome translocations, which disrupt the dystrophin gene. DMD may also be observed in females with Turner syndrome (45,X), if the remaining X chromosome carries a DMD mutation. We report here the case of a karyotypically normal female affected with DMD as a result of homozygosity for a deletion of exon 50 of the dystrophin gene. PCR analysis of microsatellite markers spanning the length of the X chromosome demonstrated that homozygosity for the dystrophin gene mutation was caused by maternal isodisomy for the entire X chromosome. This finding demonstrates that uniparental isodisomy of the X chromosome is an additional mechanism for the expression of X-linked recessive disorders. The proband's clinical presentation is consistent with the absence of imprinted genes (i.e., genes that are selectively expressed based on the parent of origin) on the X chromosome.

Binding of sequence-specific proteins to the 3'-untranslated region of vasoactive intestinal peptide mRNA.

Multimeric AUUUA elements in an AU-rich 3'-untranslated region (3'-UTR) have been shown to confer message instability to numerous ephemeral transcripts through the formation of RNA-protein complexes. We show here that the 3'-UTR of VIP mRNA, which contains 3 AUUUA motifs in an AU-rich context, forms specific complexes with cytoplasmic proteins in a concentration-dependent, tissue-specific manner. We also demonstrate that an AU-rich segment of c-fos mRNA can successfully compete with VIP mRNA for binding with cytoplasmic proteins. These studies provide the first evidence for a mechanism by which VIP is post-transcriptionally regulated through specific sequences in its 3'-UTR.