Genetic Variation is the Major Determinant of Individual Differences in Leukocyte Endothelial Adhesion

Abstract

Purpose: Purpose: Diabetic retinopathy (DR) is a leading cause of irreversible blindness worldwide. Enhanced adhesion of leukocytes to endothelium is implicated in vascular dysfunction in DR. LEA is an important therapeutic target and a potential quantitative biomarker for DR. Understanding the underlying genetic basis of LEA could have a significant biomedical impact as it can improve the understanding of molecular mechanisms underlying LEA, enable therapeutic targeting, specificity for treating DR. The goal of our study was to determine the genetic contribution to LEA.

Methods: Methods: Heritability was determined using Twin study. Lymphobalstoid cell lines (LCL’s) derived from leukocytes of 23 monozygotic twin and 23 sibling pairs were used in study. LEA was assessed through a novel cell-based high-throughput assay. Calcein AM labeled LCL’s were added at a density of 50,000 cells/well on confluent monolayer of vascular endothelial cells in 96 well plates and incubated for 30 min at 37 deg C. All plates included T21 and Ramos LCL as positive and negative controls respectively. Nonadherant cells were removed by an automated wash protocol. Relative fluorescence units (RFU) of adherent cells were assessed by Acumen. LEA was calculated as (%) of input RFU. Intra-pair similarity was determined for monozygotic twins and siblings using interclass correlation coefficient (ICC) and heritability estimated using twinan90 function in R package. The significance of heritability determined by randomizing the samples with 10000 simulations to generate a null distribution. Linear Pearson’s correlation coefficient (r) was used to determine the contributions of potential confounders.

Results: RESULTS: The assay was sensitive to detect inter individual differences in LEA. The assay had a CV of 8.4, Z’-factor of 0.55, SNR of 12.5. Potential confounders, including subject age (p-value = 0.71), gender (p-value = 0.23), transformation time (p-value = 0.49) and LCL cell growth rate (p-value = 0.94) had no correlation. A higher intra -pair correlation was found in adhesion between the twins (0.60) than siblings (0.25). The heritability of LEA was estimated to be 69.66% with an empirical p value of < 0.0001

Conclusions: CONCLUSIONS: Our study is the first to demonstrate that there is a heritable component to leukocyte endothelial adhesion and genetic predisposition significantly contributes to LEA.

Keywords: 499 diabetic retinopathy • 459 clinical (human) or epidemiologic studies: biostatistics/epidemiology methodology • 539 genetics