Investigation of the relationship between clusterin genetic polymorphisms and clusterin level of tear and aqueous humor in pseudoexfoliation syndrome and glaucoma

Psödoeksfoliasyon sendromu (PES), yaşa bağlı olarak gelişen, hücre dışı matrikste lifsi materyallerin hem oküler dokularda hem de çeşitli iç organlarda birikmesi ile karakterize edilen sistemik bir bozukluktur. Psödoeksfoliasyon sebebiyle oluşan glokoma psödoeksfoliatif glokom (PEG) adı verilir ve PEG tedavi edilmediğinde körlüğe sebep olabilmektedir. Bu nedenle PEG ve PES hastalığına yol açan potansiyel faktörlerin bulunması ve PEG gelişme riski olan PES hastalarının tespit edilmesi büyük önem taşımaktadır. Clusterin proteini hücresel stres durumlarında, yanlış katlanmış proteinlerin stresle tetiklenen çökelmesi ve yığışmasının önlenmesini sağlar. Isı şoku ve oksidatif stres gibi hücresel stres durumlarında clusterin ifadesi indüklenmektedir. PES hastalarında gözün ön kamarasındaki oksidatif ortam nedeniyle protein yanlış katlanmalarının meydana gelmesine rağmen PEG ve PES hastalarının aköz humor örneklerinde clusterin ifadesi azalmıştır. Clusterin ifadesindeki azalmanın sebeplerinden biri genetik polimorfizmler olabilir. Bu çalışma PEG ve PES riski ile clusterin genetik polimorfizmleri ve aköz humor ile gözyaşındaki clusterin protein seviyeleri arasındaki ilişkiyi incelemeyi amaçlamıştır. Bu çalışma kapsamındaki 213 PEG hastası, 214 PES hastası ve 215 kontrol bireyin tam kan örnekleri ile clusterin genetik polimorfizmlerinin genotiplemeleri PCR-RFLP ve real-time PCR yöntemleri ile yapılmıştır. 12 PEG hastası, 17 PES hastası ve 22 kontrolün aköz humor örneği ile 80 PEG hastası, 81 PES hastası ve 80 kontrolün gözyaşı örneğinin clusterin ve total protein konsantrasyonu analizleri ise ELISA ve Bradford yöntemleri ile yapılmıştır. Tüm örnekler Sağlık Bilimleri Üniversitesi, Tıp Fakültesi, Gülhane Eğitim ve Araştırma Hastanesi Göz Hastalıkları Servisi'nden (Ankara) alınmıştır. Bu çalışma erkek olmanın PEG riskini ~2 kat arttırdığını göstermiştir. Clusterin rs11136000 C/T, rs1532278 C/T ve rs2279590 C/T için TT genotipi ~3 kat koruyucu, rs3087554 A/G için ise AA genotipi PEG hastalığında ~2.4 kat koruyucu görünmektedir. Birleştirilmiş haplotip analizine göre, clusterin rs11136000 C/T, rs1532278 C/T ve rs2279590 C/T için CC genotipi ile rs3087554 A/G için GA genotipine sahip olmak PEG ile PES hastalıkları için ayırt edici olabilir gibi görünmektedir. Aköz humor örneklerindeki clusterin ve total protein konsantrasyonu ile gözyaşı örneklerindeki total protein konsantrasyonu PEG hastalarında PES hastalarına ve kontrollere kıyasla istatistiksel olarak anlamlı seviyede yüksektir. Gözyaşında clusterin seviyesini ve Türk toplumunda aköz humordaki clusterin seviyesini inceleyen herhangi bir çalışma bulunmamaktadır ve bu yönüyle bu çalışma ilktir. Clusterin rs1532278 C/T PEG ve PES kapsamında, clusterin rs3087554 A/G Türk popülasyonunda, clusterin rs2279590 C/T ise PEG ve PES kapsamında Türk popülasyonunda ilk kez bu çalışmada incelenmiştir. İleride bu konularda daha fazla çalışma yapılarak bu sonuçlar onaylanabilir ve biyobelirteç olarak kullanılacak bir parametre geliştirilebilir. Bu çalışma bu yönüyle Biyomedikal Mühendisliği'ne katkı sağlayan, ileride geliştirilecek tanı ve tedavi araçları için alt yapı hazırlayan bir proje özelliği taşımaktadır. Anahtar Kelimeler: Psödoeksfoliasyon, Glokom, Clusterin, rs11136000 C/T, rs3087554 A/G, rs1532278 C/T, rs2279590 C/T, Genotip, Polimorfizm, Aköz humor, Gözyaşı.Pseudoexfoliation syndrome (PES) is an age-related systemic disorder of extracellular matrix characterized by the presence of fibrillar deposits in both ocular tissues and many internal organs. Glaucoma that occurs due to pseudoexfoliation is called pseudoexfoliative glaucoma (PEG) and can cause blindness if it is not treated. Therefore, in order to find potential factors causing PEG and PES diseases, and the identification of PES patients who are at risk of developing PEG is of great importance. Clusterin protein prevents the stress-induced precipitation and aggregation of misfolded proteins under cellular stress conditions. Clusterin expression is induced in cellular stress conditions such as heat shock and oxidative stress. Despite the formation of misfolded proteins due to the oxidative environment in the anterior chamber of the eye in PES patients, clusterin expression level is unexpectedly low in aqueous humor of PEG and PES patients. Genetic polymorphisms may be one of the factors causing low clusterin expression. This study aimed to investigate the association of clusterin genetic polymorphisms and clusterin protein levels of aqueous humor and tears in PEG and PES risk. The genotyping of clusterin genetic polymorphisms from whole blood samples of 213 PEG patients, 214 PES patients and 215 control individuals was performed by PCR-RFLP and real-time PCR methods. Clusterin and total protein concentrations of aqueous humor samples of 12 PEG patients, 17 PES patients, 22 controls and of tear samples of 80 PEG patients, 81 PES patients and 80 controls were analyzed by ELISA and Bradford methods. All samples were obtained from University of Health Sciences, Faculty of Medicine, Gülhane Training and Research Hospital, Ophthalmology Unit, Ankara, Turkey. This study shows that being male significantly increased PEG risk by ~2-fold. TT genotype in clusterin rs11136000 C/T, rs1532278 C/T and rs2279590 C/T is ~3-fold protective and AA genotype in rs3087554 A/G is ~2.4-fold protective against PEG disease. According to combined genotype analysis, having CC genotype in clusterin rs11136000 C/T, rs1532278 C/T and rs2279590 C/T and having AG genotype in rs3087554 A/G seems to be distinctive for PEG and PES diseases. Clusterin and total protein concentration in aqueous humor samples and total protein concentration in tear samples were significantly higher in PEG patients than PES patients and controls. To the best of our knowledge, this study is the first investigation of clusterin levels in tears and of clusterin levels in aqueous humor in Turkish population. Clusterin rs1532278 C/T, clusterin rs3087554 A/G and clusterin rs2279590 C/T are studied for the first time in PEG and PES risk, in Turkish population and in Turkish population for PEG and PES risk, respectively. These results can be confirmed in the future by further studies on these issues and a parameter to be used as a biomarker can be developed. In this respect, this study is a project that contributes to Biomedical Engineering and prepares background for diagnostic and therapeutic tools that will be developed in the future. Keywords: Pseudoexfoliation, Glaucoma, Clusterin, rs11136000 C/T, rs3087554 A/G, rs1532278 C/T, rs2279590 C/T, Genotype, Polymorphism, Aqueous humor, Tear

Paraoxonase 1 (PON1) promoter (−107T/C) and coding region (192Q/R and 55L/M) genetic variations in pseudoexfoliation syndrome and pseudoexfoliative glaucoma risk

Purpose Pseudoexfoliation syndrome (PEX) is characterized by the accumulation of microscopic extracellular material in the anterior chamber of the eye and can lead to the development of pseudoexfoliative glaucoma (PEG) in some patients. The pathogenesis of PEX is not fully understood, and there are no objective biomarkers for its early diagnosis. Recent research has indicated that oxidative stress and inflammation might play a role in the pathophysiology of the production of pseudoexfoliation material. Therefore, in the present study, we aimed to analyze the possible association between three genetic variants of paraoxonase 1 (PON1), a well-recognized antioxidant and anti-inflammatory enzyme, and PEX/PEG. Methods The study population consisted of patients with PEX (n = 150), patients with PEG (n = 150), and control subjects (n = 150). PON1 −107T/C, 192Q/R, and 55L/M genotypes were determined using PCR followed by restriction fragment length polymorphism analysis. The correlation between these genetic alterations and clinical visual characteristics was also investigated. Results The minor allele frequencies and genotype distributions of PON1 did not differ significantly between the PEG, PEX, and control groups. Moreover, PON1 genotypes did not significantly influence visual clinical parameters in stratification analysis. On the other hand, in correlation analysis, pattern standard deviation was significantly correlated with the −107T/C genotypes in PEX group. In addition, intraocular pressure was correlated with the 55L/M genotypes and mean deviation was correlated with the −107T/C genotypes in the control group. Furthermore, intraocular pressure was significantly inversely correlated with sex (r =  − 0.116, P = 0.011) in the overall study group. Logistic regression analysis showed that having a PON1 −107TC or CC genotype is significantly associated with PEX (OR = 1.909, P = 0.020). Conclusions This study, for the first time, analyzed the relationship between PON1 genetic variants, clinical visual parameters, and PEX/PEG. The results indicated a possible role for the PON1 promoter variant in PEX

precisionFDA Truth Challenge V2: Calling variants from short- and long-reads in difficult-to-map regions

The precisionFDA Truth Challenge V2 aimed to assess the state-of-the-art of variant calling in difficult-to-map regions and the Major Histocompatibility Complex (MHC). Starting with FASTQ files, 20 challenge participants applied their variant calling pipelines and submitted 64 variant callsets for one or more sequencing technologies (~35X Illumina, ~35X PacBio HiFi, and ~50X Oxford Nanopore Technologies). Submissions were evaluated following best practices for benchmarking small variants with the new GIAB benchmark sets and genome stratifications. Challenge submissions included a number of innovative methods for all three technologies, with graph-based and machine-learning methods scoring best for short-read and long-read datasets, respectively. New methods out-performed the 2016 Truth Challenge winners, and new machine-learning approaches combining multiple sequencing technologies performed particularly well. Recent developments in sequencing and variant calling have enabled benchmarking variants in challenging genomic regions, paving the way for the identification of previously unknown clinically relevant variants

PrecisionFDA Truth Challenge V2: Calling variants from short and long reads in difficult-to-map regions

The precisionFDA Truth Challenge V2 aimed to assess the state of the art of variant calling in challenging genomic regions. Starting with FASTQs, 20 challenge participants applied their variant-calling pipelines and submitted 64 variant call sets for one or more sequencing technologies (Illumina, PacBio HiFi, and Oxford Nanopore Technologies). Submissions were evaluated following best practices for benchmarking small variants with updated Genome in a Bottle benchmark sets and genome stratifications. Challenge submissions included numerous innovative methods, with graph-based and machine learning methods scoring best for short-read and long-read datasets, respectively. With machine learning approaches, combining multiple sequencing technologies performed particularly well. Recent developments in sequencing and variant calling have enabled benchmarking variants in challenging genomic regions, paving the way for the identification of previously unknown clinically relevant variants