Unraveling the genetic complexities of combined retinal dystrophy and hearing impairment.

Usher syndrome, the most prevalent cause of combined hereditary vision and hearing impairment, is clinically and genetically heterogeneous. Moreover, several conditions with phenotypes overlapping Usher syndrome have been described. This makes the molecular diagnosis of hereditary deaf-blindness challenging. Here, we performed exome sequencing and analysis on 7 Mexican and 52 Iranian probands with combined retinal degeneration and hearing impairment (without intellectual disability). Clinical assessment involved ophthalmological examination and hearing loss questionnaire. Usher syndrome, most frequently due to biallelic variants in MYO7A (USH1B in 16 probands), USH2A (17 probands), and ADGRV1 (USH2C in 7 probands), was diagnosed in 44 of 59 (75%) unrelated probands. Almost half of the identified variants were novel. Nine of 59 (15%) probands displayed other genetic entities with dual sensory impairment, including Alström syndrome (3 patients), cone-rod dystrophy and hearing loss 1 (2 probands), and Heimler syndrome (1 patient). Unexpected findings included one proband each with Scheie syndrome, coenzyme Q10 deficiency, and pseudoxanthoma elasticum. In four probands, including three Usher cases, dual sensory impairment was either modified/aggravated or caused by variants in distinct genes associated with retinal degeneration and/or hearing loss. The overall diagnostic yield of whole exome analysis in our deaf-blind cohort was 92%. Two (3%) probands were partially solved and only 3 (5%) remained without any molecular diagnosis. In many cases, the molecular diagnosis is important to guide genetic counseling, to support prognostic outcomes and decisions with currently available and evolving treatment modalities.

The Genetic Landscape of Inherited Retinal Diseases in a Mexican Cohort: Genes, Mutations and Phenotypes.

In this work, we aimed to provide the genetic diagnosis of a large cohort of patients affected with inherited retinal dystrophies (IRDs) from Mexico. Our data add valuable information to the genetic portrait in rare ocular diseases of Mesoamerican populations, which are mostly under-represented in genetic studies. A cohort of 144 unrelated probands with a clinical diagnosis of IRD were analyzed by next-generation sequencing using target gene panels (overall including 346 genes and 65 intronic sequences). Four unsolved cases were analyzed by whole-exome sequencing (WES). The pathogenicity of new variants was assessed by in silico prediction algorithms and classified following the American College of Medical Genetics and Genomics (ACMG) guidelines. Pathogenic or likely pathogenic variants were identified in 105 probands, with a final diagnostic yield of 72.9%; 17 cases (11.8%) were partially solved. Eighteen patients were clinically reclassified after a genetic diagnostic test (17.1%). In our Mexican cohort, mutations in 48 genes were found, with ABCA4, CRB1, RPGR and USH2A as the major contributors. Notably, over 50 new putatively pathogenic variants were identified. Our data highlight cases with relevant clinical and genetic features due to mutations in the RAB28 and CWC27 genes, enrich the novel mutation repertoire and expand the IRD landscape of the Mexican population.

Autofluorescence in female carriers with choroideremia: A familial case with a novel mutation in the CHM gene.

BACKGROUND: Choroideremia is an X-linked chorioretinal dystrophy caused by mutations in the CHM gene. The main differential diagnosis is X-linked retinitis pigmentosa. Clinically, male patients that are affected by these two diseases have similar symptoms. This work aims to report a familial case of choroideremia initially diagnosed as X-linked retinitis pigmentosa with a novel mutation in the CHM gene, and the relevance of fundus autofluorescence (FAF) in female carriers. MATERIALS AND METHODS: A complete ophthalmological evaluation was done in a 37-year-old woman and her 53-year-old maternal uncle; the uncle had been diagnosed previously with X-linked retinitis pigmentosa. A visual field test, FAF imaging, full-field electroretinography, and a genetic test were performed. RESULTS: In the proband, the fundoscopy revealed diffuse changes in the retinal pigment epithelium in both eyes, and the FAF showed a speckled pattern of low- and high-density. The maternal uncle's ophthalmological evaluation showed choroidal and retinal atrophy consistent with choroideremia. The molecular analysis revealed a pathogenic variant in the CHM gene, c.190-1 G > T. CONCLUSIONS: In female carriers of choroideremia and X-linked retinitis pigmentosa, differential diagnosis may be challenging. A speckled pattern of low- and high-density in autofluorescence is commonly found in female carriers of choroideremia. FAF is a powerful tool for making a correct clinical diagnosis because the pattern in FAF is much more apparent than the visible retinal changes obtained by fundoscopy. Although it is crucial to perform molecular analysis to confirm the diagnosis, FAF is useful when genetic testing may not be readily available.

Novel mutations in the PITX2 gene in Pakistani and Mexican families with Axenfeld-Rieger syndrome.

PURPOSE: Axenfeld-Rieger syndrome (ARS) is a rare autosomal dominant disorder that affects the anterior segment of the eye. The aim of this study was to examine the PITX2 gene to identify possible novel mutations in Pakistani and Mexican families affected by the ARS phenotype. METHODS: Three unrelated probands with a diagnosis of ARS were recruited for this study. Genomic DNA was isolated from the peripheral blood of the probands and their family members. Polymerase chain reaction and Sanger sequencing were used for the analysis of coding exons and the flanking intronic regions of the PITX2 gene. Bioinformatics tools and database (VarSome, Provean, and MutationTaster, SIFT, PolyPhen-2, and HOPE) were evaluated to explore missense variants. RESULTS: We identified novel heterozygous variations in the PITX2 gene that segregated with the ARS phenotype within the families. The variant NM_153426.2(PITX2):c.226G > T or p.(Ala76Ser) and the mutation NM_153426.2(PITX2):c.455G > A or p.(Cys152Tyr) were identified in two Pakistani pedigrees, and the mutation NM_153426.2(PITX2):c.242_265del or p.(Lys81_Gln88del), segregated in a Mexican family. CONCLUSION: Our study extends the spectrum of PITX2 mutations in individuals with ARS, enabling an improved diagnosis of this rare but serious syndrome.

Increasing the Genetic Diagnosis Yield in Inherited Retinal Dystrophies: Assigning Pathogenicity to Novel Non-canonical Splice Site Variants.

AIMS: We aimed to validate the pathogenicity of genetic variants identified in inherited retinal dystrophy (IRD) patients, which were located in non-canonical splice sites (NCSS). METHODS: After next generation sequencing (NGS) analysis (target gene panels or whole exome sequencing (WES)), NCSS variants were prioritized according to in silico predictions. In vivo and in vitro functional tests were used to validate their pathogenicity. RESULTS: Four novel NCSS variants have been identified. They are located in intron 33 and 34 of ABCA4 (c.4774-9G>A and c.4849-8C>G, respectively), intron 2 of POC1B (c.101-3T>G) and intron 3 of RP2 (c.884-14G>A). Functional analysis detected different aberrant splicing events, including intron retention, exon skipping and intronic nucleotide addition, whose molecular effect was either the disruption or the elongation of the open reading frame of the corresponding gene. CONCLUSIONS: Our data increase the genetic diagnostic yield of IRD patients and expand the landscape of pathogenic variants, which will have an impact on the genotype-phenotype correlations and allow patients to opt for the emerging gene and cell therapies.

Expansion of the phenotypic spectrum and description of molecular findings in a cohort of patients with oculocutaneous mosaic RASopathies.

BACKGROUND: Postzygotic KRAS, HRAS, NRAS, and FGFR1 mutations result in a group of mosaic RASopathies characterized by related developmental anomalies in eye, skin, heart, and brain. These oculocutaneous disorders include oculoectodermal syndrome (OES) encephalo-cranio-cutaneous lipomatosis (ECCL), and Schimmelpenning-Feuerstein-Mims syndrome (SFMS). Here, we report the results of the clinical and molecular characterization of a novel cohort of patients with oculocutaneous mosaic RASopathies. METHODS: Two OES, two ECCL, and two SFMS patients were ascertained in the study. In addition, two subjects with unilateral isolated epibulbar dermoids were also enrolled. Molecular analysis included PCR amplification and Sanger sequencing of KRAS, HRAS, NRAS, and FGFR1 genes in DNA obtained from biopsies (skin/epibulbar dermoids), buccal mucosa, and blood leukocytes. Massive parallel sequencing was employed in two cases with low-level mosaicism. RESULTS: In DNA from biopsies, mosaicism for pathogenic variants, including KRAS p.Ala146Thr in two OES subjects, FGFR1 p.Asn546Lys and KRAS p.Ala146Val in ECCL patients, and KRAS p.Gly12Asp in both SFMS patients, was demonstrated. No mutations were shown in DNA from conjunctival lesions in two subjects with isolated epibubar dermoids. CONCLUSION: Our study allowed the expansion of the clinical spectrum of mosaic RASopathies and supports that mosaicism for recurrent mutations in KRAS and FGFR1 is a commonly involved mechanism in these rare oculocutaneous anomalies.

Mutation update: TGFBI pathogenic and likely pathogenic variants in corneal dystrophies.

Human transforming growth factor ß-induced (TGFBI), is a gene responsible for various corneal dystrophies. TGFBI produces a protein called TGFBI, which is involved in cell adhesion and serves as a recognition sequence for integrins. An alteration in cell surface interactions could be the underlying cause for the progressive accumulation of extracellular deposits in different layers of the cornea with the resulting changes of refractive index and transparency. To this date, 69 different pathogenic or likely pathogenic variants in TGFBI have been identified in a heterozygous or homozygous state in various corneal dystrophies, including a novel variant reported here. All disease-associated variants were inherited as autosomal-dominant traits but one; this latter was inherited as an autosomal recessive trait. Most corneal dystrophy-associated variants are located at amino acids Arg124 and Arg555. To keep the list of corneal dystrophy-associated variant current, we generated a locus-specific database for TGFBI (http://databases.lovd.nl/shared/variants/TGFBI) containing all pathogenic and likely pathogenic variants reported so far. Non-disease-associated variants are described in specific databases, like gnomAD and ExAC but are not listed here. This article presents the most recent up-to-date list of disease-associated variants.

[Extension of TORCH spectrum in ophthalmology: congenital Zika, case report]. / Ampliación del espectro TORCH en oftalmología: Zika congénito, reporte de un caso.

BACKGROUND: Zika is a flavivirus that can be transmitted transplacentally. Eye abnormalities have been reported in 70% of Zika cases, and 41.7% of them can occur in the absence of microcephaly. The most common ocular abnormalities are macular atrophy, optic atrophy and chorioretinal coloboma. The objective was to report the case of eye disorders associated with Zika, acquired transplacentally, despite negative results for TORCH, and serology and PCR analyses for Zika. CLINICAL CASE: 9-month-old female patient, born in Chiapas, Mexico, brought to an ophthalmologic evaluation because she did not follow objects. As family background patient's mother had Zika, confirmed serologically at 9 weeks gestation. Physical examination revealed microcephaly, redundant skin on neck, joint stiffness and delayed psychomotor development. Ophthalmological examination revealed in right eye atrophy of the optic nerve, and left eye with exotropia, macular scar and optic nerve aplasia. TORCH profile and serology and PCR for Zika were negative. CONCLUSIONS: Despite the negative serology for Zika, given the history of pregnancy and the pre and post-natal clinical manifestations, diagnosis of embryopathy secondary to Zika infection with optic nerve aplasia, chorioretinal atrophy, macular scar, microcephaly and global neurodevelopmental delay was made.

INTRODUCCIÓN: el Zika es un flavivirus que puede ser transmitido de forma transplacentaria. Las anomalías oculares han sido reportadas en un 70% de los casos y se ha visto que 41.7% de ellas pueden ocurrir en ausencia de microcefalia. Las alteraciones oculares más comunes son: atrofia macular, atrofia óptica y coloboma coriorretiniano. El objetivo de este estudio fue reportar un caso de alteraciones oculares asociadas a Zika, adquirido de forma transplacentaria, a pesar de los resultados negativos para el perfil TORCH y Zika de los análisis de serología y PCR. CASO CLÍNICO: paciente femenina de nueve meses de edad, originaria de Chiapas, México, traída a revisión oftalmológica porque no seguía objetos. Como antecedentes, la paciente tenía madre con diagnóstico de Zika confirmado serológicamente a las nueve semanas de gestación. A la exploración física se encontró microcefalia, piel redundante en cuello, rigidez articular y retraso en el desarrollo psicomotor. A la exploración oftalmológica fueron evidentes atrofia del nervio óptico de ojo derecho, ojo izquierdo con exotropía, cicatriz macular y aplasia del nervio óptico. Tanto el perfil TORCH como la serología y la PCR para Zika fueron negativos. CONCLUSIONES: a pesar de la serología negativa para Zika, dados los antecedentes de la gestación y las manifestaciones clínicas pre y postnatales se integró el diagnóstico de embriopatía secundaria a infección por Zika con aplasia del nervio óptico, atrofia coriorretiniana, cicatriz macular, microcefalia y retraso global del neurodesarrollo.

Extension of the mutational and clinical spectrum of SOX2 related disorders: Description of six new cases and a novel association with suprasellar teratoma.

SOX2 is a transcription factor that is essential for maintenance of pluripotency and has several conserved roles in early embryonic development. Heterozygous loss-of-function variants in SOX2 are identified in approximately 40% of all cases of bilateral anophthalmia/micropthalmia (A/M). Increasingly SOX2 mutation-positive patients without major eye findings, but with a range of other developmental disorders including autism, mild to moderate intellectual disability with or without structural brain changes, esophageal atresia, urogenital anomalies, and endocrinopathy are being reported, suggesting that the clinical phenotype associated with SOX2 loss is much broader than previously appreciated. In this report we describe six new cases, four of which carry novel pathogenic SOX2 variants. Four cases presented with bilateral anophthalmia in addition to extraocular involvement. Another individual presented with only unilateral anophthalmia. One individual did not have any eye findings but presented with a suprasellar teratoma in infancy and was found to have the recurrent c.70del20 mutation in SOX2 (c.70_89del, p.Asn24Argfs*65). This is this first time this tumor type has been reported in the context of a de novo SOX2 mutation. Notably, individuals with hypothalamic hamartomas and slow-growing hypothalamo-pituitary tumors have been reported previously, but it is still unclear how SOX2 loss contributes to their formation.

Severe Phenotype of Keratitis-Ichthyosis-Deafness Syndrome With Presumed Ocular Surface Squamous Neoplasia.

PURPOSE: The aim of this study was to describe a case of severe keratitis-ichthyosis-deafness (KID) syndrome with ocular surface squamous neoplasia. METHODS: The affected patient underwent complete ocular and systemic examinations. The molecular studies included polymerase chain reaction amplification and automated DNA sequencing of the complete gap junction beta-2 (GJB2) gene coding sequence. RESULTS: A 30-year-old man presented with generalized erythro-hyperkeratosis and deafness and complaints of decreased visual acuity, tearing, and photophobia. Ophthalmic examination showed corneal erosion, vascularization, and a gray gelatinous lesion partially covering the right cornea, suggestive of squamous neoplasia. The clinical features were characteristic of KID syndrome. This diagnosis was confirmed with a DNA analysis showing the pathogenic variant p.D50N in the GJB2 gene. Presumed squamous neoplasia was treated with topical interferon α2b. CONCLUSIONS: KID syndrome is a very rare disease that has been reported with an incremental incidence of squamous cell carcinoma of the mucous membranes and skin (12%-15%). Here, we presented a case of severe systemic KID syndrome with ocular surface squamous neoplasia.

Tietz/Waardenburg type 2A syndrome associated with posterior microphthalmos in two unrelated patients with novel MITF gene mutations.

Tietz syndrome and Waardenburg syndrome type 2A are allelic conditions caused by MITF mutations. Tietz syndrome is inherited in an autosomal dominant pattern and is characterized by congenital deafness and generalized skin, hair, and eye hypopigmentation, while Waardenburg syndrome type 2A typically includes variable degrees of sensorineural hearing loss and patches of de-pigmented skin, hair, and irides. In this paper, we report two unrelated families with MITF mutations. The first family showed an autosomal dominant pattern and variable expressivity. The second patient was isolated. MITF gene analysis in the first family demonstrated a c.648A>C heterozygous mutation in exon 8 c.648A>C; p. (R216S), while in the isolated patient, an apparently de novo heterozygous c.1183_1184insG truncating mutation was demonstrated in exon 10. All patients except one had bilateral reduced ocular anteroposterior axial length and a high hyperopic refractive error corresponding to posterior microphthalmos, features that have not been described as part of the disease. Our results suggest that posterior microphthalmos might be part of the clinical characteristics of Tietz/Waardenburg syndrome type 2A and expand both the clinical and molecular spectrum of the disease. © 2016 Wiley Periodicals, Inc.

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.