Search

Purpose To investigate the effectiveness of phenotype-based search approaches using publicly available online databases. Methods We included consecutively solved cases from our exome database. For each case, the combination of Human Phenotype Ontology terms reported by the referring clinician was used to perform a search in three commonly used databases: OMIM (first 300 results), Phenolyzer (first 300 results), and Mendelian (all 100 results). Results One hundred cases were included (43 females; mean age: 10 years). The actual molecular diagnosis identified through exome sequencing was not included in the search results of any of the queried databases in 33% of cases. In 85% of cases it was not found within the top five search results. When included, its median rank was 61 (range: 1–295), 21 (1–270), and 29 (1–92) in OMIM, Phenolyzer and Mendelian, respectively. Conclusion This study demonstrates that, in most cases, phenotype-based search approaches using public online databases is ineffective in providing a probable diagnosis for Mendelian conditions. Genotype-first approach through molecular-guided diagnostics with backward phenotyping may be a more appropriate approach for these disorders, unless a specific diagnosis is considered a priori based on highly unique phenotypic features or a specific facial gestalt.

While the samples and data from the Pima Indians of the Gila River Indian Community have been included in many international HLA workshops and conferences and have been the focus of numerous population reports and the source of novel alleles at the classical HLA loci, they have not been studied for the non-classical loci. In order to expand our HLA-disease association studies, we typed over 300 whole genome sequences from full Pima heritage members, controlled for first degree relationship, and employed recently developed computer algorithms to resolve HLA alleles. Both classical-HLA-A, -B, and -C- and non-classical- HLA-E, -F, -G, -J, -L, -W, -Y, -DPA2, -DPB2, -DMA, -DMB, -DOA, -DRB2, -DRB9, TAP1- loci were typed at the 4-field level of resolution. We present allele and selected haplotype frequencies, test the genotype distributions for population structure, discuss the issues that are created for tests of Hardy-Weinberg equilibrium over the four sample spaces of high resolution HLA typing, and address the implications for the evolution of non-classical pseudogenes that are no longer expressed in a phenotype subject to natural selection.

PURPOSE Clinical data provided to genetic testing laboratories are frequently scarce. Our purpose was to evaluate clinical scenarios where phenotypic refinement in proband's family members might impact exome data interpretation. METHODS Of 614 exomes, 209 were diagnostic and included in this study. Phenotypic information was gathered by the variant interpretation team from genetic counseling letters and images. If a discrepancy between reported clinical findings and presumably disease-causing variant segregation was observed, referring clinicians were contacted for phenotypic clarification. RESULTS In 16/209 (7.7%) cases, phenotypic refinement was important due to (1) lack of cosegregation of disease-causing variant with the reported phenotype; (2) identification of different disorders with overlapping symptoms in the same family; (3) similar features in proband and family members, but molecular cause identified in proband only; and (4) previously unrecognized maternal condition causative of child's phenotype. As a result of phenotypic clarification, in 12/16 (75%) cases definition of affected versus unaffected status in one of the family members has changed, and in one case variant classification has changed. CONCLUSION Detailed description of phenotypes in family members including differences in clinical presentations, even if subtle, are important in exome interpretation and should be communicated to the variant interpretation team.

Applying exome sequencing to populations with unique genetic architecture has the potential to reveal novel genes and variants associated with traits and diseases. We sequenced and analyzed the exomes of 6,716 individuals from a Southwestern American Indian (SWAI) population with well-characterized metabolic traits. We found that the SWAI population has distinct allelic architecture compared to populations of European and East Asian ancestry, and there were many predicted loss-of-function (pLOF) and nonsynonymous variants that were highly enriched or private in the SWAI population. We used pLOF and nonsynonymous variants in the SWAI population to evaluate gene-burden associations of candidate genes from European genome-wide association studies (GWASs) for type 2 diabetes, body mass index, and four major plasma lipids. We found 19 significant gene-burden associations for 11 genes, providing additional evidence for prioritizing candidate effector genes of GWAS signals. Interestingly, these associations were mainly driven by pLOF and nonsynonymous variants that are unique or highly enriched in the SWAI population. Particularly, we found four pLOF or nonsynonymous variants in APOB, APOE, PCSK9, and TM6SF2 that are private or enriched in the SWAI population and associated with low-density lipoprotein (LDL) cholesterol levels. Their large estimated effects on LDL cholesterol levels suggest strong impacts on protein function and potential clinical implications of these variants in cardiovascular health. In summary, our study illustrates the utility and potential of exome sequencing in genetically unique populations, such as the SWAI population, to prioritize candidate effector genes within GWAS loci and to find additional variants in known disease genes with potential clinical impact.

The Pima Indians of the Gila River Indian Community in Arizona have participated in a long-range study of type 2 diabetes mellitus since 1965 and have been the subject of HLA typing and population studies since the early days of serological assays. These data have been in numerous HLA workshops and conferences and have been the source of at least five novel alleles at the classical HLA loci. In recent time nearly the entire study group was subject to next generation sequencing by whole genome or exome technologies, which has allowed us to HLA type over 3000 full heritage persons with recently developed computer algorithms. We present here the results for the classical HLA Loci: HLA-A, B, C, DRA, DRB1, DRB3, DRB4, DRB5, DPA1, DPB1, DQA1, and DQB1 to the third field of resolution for synonymous alleles and type the likely four field resolution alleles from the subset of whole genome sequences. Allele frequencies, and haplotype frequencies at up to five loci, are presented as well as measures of population structure and heterozygosity. We define a core set of HLA variation that approximates the distribution for the Paleo-Indians and impute nine-locus, 4-field haplotypes that are expected to be common in full heritage peoples.

Objective: To present a case of adult-onset familial diabetes mellitus in which a genetic etiology typical for neonatal diabetes was identified.Methods: We conducted whole-exome and Sanger sequencing, assessed clinical presentation and family history, and performed a literature review.Results: A 40-year-old, thin woman presented with a 10-year history of mildly elevated fasting glucose and A1C levels. Her mother had diabetes mellitus since her 40s and is on insulin, and her daughter presented with diabetes mellitus at age 21. The patient and her daughter underwent whole-exome sequencing and were found to have a mutation in the KCNJ11 gene, c.G685A, p.E229K. This mutation is known to be associated with neonatal presentation of diabetes mellitus, which neither of these family members had a history of. Given her known mutation and postprandial hyperglycemia, a trial of low-dose sulfonylurea was initiated in the daughter but failed due to hypoglycemia. She was found to have a CYP2C9 genotype associated with poor oral drug clearance.Conclusion:KCNJ11 mutation should be screened for in patients and families meeting criteria for maturity-onset diabetes of the young, even without evidence of neonatal onset in the family. Furthermore, even though sulfonylurea therapy is successful in the majority of patients with KCNJ11 mutations, there may be a role for other interacting environmental or genetic modifiers, such as CYP2C9 geno-type, that affect sulfonylurea metabolism. Those patients who have delayed onset of disease and milder courses may be less ideal candidates for this treatment.Abbreviations: BMI = body mass index;KATP = ATP-sensitive potassium channel;MODY = maturity-onset diabetes of the young;NDM = neonatal diabetes mellitus

PurposeMonogenic diabetes accounts for 1-2% of diabetes cases. It is often undiagnosed, which may lead to inappropriate treatment. This study was performed to estimate the prevalence of monogenic diabetes in a cohort of overweight/obese adolescents diagnosed with type 2 diabetes (T2D).MethodsSequencing using a custom monogenic diabetes gene panel was performed on a racially/ethnically diverse cohort of 488 overweight/obese adolescents with T2D in the Treatment Options for Type 2 Diabetes in Adolescents and Youth (TODAY) clinical trial. Associations between having a monogenic diabetes variant and clinical characteristics and time to treatment failure were analyzed.ResultsMore than 4% (22/488) had genetic variants causing monogenic diabetes (seven GCK, seven HNF4A, five HNF1A, two INS, and one KLF11). Patients with monogenic diabetes had a statistically, but not clinically, significant lower body mass index (BMI) z-score, lower fasting insulin, and higher fasting glucose. Most (6/7) patients with HNF4A variants rapidly failed TODAY treatment across study arms (hazard ratio = 5.03, P = 0.0002), while none with GCK variants failed treatment.ConclusionThe finding of 4.5% of patients with monogenic diabetes in an overweight/obese cohort of children and adolescents with T2D suggests that monogenic diabetes diagnosis should be considered in children and adolescents without diabetes-associated autoantibodies and maintained C-peptide, regardless of BMI, as it may direct appropriate clinical management.

Moderate alcohol consumption has been associated with a lower risk of coronary artery disease (CAD) in the general population but has not been well studied in US veterans. We obtained self-reported alcohol consumption from Million Veteran Program participants. Using electronic health records, CAD events were defined as 1 inpatient or 2 outpatient diagnosis codes for CAD, or 1 code for a coronary procedure. We excluded participants with prevalent CAD (n = 69,995) or incomplete alcohol information (n = 8,449). We used a Cox proportional hazards model to estimate hazard ratios and 95% confidence intervals for CAD, adjusting for age, gender, body mass index, race, smoking, education, and exercise. Among 156,728 participants, the mean age was 65.3 years (standard deviation = 12.1) and 91% were men. There were 6,153 CAD events during a mean follow-up of 2.9 years. Adjusted hazard ratios (95% confidence intervals) for CAD were 1.00 (reference), 1.02 (0.92 to 1.13), 0.83 (0.74 to 0.93), 0.77 (0.67 to 0.87), 0.71 (0.62 to 0.81), 0.62 (0.51 to 0.76), 0.58 (0.46 to 0.74), and 0.95 (0.85 to 1.06) for categories of never drinker; former drinker; current drinkers of ≤0.5 drink/day, >0.5 to 1 drink/day, >1 to 2 drinks/day, >2 to 3 drinks/day, and >3 to 4 drinks/day; and heavy drinkers (>4 drinks/day) or alcohol use disorder, respectively. For a fixed amount of ethanol, intake at ≥3 days/week was associated with lower CAD risk compared with ≤1 day/week. Beverage preference (beer, wine, or liquor) did not influence the alcohol-CAD relation. Our data show a lower risk of CAD with light-to-moderate alcohol consumption among US veterans, and drinking frequency may provide a further reduction in risk.

Large-scale human genetics studies are ascertaining increasing proportions of populations as they continue growing in both number and scale. As a result, the amount of cryptic relatedness within these study cohorts is growing rapidly and has significant implications on downstream analyses. We demonstrate this growth empirically among the first 92,455 exomes from the DiscovEHR cohort and, via a custom simulation framework we developed called SimProgeny, show that these measures are in-line with expectations given the underlying population and ascertainment approach. For example, we identified ∼66,000 close (first- and second-degree) relationships within DiscovEHR involving 55.6% of study participants. Our simulation results project that >70% of the cohort will be involved in these close relationships as DiscovEHR scales to 250,000 recruited individuals. We reconstructed 12,574 pedigrees using these relationships (including 2,192 nuclear families) and leveraged them for multiple applications. The pedigrees substantially improved the phasing accuracy of 20,947 rare, deleterious compound heterozygous mutations. Reconstructed nuclear families were critical for identifying 3,415 de novo mutations in ∼1,783 genes. Finally, we demonstrate the segregation of known and suspected disease-causing mutations through reconstructed pedigrees, including a tandem duplication in LDLR causing familial hypercholesterolemia. In summary, this work highlights the prevalence of cryptic relatedness expected among large healthcare population genomic studies and demonstrates several analyses that are uniquely enabled by large amounts of cryptic relatedness.

PurposeWe integrated whole-exome sequencing (WES) and chromosomal microarray analysis (CMA) into a clinical workflow to serve an endogamous, uninsured, agrarian community.MethodsSeventy-nine probands (newborn to 49.8 years) who presented between 1998 and 2015 remained undiagnosed after biochemical and molecular investigations. We generated WES data for probands and family members and vetted variants through rephenotyping, segregation analyses, and population studies.ResultsThe most common presentation was neurological disease (64%). Seven (9%) probands were diagnosed by CMA. Family WES data were informative for 37 (51%) of the 72 remaining individuals, yielding a specific genetic diagnosis (n = 32) or revealing a novel molecular etiology (n = 5). For five (7%) additional subjects, negative WES decreased the likelihood of genetic disease. Compared to trio analysis, "family" WES (average seven exomes per proband) reduced filtered candidate variants from 22 ± 6 to 5 ± 3 per proband. Nineteen (51%) alleles were de novo and 17 (46%) inherited; the latter added to a population-based diagnostic panel. We found actionable secondary variants in 21 (4.2%) of 502 subjects, all of whom opted to be informed.ConclusionCMA and family-based WES streamline and economize diagnosis of rare genetic disorders, accelerate novel gene discovery, and create new opportunities for community-based screening and prevention in underserved populations.

Context Sulfate is critical in the biotransformation of multiple compounds via sulfation. These compounds include neurotransmitters, proteoglycans, xenobiotics, and hormones such as dehydroepiandrosterone (DHEA). Sulfation reactions are thought to be rate-limited by endogenous sulfate concentrations. The gene, SLC13A1, encodes the sodium-sulfate cotransporter NaS1, responsible for sulfate (re)absorption in the intestines and kidneys. We previously reported two rare, non-linked, nonsense variants in SLC13A1 (R12X and W48X) associated with hyposulfatemia (P = 9 × 10− 20). Objective To examine the effect of serum sulfate concentration and sulfate-lowering genotype on DHEA homeostasis. Design Retrospective cohort study. Setting Academic research. Patients Participants of the Amish Pharmacogenomics of Anti-Platelet Intervention (PAPI) Study and the Amish Hereditary and Phenotype Intervention (HAPI) Study. Main outcome measures DHEA, DHEA-S, and DHEA-S/DHEA ratio. Results Increased serum sulfate was associated with decreased DHEA-S (P = 0.03) and DHEA-S/DHEA ratio (P = 0.06) in males but not females. Female SLC13A1 nonsense variant carriers, who had lower serum sulfate (P = 9 × 10− 13), exhibited 14% lower DHEA levels (P = 0.01) and 7% higher DHEA-S/DHEA ratios compared to female non-carriers (P = 0.002). Consistent with this finding, female SLC13A1 nonsense variant carriers also had lower total testosterone levels compared to non-carrier females (P = 0.03). Conclusions Our results demonstrate an inverse relationship between serum sulfate, and DHEA-S and DHEA-S/DHEA ratio in men, while also suggesting that the sulfate-lowering variants, SLC13A1 R12X and W48X, decrease DHEA and testosterone levels, and increase DHEA-S/DHEA ratio in women. While paradoxical, these results illustrate the complexity of the mechanisms involved in DHEA homeostasis and warrant additional studies to better understand sulfate's role in hormone physiology.

Summary Parent-of-origin (PoO) effects refer to the differential phenotypic impacts of genetic variants dependent on their parental inheritance due to imprinting. While PoO effects can influence complex traits, they may be poorly captured by models that do not differentiate the parental origin of the variant. The aim of this study was to conduct a genome-wide screen for PoO effects on a broad range of clinical traits derived from electronic health records (EHR) in the DiscovEHR study enriched with familial relationships. Using pairwise kinship estimates from genetic data and demographic data, we identified 22,051 offspring among 134,049 individuals in the DiscovEHR study. PoO of ~9 million variants was assigned in the offspring by comparing offspring and parental genotypes and haplotypes. We then performed genome-wide PoO association analyses across 154 quantitative and 611 binary traits extracted from EHR. Of the 732 significant PoO associations identified (p < 5 × 10−8), we attempted to replicate 274 PoO associations in the UK Biobank study with 5,015 offspring and replicated 9 PoO associations (p < 0.05). In summary, our study implements a bioinformatic and statistical approach to examine PoO effects genome-wide in a large population study enriched with familial relationships and systematically characterizes PoO effects on hundreds of clinical traits derived from EHR. Our results suggest that, while the statistical power to detect PoO effects remains modest yet, accurately modeling PoO effects has the potential to find new associations that may have been missed by the standard additive model, further enhancing the mechanistic understanding of genetic influence on complex traits., Genetic variants can have differential phenotypic effects based on parent-of-origin (PoO) due to imprinting. PoO of variants can be inferred by comparing parental and offspring genetic data. Genome-wide PoO-aware association testing reveals PoO effects across numerous clinical traits. PoO statistical models can identify novel associations undetected by standard additive models.

BackgroundGenetic factors that influence kidney traits have been understudied for low frequency and ancestry-specific variants.MethodsWe combined whole genome sequencing (WGS) data from 23,732 participants from 10 NHLBI Trans-Omics for Precision Medicine (TOPMed) Program multi-ethnic studies to identify novel loci for estimated glomerular filtration rate (eGFR). Participants included European, African, East Asian, and Hispanic ancestries. We applied linear mixed models using a genetic relationship matrix estimated from the WGS data and adjusted for age, sex, study, and ethnicity.FindingsWhen testing single variants, we identified three novel loci driven by low frequency variants more commonly observed in non-European ancestry (PRKAA2, rs180996919, minor allele frequency [MAF] 0.04%, P=6.1×10-11; METTL8, rs116951054, MAF 0.09%, P=4.5×10-9; and MATK, rs539182790, MAF 0.05%, P=3.4×10-9). We also replicated two known loci for common variants (rs2461702, MAF=0.49, P=1.2×10-9, nearest gene GATM, and rs71147340, MAF=0.34, P=3.3×10-9, CDK12). Testing aggregated variants within a gene identified the MAF gene. A statistical approach based on local ancestry helped to identify replication samples for ancestry-specific variants.InterpretationThis study highlights challenges in studying variants influencing kidney traits that are low frequency in populations and more common in non-European ancestry.

The triglyceride (TG) response to a high-fat meal (postprandial lipemia, PPL) affects cardiovascular disease risk and is influenced by genes and environment. Genes involved in lipid metabolism have dominated genetic studies of PPL TG response. We sought to elucidate common genetic variants through a genome-wide association (GWA) study in the Genetics of Lipid Lowering Drugs and Diet Network (GOLDN).The GOLDN GWAS discovery sample consisted of 872 participants within families of European ancestry. Genotypes for 2,543,887 variants were measured or imputed from HapMap. Replication of our top results was performed in the Heredity and Phenotype Intervention (HAPI) Heart Study (n = 843). PPL TG response phenotypes were constructed from plasma TG measured at baseline (fasting, 0 hour), 3.5 and 6 hours after a high-fat meal, using a random coefficient regression model. Association analyses were adjusted for covariates and principal components, as necessary, in a linear mixed model using the kinship matrix; additional models further adjusted for fasting TG were also performed. Meta-analysis of the discovery and replication studies (n = 1715) was performed on the top SNPs from GOLDN.GOLDN revealed 111 suggestive (p1E-05) associations, with two SNPs meeting GWA significance level (p5E-08). Of the two significant SNPs, rs964184 demonstrated evidence of replication (p = 1.20E-03) in the HAPI Heart Study and in a joint analysis, was GWA significant (p = 1.26E-09). Rs964184 has been associated with fasting lipids (TG and HDL) and is near ZPR1 (formerly ZNF259), close to the APOA1/C3/A4/A5 cluster. This association was attenuated upon additional adjustment for fasting TG.This is the first report of a genome-wide significant association with replication for a novel phenotype, namely PPL TG response. Future investigation into response phenotypes is warranted using pathway analyses, or newer genetic technologies such as metabolomics.

Objectives This study sought to identify loci for coronary artery calcification (CAC) in patients with chronic kidney disease (CKD). Background CKD is associated with increased CAC and subsequent coronary heart disease (CHD), but the mechanisms remain poorly defined. Genetic studies of CAC in CKD may provide a useful strategy for identifying novel pathways in CHD. Methods We performed a candidate gene study (∼2,100 genes; ∼50,000 single nucleotide polymorphisms [SNPs]) of CAC within the CRIC (Chronic Renal Insufficiency Cohort) study (N = 1,509; 57% European, 43% African ancestry). SNPs with preliminary evidence of association with CAC in CRIC were examined for association with CAC in the PennCAC (Penn Coronary Artery Calcification) (N = 2,560) and AFCS (Amish Family Calcification Study) (N = 784) samples. SNPs with suggestive replication were further analyzed for association with myocardial infarction (MI) in the PROMIS (Pakistan Risk of Myocardial Infarction Study) (N = 14,885). Results Of 268 SNPs reaching p −4 for CAC in CRIC, 28 SNPs in 23 loci had nominal support (p chr9p21 and COL4A1 , known loci for CHD, these included SNPs having reported genome-wide association study association with hypertension (e.g., ATP2B1 ). In PROMIS, 4 of the 23 suggestive CAC loci ( chr9p21 , COL4A1 , ATP2B1 , and ABCA4 ) had significant associations with MI, consistent with their direction of effect on CAC. Conclusions We identified several loci associated with CAC in CKD that also relate to MI in a general population sample. CKD imparts a high risk of CHD and may provide a useful setting for discovery of novel CHD genes and pathways.

We previously detected strong evidence for linkage of forearm bone mineral density (BMD) to chromosome 4p (lod = 4.3) in a set of 29 large Mexican American families. Fibroblast growth factor binding protein 1 (FGFBP1) is a strong candidate gene for bone homeostasis in this region. We sequenced the coding region of FGFBP1 in a subset of our Mexican American study population and performed association studies with BMD on SNPs genotyped in the entire cohort. We then attempted to replicate these findings in an independent study cohort and performed invitro functional studies on replicated, potentially functional polymorphisms using a luciferase reporter construct to evaluate influence on gene expression. Several SNPs spanning the gene, all in one large block of linkage disequilibrium, were significantly associated with BMD at various skeletal sites (n=872, p = 0.001-0.04). The associations were then replicated in an independent population of European ancestry (n = 972; p = 0.02-0.04). Sex-stratified association analyses in both study populations suggest this association is much stronger in men. Subsequent luciferase reporter gene assays revealed marked differences in FGFBP1 expression among the three common haplotypes. Further experiments revealed that a promoter polymorphism, rs12503796, results in decreased expression of FGFBP1 and inhibits upregulation of the gene by testosterone in vitro. Collectively, these findings suggest that sequence variation in FGFBP1 may contribute to variation in BMD, possibly influencing osteoporosis risk.

The loss-of-function hepatic cytochrome P450 (CYP) 2C19*2 allele has been associated with reduced clopidogrel active metabolite generation and higher ex vivo platelet reactivity to adenosine diphosphate. Independently, in post hoc analyses, CYP2C19*2 has been associated with worse clinical outcomes during clopidogrel therapy. The controversy surrounding the diminished effectiveness of clopidogrel in poor metabolizers, those having 2 loss-of-function alleles, has been recently highlighted in the “boxed warning” issued by the U.S. Food and Drug Administration. However, much of the variation in clopidogrel response is not explained by the CYP2C19*2 allele (the most frequent loss-of-function allele), and other factors, both genetic and nongenetic, are likely to be important contributors. High on-treatment platelet reactivity to adenosine diphosphate during clopidogrel therapy is a well-documented predictor of recurrent ischemic events in the percutaneous coronary intervention population. While platelet function is dynamic in individual patients because of the influence of variable external factors, the influence of the CYP2C19*2 allele is intrinsically constant. Thus, it may be reasonable to consider both genotyping and platelet function measurement to assess ischemic risk and to guide antiplatelet therapy. Prospective clinical trials to test new algorithms for optimal personalized antiplatelet therapy are needed to provide the evidence base required for the routine adoption of genotyping into clinical practice.

Background : A common functional missense mutation [Ala54Thr of the fatty acid-binding protein 2 gene (FABP2)] has previously been studied for associations with glucoregulation, postprandial lipemia, and lipid oxidation rates. However, most of those studies have not accounted for the interactive and potentially confounding effects of habitual physical activity and diet. Objective : We tested the hypothesis that, in sedentary nondiabetic subjects following a low-fat diet, Thr54 FABP2 carriers have lower glucoregulatory function, greater postprandial lipemia, and greater lipid oxidation rates than do their Ala54 FABP2-homozygous counterparts. Design : Men and women (n = 122) aged 50-75 y who were following a low-fat diet were genotyped and underwent oral-glucose-tolerance tests. A subgroup (n = 36) also underwent postprandial lipemia tests with lipid oxidation rate measurements. Results : Thr54 carriers were less likely to have normal glucose tolerance (P = 0.05) and had higher fasting glucose concentrations (P = 0.003) than did Ala54 homozygotes. In Thr54 carriers, the insulin sensitivity index was lower (P = 0.02), and the fasting insulin and the oral-glucose-tolerance test insulin area under the curve were higher (P = 0.05 and 0.03, respectively) than in Ala54 homozygotes. FABP2 genotype was not associated with fasting or postprandial lipemia test triacylglycerol or free fatty acids (P ≥0.22 for all), but postprandial lipid oxidation rates were higher (P = 0.01), which suggests that fat absorption is higher in Thr54 carriers than in Ala54 homozygotes. Conclusions : In sedentary nondiabetic persons following a low-fat diet, FABP2 Thr54 carriers have lower glucose tolerance and lower insulin action than do Ala54-homozygous persons. Furthermore, FABP Thr54 carriers have higher lipid oxidation rates, which may be the mechanism of glucoregulatory dysfunction.

Objective The vesicle-associated membrane protein-4 ( VAMP4 ) gene is an excellent type 2 diabetes (T2DM) positional candidate gene. It is located on chromosome 1q24-q25, a region of linkage to T2DM in the Amish and several other populations. VAMP4 is expressed in liver and skeletal muscle and participates in intracellular trafficking of secreted and membrane-associated proteins. Design and methods We sequenced VAMP4 in 20 Amish subjects. Polymorphisms in and around VAMP4 were genotyped in 65 Amish subjects with T2DM, 64 subjects with impaired glucose homeostasis (IGH), and 126 normal glucose tolerant controls, as well as in an expanded set of 749 participants of the Amish Family Diabetes Study for whom glucose and insulin levels during an oral glucose tolerance test (OGTT) and other quantitative traits related to diabetes were available. Case-control and quantitative trait association analyses were performed. Results We found three common non-coding intragenic polymorphisms: a 23bp insertion/deletion (I/D) in the 5′ untranslated region (UTR) in exon 1 at position 73127, and G35319T and C335296T single nucleotide polymorphisms (SNPs) in the 3′ UTR (NCBI Accession No. Z98751). The two 3′ UTR SNPs were in complete linkage disequilibrium (LD) and both were in strong LD with the exon 1 I/D polymorphism (|D′|=0.82). Similarly, three extragenic flanking SNPs (rs978985, rs203255, and rs1023479) showed moderate LD with the neighboring intragenic SNPs (|D′|=0.23–0.69). None of the SNPs individually nor any of the 2-, 3-, 4-, or 5-polymorphism haplotypes were associated with T2DM or IGH. The exon 1 I/D polymorphism was not associated with significant differences in mean fasting or stimulated glucose or insulin levels during an OGTT or other diabetes-related quantitative traits in the expanded set of 749 subjects. Conclusion Variation in VAMP4 does not significantly influence risk of T2DM or IGH in the Amish.

Elevated serum low density lipoprotein cholesterol (LDL-C) and triglyceride (TG) and decreased high density lipoprotein cholesterol (HDL-C) levels are established risk factors for cardiovascular disease (CVD). To identify quantitative trait loci influencing lipid levels, we conducted genome-wide linkage analyses of total serum cholesterol (TSC), HDL-C, ln-transformed TG (LNTG) and LDL-C levels in 612 individuals from 28 families of the Amish Family Diabetes Study (AFDS). Subjects were genotyped for 373 microsatellite markers covering all 22 autosomes and the X chromosome at an average density of 9.7 centimorgans. All lipid traits exhibited moderate estimated heritability (h2 +/- S.E.): TSC, 0.63 +/- 0.11; HDL-C, 0.54 +/- 0.08; LNTG, 0.37 +/- 0.08; LDL-C, 0.62 +/- 0.10. The highest logarithm of the odds (LOD) score observed was 2.47 (P = 0.0003), at 3p25 for LDL-C. LOD scores exceeding 2.0 (P0.001) were also observed at 2p23 (LOD = 2.17) and 19p13 (LOD = 2.23) for LDL-C, and at 11q23 (LOD = 2.03) for LNTG. Three additional regions exhibited LOD scores greater than 1.5, corresponding to a P-value of0.005. Many of the regions suggestively linked in this genome-wide scan contain genes encoding proteins with established roles in lipid metabolism, including apolipoproteins, peroxisome proliferater-activated receptor-gamma and the LDL receptor.

Obesity prevalence has increased markedly over the past few decades. The obesity pandemic has huge implications for public health and our society. Although multiple studies show that the genetic contribution to obesity is significant, our genes have not changed appreciably over this time period. It was hypothesized that natural selection favors genotypes that result in a thrifty metabolism because individuals who carry these genotypes would be more likely to survive times of nutrient scarcity and to pass these genotypes to successive generations. Now that most of the world has adopted an increasingly "obesigenic" lifestyle of excess caloric intake and decreased physical activity, these same genes contribute to obesity and poor health. With the exception of the rare mutations that cause severe morbid obesity, it seems that numerous genes, each with modest effect, contribute to an individual's predisposition toward the more common forms of obesity. Variants in several candidate genes have been identified: association analyses and functional studies show that they contribute to modest obesity and related phenotypes. More recently, insights regarding gene-gene interactions have begun to emerge. Genome-wide scans for obesity phenotypes have led to the identification of several chromosome regions that are likely to harbor obesity susceptibility genes. Because of the increasing number of genome scans, several regions of replication have emerged. Positional cloning of these genes will undoubtedly unveil new insights into the molecular and pathophysiologic mechanisms of energy homeostasis and obesity.

The Trp64Arg polymorphism in the [beta ] 3 -adrenoceptor gene has been associated with increased prevalence of obesity, type 2 diabetes, and low rates of energy expenditure, although these findings are not unanimous. It is currently unknown if the presence of the Trp64Arg gene variant impedes the loss of body weight in obese, postmenopausal women via a reducing effect on energy expenditure. The objective of this study was to compare body composition and energy expenditure in carriers and noncarriers of the Trp64Arg variant in the [beta ] 3 -adrenoceptor before and after weight loss. We measured body composition, total daily energy expenditure (TEE), resting metabolic rate (RMR), physical activity energy expenditure (PAEE), thermic effect of feeding (TEF), and respiratory quotient (RQ) in 34 obese, postmenopausal women (19 carriers and 15 noncarriers for the Trp64Arg variant) before and after a weight loss intervention. There were no differences in body composition or daily energy expenditure and its components between the 2 groups at baseline. There were significant reductions in body mass, body mass index (BMI), percent body fat, fat-free mass, and fat mass (main effect, all P [lt ] .0001) when analyzed with the 2 genotypes combined, but no significant differences between carriers and noncarriers with respect to change in these variables (group [times ] time interaction term, all P [gt ] .05). Total energy expenditure tended to be reduced (490 kJ [middot] d [minus ]1 , P = .13) in both groups following weight loss, but there was no significant group [times ] time interaction term ( P = .78), indicating no difference in the response of the 2 genotypes. There was a 9% reduction in RMR (611 kJ [middot] d [minus ]1 , P [lt ] .001) when both groups were considered together, but no significant group [times ] time interaction term ( P = .84), suggesting that both groups responded in a similar manner to the weight loss intervention. PAEE and the TEF were not different following weight loss (both P [gt ] .60). There was a trend for RQ to be reduced after weight loss ( P = .07), but there was no difference between carriers or noncarriers of the Trp64Arg variant ( P = .58). In summary, we found that obese postmenopausal women who carry the Trp64Arg variant in the [beta ] 3 -adrenoceptor had similar changes in body composition and energy expenditure to noncarriers of the variant in response to prolonged caloric restriction. These results suggest that the presence of the Trp64Arg variant in the [beta ] 3 -adrenoceptor should not be a hindrance to weight reduction.

This study determined whether sequence variations in genes related to glucose and insulin metabolism are associated with insulin sensitivity in postmenopausal women after accounting for habitual physical activity levels, body composition, and hormone-replacement therapy (HRT). Eighteen sedentary, 19 physically active, and 23 athletic postmenopausal white women underwent a frequently sampled intravenous glucose tolerance test to determine insulin sensitivity (SI) and dual-energy x-ray absorptiometry to determine body composition. After accounting for the effects of body composition, habitual physical activity levels, and HRT status, SI was 26% lower in subjects with the Thr54 fatty acid[ndash ]binding protein 2 (FABP2) allele compared with Ala54 homozygotes (4.3 [plusmn] 0.5 v 5.8 [plusmn] 0.6 [mu ]U [times ] 10[minus ]4/min/mL; P [lt ] .05). Angiotensin-converting enzyme genotype was not significantly associated with SI. There were no significant associations between Gln27Glu [beta ]2-adrenergic receptor or Pro12Ala peroxisome proliferator-activated receptor [gamma ] variants and glucose or insulin kinetic parameters. It was concluded that FABP2 genotype influences insulin sensitivity independent of body composition, habitual physical activity levels, and HRT status in postmenopausal white women.

Pancreatic regenerating protein (reg I) is expressed in acinar cells and is mitogenic to β- and ductal cells. Isolation of large amounts of endogenous reg I for in vivo and in vitro experiments has been difficult. The aim of this study was to develop a recombinant protein and determine its bioactivity on rat pancreatic derived cells. cDNA of the rat reg I coding region was created with unique BamHI flanking sequences using reverse transcriptase PCR. The coding region was then cloned into a bacterial expression vector in which expression is controlled by a T7 promoter. After transformation into the Escherichia coli strain B21(DE3) and induction by isopropyl-β- d -thiogalactopyranoside, a fusion protein of 24 kDa in size, named reg-PET, was noted in the bacterial lysate. This protein contained a polyhistidine and S-peptide sequence to facilitate isolation and identification, respectively. This protein was purified using affinity chromatography, and identity was confirmed with gel electrophoresis and Western analysis. The reg-PET protein was mitogenic to both ARIP and RIN cells, rat pancreatic ductal and β-cell lines, respectively. Antibodies raised to the protein reacted against rat reg I in pancreas. The purified recombinant reg I fusion protein, like endogenous reg I, is mitogenic to pancreatic derived cells. It is more potent than reg I isolated from pancreatic tissue. This protein can be isolated rapidly, easily, and with a high amount of purity.

Arguably, antiplatelet agents are the most important therapy we administer to stented patients. Antiplatelet agents are given to prevent the most dreaded event that often has catastrophic consequences, coronary thrombosis. It is well established that platelet reactivity to adenosine diphosphate (ADP

BACKGROUND & AIMS: Pancreatic thread proteins (PTPs) are acinar cell products and members of the regenerating gene (reg) family. reg expression increases during islet regeneration, is depressed during aging-related islet dysfunction, and may be important in beta-cell growth and maintenance. The aim of this study was to examine the genetic expression of reg in pancreatic-derived cells in vitro and the mitogenic effect of PTP/reg protein on these cells. METHODS: reg gene expression was measured by Northern analysis in three rat pancreatic cell lines: ARIP (ductal), AR42J (acinar), and RIN (beta-cell). PTP/reg protein was isolated from bovine and human pancreas. Cells were cultured with PTP/reg for 72 hours, and thymidine incorporation was measured. RESULTS: reg messenger RNA was detected in AR42J but not in ARIP or RIN. PTP/reg protein was mitogenic to RIN and ARIP in a dose- related fashion but not to AR42J. It was not mitogenic to cultured mature rat islets. CONCLUSIONS: reg messenger RNA is expressed in acinar but not in beta-cell or ductal pancreatic cell lines. PTP/reg protein was mitogenic to both beta-cell and ductal cell lines but not to mature, nondividing islets. This supports the hypothesis that PTP/reg protein is an acinar cell-derived mediator of beta-cell growth and may be involved in modulating the duct-to-islet axis. (Gastroenterology 1996 Apr;110(4):1208-14)

The Trp 64 Arg mutation in the beta 3-adrenergic receptor (beta 3AR) gene was investigated in 350 Japanese subjects. This mutation was not associated with non-insulin-dependent diabetes mellitus (NIDDM). In 191 subjects without NIDDM, body mass index (BMI) was significantly higher in subjects homozygous for this mutation than in those homozygous for the normal allele (24.7 +/- 1.4 vs 22.1 +/- 0.2 kg/m2, p = 0.009). Moreover, the frequency of the mutant allele in obese subjects (BMI > 26.4) was significantly higher than that in non-obese subjects (BMI < 22) (0.37 vs 0.15, p = 0.009). The presence of this mutation was also accompanied by significantly higher fasting (p = 0.000) and 2 hrs (p = 0.018) serum insulin levels during an oral glucose tolerance test. The beta 3AR may be one of the loci contributing to obesity and hyperinsulinemia/insulin resistance in Japanese subjects.

We have previously cloned cDNAs that correspond to two different nonallelic preproinsulin genes in the amphibian, Xenopus laevis (Shuldiner et al. , 1989, J. Biol. Chem. 264, 9428-9432). The coding regions of the two genes are very similar (i.e., 93% amino acid identity). While both preproinsulin genes are expressed coordinately in the adult pancreas, they are differentially expressed in prepancreatic embryos during neurulation (Shuldiner et al. , 1991, Proc. Natl. Acad. Sci. USA 88, 7679-7683). We now report the use of the polymerase chain reaction (PCR) to investigate the genomic structures of both Xenopus preproinsulin genes. First, the nucleotide sequence of a portion of the 5′ untranslated legion that was lacking in our cDNA clones was obtained using rapid amplification of cDNA ends (5′ RACE). Then, oligonucleotide primers were designed that flanked putative exon-intron splice boundaries, and intronic sequences in each of the two nonallelic genes were amplified. We determined that both Xenopus preproinsulin genes contain two introns, one interrupting the 5′ untranslated region, and the second interrupting the region encoding the C-peptide. The introns are similar in position, but of greater length than those reported in most other species. Interestingly, dideoxy sequence analysis of the PCR-amplified introns revealed that the exon-intron splice junctions are well conserved between the two nonallelic genes, but internal to these junctions, the respective introns diverge significantly from each other. Thus, ample time has elapsed since the duplication of these two genes for marked divergence to occur within the introns suggesting that these regions are not important for expression in the adult pancreas. From these studies, we predict that elucidation of the sequences of the 5′ flanking regions of the two nonallelic preproinsulin genes will reveal conserved regions that will be important for coordinate expression, as well as less conserved regions that will either be unimportant for coordinate expression (i.e., pancreatic expression) or important for differential expression (i.e., prepancreatic expression).

Using the polymerase chain reaction (PCR), we have amplified and characterized partial nucleotide sequences of two distinct insulin-like growth factor-I genes (designated IGF-I′ and IGF-I″) from the amphibian, Xenopus laevis . The amplified fragments encoded much of the coding region of the mature peptide (exon III in mammalian IGF-I genes), and exhibited 93% similarity to each other, and 68–82% similarity to mammalian IGF-I amino acid sequences. Southern blot analysis using genomic DNA from a homozygous frog revealed that these two genes are nonallelic in a single organism, like the two nonallelic genes encoding Xenopus insulins that we have characterized previously. Furthermore, both IGF-I mRNAs are expressed in similar quantities in adult liver.

Background Complementary and alternative medicine (CAM) in the form of vitamin and supplement use is increasingly prevalent in the United States. The interplay between CAM use and use of conventional medications is not well studied. We examined this issue in Old Order Amish (OOA), a population lacking several factors known to influence supplement use, whose culture and barriers to conventional medications may result in high rates of supplement use. Objective We characterized the patterns of supplement use in OOA, including the extent to which CAM use aggregates in families, and assessed whether higher use of supplements is associated with lower medication use. Design We conducted a cross-sectional study of conventional medications and supplements in 2,372 adult Amish from the Lancaster County, PA, area. Data were collected through face-to-face interviews. Supplements were subcategorized as herbal vs vitamin/mineral supplements. Results Seventy-seven percent of all Amish adults reported current supplement use, whereas 22% reported medication use. Women used supplements more often and used more supplements than men, and familial aggregation of supplement use was stronger in family pairs involving women. Supplement use was associated with less medication use after controlling for age, sex, body mass index, and self-reported histories of hypertension, diabetes, and hyperlipidemia (adjusted odds ratio [OR] 0.96, 95% CI 0.92 to 1.00; P =0.047). This association was driven primarily by use of herbal supplements (adjusted OR 0.94, 95% CI 0.89 to 0.99; P =0.025) as vitamin/mineral supplements were not associated with different use of medication (adjusted OR 0.99, 95% CI 0.90 to 1.09; P =0.8). In analyses limited to cardiovascular medications and cardiovascular supplements in participants with hyperlipidemia, hypertension, or diabetes, supplement use was not associated with conventional medication use. Conclusions OAA, particularly women, take dietary supplements much more frequently than they use conventional medications. Use of herbal supplements is associated with less use of conventional medications, whereas vitamin/mineral supplement use is not.

In rodents, administration of leptin promotes beta3-adrenergic stimulation of thermogenesis in brown adipose tissue. Conversely, administration of a beta3-adrenoceptor (beta3-AR) agonist decreases leptin mRNA expression and secretion, suggesting that leptin and sympathetic nervous system activity mediated through the beta3-AR comprise a negative-feedback loop. It has recently been proposed that a defect in the beta3-AR in humans may contribute to a resistance to the sympathetically mediated effects of leptin on thermogenesis and lipolysis, thus leading to obesity and type 2 diabetes mellitus. We thus hypothesized that the Trp64Arg variant in the human beta3-AR would be associated with elevated plasma leptin concentrations. We studied 101 healthy nondiabetic Pima Indians: 11 Arg64 homozygotes, 35 Trp64 homozygotes, and 55 heterozygotes. The fasting plasma leptin concentration as an absolute value or after adjustment for percent body fat and sex was not associated with the beta3-AR genotype. Thus, the data do not support an influence of the Trp64Arg variant on the plasma leptin concentration.

We undertook the cloning of preproinsulin cDNAs from the South African clawed toad, Xenopus laevis, in order to study the role of insulin during embryogenesis in this species. We found that X. laevis contains two different preproinsulin cDNAs, both of which code for peptides containing 106 amino acids of typical structure but which differ by eight amino acids: one in the signal peptide, two in the B-chain, four in the C-peptide, and one in the A-chain. Southern blot analysis indicates that the two preproinsulin cDNAs identified correspond to two different nonallelic genes which we believe arose through a recent gene duplication within the amphibian radiation possibly during the development of tetraploidy in this species. Both genes are expressed, since we have recently identified the two corresponding insulins in pancreatic extracts of adult toads (Shuldiner, A.R., Bennett, C., Robinson, E.A., and Roth, J. (1989) Endocrinology, in press). These cDNAs represent the first amphibian preproinsulin sequences to be elucidated.