Targeted genetic analysis unveils novel associations between ACE I/D and APO T158C polymorphisms with D-dimer levels in severe COVID-19 patients with pulmonary embolism

Only a percentage of COVID-19 patients develop thrombotic complications. We hypothesized that genetic profiles may explain part of the inter-individual differences. Our goal was to evaluate the genotypic distribution of targeted DNA polymorphisms in COVID-19 patients complicated (PE+) or not (PE−) by pulmonary embolism. We designed a retrospective observational study enrolling N = 94 consecutive patients suffering severe COVID-19 with pulmonary embolism (PE+, N = 47) or not (PE−, N = 47) during hospitalization. A panel of N = 13 prothrombotic DNA polymorphisms (FV R506Q and H1299R, FII G20210A, MTHFR C677T and A1298C, CBS 844ins68, PAI-1 4G/5G, GPIIIa HPA-1 a/b, ACE I/D, AGT T9543C, ATR-1 A1166C, FGB − 455G > A, FXIII103G > T) and N = 2 lipid metabolism-related DNA polymorphisms (APOE T 112C and T158C) were investigated using Reverse Dot Blot technique. Then, we investigated possible associations between genotypic subclasses and demographic, clinical, and laboratory parameters including age, obesity, smoking, pro-inflammatory cytokines, drug therapy, and biomarkers of thrombotic risk such as D-dimer (DD). We found that 58.7% of PE+ had homozygous mutant D/D genotype at ACE I/D locus vs. PE− (40.4%) and 87% of PE+ had homozygous mutant C/C genotype at APOE T158C locus vs. PE− (68.1%). In PE+ group, DD levels were significantly higher in D/D and I/D genotypes at ACE I/D locus (P = 0.00066 and P = 0.00023, respectively) and in C/C and T/C genotypes at APOE T158C locus (P = 1.6e−06 and P = 0.0012, respectively) than PE− group. For the first time, we showed significant associations between higher DD levels and ACE I/D and APOE T158C polymorphisms in PE+ vs. PE− patients suggesting potential useful biomarkers of poor clinical outcome. Supplementary Information The online version contains supplementary material available at 10.1007/s11239-022-02728-z.


Introduction
Complex genetic and epigenetic molecular circuits can underlie the strong propensity to thrombotic events in patients with coronavirus disease 2019 (COVID- 19), especially in those patients who are affected by the severe form of disease; however, the molecular components of the individual risk predisposing to thrombosis have not been fully established yet [1][2][3][4][5]. COVID-19 has a wide spectrum of possible clinical features ranging from asymptomatic disease to severe interstitial pneumoniae and acute respiratory distress syndrome (ARDS) [2,3]. Retrospective studies showed that increased levels of D-dimer (DD), procalcitonin, and C Reactive Protein (CRP) may be prognostic biomarkers useful to predict the onset of thrombotic complications [6]. Moreover, the incidence of thrombotic events (about 31%) [7] was associated with increased morbidity and mortality in critically ill patients [8]. At molecular level, the thrombotic manifestations of severe COVID-19 patients seem to be consequences of direct SARS-CoV-2 cytotoxic effects, endothelialitis, dysregulated immune response, platelet aggregation, and complement and coagulation cascade activation [1,8]. But none of these molecular pathways is sufficient to explain why some COVID-19 patients develop thrombotic manifestations and other do not.
Genetic risk factors related to a pre-existing prothrombotic state seem to play a key role in determining the individual susceptibility both to SARS-CoV-2 infection and clinical course of disease [9,10]. We conducted a retrospective, single-centre, observational study to evaluate the genotypic distribution of N = 15 polymorphisms involved in thrombotic risk and lipid metabolism in COVID-19 patients who developed acute pulmonary embolism (PE+) as compared to those who did not develop acute pulmonary embolism (PE−).

Ethics statement
This study was approved by the local ethics committee (AOC-0017432-2020). Each study participant provided written informed consent prior to study enrolment, collection of samples and subsequent genetic analysis. This study was conducted according to the principles and guidelines expressed in the Declaration of Helsinki.

Statistical analysis
A statistical post-hoc power analysis was performed to compute power values for given sample sizes, effect size, and alpha level. By selecting one-tailed Wilcoxon-Mann-Whitney test with 2 groups (PE+ vs. PE−), with a sample size of 47 for both groups, a medium effect size (f = 0.5) and an alpha level of 0.05, the power resulted around 0.76. Thus, our sample size was more than adequate for the main objective of this study and for possible subgroup analysis. G*Power software version 3.1 was used to compute the power analysis.
Statistical analysis was performed using R Core Team (version 4.0. 0). Continuous variables were expressed as mean and standard deviation (SD). Data distribution was tested for normality through the Shapiro-Wilk test. Unpaired Student's t-test or Wilcoxon rank-sum test, as required, were performed for comparison between two groups. Categorical variables were expressed as a percentage and were compared using the Chi-Square test or the Fisher's exact test. A P < 0.05 was considered significant. Bonferroni's correction

Clinical characteristics of study population
Clinical characteristics of COVID-19 patients are summarized in Table 2 (Table 3 and Supplementary Fig. 1). Otherwise,

Genotyping
We evaluated a panel of 15 polymorphisms already known to be associated with increased thrombotic risk and lipid metabolism dysfunction (

Associations between ACE I/D polymorphism and clinical parameters
Based on our interest on ACE I/D polymorphism and its documented association with the severity of COVID-19 in Europe [11][12][13][14][15], we investigated potential associations between genotypic subclasses and clinical and laboratory parameters. As showed in Supplementary Fig. 2 levels (ρ = 1, P < < 0.01; ρ = 0.9, P = 0.04) as well as between IL6 and DD levels (ρ = 0.9, P = 0.04) for I/I genotype.

Association of DD levels with ACE I/D and APOE T158C polymorphisms
We investigated the distribution of DD levels according to the ACE I/D and APOE T158C (Fig. 1, on the left panel) genotypic subclasses. First, in PE+ patients DD levels were significantly higher in carriers of D/D and I/D genotypes at ACE I/D locus (respectively, P = 0.00066 and P = 0.00023) (Fig. 1, on the right panel) and in carriers of C/C and T/C genotypes at APOE T158C locus (respectively P = 1.6e−06 and P = 0.0012) as compared to PE− patients. No significant difference was observed in DD levels between the three genotypic subclasses at ACE I/D and APOE T158C loci both in PE+ and PE− patients ( Supplementary Fig. 4).

Discussion
Our major findings were as follow: (1)  Previous data showed a higher prevalence of ACE D/D genotype in severe COVID-19 patients as compared to those with less severe disease [12,13,16]. At genetic level, the ACE I/D polymorphism is distinguished by either an insertion (I) or deletion (D) of 287 base pairs (Alu repeat segment) in the intron 16. In the general population, the carriers of the mutant D allele had higher ACE protein plasma and tissue levels (ACE levels in D/D carriers were approximately twice that in I/I carriers) as well as elevated levels of the vasopressor angiotensin II and reduced half-life of the vasodilator bradykinin as compared to carriers of the I allele [17,18]. Beyond the systemic hypertension, ACE D/D genotype was significantly associated to cardiometabolic diseases, such as obesity and T2D, which are known risk factors for COVID-19 [19]. Therefore, it is plausible that ACE I/D polymorphism may play a key role in COVID-19 patients who are susceptible to develop severe lung injury or ARDS. Also, the racial difference of ACE I/D polymorphism is well established. In European populations (Italy, Spain, and France), there is a higher frequency of D allele up to 82% to 87% [20] than Eastern Asian populations (Chinese, Korean, Taiwanese, and Japanese) which have a higher frequency of ACE I allele (33% to 51%) [21]. Globally, it seems that the racial variance of ACE I/D genotype coincides with the differences of outcome; in fact, populations with higher D allele frequency (e.g., Italian) experienced higher fatality (https:// www. world omete rs. info/ coron aviru s/# count ries). ACE gene shows potent vasoconstrictive effects, attenuation of fibrinolysis, and platelet activation and aggregation, and ACE I/D polymorphism represents a susceptibility risk biomarker for thrombosis. In particular, ACE D/D genotype was associated to thromboembolic manifestations in patients affected by other diseases with no pre-existing risk factors and traditional thrombophilia-related polymorphisms [22], increased venous thromboembolism risk in patients with a thrombogenic condition [23,24], and hypercoagulability and endothelial damage in hypertensive patients [25]. At global level, our data corroborated previous evidence about the increased DD plasma levels in severe COVID-19 patients with thrombotic complications [6,26]. Screening the three genotypic subclasses, we found for the first time a statistically significant association between increased plasma DD levels and carriers both of D/D and I/D genotypes in PE+ vs. PE− patients. Despite there is need to validate molecular mechanisms underlying this association, we hypothesized that the unbalance of ACE/ACE2 levels characterizing the D/D and I/D genotypes might induce apoptotic processes which target the endothelial cells of the vascular structure leading to coagulopathy and thus increased DD levels in COVID-19 patients.
The APOE locus has been associated with increased vulnerability to severe COVID-19 and mortality, especially for the APOE4 homozygous genotype (ε4/ε4) [27], which is the strongest genetic risk factor for sporadic Alzheimer's disease. The APOE ε4 allele is characterized by the presence of a C at the APOE T112C locus and a C at the APOE T158C [28]. Our analysis showed that PE+ patients had a higher percentage of homozygous mutant C/C genotype at APOE T158C locus vs. PE− patients, even if we did not find statistically significant difference for the APOE T 112C locus. Also, DD levels were significantly higher in carriers of C/C and T/C genotypes at APOE T158C locus.
During pandemic, inherited thrombophilia has been associated with severe COVID-19 [29][30][31]. However, there is not a rigorous consensus regarding the genes which are truly involved in inherited thrombophilia. For example, the MTHFR polymorphism is considered as "benign" without a validated link to thrombosis thus questioning its inclusion in the current thrombophilia laboratory test panels [32]. In our study, we did not find statistical differences for MTHFR C677T polymorphism in PE+ vs. PE−, as well as for the other polymorphisms included in the panel. Considering the MTHFR C677T polymorphism, the prevalence of the homozygous TT genotype was 25.5% and 19.6% in PE− vs. PE+ patients (Table 1). These distributions are quite similar to known global levels of the MTHFR C677T polymorphism, for which the prevalence of the homozygous TT genotype was about 26% and 20% in Campania and Sicily (South Italy), respectively [33].
Nevertheless, our small sample size did not allow a secure finding but limited our observation to a hypothesis generating study.
The unique exception was the CBS 844ins68 polymorphism. Surprisingly, D/D homozygous wild-type genotype was most represented in PE+ (95.7%) vs. PE− (80.9%) patients. This might be due to the relatively small number of patients recruited in the study. As in oncology and cardiovascular diseases, the study of genetic regulation should be coupled with the investigation of epigenetic regulation of prothrombotic genes in severe COVID-19 patients and their possible involvement in immune reactivity [5,[34][35][36][37][38][39][40][41][42].
Our study has some limitations. First, this is a retrospective study. Second, we focused on COVID-19 patients with and without PE, with no control group (non-COVID-19 patients and/or healthy subjects) limiting the attribution of results to a specific association with COVID-19. Second, PE+ patients showed a higher prevalence of homozygous mutant ACE D/D genotype and APOE C/C genotype vs. PE− patients; but homozygous mutant ACE D/D genotype and APOE C/C genotype were also found in 40.4% and 68.1% of PE− patients (Table 1). This suggests a quite high absolute prevalence of the homozygous mutant polymorphisms in these genes. This somehow limits the predictivity of the mutant genotype for PE+.

Conclusions
Our data documented a differential distribution of homozygous mutant ACE D/D and APOE C/C genotypes between PE+ and PE− patients. Higher DD plasma levels significantly associated with carriers of D/D and I/D genotypes at ACE I/D locus as well as with carriers of C/C and T/C genotypes at APOE T158C locus in PE+ vs. PE− patients. These associations and the potential pathogenic role of these polymorphisms needs to be further evaluated in larger prospective studies. Funding Open access funding provided by Università degli Studi della Campania Luigi Vanvitelli within the CRUI-CARE Agreement. There are no funding supporting this study.

Data availability
The data that support the findings of this study are available from the corresponding author, [GB], upon reasonable request.

Conflict of interest The Authors declare no conflict of interest.
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