Amit Majithia

Rare variants in PPARG with decreased activity in adipocyte differentiation are associated with increased risk of type 2 diabetes

Significance Genome sequencing of individuals in the population reveals new mutations in almost every protein coding gene; interpreting the consequence of these mutations for human health and disease remains challenging. We sequenced the gene PPARG, a target of antidiabetic drugs, in nearly 20,000 individuals with and without type 2 diabetes (T2D). We identified 49 previously unidentified protein-altering mutations, characterized their cellular function in human cells, and discovered that nine of these mutations cause loss-of-function (LOF). The individuals who carry these nine LOF mutations have a sevenfold increased risk of T2D, whereas individuals carrying mutations we classify as benign have no increased risk of T2D. 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.

Prospective functional classification of all possible missense variants in PPARG

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.

IGF2BP2/IMP2-Deficient Mice Resist Obesity through Enhanced Translation of Ucp1 mRNA and Other mRNAs Encoding Mitochondrial Proteins

Although variants in the IGF2BP2/IMP2 gene confer risk for type 2 diabetes, IMP2, an RNA binding protein, is not known to regulate metabolism. Imp2(-/-) mice gain less lean mass after weaning and have increased lifespan. Imp2(-/-) mice are highly resistant to diet-induced obesity and fatty liver and display superior glucose tolerance and insulin sensitivity, increased energy expenditure, and better defense of core temperature on cold exposure. Imp2(-/-) brown fat and Imp2(-/-) brown adipocytes differentiated in vitro contain more UCP1 polypeptide than Imp2(+/+) despite similar levels of Ucp1 mRNA; the Imp2(-/-)adipocytes also exhibit greater uncoupled oxygen consumption. IMP2 binds the mRNAs encoding Ucp1 and other mitochondrial components, and most exhibit increased translational efficiency in the absence of IMP2. In vitro IMP2 inhibits translation of mRNAs bearing the Ucp1 untranslated segments. Thus IMP2 limits longevity and regulates nutrient and energy metabolism in the mouse by controlling the translation of its client mRNAs.

Clinical translation of genetic predictors for type 2 diabetes.

Purpose of reviewTo highlight recent type 2 diabetes (T2D)-associated genetic discoveries and their potential for clinical application. Recent findingsThe advent of genome-wide association screening has uncovered many loci newly associated with T2D. This review describes the techniques applied to discover novel T2D genes and compares their relative strengths, biases, and findings to date. The results of large-scale genome-wide association studies carried out since 2007 are summarized, and limitations of interpreting this preliminary data are offered. Recent studies exploring the clinical potential of these discoveries are reviewed, focusing on insights into T2D pathogenesis, risk prediction of future diabetes, and utility in guiding pharmacotherapy. The new T2D-associated loci have been implicated in β-cell development and function, highlighting insulin secretion in the disease process. Preliminary risk prediction studies show that more loci are needed to improve T2D risk indices. Studies have also revealed that genes may play a role in the pharmacologic response to antidiabetic medications. SummarySince 2007, genome-wide association studies have rapidly increased the number of T2D-associated loci. This review summarizes the history of genetic association studies, the results from the new genome-wide association studies, and the clinical application of these findings.

TOR complex 2 (TORC2) in Dictyostelium suppresses phagocytic nutrient capture independently of TORC1-mediated nutrient sensing.

The TOR protein kinase functions in two distinct complexes, TOR complex 1 (TORC1) and 2 (TORC2). TORC1 is required for growth in response to growth factors, nutrients and the cellular energy state; TORC2 regulates AKT signaling, which can modulate cytoskeletal polarization. In its ecological niche, Dictyostelium engulf bacteria and yeast for nutrient capture. Despite the essential role of TORC1 in control of cellular growth, we show that nutrient particle capture (phagocytosis) in Dictyostelium is independent of TORC1-mediated nutrient sensing and growth regulation. However, loss of Dictyostelium TORC2 components Rictor/Pia, SIN1/RIP3 and Lst8 promotes nutrient particle uptake; inactivation of TORC2 leads to increased efficiency and speed of phagocytosis. In contrast to phagocytosis, we show that macropinocytosis, an AKT-dependent process for cellular uptake of fluid phase nutrients, is not regulated by either of the TOR complexes. The integrated and balanced regulation of TORC1 and TORC2 might be crucial in Dictyostelium to coordinate growth and energy needs with other essential TOR-regulated processes.

Combinatorial cell-specific regulation of GSK3 directs cell differentiation and polarity in Dictyostelium

In Dictyostelium, the interaction of secreted cAMP with specific cell surface receptors regulates the activation/de-activation of GSK3, which mediates developmental cell patterning. In addition, Dictyostelium cells polarize in response to extracellular cAMP, although a potential role for GSK3 in this pathway has not been investigated. Previously, we had shown that ZAK1 was an activating tyrosine kinase for GSK3 function in Dictyostelium and we now identify ZAK2 as the other tyrosine kinase in the cAMP-activation pathway for GSK3; no additional family members exist. We also now show that tyrosine phosphorylation/activation of GSK3 by ZAK2 and ZAK1 separately regulate GSK3 in distinct differentiated cell populations, and that ZAK2 acts in both autonomous and non-autonomous pathways to regulate these cell-type differentiations. Finally, we demonstrate that efficient polarization of Dictyostelium towards cAMP depends on ZAK1-mediated tyrosine phosphorylation of GSK3. Combinatorial regulation of GSK3 by ZAK kinases in Dictyostelium guides cell polarity, directional cell migration and cell differentiation, pathways that extend the complexity of GSK3 signaling throughout the development of Dictyostelium.

Gene family information facilitates variant interpretation and identification of disease-associated genes

Differentiating risk-conferring from benign missense variants, and therefore optimal calculation of gene-variant burden, represent a major challenge in particular for rare and genetic heterogeneous disorders. While orthologous gene conservation is commonly employed in variant annotation, approximately 80% of known disease-associated genes are paralogs and belong to gene families. It has not been thoroughly investigated how gene family information can be utilized for disease gene discovery and variant interpretation. We developed a paralog conservation score to empirically evaluate whether paralog conserved or nonconserved sites of in-human paralogs are important for protein function. Using this score, we demonstrate that disease-associated missense variants are significantly enriched at paralog conserved sites across all disease groups and disease inheritance models tested. Next, we assessed whether gene family information could assist in discovering novel disease-associated genes. We subsequently developed a gene family de novo enrichment framework that identified 43 exome-wide enriched gene families including 98 de novo variant carrying genes in more than 10k neurodevelopmental disorder patients. 33 gene family enriched genes represent novel candidate genes which are brain expressed and variant constrained in neurodevelopmental disorders.