Diseño y validación de un ensayo de minisecuenciación múltiple para detectar polimorfismos asociados con Síndrome Metabólico / Design and validation of a multiple minisequencing assay to detect polymorphisms associated with Metabolic Syndrome

Resumen Introducción: Es importante identificar los polimorfismos de interés clínico en patologías complejas como el Síndrome Metabólico. Por esto, las metodologías para su evaluación deben estar diseñadas y validadas correctamente, esto permite optimizar recursos y tiempo en la genotipificación y detección correcta de los alelos presentes en los individuos. Objetivo: Diseñar y validar una PCR múltiple, seguida de detección por minisecuenciación, para la genotipificación de ocho polimorfismos de nucleótido simple ubicados en el gen del Receptor Beta 3-Adrenérgico (rs4994 y rs4998), gen de la Apolipoproteina A5 (rs3135506 y rs2075291), gen de la Adiponectina (rs1501299 y rs2241766) y gen del Receptor Activador de la Proliferación de los Peroxisomas tipo gamma (rs1801282 y rs1800571), asociados con el síndrome metabólico. Materiales y métodos: Se diseñaron 24 cebadores para la amplificación y detección de ocho polimorfismos de nucleótido sencillo ubicados en cuatro genes candidatos a estar asociados con el síndrome metabólico, usando el software Primer3®. Dieciséis fueron diseñados para amplificar los polimorfismos y ocho para detectarlos por minisecuenciación. Las estructuras secundarias entre los cebadores se verificaron con el software Autodimer. Los polimorfismos se amplificaron simultáneamente y los fragmentos amplificados se acoplaron a las sondas diseñadas para detectar por minisecuenciación el alelo presente, por medio de bases marcadas con fluorocromos. Finalmente, los alelos fueron detectados por electroforesis capilar en un analizador genético ABI 310 y se interpretaron con el software GeneMapper®. La validación del multiplex se realizó genotipando 20 muestras de individuos, cada uno de ellos autorizó este procedimiento por medio del consentimiento informado. Resultados: Se obtuvieron los perfiles genéticos de los 20 controles genotipados, a partir de la amplificación múltiple, seguida de minisecuenciación, diseñada y validada para detectar los ocho polimorfismos. Conclusión: Se diseñó y validó un ensayo para la detección simultánea de los polimorfismos, ubicados en cuatro genes asociados con el Síndrome metabólico. Los cuales pueden ser empleados como referencia para futuros estudios poblacionales.

Abstract Introduction: It is important to identify the polymorphisms of clinical interest in complex pathologies such as Metabolic Syndrome. Therefore, the methodologies for its evaluation must be designed and validated correctly, this permits optimization of resources and time in genotyping and correct detection of the alleles present in individuals. Objective: To design and validate a multiplex PCR, followed by detection by minisequencing, for the genotyping of eight single nucleotide polymorphisms located in the Beta 3-Adrenergic Receptor gene (rs4994 and rs4998), Apolipoprotein A5 gene (rs3135506 and rs2075291), Adiponectin gene (rs1501299 and rs2241766) and gamma-type Peroxisome Proliferation Activating Receptor gene (rs1801282 and rs1800571), associated with metabolic syndrome. Materials and methods: Twenty-four primers were designed for the amplification and detection of eight single nucleotide polymorphisms located in four candidate genes to be associated with the metabolic syndrome, using the Primer3® software. Sixteen were designed to amplify the polymorphisms and eight to detect them by minisequencing. The secondary structures between the primers were verified with Autodimer software. The polymorphisms were simultaneously amplified, and the amplified fragments were coupled to probes designed to minisequence the present allele using fluorochrome-labeled bases. Finally, the alleles were detected by capillary electrophoresis using an ABI 310 genetic analyzer and analyzed with the GeneMapper® software. The validation of the multiplex was performed by genotyping 20 individual samples, each of them authorized this procedure through informed consent. Results: The genetic profiles of the 20 genotyped controls were obtained, from multiple amplification, followed by minisequencing, designed and validated to detect the eight polymorphisms. Conclusion: An essay was designed and validated for the simultaneous detection of polymorphisms, located in four genes associated with metabolic syndrome, and can used as a reference for future population studies.

Variants of Lipolysis-Related Genes in Korean Patients with Very High Triglycerides

We investigated the prevalence and characteristics of variants of five lipolysis-related genes in Korean patients with very high triglycerides (TGs). Twenty-six patients with TG levels >885 mg/dL were selected from 13545 Korean subjects. Five candidate genes, LPL, APOC2, GPIHBP1, APOA5, and LMF1, were sequenced by targeted next-generation sequencing. Predictions of functional effects were performed and matched against public databases of variants. Ten rare variants of three genes were found in nine (34.6%) patients (three in LPL, four in APOA5, and three in LMF1). Five were novel and all variants were suspected of being disease-causing. Nine were heterozygous, and one (3.8%) had a homozygous rare variant of LPL. Six common variants of four genes were observed in 25 (96.2%) patients (one in LPL, one in GPIHBP1, two in APOA5, and two in LMF1). The c.G41T variant of GPIHBP1 and c.G533T variant of APOA5 were most frequent and found in 15 (57.7%) and 14 (53.8%) patients, respectively. Rare homozygous variants of the genes were very uncommon, while diverse rare heterozygous variants were commonly identified. Taken together, most study subjects may be manifesting the combined effects of rare heterozygous variants and common variants.

Polimorfismo S19W (Ser19Ter) de la APOA5 y su relación con la hipertrigliceridemia en una población de Colombia / S19W polymorphism (Ser19Ter) of APOA5 and its relationship with hypertriglyceridemia in a Colombian population

RESUMEN Introducción: Las enfermedades cardiovasculares (ECV) son responsables del 29,69% de las muertes en Colombia. Se ha encontrado la hipertrigliceridemia, en diversos estudios como factor de riesgo independiente para la ECV. El polimorfismo S19W (Ser19Ter) de la ApoA5 se ha asociado en algunas poblaciones con la hipertrigliceridemia. Sin embargo, en Colombia esto ha sido poco estudiado. Objetivo: Estimar la asociación entre el polimorfismo S19W y la hipertrigliceridemia en población colombiana. Metodología: Estudio tipo Corte Transversal, con 400 individuos provenientes de Bucaramanga, Colombia. Se cuantificó los TAG y se genotipificaron mediante la técnica de SNaPshot y mini secuenciación. Los resultados fueron analizados utilizando el software de análisis genético Arlequín 3.5.1.2. Resultados: El polimorfismo S19W (Ser19Ter) mostró tres perfiles, CC, GG y CG. El polimorfismo S19W se caracterizó tanto en afectados como en no afectados, mostrando que no existen diferencias significativas en esta distribución cuando se comparan los dos grupos. Discusión: Diversos mecanismos se han propuesto para sustentar la hipertrigliceridemia como un factor de riesgo para ECV, entre los que se cuenta la APOA5. El estudio comprobó que la población estudiada se encuentra en equilibrio de Hardy Weinberg y al genotipo CC como el más frecuente. Los genotipos GG y el GC presentaron valores significativos en el grupo de sujetos afectados, (p<0,01 y p=0,03; respectivamente). Se demostró la existencia de una estrecha relación entre el polimorfismo Ser19Trp y la hipertrigliceridemia (p<0,01). Conclusión: Se pudo demostrar la existencia de una relación entre el polimorfismo Ser19Trp de la Apo A5 con los niveles elevados de TAG (p<0,01).

ABSTRACT Introduction: Cardiovascular diseases (CVD) are responsible for 29.69% of the deaths in Colombia. Several studies have shown that hypertriglyceridemia is an independent risk factor for CVD. ApoA5 gene S19W (Ser19Ter) polymorphism has been associated with hypertriglyceridemia in some populations; however, their influence in Colombia is unknown. Objective: To determine the relationship between S19W polymorphism and hypertriglyceridemia in Colombian population. Methodology: Transversal crossover Studio, included a total of 400 individuals. TAG was quantified and genotyped using the technique SnapShot and mini sequencing. The results were analyzed using genetic analysis software Arlequin 3.5.1.2. Results: S19W (Ser19Ter) polymorphism showed three profiles, CC, GG and CG. The S19W polymorphism was characterized both affected and not affected. There wasn´t significant differences in the distribution when the two groups are compared. Discussion: Various mechanisms have been proposed to support hypertriglyceridemia as a risk factor for CVD, including the APOA5 counted. The study found that the study population is in Hardy Weinberg and CC genotype as the most frequent. The GG and GC genotypes showed significant values in the group of affected subjects (p = 0.002 and 0.03). It demonstrated the existence of a close relationship between the Ser19Trp polymorphism and hypertriglyceridemia (p<0,01). Conclusion: It was possible to demonstrate the existence of a relationship between polymorphism Ser19Trp of ApoA5 with elevated levels of TAG (p<0,01).

Relationship between apolipoprotein A5gene-1131T＞C and fat content on adult Tibetans in Tibetarea and Han nationality population in Liaoning province / 安徽医科大学学报

Objective To explore the gene frequency of apolipoprotein A5(APOA5)-1131T>C single nucleotide polymorphisms and its relationship with the fat contents of adult Tibetans in Tibet and Han nationality population in Liaoning province.Methods In 100 Tibetan nationality(the study group) and 100 Han nationality individuals(the control group), the polymorphism of apoa5-1131T>C genotypes was detected by polymerase chain reaction-restricted fragments length polymorphism(PCR-RFLP), and fat contents were got by using bioelectrical impedance measurement.Results Results of fat contents of body, trunk and limbs(left upper limbs, left lower limbs, right upper limbs, right lower limbs and total limbs) were respectively below:① There was significant difference between male TC+CC genotype and TT genotype of Tibetan nationality.There was significant difference between male TT genotype of Tibetan nationality and of Han nationality.② There was significant difference between female TT genetype of Tibetan nationality and of Han nationality except result of the body fat contents.③ There was significant difference between the trunk and total limbs fat contents in male TT genotype, and male TC+CC genotype of Tibetan nationality and of Han nationality, and female TT genotype of Tibetan nationality.Conclusion APOA5 gene-1131T>C affects the body fat contents of Tibetans and Han nationality population, and APOA5 gene-1131T>C probably affects lipopexia in trunk.

Association of APOA5 Polymorphisms with Plasma Lipid Levels and Severity of Coronary Stenosis in Patients with Coronary Heart Disease / 中山大学学报(医学科学版)

[Objective]To explore the possible effects of the apolipoprotein A5(APOA5)gene rs2075291 and rs3135507 poly-morphisms on plasma lipid levels and the severity of coronary stenosis in patients with coronary heart disease(CHD)in Chinese Han people.[Methods]Polymerase chain reaction-restriction fragment length polymorphism method(PCR-RFLP)was used to identify the genotypes of the rs2075291 or rs3135507 polymorphism for the 324 patients with CHD and the 152 CHD-free controls,and the lipid levels between the genotypes were compared. The severity of coronary stenosis was assessed by the Gensini scoring system ,and the associations of the rs2075291 and rs3135507 polymorphisms as well as other factors with the Gensini scores were analyzed by mul-tivariate linear regression.[Results]The CHD patients had higher prevalence of hypertension ,and higher levels of triglycerides (TG),total cholesterol(TC),apolipoprotein B100(APOB100),lipoprotein(a)[Lp(a)],TG/high-density lipoprotein cholesterol (HDL-C),TC/HDL-C,low-density lipoprotein cholesterol(LDL-C)/HDL-C and APOB100/apolipoprotein AI(APOAI),and low-er levels of HDL-C and APOAI than the controls(P 0.05).[Conclusion]The APOA5 rs2075291 polymorphism had significant effects on plasma lipid levels,but no effects on the occurrence and development of CHD. Further multicenter case-control studies with large samples are needed to validate these findings.

The interaction of Apolipoprotein A5 gene promoter region T-1131C polymorphism (rs12286037) and lifestyle modification on plasma triglyceride levels in Japanese

BACKGROUND/OBJECTIVE: Apolipoprotein A5 gene promoter region T-1131C polymorphism (APOA5 T-1131C) is known to be associated with elevated plasma TG levels, although little is known of the influence of the interaction between APOA5 T-1131C and lifestyle modification on TG levels. To investigate this matter, we studied APOA5 T-1131C and plasma TG levels of subjects participating in a three-month lifestyle modification program. SUBJECTS/METHODS: A three-month lifestyle modification program was conducted with 297 participants (Age: 57 +/- 8 years) in Izumo City, Japan, from 2001-2007. Changes in energy balance (the difference between energy intake and energy expenditure) and BMI were used to evaluate the participants' responses to the lifestyle modification. RESULTS: Even after adjusting for confounding factors, plasma TG levels were significantly different at baseline among three genotype subgroups: TT, 126 +/- 68 mg/dl; TC, 134 +/- 74 mg/dl; and CC, 172 +/- 101 mg/dl. Lifestyle modification resulted in significant reductions in plasma TG levels in the TT, TC, and CC genotype subgroups: -21.9 +/- 61.0 mg/dl, -20.9 +/- 51.0 mg/dl, and -42.6 +/- 78.5 mg/dl, respectively, with no significant differences between them. In a stepwise regression analysis, age, APOA5 T-1131C, body mass index (BMI), homeostasis model assessment-insulin resistance (HOMA-IR), and the 18:1/18:0 ratio showed independent association with plasma TG levels at baseline. In a general linear model analysis, APOA5 T-1131C C-allele carriers showed significantly greater TG reduction with decreased energy balance than wild type carriers after adjustment for age, gender, and baseline plasma TG levels. CONCLUSIONS: The genetic effects of APOA5 T-1131C independently affected plasma TG levels. However, lifestyle modification was effective in significantly reducing plasma TG levels despite the APOA5 T-1131C genotype background.

The study of apolipoprotein a5 gene polymorphisms in relation tolipidslevel in people with metabolic syndrome / Монголын Анагаах Ухаан

Background: Epidemiologic studies have shown a higher prevalenceof hypertriglyceridemia among patients with CHDthan among unaffected populations. Dozens of polymorphisms in different genesthat could have some effect on plasma TG levels havebeen analyzed. The most promising results are connected withvariants within the apolipoproteins (APO) APOA1/APOC3/ APOA4 gene cluster. Transgenic mice overexpressing human apolipoprotein A5decreased plasma triglyceride concentrations to one-third of those in control mice; conversely, knockout mice lacking APOA5 had four times as much plasma triglycerides as controls.The human APOA5 gene consistsof 4 exons and codes 369 aminoacidprotein, which is expressed almost exclusively in the liver.A minor allele of APOA5 (1259C, IVS3+476A and 1131C) which was independently associated with high plasma triglyceride levels in African-American, non Hispanic whites, Hispanic, Caucasians and Japanese were reported. Four polymorphisms in ApoA5 (1259T>C, IVS3+476G>A, S19W and 1131T>C) has been correlatedwith high TG levels in diabetic women. Materials and Methods: 162 people with MS for case group and 144 people for control group were selected in this study. MS was diagnosed according to IDF criteria and serum triglyceride, total cholesterol and HDL levels were determined. DNA from both case and control subjects were extracted from blood samples (20μL) using “G-spin™ Total DNA Extraction Kit”(iNtRON Biotechnology, Inc).The genotypes for fourpolymorphisms of ApoA5 were determined using a combination of PCR and sequence-specific oligonucleotide probes. Results: There were 304 total subjects included males 50.3% (153) and female 49.7% (151) in our study. The appearance of risk genotypes of 1177C>T, 1259T>C, IVS3+476G>A and 1131T>C polymorphisms in ApoA5 gene were higher in MS group than control group.Serum levels of triglycerides and total cholesterol differed significantly (pC genotypes. Conclusion: TAG and TC level was higher in people with 1131T>C-CC genotype than other genotypes in both groups (p=0.010, p=0.001). We determined that the odds ratio for the hypertriglyceridemia was 5.98 for ApoA5-1131T>C CC-genotype carriers.

The study of apolipoprotein A5 gene polymorphism in relation to serum triglyceride level in people with metabolic syndrome / Монголын Анагаах Ухаан

Background. A large number of longitudinal studies indicate significantly increased risk of cardiovascular events and death in people with the MetSyn and high plasma levels of triglycerides are an independent risk factor for the development of cardiovascular disease. Apolipoprotein A5 (APOA5) gene, a new member of the APOA1/C3/A4 gene cluster, was identified by comparative sequencing of human and mice DNA by Pennacchio and co-workers in 2001. Since this discovery, variants of ApoA5 gene have been independently assiociated with level of plasma triglyceride in many countries. Human ApoA5 is expressed in the liver then appears in plasma in association with VLDL and HDL and plays a major role in TG catabolism. Variant at ApoA5 gene locus, 1177C>T is located in 3’ UTR which often contains regulatory regions that influence post-transcriptional gene expression. One alteration can be responsible for the altered expression of many genes. Materials and Methods. 152 people with MS for case group and 152 people for control group were selected in this study. MS was diagnosed according to IDF criteria and serum triglyceride levels were determined. DNA from both case and control subjects were extracted from blood samples (200 ml) using “G-spin™ Total DNA Extraction Kit”(iNtRON Biotechnology, Inc). To detect the 1177C>T variation of ApoA5 gene, using High Pure PCR Template Preparation Kits, a forward primer 5’-CTCTGAGCCTCTAGCATGGTTGAGT- 3’ and the mismatch reverse primer 5’-GAGCATTCCCAAATGAGCAC-3’ were used to create the HinfI restriction site. Results. There were 304 total subjects included males 50.3% (153) and female 49.7% (151) in our study. Incident of CC genotype was 71.1% (216), CT genotype was 25% (76) and TT genotype was 3.9%, TAG level was higher in males than females in both groups (p=0.016, ð=0.001) for CC genotype and also, higher with MS in males for CT genotype (p=). But, TAG level was no significant difference among three genotypes in group with MS subjects (male p=0.236, female p=0.881). Conclusion: The TT genotype of the ApoA5 gene 1177C>T polymorphism frequency was 2.9% in control subjects and 4.9% in subjects with MS. However, TG level was not differ in both groups for TT genotype, TAG level in males was higher compared with females (p=0.016 in control, p=0.001 in group with MS).

Impacto de la apolipoproteína A5 en el riesgo cardiovascular: Modulaciones genéticas y ambientales / Impact of apolipoprotein A5 on cardiovascular risk: Genetic and environmental modulation

Triglyceride concentrations are an independent risk factor for coronary heart disease. Apolipoprotein A5 gene (APOA5) has an important role determining triglyceride metabolism and it is a potential cardiovascular risk. However the mechanisms for these actions are not well-known. Despite the different allelic frequency of its major polymorphisms in different populations, multiple studies have shown consistent associations between these variants and fasting triglycerides. Variations in the APOA5 gene have also been associated with postprandial triglycerides, as well as with different sizes of lipoproteins and other markers. Moreover, some of the APOA5 gene variants have been associated with ischemic heart disease, stroke, and carotid intima media thickness, although the references on this issue are scanty and contradictory. This may be due to the presence of gene-environment interactions that have been poorly studied until now. Among the few studies that have examined the infuence of environmental factors on possible genetic variations, the most important are those that contemplate possible gene-diet interactions. However, the evidence is still scarce and more research is required in the feld of nutrigenomics. To understand the impact of this gene on cardiovascular disease, we review the genetic functionality and variability of APOA5, its associations with intermediate and fnal phenotypes and gene-environment interactions detected.

Polimorfismos genéticos de APOA5 se asocian a hipertrigliceridemia e hiperglicemia en individuos chilenos con enfermedad coronaria y controles / Genetic polymorphisms of APOA5 are associated with hypertriglyceridemia and hyperglycemia in Chilean subjects with coronary artery disease and controls

Introducción: Diversas variantes genéticas han sido relacionadas al desarrollo de enfermedad coronaria y/o sus factores de riesgo; entre ellas, los polimorfismos S19W y -1131T>C del gen que codifica para la apolipoproteína A5 (APOA5). Así, el objetivo del presente estudio fue investigar la posible asociación entre las variantes S19W y -1131T>C del gen APOA5 y enfermedad coronaria en individuos chilenos. Métodos: Se evaluaron 425 sujetos adultos, no relacionados; 209 pacientes con enfermedad coronaria (EC) comprobada por angiografía (estenosis→ 70 por ciento), con edades entre 33 y 74 años, y 216 individuos controles (30 a 68 años). La genotipificación de los polimorfismos S19Wy -1131T>C del gen APOA5 fue realizada mediante la técnica de PCR-RFLP Resultados: La distribución de los genotipos para el polimorfismo S19W del gen APOA5 en el grupo casos (SS: 80 por ciento, SW: 19 por ciento y WW: 1 por ciento) y en el grupo control (SS: 82 por ciento, SW: 17 por ciento y WW: 1 por ciento) fue semejante (p=NS). La distribución genotípica para el polimorfismo -1131T>C en pacientes con EC (TT: 56 por ciento, TC: 37 por ciento, y CC: 7 por ciento) y controles (TT: 63 por ciento, TC: 30 por ciento y CC: 7 por ciento) fue similar (p=NS). Las ORs relacionadas a los alelos mutados 19W (1.12; I.C.95 por ciento, 0.72- 1.74, p=NS)y-1131C (1.19; I.C.95 por ciento,, 0.87- 1.63, p=NS), confirman la ausencia de asociación. Por otro lado, las concentraciones de triglicéridos y glucosa en ayunas fueron significativamente más elevadas en los sujetos portadores de los alelos 19Wy -1131C, tanto en casos como en controles (p<0.05). Conclusión: La asociación observada entre las variantes genéticas de APOA5 y las altas concentraciones séricas de triglicéridos y glucosa, en ambos grupos, sugiere que estos polimorfismos podrían contribuir al desarrollo de la dislipidemia diabética; un reconocido factor de riesgo para enfermedad arterial coronaria.

Background: Several genetic variants have been linked to the development of coronary heart disease and/or their risk factors, including the S19Wand-1131T> C polymorphisms of the gene that encodes apolipoprotein A5 (APOA5). Thus, the objective of this study was to investigate the possible association between S19W and -1131T>C genetic variants ofAPOA5 and coronary disease in Chilean individuals. Methods: We evaluated 425 not related subjects; 209 patients with coronary artery disease (CAD) confirmed by angiography (stenosis→ 70 percent,), aged between 33 and 74 years, and 216 control individuals (30 to 68 years). The genotyping of S19W and -1131T>C polymorphisms of APOA5 gene was evaluated by PCR-RFLP. Results: The genotype distribution of S19W polymorphism of APOA5 gene in CAD patients (SS: 80 percent,, SW: 19 percent, WW: 1 percent>) and controls (SS: 82 percent,, SW: 17 percent, WW: 1 percent>) was similar (p = NS). In the same way the genotype distribution of-1131T>C genetic variant in CAD subjects (TT: 56 percent,, TC: 37 percent,, and CC: 7 percent>) and controls (TT: 63 percent,, TC: 30 percent, and CC: 7 percent) was equivalent (p = NS). The Odds ratios related to the mutant alleles 19W (1.12, 95 percent, Cl, 0.72 - 1.74, p = NS) and -1131C (1.19, 95 percent, Cl, 0.87 -1.63, p = NS) confirms the absence of association. On the other hand, the triglycerides and fasting glucose concentrations were significantly higher in subjects carrying the alleles 19W and -1131C, in both groups, CAD patients and controls (p <0.05). Conclusion: The observed association between genetic variants of APOA5 and higher serum levels of triglycerides and glucose, in both groups, suggesting that these polymorphisms could be contribute to the development of diabetic dyslipidemia, a known risk factor for coronary artery disease.

Expression and preparation of polyclonal antibodies against human apolipoprotein A5 and tissue distribution / 中国实用内科杂志

Objective To prepare the recombination protein and polyclonal antibodies against human apolipoprotein A5(ApoA5)and detect the expression of ApoA5 in human tissues with the antibody,then further study the fuction and serum level of ApoA5.Methods With PCR and gene recombinated techniques,the prokaryotic ApoA5 expression vector was constructed.The His-ApoA5 fusion protein was expressed in E.ColiM15,and the fusion protein was injected into New Zealand rabbits to prepare polyclonal antibody.The antibody was tested for their specificity with ELISA and Western-blot,then it was used to detect the tissue distribution of ApoA5.Results It was found that there was a specific brand at 40 ku of the recombination ApoA5(rApoA5);the polyclonal antibody had high immunoreactivity and specificity.ApoA5 was expressed in human sera and hepatic tissue,not in other tissues(heart,vessel,spleen,intestine and lung),regardless of the lipid level.Conclusion The pQE30-ApoA5 is constructed successfully,the rApoA5 can be expressed in E.Coli M15;ApoA5 antibody has highly specifity.It can be used in clinical test.There is ApoA5 in human serum,and ApoA5 is expressed in liver which may be significant in lipid metabolism.