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Epithelial ovarian cancer (EOC) is the fifth leading cause of cancer mortality in American women. Normal ovarian physiology is intricately connected to small GTP binding proteins of the Ras superfamily (Ras, Rho, Rab, Arf, and Ran) which govern processes such as signal transduction, cell proliferation, cell motility, and vesicle transport. We hypothesized that common germline variation in genes encoding small GTPases is associated with EOC risk. We investigated 322 variants in 88 small GTPase genes in germline DNA of 18,736 EOC patients and 26,138 controls of European ancestry using a custom genotype array and logistic regression fitting log-additive models. Functional annotation was used to identify biofeatures and expression quantitative trait loci that intersect with risk variants. One variant, ARHGEF10L (Rho guanine nucleotide exchange factor 10 like) rs2256787, was associated with increased endometrioid EOC risk (OR = 1.33, p = 4.46 x 10-6). Other variants of interest included another in ARHGEF10L, rs10788679, which was associated with invasive serous EOC risk (OR = 1.07, p = 0.00026) and two variants in AKAP6 (A-kinase anchoring protein 6) which were associated with risk of invasive EOC (rs1955513, OR = 0.90, p = 0.00033; rs927062, OR = 0.94, p = 0.00059). Functional annotation revealed that the two ARHGEF10L variants were located in super-enhancer regions and that AKAP6 rs927062 was associated with expression of GTPase gene ARHGAP5 (Rho GTPase activating protein 5). Inherited variants in ARHGEF10L and AKAP6, with potential transcriptional regulatory function and association with EOC risk, warrant investigation in independent EOC study populations. Show less

Achalasia is a rare motility disorder characterized by myenteric neuron and interstitial cells of Cajal (ICC) abnormalities leading to deranged/absent peristalsis and lack of relaxation of the lower esophageal sphincter. The mechanisms contributing to neuronal and ICC changes in achalasia are only partially understood. Our goal was to identify novel molecular features occurring in patients with primary achalasia. Esophageal full-thickness biopsies from 42 (22 females; age range: 16-82 years) clinically, radiologically, and manometrically characterized patients with primary achalasia were examined and compared to those obtained from 10 subjects (controls) undergoing surgery for uncomplicated esophageal cancer (or upper stomach disorders). Tissue RNA extracted from biopsies of cases and controls was used for library preparation and sequencing. Data analysis was performed with the "edgeR" option of R-Bioconductor. Data were validated by real-time RT-PCR, western blotting and immunohistochemistry. Quantitative transcriptome evaluation and cluster analysis revealed 111 differentially expressed genes, with a P ≤ 10 The identification of altered gene expression, including INPP4B, a regulator of the Akt pathway, highlights novel signaling pathways involved in the neuronal and ICC changes underlying primary achalasia. Show less

A major flaw in autism spectrum disorder (ASD) management is late diagnosis. Activity-dependent neuroprotective protein (ADNP) is a most frequent de novo mutated ASD-related gene. Functionally, ADNP protects nerve cells against electrical blockade. In mice, complete Adnp deficiency results in dysregulation of over 400 genes and failure to form a brain. Adnp haploinsufficiency results in cognitive and social deficiencies coupled to sex- and age-dependent deficits in the key microtubule and ion channel pathways. Here, collaborating with parents/caregivers globally, we discovered premature tooth eruption as a potential early diagnostic biomarker for ADNP mutation. The parents of 44/54 ADNP-mutated children reported an almost full erupted dentition by 1 year of age, including molars and only 10 of the children had teeth within the normal developmental time range. Looking at Adnp-deficient mice, by computed tomography, showed significantly smaller dental sacs and tooth buds at 5 days of age in the deficient mice compared to littermate controls. There was only trending at 2 days, implicating age-dependent dysregulation of teething in Adnp-deficient mice. Allen Atlas analysis showed Adnp expression in the jaw area. RNA sequencing (RNAseq) and gene array analysis of human ADNP-mutated lymphoblastoids, whole-mouse embryos and mouse brains identified dysregulation of bone/nervous system-controlling genes resulting from ADNP mutation/deficiency (for example, BMP1 and BMP4). AKAP6, discovered here as a major gene regulated by ADNP, also links cognition and bone maintenance. To the best of our knowledge, this is the first time that early primary (deciduous) teething is related to the ADNP syndrome, providing for early/simple diagnosis and paving the path to early intervention/specialized treatment plan. Show less

Particulate matter (PM) exposure leads to premature death, mainly due to respiratory and cardiovascular diseases. Identification of transcriptomic biomarkers of air pollution exposure and effect in a healthy adult population. Microarray analyses were performed in 98 healthy volunteers (48 men, 50 women). The expression of eight sex-specific candidate biomarker genes (significantly associated with PM Average long-term PM Expression of the sex-specific candidate genes identified in the discovery population predicted PM Show less

Atherosclerosis in the carotid arteries is a major cause of ischemic stroke, which accounts for 85% of all stroke cases. Genetic factors contributing to carotid atherosclerosis remain poorly understood. The aim of this study was to identify chromosomal regions harboring genes contributing to carotid atherosclerosis in mice. From an intercross between BALB/cJ (BALB) and SM/J (SM) apolipoprotein E-deficient ( Show less

In this study, we report the experience of the only reference clinical next-generation sequencing lab in Saudi Arabia with the first 1000 families who span a wide-range of suspected Mendelian phenotypes. A total of 1019 tests were performed in the period of March 2016-December 2016 comprising 972 solo (index only), 14 duo (parents or affected siblings only), and 33 trio (index and parents). Multigene panels accounted for 672 tests, while whole exome sequencing (WES) represented the remaining 347 tests. Pathogenic or likely pathogenic variants that explain the clinical indications were identified in 34% (27% in panels and 43% in exomes), spanning 279 genes and including 165 novel variants. While recessive mutations dominated the landscape of solved cases (71% of mutations, and 97% of which are homozygous), a substantial minority (27%) were solved on the basis of dominant mutations. The highly consanguineous nature of the study population also facilitated homozygosity for many private mutations (only 32.5% of the recessive mutations are founder), as well as the first instances of recessive inheritance of previously assumed strictly dominant disorders (involving ITPR1, VAMP1, MCTP2, and TBP). Surprisingly, however, dual molecular diagnosis was only observed in 1.5% of cases. Finally, we have encountered candidate variants in 75 genes (ABHD6, ACY3, ADGRB2, ADGRG7, AGTPBP1, AHNAK2, AKAP6, ASB3, ATXN1L, C17orf62, CABP1, CCDC186, CCP110, CLSTN2, CNTN3, CNTN5, CTNNA2, CWC22, DMAP1, DMKN, DMXL1, DSCAM, DVL2, ECI1, EP400, EPB41L5, FBXL22, GAP43, GEMIN7, GIT1, GRIK4, GRSF1, GTRP1, HID1, IFNL1, KCNC4, LRRC52, MAP7D3, MCTP2, MED26, MPP7, MRPS35, MTDH, MTMR9, NECAP2, NPAT, NRAP, PAX7, PCNX, PLCH2, PLEKHF1, PTPN12, QKI, RILPL2, RIMKLA, RIMS2, RNF213, ROBO1, SEC16A, SIAH1, SIRT2, SLAIN2, SLC22A20, SMDT1, SRRT, SSTR1, ST20, SYT9, TSPAN6, UBR4, VAMP4, VPS36, WDR59, WDYHV1, and WHSC1) not previously linked to human phenotypes and these are presented to accelerate post-publication matchmaking. Two of these genes were independently mutated in more than one family with similar phenotypes, which substantiates their link to human disease (AKAP6 in intellectual disability and UBR4 in early dementia). If the novel candidate disease genes in this cohort are independently confirmed, the yield of WES will have increased to 83%, which suggests that most "negative" clinical exome tests are unsolved due to interpretation rather than technical limitations. Show less

Genetic factors make a substantial contribution to inter-individual variability in cognitive function. A recent meta-analysis of genome-wide association studies identified two loci, AKAP6 and MIR2113, that are associated with general cognitive function. Here, we extend this previous research by investigating the association of MIR2113 and AKAP6 with baseline and longitudinal non-linear change across a broad spectrum of cognitive domains in a community-based cohort of older adults without dementia. Two single nucleotide polymorphisms (SNPs), MIR211-rs10457441 and AKAP6-rs17522122 were genotyped in 1570 non-demented older Australians of European ancestry, who were examined up to 4 times over 12 years. Linear mixed effects models were used to examine the association between AKAP6 and MIR2113 with cognitive performance in episodic memory, working memory, vocabulary, perceptual speed and reaction time at baseline and with linear and quadratic rates of change. AKAP6-rs17522122*T was associated with worse baseline performance in episodic memory, working memory, vocabulary and perceptual speed, but it was not associated with cognitive change in any domain. MIR2113-rs10457441*T was associated with accelerated decline in episodic memory. No other associations with baseline cognitive performance or with linear or quadratic rate or cognitive changes were observed for this SNP. These results confirm the previous finding that AKAP6 is associated with performance across multiple cognitive domains at baseline but not with cognitive decline, while MIR2113 primarily affects the rate at which memory declines over time. Show less

Genome-wide association studies (GWAS) have identified several genetic regions involved in immune-regulatory mechanisms to be associated with celiac disease. Previous GWAS also revealed an over-representation of genes involved in type 2 diabetes and anorexia nervosa associated with celiac disease, suggesting involvement of common metabolic pathways for development of these chronic diseases. The aim of this study was to extend these previous analyses to study the gene expression in the gut from children with active celiac disease. Thirty six target genes involved in type 2 diabetes and four genes associated with anorexia nervosa were investigated for gene expression in small intestinal biopsies from 144 children with celiac disease at median (range) age of 7.4 years (1.6-17.8) and from 154 disease controls at a median (range) age 11.4.years (1.4-18.3). A total of eleven of genes were differently expressed in celiac patients compared with disease controls of which CD36, CD38, FOXP1, SELL, PPARA, PPARG, AGT previously associated with type 2 diabetes and AKAP6, NTNG1 with anorexia nervosa remained significant after correction for multiple testing. Shared genetic factors involved in celiac disease, type 2 diabetes and anorexia nervosa suggest common underlying molecular pathways for these diseases. Show less

We reported earlier that the endocochlear potential (EP) differs between C57BL/6J (B6) and BALB/cJ (BALB) mice, being lower in BALBs by about 10 mV (Ohlemiller et al. Hear Res 220: 10-26, 2006). This difference corresponds to strain differences with respect to the density of marginal cells in cochlear stria vascularis. After about 1 year of age, BALB mice also tend toward EP reduction that correlates with further marginal cell loss. We therefore suggested that early sub-clinical features of the BALB stria vascularis may predispose these mice to a condition modeling Schuknecht's strial presbycusis. We further reported (Ohlemiller et al. J Assoc Res Otolaryngol 12: 45-58, 2011) that the acute effects of a 2-h 110 dB SPL noise exposure differ between B6 and BALB mice, such that the EP remains unchanged in B6 mice, but is reduced by 40-50 mV in BALBs. In about 25 % of BALBs, the EP does not completely recover, so that permanent EP reduction may contribute to noise-induced permanent threshold shifts in BALBs. To identify genes and alleles that may promote natural EP variation as well as noise-related EP reduction in BALB mice, we have mapped related quantitative trait loci (QTLs) using 12 recombinant inbred (RI) strains formed from B6 and BALB (CxB1-CxB12). EP and strial marginal cell density were measured in B6 mice, BALB mice, their F1 hybrids, and RI mice without noise exposure, and 1-3 h after broadband noise (4-45 kHz, 110 dB SPL, 2 h). For unexposed mice, the strain distribution patterns for EP and marginal cell density were used to generate preliminary QTL maps for both EP and marginal cell density. Six QTL regions were at least statistically suggestive, including a significant QTL for marginal cell density on chromosome 12 that overlapped a weak QTL for EP variation. This region, termed Maced (Marginal cell density QTL) supports the notion of marginal cell density as a genetically influenced contributor to natural EP variation. Candidate genes for Maced notably include Foxg1, Foxa1, Akap6, Nkx2-1, and Pax9. Noise exposure produced significant EP reductions in two RI strains as well as significant EP increases in two RI strains. QTL mapping of the EP in noise-exposed RI mice yielded four suggestive regions. Two of these overlapped with QTL regions we previously identified for noise-related EP reduction in CBA/J mice (Ohlemiller et al. Hear Res 260: 47-53, 2010) on chromosomes 5 and 18 (Nirep). The present map may narrow the Nirep interval to a ~10-Mb region of proximal Chr. 18 that includes Zeb1, Arhgap12, Mpp7, and Gjd4. This study marks the first exploration of natural gene variants that modulate the EP. Their orthologs may underlie some human hearing loss that originates in the lateral wall. Show less

Gastric cancer is not a single disease, and its subtype classification is still evolving. Next-generation sequencing studies have identified novel genetic drivers of gastric cancer, but their use as molecular classifiers or prognostic markers of disease outcome has yet to be established. In this study, we integrated somatic mutational profiles and clinicopathologic information from 544 gastric cancer patients from previous genomic studies to identify significantly mutated genes (SMG) with prognostic relevance. Gastric cancer patients were classified into regular (86.8%) and hypermutated (13.2%) subtypes based on mutation burden. Notably, TpCpW mutations occurred significantly more frequently in regular, but not hypermutated, gastric cancers, where they were associated with APOBEC expression. In the former group, six previously unreported (XIRP2, NBEA, COL14A1, CNBD1, ITGAV, and AKAP6) and 12 recurrent mutated genes exhibited high mutation prevalence (≥3.0%) and an unexpectedly higher incidence of nonsynonymous mutations. We also identified two molecular subtypes of regular-mutated gastric cancer that were associated with distinct prognostic outcomes, independently of disease staging, as confirmed in a distinct patient cohort by targeted capture sequencing. Finally, in diffuse-type gastric cancer, CDH1 mutation was found to be associated with shortened patient survival, independently of disease staging. Overall, our work identified previously unreported SMGs and a mutation signature predictive of patient survival in newly classified subtypes of gastric cancer, offering opportunities to stratify patients into optimal treatment plans based on molecular subtyping. Cancer Res; 76(7); 1724-32. ©2016 AACR. Show less

Neurotrophic factor and cAMP-dependent signaling promote the survival and neurite outgrowth of retinal ganglion cells (RGCs) after injury. However, the mechanisms conferring neuroprotection and neuroregeneration downstream to these signals are unclear. We now reveal that the scaffold protein muscle A-kinase anchoring protein-α (mAKAPα) is required for the survival and axon growth of cultured primary RGCs. Although genetic deletion of mAKAPα early in prenatal RGC development did not affect RGC survival into adulthood, nor promoted the death of RGCs in the uninjured adult retina, loss of mAKAPα in the adult increased RGC death after optic nerve crush. Importantly, mAKAPα was required for the neuroprotective effects of brain-derived neurotrophic factor and cyclic adenosine-monophosphate (cAMP) after injury. These results identify mAKAPα as a scaffold for signaling in the stressed neuron that is required for RGC neuroprotection after optic nerve injury. Show less

Skeletal muscle regeneration occurs continuously to repair muscle damage incurred during normal activity and in chronic disease or injury. Herein, we report that A-kinase anchoring protein 6 (AKAP6) is important for skeletal myoblast differentiation and muscle regeneration. Compared with unstimulated skeletal myoblasts that underwent proliferation, differentiated cells show significant stimulation of AKAP6 expression. AKAP6 knockdown with siRNA effectively halts the formation of myotubes and decreases the expression of the differentiation markers myogenin and myosin heavy chain. When shAKAP6-lentivirus is delivered to mice with cardiotoxin (CTX)-induced muscle injury, muscle regeneration is impaired compared with that of mice injected with control shMock-lentivirus. The motor functions of mice infected with shAKAP6-lentivirus (CTX+shAK6) are significantly worse than those of mice infected with shMock-lentivirus (CTX+shMock). Mechanistic analysis showed that AKAP6 promotes myogenin expression through myocyte enhancer factor 2A (MEF2A). Notably, myogenin increases AKAP6 expression as well. The results of chromatin immunoprecipitation and luciferase assays showed that myogenin binds to an E-box site on the AKAP6 promoter. Taken together, our findings demonstrate a novel interplay between AKAP6 and myogenin, and we suggest that AKAP6 is an important regulator of myoblast differentiation, myotube formation, and muscle regeneration. Show less

General cognitive function is substantially heritable across the human life course from adolescence to old age. We investigated the genetic contribution to variation in this important, health- and well-being-related trait in middle-aged and older adults. We conducted a meta-analysis of genome-wide association studies of 31 cohorts (N=53,949) in which the participants had undertaken multiple, diverse cognitive tests. A general cognitive function phenotype was tested for, and created in each cohort by principal component analysis. We report 13 genome-wide significant single-nucleotide polymorphism (SNP) associations in three genomic regions, 6q16.1, 14q12 and 19q13.32 (best SNP and closest gene, respectively: rs10457441, P=3.93 × 10(-9), MIR2113; rs17522122, P=2.55 × 10(-8), AKAP6; rs10119, P=5.67 × 10(-9), APOE/TOMM40). We report one gene-based significant association with the HMGN1 gene located on chromosome 21 (P=1 × 10(-6)). These genes have previously been associated with neuropsychiatric phenotypes. Meta-analysis results are consistent with a polygenic model of inheritance. To estimate SNP-based heritability, the genome-wide complex trait analysis procedure was applied to two large cohorts, the Atherosclerosis Risk in Communities Study (N=6617) and the Health and Retirement Study (N=5976). The proportion of phenotypic variation accounted for by all genotyped common SNPs was 29% (s.e.=5%) and 28% (s.e.=7%), respectively. Using polygenic prediction analysis, ~1.2% of the variance in general cognitive function was predicted in the Generation Scotland cohort (N=5487; P=1.5 × 10(-17)). In hypothesis-driven tests, there was significant association between general cognitive function and four genes previously associated with Alzheimer's disease: TOMM40, APOE, ABCG1 and MEF2C. Show less

Cardiac remodeling is regulated by an extensive intracellular signal transduction network. Each of the many signaling pathways in this network contributes uniquely to the control of cellular adaptation. In the last few years, it has become apparent that multimolecular signaling complexes or "signalosomes" are important for fidelity in intracellular signaling and for mediating crosstalk between the different signaling pathways. These complexes integrate upstream signals and control downstream effectors. In the cardiac myocyte, the protein mAKAPβ serves as a scaffold for a large signalosome that is responsive to cAMP, calcium, hypoxia, and mitogen-activated protein kinase signaling. The main function of mAKAPβ signalosomes is to modulate stress-related gene expression regulated by the transcription factors NFATc, MEF2, and HIF-1α and type II histone deacetylases that control pathological cardiac hypertrophy. Show less

The c-Jun N-terminal Kinases (JNKs) play important roles in cell responses to stress or growth factor stimulation. The JNK1α1 isoform shares >90% identity with a predominantly neuronal JNK3α1 isoform, but JNK3α1 also includes a distinctive 38 amino acid N-terminal sequence. To address the outstanding question of the potential for these JNK isoforms to have different binding partners that mediate different biological actions, the work presented here refined the yeast two-hybrid approach to identify and categorize binding partners for JNK1α1 and JNK3α1. Specifically, site-directed mutagenesis of the JNK1α1 common docking (CD) domain that mediates typical JNK-binding domain (JBD)-dependent interactions, truncation of the distinctive JNK3 N-terminal domain (i.e. ΔN JNK3α1), and interaction evaluation in the yeast two-hybrid system defined the interacting partners as either JNK1-specific interactors (ATF7, FUS, KCNE4, PIAS1, SHANK1, TKT), typical JBD-dependent interactors shared by JNK1α1 and JNK3α1 (AKAP6, BMPR2, EEF1A1, GFAP, GRIP2, GTF2F1, HDAC2, MAP1B, MYO9B, PTPN2, RABGAP1, RUSC2, SUMO1, SYPL1, TOPBP1, ZNF668), or JNK3-specific partners (ATXN1, NNAT, PTGDS) dependent on interaction with the JNK3 N-terminal extension. The interacting partners ATF7, AKAP6, and ATXN1 were explored further as representatives of these different classes. Two potential JBDs were identified in ATF7 as important for its interaction with JNK1α1, but additionally an interaction between ATF7 and ΔN JNK3α1 was shown to be JBD-dependent, suggesting that the JNK3α1 N-terminus prevents interaction with some proteins. For the shared partner AKAP6, one of the multiple potential JBDs predicted by sequence analysis was important for the AKAP6-JNK interaction in the yeast screening system as well as in mammalian cells. Finally, the ATXN1-JNK3α1 interaction was dependent on the JNK3α1 N-terminus in a mammalian cell context. These studies therefore highlight a diversity of potential JNK-interacting partners with both JBD-dependent as well as JBD-independent modes of interaction. Show less

The calcium/calmodulin-dependent protein phosphatase calcineurin is required for the induction of transcriptional events that initiate and promote myogenic differentiation. An important effector for calcineurin in striated muscle is the transcription factor myocyte enhancer factor 2 (MEF2). The targeting of the enzyme and substrate to specific intracellular compartments by scaffold proteins often confers specificity in phosphatase activity. We now show that the scaffolding protein mAKAP organizes a calcineurin/MEF2 signaling complex in myocytes, regulating gene transcription. A calcineurin/mAKAP/MEF2 complex can be isolated from C2C12 cells and cardiac myocytes, and the calcineurin/MEF2 association is dependent on mAKAP expression. We have identified a peptide comprising the calcineurin binding domain in mAKAP that can disrupt the binding of the phosphatase to the scaffold in vivo. Dominant interference of calcineurin/mAKAP binding blunts the increase in MEF2 transcriptional activity seen during myoblast differentiation, as well as the expression of endogenous MEF2-target genes. Furthermore, disruption of calcineurin binding to mAKAP in cardiac myocytes inhibits adrenergic-induced cellular hypertrophy. Together these data illustrate the importance of calcineurin anchoring by the mAKAP scaffold for MEF2 regulation. Show less

Protein kinase A (PKA) substrate phosphorylation is facilitated through its co-localization with its signaling partner by A-kinase anchoring proteins (AKAPs). mAKAP (muscle-selective AKAP) localizes PKA and its substrates such as phosphodiesterase-4D3 (PDE4D3), ryanodine receptor, and protein phosphatase 2A (PP2A) to the sarcoplasmic reticulum and perinuclear space. The genetic role of mAKAP, in modulating PKA/PDE4D3 molecular signaling during cardiac diseases, remains unclear. The purpose of this study was to examine the effects of naturally occurring mutations in human mAKAP on PKA and PDE4D3 signaling. We have recently identified potentially important human mAKAP coding non-synonymous polymorphisms located within or near key protein binding sites critical to β-adrenergic receptor signaling. Three mutations (P1400S, S2195F, and L717V) were cloned and transfected into a mammalian cell line for the purpose of comparing whether those substitutions disrupt mAKAP binding to PKA or PDE4D3. Immunoprecipitation study of mAKAP-P1400S, a mutation located in the mAKAP-PDE4D3 binding site, displayed a significant reduction in binding to PDE4D3, with no significant changes in PKA binding or PKA activity. Conversely, mAKAP-S2195F, a mutation located in mAKAP-PP2A binding site, showed significant increase in both binding propensity to PKA and PKA activity. Additionally, mAKAP-L717V, a mutation flanking the mAKAP-spectrin repeat domain, exhibited a significant increase in PKA binding compared to wild type, but there was no change in PKA activity. We also demonstrate specific binding of wild-type mAKAP to PDE4D3. Binding results were demonstrated using immunoprecipitation and confirmed with surface plasmon resonance (Biacore-2000); functional results were demonstrated using activity assays, Ca(2+) measurements, and Western blot. Comparative analysis of the binding responses of mutations to mAKAP could provide important information about how these mutations modulate signaling. Show less

Metabolic syndrome (MetS) is an aberration associated with increased risk for cancer and inflammation. Adiponectin, an adipocyte-produced abundant protein hormone, has countering effect on the diabetogenic and atherogenic components of MetS. Plasma levels of adiponectin are negatively correlated with onset of cancer and cancer patient mortality. We previously performed microsatellite linkage analyses using adiponectin as a surrogate marker and revealed two QTLs on chr5 (5p14) and chr14 (14q13). Using individuals from 85 extended families that contributed to the linkage and who were measured for 42 clinical and biologic MetS phenotypes, we tested QTL-based SNP associations, peripheral white blood cell (PWBC) gene expression, and the effects of cis-acting SNPs on gene expression to discover genomic elements that could affect the pathophysiology and complications of MetS. Adiponectin levels were found to be highly intercorrelated phenotypically with the majority of MetS traits. QTL-specific haplotype-tagging SNPs associated with MetS phenotypes were annotated to 14 genes whose function could influence MetS biology as well as oncogenesis or inflammation. These were mechanistically categorized into four groups: cell-cell adhesion and mobility, signal transduction, transcription and protein sorting. Four genes were highly prioritized: cadherin 18 (CDH18), myosin X (MYO10), anchor protein 6 of AMPK (AKAP6), and neuronal PAS domain protein 3 (NPAS3). PWBC expression was detectable only for the following genes with multi-organ or with multi-function properties: NPAS3, MARCH6, MYO10 and FBXL7. Strong evidence of cis-effects on the expression of MYO10 in PWBC was found with SNPs clustered near the gene's transcription start site. MYO10 expression in PWBC was marginally correlated with body composition (p = 0.065) and adipokine levels in the periphery (p = 0.064). Variants of genes AKAP6, NPAS3, MARCH6 and FBXL7 have been previously reported to be associated with insulin resistance, inflammatory markers or adiposity studies using genome-wide approaches whereas associations of CDH18 and MYO10 with MetS traits have not been reported before. Adiponectin QTLs-based SNP association and mRNA expression identified genes that could mediate the association between MetS and cancer or inflammation. Show less

Cardiac myocyte hypertrophy is the main compensatory response to chronic stress on the heart. p90 ribosomal S6 kinase (RSK) family members are effectors for extracellular signal-regulated kinases that induce myocyte growth. Although increased RSK activity has been observed in stressed myocytes, the functions of individual RSK family members have remained poorly defined, despite being potential therapeutic targets for cardiac disease. To demonstrate that type 3 RSK (RSK3) is required for cardiac myocyte hypertrophy. RSK3 contains a unique N-terminal domain that is not conserved in other RSK family members. We show that this domain mediates the regulated binding of RSK3 to the muscle A-kinase anchoring protein scaffold, defining a novel kinase anchoring event. Disruption of both RSK3 expression using RNA interference and RSK3 anchoring using a competing muscle A-kinase anchoring protein peptide inhibited the hypertrophy of cultured myocytes. In vivo, RSK3 gene deletion in the mouse attenuated the concentric myocyte hypertrophy induced by pressure overload and catecholamine infusion. Taken together, these data demonstrate that anchored RSK3 transduces signals that modulate pathologic myocyte growth. Targeting of signaling complexes that contain select kinase isoforms should provide an approach for the specific inhibition of cardiac myocyte hypertrophy and for the development of novel strategies for the prevention and treatment of heart failure. Show less

Multiple musculoskeletal traits assessed by various methods at different skeletal sites serve as surrogates for osteoporosis risk. However, it is a challenge to select the most relevant phenotypes for genetic study of fractures. Principal component analyses (PCA) were conducted in participants of the Framingham Osteoporosis Study on 17 measures including bond mineral density (BMD) (hip and spine), heel ultrasound, leg lean mass (LLM), and hip geometric indices, adjusting for covariates (age, height, body mass index [BMI]), in a combined sample of 1180 men and 1758 women, as well as in each sex. Four principal components (PCs) jointly explained ~69% of the total variability of musculoskeletal traits. PC1, explaining ~33% of the total variance, was referred to as the component of "Bone strength," because it included the hip and spine BMD as well as several hip cross-sectional properties. PC2 (20.5% variance) was labeled as "Femoral cross-sectional geometry;" PC3 (~8% variance) captured only ultrasound measures; PC4, explaining ~7% variance, was correlated with LLM and hip geometry. We then evaluated ~2.5 mil SNPs for association with PCs 1, 2, and 4. There were genome-wide significant associations (p < 5 × 10⁻⁸) between PC2 and HTR1E (that codes for one of the serotonin receptors) and PC4 with COL4A2 in women. In the sexes-combined sample, AKAP6 was associated with PC2 (p = 1.40 × 10⁻⁷). A single nucleotide polymorphism (SNP) in HTR1E was also associated with the risk of nonvertebral fractures in women (p = 0.005). Functions of top associated genes were enriched for the skeletal and muscular system development (p < 0.05). In conclusion, multivariate combination provides genetic associations not identified in the analysis of primary phenotypes. Genome-wide screening for the linear combinations of multiple osteoporosis-related phenotypes suggests that there are variants with potentially pleiotropic effects in established and novel pathways to be followed up to provide further evidence of their functions. Show less

Differentiation of skeletal myoblast cells to functional myotubes involves highly regulated transcriptional dynamics. The myocyte enhancer factor 2 (MEF2) transcription factors are critical to this process, synergizing with the master regulator MyoD to promote muscle specific gene transcription. MEF2 is extensively regulated by myogenic stimuli, both transcriptionally and post-translationally, but to date there has been little progress in understanding how signals upstream of MEF2 are coordinated to produce a coherent response. In this study, we define a novel interaction between the muscle A-kinase anchoring protein (mAKAP) and MEF2 in skeletal muscle. Discrete domains of MEF2 and mAKAP bind directly. Their interaction was exploited to probe the function of mAKAP-tethered MEF2 during myogenic differentiation. Dominant interference of MEF2/mAKAP binding was sufficient to block MEF2 activation during the early stages of differentiation. Furthermore, extended expression of this disrupting domain effectively blocked myogenic differentiation, halting the formation of myotubes and decreasing expression of several differentiation markers. This study expands our understanding of the regulation of MEF2 in skeletal muscle and identifies the mAKAP scaffold as a facilitator of MEF2 transcription and myogenic differentiation. Show less

Anorexia nervosa (AN) is a mental illness with high mortality that most commonly afflicts adolescent female individuals. Clinical symptoms include chronic food refusal, weight loss and body image distortions. We carried out a genome-wide association study on 1033 AN cases and 3733 pediatric control subjects, all of whom were of European ancestry and were genotyped on the Illumina HumanHap610 platform (Illumina, San Diego, CA, USA). We confirmed that common single-nucleotide polymorphisms (SNPs) within OPRD1 (rs533123, P=0.0015) confer risk for AN, and obtained suggestive evidence that common SNPs near HTR1D (rs7532266, P=0.04) confer risk for restricting-type AN specifically. However, no SNPs reached genome-wide significance in our data, whereas top association signals were detected near ZNF804B, CSRP2BP, NTNG1, AKAP6 and CDH9. In parallel, we performed genome-wide analysis on copy number variations (CNVs) using the signal intensity data from the SNP arrays. We did not find evidence that AN cases have more CNVs than control subjects, nor do they have over-representation of rare or large CNVs. However, we identified several regions with rare CNVs that were only observed in AN cases, including a recurrent 13q12 deletion (1.5 Mb) disrupting SCAS in two cases, and CNVs disrupting the CNTN6/CNTN4 region in several AN cases. In conclusion, our study suggests that both common SNPs and rare CNVs may confer genetic risk to AN. These results point to intriguing genes that await further validation in independent cohorts for confirmatory roles in AN. Show less

mAKAPbeta is the scaffold for a multimolecular signaling complex in cardiac myocytes that is required for the induction of neonatal myocyte hypertrophy. We now show that the pro-hypertrophic phosphatase calcineurin binds directly to a single site on mAKAPbeta that does not conform to any of the previously reported consensus binding sites. Calcineurin-mAKAPbeta complex formation is increased in the presence of Ca(2+)/calmodulin and in norepinephrine-stimulated primary cardiac myocytes. This binding is of functional significance because myocytes exhibit diminished norepinephrine-stimulated hypertrophy when expressing a mAKAPbeta mutant incapable of binding calcineurin. In addition to calcineurin, the transcription factor NFATc3 also associates with the mAKAPbeta scaffold in myocytes. Calcineurin bound to mAKAPbeta can dephosphorylate NFATc3 in myocytes, and expression of mAKAPbeta is required for NFAT transcriptional activity. Taken together, our results reveal the importance of regulated calcineurin binding to mAKAPbeta for the induction of cardiac myocyte hypertrophy. Furthermore, these data illustrate how scaffold proteins organizing localized signaling complexes provide the molecular architecture for signal transduction networks regulating key cellular processes. Show less

Epidemiological studies have suggested an association between selenium intake and protection from a variety of cancer. Considering this clinical importance of selenium, we aimed to identify the genes associated with resistance to selenium treatment. We have applied a previous methodology developed by our group, which is based on the genetic and pharmacological data publicly available for the NCI60 cancer cell line panel. In short, we have categorized the NCI60 cell lines as selenium resistant and sensitive based on their growth inhibition (GI50) data. Then, we have utilized the Affymetrix 125K SNP chip data available and carried out a genome-wide case-control association study for the selenium sensitive and resistant NCI60 cell lines. Our results showed statistically significant association of four SNPs in 5q33-34, 10q11.2, 10q22.3 and 14q13.1 with selenium resistance. These SNPs were located in introns of the genes encoding for a kinase-scaffolding protein (AKAP6), a membrane protein (SGCD), a channel protein (KCNMA1), and a protein kinase (PRKG1). The knock-down of KCNMA1 by siRNA showed increased sensitivity to selenium in both LNCaP and PC3 cell lines. Furthermore, SNP-SNP interaction (epistasis) analysis indicated the interactions of the SNPs in AKAP6 with SGCD as well as SNPs in AKAP6 with KCNMA1 with each other, assuming additive genetic model. These genes were also all involved in the Ca(2+) signaling, which has a direct role in induction of apoptosis and induction of apoptosis in tumor cells is consistent with the chemopreventive action of selenium. Once our findings are further validated, this knowledge can be translated into clinics where individuals who can benefit from the chemopreventive characteristics of the selenium supplementation will be easily identified using a simple DNA analysis. Show less

A-kinase (PKA) anchoring proteins (AKAPs) are essential for targeting type II PKA to specific locales in the cell to control function. In the present study, AKAP5 (formerly AKAP150) and AKAP6 were identified in mouse parotid acini by type II PKA regulatory subunit (RII) overlay assay and Western blot analysis of mouse parotid cellular fractions, and the role of AKAP5 in mouse parotid acinar cell secretion was determined. Mice were euthanized with CO(2). Immunofluorescence staining of acinar cells localized AKAP5 to the basolateral membrane, whereas AKAP6 was associated with the perinuclear region. In functional studies, amylase secretion from acinar cells of AKAP5 mutant [knockout (KO)] mice treated with the beta-adrenergic agonist, isoproterenol, was reduced overall by 30-40% compared with wild-type (WT) mice. In contrast, amylase secretion in response to the adenylyl cyclase (AC) activator, forskolin, and the cAMP-dependent protein kinase (PKA) activator, N(6)-phenyl-cAMP, was not statistically different in acini from WT and AKAP5 KO mice. Treatment of acini with isoproterenol mimicked the effect of the Epac activator, 8-(4-methoxyphenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate (8-pMeOPT-2'-O-Me-cAMP), in stimulating Rap1. However, in contrast to isoproterenol, treatment of acini with 8-pMeOPT-2'-O-Me-cAMP resulted in stimulation of amylase secretion from both AKAP5 KO and WT acinar cells. As a scaffolding protein, AKAP5 was found to coimmunoprecipitate with AC6, but not AC8. Data suggest that isoproterenol-stimulated amylase secretion occurs via both an AKAP5/AC6/PKA complex and a PKA-independent, Epac pathway in mouse parotid acini. Show less

The concentration of the second messenger cAMP is tightly controlled in cells by the activity of phosphodiesterases. We have previously described how the protein kinase A-anchoring protein mAKAP serves as a scaffold for the cAMP-dependent protein kinase PKA and the cAMP-specific phosphodiesterase PDE4D3 in cardiac myocytes. PKA and PDE4D3 constitute a negative feedback loop whereby PKA-catalyzed phosphorylation and activation of PDE4D3 attenuate local cAMP levels. We now show that protein phosphatase 2A (PP2A) associated with mAKAP complexes is responsible for reversing the activation of PDE4D3 by catalyzing the dephosphorylation of PDE4D3 serine residue 54. Mapping studies reveal that a C-terminal mAKAP domain (residues 2085-2319) binds PP2A. Binding to mAKAP is required for PP2A function, such that deletion of the C-terminal domain enhances both base-line and forskolin-stimulated PDE4D3 activity. Interestingly, PP2A holoenzyme associated with mAKAP complexes in the heart contains the PP2A targeting subunit B56delta. Like PDE4D3, B56delta is a PKA substrate, and PKA phosphorylation of mAKAP-bound B56delta enhances phosphatase activity 2-fold in the complex. Accordingly, expression of a B56delta mutant that cannot be phosphorylated by PKA results in increased PDE4D3 phosphorylation. Taken together, our findings demonstrate that PP2A associated with mAKAP complexes promotes PDE4D3 dephosphorylation, serving both to inhibit PDE4D3 in unstimulated cells and also to mediate a cAMP-induced positive feedback loop following adenylyl cyclase activation and B56delta phosphorylation. In general, PKA.PP2A.mAKAP complexes exemplify how protein kinases and phosphatases may participate in molecular signaling complexes to dynamically regulate localized intracellular signaling. Show less

Protein kinase A-anchoring proteins (AKAPs) play important roles in the compartmentation of cAMP signaling, anchoring protein kinase A (PKA) to specific cellular organelles and serving as scaffolds that assemble localized signaling cascades. Although AKAPs have been recently shown to bind adenylyl cyclase (AC), the functional significance of this association has not been studied. In cardiac myocytes, the muscle protein kinase A-anchoring protein beta (mAKAPbeta) coordinates cAMP-dependent, calcium, and MAP kinase pathways and is important for cellular hypertrophy. We now show that mAKAPbeta selectively binds type 5 AC in the heart and that mAKAPbeta-associated AC activity is absent in AC5 knock-out hearts. Consistent with its known inhibition by PKA phosphorylation, AC5 is inhibited by association with mAKAPbeta-PKA complexes. AC5 binds to a unique N-terminal site on mAKAP-(245-340), and expression of this peptide disrupts endogenous mAKAPbeta-AC association. Accordingly, disruption of mAKAPbeta-AC5 complexes in neonatal cardiac myocytes results in increased cAMP and hypertrophy in the absence of agonist stimulation. Taken together, these results show that the association of AC5 with the mAKAPbeta complex is required for the regulation of cAMP second messenger controlling cardiac myocyte hypertrophy. Show less

The activity of the transcription factor hypoxia-inducible factor 1alpha (HIF-1alpha) is increased in response to reduced intracellular oxygen. Enzymes of the protein ubiquitin machinery that signal the destruction or stabilization of HIF-1alpha tightly control this transcriptional response. Here, we show that muscle A kinase-anchoring protein (mAKAP) organized ubiquitin E3 ligases that managed the stability of HIF-1alpha and optimally positioned it close to its site of action inside the nucleus. Functional experiments in cardiomyocytes showed that depletion of mAKAP or disruption of its targeting to the perinuclear region altered the stability of HIF-1alpha and transcriptional activation of genes associated with hypoxia. Thus, we propose that compartmentalization of oxygen-sensitive signaling components may influence the fidelity and magnitude of the hypoxic response. Show less

Common challenges to any cell are the processing of the extracellular stimuli it receives into intracellular signaling cascades that initiate a multitude of diverse biological functions. However, many of these stimuli act via a common signaling pathway, suggesting the cell must somehow discriminate between different stimuli and respond accordingly. Subcellular targeting through the association with adaptor and scaffolding proteins has emerged as a key mechanism by which cells maintain signaling specificity. Compartmentation of cAMP signaling is maintained by the clustering of cAMP signaling enzymes in discrete units by the scaffolding protein A-kinase anchoring proteins (AKAP). In doing so, AKAPs provide the molecular architecture for the cAMP micordomains that underlie the spacial-temporal control of cAMP signaling. Show less