Identification of a Novel ZNF469 Mutation in a Pakistani Family With Brittle Cornea Syndrome.

PURPOSE: Brittle cornea syndrome (BCS) is a rare recessive disorder affecting connective tissues, most prominently in the eye. Pathogenic mutations causing BCS have been identified in PRDM5 and ZNF469 genes. This study investigates the genetic cause of BCS in a large, consanguineous Pakistani family with 4 affected and 3 unaffected individuals. METHODS: The coding region and exon-intron splice junctions of PRDM5 and ZNF469 genes were amplified by polymerase chain reaction, and bidirectional Sanger sequencing was performed to find the pathogenic change responsible for causing the disease in the family. RESULTS: A novel homozygous duplication c.9831dupC (p.Arg3278GlnfsX197) in the ZNF469 gene was identified, which was found to be co-segregating with the disease in the family. CONCLUSIONS: This is the first report of a ZNF469 homozygous mutation causing a BCS phenotype in a consanguineous Pakistani family. Our data extend the mutation spectrum of ZNF469 variants implicated in BCS.

Mutational analysis of the CYP1B1 gene in Pakistani primary congenital glaucoma patients: Identification of four known and a novel causative variant at the 3' splice acceptor site of intron 2.

Primary congenital glaucoma (PCG) causes blindness in early age. It has an autosomal recessive pattern of inheritance, hence is more prevalent in populations with frequent consanguineous marriages that occur in the Pakistani population. Mutations in the CYP1B1 gene are commonly associated with PCG. The aim of the present study was to identify genetic mutations in the CYP1B1 gene in PCG cases belonging to 38 Pakistani families. DNA was extracted using blood samples collected from all enrolled patients, their available unaffected family members and controls. Direct sequencing of the CYP1B1 gene revealed a novel 3' splice acceptor site causative variant segregating in an autosomal recessive manner in a large consanguineous family with four PCG-affected individuals. The novel variant was not detected in 93 ethnically matched controls. Furthermore, four already reported mutations, including p.G61E, p.R355X, p.R368H, and p.R390H were also detected in patients belonging to nine different families. All identified causative variants were evaluated by computational programs, that is, SIFT, PolyPhen-2, and MutationTaster. Pathogenicity of the novel splice site variant identified in this study was analyzed by Human Splicing Finder and MaxEntScan. Ten out of 38 families with PCG had the disease due to CYP1B1 mutations, suggesting CYP1B1 was contributing to PCG in these Pakistani patients. Identification of this novel 3' splice acceptor site variant in intron 2 is the first report for the CYP1B1 gene contributing to genetic heterogeneity of disease.

Correction: Loss of the BMP Antagonist, SMOC-1, Causes Ophthalmo-Acromelic (Waardenburg Anophthalmia) Syndrome in Humans and Mice.

[This corrects the article DOI: 10.1371/journal.pgen.1002114.].

Delineation of Novel Autosomal Recessive Mutation in GJA3 and Autosomal Dominant Mutations in GJA8 in Pakistani Congenital Cataract Families.

Congenital cataract is a clinically and genetically heterogeneous disease. The present study was undertaken to find the genetic cause of congenital cataract families. DNA samples of a large consanguineous Pakistani family were genotyped with a high resolution single nucleotide polymorphism Illumina microarray. Homozygosity mapping identified a homozygous region of 4.4 Mb encompassing the gene GJA3. Sanger sequence analysis of the GJA3 gene revealed a novel homozygous variant c.950dup p.(His318ProfsX8) segregating in an autosomal recessive (AR) manner. The previously known mode of inheritance for GJA3 gene mutations in cataract was autosomal dominant (AD) only. The screening of additional probands (n = 41) of cataract families revealed a previously known mutation c.56C>T p.(Thr19Met) in GJA3 gene. In addition, sequencing of the exon-intron boundaries of the GJA8 gene in 41 cataract probands revealed two additional mutations: a novel c.53C>T p.(Ser18Phe) and a known c.175C>G p.(Pro59Ala) mutation, both co-segregating with the disease phenotype in an AD manner. All these mutations are predicted to be pathogenic by in silico analysis and were absent in the control databases. In conclusion, results of the current study enhance our understanding of the genetic basis of cataract, and identified the involvement of the GJA3 in the disease etiology in both AR and AD manners.

Variants in the PRPF8 Gene are Associated with Glaucoma.

Glaucoma is the cause of irreversible blindness worldwide. Mutations in six genes have been associated with juvenile- and adult-onset familial primary open angle glaucoma (POAG) prior to this report but they explain only a small proportion of the genetic load. The aim of the study is to identify the novel genetic cause of the POAG in the families with adult-onset glaucoma. Whole exome sequencing (WES) was performed on DNA of two affected individuals, and predicted pathogenic variants were evaluated for segregation in four affected and three unaffected Dutch family members by Sanger sequencing. We identified a pathogenic variant (p.Val956Gly) in the PRPF8 gene, which segregates with the disease in Dutch family. Targeted Sanger sequencing of PRPF8 in a panel of 40 POAG families (18 Pakistani and 22 Dutch) revealed two additional nonsynonymous variants (p.Pro13Leu and p.Met25Thr), which segregate with the disease in two other Pakistani families. Both variants were then analyzed in a case-control cohort consisting of Pakistani 320 POAG cases and 250 matched controls. The p.Pro13Leu and p.Met25Thr variants were identified in 14 and 20 cases, respectively, while they were not detected in controls (p values 0.0004 and 0.0001, respectively). Previously, PRPF8 mutations have been associated with autosomal dominant retinitis pigmentosa (RP). The PRPF8 variants associated with POAG are located at the N-terminus, while all RP-associated mutations cluster at the C-terminus, dictating a clear genotype-phenotype correlation.

Retinoblastoma presenting as total hyphema: Three year follow-up.

Retinoblastoma very rarely presents as total hyphema. Our patient presented at an early age of 7 months. Follow-up of 3 years shows that unilateral group E retinoblastoma was treated successfully with enucleation and adjuvant chemotherapy. The fellow eye remained normal during this period. The factors associated with delay in treatment are also described. Reports like the present case add to the information available about advanced staging of retinoblastoma at the time of presentation, seen in cases with spontaneous hyphema due to the tumor.

Tumor Regression Patterns in Retinoblastoma.

OBJECTIVE: To observe the types of tumor regression after treatment, and identify the common pattern of regression in our patients. STUDY DESIGN: Descriptive study. PLACE AND DURATION OF STUDY: Department of Pediatric Ophthalmology and Strabismus, Al-Shifa Trust Eye Hospital, Rawalpindi, Pakistan, from October 2011 to October 2014. METHODOLOGY: Children with unilateral and bilateral retinoblastoma were included in the study. Patients were referred to Pakistan Institute of Medical Sciences, Islamabad, for chemotherapy. After every cycle of chemotherapy, dilated fundus examination under anesthesia was performed to record response of the treatment. Regression patterns were recorded on RetCam II. RESULTS: Seventy-four tumors were included in the study. Out of 74 tumors, 3 were ICRB group A tumors, 43 were ICRB group B tumors, 14 tumors belonged to ICRB group C, and remaining 14 were ICRB group D tumors. Type IV regression was seen in 39.1% (n=29) tumors, type II in 29.7% (n=22), type III in 25.6% (n=19), and type I in 5.4% (n=4). All group A tumors (100%) showed type IV regression. Seventeen (39.5%) group B tumors showed type IV regression. In group C, 5 tumors (35.7%) showed type II regression and 5 tumors (35.7%) showed type IV regression. In group D, 6 tumors (42.9%) regressed to type II non-calcified remnants. CONCLUSION: The response and success of the focal and systemic treatment, as judged by the appearance of different patterns of tumor regression, varies with the ICRB grouping of the tumor.

Anisometropia and Ptosis in Patients with Monocular Elevation Deficiency.

OBJECTIVE: To determine the effect of ptosis on the refractive error in eyes having monocular elevation deficiency. STUDY DESIGN: Case series. PLACE AND DURATION OF STUDY: Al-Shifa Trust Eye Hospital, Rawalpindi, from January 2011 to January 2014. METHODOLOGY: Visual acuity, refraction, orthoptic assessment and ptosis evaluation of all patients having monocular elevation deficiency (MED) were recorded. Shapiro-Wilk test was used for tests of normality. Median and interquartile range (IQR) was calculated for the data. Non-parametric variables were compared, using the Wilcoxon signed ranks test. P-values of <0.05 were considered significant. RESULTS: Atotal of of 41 MED patients were assessed during the study period. Best corrected visual acuity (BCVA) and refractive error was compared between the eyes having MED and the unaffected eyes of the same patient. The refractive status of patients having ptosis with MED were also compared with those having MED without ptosis. Astigmatic correction and vision had significant difference between both the eyes of the patients. Vision was significantly different between the two eyes of patients in both the groups having either presence or absence of ptosis (p=0.04 and p < 0.001, respectively). CONCLUSION: Significant difference in vision and anisoastigmatism was noted between the two eyes of patients with MED in this study. The presence or absence of ptosis affected the vision but did not have a significant effect on the spherical equivalent (SE) and astigmatic correction between both the eyes.

Identification of Novel Variants in LTBP2 and PXDN Using Whole-Exome Sequencing in Developmental and Congenital Glaucoma.

BACKGROUND: Primary congenital glaucoma (PCG) is the most common form of glaucoma in children. PCG occurs due to the developmental defects in the trabecular meshwork and anterior chamber of the eye. The purpose of this study is to identify the causative genetic variants in three families with developmental and primary congenital glaucoma (PCG) with a recessive inheritance pattern. METHODS: DNA samples were obtained from consanguineous families of Pakistani ancestry. The CYP1B1 gene was sequenced in the affected probands by conventional Sanger DNA sequencing. Whole exome sequencing (WES) was performed in DNA samples of four individuals belonging to three different CYP1B1-negative families. Variants identified by WES were validated by Sanger sequencing. RESULTS: WES identified potentially causative novel mutations in the latent transforming growth factor beta binding protein 2 (LTBP2) gene in two PCG families. In the first family a novel missense mutation (c.4934G>A; p.Arg1645Glu) co-segregates with the disease phenotype, and in the second family a novel frameshift mutation (c.4031_4032insA; p.Asp1345Glyfs*6) was identified. In a third family with developmental glaucoma a novel mutation (c.3496G>A; p.Gly1166Arg) was identified in the PXDN gene, which segregates with the disease. CONCLUSIONS: We identified three novel mutations in glaucoma families using WES; two in the LTBP2 gene and one in the PXDN gene. The results will not only enhance our current understanding of the genetic basis of glaucoma, but may also contribute to a better understanding of the diverse phenotypic consequences caused by mutations in these genes.

A Novel Homozygous Mutation in FOXC1 Causes Axenfeld Rieger Syndrome with Congenital Glaucoma.

BACKGROUND: Anterior segment dysgenesis (ASD) disorders are a group of clinically and genetically heterogeneous phenotypes in which frequently cornea, iris, and lens are affected. This study aimed to identify novel mutations in PAX6, PITX2 and FOXC1 in families with anterior segment dysgenesis disorders. METHODS: We studied 14 Pakistani and one Mexican family with Axenfeld Rieger syndrome (ARS; n = 10) or aniridia (n = 5). All affected and unaffected family members underwent full ophthalmologic and general examinations. Total genomic DNA was isolated from peripheral blood. PCR and Sanger sequencing were performed for the exons and intron-exon boundaries of the FOXC1, PAX6, and PITX2 genes. RESULTS: Mutations were identified in five of the 15 probands; four variants were novel and one variant was described previously. A novel de novo variant (c.225C>A; p.Tyr75*) was identified in the PAX6 gene in two unrelated probands with aniridia. In addition, a known variant (c.649C>T; p.Arg217*) in PAX6 segregated in a family with aniridia. In the FOXC1 gene, a novel heterozygous variant (c.454T>C; p.Trp152Arg) segregated with the disease in a Mexican family with ARS. A novel homozygous variant (c.92_100del; p.Ala31_Ala33del) in the FOXC1 gene segregated in a Pakistani family with ARS and congenital glaucoma. CONCLUSIONS: Our study expands the mutation spectrum of the PAX6 and FOXC1 genes in individuals with anterior segment dysgenesis disorders. In addition, our study suggests that FOXC1 mutations, besides typical autosomal dominant ARS, can also cause ARS with congenital glaucoma through an autosomal recessive inheritance pattern. Our results thus expand the disease spectrum of FOXC1, and may lead to a better understanding of the role of FOXC1 in development.

Congenital Cataract, Nasolacrimal Duct Obstruction, and Methicillin-Resistant Staphylococcus aureus Conjunctivitis: When to Operate?

Methicillin-resistant Staphylococcus aureus is one of the toughest organisms to treat, especially in cases where intraocular surgery is contemplated, because the risks are aggravated. Conjunctival swab culture and sensitivity tests are significant when there is history of recent hospitalization. In this report, an infant with successful cataract surgery after elimination of the organism is presented.

Whole exome sequencing identifies a heterozygous missense variant in the PRDM5 gene in a family with Axenfeld-Rieger syndrome.

Axenfeld-Rieger syndrome (ARS) is a disorder affecting the anterior segment of the eye, often leading to secondary glaucoma and several systemic malformations. It is inherited in an autosomal dominant fashion that has been associated with genetic defects in PITX2 and FOXC1. Known genes CYP1b1, PITX2, and FOXC1 were excluded by Sanger sequencing. The purpose of current study is to identify the underlying genetic causes in ARS family by whole exome sequencing (WES). WES was performed for affected proband of family, and variants were prioritized based on in silico analyses. Segregation analysis of candidate variants was performed in family members. A novel heterozygous PRDM5 missense variant (c.877A>G; p.Lys293Glu) was found to segregate with the disease in an autosomal dominant fashion. The novel missense variant was absent from population-matched controls, the Exome Variant Server, and an in-house exome variant database. The Lys293Glu variant is predicted to be pathogenic and affects a lysine residue that is conserved in different species. Variants in the PRDM5 gene were previously identified in anterior segment defects, i.e., autosomal recessive brittle cornea syndrome and keratoconus. The results of this study suggest that genetic variants in PRDM5 can lead to various syndromic and nonsyndromic disorders affecting the anterior segment of the eye.

Ophthalmic manifestations of linear nevus sebaceous/organoid nevus syndrome.

Linear Nevus Sebaceous Syndrome (LNSS) is a rare sporadic oculoneurocutaneous disorder, also classified as Organoid Nevus Syndrome. It consists of a triad of midline facial linear nevus sebaceous, central nervous system and ocular abnormalities. To the best of authors' knowledge ophthalmic features of LNSS have never been reported in Pakistani population. We report two cases of LNSS, associated with multiple cutaneous nevus sebaceous lesions, complex ocular choristomas and rare bilateral presentation in one patient. Ocular choristomas included limbal dermoids, dermolipomas at superior fornices and chroidal choristoma. Ocular surface was successfully reconstructed by excision of limbal dermoids, partial keratectomy and amniotic membrane transplant.

Identification of novel CYP1B1 gene mutations in patients with primary congenital and primary open-angle glaucoma.

BACKGROUND: CYP1B1 is the most commonly mutated gene in primary congenital glaucoma (PCG), and mutations have also been identified in primary open-angle glaucoma (POAG). This study was undertaken to describe mutations in CYP1B1 in patients and families with PCG and POAG from Pakistan. DESIGN: Case-control series. PARTICIPANTS: Forty families, 190 sporadic POAG cases and 140 controls from Pakistan. METHODS: Patients and healthy individuals of one consanguineous Pakistani family were genotyped with high-resolution single nucleotide polymorphism microarrays. Homozygosity mapping was performed using HomozygosityMapper. Direct sequencing of CYP1B1 gene was performed in probands of the families, sporadic POAG cases and control individuals. MAIN OUTCOME MEASURES: Mutations in the CYP1B1 gene in PCG and POAG patients. RESULTS: Homozygosity mapping in a consanguineous Pakistani family revealed one 11-Mb homozygous region encompassing the CYP1B1 gene. A homozygous CYP1B1 missense mutation (p.Arg390His) was identified in this family. Sequence analysis of CYP1B1 in 39 additional families revealed one known and three novel homozygous mutations in PCG (p.Ala288Pro, p.Asp242Ala, p.Arg355* and p.Arg290Profs*37). In POAG, one novel heterozygous missense mutation (p.Asp316Val) was identified in one family and a previously reported mutation (p.Glu229Lys) was identified in three families. Analysis of CYP1B1 in a panel of 190 sporadic POAG patients revealed three novel heterozygous variants (p.Thr234Lys, p.Ala287Pro and p.Gln362*) and three previously reported heterozygous variants (p.Gly61Glu, p.Glu229Lys and p.Arg368His). The p.Glu229Lys variant was significantly associated with POAG (P = 0.03; odds ratio 2.49). CONCLUSIONS: This study confirms that CYP1B1 mutations are associated with POAG and PCG in the Pakistani population.

Duane's syndrome: surgical outcome and non ophthalmologic associations.

BACKGROUND: Duane retraction syndrome (DRS) is the most common of the ocular congenital cranial dysinnervation disorders .This study evaluates the types of Duane syndrome and its management in patients presenting to the paediatric and strabismus unit of a tertiary care eye hospital. METHODS: This case series study involved 41 patients diagnosed with Duane syndrome between January 2007 and December 2009. History of presenting complaints, past treatment and family history were recorded. Ocular examination and orthoptic assessment was carried out RESULTS: Forty one patients were included in this case series study. It involved 10 right eyes, 27 left eyes and both eyes of 4 patients. There were 26 females and 15 males. Type-1 Duane syndrome was present in 28 (68.3%), type 2 in 8 (19.5%), Type-3 in 4 (9.8%) and type-4 with synergistic divergence was present in 1 (2.4%) patient. Comorbidity was present in 6(14.6%) patients. Surgery was carried out in 26 (63.4%) patients either for  abnormal head posturing or significant upshoots or down shoots. Upshoots noted in 21 eyes, were completely or partially resolved in 15 cases. Among 4 patients with down shoots on adduction, complete resolution was seen in 1. The pre and post-operative measurements of horizontal deviation showed statistically significant difference in Duane type-1 and 2, where as in Duane type-3 it was not significant. One patient with type-4 Duane did not undergo surgery. CONCLUSIONS: Recession of the horizontal recti is more effective in treating the upshoot or down shoot associated with DRS as compared to recession and y-split of the horizontal muscle.

Monocular elevation deficiency with associated dextrocardia and situs inversus.

A patient having monocular elevation deficiency with associated dextrocardia and situs inversus is reported. Review of the literature regarding ocular features described in association with dextrocardia is also presented.

Retinoblastoma in an adult.

BACKGROUND: Retinoblastoma is the most common pediatric ocular tumour. It may rarely present in adults. The present case adds to the number of 26 cases already published in literature since 1919 till 2013. Our aim is to highlight the rare occurrence of retinoblastoma in adults along with its features which differentiate it from paediatric retinoblastoma. CASE PRESENTATION: We describe a case of adult onset retinoblastoma (group E, according to the international classification of retinoblastoma) occurring in a 25 year old male. He presented with decreasing visual acuity in the right eye of 4 months duration. He had neo-vascular glaucoma and pseudohypopyon. B scan ultrsonography of his right eye showed intraocular growth without any calcification. The CT scan of the orbits and brain showed intraocular growth in the right eye with no calcification. Enucleation of the right eye was carried out. Retinoblastoma was confirmed on histopathology of the enuleated globe. CONCLUSIONS: The present case adds to the number of adult Rb patients reported in literature. Early detection to salvage the life can be made possible if the clinician keeps a high index of suspicion when observing retinal mass of adult onset. Proper counselling of the patient in order to seek his full involvement in management may help in improving the prognosis of the disease.

Clinical course of retinoblastoma.

OBJECTIVE: To determine the clinical manifestations and results of current treatment for patients with retinoblastoma (Rb) in a tertiary care eye hospital in the north west of Pakistan. STUDY DESIGN: Case series. PLACE AND DURATION OF STUDY: Al-Shifa Trust Eye Hospital, Rawalpindi, Pakistan, from January 2006 and December 2009. METHODOLOGY: The data of 139 patients diagnosed as having retinoblastoma was collected. Gender, age at diagnosis, laterality, presenting sign, classification of tumour, treatment modality and outcome were noted. RESULTS: The mean age of presentation in this patients ranged from 6 to 50 months (mean: 24.05 ± 10.74 months). The most common presenting sign was leucocoria in 78 eyes (44.1%). One hundred and one (72.7%) patients had unilateral retinoblastoma. Using the International Classification of Retinoblastoma (ICRB), 135 (76.3%) eyes were placed in group-E. one hundred and twenty four (77.5%) eyes were enucleated or exenterated while globe preservation was achieved by chemoreduction and/or focal therapy in the rest of the treated eyes (n = 36, 22.5%). Twenty three (16.5%) cases were lost to follow-up before one year. Ninety two (66.2%) patients survived, being free of tumour, at least one year after the completion of treatment. CONCLUSION: Most children with Rb showed an advanced stage of tumour at the time of diagnosis. Measures to improve the rate of globe preservation and patient survival by early diagnosis and intervention are the need of the hour.

Effects of Artisan aphakic intraocular lens on central corneal thickness and intra ocular pressure in pediatric eyes with crystalline subluxated lenses.

PURPOSE: To study effects of Artisan iris fixated intraocular lens (IOL) on central corneal thickness (CCT) and intraocular pressure (IOP) in pediatric eyes with crystalline subluxated lenses. MATERIALS AND METHODS: The study included 17 eyes undergoing Artisan aphakic IOL implantation after lensectomy for subluxated crystalline lenses. CCT and IOP measurements were recorded pre-operatively and post-operatively taking the mean of 4 post-operative visits. Patients were divided into Group A (n = 8) including patients with lensectomy and iris fixation of Artisan IOL as a primary procedure and Group B (n = 9) including patients in which lensectomy was carried out as a primary surgery and Artisan IOL fixation as a secondary procedure. RESULTS: Children ranged in age from 08 years to 16 years, mean 11.59 ± 2.96 years. Follow-up period ranged from 7 months to 16 months, mean 11.24 months ± 4.27. Mean pre-operative and post-operative IOP in Group A was 14.88 ± 2.80 and 14.16 ± 0.59 respectively (P = 0.528). In Group B it was 12.44 ± 2.79 and 14.44 ± 1.15 respectively (P = 0.080). Mean pre-operative and post-operative CCT in Group A was 529.13 ± 24.23 and 529.87 ± 17.46 respectively (P = 0.674). In Group B it was 567.33 ± 29.13 and 568.83 ± 25.69 respectively (P = 0.859). CONCLUSIONS: Primary and secondary Artisan aphakic IOL implantation did not cause any significant changes in corneal thickness or IOP during the follow-up period.

Retinal detachment in paediatric patients.

OBJECTIVE: To assess the causes of retinal detachment in children and the various operative procedures requiring vitreoretinal surgical intervention for the same. STUDY DESIGN: Case series. PLACE AND DURATION OF STUDY: Department of Ophthalmology, Al-Shifa Trust Eye Hospital, Rawalpindi, from January 2006 to May 2009. METHODOLOGY: A total of 281 eyes of 258 patients, (aged 0-18 years) who underwent vitreo-retinal surgical intervention for retinal detachment were included. Surgical log was searched for the type of retinal detachment and its causes. Frequencies of various interventions done in these patients viz. vitrectomy, scleral buckle, use of tamponading agents, laser photocoagulation and cryotherapy were noted. Results were described as descriptive statistics. RESULTS: Myopia was the cause in 62 (22.1%) and trauma in 51 (18.1%) of the eyes. Total retinal detachment (RD) was treated in 94 (33.5%) eyes, sub total RD in 36 (12.8%), recurrent RD in 32 (11.4%), giant retinal tear in 28 (10%), tractional RD in 15 (5.3%) and exudative RD in 2 (0.7%). Prophylactic laser or cryotherapy was applied in 74 (26.3%) of the eyes. Pars plana vitrectomy (PPV) was carried out in 159 (56.6%) eyes while scleral buckle procedure was done in 129 (45.9%) eyes. Silicon oil was used in 149 (53%), perfluorocarbon liquid in 32 (11.4%) and gas tamponade in 20 (7.1%) eyes. CONCLUSION: The most common cause of retinal detachment in paediatric patients was myopia, followed by trauma. Total RD was more common as compared to the other types. The most common procedure adopted was pars plana vitrectomy followed by scleral buckle procedure.