Arq Bras Cardiol abc Arquivos Brasileiros de Cardiologia Arq. Bras. Cardiol. 0066-782X 1678-4170 Sociedade Brasileira de Cardiologia - SBC dgS647T7f6LSfJCP8LqTzRJ S0066-782X2022005013401 S0066-782X2022001000593 10.36660/abc.20210422 Artigo Original Associação do Genótipo e Fenótipo da Paraoxonase-1 com Angiografia Positiva para Doença Arterial Coronariana Soflaei Sara Saffar 1 * Baktashian Mojtaba 1 * Moghaddam Kiana Hosseinpour 2 Saberi-Karimian Maryam 3 Kosari Negin 1 Hashemi Seyed Mohammad 4 Mouhebati Mohsen 5 Amini Mahsa 1 Dehghani Mashallah 5 Esmaily Habibollah 6 Ebrahimi Mahmoud 5 Falsoleiman Homa 5 Nosrati-Tirkani Abolfazl 7 Sadabadi Fatemeh 1 Ferns Gordon A. 8 Salehi Mansoor 9 Pasdar Alireza 1 10 0000-0002-1081-6754 Ghayour-Mobarhan Majid 3 5 International UNESCO Center for Health-Related Basic Sciences and Human Nutrition Mashhad University of Medical Sciences Mashhad Irã International UNESCO Center for Health-Related Basic Sciences and Human Nutrition , Mashhad University of Medical Sciences , Mashhad – Irã Department of Biology Faculty of Sciences Ferdowsi University of Mashhad Mashhad Irã Department of Biology , Faculty of Sciences , Ferdowsi University of Mashhad , Mashhad – Irã Metabolic Syndrome Research Center Faculty of Medicine Mashhad University of Medical Sciences Mashhad Irã Metabolic Syndrome Research Center , Faculty of Medicine , Mashhad University of Medical Sciences , Mashhad – Irã Department of Cardiology School of Medicine Isfahan University of Medical Sciences Isfahan Irã Department of Cardiology , Chamran Hospital, School of Medicine , Isfahan University of Medical Sciences , Isfahan – Irã Cardiovascular Research Center School of Medicine Mashhad University of Medical Sciences Mashhad Irã Cardiovascular Research Center , School of Medicine , Mashhad University of Medical Sciences , Mashhad – Irã Department of Biostatistics and Epidemiology School of Health Mashhad University of Medical Sciences Mashhad Irã Department of Biostatistics and Epidemiology , School of Health , Mashhad University of Medical Sciences , Mashhad – Irã Biochemistry of Nutrition Research Center Faculty of Medicine Mashhad University of Medical Sciences Mashhad Irã Biochemistry of Nutrition Research Center , Faculty of Medicine , Mashhad University of Medical Sciences , Mashhad – Irã Brighton & Sussex Medical School Division of Medical Education Brighton Reino Unido Brighton & Sussex Medical School , Division of Medical Education , Falmer, Brighton – Reino Unido Department of genetics Faculty of medicine and genetics laboratory AL Zahra hospital Isfahan University of Medicine Isfahan Irã Department of genetics , Faculty of medicine and genetics laboratory AL Zahra hospital , Isfahan University of Medicine , Isfahan – Irã Division of Applied Medicine Medical School University of Aberdeen Aberdeen Reino Unido Division of Applied Medicine , Medical School , University of Aberdeen , Foresterhill, Aberdeen – Reino Unido Correspondência: Majid Ghayour-Mobarhan • Metabolic Syndrome Research Center, School of Medicine, Mashhad – University of Medical Sciences, 99199-91766, Mashhad – Irã E-mail: GhayourM@mums.ac.ir

Contribuição dos autores

Concepção e desenho da pesquisa: Soflaei SS, Baktashian M, Hashemi SM, Mouhebati M, Dehghani M, Ebrahimi M, Falsoleiman H, Ferns GA, Salehi M, Pasdar A, Ghayour-Mobarhan M; Obtenção de dados: Soflaei SS, Baktashian M, Moghaddam KH, Kosari N, Amini M, Dehghani M, Ebrahimi M, Falsoleiman H, Nosrati-Tirkani A, Sadabadi F; Análise e interpretação dos dados: Soflaei SS, Moghaddam KH, Saberi-Karimian M, Amini M, Nosrati-Tirkani A, Pasdar A; Análise estatística: Esmaily H; Obtenção de financiamento: Hashemi SM, Salehi M, Ghayour-Mobarhan M; Redação do manuscrito: Soflaei SS, Saberi-Karimian M, Nosrati-Tirkani A; Revisão crítica do manuscrito quanto ao conteúdo intelectual importante: Mouhebati M, Falsoleiman H, Ferns GA, Salehi M, Pasdar A, Ghayour-Mobarhan M.

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 02 09 2022 10 2022 119 4 593 601 12 06 2021 02 03 2022 06 04 2022 This is an Open Access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Resumo Fundamento

Tem sido demonstrado que um aumento dos níveis séricos de PON1 é protetor contra vários distúrbios. Foi relatado que vários polimorfismos de nucleotídeo único (SNPs, single nucleotide polymorphisms ) do gene PON1 estão associados a níveis e atividade de proteínas enzimáticas séricas.

 Objetivos

Investigar a associação de SNPs do PON1 e atividade da paraoxonase sérica com a doença arterial coronariana (DAC).

 Métodos

Foram estudados 601 pacientes não relacionados submetidos à angiografia coronária, incluindo aqueles com estenose >50% (N=266) e aqueles com estenose <30% (N=335). Os SNPs rs662 e rs840560 do gene da paraoxonase foram determinados utilizando o método ARMS-PCR e o SNP rs705379 foi genotipado utilizando análise de PCR-RFLP. A atividade da paraoxonase sérica foi medida utilizando paraoxon como substrato. O valor de p<0,05 foi considerado significante.

 Resultados

A atividade da paraoxonase sérica não foi significativamente diferente entre os grupos de estudo. Após ajuste para idade, sexo, hipertensão, diabetes mellitus e dislipidemia, o genótipo GG e o modelo codominante de rs662 foram positivamente associados a uma angiografia positiva (respectivamente, OR = 2,424, IC 95% [1,123-5,233], p <0,05, OR = 1,663, IC 95% [1,086-2,547]). A atividade da paraoxonase sérica foi significativamente maior no alelo G e variante GG do polimorfismo rs662, alelo A e variante AA de rs854560 e alelo C e variante CC de rs705379. A análise de haplótipos mostrou que o haplótipo ATC foi significativamente mais prevalente no grupo com angiografia negativa. A análise entre os grupos indicou que o alelo A de rs662 foi significativamente associado à menor atividade da paraoxonase no grupo com angiografia positiva (p=0,019).

 Conclusões

A presença do alelo G do polimorfismo de nucleotídeo único rs662 está independentemente associada ao aumento do risco de DAC.

 Doença da Artéria Coronariana Angiografia Arildialquilfosfatase Introdução

A Organização Mundial da Saúde (OMS) relata que 71% das mortes a cada ano são devido a doenças não transmissíveis, das quais, 43% são devido à doença arterial coronariana (DAC).1 Isso também foi relatado como sendo de aproximadamente 46% no Irã em 2019,2 onde a prevalência de DAC está aumentando.3 , 4

O estresse oxidativo tem um papel fundamental no início e progressão da aterosclerose,5 bem como na patogênese da DAC e seus desfechos relacionados.6 Tem sido demonstrado que a lipoproteína de alta densidade (HDL, high-density lipoprotein ) possui propriedades antioxidantes e anti-inflamatórias nas quais a paraoxonase 1 (PON1) pode desempenhar um papel, diminuindo a produção de lipoproteína de baixa densidade (LDL, low-density lipoprotein ) oxidada durante o processo de peroxidação lipídica. A PON1 é uma esterase cálcio-dependente, cuja concentração sérica difere por etnia e geograficamente. Foi demonstrado que a diminuição da atividade de PON1 está associada a condições em que há estresse oxidativo, incluindo síndrome metabólica, DAC, Alzheimer e envelhecimento, e nesse caso, níveis aumentados de PON1 podem ser protetores.7 O cluster de genes da paraoxonase codifica três membros distintos, PON1-PON2 e PON3, localizados no cromossomo 7q21.3. Mais de 160 polimorfismos de nucleotídeo único (SNPs, single nucleotide polymorphisms ) foram identificados para o gene PON1,8 dos quais rs662, rs854560, rs705379 são descritos como associados aos níveis e atividade de proteínas enzimáticas séricas. Os SNPs rs662 e rs850560 estão localizados dentro de regiões codificadoras e resultam em uma substituição de aminoácidos9 , 10 enquanto o terceiro polimorfismo, rs705379, está localizado na região promotora.11 A presença do rs662 resulta em uma substituição de glutamina por arginina, aumenta a taxa de hidrólise de paraoxon e clorpirifós-oxon. Enquanto o polimorfismo rs850560, uma substituição do aminoácido leucina para metionina, também está associado à diminuição da PON12 sérica. O SNP rs705379 ocorre no local de ligação do fator de transcrição Sp1, e tem o maior efeito sobre a expressão de PON.11 Esse polimorfismo é responsável por aproximadamente 30% das variações nos níveis plasmáticos de PON1. O alelo C de rs705379 está associado a um aumento da atividade do promotor e, portanto, a expressão do gene PON1 é aumentada.13 Além disso, vários estudos mostraram que esse polimorfismo está associado a um risco aumentado de DAC, especialmente em jovens14 - 16 e em indivíduos com diabetes tipo 2.17

Como a DAC é uma doença importante em relação à mortalidade e os estudos mostram que existe uma associação entre PON1 e DAC, decidiu-se investigar essa associação na sociedade iraniana, principalmente no nordeste do país.18 Existem poucos estudos sobre a associação entre o genótipo ou fenótipo de PON1 e DAC no nordeste do Irã,19 - 21 e a atividade enzimática sérica não foi estudada juntamente com polimorfismos. O objetivo do presente estudo foi avaliar a associação entre polimorfismos de PON1 e atividade da paraoxonase com DAC em adultos iranianos que vivem no nordeste do país.

 Material e Métodos Desenho e população do estudo

Esse estudo de caso-controle foi realizado entre dezembro de 2014 e abril de 2017; foram recrutados 601 pacientes iranianos não relacionados que foram submetidos à angiografia coronária eletiva. Os pacientes foram encaminhados para angiografia devido a dor torácica ou sintomas equivalentes, como dispneia aos esforços. Com base nos resultados da angiografia, os pacientes foram divididos em dois grupos: aqueles com doença arterial coronariana obstrutiva com estenose coronariana >50% em pelo menos uma artéria coronária (N=266) (angiografia positiva) e pacientes com doença arterial coronariana não obstrutiva com estenose <30% nas artérias coronárias (N=335) (angiografia negativa).

Os dados demográficos incluindo sexo, idade, histórico de tabagismo, histórico pregresso de diabetes mellitus (DM), hipertensão (HAS) e dislipidemia foram coletados dos prontuários médicos. A pressão arterial sistólica (PAS) e a pressão arterial diastólica (PAD) foram medidas imediatamente antes do procedimento. Pacientes com doença autoimune, câncer ativo, trombofilia ou doença renal crônica foram excluídos.

As amostras de sangue foram coletadas antes do procedimento em tubos contendo EDTA para extração de DNA e em tubos sem anticoagulante para medida da atividade da paraoxonase. O soro foi separado por centrifugação do sangue por 15 min a 1000 rpm, que é velocidade recomendada pelo fabricante (BEHDAD, Irã) e armazenado a -80°C.

 Genotipagem

O DNA foi extraído do sangue com EDTA, utilizando um kit de isolamento de DNA genômico (Genet bio, Coréia) com base nas instruções do fabricante. A pureza e a quantificação do DNA foram determinadas por espectrofotometria UV (Infinite 200PRONanoQuant, Tecan).

Três SNPs dos genes PON1 foram genotipados. Utilizamos o método duplo ARMS-PCR para os SNPs rs662 e rs854560 e o método PCR-RFLP para o SNP rs705379. Detalhes sobre os primers utilizados e as condições de PCR são detalhados no Suplemento 1. A eletroforese em gel foi realizada utilizando agarose 2% com tampão Tris-Borato-EDTA (TBE) para os três SNPs. Para determinar os genótipos de rs705379, foram utilizadas 5 unidades de Bsh1236I (Thermo Scientific) por 16 horas a 37°C. O produto da PCR 109bp foi cortado em fragmentos de 67pb e 42pb e visualizado com Transiluminador UV. O sequenciamento foi realizado para confirmar a precisão das técnicas de genotipagem.

 Atividade da paraoxonase

A atividade da paraoxonase foi medida pela adição de 10 μL de soro a 290 μL de tampão Tris-HCl (100mmol/l, pH=8,0) contendo 1mmol/Lde CaCl2 e 1mmol/L de paraoxon (D9286, Sigma Chemical Company, Deisenhofen, Alemanha). A geração de p-nitrofenol foi medida a 405 nm e à temperatura ambiente, utilizando um leitor de placas (EPOCH, EUA) 3 e 6 minutos após a adição de paraoxon como substrato. A atividade da paraoxonase foi relatada em Unidades por litro de soro por minuto.

 Considerações éticas

Todos os participantes preencheram um formulário de consentimento informado por escrito. O Comitê de Ética da Mashhad University of Medical Sciences aprovou o protocolo do estudo (número de identificação: 930834).

 Análise estatística

Todos os dados foram analisados estaticamente utilizando o software Statistical Package for Social Sciences (SPSS Inc., IL, EUA). A normalidade dos dados foi verificada pelo teste de Kolmogorov-Smirnov. As variáveis com distribuição normal foram expressas em média ± desvio padrão (DP) e as variáveis sem distribuição normal foram descritas por mediana e intervalo interquartil. As variáveis categóricas foram expressas por número e porcentagem. Para a análise entre grupos, o teste de qui-quadrado foi usado para dados categóricos, e para dados quantitativos, utilizou-se o teste t para amostras independentes (para dados normalmente distribuídos) ou teste de Mann-Whitney e Kruskal-Wallis (para dados não distribuídos normalmente), respectivamente. Para indicar a associação entre SNPs e angiografia positiva, foram realizadas as análises univariada e multivariada com regressão logística binária, sendo expressas em OR (IC95%). A significância estatística foi estabelecida em p< 0,05. A análise de haplótipos foi realizada utilizando o SNPAlyze (versão demo, V8.1.1).

 Resultados

As diferenças nas características basais entre os grupos de estudo são mostradas na Tabela 1 . As diferenças entre a frequência dos três genótipos e a positividade da angiografia são mostradas na Tabela 2 . Após ajuste para idade, sexo, hipertensão, DM e dislipidemia, um modelo recessivo para o genótipo GG de rs662 foi significante entre as populações do estudo. Além disso, um resultado significante foi observado no modelo codominante para rs662.

Características basais da população do estudo
Variável Angiografia negativa (N=266) Angiografia positiva (N=335) p Valor
Idade (anos) (Média±DP)1 55,70±10,96 61,53±8,91 <0,001
Sexo (N%)2 Masculino 130 (48,9%) 227 (69,8%) <0.001
Feminino 136 (51,1%) 98 (30,2%)
Histórico de tabagismo (N %)2 42 (16,0%) 52 (16,5%) 0,890
Histórico de HTN (N %)2 121 (45,5%) 192 (59,3%) 0,001
Histórico de DM (N %)2 70 (26,3%) 130 (40,5%) <0,001
Histórico de dislipidemia (N %)2 93 (35,1%) 178 (55,3%) <0,001
IMC (N %)2 Normal (BMI<25) 87 (37,5%) 104 (35,6%) 0.683
Sobrepeso (25≤BMI<30) 101 (43,5%) 138 (47,3%)
Obeso (BMI≥30) 44 (19,0%) 50 (17,1%)
PAS (Média ± DP)1 121,81±17,37 124,93±15,44 0,035
PAD (Média ± DP)1 76,12±10,42 77,75±8,75 0,063
Atividade da paraoxonase sérica (U/L) (Mediana (IQR))1 57,60(32,70-105,15) 52,20(30,38-95,18) 0,237

HTN: hipertensão; DM: diabetes mellitus; IMC: índice de massa corporal; PAS: pressão arterial sistólica; PAD: pressão arterial diastólica. 1 Análise por teste t ou teste de Mann-Whitney quando necessário. 2 Análise pelo teste do qui-quadrado.

Associação entre polimorfismos de PON1 e DAC
Genótipos Angiografia negativa (N=266) Angiografia positiva (N=335) Regressão univariada Regressão multivariada1
rs662 A 0,70 0,68 1,093 (0,847-1,410) 1,062 (0,773-1,460)
G 0,30 0,32
AA 112(44,8%) 150(48,1%) Ref, Ref,
AG 121(48,4%) 121(38,8%) 0,747 (0,525-1,061) 0,685 (0,439-1,069)
GG 17(6,8%) 41(13,1%) 1,913 (1,022-3,583)* 1,802 (0,827-3,925)
Modelo dominante AA 112(44,8%) 150(57,3%) 0,833 (0,632-1,233) 0,818 (0,535-1,250)
AG+GG 138(55,2%) 162(51,9%)
Modelo recessivo AA+AG 234(93,6%) 271(86,9%) 2,360 (1,274-4,373)** 2,424 (1,123-5,233)*
GG 16(6,4%) 41(13,1%)
Modelo aditivo AA 112(87,5%) 150(78,5%) 2,041 (1,076-3,871)* 2,080 (0,900-4,810)
GG 16(12,5%) 41(21,5%)
Modelo codominante AG 122(48,8%) 121(38,9%) 1,508 (1,077-2,113)* 1,663 (1,086-2,547)*
AA+GG 128(51,2%) 190(61,1%)
rs854560 A 0,62 0,67 0,944 (0,682-1,309) 0,836 (0,583-1,200)
T 0,38 0,33
AA 88(35,5%) 131(42,3%) Ref, Ref,
AT 131(52,8%) 149(48,1%) 1,053 (0,692-1,603) 0,918 (0,550-1,533)
TT 29(11,7%) 30(9,7%) 0,299(0,694-1,383) 0,603(0,260-1,399)
Modelo dominante AA 86(34,7%) 130(41,9%) 1,004(0,643-1,568) 0,854(0,523-1,393)
AT+TT 162(65,3%) 180(58,1%)
Modelo recessivo AA+AT 220(88,7%) 279(90,0%) 0,770(0,381-1,556) 0,631(0,285-1,398)
TT 28(11,3%) 31(10,0%)
Modelo aditivo AA 86(75,4%) 130(81,2%) 0,789(0,374-1,665) 0,601(0,259-1,396)
TT 28(24,6%) 30(18,8%)
Modelo codominante AT 134(54,0%) 149(48,2%) 0,909(0,586-1,412) 0,990 (0,610-1,607)
AA+TT 114(46,0%) 160(51,8%)
rs705379 C 0,50 0,49 1,055 (0,798-1,394) 0,941(0,669-1,322)
T 0,50 0,51
CC 49(24,1%) 68(21,9%) Ref, Ref,
CT 109(53,7%) 171(55,0%) 0,941(0,574-1,544) 1,079(0,589-1,975)
TT 45(22,2%) 72(23,2%) 1,098 (0,603-2,000) 0,585(0,418-1,761)
Modelo dominante CC 52 (25,6%) 67(21,4%) 1,086 (0,642-1,838) 1,143 (0,645-2,028)
CT+TT 151(74,4%) 246(78,6%)
Modelo recessivo CC+CT 159(78,3%) 242(77,3%) 1,001 (0,600-1,703) 0,820 0,460-1,462)
TT 44(21,7%) 71(22,7%)
Modelo aditivo CC 52(54,2%) 67(48,6%) 1,075 (0,561-2,063) 0,939 (0,458-1,924)
TT 44(45,8%) 71(51,4%)
Modelo codominante CT 107(52,7%) 175(55,9%) 0,952 (0,613-1,478) 0,791 (0,486-1,288)
CC+TT 96(47,3%) 138(44,1%)

A análise foi realizada usando regressão univariada e multivariada. * Valor p ≤ 0,05. **Valor p ≤ 0,01. 1 Ajustado para idade, sexo, hipertensão, diabetes mellitus, dislipidemia.

A análise do haplótipo mostrou que o haplótipo “ATC” apresentou diferença significante entre os dois grupos analisados (p=0,017) ( Tabela 3 ).

Diferença entre frequências de haplótipos nos grupos de estudo
Haplótipos 1 Total (N=591) Angiografia negativa (N=266) Angiografia positiva (N=335) p valor
AAC 0,241 0,226 0,248 0,509
ATT 0,205 0,200 0,209 0,781
AAT 0,150 0,159 0,144 0,639
GAC 0,140 0,134 0,147 0,682
GAT 0,120 0,110 0,126 0,549
ATC 0,095 0,133 0,072 0,017
GTT 0,024 0,021 0,026 0,742
GTC 0,025 0,017 0,029 0,437

1 Teste de Qui-quadrado usado para análise.

A Tabela 4 mostra a diferença entre os genótipos e a atividade da paraoxonase nos grupos de estudo. No total e em ambos os casos e controles, a atividade da paraoxonase estava aumentada na presença do alelo G em comparação com a presença do alelo A. Além disso, a atividade da paraoxonase foi significantemente maior na presença do alelo A de rs850560 em comparação com a presença do alelo T nesse locus e do alelo C de rs705379 no promotor de PON1. As comparações entre os grupos indicaram que a atividade da paraoxonase foi significantemente menor para o genótipo AA do polimorfismo rs662 na DAC em comparação aos controles (p=0,019).

Análise entre grupos de diferentes genótipos de PON1 e atividade de paraoxonase sérica
SNPs Total (N=591) p valor1 Angiografia negativa (N=266) p valor2 Angiografia positiva (N=335) p valor3 p valor4
rs662 A 45,45 (27,9-78,75) <0,001 46,80 (28,35-87,75) <0,001 45,00 (26,55-67,50) <0,001 0,019
G 94,28 (48,26-148,95) 95,85 (60,19-158,44) 94,28 (48,26(147,15) 0,527
AA 40,05 (25,2-57,60) <0,001*,**,*** 41,40 (26,55-58,05) <0,001*,**,*** 40,05 (23,40-57,15) <0,001*,**,*** 0,345
AG 81,90 (43,76-126,79) 85,63 (44,55-141,08) 80,10 (43,65-122,85) 0,084
GG 129,60 (80,21-175,73) 142,65 (84,60-176,40) 127,35 (62,21-175,28) 0,933
rs854560 A 60,53 (35,51-117,56) <0,001 65,70 (34,65-119,35) 0,001 59,40 (36,90-110,59) <0,001 0,162
T 41,85 (22,95-79,54) 43,00 (26,55-84,71) 42,08 (22,50-70,88) 0,585
AA 67,95 (42,41-129,49) <0,001*,**,*** 80,10 (38,40-130,28) <0,001*,** 65,48 (43,20(128,81) <0,001*,**,*** 0,228
AT 49,50 (28,30-87,75) 57,10 (31,95-95,40) 47,70 (26,55(83,25) 0,487
TT 29,25 (17,10-46,80) 30,15 (17,55-43,50) 29,25 (17,10-48,15) 0,888
rs705379 C 65,25 (34,20-122,51) <0,001 78,08 (38,40-133,54) 0,003 63,00 (32,85-111,60) 0,001 0,180
T 47,70 (27,00-87,75) 45,46 (26,55-91,05) 47,70 (29,25-86,85) 0,530
CC 75,38 (40,50-144,00) <0,001*,**,*** 83,93 (44,59-156,83) <0,001*,** 72,90 (36,90-127,35) <0,001** 0,329
CT 54,90 (31,30-98,10) 56,70 (31,46-103,24) 54,90 (31,16-94,40) 0,299
TT 41,40 (22,89-65,93) 37,35 (17,55-73,80) 45,45 (24,98-60,30) 0,770

Para a análise estatística, teste de Kruskal-Wallis foi usado para comparação entre mais de 2 grupos e teste de Mann-Whitney para comparação entre 2 grupos. 1 status de atividade da paraoxonase sérica e SNPs no total. 2 status de atividade da paraoxonase sérica e SNPs no grupo angiografia negativa. 3 status de atividade da paraoxonase sérica e SNPs no grupo angiografia positiva. 4 Comparação entre a atividade da paraoxonase sérica e diferentes genótipos nos grupos angiografia positiva e negativa. *Diferença significante entre homozigotos dominantes e heterozigotos. ** Diferença significante entre dois homozigotos. ***Diferença significante entre homozigotos recessivos e heterozigotos.

 Discussão

Não foi possível demonstrar qualquer associação significante entre os polimorfismos rs850560 e rs705379 e CAD definida angiograficamente em adultos iranianos, enquanto a análise do polimorfismo rs662 mostrou que a homozigosidade para a variante GG vs. AA e AG totais estava associada a uma prevalência maior do que 2 vezes na angiografia positiva em comparação com indivíduos com angiografia negativa. Além disso, observamos que a atividade da paraoxonase sérica estava associada a todos os três SNPs avaliados em ambos os grupos de indivíduos. Pode-se dizer que a atividade da enzima paraoxonase era maior nos portadores dos alelos R de rs662, alelo A de rs850560 e alelo C de rs705379. Não houve relação significante entre a positividade da angiografia e a atividade da paraoxonase sérica, embora uma atividade sérica média mais baixa de PON1 tenha sido observada nos pacientes com angiografia positiva.

Em uma metanálise de 17 estudos de diferentes cidades e estados do México realizados em 2018, os genótipos mais associados com atividade enzimática diminuída foram AT/TT de rs850560 e AA de rs662.22 Esses resultados foram compatíveis com nossos achados, embora tenha sido uma metanálise nacional conduzida na população do México e, portanto, a etnia era diferente.

Vários estudos investigaram a relação entre os dois SNPs codificadores (rs662 e rs850560) e DAC. A metanálise sugere uma associação entre DAC e PON1. Qinghua Zeng e Juan Zeng sugeriram que o polimorfismo rs662 poderia ser usado para identificar indivíduos altamente suscetíveis a DAC.23 Em uma metanálise de 43 estudos que avaliaram 11.000 casos e 13.000 controles. Wheeler et al., mostraram que houve uma relação significante entre o alelo G de rs662 e a DAC, mas não houve associação entre o polimorfismo rs850560 e DAC,24 semelhante aos nossos achados. Uma metanálise realizada por Wang et al., de 88 estudos de caso-controle em 2011, relatou resultados semelhantes.25

Vaisi-Raygani et al. também encontraram uma relação significante entre o polimorfismo rs662 e DAC no oeste do Irã. Assim, indivíduos com alelo G têm uma chance 1,6 vezes maior de apresentarem DAC.26 Por outro lado, Ahmad et al. afirmaram que o alelo G de rs662 está associado ao risco de DAC.27 Nossos resultados também são consistentes com outro estudo realizado no norte do Irã, que relatou uma relação entre rs705379 e DAC. Najafi et al. mostraram que, embora esse polimorfismo esteja associado à atividade enzimática sérica, não houve relação significante com a ocorrência de DAC quando comparado ao grupo controle.28

Tang et al., avaliaram as atividades séricas da PON-1 e seus determinantes genéticos relacionados em 3.668 indivíduos estáveis (média de idade de 63±11 anos) submetidos à angiografia coronária eletiva (ACE) sem síndrome coronariana aguda e que foram acompanhados prospectivamente para eventos cardiovasculares adversos maiores por um período de 3 anos. Os resultados mostraram que as variantes rs854560 e rs662 tinham fortes efeitos genéticos na atividade sérica de PON1, mas não estavam associadas ao risco de eventos cardíacos adversos maiores (ECAM). Eles sugeriram que os efeitos genéticos desse SNP sobre a atividade da arilesterase são muito fracos para serem observados, especialmente se um limiar biológico mínimo de mudança de atividade for necessário para influenciar o risco de incidentes de ECAM.29

Da mesma forma, o estudo GeneBank, um estudo prospectivo com 1.399 indivíduos submetidos à coronariografia diagnóstica eletiva com (idade de 65±11 anos) e sem (idade de 57±12 anos) DAC, mostrou que o polimorfismo rs662 é responsável por cerca de 60% das variações na atividade da PON1. Além disso, a diminuição da atividade sérica de PON1 e seu genótipo AA foram associados a um aumento do estresse oxidativo. Esse genótipo está associado a um aumento na mortalidade e desfechos adversos de eventos cardiovasculares, incluindo infarto do miocárdio e acidente vascular cerebral. Seus resultados mostraram que a incidência desses efeitos adversos foi significantemente menor em indivíduos com maior atividade de PON1.30

Um estudo de Ochoa-Martínez et al. observou que os portadores do alelo G do polimorfismo rs662 apresentavam níveis mais altos de biomarcadores de DAC do que os portadores do alelo A.31

Liu et al., relataram que o polimorfismo rs662 está significantemente associado à DAC. Em consonância com nossos achados, esse estudo mostrou que os portadores do alelo G desse polimorfismo estão significantemente mais expostos à DAC, e que a atividade e concentração de PON1 estavam positivamente associadas ao alelo G. Além disso, a concentração e a atividade dessa enzima estavam diminuídas nos pacientes com DAC em relação aos controles,18 o que não foi encontrado em nosso estudo. Há um paradoxo que já foi explicado por Aviram et al., que relataram que o sítio ativo de PON1 necessário para a proteção contra a oxidação de LDL é diferente daquele necessário para sua atividade da paraoxonase, sugerindo que o alelo R, apesar de apresentar maior atividade em relação ao paraoxon, é defeituoso, pois impede sua atividade antioxidante em relação ao LDL devido ao seu efeito modulador do sítio ativo.32 Assim, os portadores do alelo G possuem capacidade reduzida de prevenir a modificação oxidativa de LDL e, consequentemente, são mais suscetíveis a desenvolver DAC do que os portadores do alelo A.30

O efeito do SNP rs705379 no promotor do gene PON1 é o outro determinante da atividade de PON1. Brophy et al. mostraram que esse polimorfismo foi responsável por 22,8% da inconsistência na atividade de PON1. Seus resultados mostraram que a atividade de PON1 estava reduzida em 376 indivíduos brancos na presença do alelo T do polimorfismo rS705379 em comparação com a presença do alelo C.33 Vários estudos anteriores relataram que o alelo T desse polimorfismo está associado a um risco aumentado de DAC em homens,14 , 15 embora não fosse possível confirmar esse resultado, o que pode ser devido a diferenças de idade, etnia, presença de doença, número de participantes inscritos em vários estudos ou diferença no grupo controle. Voetsch et al., mostraram resultados semelhantes em 118 pacientes jovens (idade <45 anos) com um primeiro AVC isquêmico arterial não fatal.16 Gupta et al., relataram que a atividade de PON1 está reduzida em pacientes com DAC demonstrada angiograficamente em comparação com indivíduos saudáveis em um grupo étnico discreto de indianos no noroeste do Punjabi, que têm alta incidência de DAC. Além disso, seus resultados mostraram que os polimorfismos rs662 nas regiões codificadoras estão todos independentemente associados à DAC.34 James et al. relataram que existe uma associação entre o alelo T de rs705379 e o risco aumentado de DAC em pacientes diabéticos tipo II.17

A presença do SNP rs662 por si só não é suficiente para o desenvolvimento de aterosclerose, pois vários estudos anteriores relataram que, além do genótipo, a atividade e as concentrações enzimática também têm papéis importantes.35 Além disso, diferenças étnicas,36 fatores dietéticos e ambientais,37 e o status do HDL38 podem afetar o fenótipo de PON1. Além disso, um estudo destacou a importância do HDL, que é um fator relacionado à DAC, e que envolve PON1.39 Foi relatado que os três genótipos AA, AG e GG apresentam valores médios semelhantes de níveis de PON1/HDL. Este resultado pode explicar por que tentativas anteriores de correlacionar o genótipo rs662 com uma predisposição para aterosclerose falharam e abre caminho para novas metodologias de fenotipagem de PON1 que podem fornecer uma melhor correlação.40 Uma limitação do nosso estudo foi o uso de indivíduos com estenose <30% como controles. Idealmente, os controles seriam indivíduos com DAC mínima definida angiograficamente, mas esses indivíduos raramente necessitam de angiografia nessa idade.

 Conclusões

Descobrimos que portadores do alelo G do polimorfismo Q192R do gene PON1 estavam independentemente associados a uma angiografia coronária positiva. Além disso, portadores do alelo G de rs662, alelo A de rs850560 e alelo C de rs705379 têm níveis aumentados de atividade sérica de PON1. Não foi possível estabelecer qualquer relação significante entre a atividade da paraoxonase sérica e uma angiografia positiva em uma amostra do nordeste do Irã.

 Agradecimento

Este estudo recebeu apoio de Mashhad e Isfahan University of Medical Sciences. Os autores agradecem aos técnicos do laboratório de cateterismo Sina, Sadi, Ghaem e aos técnicos do laboratório de genética Isfahan Alzahra.

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Bhattacharyya T Nicholls SJ Topol EJ Zhang R Yang X Schmitt D et al Relationship of Paraoxonase 1 (PON1) Gene Polymorphisms and Functional Activity with Systemic Oxidative Stress and Cardiovascular Risk JAMA 2008 299 11 1265 1276 10.1001/jama.299.11.1265 31. Ochoa-Martínez ÁC, Araiza-Gamboa Y, Varela-Silva JA, Orta-García ST, Carrizales-Yáñez L, Pérez-Maldonado IN. Effect of Gene-environment Interaction (arsenic exposure - PON1 Q192R polymorphism) on Cardiovascular Disease Biomarkers in Mexican Population. Environ Toxicol Pharmacol. 2021;81:103519. doi: 10.1016/j.etap.2020.103519. Ochoa-Martínez ÁC Araiza-Gamboa Y Varela-Silva JA Orta-García ST Carrizales-Yáñez L Pérez-Maldonado IN Effect of Gene-environment Interaction (arsenic exposure - PON1 Q192R polymorphism) on Cardiovascular Disease Biomarkers in Mexican Population Environ Toxicol Pharmacol 2021 81 103519 10.1016/j.etap.2020.103519 32. Aviram M, Billecke S, Sorenson R, Bisgaier C, Newton R, Rosenblat M, et al. Paraoxonase Active Site Required for Protection Against LDL Oxidation Involves its Free Sulfhydryl Group and is Different from that Required for its Arylesterase/paraoxonase Activities: Selective Action of Human Paraoxonase Allozymes Q and R. Arterioscler Thromb Vasc Biol. 1998;18(10):1617-24. doi: 10.1161/01.atv.18.10.1617. Aviram M Billecke S Sorenson R Bisgaier C Newton R Rosenblat M et al Paraoxonase Active Site Required for Protection Against LDL Oxidation Involves its Free Sulfhydryl Group and is Different from that Required for its Arylesterase/paraoxonase Activities: Selective Action of Human Paraoxonase Allozymes Q and R Arterioscler Thromb Vasc Biol 1998 18 10 1617 1624 10.1161/01.atv.18.10.1617 33. Brophy VH, Jampsa RL, Clendenning JB, McKinstry LA, Jarvik GP, Furlong CE. Effects of 5’ Regulatory-region Polymorphisms on Paraoxonase-gene (PON1) Expression. Am J Hum Genet. 2001;68(6):1428-36. doi: 10.1086/320600. Brophy VH Jampsa RL Clendenning JB McKinstry LA Jarvik GP Furlong CE Effects of 5’ Regulatory-region Polymorphisms on Paraoxonase-gene (PON1) Expression Am J Hum Genet 2001 68 6 1428 1436 10.1086/320600 34. Gupta N, Singh S, Maturu VN, Sharma YP, Gill KD. Paraoxonase 1 (PON1) Polymorphisms, Haplotypes and Activity in Predicting Cad Risk in North-West Indian Punjabis. PLoS One. 2011;6(5):e17805. doi: 10.1371/journal.pone.0017805. Gupta N Singh S Maturu VN Sharma YP Gill KD Paraoxonase 1 (PON1) Polymorphisms, Haplotypes and Activity in Predicting Cad Risk in North-West Indian Punjabis PLoS One 2011 6 5 e17805 10.1371/journal.pone.0017805 35. Mackness M, Mackness B. Paraoxonase 1 and Atherosclerosis: Is the Gene or the Protein More Important? Free Radic Biol Med. 2004;37(9):1317-23. doi: 10.1016/j.freeradbiomed.2004.07.034. Mackness M Mackness B Paraoxonase 1 and Atherosclerosis: Is the Gene or the Protein More Important? Free Radic Biol Med 2004 37 9 1317 1323 10.1016/j.freeradbiomed.2004.07.034 36. La Du B. Human Serum Paraoxonase/arylesterase. Amsterdam: Elsevier; 1992. La Du B Human Serum Paraoxonase/arylesterase Amsterdam Elsevier 1992 37. Aviram M, Rosenblat M, Gaitini D, Nitecki S, Hoffman A, Dornfeld L, et al. Pomegranate Juice Consumption for 3 Years by Patients with Carotid Artery Stenosis Reduces Common Carotid Intima-media Thickness, Blood Pressure and LDL Oxidation. Clin Nutr. 2004;23(3):423-33. doi: 10.1016/j.clnu.2003.10.002. Aviram M Rosenblat M Gaitini D Nitecki S Hoffman A Dornfeld L et al Pomegranate Juice Consumption for 3 Years by Patients with Carotid Artery Stenosis Reduces Common Carotid Intima-media Thickness, Blood Pressure and LDL Oxidation Clin Nutr 2004 23 3 423 433 10.1016/j.clnu.2003.10.002 38. Cabana VG, Reardon CA, Feng N, Neath S, Lukens J, Getz GS. Serum Paraoxonase: Effect of the Apolipoprotein Composition of HDL and the Acute Phase Response. J Lipid Res. 2003;44(4):780-92. doi: 10.1194/jlr.M200432-JLR200. Cabana VG Reardon CA Feng N Neath S Lukens J Getz GS Serum Paraoxonase: Effect of the Apolipoprotein Composition of HDL and the Acute Phase Response J Lipid Res 2003 44 4 780 792 10.1194/jlr.M200432-JLR200 39. Mahrooz A, Mackness M, Bagheri A, Ghaffari-Cherati M, Masoumi P. The Epigenetic Regulation of Paraoxonase 1 (PON1) as an Important Enzyme in HDL Function: The Missing Link Between Environmental and Genetic Regulation. Clin Biochem. 2019;73:1-10. doi: 10.1016/j.clinbiochem.2019.07.010. Mahrooz A Mackness M Bagheri A Ghaffari-Cherati M Masoumi P The Epigenetic Regulation of Paraoxonase 1 (PON1) as an Important Enzyme in HDL Function: The Missing Link Between Environmental and Genetic Regulation Clin Biochem 2019 73 1 10 10.1016/j.clinbiochem.2019.07.010 40. Gaidukov L, Rosenblat M, Aviram M, Tawfik DS. The 192R/Q Polymorphs of Serum Paraoxonase PON1 Differ in HDL Binding, Lipolactonase Stimulation, and Cholesterol Efflux. J Lipid Res. 2006;47(11):2492-502. doi: 10.1194/jlr.M600297-JLR200. Gaidukov L Rosenblat M Aviram M Tawfik DS The 192R/Q Polymorphs of Serum Paraoxonase PON1 Differ in HDL Binding, Lipolactonase Stimulation, and Cholesterol Efflux J Lipid Res 2006 47 11 2492 2502 10.1194/jlr.M600297-JLR200

https://www.who.int/news-room/fact-sheets/detail/noncommunicable-diseases

Vinculação acadêmica

Este artigo é parte de tese de doutorado de Majid Ghayour-Mobarhan pelo Mashhad University of Medical Sciences.

Fontes de financiamento: O presente estudo foi financiado por Mashhad and Isfahan University of Medical Sciences.

 * Material suplementar

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 Original Article Association of Paraoxonase-1 Genotype and Phenotype with Angiogram Positive Coronary Artery Disease Soflaei Sara Saffar 1 * Baktashian Mojtaba 1 * Moghaddam Kiana Hosseinpour 2 Saberi-Karimian Maryam 3 Kosari Negin 1 Hashemi Seyed Mohammad 4 Mouhebati Mohsen 5 Amini Mahsa 1 Dehghani Mashallah 5 Esmaily Habibollah 6 Ebrahimi Mahmoud 5 Falsoleiman Homa 5 Nosrati-Tirkani Abolfazl 7 Sadabadi Fatemeh 1 Ferns Gordon A. 8 Salehi Mansoor 9 Pasdar Alireza 1 10 0000-0002-1081-6754 Ghayour-Mobarhan Majid 3 5 Iran International UNESCO Center for Health-Related Basic Sciences and Human Nutrition, Mashhad University of Medical Sciences, Mashhad – Iran Iran Department of Biology, Faculty of Sciences, Ferdowsi University of Mashhad, Mashhad – Iran Iran Metabolic Syndrome Research Center, Faculty of Medicine, Mashhad University of Medical Sciences,Mashhad – Iran Iran Department of Cardiology, Chamran Hospital, School of Medicine, Isfahan University of Medical Sciences,Isfahan – Iran Iran Cardiovascular Research Center, School of Medicine, Mashhad University of Medical Sciences, Mashhad – Iran Iran Department of Biostatistics and Epidemiology, School of Health, Mashhad University of Medical Sciences,Mashhad – Iran Iran Biochemistry of Nutrition Research Center, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad – Iran UK Brighton & Sussex Medical School, Division of Medical Education, Falmer, Brighton – UK Iran Department of genetics, Faculty of medicine and genetics laboratory AL Zahra hospital, Isfahan University of Medicine, Isfahan – Iran UK Division of Applied Medicine, Medical School, University of Aberdeen, Foresterhill, Aberdeen – UK Mailing Address: Majid Ghayour-Mobarhan • Metabolic Syndrome Research Center, School of Medicine, Mashhad – University of Medical Sciences, 99199-91766, Mashhad – Iran E-mail: GhayourM@mums.ac.ir

These authors contributed equally

Author Contributions

Conception and design of the research: Soflaei SS, Baktashian M, Hashemi SM, Mouhebati M, Dehghani M, Ebrahimi M, Falsoleiman H, Ferns GA, Salehi M, Pasdar A, Ghayour-Mobarhan M; Acquisition of data: Soflaei SS, Baktashian M, Moghaddam KH, Kosari N, Amini M, Dehghani M, Ebrahimi M, Falsoleiman H, Nosrati-Tirkani A, Sadabadi F; Analysis and interpretation of the data: Soflaei SS, Moghaddam KH, Saberi-Karimian M, Amini M, Nosrati-Tirkani A, Pasdar A; Statistical analysis: Esmaily H; Obtaining financing: Hashemi SM, Salehi M, Ghayour-Mobarhan M; Writing of the manuscript: Soflaei SS, Saberi-Karimian M, Nosrati-Tirkani A; Critical revision of the manuscript for important intellectual content: Mouhebati M, Falsoleiman H, Ferns GA, Salehi M, Pasdar A, Ghayour-Mobarhan M.

Potential Conflict of Interest

No potential conflict of interest relevant to this article was reported.

 Abstract Background

It has been shown that increased serum PON1 levels are protective against several disorders. Several single nucleotide polymorphisms (SNPs) of the PON1 gene have been reported to be associated with serum enzyme protein levels and activity.

 Objective

To investigate the association of SNPs of PON1 and serum paraoxonase activity with coronary artery disease (CAD).

 Methods

A total of 601 unrelated patients who underwent coronary angiography including those who had >50% stenosis (N=266) and those with <30% stenosis (N=335) were studied. The Paraoxonase gene rs662 and rs840560 SNPs were determined using the ARMS-PCR method and the rs705379 SNP was genotyped using PCR-RFLP analysis. Serum paraoxonase activity was measured using paraoxon as a substrate. A p value of p<0.05 was considered as significant.

 Results

Serum paraoxonase activity was not significantly different between the study groups. After adjustment for age, sex, hypertension, diabetes mellitus and dyslipidemia, the GG genotype and co-dominant model of rs662 was positively associated with a positive angiogram (respectively, OR=2.424, 95%CI [1.123-5.233], p<0.05, OR=1.663, 95%CI [1.086-2.547]). Serum paraoxonase activity was significantly higher in the G allele and GG variant of rs662, A allele and AA variant of rs854560 and C allele and CC variant of rs705379. The haplotype analysis has shown that the ATC haplotype was significantly more prevalent among the angiogram negative group. The analysis between groups indicated that the A allele of rs662 was significantly associated with lower paraoxonase activity in the positive angiogram group (p=0.019).

 Conclusions

The presence of the G allele of the rs662 single nucleotide polymorphism is independently associated to increased risk of CAD.

 Coronary Artery Disease Angiography Aryldialkylphosphatase Introduction

The World Health Organization (WHO) reports that 71% of deaths each year are due to non-communicable diseases, of which 43% are due to coronary artery disease (CAD).1 This was also reported to be approximately 46% in Iran in 2019,2 where the prevalence of CAD is increasing.3 , 4

Oxidative stress has a key role in the initiation and progression of atherosclerosis,5 as well as in the pathogenesis of CAD and its related outcomes.6 It has been shown that high density lipoprotein (HDL) has antioxidant and anti-inflammatory properties, in which paraoxonase 1 (PON1) may play a role by decreasing the production of oxidized low density lipoprotein (LDL) during the process of lipid peroxidation. PON1 is a calcium-dependent esterase, of which serum concentration differs by ethnicity and geographically. It has been shown that a decreased PON1 activity is associated with conditions in which there is oxidative stress, including metabolic syndrome, CAD, Alzheimer and aging, and in this case, increased PON1 levels may be protective.7 The paraoxonase gene cluster encodes for three distinct members: PON1-PON2 and PON3, which are located on chromosome 7q21.3. More than 160 single nucleotide polymorphisms (SNPs) have been identified for the PON1 gene,8 of which the rs662, rs854560, rs705379 are reported to be associated with serum enzyme protein levels and activity. The rs662 and rs850560 SNPs are located within coding regions and exert an amino acid substitution,9 , 10 whereas the third polymorphism, rs705379, is located on the promoter region.11 The presence of rs662 results in a glutamine-to-arginine substitution, increases the rate of hydrolysis of paraoxon and chlorpyrifos-oxon. While the rs850560 polymorphism, which results in a leucine-to-methionine amino acid substitution, is also associated with decreased serum PON1.12 The rs705379 SNP occurs at the binding site of the transcription factor Sp1, and has the greatest effect on the expression of PON1.11 This polymorphism accounts for approximately 30% of variations in plasma PON1 levels. The C allele of rs705379 is associated with an increased promoter activity, and therefore the expression of the PON1 gene is augmented.13 Moreover, several studies have shown that this polymorphism is associated with an increased risk of CAD, especially in young people14 - 16 and in individuals with type 2 diabetes.17

Since CAD is an important disease in relation to mortality and studies have shown that there is an association between PON1 and CAD, it was decided to investigate this association in the Iranian society, especially in the northeast of the country.18 There are few studies on the association between PON1 genotype, or phenotype and CAD in northeastern Iran19 - 21 and serum enzyme activity was not studied along with the polymorphisms. The aim of current study was to assess the association between PON1 polymorphisms and paraoxonase activity with CAD among Iranian adults living in northeastern Iran.

 Material and methods Study design and population

This case-control study was carried out between December 2014 and April 2017; 601 unrelated Iranian patients who underwent elective coronary angiography were recruited. Patients were referred for angiography because of chest pain or equivalent symptoms, such as dyspnea on exertion. Based on the results of the angiography, the patients were divided into two groups: those with obstructive coronary artery disease with coronary stenosis >50% in at least one coronary artery (N=266) (angiogram positive) and patients with non-obstructive coronary artery disease with stenosis <30% in coronary arteries (N=335) (angiogram negative).

Demographic data including sex, age, smoking history, past history of diabetes mellitus (DM), hypertension (HTN), and dyslipidemia were collected from the medical records. Systolic blood pressure (SBP) and diastolic blood pressure (DBP) were measured right before the procedure. Patients with autoimmune disorders, active cancer, thrombophilia or chronic kidney disease were excluded.

Blood samples were taken before the procedure into EDTA tubes for DNA extraction and into tubes with no anticoagulant for paraoxonase activity measurement. Serum was separated by centrifuging the blood for 15 min at 1000 rpm speed (manufacturer’s recommended speed) (BEHDAD, Iran) and stored at -80°C.

 Genotyping

DNA was extracted from EDTA blood, using a genomic DNA isolation kit (Genet bio, Korea) based on the manufacturer’s instructions. DNA Purity and quantification were determined by UV spectrophotometry (Infinite 200PRONanoQuant, Tecan).

Three SNPs of PON1 genes were genotyped. We used the double ARMS-PCR method for the rs662 and rs854560 SNPs and the PCR-RFLP method for rs705379 SNP. Details on the primers used and PCR conditions are detailed in Supplement 1. Gel electrophoresis was performed using 2% agarose in TBE for the three SNPs. To determine the genotypes of rs705379, we used 5 units of Bsh1236I (Thermo Scientific) for 16 hours at 37°C. The 109bp PCR product was cut into 67bp and 42bp fragments and visualized with UV-Trans-illuminator. Sequencing was performed to confirm the accuracy of the genotyping techniques.

 Paraoxonase activity

Paraoxonase activity was measured by adding 10μL of serum to 290 μL of Tris-HCl buffer (100mmol/L, pH=8.0) containing 1mmol/L CaCl2and 1mmol/L paraoxon (D9286, Sigma Chemical Company, Deisenhofen, Germany). The generation of p-nitrophenol was measured at 405 nm and at room temperature using a plate reader (EPOCH, USA) 3 and 6 minutes after adding paraoxon as the substrate. Paraoxonase activity was reported in Units per liter of serum per minute.

 Ethical considerations

All participants filled out a written informed consent form. The Ethics Committee at Mashhad University of Medical Sciences approved the study protocol (ID Number: 930834).

 Statistical analysis

All data were statistically analyzed using the Statistical Package for Social Sciences software (SPSS Inc., IL, USA). Data normality was checked by the Kolmogorov-Smirnov test. Normally distributed variables were expressed in Mean ± standard deviation (SD) and variables without a normal distribution were described using median and interquartile range. Categorical variables were reported by number and percentage. For the analysis between groups, the chi-square test was used for categorical data, and the independent sample t-test for quantitative data (for normally distributed data) or Mann-Whitney and Kruskal-Wallis (for non-normally distributed data), respectively. Univariate and multivariate analyses with binary logistic regression were performed to indicate the association between SNPs and positive angiography, being expressed in OR (95%CI). Statistical significance was set at p <0.05. Haplotype analysis was performed using SNPAlyze (demo version, V8.1.1).

 Results

Differences in baseline characteristics between the study groups are shown in table 1 . The differences between the frequencies of the three genotypes and angiogram positivity are shown in Table 2 . After adjusting for age, sex, HTN, DM and dyslipidemia, a recessive model for GG genotype of rs662 was significant between the study populations. Also, a significant result was observed in the co-dominant model for rs662.

Baseline characteristics of the study population
Variable Angiogram negative (N=266) Angiogram positive (N=335) p Value
Age (y) (Mean±SD)1 55.70±10.96 61.53±8.91 <0.001
Sex (N%)2 Male 130 (48.9%) 227 (69.8%) <0.001
Female 136 (51.1%) 98 (30.2%)
Positive smoking history (N %)2 42 (16.0%) 52 (16.5%) 0.890
HTN history (N %)2 121 (45.5%) 192 (59.3%) 0.001
DM history (N %)2 70 (26.3%) 130 (40.5%) <0.001
Dyslipidemia history (N %)2 93 (35.1%) 178 (55.3%) <0.001
BMI (N %)2 Normal (BMI<25) 87 (37.5%) 104 (35.6%) 0.683
Overweight (25≤BMI<30) 101 (43.5%) 138 (47.3%)
Obese (BMI≥30) 44 (19.0%) 50 (17.1%)
SBP (Mean ± SD)1 121.81±17.37 124.93±15.44 0.035
DBP (Mean ± SD)1 76.12±10.42 77.75±8.75 0.063
Serum Paraoxonase activity (U/L) (Median(IQR))1 57.60(32.70-105.15) 52.20(30.38-95.18) 0.237

HTN: hypertension; DM: diabetes mellitus; BMI: body mass index; SBP: systolic blood pressure; DBP: diastolic blood pressure.1Analysis by t-test or Mann-Whitney test, when necessary.2Analysis by Chi-square test.

Association between PON1 polymorphisms and CAD
Genotypes Angiogram negative (N=266) Angiogram positive (N=335) Univariate regression Multivariate regression1
rs662 A 0.70 0.68 1.093 (0.847-1.410) 1.062 (0.773-1.460)
G 0.30 0.32
AA 112(44.8%) 150(48.1%) Ref. Ref.
AG 121(48.4%) 121(38.8%) 0.747 (0.525-1.061) 0.685 (0.439-1.069)
GG 17(6.8%) 41(13.1%) 1.913 (1.022-3.583)* 1.802 (0.827-3.925)
Dominant model AA 112(44.8%) 150(57.3%) 0.833 (0.632-1.233) 0.818 (0.535-1.250)
AG+GG 138(55.2%) 162(51.9%)
Recessive model AA+AG 234(93.6%) 271(86.9%) 2.360 (1.274-4.373)** 2.424 (1.123-5.233)*
GG 16(6.4%) 41(13.1%)
Additive model AA 112(87.5%) 150(78.5%) 2.041 (1.076-3.871)* 2.080 (0.900-4.810)
GG 16(12.5%) 41(21.5%)
Co-dominant model AG 122(48.8%) 121(38.9%) 1.508 (1.077-2.113)* 1.663 (1.086-2.547)*
AA+GG 128(51.2%) 190(61.1%)
rs854560 A 0.62 0.67 0.944 (0.682-1.309) 0.836 (0.583-1.200)
T 0.38 0.33
AA 88(35.5%) 131(42.3%) Ref. Ref.
AT 131(52.8%) 149(48.1%) 1.053 (0.692-1.603) 0.918 (0.550-1.533)
TT 29(11.7%) 30(9.7%) 0.299(0.694-1.383) 0.603(0.260-1.399)
Dominant model AA 86(34.7%) 130(41.9%) 1.004(0.643-1.568) 0.854(0.523-1.393)
AT+TT 162(65.3%) 180(58.1%)
Recessive model AA+AT 220(88.7%) 279(90.0%) 0.770(0.381-1.556) 0.631(0.285-1.398)
TT 28(11.3%) 31(10.0%)
Additive model AA 86(75.4%) 130(81.2%) 0.789(0.374-1.665) 0.601(0.259-1.396)
TT 28(24.6%) 30(18.8%)
Co-dominant model AT 134(54.0%) 149(48.2%) 0.909(0.586-1.412) 0.990 (0.610-1.607)
AA+TT 114(46.0%) 160(51.8%)
rs705379 C 0.50 0.49 1.055 (0.798-1.394) 0.941(0.669-1.322)
T 0.50 0.51
CC 49(24.1%) 68(21.9%) Ref. Ref.
CT 109(53.7%) 171(55.0%) 0.941(0.574-1.544) 1.079(0.589-1.975)
TT 45(22.2%) 72(23.2%) 1.098 (0.603-2.000) 0.585(0.418-1.761)
Dominant model CC 52 (25.6%) 67(21.4%) 1.086 (0.642-1.838) 1.143 (0.645-2.028)
CT+TT 151(74.4%) 246(78.6%)
Recessive model CC+CT 159(78.3%) 242(77.3%) 1.001 (0.600-1.703) 0.820 0.460-1.462)
TT 44(21.7%) 71(22.7%)
Additive model CC 52(54.2%) 67(48.6%) 1.075 (0.561-2.063) 0.939 (0.458-1.924)
TT 44(45.8%) 71(51.4%)
Co-dominant model CT 107(52.7%) 175(55.9%) 0.952 (0.613-1.478) 0.791 (0.486-1.288)
CC+TT 96(47.3%) 138(44.1%)

Analysis was performed using univariate and multivariate regression. *P value ≤ 0.05. **P value ≤ 0.01.1Adjusted for age, sex, hypertension, diabetes mellitus, dyslipidemia.

The haplotype analysis showed that the “ATC” haplotype showed a significant difference between the two analyzed groups (p=0.017) ( Table3 ).

Difference between haplotype frequencies in the study groups
Haplotypes1 Total (N=591) Angiogram negative (N=266) Angiogram positive (N=335) p value
AAC 0,241 0,226 0,248 0,509
ATT 0,205 0,200 0,209 0,781
AAT 0,150 0,159 0,144 0,639
GAC 0,140 0,134 0,147 0,682
GAT 0,120 0,110 0,126 0,549
ATC 0,095 0,133 0,072 0,017
GTT 0,024 0,021 0,026 0,742
GTC 0,025 0,017 0,029 0,437

1Chi-square test used for the analysis.

Table 4 shows the difference between genotypes and paraoxonase activity in the study groups. In total and in both cases and controls, paraoxonase activity was increased in the presence of the G allele in comparison with the presence of the A allele. Moreover, paraoxonase activity was significantly higher in the presence of the A allele of rs850560 in comparison with the presence of the T allele at this locus and the C allele of rs705379 in PON1 promoter. Comparisons between the groups indicated that paraoxonase activity was significantly lower for the AA genotype of the rs662 polymorphism in CAD when compared to the controls (p=0.019).

Analysis between groups of different PON1 genotypes and serum paraoxonase activity
SNPs Total (N=591) p value1 Angiogram negative (N=266) p value2 Angiogram positive (N=335) p value3 p value4
rs662 A 45.45 (27.9-78.75) <0.001 46.80 (28.35-87.75) <0.001 45.00 (26.55-67.50) <0.001 0.019
G 94.28 (48.26-148.95) 95.85 (60.19-158.44) 94.28 (48.26(147.15) 0.527
AA 40.05 (25.2-57.60) <0.001*,**,*** 41.40 (26.55-58.05) <0.001*,**,*** 40.05 (23.40-57.15) <0.001*,**,*** 0.345
AG 81.90 (43.76-126.79) 85.63 (44.55-141.08) 80.10 (43.65-122.85) 0.084
GG 129.60 (80.21-175.73) 142.65 (84.60-176.40) 127.35 (62.21-175.28) 0.933
rs854560 A 60.53 (35.51-117.56) <0.001 65.70 (34.65-119.35) 0.001 59.40 (36.90-110.59) <0.001 0.162
T 41.85 (22.95-79.54) 43.00 (26.55-84.71) 42.08 (22.50-70.88) 0.585
AA 67.95 (42.41-129.49) <0.001*,**,*** 80.10 (38.40-130.28) <0.001*,** 65.48 (43.20(128.81) <0.001*,**,*** 0.228
AT 49.50 (28.30-87.75) 57.10 (31.95-95.40) 47.70 (26.55(83.25) 0.487
TT 29.25 (17.10-46.80) 30.15 (17.55-43.50) 29.25 (17.10-48.15) 0.888
rs705379 C 65.25 (34.20-122.51) <0,001 78.08 (38.40-133.54) 0,003 63.00 (32.85-111.60) 0.001 0.180
T 47.70 (27.00-87.75) 45.46 (26.55-91.05) 47.70 (29.25-86.85) 0.530
CC 75.38 (40.50-144.00) <0.001*,**,*** 83.93 (44.59-156.83) <0.001*,** 72.90 (36.90-127.35) <0.001** 0.329
CT 54.90 (31.30-98.10) 56.70 (31.46-103.24) 54.90 (31.16-94.40) 0.299
TT 41.40 (22.89-65.93) 37.35 (17.55-73.80) 45.45 (24.98-60.30) 0.770

For statistical analysis, Kruskal–Wallis test was used for comparison between more than 2 groups and Mann-Whitney test for comparison between 2 groups.1serum paraoxonase activity status and SNPs in total.2serum paraoxonase activity status and SNPs in the angiogram negative group.3serum paraoxonase activity status and SNPs in the angiogram positive group.4Comparison between serum paraoxonase activity and different genotypes in angiogram positive and negative groups. *Significant difference between dominant homozygous and heterozygous. **Significant difference between two homozygous. ***Significant difference between recessive homozygous and heterozygous.

 Discussion

We could not show any significant association between rs850560 and rs705379 polymorphisms and angiographically defined CAD in Iranian adults, whereas the analysis of the rs662 polymorphism showed that homozygosity for the GG variant vs. total AA and AG was associated with a more than a 2-fold higher prevalence in the positive angiogram when compared to the negative angiogram subjects. Moreover, we found that serum paraoxonase activity was associated with all three assessed SNPs in both groups of subjects. It can be said that the paraoxonase enzyme activity was higher in carriers of R alleles of rs662, A allele of rs850560 and C Allele of rs705379. There was no significant relationship between angiogram positivity and serum paraoxonase activity, although a lower mean serum PON1 activity was observed in the angiogram positive patients.

In a meta-analysis on 17 studies carried out in different cities and states of Mexico conducted in 2018, the most frequently associated genotypes with decreased enzyme activity were AT/TT of rs850560 and AA of rs662.22 These results were compatible with our findings, although this was a national meta-analysis conducted in the Mexico population and, therefore, the ethnicity was different.

Several studies have investigated the relationship between the two coding SNPs (rs662 and rs850560) and CAD. The meta-analysis suggests an association between CAD and PON1. Qinghua Zeng and Juan Zeng suggested that the rs662 polymorphism could be used to identify individuals that are highly susceptible to CAD.23 In a meta-analysis on 43 studies that assessed 11,000 cases and 13,000 controls, Wheeler et al. showed there was a significant relationship between the G allele of the rs662 polymorphism and CAD, but there was no association between the rs850560 polymorphism and CAD,24 similar to our findings. A meta-analysis conducted by Wang et al., of 88 case-control studies in 2011 reported similar results.25

Vaisi-Raygani et al., also found a significant relationship between the rs662 polymorphism and CAD in western Iran. Thus, individuals with the G allele have a 1.6-fold chance of having CAD.26 On the other hand, Ahmad et al. stated that the G allele of rs662 is associated with the risk of developing CAD.27 Our results are also consistent with another study carried out in the northern Iran, which reported that there was a relationship between rs705379 and CAD. Najafi et al. showed that, while this polymorphism is associated with serum enzyme activity, there was no significant relationship with the occurrence of CAD when compared to the control group.28

Tang et al., evaluated serum PON-1 activity and its related genetic determinants in 3,668 stable subjects (mean age of 63±11) undergoing elective coronary angiography (ECA) without acute coronary syndrome and were prospectively followed for major adverse cardiovascular events for a period of 3 years. Their results showed that the rs854560 and rs662 variants had strong genetic effects on serum PON1 activity but were not associated with the risk of major adverse cardiac events (MACE). They suggested that the genetic effects of this SNP on arylesterase activity are too weak to be observed, especially if a minimum biological threshold of activity change is needed to influence the risk of MACE incidents.29

Similarly, the GeneBank study, a prospective study on 1,399 individuals undergoing elective diagnostic coronary angiography with (aged 65±11 years) and without (aged 57±12 years) CAD, showed that the rs662 polymorphism accounts for about 60% of the variations in PON1 activity. Moreover, decreased serum activity of PON1 and its AA genotype were associated with an increase in oxidative stress. This genotype is associated with an increase in mortality and adverse outcomes of cardiovascular events, including myocardial infarction and stroke. Their results showed that the incidence of these adverse effects was significantly lower in individuals with higher PON1 activity.30

A study by Ochoa-Martínez et al. found that carriers of the G allele of the rs662 polymorphism had higher levels of CAD biomarkers than carriers of the A allele.31

Liu et al., have reported that the rs662 polymorphism is significantly associated with CAD. In line with our findings, this study showed that the carriers of this polymorphism G allele are significantly more exposed to CAD and the activity and concentration of PON1 have been positively associated with the G allele. In addition, the concentration and activity of this enzyme was decreased in patients with CAD compared to the controls,18 which was not found in our study. There is a paradox, which was previously explained by Aviram et al., who reported that the PON1 active site required for protection against LDL oxidation is different from the one required for its paraoxonase activity, suggesting that the R allele, despite showing higher activity toward paraoxon, is defective as it prevents its antioxidant activity toward LDL due to its active site modulating effect.32 Thus, carriers of the G allele have reduced capacity to prevent the oxidative modification of LDL and, consequently, are more susceptible to develop CAD than carriers of the A allele.30

The rs705379 SNP effect on the PON1 gene promoter is the other determinant of PON1 activity. Brophy et al., have shown that this polymorphism accounted for 22.8% of the inconsistency in PON1 activity. Their results showed that PON1 activity was decreased in 376 white individuals in the presence of the T allele of the rS705379 polymorphism in comparison with presence of the C allele.33 Several previous studies have reported that the T allele of this polymorphism is associated with an increased risk of CAD in males,14 , 15 although we could not confirm this result, which may be due to differences in age, ethnicity, presence of disease, the number of enrolled participants in various studies or difference in control group. Voetsch et al., have shown similar results in a group of 118 young patients (aged <45 years) with a first nonfatal arterial ischemic stroke.16 Gupta et al. have reported that PON1 activity is reduced in angiographically CAD patients when compared to healthy subjects in a discretely ethnic Indian group in northwest Punjabi who have high incidence of CAD. Moreover, their results showed that the rs662 polymorphisms in the coding regions are all independently associated with CAD.34 James et al. have reported that there is an association between the T allele of rs705379 and increased risk of CAD in type II diabetic patien.17

The presence of the rs662 SNP alone is not enough for atherosclerosis development, because several previous studies have reported that in addition to genotype, enzyme activity and concentrations have important roles, too.35 Moreover, ethnic differences,36 dietary and environmental factors,37 and HDL status38 can all have an effect on the PON1 phenotype. In addition, a study highlighted the importance of HDL, which is a CAD-related factor, in that PON1 is involved.39 It has been reported that all three genotypes AA, AG, and GG result in similar mean values of PON1/HDL levels. This result may explain why previous attempts to correlate the rs662 genotype with a predisposition for atherosclerosis failed and opens the road for new PON1 phenotyping methodologies that may provide a better correlation.40 One limitation of our study was the use of subjects with <30% stenosis as controls. Ideally, the controls would be individuals with minimal angiographically-defined CAD, but these individuals uncommonly require angiography at this age.

 Conclusions

We have found that carriers of the G allele of the Q192R polymorphism of the PON1 gene were independently associated with a positive coronary angiogram. Moreover, carriers of G allele rs662, A allele of rs850560 and C allele of rs705379 have increased levels of serum PON1 activity. We could not establish any significant relationship between serum paraoxonase activity and a positive angiogram in a sample from northeastern Iran.

 Acknowledgment

This study was supported by Mashhad and Isfahan University of Medical Sciences. The authors would like to thank technicians of Sina, Sadi, Ghaem catheterization laboratory and technicians of Isfahan Alzahra genetics laboratory.

https://www.who.int/news-room/fact-sheets/detail/noncommunicable-diseases

Study Association

This article is part of the thesis of doctoral submitted by Majid Ghayour-Mobarhan, from Mashhad University of Medical Sciences.

Sources of Funding: This study was funded by Mashhad and Isfahan University of Medical Sciences.

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