Study Organization
The Eyes of Africa study was funded as a Collaborative Network by the National Institutes of Health through the Human Heredity and Health in Africa (H3Africa) program (https://h3africa.org/). There are six collaborating clinical ascertainment centers across Sub Saharan Africa: three in Nigeria, and one each in Ghana, Malawi and South Africa. The collaborating institutions in Nigeria are the College of Medicine of the University of Ibadan, the College of Medicine University of Nigeria Nsukka, Enugu, and the Navy Hospital in Lagos. Other collaborating institutions are the Korle Bu Teaching Hospital at the University of Ghana in Accra, the Eyes of Africa clinic in Lilongwe, Malawi, and the Eye Center East London South Africa. Duke University plays an advisory role and assists with data analysis. The Principal Investigator of the study (AA) is located at the University of Ibadan, where she directs the study through the Administrative Core.
Ethical Considerations
The study protocol was reviewed and approved by the University of Ibadan/ University College Hospital Research Ethics Committee. Written informed consent was obtained from all participants before recruitment into the study.
Participants
Written consent to participate was obtained from all potential participants in their local language prior to screening and before any study-related interventions (including examination for eligibility). Eligible subjects who wished to participate were enrolled. The study used broad consent to enable use of the samples and associated clinical data in as many of the H3Africa-sponsored research projects as possible.
Study Design
There are three components to the Eyes of Africa study: (1) A Genome Wide Association Study (GWAS) to identify new POAG susceptibility genes through collection and analysis of a large, well-phenotyped set of POAG cases and unaffected controls; (2) Sequence analysis of multiplex pedigrees to identify Mendelian forms of early onset glaucoma; and (3) A series of qualitative research studies to understand knowledge and attitudes about glaucoma, to educate and inform community members about glaucoma, and to conduct clinical screening of high-risk families and individuals. The design of the latter component (qualitative studies) will be reported separately.
GWAS
Participants in the GWAS study were recruited through regularly scheduled glaucoma clinics (cases) or general medicine clinics (controls) at the six sites listed above. In general, POAG cases were defined as all persons above the age of 40 years, with typical glaucomatous optic neuropathy characterized by cupping, pallor, thinning or loss of retinal nerve fiber layer on optic disc examination and/or with optical coherence tomography (OCT), with characteristic visual field (VF) loss and open angles on gonioscopy, and had no other ocular abnormality (secondary causes) to account for these changes. These criteria are consistent with the International Society of Geographical and Epidemiologic Ophthalmology (ISGEO) classification system [14]:
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1.
The highest level of evidence is eyes with optic disc abnormalities: vertical cup to disc ratio (VCDR) in the 97.5th percentile in the normal population and VF defects compatible with glaucoma, e.g. eyes with VCDR ≥ 0.7 and/or VCDR asymmetry ≥ 0.2 or a neuro-retinal rim width reduced to ≤ 0.1 CDR (between 11 and 1 o’clock or 5 to 7 o’clock) that also shows a definite visual field defect consistent with glaucoma.
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2.
A severely damaged optic disc (VCDR 99.5th percentile of the hyper-normal population) was sufficient to make the diagnosis if a VF test could not be performed satisfactorily (e.g. VCDR ≥ 0.9 in which a visual field could not be performed satisfactorily.)
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3.
In the presence of media opacity preventing optic disc exam and VF testing, then a visual acuity 20/400, plus any of the following: evidence of glaucoma filtering surgery, current use of anti-glaucoma medications or medical records showing a history of glaucoma-related vision loss.
All individuals with other forms of glaucoma apart from POAG were excluded from this study. This included the presence of any potential secondary causes of glaucoma, such as pigment dispersion, narrow angles, or previous ocular injury. All individuals with greater than 8 diopters of myopia or hyperopia were excluded from both cases and controls.
Controls were defined as individuals over the age of 40, with no history of IOP > 22mmHg, who were not affected by primary or secondary glaucoma.
Juvenile Open Angle Glaucoma (JOAG) Sequence Scan
Index cases and their family members participating in the JOAG study were recruited from the University of Ibadan site. All glaucoma cases younger than 40 years with all the above characteristic features of POAG were classified as Juvenile Open angle glaucoma (JOAG). Family history of vision difficulties was obtained, and if other family members were suspected of being glaucoma cases, a field visit to examine and recruit additional family members was undertaken.
Study Assessments
Basic demographic information was collected, including ethnicity and native language of both participants and parents. General health information has been collected by all H3Africa-sponsored studies, including body mass index, blood pressure, medication history, smoking and alcohol use, and general medical history. All study participants underwent a comprehensive ocular examination. Slit lamp bio-microscopy was used to examine the conjunctiva, cornea, anterior chamber, iris, and lens. Automated refraction and keratometry (Auto Refractometer KR-800, Topcon Ltd, Tokyo, Japan) measurements, central corneal thickness, axial length and anterior chamber depth, lens diameter (PacScan plus:Sonomed model 300AP) measurements were performed. Using the results of the automated refractometer, best corrected distant visual acuity was determined for a distance of 6 m, and as near vision at a distance 25–30 cm using Jaeger charts, uncorrected and corrected using an addition for near vision. Perimetry was then performed using the Humphrey matrix visual field perimeter (Carl Zeiss Meditec, Inc., Dublin, CA), with the 24 − 2/10 − 2 SITA-standard program. Optical coherence tomography of the retinal nerve fiber layer was obtained using the Optovue machine (iscan Optovue OCT model: Ivue 500). The anterior chamber depth was evaluated and graded using Van Herrick’s system. Intraocular pressure (IOP) was measured with the Goldmann applanation tonometer.
Gonioscopy was performed on all subjects using the Posner 4-mirror lens and the angles were graded according to the Shaffer’s grading system. Only patients with open angles were recruited into the study. All eyes with open angles were dilated after gonioscopy. The optic disc was examined using a handheld, 78-D lens and 10x eye piece of the slit lamp for stereoscopic evaluation of the vertical optic disc and cup diameters with an eyepiece micrometer scale. Notching of the disc rim (defined as partial or complete loss of neuro-retinal rim over one or more clock hours in the superior or inferior quadrants), or optic pits, disc drusen, and disc hemorrhage were noted.
A hard-copy clinical report form was used to record all data collected during subject examinations. As described below, the data are subsequently entered into a REDCap (Research Electronic Data Capture) database hosted at Duke University, Durham, North Carolina.
Sample Management
Sample Collection
After the examination stage, blood samples were collected from eligible cases and controls by research assistants and medical laboratory scientists trained on the biorepository process of the project before commencement of study and sample collection. Kits for the sample collection were centrally procured by the Eyes of Africa administrative core and distributed to all the other centers. Prior to collecting the samples, strips of barcode labels were applied to the tubes, and to informed consent and case report forms. A 10 ml sample of venous blood was collected in EDTA tubes from cases and controls for DNA extraction and genetic analysis. This bottle was covered, sealed and placed in a small sized cooler containing ice packs. Blood samples were kept in these ice packs for a maximum of 2 h to maintain proper cold chain system during transit to the laboratory. The box was sealed and clearly marked biohazard material.
Sample Processing
Each transported sample was accompanied by relevant identification details and duly filled questionnaire. The laboratory was notified ahead of sample collection. Blood sample bottles collected were centrifuged and separated into different fractions (two plasma aliquots, one buffy coat, and one red cell concentrate, the latter of which contains small amounts of white cells and could be used as a backup source of DNA should extraction of the buffy coat fail). All four sample tubes were immediately frozen in barcoded cryo-vials in -80 C at the coordinating centers where ultra-low temp freezers are available. In collaboration with the H3 Africa biobank in Abuja [15], we developed a protocol for collection, shipping, and processing of blood samples to ensure an uninterrupted cold chain. This primarily involved the shipment of samples in credo boxes—Styrofoam-insulated cardboard boxes lined on all 6 sides with cold packs chilled to -80 C or -20 C prior to shipment. The Abuja biobank has demonstrated that this system enables overnight shipping without sample thaw [15].
At two sites within Nigeria (Enugu and Lagos) no -80 C storage was available to the study. Accordingly, our study protocol was modified. After blood centrifugation, sample aliquots were stored locally in -20 C for a maximum of 2 weeks and subsequently shipped in credo boxes to the central biorepository in Ibadan. Samples that arrived frozen were shifted to -80 C, while those that had thawed en-route were immediately extracted for DNA. Temperature of all the freezers (-20 degrees and − 80 degrees) were monitored daily with a temperature log. Batches of buffy coats stored at -80 C were shipped periodically from Ibadan to the Abuja Biobank for DNA extraction and sample quality control testing. DNA aliquots were shipped from Abuja to the core laboratories where downstream DNA testing was performed.
Data Management
At the time of subject ascertainment, all study subject data were entered onto a paper case record form (CRF). Subsequently, a password protected and encrypted REDCap Electronic database was set up at Duke University and was used to archive the subject data. In order to ensure data accuracy and enhance data completeness, the database incorporated value range checks and logic checks to ensure mandatory fields were not skipped and data entered were appropriate for the field. The study Biostatistician performed intermittent real time data management by generating data quality control queries including valid ranges, consistency checking, completeness etc. Only staff listed in the delegation log were provided with unique individual password to access the REDCap platform for data entry from multiple study sites. Only the Biostatistician was provided with permission for data download, upload, editing and overall management.
Statistical Analysis Plan
The primary analysis is designed to assess association between exposure variables (including central corneal thickness, mean keratometric readings, mean anterior chamber depth, mean lens diameter, optical coherence tomogram etc.) and case control status using generalized linear (regressions) models with binomial link function. Odds ratios (with their respective 95 % confidence intervals [CI]) will be estimated to test association between secondary exposure variables such as sociodemographic characteristics, hypertension status (or blood pressure), lifestyle factors (cigarette smoking, alcohol use etc.) and case-control status using logistic regression with and without adjustments for confounding factors. Adjusted population attributable risks (PARs) with their respective 95 % CIs for each exposure variable included in the best-fitted adjusted models will be calculated [16, 17]. The PARs will be estimated as the proportion of the risk of POAG in the population that is attributable to the individual risk factors.
Power/Sample Size Justification for the Genomic Data
We will utilize GWAS in a case-control study of 4000 POAG cases and 4000 controls. Based on reported single nucleotide polymorphisms (SNPs) associated with POAG, a sample size of at least 4000 case-control pairs provides > 90 % power to detect a SNP with genetic odds ratio of at least 1.2 and minor allele frequency of 0.2.
Power Justification for the Phenomic Data
A sample size of 8000 (4000 case and 4000 controls) provides substantial power to quantify the contributions of traditional and novel risk factors for Primary Open Angle Glaucoma (POAG). Assuming at least 5 % probability of exposure in the control population and a 0.75 correlation coefficient for exposure between cases and controls, a sample size of 4000 in each group will allow detecting an odds ratio of ≥ 1.0035 with a power of ≥ 80 % using logistic regression [18].