Genetic Diversity of the ACE2 Gene and Susceptibility to COVID-19 in Morocco Population

Introduction

The COVID-19 pandemic, caused by SARS-CoV2, has been an unprecedented event that has impacted the entire world since its emergence in December 2019 (Atzrodt et al., 2020; Farhane et al., 2021). According to World Health Organization statistics, in December 2024, the virus had yet caused over 776.89 million infections (near to 10%) and over 7.07 million deaths worldwide (over 1%). However, these statistics, while global, vary considerably across regions and populations. In Morocco, located in Northwest Africa, the pandemic has caused over 1,279,450 confirmed cases and 16,310 deaths (in December 2024), with an infection rate of 3.43% and a mortality rate of 1.27%. Notwithstanding, these data show marked variations across different regions of the country.

Infection rates and clinical outcomes differ significantly between individuals and also between countries, raising questions about the factors influencing susceptibility to SARS-CoV-2 (Ovsyannikova et al., 2020). Although elements such as comorbidities, access to care, and public health policies are obvious determinants, genetic factors deserve particular attention (Phuong et al., 2022). Many studies established that some genetic variations could explain the observed differences in immune response and disease severity (Choudhary et al., 2021). Among them, the angiotensin-converting enzyme 2 (ACE2) gene is particularly central in these investigations (Fawzy et al., 2022).

ACE2, a type I transmembrane enzyme belonging to the metallocarboxypeptidase family, shares similarities with ACE (Clarke et al., 2012). The latter, essential in the renin-angiotensin system, is an important therapeutic target in the management of hypertension (Su et al., 2021). ACE2 catalyzes the terminal cleavage of angiotensin I to form angiotensin 1–9 and angiotensin II to produce angiotensin 1–7 (Liu et al., 2020). Its expression is predominant in vascular endothelial cells, renal tubular epithelium, and testicular Leydig cells, as well as in the lung, kidney, gastrointestinal tract, blood vessels, and oral mucosa, which plays a key role in the entry of SARS-CoV-2 into human cells (Beyerstedt et al., 2021). ACE2 is crucial for blood volume regulation, systemic vascular resistance, and cardiovascular homeostasis (Galán and Jiménez-Altayó, 2020). It is implicated in various pathologies, including hypertension, stroke, dyslipidemia, cardiovascular diseases, type 1 or 2 diabetes, and renal disorders, suggesting increased susceptibility to SARS-CoV-2 (El-Arif et al., 2021).

Some recent genetic studies have shown that some ACE2 SNPs could influence the efficiency of the SARS-CoV-2 virus binding to its receptor and the modulation of immune response (Masilela et al., 2021; Ovsyannikova et al., 2020; Suryamohan et al., 2021). In this way, notable differences in allele frequency distribution of ACE2 SNPs across populations and ethnic groups have been linked to a wide diversity of their expression patterns (Cao et al., 2020). For example, the alternative allele “G” of rs4646127 was associated with increased expression in various tissues (Choudhary et al., 2021; Strafella et al., 2020). This allele is distinguished by particularly high frequencies in Asian populations and by lower ones in European and American populations (Cao et al., 2020). Others studies aiming to assess the association of ACE2 rs2285666 with COVID-19, have shown that the alternative “T” allele in the Indian population was associated with lower COVID-19 infection rate and reduced mortality (protective effect) (Srivastava et al., 2020a). Similarly, in the Iranian population, higher frequencies of the “C” reference allele were associated with higher infection rate and mortality (Khalilzadeh et al., 2022).

However, although these variations have been studied in various populations, research on their specific role in the Moroccan population remains limited. Given Morocco’s genetic diversity, shaped by centuries of ethnic mixing (Harich et al., 2026), it is essential to explore how the present genetic variations might influence vulnerability to SARS-CoV-2 infection. In this context, the present study aimed to analyze the distribution of allele frequencies of four SNPs of the ACE2 gene and to examine their genetic association with COVID-19 infection rates in Morocco.

Materials and Methods

SNPs genotyping

ACE2 gene polymorphisms (rs4646127, rs2074192, rs2285666, and rs112171234) were genotyped using specific Human tests TaqMan 40x SNP (ThermoFisher Scientific^©). Real-time PCR reactions were performed using TaqPath™ ProAmp™ Master Mix (ThermoFisher Scientific^©) on the QuantStudio 5 Real-Time PCR System (0.2 mL), following the manufacturer’s standard PCR protocol. The results were then analyzed using QuantStudio™ Design & Analysis software. Each genotyping test TaqMan The predesigned primer contained two distinct primers, forward and reverse, that flanked each SNP, as well as two TaqMan probes labeled with VIC and FAM fluorochromes, differentiated only at the SNP site. One probe was complementary to the wild-type allele, while the other corresponded to the variant allele (Figs. 1 and 2).

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FIG. 1.

Real-time PCR of the three genotypes of (rs4646127 [A > G]): (A) homozygous wild-type AA genotype. (B) homozygous variant GG genotype. (C) heterozygous AG genotype.

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FIG. 2.

Allelic discrimination diagram of the three genotypes (rs4646127 (A > G). Red dots represent homozygous wild-type AA genotype, blue dots homozygous GG variant genotype, green dots heterozygous AG genotype.

Statistical analysis

Deviations from Hardy-Weinberg equilibrium and differences in allele frequencies between population groups were assessed using the chi-square goodness-of-fit test (χ²).

The infection rates (I.R.) was calculated for each region as follows:

I . R . = ( number of cases ) / ( total population ) × 1 00

Data on the number of cases per region were collected from the official website of the Moroccan Ministry of Health (https://www.covidmaroc.ma/Pages/Accueilfr.aspx, December 2024), while information on the population was obtained from the official website of the High Commission for Planning of Morocco (https://www.hcp.ma; December 2024). The relationships between the COVID-19 infection rate and the alternative allele frequency of genetic polymorphisms were assessed using Pearson correlation analysis, and robustness of results was estimated by bootstrapping. All statistical analyses were performed using SPSS version 26 software (SPSS Inc., Chicago, IL, USA), with a significance threshold set at p < 0.05. The QGIS 3.16 software (http://www.qgis.org) was used to generate interpolation maps, allowing the visualization of the geographical distribution of allele frequencies and the infection rate for each region.

Regarding the distribution of alternative allele frequency in global populations, we queried the 1000 Genomes Project (2015), Phase 3, online database to obtain alternative allele frequency distributions for the four SNPs (rs4646127, rs2074192, rs2285666, and rs112171234) in populations from Africa, Europe, South Asia, Southeast Asia, and Latino-America. Concerning the allele frequency in the Egyptian population, it was obtained from the Egypt Biobank website (https://www.egyptian-genome.org/).

Results

Distribution of allele frequencies across regions of Morocco

Our spatial study highlighted a notable variation in the heatmap of alternative allele frequencies for all SNPs among the four Moroccan regions (FIG. 3.). For rs4646127, the frequency of the alternative allele exceeds that of the reference allele, ranging from 58.2% to 74% (FIG. 3A). An increasing gradient is observed, ranging from the Ouarzazate and Rif regions to those of Doukkala and Guelmim. The comparative analysis (Table 1) reveals only one significant difference between Doukkala and Ouarzazate (p = 0.0185).

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FIG. 3.

Heatmap frequency distribution of alternative alleles of the four SNPs in the four samples from the Moroccan population. (A: rs4646127, B: rs2074192, C: rs2285666, D: rs112171234).

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Table 1.

Distribution and Comparison of Allele Frequencies in Moroccan Populations

SNPs	Allele	Doukkala	Guelmim	Ouarzazate	Rif
	N	50	51	49	50
rs4646127 (A>G)	A	0.260	0.314	0.418	0.370
	G	0.740	0.686	0.582	0.630
rs2074192 (C>T)	C	0.630	0.578	0.592	0.580
	T	0.370	0.422	0.408	0.420
rs2285666 (C>T)	C	0.700	0.657	0.878	0.880
	T	0.300	0.343	0.122	0.120
rs112171234 (T>G)	T	0.980	1.000	0.980	1.000
	G	0.020	0.000	0.020	0.000

	Doukkala vs. Guelmim	Doukkala vs. Ourzazate	Doukkala vs. Rif	Guelmim vs. Ourzazate	Guelmim vs. Rif	Ourzazate vs. Rif
SNPs	p-value	p-value	p-value	p-value	p-value	p-value
rs4646127 (A>G)	0.398	0.018*	0.094	0.124	0.399	0.486
rs2074192 (C>T)	0.677	0.060	0.022*	0.021*	0.006**	0.687
rs2285666 (C>T)	0.511	0.002**	0.001**	0.0002***	0.0001***	0.957
rs112171234 (T>G)	0.151	0.983	0.155	0.147	1.00	1.51

The data in bold correspond to the frequencies of the alternative alleles in the different regions.

p value (χ²): Comparison of allele frequencies between the Moroccan populations.

Significance of the difference: ^*p < 0.05; ^**p < 0.01; ^***p < 0.001.

Concerning the rs2074192, the frequencies of the alternative allele vary between 37% and 42.2% (FIG. 3B). Significant color differences are observed between the regions of Doukkala and Rif (p = 0.022) and Guelmim and Ouarzazate (p = 0.021), as well as Guelmim and Rif (p = 0.006) (Table 1).

For rs2285666, the frequency of the alternative allele ranges from 12.2% to 34.3% (FIG. 3C). Significant differences appear between several pairs of regions (Table 1): Doukkala and Ouarzazate (p = 0.0022), Doukkala and Rif (p = 0.0018), Guelmim and Ouarzazate (p = 0.0023), and Guelmim and Rif (p = 0.0001).

Finally, for the rs112171234, the frequency of the alternative allele is extremely low, ranging from 0% to 2% (FIG. 3D). No significant differences were detected between the studied populations, with all comparisons being nonsignificant (Table 1).

Correlation between allele frequencies and infection rate

Figure 4 shows the distribution of infection rates across the 12 regions of Morocco. The highest rate was recorded in the Dakhla-Oued Eddahab region (6.93%), while the lowest rate was found in the Beni Mellal-Meknès region (1.79%). However, the national average infection rate was 3.43%.

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FIG. 4.

Interpolation map of infection rates in the 12 regions of Morocco Data recorded in December 2024, from the Moroccan Ministry of Health and the High Commission for Planning (HCP) of Morocco.

Regarding the regions considered in our study, the infection rates were 6.41% for the Casablanca-Settat region, which includes the Doukkala sample; 3.98% for the Guelmim-Oued Noun region; 2.82% for the Tangier-Tetouan-Al Hoceima region, which includes the Rif sample; and finally, 2.11% for the Daraa-Tafilalet region, which includes the Ouarzazate sample (Fig. 4).

To study the relationship between allele frequency and infection rate in Moroccan populations, we performed linear regression and Pearson correlation coefficient analyses for the four genetic variants and the infection rate. The results of the regression analysis (Table 2) revealed a significant positive correlation between the frequency of the alternative allele (G) of rs4646127 and the infection rate (Pearson’s r = 0.966; p = 0.034). This analysis was performed using four regional observations (n = 4), corresponding to the four sampled Moroccan populations. A higher number of cases of infection is observed when the frequency of this allele increases. The goodness of fit (R²) explains 93.3% of the observed variation, suggesting that the effect of this allele is particularly important for Moroccan populations infections’ rates (Fig. 5). In contrast, the other SNPs studied did not show a significant association between the frequencies of the alternative allele and the infection rate.

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Table 2.

Correlation Between Alternative Allele Frequency and COVID-19 Infection Rate (n = 4 Regions)

	Linear Regression Analysis Parameters			Pearson correlation
SNP	R²	F	p	r	p
rs4646127 (A>G)	0.933	27.8	0.0342	0.966	0.034
rs2074192 (C>T)	0.636	3.50	0.2024	−0.798	0.202
rs2285666 (C>T)	0.586	2.83	0.2346	0.765	0.235
rs112171234 (T>G)	0.070	0.152	0.7342	0.266	0.734

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FIG. 5.

Linear regression line of the infection rate as a function of the G allele frequency. (SNP rs4646127), (n = 4 regions).

Comparison of Allele Frequencies Among Global Populations

Comparative analyses of allele frequencies of the four SNPs analyzed reveal that the Moroccan population formed by the samples of the four regions exhibits significant genetic differences compared with several global populations (Table 3). Alternative allele ranges of variation among the analyzed global populations are the following:

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Table 3.

Comparison of Allele Frequencies Between the Moroccan Population and Different Populations Worldwide

	European	African	East Asian	America	South Asian	Egypt	Morocco
rs4646127 (A>G)
n	14,092	2,898	86	146	98	190	200
A	0.375	0.210	0.020	0.295	0.230	0.437	0.340
G	0.625	0.790	0.980	0.705	0.770	0.563	0.660
p-value (χ2)	0.151	0.0001***	0.0001***	0.206	0.006**	0.006**
rs2074192 (C>T)
n	41,772	5,109	388	594	180	204	200
C	0.553	0.684	0.575	0.549	0.761	0.623	0.595
T	0.447	0.316	0.425	0.451	0.239	0.377	0.405
p-value (χ2)	0.092	0.0001***	0.333	0.108	0.0001**	0.389
rs2285666 (C>T)
n	100,440	6,869	416	600	178	218	200
C	0.796	0.769	0.438	0.805	0.494	0.844	0.778
T	0.204	0.232	0.562	0.195	0.506	0.156	0.223
p-value (χ2)	0.359	0.665	0.0001***	0.235	0.0001***	0.139
rs112171234 (T>G)
n	9,684	2,226	86	134	98	210	200
T	1.000	0.941	1.000	1.000	1.000	0.957	0.990
G	0.000	0.059	0.000	0.000	0.000	0.043	0.010
p-value (χ2)	0.0001***	0.0001***	0.188	0.101	0.160	0.004**

p value (χ²): Comparison of allele frequencies between the Moroccan population and different populations worldwide.

Significance of the difference: ^*p < 0.05; ^**p < 0.01; ^***p < 0.001.

Discussion

Various virus-related, host-related, and environmental factors have been proposed to influence susceptibility to COVID-19 disease and its severity (Sukocheva et al., 2022). Among these factors, genetics plays a key role due to its complex relationship with health and disease (Abdelzaher et al., 2020; Anastassopoulou et al., 2020). According to the literature, genetic architecture explains approximately half of the variations observed in COVID-19 clinical manifestations (Anastassopoulou et al., 2020; Barros et al., 2024; Cao et al., 2020). In this way, some ACE2 gene polymorphisms have been associated with COVID-19 and with also its severity (Cao et al., 2020; Suryamohan et al., 2021). Several studies have highlighted the impact of its genetic variants on severity phenotypes such as respiratory failure, hospitalization, or death (Srivastava et al., 2020b; Vadgama et al., 2022).

This study is the first of its kind conducted in Morocco. It analyzed the allele frequency distribution of four SNPs in the ACE2 gene (rs4646127, rs2074192, rs2285666, and rs112171234) in four anthropologically well-characterized and apparently healthy samples from the Moroccan population, originating from the four cardinal regions of the country. The study also examined the genetic association between these polymorphisms and regional COVID-19 infection rates. The results showed significant spatial variations in the allelic frequencies of the four SNPs between the four Moroccan regions, in the same way that studies using other types of genetic polymorphisms have highlighted a fairly significant genetic heterogeneity between regions of Morocco and also with other Mediterranean and global populations (Harich et al., 2026,2002). The most relevant finding of our study was the significant positive correlation observed between the SNP rs4646127 and the infection rate in all Moroccan regions, and also the absence of positive correlation with the three other SNPs analyzed. This result can be corroborated by the increased ACE2 expression which has been associated with greater susceptibility to SARS-CoV-2 infection and more severe forms of COVID-19 (Zheng, 2022; Pecoraro et al., 2023; Sheikhian et al., 2023; de Sousa et al., 2023). Mendelian randomization analyses based on eQTL and GWAS data have also demonstrated a causal effect of ACE2 expression on the risk of COVID-19 (Zheng, 2022). The ACE2 protein is present in large quantities in various tissues of the body, including lung and colon, with a particularly high concentration in the kidneys, heart, and testicles (Beyerstedt et al., 2021; Bourgonje et al., 2020). The most vulnerable apparently healthy populations to the disease were the ones who exhibit higher levels of pulmonary ACE2 expression (Zheng, 2022 ;Pecoraro et al., 2023 ; Sheikhian et al., 2023 ; Sousa et al., 2023).

ACE2 gene is located on chromosome Xp22 and is characterized by a significant polymorphism (nearly 140 SNPs), some of which were associated with COVID-19 (Alimoradi et al., 2022; Chaoxin et al., 2013; Kotsev et al., 2021). ACE2 gene expression varies from individual to individual, partly due to genetic diversity of SNPs, which could lead to the infection’s rates varying between populations (Alimoradi et al., 2022; Cao et al., 2020). These significant population variation in the association between ACE2 SNPs and public health indicators, such as infection and case fatality rates, could be useful for developing appropriate prevention and treatment strategies.

The alternative allele (G) of the rs4646127 SNP has been associated with increased ACE2 expression, that could influence host responses to SARS-CoV-2 (Zhang et al., 2022), and also a potential role in cumulative susceptibility to SARS-CoV-2 infection. In the Moroccan population, the strong positive correlation observed between the frequency of the G allele of rs4646127 and regional rates of COVID-19 infection (r = 0.966; p = 0.034; n = 4) suggests the existence of a stratified association at the regional level.

Correlation analyses conducted for the other SNPs did not reveal a significant association with the infection rate in the Moroccan population (rs2074192, p = 0.202; rs112171234, p = 0.734; rs2285666, p = 0.235). Nevertheless, the observed differences between populations could be explained by specific genetic factors, distinct environmental influences, and complex interactions between these two dimensions. Thus, genetic diversity plays a major role in modulating host responses to SARS-CoV-2 infection and in the clinical course of the disease (Cao et al., 2020; Niemi et al., 2022; Suryamohan et al., 2021).

This study revealed significant gradients of alternative allele frequencies for several SNPs among the studied regions of Morocco, modulating the global regional genetic differences. SNP rs4646127 shows a clear geographic gradient, with higher frequencies in the Doukkala and Guelmim regions compared with Ouarzazate and Rif, mainly the gradient from East to West, and a statistically significant difference between some of these regions. For rs2074192 SNP, lower variations were recorded, with a slight gradient from West to East. SNP rs2285666, with more variable frequencies, shows marked differences between several pairs of regions, especially between Doukkala and the other regions and also a slight gradient from West to east. In contrast, SNP rs112171234 with null or very low frequencies of the alternative allele did not display any significant difference. These results highlight regional genetic diversity that could reflect specific environmental, historical, or adaptive processes.

Our analysis of the allelic frequencies of ACE2 gene polymorphisms reveals a distinct genetic profile in the Moroccan population, characterized by significant differences from African, North African, and Asian populations while also exhibiting some similarities with European and Latino-American populations. This structure suggests a specific allelic composition of their genetic pool that may modulate ACE2 expression and, consequently, influence susceptibility to SARS-CoV-2 infection. The most marked differences concern the SNPs rs4646127 and rs112171234, with respective divergence rates of 66.66% and 50%, indicating a highly differentiated allelic distribution within the Moroccan population. Interpopulation variations in the ACE2 gene, particularly intronic polymorphisms acting as eQTLs such as rs4646127, have been associated with increased ACE2 expression in some Asian populations (Cao et al., 2020). Such modulation of expression could influence the efficiency of viral entry and contribute to the observed differences in susceptibility, transmission, and severity of COVID-19 between populations.

The overall allelic differentiation rate of 54.17% observed between the Moroccan population and the other worldwide studied populations underscores the existence of a distinct genetic background, which must be taken into account when assessing infectious risk and designing appropriate prevention strategies. A better understanding of the distribution of genetic polymorphisms, such as ACE2 gene SNPs, could thus help explain interethnic and geographic variations in the incidence and severity of COVID-19 and promote the development of genetically based risk stratification approaches (Vadgama et al., 2022).

The limitations of this study include the absence of analyses related to mortality rates due to a lack of data on deaths in each region of Morocco. Furthermore, the small sample size may sometimes not be representative of the total population, which makes it difficult to draw reliable conclusions or to perform stratified analyses (e.g., sex or age). Finally, the correlation and regression analyses are based on only four regional observations (n = 4), which can limit the statistical power of our findings.

In conclusion, our results highlight the importance of genetic factors in susceptibility and vulnerability to COVID-19 in Morocco. In this regard, the rs4646127 polymorphism of the ACE2 gene could be considered with great caution as a candidate genetic marker associated with regional variations in COVID-19 infection rates. However, these results must be confirmed by larger studies integrating individual genetic and clinical data before considering its use as a susceptibility biomarker. In the same way, the results obtained for the non-association on the other SNPs must be confirmed.

Genetic characteristics could ultimately help health care professionals adapt prevention strategies, optimize epidemiological surveillance, and support the implementation of targeted therapeutic approaches for vulnerable populations. Thus, the exploration of other polymorphisms of the ACE2 gene, as well as variants located in other genes involved in viral entry, immune response, or inflammation, appears necessary to broaden the profile of genetic determinants likely to influence susceptibility and vulnerability to COVID-19 in the Moroccan population.

Authors’ Contributions

N.H.: Conceptualization, data curation, analysis, methodology, supervision, validation, and writing—review and editing. H.F.: Design, sampling, methodology, analysis, and writing–original draft. S.K.: Analysis and methodology. M.M., F.-E.A., S.N.A., O.A., and A.H.: Methodology.

Ethics Approval Statement

This study was carried out in accordance with the Declaration of Helsinki. The study was ensured authorization from the Moroccan Ministry of Health, and approval from the Ethics Committee of the Directorate of Epidemiology and Disease Control (reference number 919 DELM/24).

Consent

All participants included in the study gave written informed consent.

Data Availability Statement

Anonymized data are available on reasonable request from the corresponding author at harichanthropo@gmail.com.