APOL1 genotype associated risk for preeclampsia in African populations: Rationale and protocol design for studies in women of African ancestry in resource limited settings

Background Women of African ancestry are highly predisposed to preeclampsia which continues to be a major cause of maternal death in Africa. Common variants in the APOL1 gene are potent risk factor for a spectrum of kidney disease. Recent studies have shown that APOL1 risk variants contribute to the risk of preeclampsia. The aim of the study is to understand the contribution of APOL1 risk variants to the development of preeclampsia in pregnant women in Ghana. Methods The study is a case-control design which started recruitment in 2019 at the Korle Bu Teaching Hospital in Ghana. The study will recruit pregnant women with a target recruitment of 700 cases of preeclampsia and 700 normotensives. Clinical and demographic data of mother- baby dyad, with biospecimens including cord blood and placenta will be collected to assess clinical, biochemical and genetic markers of preeclampsia. The study protocol was approved by Korle Bu Teaching Hospital Institutional Review Board (Reference number: KBTH-IRB/000108/2018) on October 11, 2018. Preliminary results As of December 2021, a total of 773 mother-baby pairs had been recruited and majority of them had complete entry of data for analysis. The participants are made up of 384 preeclampsia cases and 389 normotensive mother-baby dyad. The mean age of participants is 30.69 ± 0.32 years for cases and 29.95 ± 0.32 for controls. Majority (85%) of the participants are between 20-30years. At booking, majority of cases had normal blood pressure compared to the time of diagnosis where 85% had a systolic BP greater than 140mmHg and a corresponding 82% had diastolic pressure greater than 90mmHg. Conclusion Our study will ultimately provide clinical, biochemical and genotypic data for risk stratification of preeclampsia and careful monitoring during pregnancy to improve clinical management and outcomes.

The work describes a case-control study design with a target number of 700 cases of preeclampsia and 700 cases of normotensive pregnant women, prospectively enrolled. Recruitment started in May 2019 involving women who are diagnosed with preeclampsia for the very first time as cases. The target population are African women; however, the only recruiting facility is KBTH in Ghana. The work will capture a significant number of Ghanaian women with longitudinal follow-up over 3 years. All pregnant women will be eligible for recruitment. Eligible women will be screened and enrolled into the study after provision of informed consent. The study protocol was approved by the Korle Bu Teaching Hospital Scientific and Technical committee/ Institutional Review Board (reference number: KBTH-STC/IRB/000108/2018). Written informed consent was obtained from participants after explaining to them about the study with the option of discontinuing without any consequences. The inclusion and exclusion criteria are shown in Table 1. Participants involved in the study went through a comprehensive clinical assessment, an overview of which is shown in Fig 1. This assessment also included each baby that was born. The recruitment team which is made up of nurses, doctors and medical laboratory scientists underwent training and familiarization with the goals of the project at the maternity block of Korle Bu Teaching Hospital (KBTH), which is a university affiliated teaching hospital. The team was then trained on how to obtain informed consent, and how to use the Redcap app to collect data. In addition, the team was taken through a practical session on how to take cord blood and placenta sample, how to snap freeze placenta sample and cord blood and how to package maternal samples for collection and processing by the laboratory technician. To ensure efficient patient recruitment and improve communication among team members, a WhatsApp group was created for the team members to alert members of potential patients for recruitment and to report issues that needed urgent attention. Our data collection instrument was transferred onto the redcap app which was initially implemented on three android tablets. After an initial pilot, we noticed that it was easier on phone so the app was installed on the phone. Each member of the recruitment team was given a unique identifier available for use from an earlier project that had been completed. Patients were assessed as potential candidates for recruitment based on their medical history. The clinical team talked to the patient in the language the patients understood and then sought consent. Once consent was given, patients were recruited into the study, given a unique code and a comprehensive questionnaire administered. Clinical information was taken from patients’ folders and then study visits scheduled based on patient’s appointment at the facility. Since study participants attended antenatal clinics, study related data collection was scheduled to coincide with the hospital visit so as not to inconvenience them. The schedules of all antenatal visits were maintained and the estimated time of delivery kept. On the day of delivery, samples were collected and newborn baby assessed. The target participants for this study include a minimum of 1400 participants of age-matched cases of patients with preeclampsia and controls of normotensive patients. The sample size has enough statistical power to allow a robust interpretation of the variables that will be collected as part of the cohort and clinical outcomes which will serve as our phenotypes. The data collected is collated and entered into Redcap by a team of clinicians, research scientists, laboratory technicians and data entry clerks. To maintain data validity and quality control, regular meetings were held with the study coordinator and recruitment team to review enrollment, data entry, missing information and specimen collection. The data are stored in a database that provides detailed recording of clinical, demographic and phenotyping characteristics of the cohort. Access to Redcap is limited to key personnel of the research team that ensures data quality and reconciliation. During the course of the study, we established an accompanying biorepository which will be available for future study. Samples collected from pregnant mothers included whole blood and urine on the day of recruitment. All samples collected are barcoded for both mother and child and stored at -80 degrees Celsius. At delivery, placenta and cord blood were collected. Placental samples were collected from a standardized location approximately 2cm beside the umbilical cord insertion, from the middle layer of placenta midway between maternal and fetal surfaces. The samples were cut into 1cm cubes and snap frozen in liquid nitrogen. Cord blood was snap frozen within 10mins of collection and then stored at -80 degree Celsius. Blood samples were processed by MDS laboratory. Laboratory tests were conducted according to local laboratory protocols. All biochemical tests were undertaken with automated analysers at the research lab of MDS Lancet laboratories, Ghana (https://www.cerbalancetafrica.com.gh/). All patients recruited into the study underwent an initial laboratory testing to obtain a baseline hematological and kidney function tests. For the mothers, hemoglobin, white blood cell and platelet counts, liver function tests, serum creatinine and urine albumin creatinine ratio were conducted. Several biomarkers have been used to diagnose preeclampsia and serve as valuable indicators. These include (i) renal impairment markers (serum creatinine, urine albumin creatinine ratio) (ii) liver dysfunction markers (AST, ALT) (iii) hypertension markers, and (iv) reduced platelets. These markers were used for phenotyping of preeclampsia and interpreted through the genetic model that will be generated. Random duplicate samples will be taken for validation of laboratory measurements periodically. We plan to measure biomarkers for preeclampsia (Table 2), conduct placental histology and also follow up both babies and their mothers to determine the incidence of chronic kidney disease and other non-communicable diseases. Preeclampsia is not an easy condition to diagnose and thus physicians typically rely on several symptoms to guide diagnosis (Fig 2). VEGF: Vascular Endothelial Growth Factor; PIGF: Placenta Growth Factor; s-FLT-1: Soluble fms-like tyrosine kinase; s-Eng: Soluble Endoglin; sCr: Serum Creatinine; UACR: Urine Albumin Creatinine Ratio; CRP: C-reactive protein; IL-6: Interleukin 6; IL-8: Interleukin 8; TNF-alpha: Tumour Necrosis Factor alpha. DNA has been isolated and currently samples are being prepared for APOL1 genotyping. In this study multiple SNP analysis will be performed to further explore the genetic predisposition to preeclampsia in West African women. Selected APOL1 SNPs to be considered are G1: rs73885319, rs60910145, and G2:rs71785313, rs12106505. Several studies on the genetic predisposition to preeclampsia have attempted to use candidate gene approach. These candidate gene approach have often focused on maternal genes as causative genes. Preeclampsia appears to be associated with APOL1 variants G1 and G2 [28, 29]. However APOL1 variants in preeclampsia are complex, as some studies have linked disease with maternal APOL1 [30], while others with fetal APOL1 variants [25, 27]. Genotyping will be undertaken in Ghana using predesigned TaqMan assays in the Pharmacogenomics and Genomic Medicine laboratory (www.pgmg-lab.com), School of Medical Sciences, University of Cape Coast.

• osafo, charlotte
• thomford, nicholas ekow
• coleman, jerry
• carboo, abraham
• guure, chris
• okyere, perditer
• adu, dwomoa
• adanu, richard
• parekh, rulan s.
• burke, david