Safety of Direct-Acting Antiviral Therapy Regarding Renal Function in Post-Liver Transplant Patients Infected with Hepatitis C Virus and a 100% 12-Week Sustained Virologic Response-A Single-Center Study.

BACKGROUND: Patients after liver transplantation (LT) with hepatitis C virus (HCV) infection often suffer from renal or hepatic impairment. Treating patients after LT with direct-acting antivirals (DAA) might result in decreasing renal function due to interaction of DAA and immunosuppressive therapy. In this single-center study we analyzed clinical parameters of 18 HCV-infected patients treated with DAA therapy after LT. METHODS: The primary end points were change of renal function (glomerular filtration rate) and sustained virologic response 12 weeks after therapy (SVR12). For secondary end points, we investigated the influence of DAA therapy on transaminases, bilirubin, international normalized ratio, noninvasive fibrosis measurement, and Model for End-Stage Liver Disease (MELD) score. RESULTS: Five out of 18 patients treated with DAA suffered from renal impairment stage 2, and 7 patients of renal impairment stage 3. Renal function at SVR12 was not influenced by preexisting renal impairment (P > .5), type of immunosuppressant (P > .5), or type of DAA regimen (P > .5). All patients reached SVR12. The levels of transaminases and bilirubin declined rapidly, as expected. Ten out of 18 patients already suffered from cirrhosis or liver fibrosis >F3 according to noninvasive measurement before initiation of treatment. Single-point acoustic radiation force impulse imaging improved in 9 patients (P = .012). In 7 patients, MELD score improved owing to the decrease of bilirubin levels. In 6 patients it worsened. CONCLUSIONS: DAA therapy in LT patients was effective and safe in this single-center real-life cohort. Renal function was not influenced by the administered drug combinations, even in patients with preexisting renal impairment.

The impact of genetic variants on BMI increase during childhood versus adulthood.

BACKGROUND: Genetic variants that predispose individuals to obesity may have differing influences during childhood versus adulthood, and additive effects of such variants are likely to occur. Our ongoing studies to identify genetic determinants of obesity in American Indians have identified 67 single-nucleotide polymorphisms (SNPs) that reproducibly associate with maximum lifetime non-diabetic body mass index (BMI). This study aimed to identify when, during the lifetime, these variants have their greatest impact on BMI increase. SUBJECTS/METHODS: A total of 5906 Native Americans of predominantly Pima Indian heritage with repeated measures of BMI between the ages of 5 and 45 years were included in this study. The association between each SNP with the rates of BMI increase during childhood (5-19 years) and adulthood (20-45 years) were assessed separately. The significant SNPs were used to calculate a cumulative allelic risk score (ARS) for childhood and adulthood, respectively, to assess the additive effect of these variants within each period of life. RESULTS: The majority of these SNPs (36 of 67) were associated with rate of BMI increase during childhood (P-value range: 0.00004-0.05), whereas only nine SNPs were associated with rate of BMI change during adulthood (P-value range: 0.002-0.02). These 36 SNPs associated with childhood BMI gain likely had a cumulative effect as a higher childhood-ARS associated with rate of BMI change (ß=0.032 kg m(-2) per year per risk allele, 95% confidence interval: 0.027-0.036, P<0.0001), such that at age 19 years, individuals with the highest number of risk alleles had a BMI of 10.2 kg m(-2) greater than subjects with the lowest number of risk alleles. CONCLUSIONS: Overall, our data indicates that genetic polymorphisms associated with lifetime BMI may influence the rate of BMI increase during different periods in the life course. The majority of these polymorphisms have a larger impact on BMI during childhood, providing further evidence that prevention of obesity will need to begin early in life.

Brain-derived neurotrophic factor in human subjects with function-altering melanocortin-4 receptor variants.

BACKGROUND: In rodents, hypothalamic brain-derived neurotrophic factor (BDNF) expression appears to be regulated by melanocortin-4 receptor (MC4R) activity. The impact of MC4R genetic variation on circulating BDNF in humans is unknown. OBJECTIVE: The objective of this study is to compare BDNF concentrations of subjects with loss-of-function (LOF) and gain-of-function (GOF) MC4R variants with those of controls with common sequence MC4R. METHODS: Circulating BDNF was measured in two cohorts with known MC4R sequence: 148 subjects of Pima Indian heritage ((mean±s.d.): age, 15.7±6.5 years; body mass index z-scores (BMI-Z), 1.63±1.03) and 69 subjects of Hispanic heritage (10.8±3.6 years; BMI-Z, 1.57±1.07). MC4R variants were characterized in vitro by cell surface expression, receptor binding and cyclic AMP response after agonist administration. BDNF single-nucleotide polymorphisms (SNPs) rs12291186, rs6265 and rs7124442 were also genotyped. RESULTS: In the Pima cohort, no significant differences in serum BDNF was observed for 43 LOF subjects versus 65 LOF-matched controls (age, sex and BMI matched; P=0.29) or 20 GOF subjects versus 20 GOF-matched controls (P=0.40). Serum BDNF was significantly associated with genotype for BDNF rs12291186 (P=0.006) and rs6265 (P=0.009), but not rs7124442 (P=0.99); BDNF SNPs did not interact with MC4R status to predict serum BDNF. In the Hispanic cohort, plasma BDNF was not significantly different among 21 LOF subjects, 20 GOF subjects and 28 controls (P=0.79); plasma BDNF was not predicted by BDNF genotype or BDNF-x-MC4R genotype interaction. CONCLUSIONS: Circulating BDNF concentrations were not significantly associated with MC4R functional status, suggesting that peripheral BDNF does not directly reflect hypothalamic BDNF secretion and/or that MC4R signaling is not a significant regulator of the bulk of BDNF expression in humans.

Human adipose microRNA-221 is upregulated in obesity and affects fat metabolism downstream of leptin and TNF-α.

AIMS/HYPOTHESIS: MicroRNAs (miRNAs) are short endogenous RNAs that regulate multiple biological processes including adipogenesis and fat metabolism. We sought to identify miRNAs that correlate with BMI and to elucidate their upstream regulation and downstream targets. METHODS: Microarray-based expression profiling of 233 miRNAs was performed on subcutaneous abdominal adipose tissue biopsies from 29 non-diabetic Pima Indian participants. Correlation of the expression levels of eight miRNAs with BMI was assessed by quantitative reverse transcription (QRT) PCR in adipose samples from 80 non-diabetic Pima Indians with a BMI of 21.6-54.0 kg/m(2). The upstream regulation of one of these miRNAs, miR-221, was tested by treating cultured human pre-adipocytes with leptin, TNF-α and insulin. Predicted targets of miR-221 were validated using QRT-PCR, immunoblots and luciferase assays. The downstream effects of miR-221 overexpression were assayed by proteomic analysis. RESULTS: Expression levels of miR-221 were positively correlated with BMI (particularly in women) and fasting insulin concentrations, while the levels of miR-193a-3p and miR-193b-5p were negatively correlated with BMI; other miRNAs did not show significant associations in the 80 samples. miR-221 was downregulated by leptin and TNF-α treatment in cultured human pre-adipocytes. Conversely, miR-221 overexpression upregulated several proteins involved in fat metabolism, mimicking peroxisome proliferator-activated receptor (PPAR) activation. Furthermore, miR-221 directly downregulated the adiponectin receptor 1 (ADIPOR1) and the transcription factor v-ets erythroblastosis virus E26 oncogene homolog 1 (ETS1). Adiponectin signalling is known to promote insulin sensitivity, and ETS1 is crucial for angiogenesis. CONCLUSIONS/INTERPRETATION: Our data suggest that miR-221 may contribute to the development of the insulin resistance that typically accompanies obesity, by affecting PPAR signalling pathways and by directly downregulating ADIPOR1 and ETS1.

HLA-DRB1 reduces the risk of type 2 diabetes mellitus by increased insulin secretion.

AIMS/HYPOTHESIS: We sought to identify the physiological implications of genetic variation at the HLA-DRB1 region in full-heritage Pima Indians in Arizona. METHODS: Single-nucleotide polymorphisms from the HLA region on chromosome 6p were tested for association with skeletal muscle mRNA expression of HLA-DRB1 and HLA-DRA, and with type 2 diabetes mellitus and prediabetic traits. RESULTS: The A allele at rs9268852, which tags HLA-DRB1 02(1602), was associated both with higher HLA-DRB1 mRNA expression (n = 133, p = 4.27 × 10(-14)) and decreased risk of type 2 diabetes (n = 3,265, OR 0.723, p = 0.002). Among persons with normal glucose tolerance (n = 266) this allele was associated with a higher mean acute insulin response during an intravenous glucose tolerance test (p = 0.005), higher mean 30 min insulin concentration during an oral glucose tolerance test (p = 0.017) and higher body fat percentage (p = 0.010). The polymorphism was not associated with HLA-DRA mRNA expression or insulin sensitivity. CONCLUSIONS/INTERPRETATION: HLA-DRB1*02 is protective for type 2 diabetes, probably by enhancing self tolerance, thereby protecting against the autoimmune-mediated reduction of insulin secretion.

Variants in ACAD10 are associated with type 2 diabetes, insulin resistance and lipid oxidation in Pima Indians.

AIMS/HYPOTHESIS: A prior genome-wide association study in Pima Indians identified a variant within the ACAD10 gene that is associated with early-onset type 2 diabetes. Acylcoenzyme A dehydrogenase 10 (ACAD10) catalyses mitochondrial fatty acid beta-oxidation, which plays a pivotal role in developing insulin resistance and type 2 diabetes. Therefore, ACAD10 was analysed as a positional and biological candidate for type 2 diabetes. METHODS: Twenty-three SNPs were genotyped in 1,500 Pima Indians to determine the linkage disequilibrium pattern across ACAD10. Association with type 2 diabetes was determined by genotyping four tag single nucleotide polymorphisms (SNPs) in a population-based sample of 3,501 full-heritage Pima Indians; two associated SNPs were further genotyped in a second population-based sample of 3,723 American Indians. Associations with quantitative traits were assessed in 415 non-diabetic full heritage Pima individuals who had been metabolically phenotyped. RESULTS: SNPs rs601663 and rs659964 were associated with type 2 diabetes in the full-heritage Pima Indian sample (p=0.04 and 0.0006, respectively), and rs659964 was further associated with type 2 diabetes in the second American Indian sample (p=0.04). Combination of these two samples provided the strongest evidence for association (p=0.009 and 0.00007, for rs601663 and rs659964, respectively). Quantitative trait analyses identified nominal associations with both lower lipid oxidation rate and larger subcutaneous abdominal adipocyte size, which is consistent with the known physiology of ACAD10, and also identified associations with increased insulin resistance. CONCLUSIONS/INTERPRETATION: We propose that ACAD10 variation may increase type 2 diabetes susceptibility by impairing insulin sensitivity via abnormal lipid oxidation.

Tackling the growth of the obesity literature: obesity evidence spreads across many journals.

OBJECTIVE: This study identified the journals with the highest yield of clinical obesity research articles and surveyed the scatter of such studies across journals. The study exemplifies an approach to establish a journal collection that is likely to contain most new knowledge about a field. DESIGN AND METHODS: All original studies that were cited in 40 systematic reviews about obesity topics ('included studies') were compiled and journal titles in which they were published were extracted. The journals were ranked by the number of included studies. The highest-yielding journals for clinical obesity and the scatter across journal titles were determined. A subset of these journals was created in MEDLINE (PubMed) to test search recall and precision for high-quality studies of obesity treatment (that is, articles that pass predetermined methodology criteria, including random allocation of participants to comparison groups, assessment of clinical outcomes, and at least 80% follow-up). RESULTS: Articles in 252 journals were cited in the systematic reviews. The three highest-yielding journals specialized in obesity, but they published only 19.2% of the research, leaving 80.8% scattered across 249 non-obesity journals. The MEDLINE journal subset comprised 241 journals (11 journals were not indexed in MEDLINE) and included 82% of the clinical obesity research articles retrieved by a search for high-quality treatment studies ('recall' of 82%). Of the articles retrieved, 11% were about clinical obesity care ('precision' of 11%), compared with precision of 6% for obesity treatment studies in the full MEDLINE database. CONCLUSION: Obesity journals captured only a small proportion of the literature on clinical obesity care. Those wishing to keep up in this field will need to develop more inclusive strategies than reading these specialty journals. A journal subset based on these findings may be useful when searching large electronic databases to increase search precision.

Protein tyrosine phosphatase 1B is not a major susceptibility gene for type 2 diabetes mellitus or obesity among Pima Indians.

AIM/HYPOTHESIS: Single-nucleotide polymorphisms (SNPs) in the protein tyrosine phosphatase 1B gene (PTPN1) have been reported to be associated with type 2 diabetes in white subjects, and insulin sensitivity and fasting glucose levels in Hispanic Americans. In this study, we determined whether SNPs in PTPN1 also have a role in type 2 diabetes susceptibility in Pima Indians, a population with the world's highest reported prevalence and incidence rates of this disease. MATERIALS AND METHODS: Thirty-one SNPs across a 161-kb region encompassing PTPN1 were genotyped in 1,037 Pima Indians for association studies with type 2 diabetes and obesity. RESULTS: Twenty-five of the SNPs had allele frequencies >0.05, and these SNPs fell into two linkage disequilibrium blocks (D' > 0.9). Block 1 contains six SNPs that span a 61-kb region upstream of PTPN1, while block 2 contains 19 SNPs that cover the entire PTPN1 gene. None of the SNPs, analysed individually or as haplotypes, was associated with either type 2 diabetes or obesity. However, three SNPs located in block 1 were nominally associated (p values ranging from 0.01 to 0.05) with insulin sensitivity as measured by the hyperinsulinaemic-euglycaemic clamp technique. CONCLUSIONS/INTERPRETATION: Based on our association results, we conclude that SNPs within PTPN1 are unlikely to have a major role in the aetiology of type 2 diabetes or obesity in Pima Indians.

Sex-specific effect of the Val1483Ile polymorphism in the fatty acid synthase gene (FAS) on body mass index and lipid profile in Caucasian children.

OBJECTIVE: A Val1483Ile polymorphism in the human fatty acid sythase gene (FAS) has recently been shown to be associated with lower percentage of body fat and substrate oxidation rates in Pima Indians, but its role in other populations has not been described. Here, we investigate the effect of this variant on obesity in Caucasian children and adolescents. SUBJECTS AND METHODS: In total, 738 Caucasian children and adolescents aged 6-17 years of the Leipzig Schoolchildren cohort, which constitutes an unselected representative German population and 205 obese children (body mass index (BMI) 2.71+/-0.04 SDS) were genotyped for genotype-phenotype associations. RESULTS: The frequency of the Ile-allele was lower in German Caucasians compared with Pima Indians (0.03 compared to 0.10). Using generalized linear regression models, there was no effect of the polymorphism on BMI in the whole normal population. However, we identified a significant interaction effect between sex and genotype (P=0.004). Subsequent sex stratified analyses revealed a lower BMI SDS in boys with Ile/Val genotype compared to Val/Val (-0.36+/-0.29 vs 0.09+/-0.05, P<0.05), while an opposite effect was observed in girls (0.48+/-0.19 vs 0.09+/-0.05, P<0.05). In genotype-phenotype associations in obese children, the polymorphism did not affect parameters of insulin, glucose, or lipid metabolism in the whole population. Again, however, obese boys with Ile/Val genotype had significantly higher high-density lipoprotein (HDL) cholesterol levels (1.46+/-0.07 vs 1.23+/-0.03 mmol/l, P<0.05). CONCLUSION: In conclusion, our findings suggest a sex-specific protective effect of the Val1483Ile polymorphism in FAS for obesity in Caucasian boys. In addition, the polymorphism may be associated with a beneficial lipid profile in obese boys.

Design and analysis of genetic association studies to finely map a locus identified by linkage analysis: sample size and power calculations.

Association (e.g. case-control) studies are often used to finely map loci identified by linkage analysis. We investigated the influence of various parameters on power and sample size requirements for such a study. Calculations were performed for various values of a high-risk functional allele (fA), frequency of a marker allele associated with the high risk allele (f1), degree of linkage disquilibrium between functional and marker alleles (D') and trait heritability attributable to the functional locus (h2). The calculations show that if cases and controls are selected from equal but opposite extreme quantiles of a quantitative trait, the primary determinants of power are h2 and the specific quantiles selected. For a dichotomous trait, power also depends on population prevalence. Power is optimal if functional alleles are studied (fA= f1 and D'= 1.0) and can decrease substantially as D' diverges from 1.0 or as f(1) diverges from fA. These analyses suggest that association studies to finely map loci are most powerful if potential functional polymorphisms are identified a priori or if markers are typed to maximize haplotypic diversity. In the absence of such information, expected minimum power at a given location for a given sample size can be calculated by specifying a range of potential frequencies for fA (e.g. 0.1-0.9) and determining power for all markers within the region with specification of the expected D' between the markers and the functional locus. This method is illustrated for a fine-mapping project with 662 single nucleotide polymorphisms in 24 Mb. Regions differed by marker density and allele frequencies. Thus, in some, power was near its theoretical maximum and little additional information is expected from additional markers, while in others, additional markers appear to be necessary. These methods may be useful in the analysis and interpretation of fine-mapping studies.

Genetic variation in UCP2 (uncoupling protein-2) is associated with energy metabolism in Pima Indians.

AIMS/HYPOTHESIS: Uncoupling protein-2 (UCP2) is thought to play a role in insulin secretion and the development of obesity. In this study, we investigated the effects of genetic variation in UCP2 on type 2 diabetes and obesity, as well as on metabolic phenotypes related to these diseases, in Pima Indians. METHODS: The coding and untranslated regions of UCP2, and approximately 1 kb of the 5' upstream region, were sequenced in DNA samples taken from 83 extremely obese Pima Indians who were not first-degree relatives. RESULTS: Five variants were identified: (1) a -866G/A in the 5' upstream region; (2) a G/A in exon 2; (3) a C/T resulting in an Ala55Val substitution in exon 4; and (4, 5) two insertion/deletions (ins/del; 45-bp and 3-bp) in the 3' untranslated region. Among the 83 subjects whose DNA was sequenced, the -866G/A was in complete genotypic concordance with the Ala55Val and the 3-bp ins/del polymorphism. The G/A polymorphism in exon 2 was extremely rare. To capture the common variation in this gene for association analyses, the -866G/A variant (as a representative of Ala55Val and the 3-bp ins/del polymorphism) and the 45-bp ins/del were also genotyped for 864 full-blooded Pima Indians. Neither of these variants was associated with type 2 diabetes or body mass index. However, in a subgroup of 185 subjects who had undergone detailed metabolic measurements, these variants were associated with 24-h energy expenditure as measured in a human metabolic chamber (p=0.007 for the 45-bp ins/del and p=0.03 for the -866G/A after adjusting for age, sex, family membership, fat-free mass and fat mass). CONCLUSIONS/INTERPRETATION: Our data indicate that variation in UCP2 may play a role in energy metabolism, but this gene does not contribute significantly to the aetiology of type 2 diabetes and/or obesity in Pima Indians.

Increased expression of inflammation-related genes in cultured preadipocytes/stromal vascular cells from obese compared with non-obese Pima Indians.

AIMS/HYPOTHESIS: The specific contributions made by the various cell types in adipose tissue to obesity, particularly obesity-related inflammation, need to be clarified. The aim of this study was to elucidate the potential role of adipocyte precursor cells (preadipocytes/stromal vascular cells [SVC]). METHODS: We performed Affymetrix oligonucleotide microarray expression profiling of cultured abdominal subcutaneous preadipocytes/SVC isolated from the adipose tissue of 14 non-obese (BMI 25+/-4 kg/m2) and 14 obese (55+/-8 kg/m2) non-diabetic Pima Indian subjects. Quantitative real-time PCR (RT-PCR) was used to verify the differential expression of several genes in an independent group of subjects. RESULTS: We identified 218 differentially expressed genes with p values less than 0.01. Microarray expression profiling revealed that the expression of inflammation-related genes was significantly upregulated in preadipocytes/SVC of obese individuals. Quantitative RT-PCR confirmed the upregulation of IL8, CTSS, ITGB2, HLA-DRA, CD53, PLA2G7 and MMP9 in preadipocytes/SVC of obese subjects. CONCLUSIONS/INTERPRETATION: The upregulation of inflammation-related genes in preadipocytes/SVC of obese subjects may increase the recruitment of immune cells into adipose tissue and may also result in changes in the extracellular matrix (tissue remodelling) to accommodate adipose tissue expansion in obesity.

11beta-Hydroxysteroid dehydrogenase Type 1: genetic polymorphisms are associated with Type 2 diabetes in Pima Indians independently of obesity and expression in adipocyte and muscle.

AIMS/HYPOTHESIS: The enzyme 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) modulates tissue-specific glucocorticoid concentrations by generating active cortisol. We have shown that adipose tissue 11beta-HSD1 mRNA levels were associated with adiposity and insulinaemia. Here we conducted further expression and genetic association studies in Pima Indians. METHODS: The 11beta-HSD1 mRNA concentrations were measured in abdominal subcutaneous adipocytes (n=61) and skeletal muscle tissues (n=64). Single nucleotide polymorphisms in the HSD11B1 gene were genotyped in a larger group of full-blooded Pima Indians. RESULTS: Two representative SNPs (SNP1, n=706; SNP5, n=839) were associated with Type 2 diabetes mellitus (p=0.01), although neither SNP was associated with obesity. Among subjects with normal glucose tolerance, SNP1 (n=127) and SNP5 (n=159) were associated with insulin-mediated glucose uptake rates (p=0.03 and p=0.04), and SNP1 was further associated with fasting, 30-min, and 2-h plasma insulin concentrations (p=0.002, p=0.002 and p=0.03). Adipocyte 11beta-HSD1 mRNA concentrations were correlated positively with adiposity and insulinaemia, and were additionally negatively correlated with insulin-mediated glucose uptake rates; nevertheless, the adipocyte 11beta-HSD1 expression did not correlate with genotypes of the donors. The muscle 11beta-HSD1 mRNA concentrations did not correlate with any anthropometric or metabolic variables. CONCLUSIONS/INTERPRETATION: We confirmed that adipocyte 11beta-HSD1 mRNA concentrations were associated with adiposity, and showed that genetic variations in the HSD11B1 gene were associated with Type 2 diabetes mellitus, plasma insulin concentrations and insulin action, independent of obesity. The variable adipose expression might not be a primary consequence of these HSD11B1 SNPs. Therefore, it is possible that the HSD11B1 gene is under tissue-specific regulation, and has tissue-specific consequences.

Variation in the FABP2 promoter affects gene expression: implications for prior association studies.

AIMS/HYPOTHESIS: An Ala54Thr polymorphism in the FABP2 gene has previously been associated with insulin resistance and lipid oxidation rates in Pima Indians. Ala54Thr functionally alters the protein's ability to bind and transport dietary fatty acids. In the current report, we sought additional functional variation in FABP2 by sequencing putative regulatory regions. METHODS: More than 1.2 Kb of the putative promoter of FABP2 was sequenced in 20 Pima subjects. Variations were genotyped in 84 additional Pima Indian subjects to assess haplotype combinations. Functional activities of variant and nonvariant promoters were compared in Caco-2 cells transfected with luciferase reporter constructs. RESULTS: Seven variations were identified in the FABP2 promoter in Pima Indians. Genotypes of these variants were in complete concordance with each other, and were in complete concordance with Ala54Thr. Therefore, only two promoter alleles were observed in Pima Indians, an Ala54-associated promoter and a Thr54-associated promoter. In contrast, genotyping of these variants in Caucasian DNA showed multiple genotypic combinations. In vitro reporter assays indicated that the Thr54-associated promoter in Pima Indians resulted in a threefold reduction in promoter activity as compared to Ala54-associated promoter. CONCLUSION/INTERPRETATION: Two functional variations exist in FABP2--the coding Ala54Thr and the variant promoter. In the Pima Indian population, but not the Caucasian population, these two functional variants are always carried on the same allele. Therefore, some of the in vivo phenotypic associations previously attributed to the Ala54Thr substitution, which alters binding characteristics of the protein, could instead be due to promoter variation, which alters expression levels.

Reduced skeletal muscle calpain-10 transcript level is due to a cumulative decrease in major isoforms.

The DNA polymorphism SNP-43 in the calpain-10 gene is associated with insulin resistance and reduced skeletal muscle transcript in Pima Indians. Alternative splicing generates transcript isoforms calpain-10a to -10h. We determined the contribution of calpain-10 mRNA isoforms to the decreased total skeletal muscle calpain-10 mRNA levels observed in the G/G homozygotes. The expression levels of the major isoforms, calpain-10a and -10f, were positively correlated with the total calpain-10 mRNA levels, indicating a cumulative effect.

A Ser311Cys mutation in the human dopamine receptor D2 gene is associated with reduced energy expenditure.

Brain dopaminergic pathways play a major role in the control of movement. Absence of the murine dopamine D2 receptor gene (drd2) produces bradykinesia and hypothermia. A Ser311Cys mutation of the human DRD2 produces a marked functional impairment of the receptor and is associated with higher BMI in some populations. We hypothesized that the Ser311Cys mutation of DRD2 may inhibit energy expenditure. Here we report that total energy expenditure (doubly labeled water) measured in 89 nondiabetic Pima Indians was 244 kcal/ day lower in homozygotes for the Cys311-encoding allele when compared with those heterozygous and homozygous for the Ser311-encoding allele (P = 0.056). The 24-h resting energy expenditure (respiratory chamber) measured in 320 nondiabetic Pimas was also 87 kcal/day lower in homozygotes for the Cys311-encoding allele when compared with those heterozygous and homozygous for the Ser311-encoding allele (P = 0.026). These findings are the first evidence that a genetic mutation is associated with reduced energy expenditure in humans. Because the impact of this mutation on human obesity is small, we suggest that either the energy deficit induced is not large enough to significantly influence body weight in this population and/or that the Cys311-encoding allele is also associated with reduced energy intake.

Effects of an Ala54Thr polymorphism in the intestinal fatty acid-binding protein on responses to dietary fat in humans.

A polymorphism in FABP2 that results in an alanine-to-threonine substitution at amino acid 54 of the intestinal fatty acid-binding protein (IFABP) is associated with insulin resistance in Pima Indians. In vitro, the threonine form (Thr54) has a higher binding affinity for long-chain fatty acids than does the alanine form (Ala54). We tested whether this polymorphism affected metabolic responses to dietary fat, in vivo. Eighteen healthy Pima Indians, half homozygous for the Thr54 form of IFABP and half homozygous for the Ala54 form, were studied. The groups were matched for sex, age, and body mass index. Plasma triglyceride, nonesterified fatty acid (NEFA), glucose, and insulin responses were measured after a mixed meal (35% of daily energy requirements, 50 g of fat) and after a high fat challenge (1362 kcal, 129 g of fat). NEFA concentrations were approximately 15% higher after the mixed meal and peaked earlier and were approximately 20% higher at 7 h in response to the high fat test meal in Thr54 homozygotes compared with Ala54 homozygotes. Insulin responses to the test meals tended to be higher in Thr54 homozygotes, but glucose and triglyceride responses were not different.The results of this study suggest that the Thr54 form of IFABP is associated with higher and prolonged NEFA responses to dietary fat in vivo. Higher NEFA concentrations may contribute to insulin resistance and hyperinsulinemia in individuals with this allele.

Association of dopamine D2 receptor polymorphisms Ser311Cys and TaqIA with obesity or type 2 diabetes mellitus in Pima Indians.

OBJECTIVES: To investigate whether (a) variants within the dopamine D2 receptor gene (DRD2) are associated with obesity and type 2 diabetes in Pima Indians, and (b) whether variation in this gene could be responsible for previously observed linkage to these phenotypes, at chromosome location 11q23-24, in this population. DESIGN: Two single nucleotide polymorphisms (SNPs), Ser311Cys and TaqIA, within the DRD2 gene were genotyped by allelic discrimination PCR in subjects who had provided evidence of linkage to diabetes and obesity in an autosome-wide scan. SUBJECTS: A total of 1,187 subjects were genotyped, including 947 full heritage Pima Indians (80%). Descriptive statistics for all subjects analyzed, for whom clinical data were available, were (mean+/-s.d.): age at time of last exam = 41 +/- 15 y; birth year=1950 +/- 14; age-sex-adjusted body mass index (BMI; adjusted to a mean age of 35y)=36 +/- 8kg/m2; male = 44%; diabetic = 57%. For full heritage Pimas only: age = 43 +/- 15 y; birth year = 1948 +/- 14; sex- age-adjusted BMI = 36 +/- 8 kg/m2; male = 43%; diabetic = 59%. RESULTS: Neither polymorphism was significantly associated with diabetes in full heritage Pimas. Individuals with a 'CG' genotype at the Ser311Cys SNP had a higher BMI than those with a 'CC' genotype (36.7 vs 35.5 kg/m2, P= 0.04). Linkage analysis of BMI, adjusted for either polymorphism, resulted in LOD scores that were similar to those obtained without adjustment. CONCLUSION: Heterozygotes at the Ser311Cys DRD2 polymorphism had a slightly higher BMI than homozygotes, however neither the Ser311Cys nor the TaqIA polymorphism accounted for the linkage with BMI on chromosome 11 in Pima Indians.

Genetic variation in the gene encoding calpain-10 is associated with type 2 diabetes mellitus.

Type 2 or non-insulin-dependent diabetes mellitus (NIDDM) is the most common form of diabetes worldwide, affecting approximately 4% of the world's adult population. It is multifactorial in origin with both genetic and environmental factors contributing to its development. A genome-wide screen for type 2 diabetes genes carried out in Mexican Americans localized a susceptibility gene, designated NIDDM1, to chromosome 2. Here we describe the positional cloning of a gene located in the NIDDM1 region that shows association with type 2 diabetes in Mexican Americans and a Northern European population from the Botnia region of Finland. This putative diabetes-susceptibility gene encodes a ubiquitously expressed member of the calpain-like cysteine protease family, calpain-10 (CAPN10). This finding suggests a novel pathway that may contribute to the development of type 2 diabetes.