Análise genética e molecular do ceratocone envolvendo os genes VSX1, SOD1, TIMP3 e LOX
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Data
2018-03-06
Autores
Lopes, Alessandro Garcia
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Universidade Estadual Paulista (Unesp)
Resumo
O ceratocone é uma doença comum que acomete a córnea, afetando ambos os gêneros em todas as etnias e que pode se manifestar bilateralmente, de forma assimétrica. Por tratar-se de uma doença degenerativa, sua progressão pode ser rápida em diversas situações, acometendo 1 a cada 2.000 pessoas e resultando em severas alterações estruturais da córnea, as quais diminuem sua espessura e modificam sua curvatura normal para um formato mais cônico. O quadro clínico é composto por sintomas variados, destacando-se miopia e astigmatismo. Atualmente sabe-se que fatores genéticos e ambientais estão envolvidos no surgimento do ceratocone. Recentemente vários estudos genéticos têm como objetivo a identificação de mutações em genes que estejam diretamente ligados à doença, bem como seus papéis fisiológicos. Contudo, apesar dos esforços direcionados para o conhecimento dos aspectos genéticos, envolvendo diversos genes, ainda existem muitas dúvidas a respeito dos papéis destes na etiologia desta anomalia ocular. Assim, neste estudo, pretendeu-se identificar a presença de mutações e/ou polimorfismos nos genes VSX1, SOD1, TIMP3 e LOX, importantes candidatos à origem e desenvolvimento desta doença, a partir da análise pareada das sequências nucleotídicas de tecido da córnea e sangue periférico dos pacientes portadores desta patologia. As avaliações oftalmológicas incluíram exame clínico, topografia e tomografia. As amostras de DNA foram extraídas de sangue periférico e de fragmentos de córnea e as regiões codificantes dos genes foram amplificadas por reação em cadeia da polimerase (PCR), desnaturadas e submetidas a eletroforese em gel de poliacrilamida. Então, o rastreamento mutacional foi realizado através da técnica de SSCP (Single-Strand Conformation Polymorphism) seguido de sequenciamento direto. A metodologia específica de pesquisa utilizando-se fragmentos de córnea, desenvolvida para este estudo, é algo inédito, diante dos trabalhos os quais utilizam apenas sangue. Alguns SNPs (polimorfismos de nucleotídeo único) foram detectados nas regiões codificantes dos genes VSX1, SOD1 e TIMP3, em ambos os tecidos, mas esses SNPs não promovem alterações patogênicas nas estruturas das proteínas codificadas por esses genes, correspondendo a apenas trocas sinônimas de aminoácidos. Quanto ao gene LOX, foi detectado a ausência de qualquer polimorfismo, tanto em gel como no sequenciamento. Em relação aos polimorfismos encontrados, eles estavam concordantes em ambos os tecidos analisados, córnea e sangue. A ausência de alterações patogênicas nestes genes, obtida por este estudo em população brasileira, aliada aos resultados obtidos em outros estudos envolvendo outras populações mundiais, os quais também não encontraram mutações nestes genes, sugerem que outros fatores genéticos possam estar implicados no surgimento do ceratocone, sendo importante ressaltar também os fatores ambientais e comportamentais envolvidos tais como atopia, ato de coçar os olhos, luz ultravioleta e o uso excessivo de lentes de contato.
Keratoconus is a common disease that affects the cornea in both genders in all ethnic groups and it can manifest asymmetrically and bilaterally in patients. Keratoconus due to a degenerative disease and its progression can be fast in various situations, affecting 1 in 2,000 people, what results in severe structural changes of the cornea, which reduce its thickness and modify its normal curvature to a more conical shape. The clinical picture consists of varied symptoms, such as myopia and astigmatism. Currently it is known that genetic and environmental factors are involved in the desease emergence. Recently several genetic studies have aimed to identify mutations in genes which are directly linked to Keratoconus, as well as their physiological roles. However, despite the efforts directed towards the understanding of the genetic aspects of this disease, involving many genes, there are still many questions about the role of these genes in Keratoconus etiology. Thus, the present study aimed to identify the presence of mutations or polymorphisms in VSX1, SOD1, TIMP3 and LOX genes, which are considered important candidates for the origin and development of this eye anomaly, using their nucleotide sequences in paired-like analysis of the corneal tissue and peripheral blood from Keratoconus patients. Ophthalmologic evaluations included a clinical examination, topography and tomography. DNA samples were extracted from peripheral blood and from corneal fragments and all coding regions of the genes were amplified by polymerase chain reaction (PCR), denatured and subjected to polyacrylamide gel electrophoresis. Thus, the mutational screening was performed by Single-Strand Conformation Polymorphism (SSCP) and direct DNA sequencing. The specific research methodology developed for this study, using cornea fragments, is something new in the field of Keatoconus research, in which only blood has been used. Some heterozygous Single-Nucleotide Polymorphisms (SNPs) were found during the screening of the coding regions of the VSX1, SOD1 and TIMP3 genes but all of them do not promote any pathogenic alteration in the protein structures, so that, corresponding to synonymous exchanges of amino acids only. In the LOX gene, no polymorphisms were detected either in the gels or in the nucleotide sequence analysis. In relation to the polymorphisms found, they were in concordance in both analyzed tissues, cornea and blood. The absence of pathogenic alterations in these genes obtained by this study of a Brazilian cohort, combined with data from other studies involving different cohorts around the world that did not identify mutations in these genes, suggest that other genetic factors may be implicated in the onset of keratoconus. It is also important to remember possible environmental and behavioral factors involved, such as atopy, eye rubbing, ultraviolet light and excessive use of contact lenses.
Keratoconus is a common disease that affects the cornea in both genders in all ethnic groups and it can manifest asymmetrically and bilaterally in patients. Keratoconus due to a degenerative disease and its progression can be fast in various situations, affecting 1 in 2,000 people, what results in severe structural changes of the cornea, which reduce its thickness and modify its normal curvature to a more conical shape. The clinical picture consists of varied symptoms, such as myopia and astigmatism. Currently it is known that genetic and environmental factors are involved in the desease emergence. Recently several genetic studies have aimed to identify mutations in genes which are directly linked to Keratoconus, as well as their physiological roles. However, despite the efforts directed towards the understanding of the genetic aspects of this disease, involving many genes, there are still many questions about the role of these genes in Keratoconus etiology. Thus, the present study aimed to identify the presence of mutations or polymorphisms in VSX1, SOD1, TIMP3 and LOX genes, which are considered important candidates for the origin and development of this eye anomaly, using their nucleotide sequences in paired-like analysis of the corneal tissue and peripheral blood from Keratoconus patients. Ophthalmologic evaluations included a clinical examination, topography and tomography. DNA samples were extracted from peripheral blood and from corneal fragments and all coding regions of the genes were amplified by polymerase chain reaction (PCR), denatured and subjected to polyacrylamide gel electrophoresis. Thus, the mutational screening was performed by Single-Strand Conformation Polymorphism (SSCP) and direct DNA sequencing. The specific research methodology developed for this study, using cornea fragments, is something new in the field of Keatoconus research, in which only blood has been used. Some heterozygous Single-Nucleotide Polymorphisms (SNPs) were found during the screening of the coding regions of the VSX1, SOD1 and TIMP3 genes but all of them do not promote any pathogenic alteration in the protein structures, so that, corresponding to synonymous exchanges of amino acids only. In the LOX gene, no polymorphisms were detected either in the gels or in the nucleotide sequence analysis. In relation to the polymorphisms found, they were in concordance in both analyzed tissues, cornea and blood. The absence of pathogenic alterations in these genes obtained by this study of a Brazilian cohort, combined with data from other studies involving different cohorts around the world that did not identify mutations in these genes, suggest that other genetic factors may be implicated in the onset of keratoconus. It is also important to remember possible environmental and behavioral factors involved, such as atopy, eye rubbing, ultraviolet light and excessive use of contact lenses.
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Palavras-chave
Ceratocone, Córnea, VSX1, TIMP3, SOD1, LOX, Miopia, Astigmatismo, Keratoconus, Cornea, Myopia, Astigmatism