CONICET | Buscador de Institutos y Recursos Humanos

IntroductionClimatic DropletKeratopathy (CDK) is an acquired degenerativedisease predominantly affecting males over 40 years old. It results inprogressive corneal opacities usually affecting both eyes. CDK is multifactorial and its etiology remainsunknown. Recent findings are consistent with CDK pathologybeing driven by environmentalfactors with oxidative stress playing an importantrole (e.g. contributing to lipid peroxidation) rather than climate factors(e.g. certain geographical regions of the world which share in common low humidity, constant winds and chronic exposure tohigh levels of ultraviolet radiation (UVR)) [1-3].We have previously found a lack of correlation between mitochondrial DNA and Y-chromosomediversity (maternal and paternal haplogroups andlineages) and the expression of CDK in populations livinginArgentine Patagonia[4]. Thepossiblecontribution of immunogenetic factors (such asHLA class I and class II genes)tothe etiopathology of CDK remainsstill understudiedas well as in many other eyediseases [5-6]. AimThe goal of this study was toinvestigate the association between HLA class I (-A, -B, -C) and HLA class II (-DPA1,-DPB1, -DQA1, -DRB1, -DRB3, -DRB4, -DRB5) genes in individualsdiagnosedwith grade 2 of Climatic Droplet Keratopathy (CDK)and healthycontrolsfrom El Cuy Department in ArgentinePatagonia. MethodsSamples CollectionThe collection ofsamples consisted of both 20 healthy controls,who did not presentany pathologyin the anterior segment of the eye (17males and 3 females), and 20 grade 2 CDK cases (16 males and 4females) from the Patagonian region of Argentina.This study was approved by the Institutional ReviewBoard of the Catholic University of Córdoba, and the Institutional ResearchEthics Committee of Health, Ministry of Health of the Province of Córdoba,Argentina (recorded in the RePIS), and carried out in accordance with theprinciples of the Statement of Helsinki. All subjects whowere over 40 years of age provided written informed consent prior to theirinclusion in the study.HLA Class I and II typing by using aNext-Generation Sequencing (NGS) based genotyping methodAll samples were genotyped for HLA-A,-B, -C,-DPA1, -DPB1,-DQA1, -DQB1, -DRB1 and -DRB3/4/5loci using the MIA FORATMNGS HLA Typing 11 Kit-96 Tests (Immucor, Inc. Norcross, GA, USA), followingmanufacturer?s automated protocol and as it is reported in Wang et al. [7]. Briefly, sequencing reads weregenerated in a high-throughput platform by using this NGS-based HLA typing method(amplicon-basedenrichment of HLA loci followed by massively parallel sequencing) that amplifies specifically and sequences these 11 HLA genes. Then,the bioinformatics pipeline (research version 3.1 of the MIA FORATMNGS HLA genotyping software) attributes (based on an internal reference databaseas well as on version 3.25 of IMGT/HLA database which contains all describedHLA reference alleles sequences) these sequencing reads to specific HLA genesand calls high-resolution (atthe 4-fieldof allele resolution level) phased genotypesfor each HLA gene. StatisticalAnalysisStatistical analyses were only performed at 2-fieldalleleresolution level as being the first minimum level of resolution to be tested inorder to see if any significant HLA allele association was possibly found inthis cohort study before proceeding with further analysis at 4-field alleleresolution level. Initial statistical analysis included Hardy-Weinberg testing (based on exact test of Guo and Thompson) [8], Ewens-Waterson homozygosity test of neutrality (using Slatkin?s Monte-Carlo implementation ofthe exact test) [9][10], determination of allele frequencies and all pairwise linkage disequilibrium (LD)estimatesusing the softwareanalysisPypop (Python for Population genomics) v.0.7.0[11]. Regardingstatistical association analyses: comparison of allelefrequenciesin 2x2 contingencytablesand the evaluation of their significance(significance of the differences wasevaluated using two-tailed Fisher?s exact test [12], and P-valuesless than 0.05 were considered significant), as well as calculation of odds ratios (OR), relative risk (RR) and respectiveconfidence intervals (with a 95% confidence interval (95%CI)) were calculated (according to Altman[13]) usingEpiInfoTM (version 7.0). Results:The observed and expected frequenciesbetween genotypes for all HLA loci showed no deviation from Hardy-Weinbergequilibrium in cases or controls, with P-values greater than 0.05. Thefrequencies of all HLA-A, -B,-C, -DPA1, -DPB1, -DQA1, -DQB1, -DRB1, -DRB3/4/5 alleleswere calculated and no significant differences were found between the twogroups studied, except for the HLA-Blocus, which showed a statistically significant difference for the HLA-B*51:01allele (2.6% CDK cases vs. 21.1% Healthy controls, P = 0.0284, OR = 0.101, and 95%CI =0.012?0.856) and also for the HLA-DRB3 locus,which in this case showed a statistically significant difference for the HLA-DRB3*01:01allele (26.5% CDK cases vs. 3.8% Healthy controls, P = 0.0329, OR = 9.000, and 95%CI = 1.060?76.427). DiscussionIn the cohort studied here,our preliminary performed analyses suggest influence of certain HLA genes onresistance and susceptibility to Climatic Droplet Keratopathy (CDK). In which wehave found associations for the allele HLA-B*51:01 (protection against developing of CDK) and for theallele HLA-DRB3*01:01 (susceptibilityfor developing of CDK).Curiously/Peculiarly,HLA-B*51:01 is the major suballele associated with Behçet?s disease (BD ) inall the populations studied [14].Regarding, HLA-DRB3*01:01, Smikle,M.F.,et al have shown a very strong association between this allele and Graves' disease in Jamaican [15].At the same time, this interpretation ofour results in this study may need to be taken carefully and in the context ofthe reduced sample size used for this study. Nevertheless, this present studyhas allowed us to report for the first time the diversity of all HLA class Iand II genes in high-resolution and phased genotypes of this cohort (of bothCDK patients and healthy controls) from this Patagonian region of Argentina. Atthe same time, the HLA typing results obtained from this study should be a goodcomplement for future studies which can continue undercover the underlying mechanisms of this corneal disease.