RESUMEN

BACKGROUND: Elevated triglyceride levels are a risk factor for cardiovascular disease. Angiopoietin-like protein 4 (Angptl4) is a metabolic factor that raises plasma triglyceride levels by inhibiting lipoprotein lipase (LPL). In non-diabetic individuals, the ANGPTL4 coding variant E40K has been associated with lower plasma triglyceride levels while the T266M variant has been associated with more modest effects on triglyceride metabolism. The objective of this study was to determine whether ANGPTL4 E40K and T266M are associated with triglyceride levels in the setting of obesity and T2D, and whether modification of triglyceride levels by these genetic variants is altered by a lifestyle intervention designed to treat T2D. METHODS: The association of ANGPTL4 E40K and T266M with fasting triglyceride levels was investigated in 2,601 participants from the Look AHEAD Clinical Trial, all of whom had T2D and were at least overweight. Further, we tested for an interaction between genotype and treatment effects on triglyceride levels. RESULTS: Among non-Hispanic White Look AHEAD participants, ANGPTL4 K40 carriers had mean triglyceride levels of 1.61 ± 0.62 mmol/L, 0.33 mmol/L lower than E40 homozygotes (p = 0.001). Individuals homozygous for the minor M266 allele (MAF 30%) had triglyceride levels of 1.75 ± 0.58 mmol/L, 0.24 mmol/L lower than T266 homozygotes (p = 0.002). The association of the M266 with triglycerides remained significant even after removing K40 carriers from the analysis (p = 0.002). There was no interaction between the weight loss intervention and genotype on triglyceride levels. CONCLUSIONS: This is the first study to demonstrate that the ANGPTL4 E40K and T266M variants are associated with lower triglyceride levels in the setting of T2D. In addition, our findings demonstrate that ANGPTL4 genotype status does not alter triglyceride response to a lifestyle intervention in the Look AHEAD study.

Asunto(s)

Angiopoyetinas/genética , Variación Genética , Triglicéridos/sangre , Anciano , Alelos , Sustitución de Aminoácidos , Proteína 4 Similar a la Angiopoyetina , Enfermedades Cardiovasculares/sangre , Enfermedades Cardiovasculares/etiología , Enfermedades Cardiovasculares/genética , Diabetes Mellitus Tipo 2/sangre , Diabetes Mellitus Tipo 2/complicaciones , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/terapia , Femenino , Homocigoto , Humanos , Estilo de Vida , Lipoproteínas HDL/sangre , Masculino , Persona de Mediana Edad , Obesidad/sangre , Obesidad/complicaciones , Obesidad/genética , Sobrepeso/sangre , Sobrepeso/complicaciones , Sobrepeso/genética , Polimorfismo de Nucleótido Simple , Factores de Riesgo , Estados Unidos , Población Blanca/genética

RESUMEN

The µ-opioid receptor (MOR) plays an important role in modulating analgesia, feeding behavior, and a range of autonomic functions. In the current study, we investigated the degree to which 13 naturally occurring missense mutations affect the pharmacological properties of the human MOR. After expression of each receptor in human embryonic kidney 293 cells, signaling (Gα(i/o)-mediated) induced by peptide agonists was assessed using luciferase reporter gene assays. Multiple mutants (S66F, S147C, R260H, R265C, R265H, and S268P) show a significant reduction in agonist potency. At the N190K variant, agonist-mediated signaling was essentially absent. Enzyme-linked immunosorbent assay, microscopic analysis, and radioligand binding assays revealed that this mutant shows markedly reduced cell-surface expression, whereas all other receptor variants were expressed at normal levels. Surface expression of the N190K variant could be increased by incubation with the alkaloid agonist buprenorphine or with either naltrexone or naloxone, structurally related MOR antagonists. We were surprised to find that both putative antagonists, despite being inactive at the wild-type MOR, triggered a concentration-dependent increase in N190K receptor-mediated signaling. In contrast, peptidic ligands failed to promote expression or rescue function of the N190K mutant. Subsequent analysis of the N190K variant in an ethnically diverse cohort identified this isoform in a subgroup of African Americans. Taken together, our studies reveal that the N190K mutation leads to severe functional alterations and, in parallel, changes the response to established MOR ligands. The extent to which this mutation results in physiological abnormalities or affects drug sensitivity in selected populations (e.g., those with chronic pain or addiction) remains to be investigated.

Asunto(s)

Péptidos/farmacología , Receptores Opioides mu/agonistas , Negro o Afroamericano , Sustitución de Aminoácidos , Línea Celular , HDL-Colesterol/sangre , Encefalina Ala(2)-MeFe(4)-Gli(5)/farmacología , Femenino , Genes Reporteros , Genotipo , Humanos , Luciferasas/biosíntesis , Luciferasas/genética , Mutagénesis Sitio-Dirigida , Mutación Missense , Naloxona/farmacología , Naltrexona/farmacología , Antagonistas de Narcóticos/farmacología , Péptidos Opioides/farmacología , Polimorfismo de Nucleótido Simple , Isoformas de Proteínas/agonistas , Isoformas de Proteínas/genética , Transporte de Proteínas , Ensayo de Unión Radioligante , Receptores Opioides mu/biosíntesis , Receptores Opioides mu/genética , Transducción de Señal , Población Blanca

RESUMEN

BACKGROUND: Sequencing of the human genome has identified numerous chromosome copy number additions and subtractions that include stable partial gene duplications and pseudogenes that when not properly annotated can interfere with genetic analysis. As an example of this problem, an evolutionary chromosome event in the primate ancestral chromosome 18 produced a partial duplication and inversion of rho-associated protein kinase 1 (ROCK1 -18q11.1, 33 exons) in the subtelomeric region of the p arm of chromosome 18 detectable only in humans. ROCK1 and the partial gene copy, which the gene databases also currently call ROCK1, include non-unique single nucleotide polymorphisms (SNPs). RESULTS: Here, we characterize this partial gene copy of the human ROCK1, termed Little ROCK, located at 18p11.32. Little ROCK includes five exons, four of which share 99% identity with the terminal four exons of ROCK1 and one of which is unique to Little ROCK. In human while ROCK1 is expressed in many organs, Little ROCK expression is restricted to vascular smooth muscle cell (VSMC) lines and organs rich in smooth muscle. The single nucleotide polymorphism database (dbSNP) lists multiple variants contained in the region shared by ROCK1 and Little ROCK. Using gene and cDNA sequence analysis we clarified the origins of two non-synonymous SNPs annotated in the genome to actually be fixed differences between the ROCK1 and the Little ROCK gene sequences. Two additional coding SNPs were valid polymorphisms selectively within Little ROCK. Little ROCK-Green Fluorescent fusion proteins were highly unstable and degraded by the ubiquitin-proteasome system in vitro. CONCLUSION: In this report we have characterized Little ROCK (ROCK1P1), a human expressed pseudogene derived from partial duplication of ROCK1. The large number of pseudogenes in the human genome creates significant genetic diversity. Our findings emphasize the importance of taking into consideration pseudogenes in all candidate gene and genome-wide association studies, as well as the need for complete annotation of human pseudogenome.

Asunto(s)

Músculo Liso/metabolismo , Polimorfismo de Nucleótido Simple , Seudogenes , Quinasas Asociadas a rho/genética , Secuencia de Bases , Cromosomas Humanos Par 18 , Duplicación de Gen , Humanos , Datos de Secuencia Molecular , Músculo Liso Vascular/metabolismo , Alineación de Secuencia , Quinasas Asociadas a rho/metabolismo

RESUMEN

Bicuspid Aortic Valve (BAV) is a highly heritable congenital heart defect. The low frequency of BAV (1% of general population) limits our ability to perform genome-wide association studies. We present the application of four a priori SNP selection techniques, reducing the multiple-testing penalty by restricting analysis to SNPs relevant to BAV in a genome-wide SNP dataset from a cohort of 68 BAV probands and 830 control subjects. Two knowledge-based approaches, CANDID and STRING, were used to systematically identify BAV genes, and their SNPs, from the published literature, microarray expression studies and a genome scan. We additionally tested Functionally Interpolating SNPs (fitSNPs) present on the array; the fourth consisted of SNPs selected by Random Forests, a machine learning approach. These approaches reduced the multiple testing penalty by lowering the fraction of the genome probed to 0.19% of the total, while increasing the likelihood of studying SNPs within relevant BAV genes and pathways. Three loci were identified by CANDID, STRING, and fitSNPS. A haplotype within the AXIN1-PDIA2 locus (p-value of 2.926x10(-06)) and a haplotype within the Endoglin gene (p-value of 5.881x10(-04)) were found to be strongly associated with BAV. The Random Forests approach identified a SNP on chromosome 3 in association with BAV (p-value 5.061x10(-06)). The results presented here support an important role for genetic variants in BAV and provide support for additional studies in well-powered cohorts. Further, these studies demonstrate that leveraging existing expression and genomic data in the context of GWAS studies can identify biologically relevant genes and pathways associated with a congenital heart defect.

Asunto(s)

Antígenos CD/genética , Válvula Aórtica/anomalías , Redes Reguladoras de Genes , Haplotipos , Cardiopatías Congénitas/genética , Proteína Disulfuro Isomerasas/genética , Receptores de Superficie Celular/genética , Proteínas Represoras/genética , Proteína Axina , Estudios de Casos y Controles , Endoglina , Estudio de Asociación del Genoma Completo , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , Polimorfismo de Nucleótido Simple

RESUMEN

OBJECTIVES: Genetic variants that influence large conductance calcium-activated potassium channel's function may alter arterial function and contribute to the known heritability of arterial stiffness and blood pressure. The beta1-subunit (KCNMB1) of the large conductance calcium-activated potassium channel includes two coding region polymorphisms. E65K, a gain-of-function polymorphism, is predicted to enhance large conductance calcium-activated potassium channel opening and vasorelaxation, whereas V110L has no known effect. We and others have reported that E65K carriers have reduced blood pressure. METHODS: To test our hypothesis that E65K has a favorable effect on arterial function, we related arterial tonometry and brachial artery phenotypes to genotypes in 1100 Framingham Offspring Study participants with available genotypes and phenotypes (53% women; mean age 61.5 +/- 9.4 years). RESULTS: The minor allele frequency was 0.10 for E65K and 0.09 for V110L; both were in Hardy-Weinberg equilibrium (chi2, P > 0.05), and haplotype analysis found R2 = 0.01. E65K was associated with lower augmented pressure (7.4 +/- 3.3 versus 9.0 +/- 3.8 mmHg, P = 0.01) and central pulse pressure (47.1 +/- 7.3 versus 50.7 +/- 7.8 mmHg, P = 0.01) in multivariable analyses. No association was noted between E65K and mean arterial pressure, carotid-femoral pulse wave velocity or brachial artery diameter, flow velocity or volume flow. V110L was not associated with tonometry or brachial measures. CONCLUSION: A diminished augmented pressure in K-carriers suggests a reduced or delayed wave reflection and supports the hypothesis that E65K reduces arterial impedance mismatch in the arterial tree. Our findings in a middle-aged community-based cohort, if replicated, would support that E65K has a favorable effect on arterial function and pulsatile hemodynamic load.

Asunto(s)

Presión Sanguínea , Subunidades beta de los Canales de Potasio de Gran Conductancia Activados por el Calcio/genética , Polimorfismo de Nucleótido Simple , Anciano , Arteria Braquial/anatomía & histología , Arteria Braquial/fisiología , Estudios de Cohortes , Femenino , Humanos , Masculino , Persona de Mediana Edad

RESUMEN

Severe factor V (FV) deficiency is a rare bleeding disorder, whose genetic bases have been characterized only in a limited number of cases. We investigated 6 unrelated patients with extremely reduced plasma FV levels, associated with a bleeding tendency ranging from moderately severe to severe. Clinical manifestations were substantially concordant with the previously established spectrum of hemorrhagic symptoms of the disease. Molecular analysis of FV gene identified 9 different mutations, 7 hitherto unknown, and 2 previously reported (Arg712ter and Tyr1702Cys). Four of 6 analyzed patients were compound heterozygotes, indicating the high allelic heterogeneity of this disease. Among novel mutations, 5 led to premature termination codons, because of nonsense (Arg1002ter, Arg1606ter, and Trp1854ter), or frameshift mutations (5127-5128insA and 6122-6123insAACAG). The remaining 2 were missense mutations (Cys472Gly and Val1813Met), located in FV A2 and A3 domains. Their effect on FV expression was studied by transient transfection experiments, demonstrating that the presence of each mutation impaired FV secretion. These data increase the number of severe FV deficiency-causing mutations by about 50%. The high number of "private" mutations identified in FV-deficient families indicates that full mutational screening of FV gene is still required for molecular diagnosis.

Asunto(s)

Deficiencia del Factor V/genética , Factor V/genética , Mutación , Adolescente , Adulto , Animales , Células COS , Clonación Molecular , Codón sin Sentido , Factor V/análisis , Factor V/aislamiento & purificación , Salud de la Familia , Femenino , Mutación del Sistema de Lectura , Hemorragia/etiología , Humanos , Masculino , Persona de Mediana Edad , Mutación Missense , Linaje , Estructura Terciaria de Proteína , Transfección

RESUMEN

Factor V (FV) deficiency is a rare bleeding disorder whose genetic basis has been described in a relatively small number of cases. Among a total of 12 genetic defects reported in severely or moderately severe deficient patients, 3 were missense mutations and in no case was the mechanism underlying the deficiency explored at the molecular level. In this study, a homozygous missense mutation at cDNA position 6394 in exon 23 of the FV gene was identified in a 22-year-old Italian patient. This mutation causes the replacement of arginine 2074 with a cysteine residue (Arg2074Cys) in the C2 domain of the protein. The effect of the Arg2074Cys mutation on FV secretion, stability, and activity was investigated. Site-directed mutagenesis of FV cDNA was used to introduce the identified mutation, and wild-type as well as mutant FV proteins were expressed by transient transfection in COS-1 cells. An enzyme immunoassay detected low FV antigen levels both in the conditioned media of cells expressing the mutant protein and in cell lysates. Metabolic labeling and pulse-chase experiments confirmed that the mutation caused an impaired secretion of FV associated with rapid intracellular degradation. In addition, evaluation of wild-type and mutant coagulant activity demonstrated that the FV molecules carrying the Arg2074Cys mutation have reduced activity. These findings, beside confirming the structural and functional importance of the arginine 2074 residue, demonstrate that its substitution with a cysteine impairs both FV secretion and activity.

Asunto(s)

Deficiencia del Factor V/genética , Factor V/genética , Mutación Missense , Adulto , Animales , Células COS , Clonación Molecular , Análisis Mutacional de ADN , Factor V/metabolismo , Femenino , Homocigoto , Humanos , Linaje , Estructura Terciaria de Proteína , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/metabolismo