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Insulin resistance causes multiple epidemic metabolic diseases, including type 2 diabetes, cardiovascular disease, and fatty liver, but is not routinely measured in epidemiological studies. To discover novel insulin resistance genes in the general population, we conducted genome-wide association studies in 382,129 individuals for triglyceride to HDL-cholesterol ratio (TG/HDL), a surrogate marker of insulin resistance calculable from commonly measured serum lipid profiles. We identified 251 independent loci, of which 62 were more strongly associated with TG/HDL compared to TG or HDL alone, suggesting them as insulin resistance loci. Candidate causal genes at these loci were prioritized by fine mapping with directions-of-effect and tissue specificity annotated through analysis of protein coding and expression quantitative trait variation. Directions-of-effect were corroborated in an independent cohort of individuals with directly measured insulin resistance. We highlight two phospholipase encoding genes, PLA2G12A and PLA2G6, which liberate arachidonic acid and improve insulin sensitivity, and VGLL3, a transcriptional co-factor that increases insulin resistance partially through enhanced adiposity. Finally, we implicate the anti-apoptotic gene TNFAIP8 as a sex-dimorphic insulin resistance factor, which acts by increasing visceral adiposity, specifically in females. In summary, our study identifies several candidate modulators of insulin resistance that have the potential to serve as biomarkers and pharmacological targets., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Loss-of-function mutations in hepatocyte nuclear factor 1A (HNF1A) are known to cause rare forms of diabetes and alter hepatic physiology through unclear mechanisms. In the general population, 1:100 individuals carry a rare, protein-coding HNF1A variant, most of unknown functional consequence. To characterize the full allelic series, we performed deep mutational scanning of 11,970 protein-coding HNF1A variants in human hepatocytes and clinical correlation with 553,246 exome-sequenced individuals. Surprisingly, we found that ∼1:5 rare protein-coding HNF1A variants in the general population cause molecular gain of function (GOF), increasing the transcriptional activity of HNF1A by up to 50% and conferring protection from type 2 diabetes (odds ratio [OR] = 0.77, p = 0.007). Increased hepatic expression of HNF1A promoted a pro-atherogenic serum profile mediated in part by enhanced transcription of risk genes including ANGPTL3 and PCSK9 . In summary, ∼1:300 individuals carry a GOF variant in HNF1A that protects carriers from diabetes but enhances hepatic secretion of atherogenic lipoproteins., Competing Interests: A.M.D. and L.K. are employees and shareholders of Alnylam Pharmaceuticals.

Adipose tissue plays a crucial role in maintaining metabolic homeostasis by storing lipids and glucose from circulation as intracellular fat. As peripheral tissues like adipose tissue become insulin resistant, decompensation of blood glucose levels occurs causing type 2 diabetes (T2D). Currently, modulating the glycocalyx, a layer of cell-surface glycans, is an underexplored pharmacological treatment strategy to improve glucose homeostasis in T2D patients. Here, we show a novel role for cell-surface heparan sulfate (HS) in establishing glucose uptake capacity and metabolic utilization in differentiated adipocytes. Using a combination of chemical and genetic interventions, we identified that HS modulates this metabolic phenotype by attenuating levels of Wnt signaling during adipogenesis. By engineering, the glycocalyx of pre-adipocytes with exogenous synthetic HS mimetics, we were able to enhance glucose clearance capacity after differentiation through modulation of Wnt ligand availability. These findings establish the cellular glycocalyx as a possible new target for therapeutic intervention in T2D patients by enhancing glucose clearance capacity independent of insulin secretion., Competing Interests: Conflict of interest P. L. S. M. G is a co-founder of Covicept Therapeutics. P. L. S. M. G and The Regents of the University of California have licensed a university invention to and have an equity interest in Covicept Therapeutics. The terms of this arrangement have been reviewed and approved by the University of California, San Diego, in accordance with its conflict-of-interest policies., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Continuous glucose monitoring (CGM) is approved for insulin dosing decisions in the ambulatory setting, but not currently for inpatients. CGM has the capacity to reduce patient-provider contact in inpatients with coronavirus disease 2019 (COVID-19), thus potentially reducing in hospital virus transmission. However, there are sparse data on the accuracy and efficacy of CGM to titrate insulin doses in inpatients., Methods: Under an emergency use protocol, CGM (Dexcom G6) was used alongside standard point-of-care (POC) glucose measurements in patients critically ill from complications of COVID-19 requiring intravenous (IV) insulin. Glycemic control during IV insulin therapy was retrospectively assessed comparing periods with and without adjunctive CGM use. Accuracy metrics were computed and Clarke Error Grid analysis performed comparing CGM glucose values with POC measurements., Results: Twenty-four critically ill patients who met criteria for emergency use of CGM resulted in 47 333 CGM and 5677 POC glucose values. During IV insulin therapy, individuals' glycemic control improved when CGM was used (mean difference -30.7 mg/dL). Among 2194 matched CGM: POC glucose pairs, a high degree of concordance was observed with a mean absolute relative difference of 14.8% and 99.5% of CGM: POC pairs falling in Zones A and B of the Clarke Error Grid., Conclusions: Continuous glucose monitoring use in critically ill COVID-19 patients improved glycemic control during IV insulin therapy. Continuous glucose monitoring glucose data were highly concordant with POC glucose during IV insulin therapy in critically ill patients suggesting that CGM could substitute for POC measurements in inpatients thus reducing patient-provider contact and mitigating infection transmission.

The seven-transmembrane superfamily member 3 protein (TM7SF3) is a p53-regulated homeostatic factor that attenuates cellular stress and the unfolded protein response. Here we show that TM7SF3 localizes to nuclear speckles; eukaryotic nuclear bodies enriched in splicing factors. This unexpected location for a trans -membranal protein enables formation of stable complexes between TM7SF3 and pre-mRNA splicing factors including DHX15, LARP7, HNRNPU, RBM14, and HNRNPK. Indeed, TM7SF3 regulates alternative splicing of >330 genes, mainly at the 3'end of introns by directly modulating the activity of splicing factors such as HNRNPK. These effects are observed both in cell lines and primary human pancreatic islets. Accordingly, silencing of TM7SF3 results in differential expression of 1465 genes (about 7% of the human genome); with 844 and 621 genes being up- or down-regulated, respectively. Our findings implicate TM7SF3, as a resident protein of nuclear speckles and suggest a role for seven-transmembrane proteins as regulators of alternative splicing., Competing Interests: All authors declare no conflicts of interest., (© 2022 The Authors.)

Purpose of Review: Type 2 diabetes (T2D) is a multifactorial, heritable syndrome characterized by dysregulated glucose homeostasis that results from impaired insulin secretion and insulin resistance. Genetic association studies have successfully identified hundreds of T2D risk loci implicating many genes in disease pathogenesis. In this review, we provide an overview of the recent T2D genetic studies from the past 3 years with particular focus on the effects of sample size and ancestral diversity on genetic discovery as well as discuss recent work on the use and limitations of genetic risk scores (GRS) for T2D risk prediction., Recent Findings: Recent large-scale, multi-ancestry genetic studies of T2D have identified over 500 novel risk loci. The genetic variants (i.e., single nucleotide polymorphisms (SNPs)) marking these novel loci in general have smaller effect sizes than previously discovered loci. Inclusion of samples from diverse ancestral backgrounds shows a few ancestry specific loci marked by common variants, but overall, the majority of loci discovered are common across ancestries. Inclusion of common variant GRS, even with hundreds of loci, does not substantially increase T2D risk prediction over standard clinical risk factors such as age and family history. Common variant association studies of T2D have now identified over 700 T2D risk loci, half of which have been discovered in the past 3 years. These recent studies demonstrate that inclusion of ancestrally diverse samples can enhance locus discovery and improve accuracy of GRS for T2D risk prediction. GRS based on common variants, however, only minimally enhances risk prediction over standard clinical risk factors., (© 2022. This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply.)

Background: The Onduo virtual care program for people with type 2 diabetes (T2D) includes a mobile app, remote lifestyle coaching, connected devices, and telemedicine consultations with endocrinologists for medication management and prescription of real-time continuous glucose monitoring (RT-CGM) devices. In a previously described 4-month prospective study of this program, adults with T2D and baseline glycated hemoglobin (HbA 1c ) ≥8.0% to ≤12.0% experienced a mean HbA 1c decrease of 1.6% with no significant increase in hypoglycemia., Objective: The objective of this analysis was to evaluate medication optimization and management in the 4-month prospective T2D study., Methods: Study participants received at least 1 telemedicine consultation with an Onduo endocrinologist for diabetes medication management and used RT-CGM intermittently to guide therapy and dosing. Medication changes were analyzed., Results: Of 55 participants, 48 (87%) had a medication change consisting of a dose change, addition, or discontinuation. Of these, 15 (31%) participants had a net increase in number of diabetes medication classes from baseline. Mean time to first medication change for these participants was 36 days. The percentage of participants taking a glucagon-like peptide-1 receptor agonist increased from 25% (12/48) to 56% (n=27), while the percentages of participants taking a sulfonylurea or dipeptidyl peptidase 4 inhibitor decreased from 56% (n=27) to 33% (n=16) and 17% (n=8) to 6% (n=3), respectively. Prescriptions of other antidiabetic medication classes including insulin did not change significantly., Conclusions: The Onduo virtual care program can play an important role in providing timely access to guideline-based diabetes management medications and technologies for people with T2D., Trial Registration: ClinicalTrials.gov NCT03865381; https://clinicaltrials.gov/ct2/show/NCT03865381., (©Amit R Majithia, David M Erani, Coco M Kusiak, Jennifer E Layne, Amy Armento Lee, Francis R Colangelo, Robert J Romanelli, Scott Robertson, Shayla M Brown, Ronald F Dixon, Howard Zisser. Originally published in JMIR Formative Research (https://formative.jmir.org), 05.04.2022.)

Non-alcoholic fatty liver disease (NAFLD) is a continuous progression of pathophysiologic stages that is challenging to diagnose due to its inherent heterogeneity and poor standardization across a wide variety of diagnostic measures. NAFLD is heritable, and several loci have been robustly associated with various stages of disease. In the past few years, larger genetic association studies using new methodology have identified novel genes associated with NAFLD, some of which have shown therapeutic promise. This mini-review provides an overview of the heterogeneity in NAFLD phenotypes and diagnostic methods, discusses genetic associations in relation to the specific stages for which they were identified, and offers a perspective on the design of future genetic mapping studies to accelerate therapeutic target identification., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Du, DeForest and Majithia.)

The Onduo Virtual Diabetes Clinic (VDC) for people with type 2 diabetes (T2D) combines a mobile app, remote lifestyle coaching, connected devices, and live video consultations with board-certified endocrinologists. Adults with T2D ( n  = 594) who were evaluated by a VDC endocrinologist, remotely prescribed and mailed a real-time continuous glucose monitoring (rtCGM) device and used ≥1 sensor completed a CGM satisfaction questionnaire. The CGM satisfaction score was 4.5 ± 0.8 out of 5. Most respondents (94.7%) agreed/strongly agreed that they were comfortable inserting the sensor remotely and that rtCGM use improved understanding of the impact of eating (97.0%), increased diabetes knowledge (95.7%), and helped improve diabetes control when not wearing the sensor (79.4%). HbA1c ( n  = 372) decreased from 7.7% ± 1.6% to 7.1% ± 1.2% ( P  < 0.001; 10.2 months). These data suggest that it is feasible to provide rtCGM directly to individuals with T2D through a VDC without in-office training. Intermittent use of rtCGM was well-received by adults with T2D and was associated with improvement in HbA1c.

The Onduo Virtual Diabetes Clinic (VDC) telehealth technology/care model for adults with type 2 diabetes (T2D) combines connected devices, remote lifestyle coaching, and clinical support with a mobile App. Key differentiating program features are the availability of live video consultations with board-certified endocrinologists for medication management and real-time continuous glucose monitor use for higher-risk participants. Preliminary data ( n = 740) suggest that participation was associated with a significant improvement in HbA1c with up to 6 months follow-up in those not meeting treatment targets. HbA1c decreased by 2.3% ± 1.9%, 0.7% ± 1.0%, and 0.2% ± 0.8% across baseline categories of >9.0%, 8.0%-9.0% and 7.0% to <8.0%, respectively (all P < .001). These findings suggest that the VDC has potential to support individuals with T2D and their clinicians in diabetes management between office visits.

Background: The Onduo virtual diabetes clinic (VDC) for people with type 2 diabetes (T2D) combines a mobile app, remote personalized lifestyle coaching, connected devices, and live video consultations with board-certified endocrinologists for medication management and prescription of real-time continuous glucose monitoring (RT-CGM) devices for intermittent use., Objective: This prospective single-arm study evaluated glycemic outcomes associated with participation in the Onduo VDC for 4 months., Methods: Adults aged ≥18 years with T2D and a baseline glycated hemoglobin (HbA1c) of ≥8% to ≤12% were enrolled from 2 primary care centers from February 2019 to October 2019. Participants were asked to engage at ≥1 time per week with their care team and to participate in a telemedicine consultation with a clinic endocrinologist for diabetes medication review. Participants were asked to use a RT-CGM device and wear six 10-day sensors (total 60 days of sensor wear) intermittently over the course of 4 months. The primary outcome was change in HbA1c at 4 months from baseline. Other endpoints included change in weight and in RT-CGM glycemic metrics, including percent time <70, 70-180, 181-250, and >250 mg/dL. Changes in blood pressure and serum lipids at 4 months were also evaluated., Results: Participants (n=55) were 57.3 (SD 11.6) years of age, body mass index 33.7 (SD 7.2), and 40% (22/55) female. HbA1c decreased significantly by 1.6% (SD 1%; P<.001). When stratified by baseline HbA1c of 8.0% to 9.0% (n=36) and >9.0% (n=19), HbA1c decreased by 1.2% (SD 0.6%; P<.001) and 2.4% (SD 1.3%; P<.001), respectively. Continuous glucose monitoring-measured (n=43) percent time in range (TIR) 70-180 mg/dL increased by 10.2% (SD 20.5%; P=.002), from 65.4% (SD 23.2%) to 75.5% (SD 22.7%), which was equivalent to a mean increase of 2.4 hours TIR per day. Percent time 181-250 mg/dL and >250 mg/dL decreased by 7.2% (SD 15.4; P=.005) and 3.0% (SD 9.4; P=.01), respectively. There was no change in percent time <70 mg/dL. Mean weight decreased by 9.0 lb (SD 10.4; P<.001). Significant improvements were also observed in systolic blood pressure, total cholesterol, low-density lipoprotein cholesterol, and triglycerides (P=.04 to P=<.001)., Conclusions: Participants in the Onduo VDC experienced significant improvement in HbA1c, increased TIR, decreased time in hyperglycemia, and no increase in hypoglycemia at 4 months. Improvements in other metabolic health parameters including weight and blood pressure were also observed. In conclusion, the Onduo VDC has potential to support people with T2D and their clinicians between office visits by increasing access to specialty care and advanced diabetes technology including RT-CGM., Trial Registration: ClinicalTrials.gov NCT03865381; https://clinicaltrials.gov/ct2/show/NCT03865381., (©Amit R Majithia, Coco M Kusiak, Amy Armento Lee, Francis R Colangelo, Robert J Romanelli, Scott Robertson, David P Miller, David M Erani, Jennifer E Layne, Ronald F Dixon, Howard Zisser. Originally published in the Journal of Medical Internet Research (http://www.jmir.org), 28.08.2020.)

The UK Biobank is an unprecedented resource for human disease research. In March 2019, 49,997 exomes were made publicly available to investigators. Here we note that thousands of variant calls are unexpectedly absent from this dataset, with 641 genes showing zero variation. We show that the reason for this was an erroneous read alignment to the GRCh38 reference. The missing variants can be recovered by modifying read alignment parameters to correctly handle the expanded set of contigs available in the human genome reference. Given the size and complexity of such population scale datasets, we propose a simple heuristic that can uncover systematic errors using summary data accessible to most investigators., (© 2020 John Wiley & Sons Ltd/University College London.)

Background: Classifying pathogenicity of missense variants represents a major challenge in clinical practice during the diagnoses of rare and genetic heterogeneous neurodevelopmental disorders (NDDs). While orthologous gene conservation is commonly employed in variant annotation, approximately 80% of known disease-associated genes belong to gene families. The use of gene family information for disease gene discovery and variant interpretation has not yet been investigated on a genome-wide scale. We empirically evaluate whether paralog-conserved or non-conserved sites in human gene families are important in NDDs., Methods: Gene family information was collected from Ensembl. Paralog-conserved sites were defined based on paralog sequence alignments; 10,068 NDD patients and 2078 controls were statistically evaluated for de novo variant burden in gene families., Results: We demonstrate that disease-associated missense variants are enriched at paralog-conserved sites across all disease groups and inheritance models tested. We developed a gene family de novo enrichment framework that identified 43 exome-wide enriched gene families including 98 de novo variant carrying genes in NDD patients of which 28 represent novel candidate genes for NDD which are brain expressed and under evolutionary constraint., Conclusion: This study represents the first method to incorporate gene family information into a statistical framework to interpret variant data for NDDs and to discover new NDD-associated genes.

Objective: Impaired expansion of peripheral fat contributes to the pathogenesis of insulin resistance and Type 2 Diabetes (T2D). We aimed to identify novel disease-gene interactions during adipocyte differentiation., Methods: Genes in disease-associated loci for T2D, adiposity and insulin resistance were ranked according to expression in human adipocytes. The top 125 genes were ablated in human pre-adipocytes via CRISPR/CAS9 and the resulting cellular phenotypes quantified during adipocyte differentiation with high-content microscopy and automated image analysis. Morphometric measurements were extracted from all images and used to construct morphologic profiles for each gene., Results: Over 10 7 morphometric measurements were obtained. Clustering of the morphologic profiles accross all genes revealed a group of 14 genes characterized by decreased lipid accumulation, and enriched for known lipodystrophy genes. For two lipodystrophy genes, BSCL2 and AGPAT2, sub-clusters with PLIN1 and CEBPA identifed by morphological similarity were validated by independent experiments as novel protein-protein and gene regulatory interactions., Conclusions: A morphometric approach in adipocytes can resolve multiple cellular mechanisms for metabolic disease loci; this approach enables mechanistic interrogation of the hundreds of metabolic disease loci whose function still remains unknown., (Copyright © 2019 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Objective: The nuclear receptor PPARγ is the master regulator of adipocyte differentiation, distribution, and function. In addition, PPARγ induces terminal differentiation of several epithelial cell lineages, including colon epithelia. Loss-of-function mutations in PPARG result in familial partial lipodystrophy subtype 3 (FPDL3), a rare condition characterized by aberrant adipose tissue distribution and severe metabolic complications, including diabetes. Mutations in PPARG have also been reported in sporadic colorectal cancers, but the significance of these mutations is unclear. Studying these natural PPARG mutations provides valuable insights into structure-function relationships in the PPARγ protein. We functionally characterized a novel FPLD3-associated PPARγ L451P mutation in helix 9 of the ligand binding domain (LBD). Interestingly, substitution of the adjacent amino acid K450 was previously reported in a human colon carcinoma cell line., Methods: We performed a detailed side-by-side functional comparison of these two PPARγ mutants., Results: PPARγ L451P shows multiple intermolecular defects, including impaired cofactor binding and reduced RXRα heterodimerisation and subsequent DNA binding, but not in DBD-LBD interdomain communication. The K450Q mutant displays none of these functional defects. Other colon cancer-associated PPARγ mutants displayed diverse phenotypes, ranging from complete loss of activity to wildtype activity., Conclusions: Amino acid changes in helix 9 can differently affect LBD integrity and function. In addition, FPLD3-associated PPARγ mutations consistently cause intra- and/or intermolecular defects; colon cancer-associated PPARγ mutations on the other hand may play a role in colon cancer onset and progression, but this is not due to their effects on the most well-studied functional characteristics of PPARγ., (Copyright © 2018 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Multi-protein complexes mTORC1 and mTORC2 are required for growth and development of eukaryotes. mTORC1 is a nutrient sensor that integrates metabolic signals and energy state to regulate cell growth/proliferation, whereas, mTORC2 primarily regulates developmental processes. Dictyostelium proliferate in rich growth media, but initiate development upon nutrient depletion. Both mTOR complexes play essential roles in Dictyostelium, where growth and developmental cycles independently require, respectively, mTORC1 or mTORC2. Many protein associations and regulatory pathways for mTORC1 and mTORC2 in Dictyostelium have context similarity to mammalian cells and specificity to inhibition by the immunosuppressive drug rapamycin. In Dictyostelium, mTORC1 function is inactivated upon starvation-induced development, but development is directly induced through rapamycin-mediated inhibition of mTORC1 activity, even in the absence of nutrient withdrawal. Pharmacologic inhibition of mTORC1, in the absence of nutrient loss, has allowed the identification of a class of essential up-regulated, developmentally-associated signaling genes and down-regulated, growth genes. We also review functional pathway regulations that integrate mTORC1/mTORC2 activities and emphasize complexity of small GTPase regulation of mTORC2 activity. Finally, epistases experiments have suggested novel upstream pathway cross-talk in Dictyostelium that requires mTORC1 and mTORC2, but for separate and independent downstream functions.

Loss-of-function mutations in PPARG cause familial partial lipodystrophy type 3 (FPLD3) and severe metabolic disease in many patients. Missense mutations in PPARG are present in ∼1 in 500 people. Although mutations are often binarily classified as benign or deleterious, prospective functional classification of all missense PPARG variants suggests that their impact is graded. Furthermore, in testing novel mutations with both prototypic endogenous (e.g., prostaglandin J2 [PGJ2]) and synthetic ligands (thiazolidinediones, tyrosine agonists), we observed that synthetic agonists selectively rescue function of some peroxisome proliferator-activated receptor-γ (PPARγ) mutants. We report on patients with FPLD3 who harbor two such PPARγ mutations (R308P and A261E). Both PPARγ mutants exhibit negligible constitutive or PGJ2-induced transcriptional activity but respond readily to synthetic agonists in vitro, with structural modeling providing a basis for such differential ligand-dependent responsiveness. Concordant with this finding, dramatic clinical improvement was seen after pioglitazone treatment of a patient with R308P mutant PPARγ. A patient with A261E mutant PPARγ also responded beneficially to rosiglitazone, although cardiomyopathy precluded prolonged thiazolidinedione use. These observations indicate that detailed structural and functional classification can be used to inform therapeutic decisions in patients with PPARG mutations., (© 2018 by the American Diabetes Association.)

Background: Patients with type 1 diabetes routinely utilize a single premeal fingerstick glucose to determine premeal insulin doses. Continuous glucose monitoring (CGM) provides much richer glycemic trend information, including glycemic slope (GS). How to incorporate this information into dosing decisions remains an open question., Methods: We examined the relationship between premeal GS and postmeal glycemic excursions in 240 individuals with type 1 diabetes receiving CGM augmented insulin pump therapy. Over 23.5 million CGM values were synchronized with 264 500 meals. CGM values were integrated 2 hours premeal to compute GS and 2 hours postmeal to compute glycemic excursion outcomes. Postmeal hyperglycemia (integrated CGM glucose >180 mg/dL*hr) and postmeal hypoglycemic events (any CGM glucose < 70 mg/dL) were tabulated according to positive/negative premeal GS and according to GS bins commonly displayed as rate-of-change arrows on CGM devices., Results: Positive versus negative premeal GS was associated with a 2.28-fold (95% CI 2.25-2.32) risk of postmeal hyperglycemia. Negative versus positive premeal GS was associated with a 2.36-fold (95% CI 2.25-2.43) increase in one or more postprandial hypoglycemic events. Premeal GS in the bin currently displayed as "no change" on existing CGM devices (-1 to 1 mg/dL/min), conferred a 1.82-fold (95% CI 1.79-1.86) risk of postprandial hyperglycemia when positive and a 2.06-fold (95% CI 1.99-2.15) increased risk of postprandial hypoglycemia when negative., Conclusion: Premeal GS predicts postmeal glycemic excursions and may help inform insulin dosing decisions. Rate-of-change arrows on existing devices obscure clinically actionable glycemic trend information from CGM users.

Clinical exome sequencing routinely identifies missense variants in disease-related genes, but functional characterization is rarely undertaken, leading to diagnostic uncertainty. For example, mutations in PPARG cause Mendelian lipodystrophy and increase risk of type 2 diabetes (T2D). Although approximately 1 in 500 people harbor missense variants in PPARG, most are of unknown consequence. To prospectively characterize PPARγ variants, we used highly parallel oligonucleotide synthesis to construct a library encoding all 9,595 possible single-amino acid substitutions. We developed a pooled functional assay in human macrophages, experimentally evaluated all protein variants, and used the experimental data to train a variant classifier by supervised machine learning. When applied to 55 new missense variants identified in population-based and clinical sequencing, the classifier annotated 6 variants as pathogenic; these were subsequently validated by single-variant assays. Saturation mutagenesis and prospective experimental characterization can support immediate diagnostic interpretation of newly discovered missense variants in disease-related genes., Competing Interests: No competing financial interests

Peroxisome proliferator-activated receptor gamma (PPARG) is a master transcriptional regulator of adipocyte differentiation and a canonical target of antidiabetic thiazolidinedione medications. In rare families, loss-of-function (LOF) mutations in PPARG are known to cosegregate with lipodystrophy and insulin resistance; in the general population, the common P12A variant is associated with a decreased risk of type 2 diabetes (T2D). Whether and how rare variants in PPARG and defects in adipocyte differentiation influence risk of T2D in the general population remains undetermined. By sequencing PPARG in 19,752 T2D cases and controls drawn from multiple studies and ethnic groups, we identified 49 previously unidentified, nonsynonymous PPARG variants (MAF < 0.5%). Considered in aggregate (with or without computational prediction of functional consequence), these rare variants showed no association with T2D (OR = 1.35; P = 0.17). The function of the 49 variants was experimentally tested in a novel high-throughput human adipocyte differentiation assay, and nine were found to have reduced activity in the assay. Carrying any of these nine LOF variants was associated with a substantial increase in risk of T2D (OR = 7.22; P = 0.005). The combination of large-scale DNA sequencing and functional testing in the laboratory reveals that approximately 1 in 1,000 individuals carries a variant in PPARG that reduces function in a human adipocyte differentiation assay and is associated with a substantial risk of T2D.

Aims/hypothesis: Individuals with impaired glucose tolerance have increased proinsulin levels, despite normal glucose or C-peptide levels. In the Diabetes Prevention Program (DPP), increased proinsulin levels predicted type 2 diabetes and proinsulin levels were significantly reduced following treatment with metformin, lifestyle modification or troglitazone compared with placebo. Genetic and physiological studies suggest a role for the zinc transporter gene SLC30A8 in diabetes risk, possibly through effects on insulin-processing in beta cells. We hypothesised that the risk allele at the type 2 diabetes-associated missense polymorphism rs13266634 (R325W) in SLC30A8 would predict proinsulin levels in individuals at risk of type 2 diabetes and may modulate response to preventive interventions., Methods: We genotyped rs13266634 in 3,007 DPP participants and examined its association with fasting proinsulin and fasting insulin at baseline and at 1 year post-intervention., Results: We found that increasing dosage of the C risk allele at SLC30A8 rs13266634 was significantly associated with higher proinsulin levels at baseline (p = 0.002) after adjustment for baseline insulin. This supports the hypothesis that risk alleles at SLC30A8 mark individuals with insulin-processing defects. At the 1 year analysis, proinsulin levels decreased significantly in all groups receiving active intervention and were no longer associated with SLC30A8 genotype (p = 0.86) after adjustment for insulin at baseline and 1 year. We found no genotype × treatment interactions at 1 year., Conclusions/interpretation: In prediabetic individuals, genotype at SLC30A8 predicts baseline proinsulin levels independently of insulin levels, but does not predict proinsulin levels after amelioration of insulin sensitivity at 1 year.

The mechanisms by which the epithelium of the digestive tract and its associated glands are specified are largely unknown. One clue is that several transcription factors are expressed in specific regions of the endoderm prior to and during organogenesis. Pdx-1, for example, is expressed in the duodenum and pancreas and Pdx-1 inactivation results in an arrest of pancreatic development after buds formation. Similarly, ngn3 is transiently expressed in the developing pancreas and a knockout results in the absence of endocrine cells. This paper focuses on the question of whether these and other transcription factors, known to be necessary for pancreatic development, are also sufficient to drive a program of pancreatic organogenesis. Using in ovo electroporation of chick embryos, we show that ectopic expression of Pdx-1 or ngn3 causes cells to bud out of the epithelium like pancreatic progenitors. The Pdx-1-expressing cells extinguish markers for other nonpancreatic regions of the endoderm and initiate, but do not complete, pancreatic cytodifferentiation. Ectopic expression of ngn3 is sufficient to turn endodermal cells of any region into endocrine cells that form islets expressing glucagon and somatostatin in the mesenchyme. The results suggest that simple gene combinations could be used in stem cells to achieve specific endodermal tissue differentiation.

We describe a multiplex nucleic acid assay that identifies and determines the abundance of four different pathogenic retroviruses (HIV-1, HIV-2, and human T-lymphotrophic virus types I and II). Retroviral DNA sequences are amplified in a single, sealed tube by simultaneous PCR assays, and the resulting amplicons are detected in real time by the hybridization of four differently colored, amplicon-specific molecular beacons. The color of the fluorescence generated in the course of amplification identifies which retroviruses are present, and the number of thermal cycles required for the intensity of each color to rise significantly above background provides an accurate measure of the number of copies of each retroviral sequence that were present originally in the sample. Fewer than 10 retroviral genomes can be detected. Moreover, 10 copies of a rare retrovirus can be detected in the presence of 100, 000 copies of an abundant retrovirus. Ninety-six samples can be analyzed in 3 hr on a single plate, and the use of a closed-tube format eliminates crossover contamination. Utilizing previously well characterized clinical samples, we demonstrate that each of the pathogenic retroviruses can be identified correctly and no false positives occur. This assay enables the rapid and reliable screening of donated blood and transplantable tissues.