Variants of WDR36 in Cameroonian glaucoma patients

Background: In Primary Open Angle Glaucoma (POAG), the most common form of glaucoma in people of African origin, it is established that retinal ganglion cells are lost due to apoptosis. Loss of WDR36 (OMIM 609669) function has been shown to result in activation of the p53 stress-response pathway, a key regulator of apoptosis. However, there is controversy surrounding its contribution in the pathogenesis of POAG. We aimed to establish an association between 3 WDR36 gene polymorphisms and POAG. Methods: We assessed 798 glaucoma medical records and selected 209 POAG cases. A total of 26 POAG cases residing in Yaoundé completed the study and 19 controls were matched for age and gender. Dried blood spots on Whatman fi lter paper grade 3 were used for DNA extraction by the Chelex method. Polymerase Chain Reaction (PCR) was conducted with two sets of primers on rs1971050, rs10038177 and rs10038058 followed by Restriction Fragment Length Polymorphism (RFLP) using restriction enzymes AluI on rs1971050, rs10038177and ApoI on rs10038058 to determine the genotypes. Results: After digestion, the homozygous mutant form of rs1971050 was evidenced in both groups of our study population T/T (100%). The T and the A alleles were the most common alleles found in our study population and were not associated with POAG. Heterozygote and homozygote mutant genotypes were obtained in both groups for rs10038177 (POAG group C/T (7.7%) and T/T (92.3%) and in the control group C/T (10.5%) and T/T (89.5%)) and rs10038058 (G/A (11.5%) and A/A (88.5%) genotypes in the POAG group and in the control group G/A (10.5%) and A/A (89.5%)). These genotypes were not associated with Primary Open Angle Glaucoma (POAG). Conclusion: Homozygous mutant genotypes of rs1971050, rs10038177 and rs10038058 may not be associated with the disease process of primary open angle glaucoma in Cameroon. Reasearch article Variants of WDR36 in Cameroonian glaucoma patients Fanny Mbacham1, Yannick Bilong1,2, Jean Paul Chedjou3,4, Arlette Nomo1,2, Chantal Nanfack Ngoune1,2, Hermann Dongmo3,4, CalvinoTAH4, Jean-Claude Katte1,5, Wilfred Mbacham1,3,4, Eugene SobngwI1,3,5 and Assumpta Lucienne Bella1,2* 1Faculty of Medicine and Biomedical Sciences, University of Yaoundé I, Cameroon 2Ophthalmology Unit, Yaoundé Gynaeco-Obstetrics and Paediatric Hospital, Cameroon 3Biotechnology Center, University of Yaoundé I, Cameroon 4Faculty of Science, University of Yaoundé I, Cameroon 5National Obesity Centre and Endocrinology and Metabolic Diseases Unit, Yaoundé Central Hospital, Cameroon Received: 11 November, 2020 Accepted: 10 December, 2020 Published: 11 December, 2020 *Corresponding author: Bella Assumpta Lucienne, MD, Professor, Department of Ophthalmology, Otorhinolaryngology and Stomatology, Yaoundé Gynaeco-Obstetric and Pediatric Hospital, Faculty of Medicine and Biomedical Sciences, University of Yaoundé I P.O Box 1937, Cameroon, Email:


Introduction
Glaucoma is an optic neuropathy associated with structural damage of the optic nerve and visual dysfunction [1]. Globally, it is the leading cause of irreversible blindness. The number of persons estimated to be blind as a result of primary glaucoma (POAG) is 4.5 million, accounting for slightly more than 12% of all global blindness [2]. In Africa, glaucoma accounts for 15% of blindness with a higher prevalence than in other regions.
The primary risk factors that are linked to the individual and enrich the study population, to determine treatment effect and detect changes early. Although there is no ideal biomarker, some of the benefi ts of integrating biomarkers into clinical disease management include: early detection of disease, severity prediction, progression and response to treatment.
Identifying these biomarkers worldwide will serve as a push to identify their biochemical pathways and molecular signifi cance in optic nerve damage.
To date 29 loci have been linked to POAG but the roles of only three genes have been identifi ed in these loci.  [6]. OPTN and MYOC have been shown to be high penetrance glaucoma causing genes. There is however great controversy among experts about the contribution of WDR36 and its variants in the pathogenesis of glaucoma. Unlike the original reports from Monemi, et al. 2005 [7] on the role of WDR36, many others followed, which failed to replicate the original studies in which the above genes were presented as glaucoma-causing genes. Data is currently inconclusive.

Objectives
The purpose of this study was to evaluate the association between POAG in a Cameroonian population and 3 Single Nucleotide Polymorphisms (SNPs) in the WDR36 genomic region.

Study population
This was a pilot case-control study over a period of 10 months (August 2018 -May 2019). Our study sample was made up of two groups. The fi rst group with participants retained from health record booklet reviews of those with primary open angle glaucoma according to the following criteria: age 40 years or more, living with primary open angle glaucoma for at least 5 years or recently diagnosed with terminal stage.
The control group was made up of participants selected during outpatient clinic consultations matched according to age, without any form of glaucoma, no family history of glaucoma or ocular hypertension, IOP < 20 mmHg, difference in IOP in both eyes not more than 3mmHg.
Sample collection and DNA extraction: Participants retained presented themselves early in the morning by appointment at the Ophthalmology unit of the Yaoundé Gynaeco-Obstetric and Paediatric Hospital. Upon arrival, for each participant, we measured far and near visual acuity in both eyes using the tumbling ''E'' eye chart and the Jaeger chart respectively. IOP was measured for both eyes using the air puff tonometer and capillary blood glucose levels were obtained using a blood glucose meter CERA-CHEK 1 Code (Model: G400) GREEN CROSS MEDIS Corps). Allfi ndings were recorded on the data sheet. Blood samples collected from participants were spotted on Whatman fi lter paper grade 3 and dried, for genomic analysis using the Chelex 100 method [8]. The resultant DNA extracts were stored at -20 °C for PCR analysis.

Molecular genotyping
The WDR36single nucleotide gene polymorphisms; rs1971050, rs10038177 and rs10038058were amplifi ed using the following primers respectively: F 5' -GCCTCTCATTTATTT-TATTTCTCAAGG -3' and R 5' -CCTCTGATACAGGGGAC-CAACTG -3'; F 5' -GCCTCTCATTTATTTTATTTCTCAAGG -3' and R 5' -CCTCTGATACAGGGGACCAACTG -3'; F 5' -GAG-GTGAAGAGCAATTGGGTTTCTC and R 5' -GCAGTGTCAG-GAAAGACACTGTACC -3'. Mookerherjee, et al. 2011 (41). The Master Mix was prepared in a 1.5ml tube according to the number of samples and spun in the centrifuge. Each PCR was carried out in a total volume of 22μl and the reaction medium was composed of 18.25 μl Nuclease free water (NFW), 2.5 μlbuffer (10X thermopol buffer), 10mM dNTPs (200 μM of each deoxyribonucleotide), (0.25 μl) μMof each primer, (0.25 μl) U/μlof Taq DNA polymerase and 3μl of DNA. The amplifi cation programs previously recorded in the thermocycler (T3 thermal cycler, Biometra, UK) proceeded as follows for the NAT2 gene amplifi cation: The amplifi cation program previously recorded in the thermocycler (T3 thermal cycler (Biometra, UK)) proceeded as follows: the pre-denaturation is carried out at 95 0 C for 4 minutes followed by 35 amplifi cation cycles. Each cycle was a succession of denaturation at 95 0 C for 30 seconds, annealing at 60 0 C for rs1971050; at 62 0 C for rs10038177 and at 50 0 C for rs10038058 for 30seconds each, elongation at 72 0 C for 30 seconds. The fi nal extension, which comes at the end of these cycles, was carried out at 72 0 C for 4 minutes.WDR36gene SNP's were determined by restriction fragment length polymorphism (RFLP) in a volume of 12μl containing the PCR water, 8.6μl buffer (10X buffer), 3.0μl of BSA, either 0.4μl of AluI restriction enzyme for rs1971050; 0.4μl of AluI restriction enzyme for rs10038177; 0.4μl ApoI restriction enzyme for rs10038058 and 8μl of WDR36 amplicon. The mixtures were then incubated at 37°C overnight for rs1971050 and rs10038177 and at 50°C overnight for rs10038058.Digested and undigested fragments of each sample were separated on a 2% agarose gel stained with ethidium bromide.

Data analysis
All data was entered and analyzed on SPSS version 25.0 (SPSS Inc., USA) statistical software and Microsoft Excel 16. The different genotypes and phenotypes of the SNPs in the study population were expressed as frequencies (%). The association between rs1971050, rs10038177 and rs10038058 SNPs of WDR36 and POAG in the study population were assessed by Chi-square analysis. All p-values <0.05 were considered statistically signifi cant on a 95% confi dence interval.

Ethical approval
The  (Table 1). Corrected visual acuity was measured in 90 eyes (45 study participants) and 74 had a visual acuity more than 3/10. In the POAG group, 6 participants had low vision and 6 others were blind.
WDR36SNP characterization results: After digestion of rs1971050, only the T allele and the T/T mutant genotype was found in our study population (Tables 2,3).
For rs10038177, the mutant heterozygote(C/T) and homozygote phenotypes (T/T) were found in our study population, with the mutant phenotype (T/T) being predominant in both the POAG group (92.3%) and the control group (89.5%) group. The heterozygote phenotypes were found in both groups at a frequency of (7.7%) for POAG group and (10.5%) for the control group (Tables 2,3).
For rs10038058, the mutant heterozygote (G/A) and homozygote (A/A) phenotypes were found in our study population. There was a predominance in both the POAG group (88.5%) and the control group (89.5%) of the mutant homozygous phenotype (A/A). The heterozygote phenotype (G/A) was found in both groups at a frequency of (11.5%) for POAG and (10.5%) for the control group (Tables 2,3). Correlation results between WDR36 SNPs and POAG: All participants showed the homozygous mutation for rs1971050. Meanwhile, the heterozygote and homozygous mutant forms were obtained in the study population for rs10038177 and rs10038058. None of these expressed phenotypes showed a signifi cant association with glaucoma (p-/, OR = 0.7 and OR = 0.9 respectively). There was no association between rs1971050,rs10038177 and rs10038058single nucleotide polymorphisms and the onset or progression of glaucoma (Table 4).