Epigenetic contribution of the myosin light chain kinase gene to the risk for acute respiratory distress syndrome

Abstract

Acute respiratory distress syndrome (ARDS) is a devastating clinical syndrome with a considerable case fatality rate (∼30%-40%). Health disparities exist with African descent (AD) subjects exhibiting greater mortality than European descent (ED) individuals. Myosin light chain kinase is encoded by MYLK, whose genetic variants are implicated in ARDS pathogenesis and may influence ARDS mortality. As baseline population-specific epigenetic changes, that is, cytosine modifications, have been observed between AD and ED individuals, epigenetic variations in MYLK may provide insights into ARDS disparities. We compared methylation levels of MYLK cytosine-guanine dinucleotides (CpGs) between ARDS patients and intensive care unit (ICU) controls overall and by ethnicity in a nested case-control study of 39 ARDS cases and 75 non-ARDS ICU controls. Two MYLK CpG sites (cg03892735 and cg23344121) were differentially modified between ARDS subjects and controls (P < 0.05; q < 0.25) in a logistic regression model, where no effect modification by ethnicity or age was found. One CpG site was associated with ARDS in patients aged <58 years, cg19611163 (intron 19, 20). Two CpG sites were associated with ARDS in EDs only, gene body CpG (cg01894985, intron 2, 3) and CpG (cg16212219, intron 31, 32), with higher modification levels exhibited in ARDS subjects than controls. Cis-acting modified cytosine quantitative trait loci (mQTL) were identified using linear regression between local genetic variants and modification levels for 2 ARDS-associated CpGs (cg23344121 and cg16212219). In summary, these ARDS-associated MYLK CpGs with effect modification by ethnicity and local mQTL suggest that MYLK epigenetic variation and local genetic background may contribute to health disparities observed in ARDS.

Figure 1. ARDS-associated and population-modified CpGs in MYLK

(A). Locations of significant CpGs after controlling for age and sex and stratifying by ethnicity (cg01894985, cg16212219), for age and ethnicity and stratifying by age (cg19611163), and for age, ethnicity and age (cg03892735, cg23344121), in logistic regression are indicated based on hg19. Vertical bars are exons; white triangles indicate significantly differentially modified CpG sites for ED ARDS cases vs. ED ICU controls; striped triangle indicates a significant CpG for less than 58 year old ARDS vs. less than 58 year old ICU controls; light gray triangles indicate significant CpG sites for ARDS cases vs. ICU controls with no effect modification by age or ethnicity; arrows indicate higher modification (up arrow) or lower modification (down arrow) for ARDS cases vs. ICU controls; numbers correlate to CpG probe ID numbers in the 450k array; white stars indicate significant mQTL detected for two target CpG sites. (B). Significantly modified CpG sites for ARDS cases vs. ICU controls controlling for age, sex and ethnicity for which no significant M-value interaction terms were identified: cg03892735, cg23344121, located in intron (4,5) and exon 18, respectively. Effect plots of the probability that a case has ARDS (y-axis) according to M-value (x-axis). Open circles indicate observed case status (1 = ARDS, 0 = ICU control), line the predicted probability across M-value, shaded area the 95% confidence limits for the predicted probabilities. (C). Significantly modified CpG sites for ARDS cases vs. ICU controls among ED, controlling for age and sex: cg01894985, cg16212219, located in intron (2,3) and intron (31,32) respectively. Effect plots of the probability that a case has ARDS (y-axis) according to M-value (x-axis). Plus signs indicate observed ED case status and open circles observed AD case status (1 = ARDS, 0 = ICU control), predicted probability across M-value is indicated by a dashed line for ED patients and a solid line for AD patients, and the shaded areas (light gray for ED, dark gray for AD) indicate the 95% confidence limits for the predicted probabilities. (D). Significantly modified CpG site for ARDS cases vs. ICU controls among those less than 58 years of age, controlling for sex and ethnicity: cg19611163, located in intron (19,20). Effect plot of the probability that a case has ARDS (y-axis) according to M-value (x-axis). Plus signs indicate observed <58 year old patient case status, open circles indicate observed ≥58 year old patient case status (1 = ARDS, 0 = ICU control), predicted probability across M-value is indicated by a dashed line for <58 year old patients and a solid line for ≥58 year old patients, and the shaded areas (dark gray for <58 year, light gray for ≥ 58 year) indicate the 95% confidence limits for the predicted probabilities.

Figure 1. ARDS-associated and population-modified CpGs in MYLK

(A). Locations of significant CpGs after controlling for age and sex and stratifying by ethnicity (cg01894985, cg16212219), for age and ethnicity and stratifying by age (cg19611163), and for age, ethnicity and age (cg03892735, cg23344121), in logistic regression are indicated based on hg19. Vertical bars are exons; white triangles indicate significantly differentially modified CpG sites for ED ARDS cases vs. ED ICU controls; striped triangle indicates a significant CpG for less than 58 year old ARDS vs. less than 58 year old ICU controls; light gray triangles indicate significant CpG sites for ARDS cases vs. ICU controls with no effect modification by age or ethnicity; arrows indicate higher modification (up arrow) or lower modification (down arrow) for ARDS cases vs. ICU controls; numbers correlate to CpG probe ID numbers in the 450k array; white stars indicate significant mQTL detected for two target CpG sites. (B). Significantly modified CpG sites for ARDS cases vs. ICU controls controlling for age, sex and ethnicity for which no significant M-value interaction terms were identified: cg03892735, cg23344121, located in intron (4,5) and exon 18, respectively. Effect plots of the probability that a case has ARDS (y-axis) according to M-value (x-axis). Open circles indicate observed case status (1 = ARDS, 0 = ICU control), line the predicted probability across M-value, shaded area the 95% confidence limits for the predicted probabilities. (C). Significantly modified CpG sites for ARDS cases vs. ICU controls among ED, controlling for age and sex: cg01894985, cg16212219, located in intron (2,3) and intron (31,32) respectively. Effect plots of the probability that a case has ARDS (y-axis) according to M-value (x-axis). Plus signs indicate observed ED case status and open circles observed AD case status (1 = ARDS, 0 = ICU control), predicted probability across M-value is indicated by a dashed line for ED patients and a solid line for AD patients, and the shaded areas (light gray for ED, dark gray for AD) indicate the 95% confidence limits for the predicted probabilities. (D). Significantly modified CpG site for ARDS cases vs. ICU controls among those less than 58 years of age, controlling for sex and ethnicity: cg19611163, located in intron (19,20). Effect plot of the probability that a case has ARDS (y-axis) according to M-value (x-axis). Plus signs indicate observed <58 year old patient case status, open circles indicate observed ≥58 year old patient case status (1 = ARDS, 0 = ICU control), predicted probability across M-value is indicated by a dashed line for <58 year old patients and a solid line for ≥58 year old patients, and the shaded areas (dark gray for <58 year, light gray for ≥ 58 year) indicate the 95% confidence limits for the predicted probabilities.

Figure 2. mQTL mapping for an ARDS-associated CpG in MYLK

(A). Box and whisker plots show the beta-values of cg16212219 and genotypes of one of its local mQTL, rs820359. The first and third quartiles are indicated at box ends, median by the horizontal line, the minimum and maximum by the end of the whiskers, and outliers by the dots. (B). The physical positions (hg19) of cg16212219 and its eight detected local mQTL on chromosome 3 are shown.