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Hypertension is a leading risk factor for stroke, heart disease, chronic kidney disease, vascular cognitive impairment, and Alzheimer’s disease. Previous genetic studies have nominated hundreds of genes linked to hypertension, and renal and cognitive diseases. Some have been advanced as candidate genes by showing that they can alter blood pressure or renal and cerebral vascular function in knockout animals; however, final validation of the causal variants and underlying mechanisms has remained elusive. This review chronicles 40 years of work, from the initial identification of adducin (ADD) as an ACTIN-binding protein suggested to increase blood pressure in Milan hypertensive rats, to the discovery of a mutation in ADD1 as a candidate gene for hypertension in rats that were subsequently linked to hypertension in man. More recently, a recessive K572Q mutation in ADD3 was identified in Fawn-Hooded Hypertensive (FHH) and Milan Normotensive (MNS) rats that develop renal disease, which is absent in resistant strains. ADD3 dimerizes with ADD1 to form functional ADD protein. The mutation in ADD3 disrupts a critical ACTIN-binding site necessary for its interactions with actin and spectrin to regulate the cytoskeleton. Studies using Add3 KO and transgenic strains, as well as a genetic complementation study in FHH and MNS rats, confirmed that the K572Q mutation in ADD3 plays a causal role in altering the myogenic response and autoregulation of renal and cerebral blood flow, resulting in increased susceptibility to hypertension-induced renal disease and cerebral vascular and cognitive dysfunction.

Objective: The expression of cytoskeleton-related protein γ-adducin (ADD3) was abnormally reduced in some tumors. Functional experiments demonstrated that it could inhibit the malignant progression of lung cancer and glioma, whereas the involvement of ADD3 in osteosarcoma was not clear. This study aimed to investigate the role of ADD3 in osteosarcoma and its upstream regulatory mechanisms. Methods: ADD3 was knocked down by siRNA transfection and the expression level of ADD3 was determined using quantitative real-time PCR assay and Western blot. CCK-8 assay and colony formation were performed to detect the capacity of cell proliferation. Transwell assay and PI and Annexin V-FITC staining were used to determine cell migration and apoptosis, respectively. Luciferase reporter experiment was performed to investigate the interaction between ADD3 and miR-23b-3p. Results: Based on gene silencing assays, we showed that knockdown of ADD3 suppressed apoptosis and promoted the proliferation and migration of osteosarcoma cells, revealing inhibitory effects of ADD3 in osteosarcoma. Luciferase reporter gene assays confirmed that miR-23b-3p could bind to the 3'-UTR of ADD3. Upregulation of miR-23b-3p not only inhibited the expression of ADD3, but also released the tumor suppressive role of ADD3 on the proliferation and migration of osteosarcoma cells. Conclusions: Our study found that ADD3 functioned as a tumor suppressor gene during osteosarcoma development. The abnormal upregulation of miR-23b-3p targeted the expression of ADD3 and resulted in accelerated osteosarcoma cell proliferation and migration. Thus, the miR-23b-3p/ADD3 axis contributes to the development of osteosarcoma and ADD3 is a key driver of malignancy.

BackgroundAcute myocardial infarction (AMI) is a major contributor of heart failure (HF). Peripheral blood mononuclear cells (PBMCs), mainly monocytes, are the essential initiators of AMI-induced HF. The powerful biomarkers for early identification of AMI patients at risk of HF remain elusive. We aimed to identify monocyte-related critical genes as predictive biomarkers for post-AMI HF.MethodsWe performed weighted gene co-expression network analysis (WGCNA) on transcriptomics of PBMCs from AMI patients who developed HF or did not. Functional enrichment analysis of genes in significant modules was performed via Metascape. Then we obtained the single-cell RNA-sequencing data of recruited monocytes/macrophages from AMI and control mice using the Scanpy and screened 381 differentially expressed genes (DEGs) between the two groups. We validated the expression changes of the 25 genes in cardiac macrophages from AMI mice based on bulk RNA-sequencing data and PBMCs data mentioned above.ResultsIn our study, the results of WGCNA showed that two modules containing 827 hub genes were most significantly associated with post-AMI HF, which mainly participated in cell migration, inflammation, immunity, and apoptosis. There were 25 common genes between DEGs and hub genes, showing close relationship with inflammation and collagen metabolism. CUX1, CTSD and ADD3 exhibited consistent changes in three independent studies. Receiver operating characteristic curve analysis showed that each of the three genes had excellent performance in recognizing post-AMI HF patients.ConclusionOur findings provided a set of three monocyte-related biomarkers for the early prediction of HF development after AMI as well as potential therapeutic targets of post-AMI HF.

Biliary atresia (BA) is an idiopathic neonatal cholestatic disease. Recent genome-wide association study (GWAS) revealed that common variation of ADD3, GPC1, ARF6, and EFEMP1 gene was associated with BA susceptibility. We aimed to evaluate the association of these genes with BA in Chinese population. Twenty single nucleotide polymorphisms (SNPs) in these four genes were genotyped in 340 BA patients and 1,665 controls. Three SNPs in ADD3 were significantly associated with BA, and rs17095355 was the top SNP (PAllele = 3.23×10-6). Meta-analysis of published data and current data indicated that rs17095355 was associated with BA susceptibility in Asians and Caucasians. Three associated SNPs were expression quantitative trait loci (eQTL) for ADD3. Two GPC1 SNPs in high linkage disequilibrium (LD) showed nominal association with BA susceptibility (PAllele = 0.03 for rs6707262 and PAllele = 0.04 for rs6750380), and were eQTL of GPC1. Haplotype harboring these two SNPs almost reached the study-wide significance (P = 0.0035). No association for ARF6 and EFEMP1 was found with BA risk in the current population. Our study validated associations of ADD3 and GPC1 SNPs with BA risk in Chinese population and provided evidence of epistatic contributions of genetic factors to BA susceptibility.

En España, más de 4000 personas serán diagnosticadas anualmente con un tumor maligno cerebral que en su mayoría recaerán en el grupo que se denomina Gliomas Malignos. La mayoría son glioblastomas (grado IV) (GB), siendo estos el tipo más agresivo. Se espera que la supervivencia media para los pacientes tratados con protocolo estándar sea de alrededor de 15 meses. La esperanza de vida tan reducida se debe a que son tumores muy proliferativos y heterogéneos. Esto subraya la necesidad de buscar alternativas para mejorar su manejo. Los estudios de esta tesis se centran en caracterizar biomarcadores y alteraciones genéticas en los diferentes estatus de presentación del EGFR que permitan abordar mejor el conocimiento de este tumor, así como confirmar su valor pronóstico o predictivo de respuesta al tratamiento. Los artículos I y II exploraron diferentes biomarcadores encontrando que la proteína Major Vault Protein (MVP) y el estado de metilación del Factor de Ribosilación de ADP 1 (ARF1) son marcadores que proporcionan información sobre los mecanismos moleculares que subyacen al comportamiento agresivo de los GB. En el artículo I se estudia el papel de la proteína MVP dentro de los diferentes estatus de EGFR y su relación con el PTEN, así como su correlación con la resistencia a múltiples fármacos al reducir la acumulación celular de agentes quimioterapéuticos en estos pacientes. Este estudio mostró que un incremento en la expresión de MVP es un factor pronóstico independiente de peor respuesta clínica a la quimioterapia y/o supervivencia global. El propósito del segundo artículo fue caracterizar el estado de metilación de genes relevantes para el GB que estuvieran asociados con alteraciones del gen EGFR, y que pudieran ser biomarcadores de mejor pronóstico, encontrándose una asociación significativa con una molécula implicada en el proceso de tráfico vesicular, el ARF1. Los resultados presentados en esta publicación proporcionan una base para una nueva hipótesis que conecta la amplificación del gen EGFR con la metilación del promotor del gen ARF1, el tráfico de vesículas, el recambio de membranas y el aumento del metabolismo tumoral. Los mecanismos subyacentes a estas conexiones y sus consecuencias funcionales quedan pendientes de establecer. En el tercer artículo se analizaron las alteraciones del número de copias somáticas (SCNA) junto con la detección de EGFRvIII y la amplificación de EGFR en una serie de 137 pacientes con GB. Se encontraron asociaciones estadísticamente significativas entre la amplificación de EGFR y/o EGFRvIII y SCNA en CDKN2A, MSH6, MTAP y ADD3 siendo tanto EGFRvIII como las pérdidas en ADD3 marcadores independientes de mal pronóstico. La mayoría de estos genes se encontraban implicados en las principales vías de señalización de EGFR. En conclusión, los resultados de estos estudios aportan nuevos conocimientos de alteraciones genéticas que probablemente están implicadas en los mecanismos que conducen al desarrollo del glioblastoma. In Spain, more than 4,000 people will be diagnosed annually with a malignant brain tumor, being the majority of them malignant gliomas. Most are glioblastomas (grade IV) (GB), these being the most aggressive type. The average survival for patients treated with the standard protocol is expected to be around 15 months. Such reduced life expectancy is due to the fact that they are highly proliferative and heterogeneous tumors. This underlines the need to look for alternatives to improve their management. The studies of this thesis focus on characterizing biomarkers and genetic alterations in the different presentation status of EGFR that can better address the knowledge of this tumor as well as confirm its prognostic or predictive value of response to treatment. Articles I and II explored different biomarkers finding that the Major Vault Protein (MVP) and the methylation status of ADP 1 Ribosylation Factor (ARF1) are markers that provide information on the molecular mechanisms underlying the aggressive behavior of GB. In article I, the role of the MVP protein within the different EGFR status and its relationship with PTEN is studied, as well as its interrelationship with resistance to multiple drugs by reducing the cellular accumulation of chemotherapeutic agents in these patients. This study showed that an increase in MVP expression is an independent prognostic factor for worse clinical response to chemotherapy and/or overall survival. The purpose of the second article was to characterize the methylation status of genes relevant to GB that would be associated with alterations in the EGFR gene and that could be biomarkers of better prognosis. A significant association with a molecule involved in the vesicular trafficking process, ARF1, was found. The results presented in this publication provided a basis for a new hypothesis connecting EGFR gene amplification with ARF1 gene promoter methylation, vesicle trafficking, membrane turnover and increased tumor metabolism. The mechanisms underlying these connections and their functional consequences remain to be established. In the third article, somatic copy number alterations (SCNA), along with EGFRvIII detection and EGFR amplification, were analyzed in a series of 137 GB patients. Statistically significant associations between the amplification of EGFR and/or EGFRvIII, and SCNA in CDKN2A, MSH6, MTAP and ADD3 were found, being both EGFRvIII and ADD3 losses independent markers of poor prognosis. Most of these genes were involved in the main EGFR signaling pathways. In conclusion, the results of these studies provide new insights into genetic alterations that are probably involved in the mechanisms that lead to the development of glioblastoma.

We compared a standard antihypertensive losartan treatment with a pharmacogenomics-guided rostafuroxin treatment in never-treated Caucasian and Chinese patients with primary hypertension. Rostafuroxin is a digitoxigenin derivative that selectively disrupts the binding to the cSrc-SH2 domain of mutant α-adducin and of the ouabain-activated Na-K pump at 10–11 M. Of 902 patients screened, 172 were enrolled in Italy and 107 in Taiwan. After stratification for country and genetic background, patients were randomized to rostafuroxin or losartan, being the difference in the fall in office systolic blood pressure (OSBP) after 2-month treatment the primary endpoint. Three pharmacogenomic profiles (P) were examined, considering: P1, adding to the gene variants included in the subsequent P2, the variants detected by post-hoc analysis of a previous trial; P2, variants of genes encoding enzymes for endogenous ouabain (EO) synthesis (LSS and HSD3B1), EO transport (MDR1/ABCB1), adducin (ADD1 and ADD3); P3, variants of the LSS gene only. In Caucasians, the group differences (rostafuroxin 50 μg minus losartan 50 mg in OSBP mmHg) were significant both in P2 adjusted for genetic heterogeneity (P2a) and P3 LSS rs2254524 AA [9.8 (0.6–19.0), P = 0.038 and 13.4 (25.4–2.5), P = 0.031, respectively]. In human H295R cells transfected with LSS A and LSS C variants, the EO production was greater in the former (P = 0.038); this difference was abolished by rostafuroxin at 10–11 M. Chinese patients had a similar drop in OSBP to Caucasians with losartan but no change in OSBP with rostafuroxin. These results show that genetics may guide drug treatment for primary hypertension in Caucasians.

Recently, we reported that a mutation in γ-Adducin (ADD3) alters the actin cytoskeleton and is associated with an impaired myogenic response of the afferent arteriole and enhanced hypertension-induced renal disease. However, it remains to be determined whether the loss of ADD3 function promotes renal injury by increasing glomerular capillary pressure (Pgc) and podocyte loss or other mechanisms. The present study compared the time course of changes in renal hemodynamics and the progression of renal injury during the development of DOCA-salt hypertension in FHH 1 BN rats (WT) with an intact myogenic response vs. FHH 1 BN Add3KO rats (Add3KO) in which the myogenic response is impaired. When transmural pressure rose from 40 to 100 mmHg, the inner diameter of the preglomerular artery constricted by 19% (47.7 ± 4.3 to 38.4 ± 3.4 μm, n = 5) in WT, but it dilated by 28% (53.0 ± 2.2 to 67.9 ± 4.3 μm, n = 7) in Add3KO. Pgc was similar (50.1 ± 0.4 vs. 51.2 ± 0.8 mmHg, n = 6) at 100 mmHg, but rose by 6 and 14 mmHg in WT vs. Add3KO when perfusion pressure rose to 150 mmHg. Mean arterial pressure increased similarly and reached 177.7 ± 3.5 vs. 182.6 ± 2.3 mmHg (n = 9) after 3 weeks of DOCA-salt hypertension in WT vs. Add3KO. After 1 week of DOCA-salt hypertension, glomerular filtration rate (GFR) increased by 38% (1.2 ± 0.1 to 1.6 ± 0.1 ml/min/kidney, n = 6) and glomerular nephrin expression decreased by 20% (165.4 ± 4.5 to 131.614 ± 5.2 RFU, n = 7) in Add3KO. Both were unaltered in WT. Proteinuria increased 2 folds in WT (56.9 ± 4.7 to 168.6 ± 26.7 mg/day, n = 12) in the first week of hypertension vs. a 6-fold increase in Add3KO (64.6 ± 4.1 to 446.0 ± 41.9 mg/day, n = 9). After 3 weeks of hypertension, the degree of glomerulosclerosis (3.4 ± 0.1 vs. 2.4 ± 0.1 glomerular injury score, n = 9~12), protein cast formation (9.0% ± 0.8% vs. 4.8% ± 0.4% of area, n = 6), epithelial-mesenchymal transition, interstitial fibrosis (17.9% ± 0.8% vs. 9.5% ± 0.3% of area, n = 6), and inflammation was significantly greater in Add3KO vs. WT. GFR and Pgc were 28% and 19% lower in Add3KO than WT. These results indicate that the impaired myogenic response increases the transmission of pressure to the glomerulus to induce the loss of podocytes, which accelerates the progression of renal injury during the development of hypertension in Add3KO.

Recently, we reported a mutation in γ-adducin (ADD3) was associated with an impaired myogenic response of the afferent arteriole and hypertension-induced chronic kidney disease (CKD) in fawn hooded hypertensive (FHH) rats. However, the mechanisms by which altered renal blood flow (RBF) autoregulation promotes hypertension-induced renal injury remain to be determined. The present study compared the time course of changes in renal hemodynamics and the progression of CKD during the development of DOCA-salt hypertension in FHH 1BNcongenic rats [wild-type (WT)] with an intact myogenic response versus FHH 1BNAdd3KO ( Add3KO) rats, which have impaired myogenic response. RBF was well autoregulated in WT rats but not in Add3KO rats. Glomerular capillary pressure rose by 6 versus 14 mmHg in WT versus Add3KO rats when blood pressure increased from 100 to 150 mmHg. After 1 wk of hypertension, glomerular filtration rate increased by 38% and glomerular nephrin expression decreased by 20% in Add3KO rats. Neither were altered in WT rats. Proteinuria doubled in WT rats versus a sixfold increase in Add3KO rats. The degree of renal injury was greater in Add3KO than WT rats after 3 wk of hypertension. RBF, glomerular filtration rate, and glomerular capillary pressure were lower by 20%, 28%, and 19% in Add3KO rats than in WT rats, which was associated with glomerular matrix expansion and loss of capillary filtration area. The results indicated that impaired RBF autoregulation and eutrophic remodeling of preglomerular arterioles increase the transmission of pressure to glomeruli, which induces podocyte loss and accelerates the progression of CKD in hypertensive Add3KO rats.

Accumulating evidence indicates that the alternative splicing program undergoes extensive changes during cancer development and progression. The RNA-binding protein QKI-5 is frequently downregulated and exhibits anti-tumor activity in lung cancer. Howeve-r, little is known about the functional targets and regulatory mechanism of QKI-5. Here, we report that upregulation of exon 14 inclusion of cytoskeletal gene Adducin 3 (ADD3) significantly correlates with a poor prognosis in lung cancer. QKI-5 inhibits cell proliferation and migration in part through suppressing the splicing of ADD3 exon 14. Through genome-wide mapping of QKI-5 binding sites in vivo at nucleotide resolution by iCLIP-seq analysis, we found that QKI-5 regulates alternative splicing of its target mRNAs in a binding position-dependent manner. By binding to multiple sites in an upstream intron region, QKI-5 represses the splicing of ADD3 exon 14. We also identified several QKI mutations in tumors, which cause dysregulation of the splicing of QKI targets ADD3 and NUMB. Taken together, our results reveal that QKI-mediated alternative splicing of ADD3 is a key lung cancer-associated splicing event, which underlies in part the tumor suppressor function of QKI.

Background Weighted co-expression network analysis (WGCNA) is a powerful systems biology method to describe the correlation of gene expression based on the microarray database, which can be used to facilitate the discovery of therapeutic targets or candidate biomarkers in diseases. Objective To explore the key genes in the development of Alzheimer's disease (AD) by using WGCNA. Methods The whole gene expression data GSE1297 from AD and control human hippocampus was obtained from the GEO database in NCBI. Co-expressed genes were clustered into different modules. Modules of interest were identified through calculating the correlation coefficient between the module and phenotypic traits. GO and pathway enrichment analyses were conducted, and the central players (key hub genes) within the modules of interest were identified through network analysis. The expression of the identified key genes was confirmed in AD transgenic mice through using qRT-PCR. Results Two modules were found to be associated with AD clinical severity, which functioning mainly in mineral absorption, NF-κB signaling, and cGMP-PKG signaling pathways. Through analysis of the two modules, we found that metallothionein (MT), Notch2, MSX1, ADD3, and RAB31 were highly correlated with AD phenotype. Increase in expression of these genes was confirmed in aged AD transgenic mice. Conclusion WGCNA analysis can be used to analyze and predict the key genes in AD. MT1, MT2, MSX1, NOTCH2, ADD3, and RAB31 are identified to be the most relevant genes, which may be potential targets for AD therapy.

Цель — изучить уровень экспрессии генов, кодирующих рецепторы TLR2 и TLR4, а также фактор некроза опухолей TNF и аддуцин ADD3, в лимфоцитах крови подростков и в подкожной жировой ткани взрослых мужчин с ожирением, как при нормальной, так и нарушенной чувствительности к инсулину.Материалы и методы. Исследования проведены на трех группах подростков в возрасте около 14 лет и трех группах взрослых мужчин в возрасте около 45 лет: нормальных без признаков ожирения (контроль) и с ожирением, которые имели нормальную чувствительность к инсулину, и с ожирением, осложненным резистентностью к инсулину. РНК выделяли из лейкоцитов крови и в подкожной жировой ткани, а уровень экспрессии генов определяли методом количественной полимеразной цепной реакции в реальном времени. Определяли также коэффициент корреляции между изменениями в экспрессии генов и индексом массы тела и резистентностью к инсулину.Результаты. Установлено, что уровень экспрессии генов TLR2 и TLR4 увеличивается, а генов TNF и ADD3, наоборот, снижается в лимфоцитах подростков при ожирении и нормальной чувствительности к инсулину по сравнению с контрольной группой. При этих условиях в подкожной жировой ткани взрослых мужчин выявлены аналогичные изменения для генов TLR2 и ADD3, в то время как уровень экспрессии гена TLR4 снижается, а гена TNF — увеличивается. Развитие резистентности к инсулину на фоне ожирения изменяет только уровень экспрессии генов ADD3 в лимфоцитах и ТNF в жировой ткани. Изменения в экспрессии исследованных генов в лимфоцитах подростков при ожирении коррелируют с изменениями индекса массы тела (TLR2 — позитивно, а TNF и ADD3 — негативно), но с резистентностью к инсулину на фоне ожирения позитивно коррелируют только изменения в экспрессии гена ADD3 в лимфоцитах подростков.Выводы. Уровень экспрессии генов TLR2, TLR4, TNF и ADD3, которые задействованы в многогранной регуляции процессов метаболизма и защитных реакциях организма, существенно нарушается в лимфоцитах подростков и подкожной жировой ткани взрослых мужчин с ожирением, как с нормальной, так и сниженной чувствительностью к инсулину, но с резистентностью к инсулину коррелируют лишь изменения в экспрессии гена ADD3 в лимфоцитах подростков., Objective. To study the expression of genes encoded TLR2 and TLR4 receptors as well as tumor necrosis factor (TNF) and adducin ADD3 in lymphocytes of obese adolescents and adult men with normal and impaired insulin sensitivity.Materials and methods. Three groups of adolescent boys with mean age 14 years and three groups of adult men with mean age 45 years were examined for this study: healthy persons with no signs of obesity (control group), obese individuals with normal insulin sensitivity and with impaired insulin sensitivity. RNA was extracted from white blood cells and subcutaneous adipose tissue, and the levels of gene expressions were studied using quantitative real-time polymerase chain reaction. Correlation coefficient between changes of gene expression and body mass index, as well as insulin resistance, was detected.Results: It was shown that the expression level of TLR2 and TLR4 genes increased, but TNF and ADD3 genes decreased in lymphocytes of obese adolescents with normal insulin sensitivity as compared to the control group. Under the same conditions in subcutaneous adipose tissue of adult men similar changes of TLR2 and ADD3 genes were detected, but the level of TLR4 gene expression decreased and TNF gene increased. The development of insulin resistance secondary to obesity changed the expression level only of ADD3 in lymphocytes and ТNF in adipose tissue. Changes of the studied gene expression in lymphocytes in obese adolescents correlated with body mass index (TLR2 - positively and TNF and ADD3 — negatively); however, in case of obesity with insulin resistance there was positive correlation with ADD3 gene expression only.Conclusions. The TLR2, TLR4, TNF та ADD3 gene expression level, which participate in the multifaceted regulation of metabolic processes and defense mechanisms of organism, is significantly deregulated in lymphocytes of adolescents and in subcutaneous adipose tissue of adult men with obesity and normal as well as suppressed insulin sensitivity. However, the insulin resistance in obese adolescents is correlated with changes in the ADD3 gene expression level in lymphocytes only., Мета: дослідити рівень експресії генів, що кодують рецептори TLR2 та TLR4, а також фактор некрозу пухлин TNF та аддуцин ADD3, у лімфоцитах крові підлітків та у підшкірній жировій тканині дорослих чоловіків з ожирінням, як за нормальної, так і порушеної чутливості до інсуліну.Пацієнти і методи. Дослідження проведені на трьох групах підлітків чоловічої статі віком біля 14 років та трьох групах дорослих чоловіків віком біля 45 років: нормальних без ознак ожиріння (контроль) та з ожирінням і нормальною чутливістю до інсуліну, а також із ожирінням, ускладненим резистентністю до інсуліну. РНК виділяли із лейкоцитів крові та підшкірної жирової тканини, а рівень експресії генів визначали методом кількісної полімеразної ланцюгової реакції у реальному часі. Визначали також коефіцієнт кореляції між змінами в експресії генів та індексом маси тіла й резистентністю до інсуліну.Результати. Встановлено, що рівень експресії генів TLR2 та TLR4 збільшується, а генів TNF та ADD3, навпаки, знижується у лімфоцитах підлітків за умов ожиріння і нормальної чутливості до інсуліну порівняно з контрольною групою. За цих самих умов у підшкірній жировій тканині дорослих чоловіків виявлені подібні зміни для генів TLR2 та ADD3, в той час як рівень експресії гена TLR4 зменшується, а гена TNF — збільшується. Розвиток резистентності до інсуліну на тлі ожиріння змінює рівень експресії лише генів ADD3 у лімфоцитах і ТNF у жировій тканині. Зміни в експресії досліджених генів у лімфоцитах підлітків за умов ожиріння корелюють зі змінами індексу маси тіла (TLR2 — позитивно, а TNF та ADD3 — негативно), але з резистентністю до інсуліну на тлі ожиріння позитивно корелюють лише зміни в експресії гена ADD3 у лімфоцитах.Висновки. Рівень експресії генів TLR2, TLR4, TNF та ADD3, які задіяні у багатогранній регуляції процесів метаболізму і захисних реакціях організму, суттєво порушується у лімфоцитах підлітків та підшкірній жировій тканині дорослих чоловіків з ожирінням, як за нормальної, так і зниженої чутливості до інсуліну, але з резистентністю до інсуліну корелюють лише зміни в експресії генів ADD3 у лімфоцитах підлітків.

Biliary atresia (BA) is an idiopathic neonatal cholestatic disease. Recent genome-wide association study (GWAS) revealed that common variation of ADD3, GPC1, ARF6, and EFEMP1 gene was associated with BA susceptibility. We aimed to evaluate the association of these genes with BA in Chinese population. Twenty single nucleotide polymorphisms (SNPs) in these four genes were genotyped in 340 BA patients and 1,665 controls. Three SNPs in ADD3 were significantly associated with BA, and rs17095355 was the top SNP (PAllele = 3.23×10-6). Meta-analysis of published data and current data indicated that rs17095355 was associated with BA susceptibility in Asians and Caucasians. Three associated SNPs were expression quantitative trait loci (eQTL) for ADD3. Two GPC1 SNPs in high linkage disequilibrium (LD) showed nominal association with BA susceptibility (PAllele = 0.03 for rs6707262 and PAllele = 0.04 for rs6750380), and were eQTL of GPC1. Haplotype harboring these two SNPs almost reached the study-wide significance (P = 0.0035). No association for ARF6 and EFEMP1 was found with BA risk in the current population. Our study validated associations of ADD3 and GPC1 SNPs with BA risk in Chinese population and provided evidence of epistatic contributions of genetic factors to BA susceptibility.

Glioblastoma multiforme (GB) is one of the most aggressive tumors. Despite continuous efforts to improve its clinical management, there is still no strategy to avoid a rapid and fatal outcome. EGFR amplification is the most characteristic alteration of these tumors. Although effective therapy against it has not yet been found in GB, it may be central to classifying patients. We investigated somatic-copy number alterations (SCNA) by multiplex ligation-dependent probe amplification in a series of 137 GB, together with the detection of EGFRvIII and FISH analysis for EGFR amplification. Publicly available data from 604 patients were used as a validation cohort. We found statistical associations between EGFR amplification and/or EGFRvIII, and SCNA in CDKN2A, MSH6, MTAP and ADD3. Interestingly, we found that both EGFRvIII and losses on ADD3 were independent markers of bad prognosis (p = 0.028 and 0.014, respectively). Finally, we got an unsupervised hierarchical classification that differentiated three clusters of patients based on their genetic alterations. It offered a landscape of EGFR co-alterations that may improve the comprehension of the mechanisms underlying GB aggressiveness. Our findings can help in defining different genetic profiles, which is necessary to develop new and different approaches in the management of our patients.

Recent evidence suggests that the presence of more than one pathogenic mutation in a single patient is more common than previously anticipated. One of the challenges hereby is to dissect the contribution of each gene mutation, for which animal models such as Drosophila can provide a valuable aid. Here, we identified three families with mutations in ADD3, encoding for adducin-γ, with intellectual disability, microcephaly, cataracts and skeletal defects. In one of the families with additional cardiomyopathy and steroid-resistant nephrotic syndrome (SRNS), we found a homozygous variant in KAT2B, encoding the lysine acetyltransferase 2B, with impact on KAT2B protein levels in patient fibroblasts, suggesting that this second mutation might contribute to the increased disease spectrum. In order to define the contribution of ADD3 and KAT2B mutations for the patient phenotype, we performed functional experiments in the Drosophila model. We found that both mutations were unable to fully rescue the viability of the respective null mutants of the Drosophila homologs, hts and Gcn5, suggesting that they are indeed pathogenic in flies. While the KAT2B/Gcn5 mutation additionally showed a significantly reduced ability to rescue morphological and functional defects of cardiomyocytes and nephrocytes (podocyte-like cells), this was not the case for the ADD3 mutant rescue. Yet, the simultaneous knockdown of KAT2B and ADD3 synergistically impaired kidney and heart function in flies as well as the adhesion and migration capacity of cultured human podocytes, indicating that mutations in both genes may be required for the full clinical manifestation. Altogether, our studies describe the expansion of the phenotypic spectrum in ADD3 deficiency associated with a homozygous likely pathogenic KAT2B variant and thereby identify KAT2B as a susceptibility gene for kidney and heart disease in ADD3-associated disorders.

A genome-wide association study in East Asians suggested a genetic association between biliary atresia (BA) and a cluster of variants within the Adducin 3 (ADD3) and ADD3 antisense RNA1 (ADD3-AS1) genes. Another study in Thai neonates reported an association between BA and rs17095355. To validate those findings, this study aimed to analyze the BA association with single nucleotide polymorphisms (SNPs) and the additive influence of ADD3 and ADD3-AS1 in Thai neonates. DNAs from 56 BA cases and 166 controls were genotyped for rs2501577, rs11194981, rs12268910 (ADD3) and rs17095355 (ADD3-AS1), using TaqMan PCR. Genotype distributions were compared between the groups, and SNP–SNP interactions were analyzed by combination of allelotypes. The risk allele frequencies of rs2501577, rs11194981, and rs17095355 in the BA group were significantly higher than in the controls. Univariate analysis showed that recessive variants in the three SNPs were associated with BA risk at ORs of 1.81 (95% CI 1.32–2.50), 1.58 (95% CI 1.14–2.20) and 1.92 (95% CI 1.39–2.66), respectively. SNP–SNP interaction analysis showed that the SNP combination of the two genes rs17095355 and rs2501577 provided an additive increase in BA risk. ADD3 and ADD3-AS1 variants increased susceptibility to BA, suggesting that these genes may play an additive role in the pathogenesis of the disease. In addition, these interactions may give a clue to the overexpression of the ADD3 protein in the liver of BA patients.

Adducin (ADD) is a family of membrane skeleton proteins including ADD1, ADD2, and ADD3 that are encoded by distinct genes on different chromosomes. Adducin is primarily responsible for the assembly of spectrin-actin network that provides physical support to the plasma membrane and mediates signal transduction in various cellular physiological processes upon regulation by protein kinase C-dependent and calcium/calmodulin-dependent pathways. Abnormal phosphorylation, genetic variations, and alternative splicing of adducin may contribute to alterations in cellular functions involved in pathogenic processes. These alterations are associated with a wide range of diseases including cancer. This paper begins with a discussion on how adducin partakes in the structural formation of membrane skeleton, its regulation, and related functional characteristics, followed by a review on the pathogenesis of hypertension, biliary atresia, and cancer with respect to increased disease susceptibility mediated by adducin polymorphism and/or dysregulation. Given the functional diversity of adducin in different cellular compartments, we aim to provide a knowledge base whereby its pathophysiological roles can be better understood. More importantly, we aim to provide novel insights that may be of significance in turning the adducin model to clinical application.

Supplemental Digital Content is available in the text, Objectives: Biliary atresia (BA) is a progressive fibroinflammatory cholangiopathy affecting the bile ducts of neonates. Although BA is the leading indication for pediatric liver transplantation, the etiology remains elusive. Adducin 3 (ADD3) and X-prolyl aminopeptidase 1 (XPNPEP1) are 2 genes previously identified in genome-wide association studies as potential BA susceptibility genes. Using zebrafish, we investigated the importance of ADD3 and XPNPEP1 in functional studies. Methods: To determine whether loss of either gene leads to biliary defects, we performed morpholino antisense oligonucleotide (MO) knockdown studies targeting add3a and xpnpep1 in zebrafish. Individuals were assessed for decreases in biliary function and the presence of biliary defects. Quantitative polymerase chain reaction was performed on pooled 5 days postfertilization larvae to assess variations in transcriptional expression of genes of interest. Results: Although both xpnpep1 and add3a are expressed in the developing zebrafish liver, only knockdown of add3a produced intrahepatic defects and decreased biliary function. Similar results were observed in homozygous add3a mutants. MO-mediated knockdown of add3a also showed higher mRNA expression of hedgehog (Hh) targets. Inhibition of Hh signaling rescued biliary defects caused by add3a knockdown. Combined knockdown of add3a and glypican-1 (gpc1), another mediator of Hh activity that is also a BA susceptibility gene, resulted in more severe biliary defects than knockdown of either alone. Conclusions: Our results support previous studies identifying ADD3 as a putative genetic risk factor for BA susceptibility. Our results also provide evidence that add3a may be affecting the Hh pathway, an important factor in BA pathogenesis.

The biological bases of cognitive impairment in schizophrenia are poorly understood and may lie in insults in neurodevelopment, leading to alterations in critical structures. Synapses proteins are claimed to have etiopathogenic roles and more direct effects on core cognitive functions. Adducins family proteins seem of great interest, as they are fundamental constituents of synapses, involved in actin cytoskeleton assembly-disassembly, responsible of synaptic plasticity. ADD2 is more prominently expressed in brain tissues and influences memory and learning, commonly impaired in schizophrenia. In the present study we tested 342 patients with schizophrenia for three common adducins genetic variants, ADD1 rs4961, ADD2 rs4984 and ADD3 rs3731566, reported to have significant effects on circulatory system in humans. Neuropsychological measures were evaluated with the Brief Assessment of Cognition in Schizophrenia (BACS), a broad battery evaluating core cognitive domains. The analysis showed significant effects of ADD2 genotype on almost every cognitive domain. Moreover, significant interactions between ADD1 and ADD3 were also observed on some BACS subtests, namely Symbol Coding and Verbal Memory. Our findings suggest that adducins are involved in cognitive impairment in schizophrenia. This effect may result both from a direct mechanism affecting synaptic building and plasticity and indirectly as a consequence of vascular insults. (C) 2015 Elsevier Masson SAS. All rights reserved.

Background Recent studies have used cluster analysis to identify phenotypic clusters of asthma with differences in clinical traits, as well as differences in response to therapy with anti-inflammatory medications. However, the correspondence between different phenotypic clusters and differences in the underlying molecular mechanisms of asthma pathogenesis remains unclear. Objective We sought to determine whether clinical differences among children with asthma in different phenotypic clusters corresponded to differences in levels of gene expression. Methods We explored differences in gene expression profiles of CD4 + lymphocytes isolated from the peripheral blood of 299 young adult participants in the Childhood Asthma Management Program study. We obtained gene expression profiles from study subjects between 9 and 14 years of age after they participated in a randomized, controlled longitudinal study examining the effects of inhaled anti-inflammatory medications over a 48-month study period, and we evaluated the correspondence between our earlier phenotypic cluster analysis and subsequent follow-up clinical and molecular profiles. Results We found that differences in clinical characteristics observed between subjects assigned to different phenotypic clusters persisted into young adulthood and that these clinical differences were associated with differences in gene expression patterns between subjects in different clusters. We identified a subset of genes associated with atopic status, validated the presence of an atopic signature among these genes in an independent cohort of asthmatic subjects, and identified the presence of common transcription factor binding sites corresponding to glucocorticoid receptor binding. Conclusion These findings suggest that phenotypic clusters are associated with differences in the underlying pathobiology of asthma. Further experiments are necessary to confirm these findings.

Genetic variants of alpha adducin (ADD1) taken alone or in interaction with those of beta (ADD2) and gamma (ADD3) subunits have been associated with primary hypertension in humans and in Milan hypertensive (MHS) rats. In this study, we report the dissection of the individual contribution of each rat Add gene to blood pressure, by congenic substitution mapping. Congenic strains were developed by introgressing Add1, Add2, and Add3 genes (and chr14, chr4, and chr1 associated segments) of MHS in the Milan normotensive rat (MNS) genetic background (MNS.H-Add1, MNS.H-Add2, and MNS.H-Add3) and vice versa (MHS.N-Add1, MHS.N-Add2, and MHS.N-Add3). Systolic blood pressure (SBP) of MNS.H-Add1 rats was significantly higher (+10 mmHg) than that of MNS, whereas SBP of MHS.N-Add1 was significantly lower (-10 mmHg) than that of MHS. The differences account for 43% of the blood pressure differences between MHS and MNS. In contrast, SBPs of Add2 and Add3 congenic strains were not different from those of the correspondent recipient parental strain. The fine mapping of chr14 congenic segment supports the identity of blood pressure QTL with Add1 gene.

Adducin is a cytoskeleton heterodimeric protein. Its subunits are encoded by three related genes (ADD1, ADD2, and ADD3) which show alternative spliced variants. Adducin polymorphisms are involved in blood pressure regulation in humans and rats. We have analyzed mRNA distribution of ADD gene family in human tissues and cells with Real-Time TaqMan RT-PCR. Whereas ADD1 is ubiquitously distributed, ADD3 is more expressed in kidney medulla and cortex than in fetal kidney, while in adult liver it is less abundant than in fetal liver. ADD2 beta1 and beta4 variants show the same pattern of distribution with the highest expression in brain, fetal liver, and kidney. Conventional RT-PCR identified new beta4 variants. Beta4a is characterized by an in-frame insertion of 21 nucleotides upstream exon 15 predicting a 7 amino acids longer protein with a similar C-terminus region. It is coexpressed with beta1 and beta4 in several tissues. Fetal kidney shows further beta4b, beta4c and beta4d variants containing internal exon deletions that enormously modify the predicted NH(2) and central regions. Our findings could help one to understand the functional role of adducin variants in specific tissues and cells.

Abstract —In a previous study, by using a candidate gene approach, we detected in both Milan hypertensive rats and humans a polymorphism in the α-adducin gene (ADD1) that was associated with blood pressure and renal sodium handling. In the present study, a genomewide search with 264 informative markers was undertaken in 251 (Milan hypertensive strain × Milan normotensive strain) F2 rats to further investigate the contribution of the adducin gene family ( Add1 , Add2 , and Add3 ) and to identify novel quantitative trait loci (QTLs) that affect blood pressure. The influence of 2 different methods of blood pressure measurement, the intracarotid catheter and the tail-cuff method, was also evaluated. We found evidence that QTLs affected systolic blood pressure (SBP) measured at the carotid (direct SBP) on rat chromosome 1 with a logarithm of the odds (LOD) score peak of 3.3 on D1Rat121 and on rat chromosome 14 on Add1 locus (LOD=3.2). A QTL for SBP measured at the tail (indirect SBP) was found on rat chromosome 10 around D10Rat33 (LOD=5.0). All of these QTLs identified chromosomal regions not detected in other rat studies and harbor genes (Na + /H + exchanger A3; α-adducin; α 1B -adrenergic receptor) that may be involved in blood pressure regulation. Therefore, these findings may be relevant to human hypertension, also in consideration of the biochemical and pathophysiological similarities between MHS and a subgroup of patients of primary hypertension, which led to the identification of α-adducin as a candidate gene in both species.

In the United States, biliary atresia (BA) is the most frequent indication for liver transplantation in pediatric patients. BA is a complex disease, with suspected environmental and genetic risk factors. A genome-wide association study in Chinese patients identified association to the 10q24.2 (hg18) genomic region. This signal was upstream of two genes, XPNPEP1 and ADD3, both expressed in intrahepatic bile ducts. We tested association to this region in 171 BA patients and 1,630 controls of European descent and found the strongest signal to be at rs7099604 (p = 2.5 × 10(-3)) in intron 1 of the ADD3 gene. Moreover, expression data suggest that ADD3, but not XPNPEP1, is differentially expressed in BA patients. The role of ADD3 in biliary development is unclear, but our findings suggest that this gene may be functionally relevant for the development of BA.

Introduction Early detection of colorectal cancer (CRC) and its precursor lesions (adenomas) is crucial to reduce mortality rates. The fecal immunochemical test (FIT) is a non-invasive CRC screening test detecting blood-derived protein hemoglobin. However, FIT sensitivity is suboptimal especially in detection of CRC precursor lesions. As adenoma-to-carcinoma progression is accompanied by alternative splicing, tumor-specific proteins derived from alternatively spliced RNA transcripts might serve as candidate biomarkers for CRC detection. Materials and methods RNA and proteins were isolated from CRC cell line SW480 before and after siRNA-mediated down-modulation of the splicing machinery: SF3B1, U2AF1, and SRSF1. To identify splice variants, mRNA was sequenced (Illumina HiSeq) and analyzed. RNA-seq analysis included quality checks (FASTQ, RSeQC), reads mapping (STAR), differential gene expression (DESeq2) and differential expression of splicing isoforms between conditions (MATS). Results from the in silico analysis were validated by qRT-PCR. Proteins were analyzed by in-depth tandem mass spectrometry (QExactive). A proteogenomic data analysis pipeline was established to enrich the sequence database, against which the mass spectra are searched, with the predicted protein splice variants and identify protein isoforms. To further extend the splice-variant database, PacBio sequencing of full-length transcripts is being performed for the SW480 siSF3B1- and control-samples. Results Differential expression analysis on RNA and protein level proved that the knock-down experiments were performed successfully. The RNA-seq analysis revealed hundreds of mRNA splice variants, including positive controls described in literature. For example, down-modulation of SF3B1 resulted in quantitative mRNA changes of spliced isoforms of ADD3 both in RNA-seq and RT-PCR data. The proteomics experiment yielded over 6000 proteins per sample, among which a number of protein isoforms resulting from alternative splicing. Conclusions and discussion We established a proteogenomic pipeline for the analysis of alternative splicing and provided experimental proof of concept. We expect, however, that the true complexity of RNA variant information remains highly underestimated. We therefore are performing PacBio long-read RNA-seq to validate our approach and to identify additional (novel) splicing events. In future studies we will apply the mRNA-seq based proteogenomic pipeline for detection of protein alterations to a series of adenomas at low- and high-risk of progression, and CRCs to validate the isoforms in a clinically relevant setting. Novel findings will be evaluated for their performance as screening markers for CRC. This work was financially supported by KWF project nr: VU 2013-6025. Citation Format: Malgorzata A. Komor, Annemieke C. Hiemstra, Thang V. Pham, Sander R. Piersma, Robert P. Sebra, Bo W. Han, Meredith Ashby, Beatriz Carvalho, Gerrit A. Meijer, Connie R. Jimenez, Remond JA Fijneman. Proteogenomic analysis of alternative splicing: the search for novel biomarkers for colorectal cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 848.

Review on ADD3 (adducin 3), with data on DNA, on the protein encoded, and where the gene is implicated.

The usefulness of the results so far published on genetics of primary hypertension for establishing the clinical impact of candidate gene polymorphisms is weakened by the scanty information regarding: a) the functional effect of the gene variants of interest in humans; b) the regulatory genetic network (RGN) where the gene is operating with all the interacting environmental–biological factors and the respective hierarchical organization; c) the consistency between the natural history of the established pathophysiological mechanisms underlying hypertension and the new molecular mechanism detected with genetics; d) the limitations regarding the translation of animal data to human due to the differences among species of the genetic molecular mechanisms underlying similar organ function changes in the different species. Of course, not all these information are available for adducin polymorphisms. In this review, being aware of their importance, the evaluation of the clinical impact of adducin has been focused on data obtained together with the interacting genetic-environmental or biological factors. Adducin polymorphisms and endogenous ouabain (EO) were detected by a top-down approach in rodents after having demonstrated, at cellular and kidney level, that an increase in tubular Na reabsorption could underlies the transition from normotension to hypertension both in rodents and humans. Therefore, we hypothesized that adducin polymorphisms and EO may operate within the triggering RGN that initiates the increase in blood pressure in both species. The distinction between triggering RGN and the secondary RGN is important both to limit the level of genetic complexity arising from secondary changes, and to detect the molecular target to develop tailored therapeutic approach. The pharmacogenomic approach, both in rodents or humans, with newly discovered and never treated hypertension, may be useful to strengthen the “causation” of genetic mechanism. Mutant adducin increases tubular reabsorption: diuretics, because of their effect on overall tubular reabsorption, or rostafuroxin, because of its selective inhibition of the adducin and ouabain effects, may be used for this purpose. Indeed the pharmacogenomic approach with both drugs have provided data consistent with the role of adducin and EO. Taken together, all these findings indicate a clear impact of adducin polymorphism and EO in a subset of patients when the appropriate environmental, biological or genetic context is taken into account. The size of this impact is variable and affected by the context.

Hypertension is an important public health problem affecting more than 50 million individuals in the USA alone. The most common form, essential hypertension, results from the complex interplay between genetic predisposition and environmental influences. Epidemiological, migration, intervention and genetic studies in humans and animals provide very strong evidence of a causal link between high salt intake and high blood pressure. One of the candidate genes for salt-sensitive hypertension is adducin. Adducin is a heterodimeric cytoskeleton protein, the three subunits of which are encoded by genes (ADD1, ADD2 and ADD3) that map to three different chromosomes. A long series of parallel studies in the Milan hypertensive rat strain model of hypertension and humans indicated that an altered adducin function might cause hypertension through enhanced constitutive tubular sodium reabsorption. An example of a prospective efficacy of pharmacogenetics and pharmacogenomics is the detection and impact of adducin polymorphisms on hypertension. In particular, the selective advantages of diuretics in preventing myocardial infarction and stroke over other antihypertensive therapies that produce a similar blood pressure reduction in carriers of the mutated adducin may support new strategies aimed at optimizing the use of new antihypertensive agents for the prevention of hypertension-associated organ damage.

There is a growing literature on the potential prospective use of genome information to enhance success in finding new medicines. An example of a prospective efficacy of pharmacogenetic and pharmacogenomics is the detection and impact of adducin polymorphism on hypertension. Adducin is a heterodimeric cytoskeleton protein, the three subunits of which are encoded by genes (ADD1, ADD2, and ADD3) that map to three different chromosomes. A long series of parallel studies in the Milan hypertensive rat strain model of hypertension and humans indicated that an altered adducin function might cause hypertension through an enhanced constitutive tubular sodium reabsorption. In particular, six linkage studies, 18 of 20 association studies, and four of five follow-up studies that measured organ damage in hypertensive patients support the clinical impact of adducing polymorphism. As many modulatory genes and environment affect the adducin activity, the context must be taken into account to measure the clinical effect size of adducins. Pharmacogenomics is giving an important contribution to this end. In particular, the selective advantages of diuretics in preventing myocardial infarction and stroke over other antihypertensive therapies that produce a similar BP reduction in carriers of the mutated adducin may support new strategies that aim to optimize the use of antihypertensive agents for the prevention of hypertension-associated organ damage.

This paper addresses the issue of collapsing dependent floating-point operations. The presentation focuses on studying the dataflow graph of benchmark involving a large number of floating-point instructions. In particular, it focuses on the relevance of new floating-point operators performing two dependent operations which are similar to "fused multiply and add". Finally, this paper examines the implementation cost and critical path reduction from this strategy.

Cet article présente une implantation d'une unité flottante capable d'effectuer deux additions flottantes consécutives. Cet opérateur prend trois nombres flottants double précision en entrée et retourne l'arrondi de la somme/soustraction de ces trois nombres. Cette unité propose pour l'unique arrondi final l'un des quatres modes d'arrondi exigés par la norme IEEE-754.