Screening for Novel LOX and SOD1 Variants in Keratoconus Patients from Brazil

Purpose: To investigate the presence of the variants of lysyl oxygenase (LOX) and superoxide dismutase 1 (SOD1) genes in Brazilian patients with advanced keratoconus. Methods: Donor genomic DNA extracted from blood samples was screened for 5’UTR, exonic LOX, and SOD1 variants in a subset of 26 patients presenting with advanced keratoconus (KISA > 1000% and I–S > 2.0) by Sanger sequencing. The impact of non-synonymous amino acid changes was evaluated by SIFT, PMUT, and PolyPhen algorithms. The Mutation Taster tool was used to evaluate the potential impact of formation of new donor and acceptor splice sites in the promoter region of affected volunteers carrying sequence variants. A 7-base SOD1 deletion (IVS2 + 50del7bp) previously associated with keratoconus was screened in 140 patients presenting classical keratoconus by gel fragment analysis, and positive samples were sequenced for confirmation. Results: We found an unreported missense variant in LOX exon 6 in one heterozygous patient, leading to substitution of proline with threonine at residue 392 (p. Thr392Pro) of LOX protein sequence. This mutation was predicted to be potentially damaging to LOX protein. Another LOX variant, Arg158Gln, was also detected in another patient but predicted to be non-pathogenic. Two additional new polymorphisms in LOX 5’UTR region (–116C > T and –58C > T) were found in two patients presenting with advanced keratoconus and were predicted to modulate or create donor/acceptor splice sites in LOX transcripts. Additionally, SOD1 deletion was detected in one patient presenting with severe keratoconus, not in control samples. Conclusion: We described three novel LOX polymorphisms identified for the first time in Brazilian patients with advanced keratoconus, as well as a previously described SOD1 deletion strongly associated with keratoconus. A possible role of these variants in modulating transcript levels in the cornea of affected individual requires further investigation

Efeito terapêutico do cross-linking corneal em portadores de ceratopatia bolhosa sintomática

Objetivo: Avaliar o efeito do cross-linking corneal (CCL) nos portadores de ceratopatia bolhosa sintomática, e seu impacto na acuidade visual, espessura corneal e sintomatologia dolorosa. Métodos: Doze pacientes com ceratopatia bolhosa sintomática foram incluídos. Exame clínico com questionário específico para a pesquisa com escala de dor (escala visual analógica numérica), acuidade visual e mensuração da espessura corneal foi realizada pré CCL, 7, 30, 60, 180 e 365 dias após. Em todos os pacientes o tratamento com UVA-crosslinking foi realizado após abrasão do epitélio corneal em lâmpada de fenda e instilação de solução de riboflavina 0,1% a cada cinco minutos por 30 minutos. Após esse período, o paciente foi submetido à exposição à luz UVA, utilizando riboflavina e anestesia tópica a cada cinco minutos por 30 minutos. Os pacientes utilizaram colírios de ofloxacina 0,3% e lágrima artificial até completa reepitelização. O teste de Friedman foi usado para comparar as médias das freqüências da acuidade visual, sintomatologia dolorosa e espessura central da córnea. p <0.05 foi utilizado para rejeitar a hipótese nula. Resultados: Doze olhos de 12 pacientes com erosões epiteliais recorrentes foram tratados. O tempo de seguimento foi de um ano. Foi observada redução significante da dor (p<0,001). As medidas de espessura corneal e da acuidade visual não sofreram alterações estatisticamente significantes. Conclusâo: Foi constatado o potencial de aplicação do cross-linking corneal no tratamento de pacientes com dor causada por ceratopatia bolhos

Autogenous fecal peritonitis in Wistar rats with permanent bilateral carotid occlusion: morbidity, mortality and microbiological response

PURPOSE: To investigate morbidity, mortality and microbiological response to fecal peritonitis induced in Wistar rats with permanent bilateral carotid ligation (PBCL). METHODS: Fecal peritonitis was induced in 30 rats, with 10 animals in each group: Group1 - normal young animals; Group2 - normal mature animals; and Group3 - rats with PBCL after four months postoperative follow-up. Peritonitis was induced with 10% stool suspension. Morbidity and mortality were evaluated. The survival animals after seven days were euthanized for tests. For microbiological studies blood were collected from the carotids and right ventricle; and fragments of lung and peritoneum. RESULTS: The morbidity and mortality of young animals were significantly lower than in mature animals with and without PBCL. There was no difference in morbidity and mortality among mature rats with and without PBCL. The diversity of microorganisms producing septicemia was similar to native micro biota of the large bowel. CONCLUSIONS: The immune response was more efficient in young animals, represented by significant less morbidity and no natural mortality. PBLC did not affect morbidity and mortality in mature rats. The immune response to fecal peritonitis has age as an independent predictor

Analysis of VSX1 variations in Brazilian subjects with keratoconus

Purpose: To screen visual system homeobox 1 (VSX1) gene in Brazilian subjects affected with keratoconus (KCN). Methods: Seventy-three patients with KCN and 106 healthy controls were enrolled in this study. Patients were diagnosed with KCN based on eye examination and corneal topographic features according to Rabinowitz's criteria (K > 47.2, I-S > 1.4 and KISA > 100%). DNA from blood samples was extracted from donors, and the exons and exon-intron boundaries of VSX1 were sequenced. The potential impact of the identified amino acid changes was assessed with Poly-Phen2, SIFT, and PMUT analysis tools. Genotyping was confirmed by RLFP technique, which was also applied to genotype non-affected individuals. Results: We found three non-synonymous substitutions (L68H, R131S, and D105E) in VSX1 exon 1, with L68H mutation as a novel variation in this gene. In silico analysis indicated that all variations found were predicted to be probably damaging to VSX1 structure and function. Examination of R131S and L68H variations segregating in one family suggested a strong effect of these variations in increasing disease severity in the proband, which presented bilateral KCN leading to corneal grafting before the age of sixteen. We found a novel synonymous substitution (P79P) and two previously described exonic polymorphisms, with unknown roles in VSX1 pathogenesis. Conclusion:VSX1 polymorphisms found in the Brazilian population support a genetic component in KCN pathogenesis. L68H is a novel mutation, and the phenotypic data suggest that this mutation might enhance disease severity when combined with other polymorphisms. However, further investigations are needed