👤 H-J Martin

The microtubule cytoskeleton regulates cell polarity by spatially organizing membrane trafficking and signaling processes. In epithelial cells, microtubules form parallel arrays aligned along the apico-basal axis, and recent work has demonstrated that the members of CAMSAP/Patronin family control apical tethering of microtubule minus ends. Here, we show that in mammalian intestinal epithelial cells, the spectraplakin ACF7 (also known as MACF1) specifically binds to CAMSAP3 and is required for the apical localization of CAMSAP3-decorated microtubule minus ends. Loss of ACF7 but not of CAMSAP3 or its homolog CAMSAP2 affected the formation of polarized epithelial cysts in three-dimensional cultures. In short-term epithelial polarization assays, knockout of CAMSAP3, but not of CAMSAP2, caused microtubule re-organization into a more radial centrosomal array, redistribution of Rab11-positive (also known as Rab11A) endosomes from the apical cell surface to the pericentrosomal region and inhibition of actin brush border formation at the apical side of the cell. We conclude that ACF7 is an important regulator of apico-basal polarity in mammalian intestinal cells and that a radial centrosome-centered microtubule organization can act as an inhibitor of epithelial polarity. Show less

The plasma proteome remains an attractive biospecimen for MS-based biomarker discovery studies. The success of these efforts relies on the continued development of quantitative MS-based proteomics approaches. Herein we report the use of the SILAC-labeled HepG2 secretome as a source for stable isotope labeled plasma proteins for quantitative LC-MS/MS measurements. The HepG2 liver cancer cell line secretes the major plasma proteins including serum albumin, apolipoproteins, protease inhibitors, coagulation factors, and transporters that represent some of the most abundant proteins in plasma. The SILAC-labeled HepG2 secretome was collected, spiked into human plasma (1:1 total protein), and then processed for LC-MS/MS analysis. A total of 62 and 56 plasma proteins were quantified (heavy:light (H/L) peptide pairs) from undepleted and depleted (serum albumin and IgG), respectively, with log2 H/L = ± 6. Major plasma proteins quantified included albumin, apolipoproteins (e.g., APOA1, APOA2, APOA4, APOB, APOC3, APOE, APOH, and APOM), protease inhibitors (e.g., A2M and SERPINs), coagulation factors (e.g., Factor V, Factor X, fibrinogen), and transport proteins (e.g., TTR). The average log2 H/L values for shared plasma proteins in both undepleted and depleted plasma samples were 0.43 and 0.44, respectively. This work further expands the SILAC strategy into MS-based biomarker discovery of clinical biospecimens. Show less

Coffee, a major dietary source of caffeine, is among the most widely consumed beverages in the world and has received considerable attention regarding health risks and benefits. We conducted a genome-wide (GW) meta-analysis of predominately regular-type coffee consumption (cups per day) among up to 91,462 coffee consumers of European ancestry with top single-nucleotide polymorphisms (SNPs) followed-up in ~30 062 and 7964 coffee consumers of European and African-American ancestry, respectively. Studies from both stages were combined in a trans-ethnic meta-analysis. Confirmed loci were examined for putative functional and biological relevance. Eight loci, including six novel loci, met GW significance (log10Bayes factor (BF)>5.64) with per-allele effect sizes of 0.03-0.14 cups per day. Six are located in or near genes potentially involved in pharmacokinetics (ABCG2, AHR, POR and CYP1A2) and pharmacodynamics (BDNF and SLC6A4) of caffeine. Two map to GCKR and MLXIPL genes related to metabolic traits but lacking known roles in coffee consumption. Enhancer and promoter histone marks populate the regions of many confirmed loci and several potential regulatory SNPs are highly correlated with the lead SNP of each. SNP alleles near GCKR, MLXIPL, BDNF and CYP1A2 that were associated with higher coffee consumption have previously been associated with smoking initiation, higher adiposity and fasting insulin and glucose but lower blood pressure and favorable lipid, inflammatory and liver enzyme profiles (P<5 × 10(-8)).Our genetic findings among European and African-American adults reinforce the role of caffeine in mediating habitual coffee consumption and may point to molecular mechanisms underlying inter-individual variability in pharmacological and health effects of coffee. Show less

Hypertrophic cardiomyopathy (HCM) results from mutations in genes encoding sarcomeric proteins, most often MYBPC3, which encodes cardiac myosin binding protein-C (cMyBP-C). A recently discovered HCM-associated 25-base pair deletion in MYBPC3 is inherited in millions worldwide. Although this mutation causes changes in the C10 domain of cMyBP-C (cMyBP-C(C10mut)), which binds to the light meromyosin (LMM) region of the myosin heavy chain, the underlying molecular mechanism causing HCM is unknown. In this study, adenoviral expression of cMyBP-C(C10mut) in cultured adult rat cardiomyocytes was used to investigate protein localization and evaluate contractile function and Ca(2+) transients, compared with wild-type cMyBP-C expression (cMyBP-C(WT)) and controls. Forty-eight hours after infection, 44% of cMyBP-C(WT) and 36% of cMyBP-C(C10mut) protein levels were determined in total lysates, confirming equal expression. Immunofluorescence experiments showed little or no localization of cMyBP-C(C10mut) to the C-zone, whereas cMyBP-C(WT) mostly showed C-zone staining, suggesting that cMyBP-C(C10mut) could not properly integrate in the C-zone of the sarcomere. Subcellular fractionation confirmed that most cMyBP-C(C10mut) resided in the soluble fraction, with reduced presence in the myofilament fraction. Also, cMyBP-C(C10mut) displayed significantly reduced fractional shortening, sarcomere shortening, and relaxation velocities, apparently caused by defects in sarcomere function, because Ca(2+) transients were unaffected. Co-sedimentation and protein cross-linking assays confirmed that C10(mut) causes the loss of C10 domain interaction with myosin LMM. Protein homology modeling studies showed significant structural perturbation in cMyBP-C(C10mut), providing a potential structural basis for the alteration in its mode of interaction with myosin LMM. Therefore, expression of cMyBP-C(C10mut) protein is sufficient to cause contractile dysfunction in vitro. Show less

Liver X Receptors (LXRs) α and β are nuclear hormone receptors that regulate multiple genes involved in reverse cholesterol transport (RCT) and are potential drug targets for atherosclerosis. However, full pan agonists also activate lipogenic genes, resulting in elevated plasma and hepatic lipids. We report the pharmacology of BMS-779788 [2-(2-(1-(2-chlorophenyl)-1-methylethyl)-1-(3'-(methylsulfonyl)-4-biphenylyl)-1H-imidazol-4-yl)-2-propanol], a potent partial LXR agonist with LXRβ selectivity, which has an improved therapeutic window in the cynomolgus monkey compared with a full pan agonist. BMS-779788 induced LXR target genes in blood in vivo with an EC50 = 610 nM, a value similar to its in vitro blood gene induction potency. BMS-779788 was 29- and 12-fold less potent than the full agonist T0901317 in elevating plasma triglyceride and LDL cholesterol, respectively, with similar results for plasma cholesteryl ester transfer protein and apolipoprotein B. However, ABCA1 and ABCG1 mRNA inductions in blood, which are critical for RCT, were comparable. Increased liver triglyceride was observed after 7-day treatment with BMS-779788 at the highest dose tested and was nearly identical to the dose response for plasma triglyceride, consistent with the central role of liver LXR in these lipogenic effects. Dose-dependent increases in biliary cholesterol and decreases in phospholipid and bile acid occurred in BMS-779788-treated animals, similar to LXR agonist effects reported in mouse. In summary, BMS-779788, a partial LXRβ selective agonist, has decreased lipogenic potential compared with a full pan agonist in cynomolgus monkeys, with similar potency in the induction of genes known to stimulate RCT. This provides support in nonhuman primates for improving LXR agonist therapeutic windows by limiting LXRα activity. Show less

The postprandial chylomicron (CM) triacylglycerol (TG) response to dietary fat, which is positively associated with atherosclerosis and cardiovascular disease risk, displays a high interindividual variability. This is assumed to be due, at least partly, to polymorphisms in genes involved in lipid metabolism. Existing studies have focused on single nucleotide polymorphisms (SNPs), resulting in only a low explained variability. We aimed to identify a combination of SNPs associated with the postprandial CM TG response. Thirty-three healthy male volunteers were subjected to 4 standardized fat tolerance test meals (to correct for intraindividual variability) and genotyped using whole-genome microarrays. The plasma CM TG concentration was measured at regular interval times after each meal. The association of SNPs in or near candidate genes (126 genes representing 6225 SNPs) with the postprandial CM TG concentration (0-8 h areas under the curve averaged for the 4 test meals) was assessed by partial least squares regression, a multivariate statistical approach. Data obtained allowed us to generate a validated significant model (P = 1.3 × 10(-7)) that included 42 SNPs in 23 genes (ABCA1, APOA1, APOA5, APOB, BET1, CD36, COBLL1, ELOVL5, FRMD5, GPAM, INSIG2, IRS1, LDLR, LIPC, LPL, LYPLAL1, MC4R, NAT2, PARK2, SLC27A5, SLC27A6, TCF7L2, and ZNF664) and explained 88% of the variance. In 39 of these SNPs, univariate analysis showed that subjects with different genotypes exhibited significantly different (q < .05) postprandial CM TG responses. Using a multivariate approach, we report a combination of SNPs that explains a significant part of the variability in the postprandial CM TG response. Show less

Plasma triglyceride levels are heritable and are correlated with the risk of coronary heart disease. Sequencing of the protein-coding regions of the human genome (the exome) has the potential to identify rare mutations that have a large effect on phenotype. We sequenced the protein-coding regions of 18,666 genes in each of 3734 participants of European or African ancestry in the Exome Sequencing Project. We conducted tests to determine whether rare mutations in coding sequence, individually or in aggregate within a gene, were associated with plasma triglyceride levels. For mutations associated with triglyceride levels, we subsequently evaluated their association with the risk of coronary heart disease in 110,970 persons. An aggregate of rare mutations in the gene encoding apolipoprotein C3 (APOC3) was associated with lower plasma triglyceride levels. Among the four mutations that drove this result, three were loss-of-function mutations: a nonsense mutation (R19X) and two splice-site mutations (IVS2+1G→A and IVS3+1G→T). The fourth was a missense mutation (A43T). Approximately 1 in 150 persons in the study was a heterozygous carrier of at least one of these four mutations. Triglyceride levels in the carriers were 39% lower than levels in noncarriers (P<1×10(-20)), and circulating levels of APOC3 in carriers were 46% lower than levels in noncarriers (P=8×10(-10)). The risk of coronary heart disease among 498 carriers of any rare APOC3 mutation was 40% lower than the risk among 110,472 noncarriers (odds ratio, 0.60; 95% confidence interval, 0.47 to 0.75; P=4×10(-6)). Rare mutations that disrupt APOC3 function were associated with lower levels of plasma triglycerides and APOC3. Carriers of these mutations were found to have a reduced risk of coronary heart disease. (Funded by the National Heart, Lung, and Blood Institute and others.). Show less

Citrulline is among the metabolites measured by expanded newborn screening (NBS). While hypocitrullinemia can be a marker for deficiency of proximal urea cycle enzymes such as ornithine transcarbamylase (OTC), only a handful of state newborn screening programs in the United States officially report a low citrulline value for further work-up due to low positive predictive value. We report a case of a male infant who was found to have hypocitrullinemia on NBS. After excluding proximal urea cycle disorders by DNA sequencing, his NBS result was felt to be a false positive. At 4 months of age, he developed poor feeding, failure to thrive, apnea and infantile spasms with a progression to intractable seizures, as well as persistent hypocitrullinemia. He was diagnosed with Leigh syndrome due to a maternally inherited homoplasmic m.8993T>G mutation in the ATPase 6 gene. His mother, who had previously been diagnosed with cerebral palsy, was concurrently diagnosed with neuropathy, ataxia, and retinitis pigmentosa (NARP) due to heteroplasmy of the same mutation. She had progressive muscle weakness, ataxia, and speech dyspraxia. The m.8993T>G mutation causes mitochondrial ATP synthase deficiency and it is hypothesized to undermine the synthesis of citrulline by CPS1. In addition to proximal urea cycle disorders, the evaluation of an infant with persistent hypocitrullinemia should include testing for the m.8993T>G mutation and other disorders that cause mitochondrial dysfunction. Show less

Obesity is a crucial public health issue in developed countries, with implications for cardiovascular and brain health as we age. A number of commonly-carried genetic variants are associated with obesity. Here we aim to see whether variants in obesity-associated genes--NEGR1, FTO, MTCH2, MC4R, LRRN6C, MAP2K5, FAIM2, SEC16B, ETV5, BDNF-AS, ATXN2L, ATP2A1, KCTD15, and TNN13K--are associated with white matter microstructural properties, assessed by high angular resolution diffusion imaging (HARDI) in young healthy adults between 20 and 30 years of age from the Queensland Twin Imaging study (QTIM). We began with a multi-locus approach testing how a number of common genetic risk factors for obesity at the single nucleotide polymorphism (SNP) level may jointly influence white matter integrity throughout the brain and found a wide spread genetic effect. Risk allele rs2815752 in NEGR1 was most associated with lower white matter integrity across a substantial portion of the brain. Across the area of significance in the bilateral posterior corona radiata, each additional copy of the risk allele was associated with a 2.2% lower average FA. This is the first study to find an association between an obesity risk gene and differences in white matter integrity. As our subjects were young and healthy, our results suggest that NEGR1 has effects on brain structure independent of its effect on obesity. Show less

The pubertal height growth spurt is a distinctive feature of childhood growth reflecting both the central onset of puberty and local growth factors. Although little is known about the underlying genetics, growth variability during puberty correlates with adult risks for hormone-dependent cancer and adverse cardiometabolic health. The only gene so far associated with pubertal height growth, LIN28B, pleiotropically influences childhood growth, puberty and cancer progression, pointing to shared underlying mechanisms. To discover genetic loci influencing pubertal height and growth and to place them in context of overall growth and maturation, we performed genome-wide association meta-analyses in 18 737 European samples utilizing longitudinally collected height measurements. We found significant associations (P < 1.67 × 10(-8)) at 10 loci, including LIN28B. Five loci associated with pubertal timing, all impacting multiple aspects of growth. In particular, a novel variant correlated with expression of MAPK3, and associated both with increased prepubertal growth and earlier menarche. Another variant near ADCY3-POMC associated with increased body mass index, reduced pubertal growth and earlier puberty. Whereas epidemiological correlations suggest that early puberty marks a pathway from rapid prepubertal growth to reduced final height and adult obesity, our study shows that individual loci associating with pubertal growth have variable longitudinal growth patterns that may differ from epidemiological observations. Overall, this study uncovers part of the complex genetic architecture linking pubertal height growth, the timing of puberty and childhood obesity and provides new information to pinpoint processes linking these traits. Show less

The posterior lateral line primordium (PLLp) migrates caudally and periodically deposits neuromasts. Coupled, but mutually inhibitory, Wnt-FGF signaling systems regulate proto-neuromast formation in the PLLp: FGF ligands expressed in response to Wnt signaling activate FGF receptors and initiate proto-neuromast formation. FGF receptor signaling, in turn, inhibits Wnt signaling. However, mechanisms that determine periodic neuromast formation and deposition in the PLLp remain poorly understood. Previous studies showed that neuromasts are deposited closer together and the PLLp terminates prematurely in lef1-deficient zebrafish embryos. It was suggested that this results from reduced proliferation in the leading domain of the PLLp and/or premature incorporation of progenitors into proto-neuromasts. We found that rspo3 knockdown reduces proliferation in a manner similar to that seen in lef1 morphants. However, it does not cause closer neuromast deposition or premature termination of the PLLp, suggesting that such changes in lef1-deficient embryos are not linked to changes in proliferation. Instead, we suggest that they are related to the role of Lef1 in regulating the balance of Wnt and FGF functions in the PLLp. Lef1 determines expression of the FGF signaling inhibitor Dusp6 in leading cells and regulates incorporation of cells into neuromasts; reduction of Dusp6 in leading cells in lef1-deficient embryos allows new proto-neuromasts to form closer to the leading edge. This is associated with progressively slower PLLp migration, reduced spacing between deposited neuromasts and premature termination of the PLLp system. Show less

Obesity is a major health issue that impedes the ability of preconditioning and postconditioning to protect the myocardium against infarction secondary to dysregulation of kinase signaling pathways. Moreover, exercise decreases cardiovascular mortality in obese patients but the mechanism remains to be established. Wild-type (WT) and obese (ob/ob) mice were assigned to sedentary conditions or regular treadmill exercise (1h/day, 5 days/7, 4 weeks, 4° slope, 10-30 cm/s) and underwent 30 min of coronary artery occlusion followed by 24h of reperfusion for infarct size measurement. In WT, exercise reduced infarct size by 60% and increased phosphorylation of kinases such as Akt, ERK 1/2, p70S6K, AMPK and GSK3β. Importantly, the level of corresponding phosphatases PTEN, MKP-3 and PP2C was decreased. Calcium concentration inducing the opening of mitochondrial permeability transition pore (mPTP) was increased by exercise. In ob/ob, regular exercise induced a robust cardioprotection by reducing infarct size (-67%), increasing kinase phosphorylation, decreasing phosphatase levels and improving the resistance to mPTP opening. However exercise did not modify hyperglycemia, hypercholesterolemia, hyperinsulinemia, fat mass and body weight in obese mice. In conclusion, regular exercise induces cardioprotection against myocardial infarction despite obesity and restores pro-survival signaling pathways with simultaneous increase in kinase phosphorylations, decreased levels of phosphatases and increased resistance of mPTP opening, independently from improvement in associated co-morbidities. Show less

Endocrine resistance and metastatic progression are primary causes of treatment failure in breast cancer. While mitogen activated protein kinases (MAPKs) are known to promote ligand-independent cell growth, the role of the MEK5-ERK5 pathway in the progression of clinical breast carcinoma remains poorly understood. Here, we demonstrated increased ERK5 activation in 30 of 39 (76.9%) clinical tumor samples, as well as across breast cancer cell systems. Overexpression of MEK5 in MCF-7 cells promoted both hormone-dependent and hormone-independent tumorigenesis in vitro and in vivo and conferred endocrine therapy resistance to previously sensitive breast cancer cells. Expression of MEK5 suppressed estrogen receptor (ER)α, but not ER-β protein levels, and abrogated downstream estrogen response element (ERE) transcriptional activity and ER-mediated gene transcription. Global gene expression changes associated with upregulation of MEK5 included increased activation of ER-α independent growth signaling pathways and promotion of epithelial-to-mesenchymal transition (EMT) markers. Taken together, our findings show that the MEK5-ERK5 pathway mediates progression to an ER(-), mesenchymal and endocrine therapy resistant phenotype. Given the need for new clinical therapeutic targets, our results demonstrate the therapeutic potential of targeting the MEK5-ERK5 pathway in breast cancer. Show less

Borderline personality disorder (BPD) and substance use disorders frequently co-occur; their dual presence predicts poor prognosis. The genetic underpinnings of BPD have not been well-characterized and could offer insight into comorbidity. The current report focuses on the association of neurexin 3 (NRXN3) single nucleotide polymorphisms (SNPs) with BPD symptoms in heroin dependent cases and controls. The sample of the Comorbidity and Trauma Study, a genetic association study of heroin dependence, consists of Australian heroin dependent cases ascertained from opioid replacement therapy clinics and controls ascertained in nearby economically disadvantaged neighborhoods. The assessment included a screening instrument for BPD, used previously in Australian population surveys. Genotypic and BPD phenotypic data were available for 1439 cases and 507 controls. We examined the association of 1430 candidate gene SNPs with BPD phenotypes. One or more NRXN3 SNPs were nominally associated with all BPD phenotypes; however, none met the conservative significance threshold we employed to correct for multiple testing. The most strongly associated SNPs included rs10144398 with identity disturbance (p=4.9×10(-5)) and rs10151731 with affective instability (p=8.8×10(-5)). The strongest association with screening positive for BPD was found for the NRXN3 SNP, rs10083466 (p=.0013). Neither the correlation of BPD phenotypes nor the linkage disequilibrium relationships of the SNPs account for the number of observed associations involving NRXN3 SNPs. Our findings provide intriguing preliminary evidence for the association of NRXN3 with BPD phenotypes. The strongest associations were found for traits (i.e., affective instability; identity disturbance) also observed with other disorders. Show less

Major depressive disorder (MDD) is a common complex disorder with a partly genetic etiology. We conducted a genome-wide association study of the MDD2000+ sample (2431 cases, 3673 screened controls and >1 M imputed single-nucleotide polymorphisms (SNPs)). No SNPs achieved genome-wide significance either in the MDD2000+ study, or in meta-analysis with two other studies totaling 5763 cases and 6901 controls. These results imply that common variants of intermediate or large effect do not have main effects in the genetic architecture of MDD. Suggestive but notable results were (a) gene-based tests suggesting roles for adenylate cyclase 3 (ADCY3, 2p23.3) and galanin (GAL, 11q13.3); published functional evidence relates both of these to MDD and serotonergic signaling; (b) support for the bipolar disorder risk variant SNP rs1006737 in CACNA1C (P=0.020, odds ratio=1.10); and (c) lack of support for rs2251219, a SNP identified in a meta-analysis of affective disorder studies (P=0.51). We estimate that sample sizes 1.8- to 2.4-fold greater are needed for association studies of MDD compared with those for schizophrenia to detect variants that explain the same proportion of total variance in liability. Larger study cohorts characterized for genetic and environmental risk factors accumulated prospectively are likely to be needed to dissect more fully the etiology of MDD. Show less

The antidiabetic drug metformin has antitumor activity in a variety of cancers because it blocks cell growth by inhibiting TORC1. Here, we show that melanoma cells that are driven by oncogenic BRAF are resistant to the growth-inhibitory effects of metformin because RSK sustains TORC1 activity even when AMP-activated protein kinase (AMPK) is activated. We further show that AMPK targets the dual-specificity protein phosphatase DUSP6 for degradation and this increases ERK activity, which then upregulates the VEGF-A protein. Critically, this drives angiogenesis and accelerates the growth of BRAF-driven tumors in mice. Unexpectedly, however, when VEGF signaling is inhibited, instead of accelerating tumor growth, metformin inhibits tumor growth. Thus, we show that BRAF-driven melanoma cells are resistant to the antigrowth effects of AMPK and that AMPK mediates cell-autonomous and cell-nonautonomous effects that accelerate the growth of these cells in vivo. Metformin inhibits the growth of most tumor cells, but BRAF-mutant melanoma cells are resistant to metformin in vitro, and metformin accelerates their growth in vivo. Unexpectedly, VEGF inhibitors and metformin synergize to suppress the growth of BRAF-mutant tumors, revealing a combination of drugs that may be effective in these patients. Show less

We implemented least absolute shrinkage and selection operator (LASSO) regression to evaluate gene effects in genome-wide association studies (GWAS) of brain images, using an MRI-derived temporal lobe volume measure from 729 subjects scanned as part of the Alzheimer's Disease Neuroimaging Initiative (ADNI). Sparse groups of SNPs in individual genes were selected by LASSO, which identifies efficient sets of variants influencing the data. These SNPs were considered jointly when assessing their association with neuroimaging measures. We discovered 22 genes that passed genome-wide significance for influencing temporal lobe volume. This was a substantially greater number of significant genes compared to those found with standard, univariate GWAS. These top genes are all expressed in the brain and include genes previously related to brain function or neuropsychiatric disorders such as MACROD2, SORCS2, GRIN2B, MAGI2, NPAS3, CLSTN2, GABRG3, NRXN3, PRKAG2, GAS7, RBFOX1, ADARB2, CHD4, and CDH13. The top genes we identified with this method also displayed significant and widespread post hoc effects on voxelwise, tensor-based morphometry (TBM) maps of the temporal lobes. The most significantly associated gene was an autism susceptibility gene known as MACROD2. We were able to successfully replicate the effect of the MACROD2 gene in an independent cohort of 564 young, Australian healthy adult twins and siblings scanned with MRI (mean age: 23.8 ± 2.2 SD years). Our approach powerfully complements univariate techniques in detecting influences of genes on the living brain. Show less

Autism spectrum disorders (ASDs) are highly heritable, yet relatively few associated genetic loci have been replicated. Copy number variations (CNVs) have been implicated in autism; however, the majority of loci contribute to <1% of the disease population. Therefore, independent studies are important to refine associated CNV regions and discover novel susceptibility genes. In this study, a genome-wide SNP array was utilized for CNV detection by two distinct algorithms in a European ancestry case-control data set. We identify a significantly higher burden in the number and size of deletions, and disrupting more genes in ASD cases. Moreover, 18 deletions larger than 1 Mb were detected exclusively in cases, implicating novel regions at 2q22.1, 3p26.3, 4q12 and 14q23. Case-specific CNVs provided further evidence for pathways previously implicated in ASDs, revealing new candidate genes within the GABAergic signaling and neural development pathways. These include DBI, an allosteric binder of GABA receptors, GABARAPL1, the GABA receptor-associated protein, and SLC6A11, a postsynaptic GABA transporter. We also identified CNVs in COBL, deletions of which cause defects in neuronal cytoskeleton morphogenesis in model vertebrates, and DNER, a neuron-specific Notch ligand required for cerebellar development. Moreover, we found evidence of genetic overlap between ASDs and other neurodevelopmental and neuropsychiatric diseases. These genes include glutamate receptors (GRID1, GRIK2 and GRIK4), synaptic regulators (NRXN3, SLC6A8 and SYN3), transcription factor (ZNF804A) and RNA-binding protein FMR1. Taken together, these CNVs may be a few of the missing pieces of ASD heritability and lead to discovering novel etiological mechanisms. Show less

A recent meta-analysis of genome-wide association (GWA) studies identified 95 loci that influence lipid traits in the adult population and found that collectively these explained about 25-30% of heritability for each trait. Little is known about how these loci affect lipid levels in early life, but there is evidence that genetic effects on HDL- and LDL-cholesterol (HDL-C, LDL-C) and triglycerides vary with age. We studied Australian adults (N = 10,151) and adolescents (N = 2,363) who participated in twin and family studies and for whom we have lipid phenotypes and genotype information for 91 of the 95 genetic variants. Heterogeneity tests between effect sizes in adult and adolescent cohorts showed an excess of heterogeneity for HDL-C (p(Het)<0.05 at 5 out of 37 loci), but no more than expected by chance for LDL-C (1 out of 14 loci), or trigycerides (0 out 24). There were 2 (out of 5) with opposite direction of effect in adolescents compared to adults for HDL-C, but none for LDL-C. The biggest difference in effect size was for LDL-C at rs6511720 near LDLR, adolescents (0.021 ± 0.033 mmol/L) and adults (0.157 ± 0.023 mmol/L), p(Het) = 0.013; followed by ZNF664 (p(Het) = 0.018) and PABPC4 (p(Het) = 0.034) for HDL-C. Our findings suggest that some of the previously identified variants associate differently with lipid traits in adolescents compared to adults, either because of developmental changes or because of greater interactions with environmental differences in adults. Show less

Concentrations of liver enzymes in plasma are widely used as indicators of liver disease. We carried out a genome-wide association study in 61,089 individuals, identifying 42 loci associated with concentrations of liver enzymes in plasma, of which 32 are new associations (P = 10(-8) to P = 10(-190)). We used functional genomic approaches including metabonomic profiling and gene expression analyses to identify probable candidate genes at these regions. We identified 69 candidate genes, including genes involved in biliary transport (ATP8B1 and ABCB11), glucose, carbohydrate and lipid metabolism (FADS1, FADS2, GCKR, JMJD1C, HNF1A, MLXIPL, PNPLA3, PPP1R3B, SLC2A2 and TRIB1), glycoprotein biosynthesis and cell surface glycobiology (ABO, ASGR1, FUT2, GPLD1 and ST3GAL4), inflammation and immunity (CD276, CDH6, GCKR, HNF1A, HPR, ITGA1, RORA and STAT4) and glutathione metabolism (GSTT1, GSTT2 and GGT), as well as several genes of uncertain or unknown function (including ABHD12, EFHD1, EFNA1, EPHA2, MICAL3 and ZNF827). Our results provide new insight into genetic mechanisms and pathways influencing markers of liver function. Show less

Dilated cardiomyopathy (DCM) in infants and children can be partially explained by genetic cause but the catalogue of known genes is limited. We reviewed our database of 41 cases diagnosed with DCM before 18 years of age who underwent detailed clinical and genetic evaluation, and summarize here the evidence for mutations causing DCM in these cases from 15 genes (PSEN1, PSEN2, CSRP3, LBD3, MYH7, SCN5A, TCAP, TNNT2, LMNA, MYBPC3, MYH6, TNNC1, TNNI3, TPM1, and RBM20). Thirty-five of the 41 pediatric cases had relatives with adult-onset DCM. More males (66%) were found among children diagnosed after 1 year of age with DCM. Nineteen mutations in 9 genes were identified among 15 out of 41 patients; 3 patients (diagnosed at ages 2 weeks, 9 and 13 years) had multiple mutations. Of the 19 mutations identified in 12 families, mutations in TPM1 (32%) and TNNT2 (21%) were the most commonly found. Of the 6 patients diagnosed before 1 year of age, 3 had mutations in TPM1 (including a set of identical twins), 1 in TNNT2, 1 in MYH7, and 1 with multiple mutations (MYH7 and TNNC1). Most DCM was accompanied by advanced heart failure and need for cardiac transplantation. We conclude that in some cases pediatric DCM has a genetic basis, which is complicated by allelic and locus heterogeneity as seen in adult-onset DCM. We suggest that future prospective comprehensive family-based genetic studies of pediatric DCM are indicated to further define mutation frequencies in known genes and to discover novel genetic cause. Show less

Liver X receptor-α (LXRA) is a nuclear receptor that regulates genes important in cholesterol homeostasis and inflammation. Several single nucleotide polymorphisms (SNPs) in the LXRA gene (NR1H3) have been earlier associated with metabolic phenotypes (dyslipidemia and elevated body mass index). Metabolic dysregulation is a major contributor to coronary disease; therefore, we assessed LXRA in International Verapamil Sustained Release SR Trandolapril Study Genetic Substudy (INVEST-GENES), a genetic-substudy of a large clinical trial in patients with hypertension and coronary artery disease. Seven tag SNPs in the LXRA gene region (NR1H3) were selected for study: rs11039149, rs12221497, rs2279238, rs7120118, rs326213, rs11039159, and rs10501321. One thousand fifty-nine patients were genotyped from the INVEST-GENES case-control set (verapamil-sustained release-based or atenolol-based treatment strategies) that comprised of 297 cases frequency matched (approximately 2.5:1) with that of event-free controls by sex and race. The primary outcome was defined as first occurrence of all-cause death, nonfatal myocardial infarction, or nonfatal stroke. Adjusted odds ratios (ORs) were calculated using logistic regression. Three of the seven SNPs were associated with significant effects on the primary outcome in nonBlacks. The variant G allele of rs11039149 and the variant A allele of rs12221497 were associated with reduced risk of experiencing the primary outcome [OR: 0.62, confidence interval (CI): 0.45-0.85, P=0.003 and OR: 0.60, CI: 0.39-0.91, P=0.016, respectively]. The rs2279238 genotype was associated with a significant increase in risk for the primary outcome (OR: 1.42, CI: 1.03-1.95, P=0.03). Furthermore, there was a significant genotype-treatment strategy interaction for carriers of the variant T allele of rs2279238 (OR for verapamil-sustained release strategy compared with atenolol strategy: 2.86, CI: 1.50-5.46, P=0.0015). Diplotype analyses showed that the SNPs are rarely coinherited and support the directionally opposite effects of the SNPs on the primary outcome. LXRA genotypes were associated with variable risk for cardiovascular outcomes and pharmacogenetic effect in INVEST-GENES. These novel findings suggest that LXRA is a genetic/pharmacogenetic target that should be further explored. Show less

Obesity is globally prevalent and highly heritable, but its underlying genetic factors remain largely elusive. To identify genetic loci for obesity susceptibility, we examined associations between body mass index and ∼ 2.8 million SNPs in up to 123,865 individuals with targeted follow up of 42 SNPs in up to 125,931 additional individuals. We confirmed 14 known obesity susceptibility loci and identified 18 new loci associated with body mass index (P < 5 × 10⁻⁸), one of which includes a copy number variant near GPRC5B. Some loci (at MC4R, POMC, SH2B1 and BDNF) map near key hypothalamic regulators of energy balance, and one of these loci is near GIPR, an incretin receptor. Furthermore, genes in other newly associated loci may provide new insights into human body weight regulation. Show less

Gprc5b, a retinoic acid-inducible orphan G protein-coupled receptor (GPCR), is a member of the group C metabotropic glutamate receptor family proteins possibly involved in non-canonical Wnt signaling. Many GPCR transcripts are alternatively spliced, which diversifies this class of proteins in their cell- and tissue-specific signaling, regulatory and/or pharmacological properties. We previously generated p97FE65 isoform-specific knockout mice that showed learning/memory deficits. In this study, we further characterized the 97FE65 null mice using cDNA microarray and RT-PCR analyses. We discovered a novel brain-specific C-terminal splice variant of Gprc5b, Gprc5b_v2, which was differentially expressed in p97FE65 wild type and null mouse brains. The null mice were generated in 129/Sv ES cells, and backcrossed to C57Bl/6J for ten generations. We found that expression of Gprc5b_v2 mRNA in the brains of p97FE65 null mice was dramatically down-regulated (more than 20 fold) compared to their wild type littermates. However, expression profiles of Gprc5b variants and SNP analysis surrounding the FE65 locus suggest that the down-regulation is unlikely due to the altered FE65 function, but rather is caused by gene retention from the 129/Sv ES cells. Consistently, in contrast to ubiquitously expressed Gprc5b_v1, Gprc5b_v2 was predominantly expressed in the brain tissues of C57Bl/6J mice. The alternative splicing of the 3' terminal exon also altered the protein coding sequences, giving rise to the characteristic C-termini. Levels of Gprc5b_v2 mRNA were increased during neuronal maturation, paralleling the expression of synaptic proteins. Overexpression of both Gprc5b variants stimulated neurite-like outgrowth in a neuroblastoma cell line. Our results suggest that Gprc5b-v2 may play a role during brain maturation and in matured brain, possibly through the regulation of neuronal morphology and protein-protein interaction. This study also highlights the fact that unexpected gene retention following repeated backcrosses can lead to important biological consequences. Show less

To analyze the mechanisms of folate uptake in retinal Müller cells. RT-PCR and Western blot analysis were performed in freshly isolated neural retina and RPE/eyecup, primary mouse Müller cells, and rMC-1 cells for the three known folate transport proteins folate receptor alpha (FRalpha), proton-coupled folate transporter (PCFT), and reduced folate carrier (RFC). Laser scanning confocal microscopy (LSCM) and immunoelectron microscopy were used to determine the subcellular location of FRalpha and PCFT in primary Müller cells. The pH dependence of the uptake of [(3)H]-methyltetrahydrofolate ([(3)H]-MTF) was assayed in Müller cells in the presence/absence of thiamine pyrophosphate, an inhibitor of RFC. FRalpha and PCFT are expressed abundantly in the retina in several cell layers, including the inner nuclear layer; they are present in primary mouse Müller cells and rMC-1 cells. LSCM localized these proteins to the plasma membrane, nuclear membrane, and perinuclear region. Immunoelectron microscopic studies revealed the colocalization of FRalpha and PCFT on the plasma membrane and nuclear membrane and within endosomal structures. Müller cell uptake of [(3)H]-MTF was robust at pH 5.0 to 6.0, consistent with PCFT activity, but also at neutral pH, reflecting RFC function. RFC was expressed in mouse Müller cells that had been allowed to proliferate in culture, but not in freshly isolated primary cells. FRalpha and PCFT are expressed in retinal Müller cells and colocalize in the endosomal compartment, suggesting that the two proteins may work coordinately to mediate folate uptake. The unexpected finding of RFC expression and activity in cultured Müller cells may reflect the upregulation of this protein under proliferative conditions. Show less

To identify loci for age at menarche, we performed a meta-analysis of 32 genome-wide association studies in 87,802 women of European descent, with replication in up to 14,731 women. In addition to the known loci at LIN28B (P = 5.4 × 10⁻⁶⁰) and 9q31.2 (P = 2.2 × 10⁻³³), we identified 30 new menarche loci (all P < 5 × 10⁻⁸) and found suggestive evidence for a further 10 loci (P < 1.9 × 10⁻⁶). The new loci included four previously associated with body mass index (in or near FTO, SEC16B, TRA2B and TMEM18), three in or near other genes implicated in energy homeostasis (BSX, CRTC1 and MCHR2) and three in or near genes implicated in hormonal regulation (INHBA, PCSK2 and RXRG). Ingenuity and gene-set enrichment pathway analyses identified coenzyme A and fatty acid biosynthesis as biological processes related to menarche timing. Show less

To analyze the aberrant expression of the GIPR and LHCGR in different forms of adrenocortical hyperplasia: ACTH-independent macronodular adrenal hyperplasia (AIMAH), primary pigmented nodular adrenocortical disease (PPNAD) and diffuse adrenal hyperplasia secondary to Cushing's disease (DAHCD). We quantified GIPR and LHCGR expressions using real time PCR in 20 patients with adrenocortical hyperplasia (seven with AIMAH, five with PPNAD, and eight with DAHCD). Normal adrenals tissues were used as control and the relative expression was compared with beta-actin. GIPR and LHCGR expressions were demonstrated in all tissues studied. Median GIPR and LHCGR mRNA levels were 1.6; 0.4; 0.5 and 1.3; 0.9; 1.0 in adrenocortical tissues from AIMAH, PPNAD and DAHCD respectively. There were no differences between GIPR and LHCGR expressions in all tissues studied. GIPR and LHCGR overexpression were not identified in the studied cases, thus suggesting that this molecular mechanism is not involved in adrenocortical hyperplasia in our patients. Show less

Glucose-dependent insulinotropic peptide receptor (GIPR) and LHCGR are G-protein-coupled receptors with a wide tissue expression pattern. Aberrant expression of these receptors has rarely been demonstrated in adult sporadic adrenocortical tumors with a lack of data on pediatric tumors. We quantified the GIPR and LHCGR expression in a large cohort of 55 patients (25 children and 30 adults) with functioning and non-functioning sporadic adrenocortical tumors. Thirty-eight tumors were classified as adenomas whereas 17 were carcinomas. GIPR and LHCGR expression were analyzed by real-time PCR and normal human pancreatic and testicular tissue samples were used as positive controls. Mean expression values were determined by fold increase in comparison with a normal adrenal pool. GIPR mRNA levels were significantly higher in adrenocortical carcinomas than in adenomas from both pediatric and adult groups. LHCGR expression was similar in both carcinomas and adenomas from the pediatric group but significantly lower in carcinomas than in adenomas from the adult group (median 0.06 and 2.3 respectively, P<0.001). GIPR was detected by immunohistochemistry in both pediatric and adult tumors. Staining and real-time PCR results correlated positively only when GIPR mRNA levels were increased at least two-fold in comparison with normal adrenal expression levels. In conclusion, GIPR overexpression was observed in pediatric and adult adrenocortical tumors and very low levels of LHCGR expression were found in all adult adrenocortical carcinomas. Show less

Recent genome-wide association (GWA) studies of lipids have been conducted in samples ascertained for other phenotypes, particularly diabetes. Here we report the first GWA analysis of loci affecting total cholesterol (TC), low-density lipoprotein (LDL) cholesterol, high-density lipoprotein (HDL) cholesterol and triglycerides sampled randomly from 16 population-based cohorts and genotyped using mainly the Illumina HumanHap300-Duo platform. Our study included a total of 17,797-22,562 persons, aged 18-104 years and from geographic regions spanning from the Nordic countries to Southern Europe. We established 22 loci associated with serum lipid levels at a genome-wide significance level (P < 5 x 10(-8)), including 16 loci that were identified by previous GWA studies. The six newly identified loci in our cohort samples are ABCG5 (TC, P = 1.5 x 10(-11); LDL, P = 2.6 x 10(-10)), TMEM57 (TC, P = 5.4 x 10(-10)), CTCF-PRMT8 region (HDL, P = 8.3 x 10(-16)), DNAH11 (LDL, P = 6.1 x 10(-9)), FADS3-FADS2 (TC, P = 1.5 x 10(-10); LDL, P = 4.4 x 10(-13)) and MADD-FOLH1 region (HDL, P = 6 x 10(-11)). For three loci, effect sizes differed significantly by sex. Genetic risk scores based on lipid loci explain up to 4.8% of variation in lipids and were also associated with increased intima media thickness (P = 0.001) and coronary heart disease incidence (P = 0.04). The genetic risk score improves the screening of high-risk groups of dyslipidemia over classical risk factors. Show less

GPR109A has been identified as a G-protein-coupled receptor for niacin. beta-hydroxybutyrate (beta-HB) is a physiologic ligand for the receptor. beta-HB, the predominate ketone body in circulation, is an important energy source for neurons, including retinal neurons, under various physiologic and pathologic conditions. The identification of GPR109A as the receptor for beta-HB suggests additional, hitherto unknown, functions for this metabolite. The circulating levels of beta-HB increase in diabetes. Since retinopathy is a serious complication associated with diabetes, we investigated GPR109A expression in retina and in different retinal cell types to determine if the receptor may have a role in the pathophysiology of diabetic retinopathy. RT-PCR, fluorescent in situ hybridization, and immunofluorescent techniques were used to analyze GPR109A expression in mouse retina and in three transformed retinal cell lines: ARPE-19 (RPE), RGC-5 (ganglion), and rMC-1 (Müller). Activation of GPR109A by niacin and beta-HB was demonstrated in ARPE-19 cells by cAMP assay. Studies conducted using mouse retinal tissues demonstrated that GPR109A is expressed in retina with its expression restricted to RPE, where it differentially polarizes to the basolateral membrane. These results were confirmed with cell lines, which demonstrated GPR109A expression in ARPE-19, but not in rMC-1 and RGC-5 cells. Primary cultures of mouse RPE also showed robust expression of GPR109A. cAMP assay demonstrated that GPR109A expressed in RPE is functional. These data represent the first report on GPR109A expression in retina. The exclusive expression of GPR109A in RPE basolateral membrane, which has access to beta-HB in blood, may have biologic importance in diabetic retinopathy. Show less