Incremental Concentrations of Tacrolimus Eye Drops as a Strategy for the Management of Severe Vernal Keratoconjunctivitis.

Purpose: To assess the effect of different concentrations of tacrolimus eye suspension on the epithelium and stromal keratocytes of human corneas and investigate whether it can be safely used for severe cases of vernal keratoconjunctivitis (VKC). Methods: Tacrolimus eye suspension was prepared in a range of concentrations of 0.005%, 0.01%, 0.05%, 0.1%, and 0.2%. Molecular analysis was performed ex vivo on human corneas (n = 18), obtained from the eye bank. Transparency and thickness of each cornea were measured while live/dead staining was performed using a triple labeling assay. An incremental concentration approach was then tested on three severe cases of VKC. Results: All tested tacrolimus concentrations showed no significant changes in corneal thickness or transparency. In corneas treated with 0.1%, rare scattered dead cells were observed, while the folds of corneal surfaces were mostly viable, unlike concentrations higher than 0.1% and lower than 0.05%. Stromal cell densities were highest in the 0.1% tacrolimus treatment condition. Incremental concentrations of tacrolimus suspension were shown to significantly improve VKC cases, where the concentration used for each case depended on the severity of the case. Conclusions: Topical administration of tacrolimus was not toxic to human corneal cells at all tested concentrations, and the 0.1% concentration has shown the best viability of the corneal tissue. Tacrolimus eye suspension was shown to be safe and effective for use in severe VKC and is proposed as a topical ocular immunosuppressant drug enabling clinicians to incrementally increase the drug concentration according to the clinical severity of the disease to achieve the optimal therapeutic response.

Frameshift variant in MITF gene in a large family with Waardenburg syndrome type II and a co-segregation of a C2orf74 variant.

Waardenburg syndrome (WS) is a hereditary disorder affecting the auditory system and pigmentation of hair, eyes, and skin. Different variants of the disease exist with the involvement of mutation in six genes. The aim of the study is to identify the genetic defects underlying Waardenburg syndrome in a large family with multiple affected individuals. Here, in this study, we recruited a large family with eleven affected individuals segregating WS type 2. We performed whole genome SNP genotyping, whole exome sequencing and segregation analysis using Sanger approach. Whole genome SNP genotyping, whole exome sequencing followed by Sanger validation of variants of interest identified a novel single nucleotide deletion mutation (c.965delA) in the MITF gene. Moreover, a rare heterozygous, missense damaging variant (c.101T>G; p.Val34Gly) in the C2orf74 has also been identified. The C2orf74 is an uncharacterized gene present in the linked region detected by DominantMapper. Variants in MITF and C2orf74 follows autosomal dominant segregation with the phenotype, however, the variant in C2orf74 is incompletely penetrant. We proposed a digenic inheritance of variants as an underlying cause of WS2 in this family.

Whole genome genotyping mapped regions on chromosome 2 and 18 in a family segregating Waardenburg syndrome type II.

OBJECTIVES: Waardenburg syndrome is a rare genetic disorder. It is characterized by sensorineural hearing impairment and pigment defects of the skin, hair and iris. In some cases abnormalities in the tissues derived from neural crest have also been reported. Mutations in several genes have been reported as an underlying cause of Waardenburg syndrome. Objective of this study is to identify the chromosomal region(s) associated with Waardenburg syndrome in an extended Saudi family. METHODS: Genomic DNA was extracted from fifteen individuals of a Saudi family segregating Waardenburg syndrome. Whole genome SNP genotyping was performed to identify common identity by descent chromosomal region(s) shared by affected individuals. RESULTS: Pedigree analysis confirm autosomal dominant inheritance of Waardenburg syndrome type II in a family. Whole genome SNP genotypes were analyzed using AutoSNPa and DominantMapper tools. Shared identity by descent chromosomal regions were identified on chromosome 2 and chromosome 18. Regions were checked for known Waardenburg syndrome genes. No known gene is present in both regions. CONCLUSIONS: In summary, we identified novel chromosomal regions associated with Waardenburg syndrome type II in a Saudi family. Deep sequencing of a complete candidate regions are required to identify the gene underlying Waardenburg syndrome in this family.

Novel homozygous loss-of-function mutations in RP1 and RP1L1 genes in retinitis pigmentosa patients.

Background: Retinitis pigmentosa (RP) is a heterogeneous group of ocular dystrophy. It is challenging to identify the underlying genetic defect in individuals with RP due to huge genetic heterogeneity. This study was designed to delineate the genetic defect(s) underlying RP in extended Saudi families and to describe the possible disease mechanism.Materials and Methods: Fundus photography and a high definition optical coherence tomography (HD-OCT) were performed in order to detect the earlier stages of macular degeneration. Genomic DNA was extracted followed by genome-wide SNP genotyping and whole exome sequencing (WES). Exome data was filtered to identify the genetic variant(s) of interest.Results: Clinical examination showed that affected individuals manifest key features of RP. The fundus exam shows pale optic disc and bone spicules at the periphery. OCT shows macular degeneration as early as at the age of 4 years. Whole genome scan by SNPs identified multiple homozygous regions. WES identified a 10 bps novel insertion mutation (c.3544_3545insAGAAAAGCTG; p.Ala1182fs) in the RP1 gene in both affected individuals of family A. Affected individual from family B showed a large insertion of 48 nucleotides in the coding part of the RP1L1 gene (c.3955_3956insGGACTAAAGTAATAGAAGGGCTGCAAGAAGAGAGGGTGCAGTTAGAGG; p.Ala1319fs). Sanger sequencing validates the autosomal recessive inheritance of the mutations.Conclusion: The results strongly suggest that the insertion mutations in the RP1 and RP1L1 genes are responsible for the retinal phenotype in affected individuals from two families. Heterozygous individuals are asymptomatic carriers. We propose that the protective allele in other homozygous regions in heterozygous carriers contribute to the phenotypic variability in asymptomatic individuals.

Whole exome sequencing identified a novel single base pair insertion mutation in the EYS gene in a six generation family with retinitis pigmentosa.

Retinitis pigmentosa (RP) is a group of inherited progressive retinal dystrophies (RD) and is characterized by photoreceptor degeneration. RP is clinically and genetically heterogeneous disorder. More than 70 genes are known and, thus, identification of causative genes and mutations in known genes is challenging. This study was designed to identify the underlying genetic defect in a large extended Saudi family with multiple RP affected members. Fundus photography, Optical Coherence Tomography (OCT) and visual field perimetry were performed for affected individuals. Whole exome sequencing was used to detect the underlying genetic defect in a large family with 12 affected individuals showing autosomal recessive isolated RP. WES data analysis identified a novel insertion mutation in the EYS (eyes shut homolog) gene (c.910_911insT; p.Trp304LeufsTer8). Sanger sequencing validates the variant discovered through exome in all 12 affected individuals and showed that this mutation is segregating with RP phenotype in an autosomal recessive manner in 51 individuals of the family tested here. Our study expands the mutation spectrum of EYS gene in RP patients and extends the body of evidence that supports the importance of EYS gene in eye development.