Screening of Factor V Leiden mutation and activated protein C resistance in Sudanese children with sickle cell disease and stroke: A descriptive cross-sectional study

Abstract

Thrombotic complications, particularly stroke, are significant causes of morbidity in children with sickle cell disease (SCD). While the Factor V Leiden (FVL) G1691A mutation is a recognized genetic risk factor for thrombophilia in Caucasian and Middle Eastern populations, its role in African pediatric SCD patients remains unclear. To determine the prevalence of the FVL G1691A mutation and its association with activated protein C resistance in Sudanese pediatric SCD patients with a history of cerebrovascular accidents. This descriptive cross-sectional study was conducted from December 2015 to May 2016 at Jaafar Ibn Auf Specialized Hospital for Children in Khartoum, Sudan. One hundred Sudanese children (<18 years) with homozygous SCD and documented cerebrovascular accidents were recruited. Genotyping for FVL was performed using allele-specific polymerase chain reaction (PCR), and activated protein C resistance was assessed using a clotting-based assay. Demographic, clinical, and familial data, including tribal affiliation and parental consanguinity, were collected. Data were analyzed using SPSS V29. Among the 100 participants (mean age 6.1 ± 3.3 years; 57% male), 2% were heterozygous for the FVL mutation (GA genotype), and none were homozygous mutants. Both heterozygous individuals demonstrated resistance to activated protein C. Parental consanguinity was reported in 79% of cases. No other FVL-associated thrombophilia was detected in the remaining 98 patients, all of whom had the wild-type genotype. The FVL mutation is rare among Sudanese pediatric SCD patients with stroke, suggesting it is unlikely to be a significant contributor to thrombosis risk in this population. These findings support the need for broader genetic and clinical investigations to identify more relevant risk factors and inform stroke prevention strategies in African children with SCD.

Key points.

• The Factor V Leiden (FVL) G1691A mutation was identified in only 2% of Sudanese children with sickle cell disease and stroke; all were heterozygous, with no homozygous cases detected.

• All FVL-positive children exhibited activated protein C resistance, indicating functional relevance of the mutation in these rare cases.

• The low prevalence of FVL suggests it is not a major contributor to stroke risk in this population.

• Routine genetic screening for FVL in Sudanese pediatric sickle cell disease patients is not recommended.

• Stroke prevention strategies should prioritize accessible and evidence-based interventions such as transcranial Doppler screening, hydroxyurea therapy, and chronic transfusions.

1. Introduction

Sickle cell disease (SCD) is the most common severe hereditary hemoglobinopathy worldwide, caused by a point mutation in the β-globin gene that produces hemoglobin S (HbS).^[1] The polymerization of deoxygenated HbS triggers erythrocyte sickling, chronic hemolytic anemia, endothelial activation, and vaso-occlusion, resulting in cumulative multiorgan damage.^[2] Every year, worldwide, more than 300,000 children are born with SCD, with over 75% of them in sub-Saharan Africa.^[3] Stroke is one of the earliest and most serious complications in SCD, with incidence rates over 10% without screening.^[4,5] In Sudan, limited access to transcranial Doppler screening and chronic transfusion programs exacerbates cerebrovascular morbidity and mortality among affected children.^[6]

In Sudanese pediatric patients, the most significant cause of stroke is SCD. Other contributing factors in the region include endemic infections, such as tuberculosis, which can cause brain tuberculomas, as well as other congenital or acquired conditions that affect blood flow and clotting.^[6]

Hypercoagulability is intrinsic to SCD, characterized by sustained platelet activation, tissue factor expression, and the release of pro-inflammatory microparticles; nevertheless, the magnitude of thrombosis risk may be modified by co-inherited thrombophilias.^[7] The G1691A mutation in the factor V gene (Factor V Leiden, FVL) is the most prevalent inherited thrombophilia among Caucasians, conferring a 3- to 7-fold higher risk of venous thromboembolism in heterozygotes and up to an 80-fold higher risk in homozygotes.^[8] Mechanistically, FVL produces an arginine-to-glutamine substitution at position 506 that renders activated factor V resistant to cleavage by activated protein C, thereby amplifying thrombin generation.^[9]

Despite extensive research in Europe and the Middle East, the distribution and clinical relevance of FVL in African populations remain poorly characterized. Previous reports from Nigeria and Sudan have reported absent or very low allele frequencies (<2%) among adults with SCD,^[10,11] whereas pediatric data, particularly in children who have already sustained stroke, are virtually non-existent. Given the high baseline stroke burden in Sudanese children with SCD, clarifying whether FVL contributes meaningfully to thrombosis risk has direct implications for targeted genetic counseling, prophylaxis, and resource allocation.

Therefore, we conducted a descriptive cross-sectional study to determine the prevalence of the FVL G1691A mutation and its functional correlate, activated protein C resistance, in Sudanese children with homozygous SCD who had experienced cerebrovascular accidents. By establishing a population-specific genetic profile, this study aims to inform locally relevant strategies for stroke prevention and to refine future research priorities in pediatric SCD thrombophilia.

2. Methods

2.1. Study design and setting

This hospital-based, descriptive cross-sectional study adheres to the STROBE guidelines of observational (cross-sectional) studies. The study was conducted at Jaafar Ibn Auf Specialized Hospital for Children in Khartoum State, Sudan, over 6 months from December 2015 to May 2016. The hospital functions as a major tertiary referral center for pediatric hematological disorders, including SCD, and receives patients from various regions of the country.

2.2. Study population and eligibility criteria

The study population included pediatric patients aged 18 years or younger with a confirmed diagnosis of homozygous sickle cell disease who had experienced at least one documented cerebrovascular accident. Diagnosis was established through hemoglobin electrophoresis and confirmed by clinical history and imaging, where available. A total of 100 patients who fulfilled these criteria were consecutively enrolled during their hospital admission.

Inclusion criteria comprised Sudanese nationality, age of 18 years or below, confirmed diagnosis of homozygous sickle cell disease, and a documented thrombotic event such as stroke. Patients were excluded if they were non-Sudanese, had sickle cell trait (HbAS) or compound heterozygous variants (e.g., HbSC, HbS/β-thalassemia), or had other known causes of thrombosis such as malignancy or nephrotic syndrome. Additionally, patients with incomplete medical records and outpatients were excluded from the analysis.

2.3. Data collection procedures

Clinical and demographic data were collected using a structured data collection checklist that incorporated information retrieved from patients’ medical records and interviews with parents or guardians. Variables collected included patient age, sex, tribal affiliation, and parental consanguinity. Relevant clinical history encompassed documentation of stroke and family history of thrombotic disorders. All data were reviewed for completeness and entered into a secure database.

2.4. Laboratory procedures

2.4.1. Blood sample collection

Venous blood samples (5 mL) were collected under aseptic conditions from each participant. Three milliliters were placed into ethylenediaminetetraacetic acid tubes for DNA extraction, while 1.8 mL were collected in 3.2% sodium citrate tubes for assessment of activated protein C resistance (APCR).

2.4.2. DNA extraction and factor V Leiden genotyping

DNA was extracted using the GF-1 Blood DNA Extraction Kit (Vivantis, Malaysia), following the manufacturer’s protocol. The purity and concentration of extracted DNA were assessed using a GeneQuant spectrophotometer, with A260/A280 ratios ranging from 1.7 to 1.9.

Genotyping for the FVL G1691A mutation was performed using allele-specific polymerase chain reaction (PCR), targeting codon 506 of the factor V gene. PCR products were resolved on a 1.5% agarose gel, stained with ethidium bromide, and visualized under UV light. Samples were classified based on banding patterns: GG (wild-type), GA (heterozygous), or AA (homozygous mutant).

2.4.3. Activated protein C resistance assay

To assess functional consequences of the FVL mutation, APCR was measured using the STA-Staclot APC-R assay on an automated STA coagulation system (Diagnostica Stago, France). Clotting times below 120 seconds were considered indicative of resistance to activated protein C, whereas clotting times equal to or above this threshold were interpreted as normal.

2.5. Statistical analysis

Data analysis was performed using IBM SPSS version 29 (Armonk). Descriptive statistics were used to summarize the data. Continuous variables such as age were expressed as means with standard deviations, while categorical variables were presented as frequencies and percentages. Genotype distributions were evaluated for deviation from Hardy–Weinberg equilibrium. Given the descriptive nature of the study and the small number of FVL-positive cases, no inferential statistical tests were conducted. There was no missing data.

2.6. Ethical considerations

The study protocol was approved by the Department of Higher Education at Sudan International University, the Khartoum State Ministry of Health, and the administration of Jaafar Ibn Auf Specialized Hospital for Children. Written informed consent was obtained from parents or legal guardians of all participating children in accordance with the principles of the Declaration of Helsinki.

3. Results

3.1. Participant flow and baseline characteristics

All 100 children with SCD and a documented cerebrovascular accident met the inclusion criteria and were enrolled; no cases were excluded, and no variables had missing data. The mean age was 6.1 ± 3.3 years (range, 0.5–17 years), and 57% were male. Consanguineous marriage of parents was common (79%) (Table 1). Thirteen tribal groups were represented, the largest being Misseria (28%), Marareet (22%), Rizegat (12%), and Bargo (8%) (Fig. 1).

Table 1.

Study variables by Factor V Leiden (FVL) mutation status (N = 100).

Variable	Category	FVL-negative n (%)	FVL-positive n (%)	Total n
Sex	Female	42 (97.7)	1 (2.3)	43
	Male	56 (98.2)	1 (1.8)	57
Age group (yr)	0–4	30 (93.8)	2 (6.2)	32
	5–9	52 (100.0)	0 (0.0)	52
	10–17	16 (100.0)	0 (0.0)	16
Tribe	Misseria	27 (96.4)	1 (3.6)	28
	Marareet	21 (95.5)	1 (4.5)	22
	Rizegat	12 (100.0)	0 (0.0)	12
	Bargo	8 (100.0)	0 (0.0)	8
	Other*	30 (100.0)	0 (0.0)	30
Parental consanguinity	Yes	77 (97.5)	2 (2.5)	79
	No	21 (100.0)	0 (0.0)	21
APCR test	Negative	98 (100.0)	0 (0.0)	98
	Positive	0 (0.0)	2 (100.0)	2

APCR = activated protein C resistance.

Figure 1.

Tribal distribution of study participants and their FVL status. FVL = Factor V Leiden.

3.2. Prevalence of factor V Leiden mutation and APCR

Genotyping showed that 98 children (98 %) carried the wild-type genotype (GG) and 2 (2%) were heterozygous GA; no homozygous mutants (AA) were detected. Both GA carriers were resistant to activated protein C (APCR-positive), whereas all GG children had normal APCR results (Table 1).

3.3. Cross-tabulation of study variables by FVL status

Table 1 details the distribution of socio-demographic and laboratory variables stratified by FVL status. The 2 mutation-positive cases comprised one male and one female aged < 5 years, with one from the Misseria and one from the Marareet tribe, each born to consanguineous parents, and both APCR-positive.

4. Discussion

4.1. Key results in context

In this hospital-based cross-sectional study involving 100 Sudanese children with homozygous SCD and documented stroke, only 2 participants (2%) were found to be heterozygous for the FVL G1691A mutation, and both exhibited activated protein C resistance. No participants carried the mutation in a homozygous state. The notably low frequency of FVL observed here supports the hypothesis that this mutation is an uncommon thrombophilic risk factor in sub-Saharan African populations and likely does not significantly contribute to the high stroke prevalence seen among Sudanese children with SCD.

4.2. Comparison with published literature

Our findings are consistent with previous studies from sub-Saharan Africa, which have reported low or absent frequencies of the FVL mutation among patients with SCD. Specifically, no carriers were identified among 120 adult Sudanese SCD patients in an earlier study.^[10] Similar findings were observed in Nigeria, Brazil, India, and the United States among African Americans, where the prevalence of this mutation did not exceed 1%, confirming a limited role for FVL in stroke risk among individuals of African ancestry.^[11–14]

Conversely, research conducted in North African and Middle Eastern populations has consistently reported significantly higher frequencies of FVL among SCD cohorts, including prevalence rates of 13% in Saudi Arabia, 14% in Iran, 14% in Tunisia, in Egypt,^[15] and among Palestinians.^[15–19] Furthermore, a recent review highlights that FVL was present in a large proportion of Caucasian populations but was nearly absent in non-Caucasian groups.^[20] These geographic variations underscore the importance of regional genetic profiles in determining thrombophilia risk, indicating that stroke prevention strategies developed for Middle Eastern populations may not be suitable or cost-effective in sub-Saharan African settings.

In our study, the 2 FVL-positive children were both younger than 5 years, born to consanguineous parents, and belonged to the Misseria and Marareet tribes. However, given the rarity of the mutation in our cohort, we were unable to assess specific demographic or tribal risk patterns meaningfully.

4.3. Biological plausibility and alternative pathways

FVL involves a single amino acid substitution (Arg506Gln), resulting in resistance to activated protein C-mediated inactivation and a consequent increase in thrombin generation by approximately 8 to 10 times.^[9] Although the biological mechanism is well-established, the very low allele frequency in West and Central African populations limits its overall impact on stroke risk in these regions. Recent studies have examined alternative thrombophilic mechanisms, including polygenic factors such as prothrombin G20210A mutations, MTHFR C677T polymorphisms, deficiencies in protein C and protein S, as well as variants in SERPINC1.^[21–23] Additionally, SCD-specific processes, such as chronic hemolysis, inflammation, splenic dysfunction, and iron overload, may collectively drive thrombotic risk in affected patients.^[22,24]

4.4. Strengths and limitations

This study provides important insights into the genetic profile of Sudanese children with SCD and stroke, using standardized genotyping and APCR testing to evaluate thrombophilia. The homogeneity of the sample, defined by confirmed diagnoses of both SCD and cerebrovascular events, strengthens the internal validity of the findings.

However, several limitations must be noted. The absence of a control group precludes comparative analysis of FVL prevalence between SCD patients with and without stroke. The sample size, while sufficient for descriptive analysis, may have been underpowered to detect rare genotypes or associations. Moreover, the study did not evaluate other prothrombotic mutations, recurrent thrombotic events, or environmental exposures that could contribute to stroke risk. Finally, as a single-center hospital-based study, its findings may not be fully generalizable to community or rural populations across Sudan.

4.5. Public-health relevance and future research

Given the very low prevalence of the FVL mutation observed in our cohort, routine genetic screening for FVL is not recommended in Sudanese pediatric SCD populations. Instead, resources should prioritize interventions with proven efficacy, including transcranial Doppler ultrasound screening, chronic transfusion programs, hydroxyurea therapy, and emerging gene-editing approaches. Future research should aim to establish multicenter, adequately powered cohorts that include stroke-negative controls, employ comprehensive genetic testing, such as whole-genome or targeted thrombophilia panels, and integrate longitudinal clinical outcome assessments. Additionally, identifying health-system barriers to implementing evidence-based stroke-prevention strategies in resource-limited settings will be essential for improving outcomes among these high-risk populations.

4.6. Generalizability

The results are likely generalizable to similar hospital-based pediatric SCD populations in Sudan and other sub-Saharan countries with comparable genetic and environmental backgrounds. However, generalizability to other ethnic groups or geographic settings, especially those with higher FVL prevalence, should be approached with caution. Future research should employ larger, multicenter cohorts and include comprehensive thrombophilia panels, control groups, and longitudinal outcome data to identify risk factors and guide targeted prevention strategies more accurately.

5. Conclusion

This study demonstrated a low prevalence (2%) of the FVL (G1691A) mutation among Sudanese pediatric patients with SCD who experienced stroke, with no cases of homozygosity observed. Although both heterozygous carriers exhibited functional resistance to activated protein C, the overall rarity of the mutation suggests that it does not significantly contribute to the thrombotic risk profile of this high-risk population.

Given the high burden of cerebrovascular complications in Sudanese children with SCD, further investigation into alternative genetic, clinical, and environmental risk factors is warranted. Broader thrombophilia screening, along with improved access to early stroke detection and prevention strategies, may offer more effective approaches to reducing thrombotic morbidity in this vulnerable group. Future research should include multicenter studies with larger sample sizes and control groups to guide evidence-based, context-specific stroke prevention policies in sub-Saharan Africa.

Acknowledgments

The authors would like to thank the staff and laboratory technicians at Jaafar Ibn Auf Specialized Hospital for Children for their cooperation and support throughout the study.

Author contributions

Conceptualization: Ashgan A. Ahmed, Sahar Siddeeg.

Data curation: Ashgan A. Ahmed, Sahar Siddeeg, Sara Alimam, Eltayeb AbdAlla.

Formal analysis: Duaa Elhag, Eltayeb AbdAlla.

Methodology: Ashgan A. Ahmed, Rehab Nasreldeen, Duaa Elhag, Eltayeb AbdAlla.

Project administration: Ashgan A. Ahmed.

Resources: Ashgan A. Ahmed.

Software: Eltayeb AbdAlla.

Supervision: Ashgan A. Ahmed.

Visualization: Eltayeb AbdAlla.

Writing – original draft: Ashgan A. Ahmed, Reem Eltayeb, Ghaida Alsaif, Sahar Siddeeg, Rehab Nasreldeen, Duaa Elhag, Sara Alimam, Eltayeb AbdAlla.

Writing – review & editing: Ashgan A. Ahmed, Reem Eltayeb, Ghaida Alsaif, Sahar Siddeeg, Rehab Nasreldeen, Eltayeb AbdAlla.