Asymmetric phenotype of Axenfeld-Rieger anomaly and aniridia associated with a novel PITX2 mutation

PurposeTo evaluate the asymmetry of the anterior segment phenotype between the two eyes of a patient with Axenfeld-Rieger syndrome (ARS).MethodsThe entire database of a tertiary glaucoma practice was screened for patients with ARS. The medical records of patients with ARS were reviewed. The clinical characteristics of ocular examination of the two eyes of each patient were recorded and compared. Dental and medical information were also reviewed where available. The anterior segment phenotype was tabulated to assess asymmetry. Asymmetric anterior segment characteristics of patients with ARS were compared with reported cases in the literature.ResultsEight patients with ARS were identified from screening of more than 5,000 patients of a tertiary glaucoma practice. All patients had Axenfeld-Rieger anomaly in both eyes except one patient presented with an asymmetric phenotype of the anterior segment with features of Axenfeld-Rieger anomaly in one eye, but aniridia in the other eye. This patient had non-ocular findings including flat midface, hypodontia with lack of an upper incisor, and redundant periumbilical skin, typical for ARS. A heterozygous C>T nucleotide substitution was identified in exon 4 of the pituitary homeobox 2 (PITX2) gene, resulting in the replacement of a glutamine codon (CAG) with a stop codon (TAG) at amino acid position 67. This mutation is denoted c.199C>T at the cDNA level or p.Gln67Stop (or Q67X) at the protein level. Only three cases with asymmetric anterior segment phenotype between the two eyes of a patient with AGS have been reported in the literature.ConclusionsVariability in phenotype may occur between the two eyes of an individual affected by ARS. The current case undermines the advantage of genetic testing to correctly diagnose a rare disease

Glaucoma spectrum and age-related prevalence of individuals with FOXC1 and PITX2 variants

Published online 3 May 2017Variation in FOXC1 and PITX2 is associated with Axenfeld-Rieger syndrome, characterised by structural defects of the anterior chamber of the eye and a range of systemic features. Approximately half of all affected individuals will develop glaucoma, but the age at diagnosis and the phenotypic spectrum have not been well defined. As phenotypic heterogeneity is common, we aimed to delineate the age-related penetrance and the full phenotypic spectrum of glaucoma in FOXC1 or PITX2 carriers recruited through a national disease registry. All coding exons of FOXC1 and PITX2 were directly sequenced and multiplex ligation-dependent probe amplification was performed to detect copy number variation. The cohort included 53 individuals from 24 families with disease-associated FOXC1 or PITX2 variants, including one individual diagnosed with primary congenital glaucoma and five with primary open-angle glaucoma. The overall prevalence of glaucoma was 58.5% and was similar for both genes (53.3% for FOXC1 vs 60.9% for PITX2, P=0.59), however, the median age at glaucoma diagnosis was significantly lower in FOXC1 (6.0±13.0 years) compared with PITX2 carriers (18.0±10.6 years, P=0.04). The penetrance at 10 years old was significantly lower in PITX2 than FOXC1 carriers (13.0% vs 42.9%, P=0.03) but became comparable at 25 years old (71.4% vs 57.7%, P=0.38). These findings have important implications for the genetic counselling of families affected by Axenfeld-Rieger syndrome, and also suggest that FOXC1 and PITX2 contribute to the genetic architecture of primary glaucoma subtypes.Emmanuelle Souzeau, Owen M Siggs, Tiger Zhou, Anna Galanopoulos, Trevor Hodson, Deepa Taranath, Richard A Mills, John Landers, John Pater, James E Smith, James E Elder, Julian L Rait, Paul Giles, Vivek Phakey, Sandra E Staffieri, Lisa S Kearns, Andrew Dubowsky, David A Mackey, Alex W Hewitt, Jonathan B Ruddle, Kathryn P Burdon and Jamie E Crai

Biallelic CPAMD8 variants are a frequent cause of childhood and juvenile open-angle glaucoma

Purpose: Developmental abnormalities of the ocular anterior segment in some cases can lead to ocular hypertension and glaucoma. CPAMD8 is a gene of unknown function recently associated with ocular anterior segment dysgenesis, myopia, and ectopia lentis. We sought to assess the contribution of biallelic CPAMD8 variants to childhood and juvenile open-angle glaucoma. Design: Retrospective, multicenter case series. Participants: A total of 268 probands and their relatives with a diagnosis of childhood or juvenile open-angle glaucoma. Methods: Patients underwent a comprehensive ophthalmic assessment, with DNA from patients and their relatives subjected to genome, exome, or capillary sequencing. CPAMD8 RNA expression analysis was performed on tissues dissected from cadaveric human eyes. Main outcome measures: Diagnostic yield within a cohort of childhood and juvenile open-angle glaucoma, prevalence and risk of ophthalmic phenotypes, and relative expression of CPAMD8 in the human eye. Results: We identified rare (allele frequency -5) biallelic CPAMD8 variants in 5.7% (5/88) of probands with childhood glaucoma and 2.1% (2/96) of probands with juvenile open-angle glaucoma. When including family members, we identified 11 individuals with biallelic variants in CPAMD8 from 7 unrelated families. Nine of these individuals were diagnosed with glaucoma (9/11, 81.8%), with a mean age at diagnosis of 9.22±14.89 years, and all individuals with glaucoma required 1 or more incisional procedures to control high intraocular pressure. Iris abnormalities were observed in 9 of 11 individuals, cataract was observed in 8 of 11 individuals (72.7%), and retinal detachment was observed in 3 of 11 individuals (27.3%). CPAMD8 expression was highest in neural crest-derived tissues of the adult anterior segment, suggesting that CPAMD8 variation may cause malformation or obstruction of key drainage structures. Conclusions: Biallelic CPAMD8 variation was associated with a highly heterogeneous phenotype and in our cohorts was the second most common inherited cause of childhood glaucoma after CYP1B1 and juvenile open-angle glaucoma after MYOC. CPAMD8 sequencing should be considered in the investigation of both childhood and juvenile open-angle glaucoma, particularly when associated with iris abnormalities, cataract, or retinal detachment

Molecular characterization of a de novo t (11;18) translocation associated with Peter´s anomaly

Dissertação para obtenção do grau de Mestre em Genética Molecular e BiomedicinaPeter’s anomaly (PA) is a congenital defect of the anterior chamber of the eye. The aim of this study is molecular characterization of a de novo balanced chromosome translocation [t(11;18)(q23.3;q11.2)] identified in a proband with syndromic form of Peter´s anomaly (ectopia lentis and mild CNS abnormalities). Chromosome breakpoints were identified at nucleotide resolution. The 11q23.3 breakpoint is at position 120,097,868 (genome assembly GRCh37/hg19) within intron 3 of out of first protein homolog gene (OAF) while, 18q11.2 breakpoint in the intergenic region between CTAGE1 and RBBP8 genes, at position 20,220,714. Although OAF is disrupted, its expression level is unchanged in proband´s lymphoblastoid cell line (LCL). Cell adhesion protein Nectin 1 or PVRL1, located 500 kb upstream from 11q23.3 breakpoint, reveal 3.5 fold increase in proband’s LCL. Expression levels of additional genes from breakpoint regions are not significantly different. Furthermore, RT-qPCR confirmed that expression level CYP1B1 from chromosome 2p22.2, and EDIL3 from chromosome 5q14 are significantly changed in proband´s LCL, 16.5 fold decrease and 126 fold increase, respectively. Alterations in CYP1B1 have been reported as PA causing mutations. Therefore, mutation screening of CYP1B1 was performed and no pathogenic mutation was identified in the proband, although, a disease-causing 13 bp, homozygous deletion was identified in a Portuguese patient with PA. In conclusion, we hypothesize that PVRL1 is a candidate gene for at least some of the observed clinical features, which is elicited in mouse models by association of its paralog PVRL3 to lens and other ocular defects involving the ciliary body. Furthermore, the involvement of the POU family domain containing transcription factor (POU2F3) from 11q23.3 and CYP1B1 or EDIL3, localized outside of the breakpoint regions, in the underlying molecular pathogenesis of syndromic PA remains to be determined

An association study of PITX2 polymorphism in a cohort of patients with primary open angle glaucoma and considerations on the genetics of glaucoma

Background: Glaucoma is a major cause of blindness world-wide. There is a need for methods to identify individuals at risk of developing glaucoma, so that early treatment can prevent visual loss. Genetic screening tests offer the prospect of pre-symptomatic diagnosis of at risk individuals. There is now strong evidence that a number of different genes are associated with glaucoma susceptibility. Mutations in the PITX2 homeobox transcription factor gene disrupt normal development of the anterior segment and cause overt structural abnormalities. It is possible that, as yet undetected mutations/polymorphisms in PITX2 may produce subtle and undetected abnormalities in anterior segment structure and function that could predispose to glaucoma. Purpose: The aim of this thesis is two fold: 1. Screening for the presence of single nucleotide polymorphisms in PITX2 gene in a cohort of 100 unrelated primary open angle glaucoma/ ocular hypertension patients, 10 Posterior embryotoxon subjects and 100 age and ethnically matched controls to establish the mutation spectrum. 2. Identification, phenotyping and recruitment for genetic studies of primary open angle glaucoma patients with strong family history of glaucoma. Materials and methods: 1. 100 primary open angle glaucoma patients and 60 age and ethnically matched controls were enrolled in the study. Patients and controls were phenotyped and a blood sample for DNA extraction collected. PITX2 exon-specific primers were used to PCR amplify patient and control DNA. Direct sequencing was used to screen for sequence alterations in the entire coding sequence of PITX2 gene. Concurrently, polymorphic sites reported in the PITX2 gene were identified from the NCBI and Ensemble databases and the frequency of polymorphic sites was investigated. The SHEsis and UNPHASED software packages were used for statistical analysis. 2. Patients diagnosed with glaucoma and strong family history were identified from Glaucoma Unit at Sunderland Eye Infirmary, phenotyped and enrolled in the study. The pedigrees were constructed and interested relatives enrolled in the study and phenotyped. A sample of blood for DNA extraction was collected from all people enrolled in the study. Results: 1. Direct sequencing did not identify any sequence variation in the coding region. 26 PITX2 polymorphic sites were identified from the internet databases, including five in the coding sequence. Sixteen non coding SNPs were confirmed within our study group and SNP frequencies were examined. None of the coding sequence SNPs was identified in our cohort, demonstrating a high degree of sequence conservation. Also, none of the SNPs confirmed in this study group showed an increased frequency in the primary open angle glaucoma group compared with the control group. 2. Thirty-three pedigrees were identified with strong family of glaucoma during the time allowed for patient recruitment. Of these, twenty-two agreed to take part in the study. Thirteen pedigrees are presented in this study, mostly demonstrating autosomal dominant inheritance. Conclusion: There is ample evidence to suggest that genetics play an important role in unravelling the pathogenesis of glaucoma. Identification and recruitment of patients for genetic studies is an essential step and the role of the clinician in this process is paramount. Also, developmental glaucoma genes are an important group of genes to be screened in primary open angle glaucoma/ocular hypertension patients, as they may play a role in the pathogenesis of this preventable blinding disease.EThOS - Electronic Theses Online ServiceRoyal College of Surgeons, Edinburgh : Glaucoma Research and Development at Sunderland (GRAD@S) fund : Pfizer OphthalmicGBUnited Kingdo

Alternative Genetic Diagnoses in Axenfeld-Rieger Syndrome Spectrum

Axenfeld–Rieger anomaly (ARA) is a specific ocular disorder that is frequently associated with other systemic abnormalities. PITX2 and FOXC1 variants explain the majority of individuals with Axenfeld–Rieger syndrome (ARS) but leave ~30% unsolved. Here, we present pathogenic/likely pathogenic variants in nine families with ARA/ARS or similar phenotypes affecting five different genes/regions. USP9X and JAG1 explained three families each. USP9X was recently linked with syndromic cognitive impairment that includes hearing loss, dental defects, ventriculomegaly, Dandy–Walker malformation, skeletal anomalies (hip dysplasia), and other features showing a significant overlap with FOXC1-ARS. Anterior segment anomalies are not currently associated with USP9X, yet our cases demonstrate ARA, congenital glaucoma, corneal neovascularization, and cataracts. The identification of JAG1 variants, linked with Alagille syndrome, in three separate families with a clinical diagnosis of ARA/ARS highlights the overlapping features and high variability of these two phenotypes. Finally, intragenic variants in CDK13, BCOR, and an X chromosome deletion encompassing HCCS and AMELX (linked with ocular and dental anomalies, correspondingly) were identified in three additional cases with ARS. Accurate diagnosis has important implications for clinical management. We suggest that broad testing such as exome sequencing be applied as a second-tier test for individuals with ARS with normal results for PITX2/FOXC1 sequencing and copy number analysis, with attention to the described genes/regions

PERIOCULAR MESENCHYME HETEROGENEITY DURING MORPHOGENESIS OF THE VERTEBRATE OCULAR ANTERIOR SEGMENT

The vertebrate eye is a complex organ, responsible for the primary sense with which we interact with our environment: vision. Development of the eye is a tightly regulated process, controlled by a vast network of genes. This process begins with eye morphogenesis, when the eye structure is formed through a series of morphogenetic movements and culminates in the creation of the optic cup, lens, and presumptive optic stalk. Next, retinal differentiation creates the critical cell layers of the retina needed to process light waves that enter the eye, including rod and cone photoreceptors, interneurons, and support cells. Failure in either one of these steps can result in developmental congenital defects or disorders which can range from relatively minor visual impairment to blindness. While extensive research has already been done to understand congenital blinding disorders, we have only begun to scratch the surface of understanding key parts of eye development and the intrinsic and extrinsic factors that go into regulating the process. While many researchers focus their work on eye morphogenesis or retinal differentiation in relation to these disorders, little attention is given to a process that overlaps with both stages: formation of the anterior segment (AS). The AS is a collection of individual structures located in the front third of the eye and has different developmental origins from the rest of the eye. AS structures are formed from a specialized group of neural crest-derived cells called Periocular Mesenchyme (POM) cells, which migrate around the forming optic cup to colonize the space between the anterior most regions of the optic cup and the surface ectoderm. Once positioned in this space, POM cells will specify into their cell types and form structures that include the corneal stroma and endothelium, the iris and ciliary body, and drainage network of the iridocorneal angle. Failure in the migration or specification of these cells can result in Anterior Segment Dysgenesis and a spectrum of visual impairment. Presently, little is understood about this transient cell population, its origins within the neural crest, its migration to the AS, or its genetic makeup during colonization and differentiation of the AS. In this dissertation, I examine in detail the POM cell population in zebrafish, focusing on its heterogeneity as well as its origins within the neural crest. In Chapter 3, I utilize several transgenic lines for POM-related transcription factors to demonstrate the heterogeneity of the POM through distribution mapping, co-expression analysis, migration behavior, and single cell RNA sequencing. I provide the first large scale evidence that the POM is a collection of subpopulations working in conjunction with one another to form the AS. In Chapter 4 I outline the role of the neural crest in POM formation and the consequences that loss of neural crest has on the development of the POM, as well as the AS. My findings provide a new lens with which to understand AS development and the cells responsible for it, and ultimately uncover new pathways for understanding the biological mechanisms involved in Anterior Segment Dysgenesis and AS-related blinding disorders

An investigation of genetic risk factors in primary open-angle glaucoma

Primary open-angle glaucoma (POAG) is a multifactorial disease with a strong genetic component. Notably however, few genes have been robustly associated with POAG in the general population. Genes in which mutation causes anterior segment angle anomalies, including LMX1B and FOXC1 are associated with a high incidence of glaucoma to about 33-75% and are strong candidates for glaucoma susceptibility. In addition, growth factors including TGFβ2 and BMP4 act in concert to maintain a balance between extracellular matrix (ECM) deposition and degradation and may play a role in glaucoma pathogenesis through misregulation of ECM synthesis of the trabecular meshwork (TM). Furthermore, OPTN E50K mutation, a known genetic locus for POAG, has been shown to account for a high percentage of 13.5% of familial normal-tension glaucoma (NTG) in individuals of white British origin. In this study, the contribution of variation at the LMX1B, FOXC1, TGFβ2 and BMP4 loci to risk of glaucoma was investigated in a case-control genetic association study in a cohort of white British descent recruited in the North-East of England comprised of 272 patients with high-tension glaucoma (HTG), 37 patients with NTG, 58 patients with ocular hypertension (OHT), and 276 normal controls. The role of OPTN E50K mutation in these unrelated white British individuals with POAG was also examined. No significant associations were identified for FOXC1, TGFβ2 and BMP4. The OPTN E50K mutation was also absent in this cohort. The study identified a significant under representation of two LMX1B haplotypes [ATG; P = 5.0E-4 (permutation P = 0.01), GCAGAC; P = 5.0E-4 (permutation P = 0.0150)] among the POAG individuals compared to the control population, consistent with a 0.3 fold decreased risk of developing POAG. A replication study involving a second cohort of 222 NTGs and 108 HTGs recruited in London showed a similar distribution of the ATG haplotype (P = 0.0047) but did not withstand permutation testing. In conclusion, LMX1B haplotypes may influence susceptibility to develop POAG in the white British population, suggesting altered LMX1B function predisposes to glaucomatous damage