👤 Kieran A Walker

Lean body mass (LM) plays an important role in mobility and metabolic function. We previously identified five loci associated with LM adjusted for fat mass in kilograms. Such an adjustment may reduce the power to identify genetic signals having an association with both lean mass and fat mass. To determine the impact of different fat mass adjustments on genetic architecture of LM and identify additional LM loci. We performed genome-wide association analyses for whole-body LM (20 cohorts of European ancestry with n = 38,292) measured using dual-energy X-ray absorptiometry) or bioelectrical impedance analysis, adjusted for sex, age, age2, and height with or without fat mass adjustments (Model 1 no fat adjustment; Model 2 adjustment for fat mass as a percentage of body mass; Model 3 adjustment for fat mass in kilograms). Seven single-nucleotide polymorphisms (SNPs) in separate loci, including one novel LM locus (TNRC6B), were successfully replicated in an additional 47,227 individuals from 29 cohorts. Based on the strengths of the associations in Model 1 vs Model 3, we divided the LM loci into those with an effect on both lean mass and fat mass in the same direction and refer to those as "sumo wrestler" loci (FTO and MC4R). In contrast, loci with an impact specifically on LM were termed "body builder" loci (VCAN and ADAMTSL3). Using existing available genome-wide association study databases, LM increasing alleles of SNPs in sumo wrestler loci were associated with an adverse metabolic profile, whereas LM increasing alleles of SNPs in "body builder" loci were associated with metabolic protection. In conclusion, we identified one novel LM locus (TNRC6B). Our results suggest that a genetically determined increase in lean mass might exert either harmful or protective effects on metabolic traits, depending on its relation to fat mass. Show less

We report changes in the genomic landscape in the development of head and neck squamous cell carcinomas HNSCC from potentially premalignant lesions (PPOLS) to malignancy and lymph node metastases. Likely pathological mutations predominantly involved a relatively small set of genes reported previously (TP53, KMT2D, CDKN2A, PIK3CA, NOTCH1 and FAT1) but also other predicted cancer drivers (MGA, PABPC3, NR4A2, NCOR1 and MACF1). Notably, all these mutations arise early and are present in PPOLs. The most frequent genetic changes, which follow acquisition of immortality and loss of senescence, are of consistent somatic copy number alterations (SCNAs) involving chromosomal regions enriched for genes in known and previously unreported cancer-related pathways. We mapped the evolution of SCNAs in HNSCC progression. One of the earliest SCNAs involved deletions of CSMD1 (8p23.2). CSMD1 deletions or promoter hypermethylation were present in all of the immortal PPOLs and occurred at high frequency in the immortal HNSCC cell lines. Modulation of CSMD1 in cell lines revealed significant suppression of proliferation and invasion by forced expression, and significant stimulation of invasion by knockdown of expression. Known cancer drivers NOTCH1, PPP6C, RAC1, EIF4G1, PIK3CA showed significant increase in frequency of SCNA in transition from PPOLs to HNSCC that correlated with their expression. In the later stages of progression, HNSCC with and without nodal metastases showed some clear differences including high copy number gains of CCND1, hsa-miR-548k and TP63 in the metastases group. Show less

Carbohydrate response element binding protein (ChREBP) is a key transcriptional regulator of de novo lipogenesis (DNL) in response to carbohydrates and in hepatic steatosis. Mechanisms underlying nutrient modulation of ChREBP are under active investigation. Here we identify host cell factor 1 (HCF-1) as a previously unknown ChREBP-interacting protein that is enriched in liver biopsies of nonalcoholic steatohepatitis (NASH) patients. Biochemical and genetic studies show that HCF-1 is O-GlcNAcylated in response to glucose as a prerequisite for its binding to ChREBP and subsequent recruitment of OGT, ChREBP O-GlcNAcylation, and activation. The HCF-1:ChREBP complex resides at lipogenic gene promoters, where HCF-1 regulates H3K4 trimethylation to prime recruitment of the Jumonji C domain-containing histone demethylase PHF2 for epigenetic activation of these promoters. Overall, these findings define HCF-1's interaction with ChREBP as a previously unappreciated mechanism whereby glucose signals are both relayed to ChREBP and transmitted for epigenetic regulation of lipogenic genes. Show less

Genome-wide association studies (GWAS) have identified >250 loci for body mass index (BMI), implicating pathways related to neuronal biology. Most GWAS loci represent clusters of common, noncoding variants from which pinpointing causal genes remains challenging. Here we combined data from 718,734 individuals to discover rare and low-frequency (minor allele frequency (MAF) < 5%) coding variants associated with BMI. We identified 14 coding variants in 13 genes, of which 8 variants were in genes (ZBTB7B, ACHE, RAPGEF3, RAB21, ZFHX3, ENTPD6, ZFR2 and ZNF169) newly implicated in human obesity, 2 variants were in genes (MC4R and KSR2) previously observed to be mutated in extreme obesity and 2 variants were in GIPR. The effect sizes of rare variants are ~10 times larger than those of common variants, with the largest effect observed in carriers of an MC4R mutation introducing a stop codon (p.Tyr35Ter, MAF = 0.01%), who weighed ~7 kg more than non-carriers. Pathway analyses based on the variants associated with BMI confirm enrichment of neuronal genes and provide new evidence for adipocyte and energy expenditure biology, widening the potential of genetically supported therapeutic targets in obesity. Show less

The heterochromatin protein 1 (HP1) family is involved in various functions with maintenance of chromatin structure. During murine somatic cell reprogramming, we find that early depletion of HP1γ reduces the generation of induced pluripotent stem cells, while late depletion enhances the process, with a concomitant change from a centromeric to nucleoplasmic localization and elongation-associated histone H3.3 enrichment. Depletion of heterochromatin anchoring protein SENP7 increased reprogramming efficiency to a similar extent as HP1γ, indicating the importance of HP1γ release from chromatin for pluripotency acquisition. HP1γ interacted with OCT4 and DPPA4 in HP1α and HP1β knockouts and in H3K9 methylation depleted H3K9M embryonic stem cell (ESC) lines. HP1α and HP1γ complexes in ESCs differed in association with histones, the histone chaperone CAF1 complex, and specific components of chromatin-modifying complexes such as DPY30, implying distinct functional contributions. Taken together, our results reveal the complex contribution of the HP1 proteins to pluripotency. Show less

Treatment of blood cholesterol is part of a strategy to lower atherosclerotic cardiovascular (ASCVD) risk. While use of HMG-CoA reductase inhibitors to modify cholesterol levels is the primary means of lowering the risk of an ASCVD event, residual risk remains. A new strategy being investigated is the use of cholesterol ester transfer protein (CETP) inhibitors to raise the levels of high-density lipoprotein cholesterol (HDL-C) and lower low-density lipoprotein cholesterol (LDL-C). While initial large-scale studies demonstrated no reduction of cardiovascular events, one CETP inhibitor, anacetrapib, has demonstrated a reduction in cardiovascular events in the REVEAL trial. Show less

Anacetrapib is a novel cholesteryl-ester transfer protein (CETP) inhibitor in late-stage clinical development, shown in preceding clinical trials to have residual pharmacological activity after prolonged washout after chronic dosing. Preclinical findings suggest that white adipose tissue is a potential depot and that accumulation into adipose tissue governs the long-term kinetics of anacetrapib in mice. A phase I study performed to test this hypothesis in humans revealed that plasma exposure was correlated with fat content in food administered with the drug. Plasma concentrations of anacetrapib seemed to reach plateau faster than adipose concentrations. Anacetrapib continued to accumulate in adipose during the treatment period despite apparent plateau in plasma with only minimal decline in adipose levels up to 1 year postdose. Because of its high lipophilicity, anacetrapib partitions into adipose tissue, this likely forms a drug reservoir that, in turn, contributes to the long residence time of the drug in plasma. Show less

Understanding the physiological mechanisms by which common variants predispose to type 2 diabetes requires large studies with detailed measures of insulin secretion and sensitivity. Here we performed the largest genome-wide association study of first-phase insulin secretion, as measured by intravenous glucose tolerance tests, using up to 5,567 individuals without diabetes from 10 studies. We aimed to refine the mechanisms of 178 known associations between common variants and glycemic traits and identify new loci. Thirty type 2 diabetes or fasting glucose-raising alleles were associated with a measure of first-phase insulin secretion at Show less

Gut microbiota may promote positive energy balance; however, germfree mice can be either resistant or susceptible to diet-induced obesity (DIO) depending on the type of dietary intervention. We here sought to identify the dietary constituents that determine the susceptibility to body fat accretion in germfree (GF) mice. GF and specific pathogen free (SPF) male C57BL/6N mice were fed high-fat diets either based on lard or palm oil for 4 wks. Mice were metabolically characterized at the end of the feeding trial. FT-ICR-MS and UPLC-TOF-MS were used for cecal as well as hepatic metabolite profiling and cecal bile acids quantification, respectively. Hepatic gene expression was examined by qRT-PCR and cecal gut microbiota of SPF mice was analyzed by high-throughput 16S rRNA gene sequencing. GF mice, but not SPF mice, were completely DIO resistant when fed a cholesterol-rich lard-based high-fat diet, whereas on a cholesterol-free palm oil-based high-fat diet, DIO was independent of gut microbiota. In GF lard-fed mice, DIO resistance was conveyed by increased energy expenditure, preferential carbohydrate oxidation, and increased fecal fat and energy excretion. Cecal metabolite profiling revealed a shift in bile acid and steroid metabolites in these lean mice, with a significant rise in 17β-estradiol, which is known to stimulate energy expenditure and interfere with bile acid metabolism. Decreased cecal bile acid levels were associated with decreased hepatic expression of genes involved in bile acid synthesis. These metabolic adaptations were largely attenuated in GF mice fed the palm-oil based high-fat diet. We propose that an interaction of gut microbiota and cholesterol metabolism is essential for fat accretion in normal SPF mice fed cholesterol-rich lard as the main dietary fat source. This is supported by a positive correlation between bile acid levels and specific bacteria of the order In conclusion, our study identified dietary cholesterol as a candidate ingredient affecting the crosstalk between gut microbiota and host metabolism. Show less

Diabetic retinopathy has recently become associated with complications similar to chronic inflammatory diseases. Although it is clear that tumor necrosis factor-α is increased in diabetes, the role of innate immunity is only recently being investigated. As such, we hypothesized that diabetes would increase Toll-like receptor 4 (TLR4) signaling, which could be inhibited by a β-adrenergic receptor agonist (Compound 49b) previously shown to have anti-inflammatory actions. In order to investigate β-adrenergic receptor signaling and TLR4 in the diabetic retina, streptozotocin-injected diabetic mice, as well as human primary retinal endothelial cells (RECs) and rat retinal Müller cells (rMC-1) exposed to high glucose (25 mM), were treated with a novel β-adrenergic receptor agonist, Compound 49b (50 nM), or phosphate-buffered saline (control). TLR4 and its downstream signaling partners (MyD88, IL-1 receptor-associated kinase 1, TNF receptor-associated factor 6 and total and phosphorylated nuclear factor-κB) were examined. In addition, we assessed high-mobility group box 1 (HMGB1) protein levels. Our data showed that diabetes or high-glucose culture conditions significantly increased TLR4 and downstream signaling partners. Compound 49b was able to significantly reduce TLR4 and related molecules in the diabetic animal and retinal cells. HMGB1 was significantly increased in RECs and Müller cells grown in high-glucose culture conditions, which was subsequently reduced with Compound 49b treatment. Our findings suggest that high glucose may increase HMGB1 levels that lead to increased TLR4 signaling. Compound 49b significantly inhibited this pathway, providing a potential mechanism for its protective actions. Show less

Apolipoprotein (apo)A-IV is a lipid emulsifying protein linked to a range of protective roles in obesity, diabetes, and cardiovascular disease. It exists in several states in plasma including lipid-bound in HDL and chylomicrons and as monomeric and dimeric lipid-free/poor forms. Our recent x-ray crystal structure of the central domain of apoA-IV shows that it adopts an elongated helical structure that dimerizes via two long reciprocating helices. A striking feature is the alignment of conserved proline residues across the dimer interface. We speculated that this plays important roles in the structure of the lipid-free protein and its ability to bind lipid. Here we show that the systematic conversion of these prolines to alanine increased the thermodynamic stability of apoA-IV and its propensity to oligomerize. Despite the structural stabilization, we noted an increase in the ability to bind and reorganize lipids and to promote cholesterol efflux from cells. The novel properties of these mutants allowed us to isolate the first trimeric form of an exchangeable apolipoprotein and characterize it by small-angle x-ray scattering and chemical cross-linking. The results suggest that the reciprocating helix interaction is a common feature of all apoA-IV oligomers. We propose a model of how self-association of apoA-IV can result in spherical lipoprotein particles, a model that may have broader applications to other exchangeable apolipoprotein family members. Show less

Abnormal accumulation of undigested macromolecules, often disease-specific, is a major feature of lysosomal and neurodegenerative disease and is frequently attributed to defective autophagy. The mechanistic underpinnings of the autophagy defects are the subject of intense research, which is aided by genetic disease models. To gain an improved understanding of the pathways regulating defective autophagy specifically in juvenile neuronal ceroid lipofuscinosis (JNCL or Batten disease), a neurodegenerative disease of childhood, we developed and piloted a GFP-microtubule-associated protein 1 light chain 3 (GFP-LC3) screening assay to identify, in an unbiased fashion, genotype-sensitive small molecule autophagy modifiers, employing a JNCL neuronal cell model bearing the most common disease mutation in CLN3. Thapsigargin, a sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) Ca(2+) pump inhibitor, reproducibly displayed significantly more activity in the mouse JNCL cells, an effect that was also observed in human-induced pluripotent stem cell-derived JNCL neural progenitor cells. The mechanism of thapsigargin sensitivity was Ca(2+)-mediated, and autophagosome accumulation in JNCL cells could be reversed by Ca(2+) chelation. Interrogation of intracellular Ca(2+) handling highlighted alterations in endoplasmic reticulum, mitochondrial, and lysosomal Ca(2+) pools and in store-operated Ca(2+) uptake in JNCL cells. These results further support an important role for the CLN3 protein in intracellular Ca(2+) handling and in autophagic pathway flux and establish a powerful new platform for therapeutic screening. Show less

We have expressed and purified three soluble fragments of the human LRIG1-ECD (extracellular domain): the LRIG1-LRR (leucine-rich repeat) domain, the LRIG1-3Ig (immunoglobulin-like) domain, and the LRIG1-LRR-1Ig fragment using baculovirus vectors in insect cells. The two LRIG1 domains crystallised so that we have been able to determine the three-dimensional structures at 2.3Å resolution. We developed a three-dimensional structure for the LRIG1-ECD using homology modelling based on the LINGO-1 structure. The LRIG1-LRR domain and the LRIG1-LRR-1Ig fragment are monomers in solution, whereas the LRIG1-3Ig domain appears to be dimeric. We could not detect any binding of the LRIG1 domains or the LRIG1-LRR-1Ig fragment to the EGF receptor (EGFR), either in solution using biosensor analysis or when the EGFR was expressed on the cell surface. The FLAG-tagged LRIG1-LRR-1Ig fragment binds weakly to colon cancer cells regardless of the presence of EGFRs. Similarly, neither the soluble LRIG1-LRR nor the LRIG1-3Ig domains nor the full-length LRIG1 co-expressed in HEK293 cells inhibited ligand-stimulated activation of cell-surface EGFR. Show less

In a screen for genes that affect the metabolic response to high-fat diet (HFD), we selected one line of N-ethyl-N-nitrosourea (ENU)-mutagenized mice, Jll, with dominantly inherited resistance to diet-induced obesity (DIO). Mutant animals had dramatically reduced body weight and fat mass, and low basal insulin and glucose levels relative to unaffected controls. Both white adipose tissue (WAT) and brown adipose tissue (BAT) depots were smaller in mutant animals. Mutant animals fed a HFD gained only slightly more weight than animals fed regular chow, and were protected from hepatic lipid accumulation. The phenotype was genetically linked to a 5.7-Mb interval on chromosome 12, and sequencing of the entire interval identified a single coding mutation, predicted to cause a methionine-to-isoleucine substitution at position 279 of the Adcy3 protein (Adcy3M279I, henceforth referred to as Adcy3Jll). The mutant protein is hyperactive, possibly constitutively so, producing elevated levels of cyclic AMP in a cell-based assay. These mice demonstrate that increased Adcy3 activity robustly protect animals from diet-induced metabolic derangements. Show less

Apolipoprotein (apo)A-IV plays important roles in dietary lipid and glucose metabolism, and knowledge of its structure is required to fully understand the molecular basis of these functions. However, typical of the entire class of exchangeable apolipoproteins, its dynamic nature and affinity for lipid has posed challenges to traditional high resolution structural approaches. We previously reported an x-ray crystal structure of a dimeric truncation mutant of apoA-IV, which showed a unique helix-swapping molecular interface. Unfortunately, the structures of the N and C termini that are important for lipid binding were not visualized. To build a more complete model, we used chemical cross-linking to derive distance constraints across the full-length protein. The approach was enhanced with stable isotope labeling to overcome ambiguities in determining molecular span of the cross-links given the remarkable similarities in the monomeric and dimeric apoA-IV structures. Using 51 distance constraints, we created a starting model for full-length monomeric apoA-IV and then subjected it to two modeling approaches: (i) molecular dynamics simulations and (ii) fitting to small angle x-ray scattering data. This resulted in the most detailed models yet for lipid-free monomeric or dimeric apoA-IV. Importantly, these models were of sufficient detail to direct the experimental identification of new functional residues that participate in a "clasp" mechanism to modulate apoA-IV lipid affinity. The isotope-assisted cross-linking approach should prove useful for further study of this family of apolipoproteins in both the lipid-free and -bound states. Show less

Genetic variation in six genes has been associated with elevated liver fat and nonalcoholic fatty liver disease in adults. The influence of these genes on liver fat and whether a genetic risk score (GRS) would improve upon the ability of common clinical risk factors to predict elevated liver fat content (ELF) in Hispanic children was determined. 223 obese Hispanic children were genotyped for six SNPs. MRI was used to measure liver fat. A GRS was tested for association with ELF using multivariate linear regression. Predictors were assessed via ROC curves and pair-wise analysis was used to determine significance alone and combined with clinical markers. Only variants in PNPLA3 and APOC3 genes were associated with liver fat (P < 0.001, P = 0.01, respectively). Subjects with a GRS = 4 had ∼3-fold higher liver fat content than subjects with GRS of 0 (15.1 ± 12.7 vs. 5.1 ± 3.7%, P = 0.03). While the addition of the GRS to a model containing BMI and liver enzymes increased ROC AUC from 0.83 to 0.85 [95% CI, 0.79-0.89], (P = 0.01), it does not improve detection of ELF from a clinical perspective. Only PNPLA3 and APOC3 were related to ELF and a GRS comprised of these susceptibility alleles did not add to the discriminatory power of traditional biomarkers for clinical assessment of liver fat. Show less

Circulating metabolites associated with insulin sensitivity may represent useful biomarkers, but their causal role in insulin sensitivity and diabetes is less certain. We previously identified novel metabolites correlated with insulin sensitivity measured by the hyperinsulinemic-euglycemic clamp. The top-ranking metabolites were in the glutathione and glycine biosynthesis pathways. We aimed to identify common genetic variants associated with metabolites in these pathways and test their role in insulin sensitivity and type 2 diabetes. With 1,004 nondiabetic individuals from the RISC study, we performed a genome-wide association study (GWAS) of 14 insulin sensitivity-related metabolites and one metabolite ratio. We replicated our results in the Botnia study (n = 342). We assessed the association of these variants with diabetes-related traits in GWAS meta-analyses (GENESIS [including RISC, EUGENE2, and Stanford], MAGIC, and DIAGRAM). We identified four associations with three metabolites-glycine (rs715 at CPS1), serine (rs478093 at PHGDH), and betaine (rs499368 at SLC6A12; rs17823642 at BHMT)-and one association signal with glycine-to-serine ratio (rs1107366 at ALDH1L1). There was no robust evidence for association between these variants and insulin resistance or diabetes. Genetic variants associated with genes in the glycine biosynthesis pathways do not provide consistent evidence for a role of glycine in diabetes-related traits. Show less

Human complex metabolic traits are in part regulated by genetic determinants. Here we applied exome sequencing to identify novel associations of coding polymorphisms at minor allele frequencies (MAFs) >1% with common metabolic phenotypes. The study comprised three stages. We performed medium-depth (8×) whole exome sequencing in 1,000 cases with type 2 diabetes, BMI >27.5 kg/m(2) and hypertension and in 1,000 controls (stage 1). We selected 16,192 polymorphisms nominally associated (p < 0.05) with case-control status, from four selected annotation categories or from loci reported to associate with metabolic traits. These variants were genotyped in 15,989 Danes to search for association with 12 metabolic phenotypes (stage 2). In stage 3, polymorphisms showing potential associations were genotyped in a further 63,896 Europeans. Exome sequencing identified 70,182 polymorphisms with MAF >1%. In stage 2 we identified 51 potential associations with one or more of eight metabolic phenotypes covered by 45 unique polymorphisms. In meta-analyses of stage 2 and stage 3 results, we demonstrated robust associations for coding polymorphisms in CD300LG (fasting HDL-cholesterol: MAF 3.5%, p = 8.5 × 10(-14)), COBLL1 (type 2 diabetes: MAF 12.5%, OR 0.88, p = 1.2 × 10(-11)) and MACF1 (type 2 diabetes: MAF 23.4%, OR 1.10, p = 8.2 × 10(-10)). We applied exome sequencing as a basis for finding genetic determinants of metabolic traits and show the existence of low-frequency and common coding polymorphisms with impact on common metabolic traits. Based on our study, coding polymorphisms with MAF above 1% do not seem to have particularly high effect sizes on the measured metabolic traits. Show less

The GPHN gene codes for gephyrin, a key scaffolding protein in the neuronal postsynaptic membrane, responsible for the clustering and localization of glycine and GABA receptors at inhibitory synapses. Gephyrin has well-established functional links with several synaptic proteins that have been implicated in genetic risk for neurodevelopmental disorders such as autism spectrum disorder (ASD), schizophrenia and epilepsy including the neuroligins (NLGN2, NLGN4), the neurexins (NRXN1, NRXN2, NRXN3) and collybistin (ARHGEF9). Moreover, temporal lobe epilepsy has been linked to abnormally spliced GPHN mRNA lacking exons encoding the G-domain of the gephyrin protein, potentially arising due to cellular stress associated with epileptogenesis such as temperature and alkalosis. Here, we present clinical and genomic characterization of six unrelated subjects, with a range of neurodevelopmental diagnoses including ASD, schizophrenia or seizures, who possess rare de novo or inherited hemizygous microdeletions overlapping exons of GPHN at chromosome 14q23.3. The region of common overlap across the deletions encompasses exons 3-5, corresponding to the G-domain of the gephyrin protein. These findings, together with previous reports of homozygous GPHN mutations in connection with autosomal recessive molybdenum cofactor deficiency, will aid in clinical genetic interpretation of the GPHN mutation spectrum. Our data also add to the accumulating evidence implicating neuronal synaptic gene products as key molecular factors underlying the etiologies of a diverse range of neurodevelopmental conditions. Show less

To summarize recent findings that have examined dietary, genetic and gene-diet interactions that contribute to fat accumulation in the liver during growth and development, with particular focus on contributions relating to dietary carbohydrate and sugar consumption. In addition, this review highlights how some of these contributions to liver fat vary across the population in terms of ethnic-specific effects. Dietary carbohydrate, and especially sugars contribute to increased liver fat accumulation due to the lipogenic potential of fructose during liver metabolism. In addition, recent genome-wide studies have identified several polymorphisms that contribute to increased liver fat accumulation, with some of these genes relating to dietary carbohydrate and sugar consumption. In particular, the patatin-like phospholipase domain-containing protein 3 (PNPLA3) gene, which is highly prevalent in Hispanics, contributes to excessive liver fat beginning at a young age, especially in the context of high sugar consumption. Dietary sugar contributes to liver fat accumulation, with this being explained by de-novo lipogenesis from fructose in the liver. Certain genetic factors, including PNPLA3, glucokinase regulatory protein and APOC3 contribute to increased liver fat accumulation, with these effects being manifested at an early age. Hispanics in particular are at elevated risk for liver fat accumulation because of the higher frequency of genetic variants such as PNPLA3 and glucokinase regulatory protein as well as an interaction between the PNPLA3 and dietary sugar. Show less

Mutations in FBN1 and TGFBR2 genes are the main causative mutations identified in Marfan syndrome (MFS). The major vascular complication of MFS is aneurysm formation. Abdominal aortic aneurysm (AAA) is an acquired disease of later life of unknown etiology. The aim of this study was to examine if genetic aberrations in MFS-related genes FBN1 and TGFBR2 are present in patients with AAA. We assessed the presence of copy number variation (CNV) in FBN1 and TGFBR2 genes in AAA biopsies from twelve patients. We also analyzed the expression of these genes in AAA biopsies compared to control biopsies from six organ donors. In addition we assessed the expression of two members of the Notch signaling pathway NOTCH3 and HEY2 as well as aortic smooth muscle cell (AoSMC) differentiation marker TAGLN in AAA and control biopsies. Loss of one copy (deletion) of the FBN1 exon 66 sequence and TGFBR2 exon 8 was identified in 7 (58%) and 11 (92%) of the 12 AAA biopsies. No copy number amplifications (duplications) were detected. Patients carrying TGFBR2 exon 8 deletion showed marked downregulation of this gene in AAA biopsies compared to control biopsies (0.699 vs. 1.765, p = 0.038). Notch signaling components NOTCH3 and HEY2 were markedly downregulated in AAA, while expression of the AoSMC differentiation marker TAGLN did not differ between AAA and control biopsies (0.468 vs. 0.486, p = 0.546). This study suggests an acquired impairment in TGF-β signaling that along with downregulation of the Notch signaling pathway may contribute to the pathogenesis of AAA. Show less

The three members of the human neurexin gene family, neurexin 1 (NRXN1), neurexin 2 (NRXN2), and neurexin 3 (NRXN3), encode neuronal adhesion proteins that have important roles in synapse development and function. In autism spectrum disorder (ASD), as well as in other neurodevelopmental conditions, rare exonic copy-number variants and/or point mutations have been identified in the NRXN1 and NRXN2 loci. We present clinical characterization of four index cases who have been diagnosed with ASD and who possess rare inherited or de novo microdeletions at 14q24.3-31.1, a region that overlaps exons of the alpha and/or beta isoforms of NRXN3. NRXN3 deletions were found in one father with subclinical autism and in a carrier mother and father without formal ASD diagnoses, indicating issues of penetrance and expressivity at this locus. Notwithstanding these clinical complexities, this report on ASD-affected individuals who harbor NRXN3 exonic deletions advances the understanding of the genetic etiology of autism, further enabling molecular diagnoses. Show less

The Hairy/Enhancer-of-split-related with YRPW-like motif (HEY) family of proteins are transcriptional repressors and downstream effectors of Notch signaling. We previously reported that HEY1 and HEY2 selectively repress androgen receptor (AR) signaling in mammalian cell lines and have shown that in human tissue HEY1 is excluded from the nuclei in prostate cancer but not benign prostatic hyperplasia. We have now characterized a third member of this family, HEYL, which is a more potent repressor of AR activity. HEYL interacted with and repressed AR activation function-1 domain and competitively inhibited SRC1e activation of AR transcriptional activity. Using a cell line inducibly expressing exogenous HEYL, we showed that HEYL represses endogenous AR-regulated genes and reduces androgen-dependent prostate cancer cell growth. Using a trans-repression assay, we identified both trichostatin-sensitive and -insensitive domains within HEYL; however, analysis of endogenous AR target genes suggested that HEYL represses AR activity through histone deacetylase I/II-independent mechanisms. Immunohistochemical analyses of tissue indicated that, in a fashion similar to that previously reported for HEY1, HEYL is excluded from the nuclei in prostate cancer but not adjacent benign tissue. This suggests that nuclear exclusion of HEY proteins may be an important step in the progression of prostate cancer. Show less

Proinsulin is a precursor of mature insulin and C-peptide. Higher circulating proinsulin levels are associated with impaired β-cell function, raised glucose levels, insulin resistance, and type 2 diabetes (T2D). Studies of the insulin processing pathway could provide new insights about T2D pathophysiology. We have conducted a meta-analysis of genome-wide association tests of ∼2.5 million genotyped or imputed single nucleotide polymorphisms (SNPs) and fasting proinsulin levels in 10,701 nondiabetic adults of European ancestry, with follow-up of 23 loci in up to 16,378 individuals, using additive genetic models adjusted for age, sex, fasting insulin, and study-specific covariates. Nine SNPs at eight loci were associated with proinsulin levels (P < 5 × 10(-8)). Two loci (LARP6 and SGSM2) have not been previously related to metabolic traits, one (MADD) has been associated with fasting glucose, one (PCSK1) has been implicated in obesity, and four (TCF7L2, SLC30A8, VPS13C/C2CD4A/B, and ARAP1, formerly CENTD2) increase T2D risk. The proinsulin-raising allele of ARAP1 was associated with a lower fasting glucose (P = 1.7 × 10(-4)), improved β-cell function (P = 1.1 × 10(-5)), and lower risk of T2D (odds ratio 0.88; P = 7.8 × 10(-6)). Notably, PCSK1 encodes the protein prohormone convertase 1/3, the first enzyme in the insulin processing pathway. A genotype score composed of the nine proinsulin-raising alleles was not associated with coronary disease in two large case-control datasets. We have identified nine genetic variants associated with fasting proinsulin. Our findings illuminate the biology underlying glucose homeostasis and T2D development in humans and argue against a direct role of proinsulin in coronary artery disease pathogenesis. Show less

OBJECTIVE Recent genome-wide association studies have revealed loci associated with glucose and insulin-related traits. We aimed to characterize 19 such loci using detailed measures of insulin processing, secretion, and sensitivity to help elucidate their role in regulation of glucose control, insulin secretion and/or action. RESEARCH DESIGN AND METHODS We investigated associations of loci identified by the Meta-Analyses of Glucose and Insulin-related traits Consortium (MAGIC) with circulating proinsulin, measures of insulin secretion and sensitivity from oral glucose tolerance tests (OGTTs), euglycemic clamps, insulin suppression tests, or frequently sampled intravenous glucose tolerance tests in nondiabetic humans (n = 29,084). RESULTS The glucose-raising allele in MADD was associated with abnormal insulin processing (a dramatic effect on higher proinsulin levels, but no association with insulinogenic index) at extremely persuasive levels of statistical significance (P = 2.1 x 10(-71)). Defects in insulin processing and insulin secretion were seen in glucose-raising allele carriers at TCF7L2, SCL30A8, GIPR, and C2CD4B. Abnormalities in early insulin secretion were suggested in glucose-raising allele carriers at MTNR1B, GCK, FADS1, DGKB, and PROX1 (lower insulinogenic index; no association with proinsulin or insulin sensitivity). Two loci previously associated with fasting insulin (GCKR and IGF1) were associated with OGTT-derived insulin sensitivity indices in a consistent direction. CONCLUSIONS Genetic loci identified through their effect on hyperglycemia and/or hyperinsulinemia demonstrate considerable heterogeneity in associations with measures of insulin processing, secretion, and sensitivity. Our findings emphasize the importance of detailed physiological characterization of such loci for improved understanding of pathways associated with alterations in glucose homeostasis and eventually type 2 diabetes. Show less

Glucose levels 2 h after an oral glucose challenge are a clinical measure of glucose tolerance used in the diagnosis of type 2 diabetes. We report a meta-analysis of nine genome-wide association studies (n = 15,234 nondiabetic individuals) and a follow-up of 29 independent loci (n = 6,958-30,620). We identify variants at the GIPR locus associated with 2-h glucose level (rs10423928, beta (s.e.m.) = 0.09 (0.01) mmol/l per A allele, P = 2.0 x 10(-15)). The GIPR A-allele carriers also showed decreased insulin secretion (n = 22,492; insulinogenic index, P = 1.0 x 10(-17); ratio of insulin to glucose area under the curve, P = 1.3 x 10(-16)) and diminished incretin effect (n = 804; P = 4.3 x 10(-4)). We also identified variants at ADCY5 (rs2877716, P = 4.2 x 10(-16)), VPS13C (rs17271305, P = 4.1 x 10(-8)), GCKR (rs1260326, P = 7.1 x 10(-11)) and TCF7L2 (rs7903146, P = 4.2 x 10(-10)) associated with 2-h glucose. Of the three newly implicated loci (GIPR, ADCY5 and VPS13C), only ADCY5 was found to be associated with type 2 diabetes in collaborating studies (n = 35,869 cases, 89,798 controls, OR = 1.12, 95% CI 1.09-1.15, P = 4.8 x 10(-18)). Show less

For most cases of colorectal cancer that arise without a family history of the disease, it is proposed that an appreciable heritable component of predisposition is the result of contributions from many loci. Although progress has been made in identifying single nucleotide variants associated with colorectal cancer risk, the involvement of low-penetrance copy number variants is relatively unexplored. We have used multiplex amplifiable probe hybridization (MAPH) in a fourfold multiplex (QuadMAPH), positioned at an average resolution of one probe per 2 kb, to screen a total of 1.56 Mb of genomic DNA for copy number variants around the genes APC, AXIN1, BRCA1, BRCA2, CTNNB1, HRAS, MLH1, MSH2, and TP53. Two deletion events were detected, one upstream of MLH1 in a control individual and the other in APC in a colorectal cancer patient, but these do not seem to correspond to copy number polymorphisms with measurably high population frequencies. In summary, by means of our QuadMAPH assay, copy number measurement data were of sufficient resolution and accuracy to detect any copy number variants with high probability. However, this study has demonstrated a very low incidence of deletion and duplication variants within intronic and flanking regions of these nine genes, in both control individuals and colorectal cancer patients. Show less

Markers at the pericentriolar material 1 gene (PCM1) have shown genetic association with schizophrenia in both a University College London (UCL) and a USA-based case-control sample. In this paper we report a statistically significant replication of the PCM1 association in a large Scottish case-control sample from Aberdeen. Resequencing of the genomic DNA from research volunteers who had inherited haplotypes associated with schizophrenia showed a threonine to isoleucine missense mutation in exon 24 which was likely to change the structure and function of PCM1 (rs370429). This mutation was found only as a heterozygote in 98 schizophrenic research subjects and controls out of 2246 case and control research subjects. Among the 98 carriers of rs370429, 67 were affected with schizophrenia. The same alleles and haplotypes were associated with schizophrenia in both the London and Aberdeen samples. Another potential aetiological base pair change in PCM1 was rs445422, which altered a splice site signal. A further mutation, rs208747, was shown by electrophoretic mobility shift assays to create or destroy a promoter transcription factor site. Five further non-synonymous changes in exons were also found. Genotyping of the new variants discovered in the UCL case-control sample strengthened the evidence for allelic and haplotypic association (P=0.02-0.0002). Given the number and identity of the haplotypes associated with schizophrenia, further aetiological base pair changes must exist within and around the PCM1 gene. PCM1 protein has been shown to interact directly with the disrupted-in-schizophrenia 1 (DISC1) protein, Bardet-Biedl syndrome 4, and Huntingtin-associated protein 1, and is important in neuronal cell growth. In a separate study we found that clozapine but not haloperidol downregulated PCM1 expression in the mouse brain. We hypothesize that mutant PCM1 may be responsible for causing a subtype of schizophrenia through abnormal cell division and abnormal regeneration in dividing cells in the central nervous system. This is supported by our previous finding of orbitofrontal volumetric deficits in PCM1-associated schizophrenia patients as opposed to temporal pole deficits in non-PCM1-associated schizophrenia patients. Caution needs to be exercised in interpreting the actual biological effects of the mutations we have found without further cell biology. However, the DNA changes we have found deserve widespread genotyping in multiple case-control populations. Show less

Deletions within the neurexin 1 gene (NRXN1; 2p16.3) are associated with autism and have also been reported in two families with schizophrenia. We examined NRXN1, and the closely related NRXN2 and NRXN3 genes, for copy number variants (CNVs) in 2977 schizophrenia patients and 33 746 controls from seven European populations (Iceland, Finland, Norway, Germany, The Netherlands, Italy and UK) using microarray data. We found 66 deletions and 5 duplications in NRXN1, including a de novo deletion: 12 deletions and 2 duplications occurred in schizophrenia cases (0.47%) compared to 49 and 3 (0.15%) in controls. There was no common breakpoint and the CNVs varied from 18 to 420 kb. No CNVs were found in NRXN2 or NRXN3. We performed a Cochran-Mantel-Haenszel exact test to estimate association between all CNVs and schizophrenia (P = 0.13; OR = 1.73; 95% CI 0.81-3.50). Because the penetrance of NRXN1 CNVs may vary according to the level of functional impact on the gene, we next restricted the association analysis to CNVs that disrupt exons (0.24% of cases and 0.015% of controls). These were significantly associated with a high odds ratio (P = 0.0027; OR 8.97, 95% CI 1.8-51.9). We conclude that NRXN1 deletions affecting exons confer risk of schizophrenia. Show less