Axenfeld-Rieger syndrome: more than meets the eye.

BACKGROUND: Axenfeld-Rieger syndrome (ARS) is characterised by typical anterior segment anomalies, with or without systemic features. The discovery of causative genes identified ARS subtypes with distinct phenotypes, but our understanding is incomplete, complicated by the rarity of the condition. METHODS: Genetic and phenotypic characterisation of the largest reported ARS cohort through comprehensive genetic and clinical data analyses. RESULTS: 128 individuals with causative variants in PITX2 or FOXC1, including 81 new cases, were investigated. Ocular anomalies showed significant overlap but with broader variability and earlier onset of glaucoma for FOXC1-related ARS. Systemic anomalies were seen in all individuals with PITX2-related ARS and the majority of those with FOXC1-related ARS. PITX2-related ARS demonstrated typical umbilical anomalies and dental microdontia/hypodontia/oligodontia, along with a novel high rate of Meckel diverticulum. FOXC1-related ARS exhibited characteristic hearing loss and congenital heart defects as well as previously unrecognised phenotypes of dental enamel hypoplasia and/or crowding, a range of skeletal and joint anomalies, hypotonia/early delay and feeding disorders with structural oesophageal anomalies in some. Brain imaging revealed highly penetrant white matter hyperintensities, colpocephaly/ventriculomegaly and frequent arachnoid cysts. The expanded phenotype of FOXC1-related ARS identified here was found to fully overlap features of De Hauwere syndrome. The results were used to generate gene-specific management plans for the two types of ARS. CONCLUSION: Since clinical features of ARS vary significantly based on the affected gene, it is critical that families are provided with a gene-specific diagnosis, PITX2-related ARS or FOXC1-related ARS. De Hauwere syndrome is proposed to be a FOXC1opathy.

Comprehensive phenotypic and functional analysis of dominant and recessive FOXE3 alleles in ocular developmental disorders.

The forkhead transcription factor FOXE3 is critical for vertebrate eye development. Recessive and dominant variants cause human ocular disease but the full range of phenotypes and mechanisms of action for the two classes of variants are unknown. We identified FOXE3 variants in individuals with congenital eye malformations and carried out in vitro functional analysis on selected alleles. Sixteen new recessive and dominant families, including six novel variants, were identified. Analysis of new and previously reported genetic and clinical data demonstrated a broad phenotypic range with an overlap between recessive and dominant disease. Most families with recessive alleles, composed of truncating and forkhead-domain missense variants, had severe corneal opacity (90%; sclerocornea in 47%), aphakia (83%) and microphthalmia (80%), but some had milder features including isolated cataract. The phenotype was most variable for recessive missense variants, suggesting that the functional consequences may be highly dependent on the type of amino acid substitution and its position. When assessed, aniridia or iris hypoplasia were noted in 89% and optic nerve anomalies in 60% of recessive cases, indicating that these defects are also common and may be underrecognized. In dominant pedigrees, caused by extension variants, normal eye size (96%), cataracts (99%) and variable anterior segment anomalies were seen in most, but some individuals had microphthalmia, aphakia or sclerocornea, more typical of recessive disease. Functional studies identified variable effects on the protein stability, DNA binding, nuclear localization and transcriptional activity for recessive FOXE3 variants, whereas dominant alleles showed severe impairment in all areas and dominant-negative characteristics.

Protein modeling and in silico analysis to assess pathogenicity of ABCA4 variants in patients with inherited retinal disease.

Purpose: The retina-specific ABCA transporter, ABCA4, plays an essential role in translocating retinoids required by the visual cycle. ABCA4 genetic variants are known to cause a wide range of inherited retinal disorders, including Stargardt disease and cone-rod dystrophy. More than 1,400 ABCA4 missense variants have been identified; however, more than half of these remain variants of uncertain significance (VUS). The purpose of this study was to employ a predictive strategy to assess the pathogenicity of ABCA4 variants in inherited retinal diseases using protein modeling and computational approaches. Methods: We studied 13 clinically well-defined patients with ABCA4 retinopathies and identified the presence of 10 missense variants, including one novel variant in the ABCA4 gene, by next-generation sequencing (NGS). All variants were structurally analyzed using AlphaFold2 models and existing experimental structures of human ABCA4 protein. The results of these analyses were compared with patient clinical presentations to test the effectiveness of the methods employed in predicting variant pathogenicity. Results: We conducted a phenotype-genotype comparison of 13 genetically and phenotypically well-defined retinal disease patients. The in silico protein structure analyses we employed successfully detected the deleterious effect of missense variants found in this affected patient cohort. Our study provides American College of Medical Genetics and Genomics (ACMG)-defined supporting evidence of the pathogenicity of nine missense ABCA4 variants, aligning with the observed clinical phenotypes in this cohort. Conclusions: In this report, we describe a systematic approach to predicting the pathogenicity of ABCA4 variants by means of three-dimensional (3D) protein modeling and in silico structure analysis. Our results demonstrate concordance between disease severity and structural changes in protein models induced by genetic variations. Furthermore, the present study suggests that in silico protein structure analysis can be used as a predictor of pathogenicity and may facilitate the assessment of genetic VUS.

SOX2 pathogenic variants with normal eyes: Expanding the phenotypic spectrum.

SOX2 pathogenic variants, though rare, constitute the most commonly known genetic cause of clinical anophthalmia and microphthalmia. However, patients without major ocular malformation, but with multi-system developmental disorders, have been reported, suggesting that the range of clinical phenotypes is broader than previously appreciated. We detail two patients with bilateral structurally normal eyes along with 11 other previously published patients. Our findings suggest that there is no obvious phenotypic or genotypic pattern that may help set apart patients with normal eyes. Our patients provide further evidence for broadening the phenotypic spectrum of SOX2 mutations and re-appraising the designation of SOX2 disorder as an anophthalmia/microphthalmia syndrome. We emphasize the importance of considering SOX2 pathogenic variants in the differential diagnoses of individuals with normal eyes, who may have varying combinations of features such as developmental delay, urogenital abnormalities, gastro-intestinal anomalies, pituitary dysfunction, midline structural anomalies, and complex movement disorders, seizures or other neurological issues.

Novel CRB1 pathogenic variant in Chuuk families with Leber congenital amaurosis.

The purpose of this article is to determine the cause of Leber congenital amaurosis (LCA) in Chuuk state, Federated States of Micronesia (FSM). In this prospective observational case series, five patients with early-onset vision loss were examined in Chuuk state, FSM, during an ocular genetics visit to study the elevated incidence of microphthalmia. Because of their low vision these patients were incorrectly assumed to have microphthalmia. A complete ophthalmological exam established a clinical diagnosis of LCA. Candidate gene exons were sequenced with a targeted retinal dystrophy panel. Five subjects in three related families were diagnosed with LCA. All five were from Tonoas Island, within the Chuuk Lagoon, with ages ranging from 6 months to 16 years. DNA sequencing of affected individuals revealed a homozygous CRB1 NM_201253.3:c.3134del pathogenic variant, which was heterozygous in their parents. CRB1 genotypes were confirmed by a PCR restriction assay. We report identification of a founder pathogenic variant in CRB1 responsible for autosomal recessive LCA in this isolated community. This discovery will lead to appropriate recurrence risk counseling.

Elucidating the clinical spectrum and molecular basis of HYAL2 deficiency.

PURPOSE: We previously defined biallelic HYAL2 variants causing a novel disorder in 2 families, involving orofacial clefting, facial dysmorphism, congenital heart disease, and ocular abnormalities, with Hyal2 knockout mice displaying similar phenotypes. In this study, we better define the phenotype and pathologic disease mechanism. METHODS: Clinical and genomic investigations were undertaken alongside molecular studies, including immunoblotting and immunofluorescence analyses of variant/wild-type human HYAL2 expressed in mouse fibroblasts, and in silico modeling of putative pathogenic variants. RESULTS: Ten newly identified individuals with this condition were investigated, and they were associated with 9 novel pathogenic variants. Clinical studies defined genotype-phenotype correlations and confirmed a recognizable craniofacial phenotype in addition to myopia, cleft lip/palate, and congenital cardiac anomalies as the most consistent manifestations of the condition. In silico modeling of missense variants identified likely deleterious effects on protein folding. Consistent with this, functional studies indicated that these variants cause protein instability and a concomitant cell surface absence of HYAL2 protein. CONCLUSION: These studies confirm an association between HYAL2 alterations and syndromic cleft lip/palate, provide experimental evidence for the pathogenicity of missense alleles, enable further insights into the pathomolecular basis of the disease, and delineate the core and variable clinical outcomes of the condition.

CNGB1-related rod-cone dystrophy: A mutation review and update.

Cyclic nucleotide-gated channel ß1 (CNGB1) encodes the 240-kDa ß subunit of the rod photoreceptor cyclic nucleotide-gated ion channel. Disease-causing sequence variants in CNGB1 lead to autosomal recessive rod-cone dystrophy/retinitis pigmentosa (RP). We herein present a comprehensive review and analysis of all previously reported CNGB1 sequence variants, and add 22 novel variants, thereby enlarging the spectrum to 84 variants in total, including 24 missense variants (two of which may also affect splicing), 21 nonsense, 19 splicing defects (7 at noncanonical positions), 10 small deletions, 1 small insertion, 1 small insertion-deletion, 7 small duplications, and 1 gross deletion. According to the American College of Medical Genetics and Genomics classification criteria, 59 variants were considered pathogenic or likely pathogenic and 25 were variants of uncertain significance. In addition, we provide further phenotypic data from 34 CNGB1-related RP cases, which, overall, are in line with previous findings suggesting that this form of RP has long-term retention of useful central vision despite the early onset of night blindness, which is valuable for patient counseling, but also has implications for it being considered a priority target for gene therapy trials.

Stargardt misdiagnosis: How ocular genetics helps.

Ocular Genetics at Wills Eye Hospital sees a wide range of rare disorders for accurate diagnosis. To demonstrate how focused consultation and genetic testing results in precise diagnoses, we investigated false diagnosis rates for patients referred with a diagnosis of Stargardt disease. This is a retrospective review of patients over a 3 year period referred to our Ocular Genetics clinic for possible Stargardt disease, or already holding a diagnosis of Stargardt disease. Results of diagnostic and genetic testing were compared to standard definition of Stargardt. Of 40 patients, 14 (35%) had been misdiagnosed. Four had non-Stargardt phenotype of which three had ABCA4 pathogenic variants with phenotypes inconsistent with Stargardt disease. Two of those with pathogenic ABCA4 variants were related. Nine had pathogenic variants in other different genes with overlapping features of Stargardt disease. One had Thioridazine maculopathy. Our study highlights the essential role of the subspecialty field of ocular genetics in obtaining accurate diagnoses for the delivery of correct counseling and interventional trial eligibility assessment.

A novel de novo intronic variant in ITPR1 causes Gillespie syndrome.

Gillespie syndrome (GLSP) is characterized by bilateral symmetric partial aplasia of the iris presenting as a fixed and large pupil, cerebellar hypoplasia with ataxia, congenital hypotonia, and varying levels of intellectual disability. GLSP is caused by either biallelic or heterozygous, dominant-negative, pathogenic variants in ITPR1. Here, we present a 5-year-old male with GLSP who was found to have a heterozygous, de novo intronic variant in ITPR1 (NM_001168272.1:c.5935-17G > A) through genome sequencing (GS). Sanger sequencing of cDNA from this individual's fibroblasts showed the retention of 15 nucleotides from intron 45, which is predicted to cause an in-frame insertion of five amino acids near the C-terminal transmembrane domain of ITPR1. In addition, qPCR and cDNA sequencing demonstrated reduced expression of both ITPR1 alleles in fibroblasts when compared to parental samples. Given the close proximity of the predicted in-frame amino acid insertion to the site of previously described heterozygous, de novo, dominant-negative, pathogenic variants in GLSP, we predict that this variant also has a dominant-negative effect on ITPR1 channel function. Overall, this is the first report of a de novo intronic variant causing GLSP, which emphasizes the utility of GS and cDNA studies for diagnosing patients with a clinical presentation of GLSP and negative clinical exome sequencing.

Ocular manifestations of Emanuel syndrome.

Emanuel syndrome is caused by a supernumerary der(22)t(11;22) and typically manifests with intellectual disability and craniofacial dysmorphism. Ocular abnormalities have infrequently been described. We report a 36-year-old man with severe intellectual disability, aphasia, and facial dysmorphism, with high myopia and juvenile open angle glaucoma (JOAG). Microarray analysis results included 47,XY,+der(22)t(11;22)(q23;q11.2), and a 269 kb deletion of 7q31.33(125,898,014-126,166,829). Two candidate genes were identified as possible etiologies for the ocular pathologies in our patient: a MFRP duplication on chromosome 11, which may play a role in high myopia and dysregulation of emmetropization, and a GRM8 deletion on chromosome 7, which may cause glutamate-induced excitotoxicity and therefore have a role in the development of JOAG, unrelated to the Emanuel syndrome genotype. We provide the first detailed description these ocular abnormalities in a patient with Emmanuel syndrome.

Early onset ectopia lentis due to a FBN1 mutation with non-penetrance.

Isolated ectopia lentis is usually autosomal dominant and commonly due to the mutations of FBN1 gene. We report on a family with ectopia lentis. The propositus is a 6-year-old boy with bilateral superior-temporal ectopia lentis. His echocardiogram was normal and he did not meet the revised Ghent criteria for Marfan syndrome. Molecular genetic testing revealed c.1948 C>T (p.Arg650Cys) in FBN1. The mother has visual acuity of 20/20 with -4.50 right eye and -2.50 left eye. She has no evidence of ectopia lentis. DNA analysis revealed that she has the same FBN1 mutation. Seven other maternal family members also have ectopia lentis. In conclusion, we report on a case of early-onset autosomal dominant isolated ectopia lentis caused by FBN1 mutation that has previously been reported only in Marfan syndrome. The child's mother presumably represents a rare case of nonpenetrance.

Anirdia-like phenotype caused by 6p25 dosage aberrations.

Axenfeld-Rieger spectrum (ARS) includes the anterior segment abnormalities posterior embryotoxon, irido-corneal adhesions, corectopia, and other abnormalities of pupil size and shape. Glaucoma occurs in approximately 50% of affected children. It is often caused by mutations of FOXC1 or PITX2. Timing of expression and dosage of these transcription factors appear to be very critical in the development of the anterior segment. We report on one child with a deletion and another with a duplication involving 6p25, causing an anirdia-like phenotype. Classic anirdia is a pan-ophthalmic disorder caused by heterozygous mutations involving the paired homeobox gene PAX6 at 11p13. It is often associated with optic nerve hypoplasia, foveal hypoplasia, corneal pannus, nystagmus, and cataract. Microdeletion of 11p13 may be associated with life threatening Wilms tumor. Distinguishing these two syndromes has critical implications for prognosis and treatment. We demonstrate how chromosomal microarray can be instrumental in differentiating these phenotypes.

Ocular manifestations of 22q11.2 microduplication.

PURPOSE: To report a new ocular manifestation of the dup22q11 syndrome and explore involved genes that may offer insight to mechanisms of pathogenesis. DESIGN: Case series. PARTICIPANTS: Two male patients with this syndrome diagnosed with dup22q11.2. METHODS: Medical records were reviewed. Duplication was detected in the oligo-single nucleotide polymorphism chromosomal microarray and duplicated genes within the segment where determined by literature and database review. Potential associations between the ophthalmologic manifestations and their physiopathology were investigated. MAIN OUTCOME MEASURES: Microarray results and identification of candidate genes within the duplicated segment. RESULTS: Our patients demonstrate previously unreported findings of dup22q11.2, including Marcus Gunn jaw winking, Duane's retraction syndrome, and other abnormal eye movements consistent with a congenital cranial dysinnervation disorder (CCDD), retinal vascular tortuosity, and primary infantile glaucoma. The duplicated segment in case 1 includes SNAP29, which could be linked with the development of retinal vascular tortuosity, and MAPK1, which seems to play a role in axonal development through the semaphorin pathway, which may serve as a candidate gene for CCDD. In case 2, the CLDN5 gene is within the duplicated segment. CLDN5 could be involved in the pathophysiology of glaucoma. CONCLUSIONS: Our cases expand the ocular phenotype for duplication of 22q11 and serve to identify potential candidate genes for the development of CCDD, retinal vascular tortuosity, and glaucoma.

The cost of genetic testing for ocular disease: who pays?

PURPOSE OF REVIEW: To facilitate ophthalmologists' understanding on the cost of genetic testing in ocular disease, the complexities of insurance coverage and its impact on the availability of testing. RECENT FINDINGS: Many insurance carriers address coverage for genetic testing in written clinical policies. They provide criteria for medically necessary testing. These policies mostly cover testing for individuals who are symptomatic and in whom testing will have a direct impact on medical treatment. In cases in which no treatments are currently available, other than research trials, patients may have difficulty in getting insurance coverage for genetic testing. SUMMARY: Genetic testing for inherited eye diseases can be costly but has many benefits to patient care, including confirmation of a diagnosis, insight into prognostic information, and identification of associated health risks, inheritance patterns, and possible current and future treatments. As gene therapy advances progress, the availability for treatment in ocular diseases, coverage for genetic testing by third-party payers could increase on the basis of current clinical policies.

Bardet-Biedl syndrome with chorioretinal coloboma: a case series and review of literature.

INTRODUCTION: Bardet-Biedl Syndrome (BBS) is a ciliopathy causing developmental defects and progressive retinal dystrophy, whereas choroidal coloboma is a developmental defect causing structural deficiency in the posterior retina. Both are rarely reported together. METHODS: Here, we describe the phenotype and genotype of three unrelated patients with co-occurrence of Bardet-Biedl Syndrome and chorioretinal coloboma and review the pertinent literature. RESULTS: We describe three unrelated patients, with variable clinical features of Bardet Biedl syndrome. None had family history of BBS or coloboma. Each carried biallelic variants in BBS1, BBS9 and TTC8 gene, respectively. Two had unilateral chorioretinal coloboma, while one had bilateral chorioretinal coloboma. DISCUSSION: Although there may be other explanatory factors yet to be revealed, our data suggests that chorioretinal coloboma may be associated with BBS. The Hedgehog (Hh) signaling pathway, an intercellular communicator for development of the eye, is dependent on the primary cilia and plays a crucial role in the closure of the optic fissure. Both disorders therefore involve disruption of primary cilia function which may explain their association.

Lyonization in ophthalmology.

PURPOSE OF REVIEW: To describe the entity of Lyonization in ocular eye diseases, along with its clinical and counseling implications. RECENT FINDINGS: Several X-linked ocular diseases such as choroideremia, X-linked retinitis pigmentosa, and X-linked ocular albinism may have signs of Lyonization on ocular examination and diagnostic testing. These findings may aid in the proper diagnosis of ocular disease in both female carriers and their affected male relatives. SUMMARY: Manifestations of Lyonization in the eye may help in the diagnosis of X-linked ocular diseases which may lead to accurate diagnosis, appropriate molecular genetic testing and genetic counseling.

Genetics of the anterior segment dysgenesis.

The anterior segment dysgeneses are a broad group of heterogeneous disorders characterized by developmental abnormalities of the anterior segment of the eye, including primary congenital aphakia, Peters sequence, aniridia, and Axenfeld-Rieger spectrum. These conditions can have overlapping phenotypes and both genotypic and phenotypic heterogeneity. This article provides a strategy for both phenotyping and then genotyping using a targeted stepwise approach.

Mutations in AGBL5 associated with Retinitis pigmentosa.

BACKGROUND: Retinitis pigmentosa (RP) is the leading cause of heritable retinal visual impairment. Clinically, it is characterized by a variable onset of progressive night blindness and visual field constriction. RP is characterized by wide genetic heterogeneity with a broad range of potential genes involved in the genesis of this disease. Very few cases have been reported of RP due to pathogenic variants in AGBL5. MATERIALS AND METHODS: We report two patients with RP and bilallelic pathogenic variants in AGBL5. RESULTS: Genetic sequencing showed one homozygous AGBL5 missense variant in one patient and a homozygous nonsense variant in the other. These patients presented with progressive peripheral vision loss and nyctalopia. Their RP phenotypes were similar to previous reports in literature. CONCLUSION: These two cases provide further evidence regarding the relationship of pathogenic variants in AGBL5 as a cause of autosomal recessive RP.

TWO CASES OF CRB1-RELATED RETINAL DYSTROPHY ASSOCIATED WITH RETINAL MASSES.

BACKGROUND/PURPOSE: Mutations in CRB1 are associated with variable severity in expression leading to apparent phenotypic diversity. We present two retinal findings. METHODS: We present two unrelated children with CRB1-related retinal dystrophy with a solitary mass visualized on fundoscopy. Both underwent a complete ophthalmologic examination including visual acuity assessment, optical coherence tomography, intravenous fluorescein angiography, and fundus autofluorescence. RESULTS: In one child, a gliotic mass was observed on the superior temporal vessel away from disk. On optical coherence tomography, the mass appeared to be located in the superficial retina and contained discrete internal moth-eaten optically empty spaces as previously reported in the astrocytic hamartomas of tuberous sclerosis. Fundus autofluorescence showed speckled hyperautofluorescence of the lesion. In the other child, there was a calcified mass within the nerve fiber layer just temporal to the optic nerve. On optical coherence tomography, this mass appeared irregular in shape, encapsulated, and had a heterogeneous disorganized interior with hyperreflective areas. CONCLUSION: In this report, we detail two presentations of CRB1-related retinal dystrophy: retinal astrocytic hamartoma and another form of superficial retinal hamartoma. We believe this may represent a manifestation of CRB1 mutations. Recognition of this finding may prevent unnecessary evaluation for tumor cause in patients with CRB1-related retinal dystrophy.

Identification and Management of a Novel PRDM5 Gene Pathologic Variant in a Family With Brittle Cornea Syndrome.

PURPOSE: The aim of this study was to report a novel PRDM5 pathologic variant and ophthalmic findings in a family with 3 children diagnosed with brittle cornea syndrome (BCS). Histopathologic findings and surgical outcome of a child with BCS who underwent full-thickness corneal transplant are described. METHODS: This is an observational case report of a nonconsanguineous Laotian family with 3 siblings diagnosed with BCS. Data collected included visual acuity, cycloplegic refraction, slit-lamp biomicroscopy, dilated fundus examination, corneal pachymetry, corneal topography, and general medical findings. Targeted testing through PRDM5 gene sequencing with copy number variation detection was conducted. RESULTS: The 3 siblings included a 12-year-old boy and 8- and 6-year-old sisters, all of whom presented with myopia, blue-tinted sclerae, thin corneas, and variable corneal scarring. All 3 affected children were found to be homozygous for the PRDM5 gene variant c.1117_1123delinsTTTAATGCTTACAAATGTTTG p.Asp373Phefs*57. Coding sequences of PRDM5 and ZNF469 genes were sequenced in their entirety, and this was the only pathologic variant present in this family. The youngest affected sister developed persistent hydrops with severely decreased vision and underwent penetrating keratoplasty. Histopathology revealed severe corneal thinning, diffuse absence of Bowman layer, and ruptured Descemet membrane scrolls. CONCLUSIONS: Three siblings with clinical signs of BCS, including corneal thinning, myopia, and blue sclerae, were found to have a novel PRDM5 gene pathologic variant. This pathologic variant has not been previously reported, although 1 downstream nonsense pathologic variant has been reported as pathogenic. The similar phenotypes in all affected patients support the pathogenicity of this variant. Surgical management of BCS presents unique challenges due to severe tissue fragility.