AUTOSOMAL DOMINANT MÜLLER CELL SHEEN DYSTROPHY: Clinical, Histopathologic, and Genetic Assessment in an Extended Family With Long Follow-Up.

BACKGROUND: Autosomal dominant Müller cell dystrophy is a rare condition we described in 1991. It is characterized by a striking sheen appearance on the retinal surface with progressive retinal changes leading to disorganization and atrophy with a decreased b-wave electroretinograms. MATERIALS AND METHODS: We examined 45 members of a 4-generation family. Fifteen subjects from three generations were found with the disease, without gender predilection. Seven patients underwent ophthalmic examination including fundus examination, intravenous fluorescein angiogram, spectral-domain optical coherence tomography, and electroretinogram. Six patients have a 30-year follow-up. Histopathology examination was performed on eyes of the eldest patient. Whole exome sequencing was done in four affected subjects. RESULTS: Findings include a decreased visual acuity, abnormal cellophane-like sheen of the vitreoretinal interface, a "plush" nerve fiber layer, and characteristic macular changes. Electroretinogram showed a selective b-wave diminution. Intravenous fluorescein angiogram presented perifoveal hyperfluorescence and capillary leakage. Spectral-domain optical coherence tomography revealed cavitations involving inner and later outer retinal layers with later disorganization. Histopathologic findings included Müller cell abnormalities with cystic disruption of inner retinal layers, pseudoexfoliation in anterior segment, and amyloidosis of extraocular vessels. Pedigree analysis suggests an autosomal dominant inheritance with late onset. DNA analysis demonstrated a previously undescribed heterozygous missense p.Glu109Val mutation in transthyretin. CONCLUSION: To the best of our knowledge, this is the first family reported with this disorder. Our data support the hypothesis that autosomal dominant Müller cell dystrophy is a distinct retinal dystrophy affecting Müller cells. Mutations in transthyretin gene may manifest as a predominantly retinal disorder.

Clinical, histopathological, and in silico pathogenicity analyses in a pedigree with familial amyloidosis of the Finnish type (Meretoja syndrome) caused by a novel gelsolin mutation.

Purpose: Familial amyloidosis of the Finnish type (FAF) is an inherited amyloidosis arising from mutations in the gelsolin protein (GSN). The disease includes facial paralysis, loose skin, and lattice corneal dystrophy. To date, FAF has been invariably associated with substitution of Asp214 in GSN. We describe the clinical, histopathological, and genetic features of a family with FAF due to a novel GSN mutation. Methods: Five affected adult individuals in a three-generation FAF pedigree were included in the study. Histopathological analysis was performed on an eyelid skin biopsy from one patient. Genetic analysis included next-generation sequencing (NGS) and Sanger sequencing for confirmation of the GSN variant. Several tools for in silico analysis of pathogenicity for the novel variant and to predict the effect of the amino acid replacement on protein stability were used. Results: Three older adult affected patients exhibited corneal lattice dystrophy, cutis laxa, and facultative peripheral neuropathy. Two younger adult individuals presented only with corneal amyloid deposits. NGS identified a heterozygous GSN c.1631T>G transversion, predicting a novel p.Met544Arg mutation. All in silico tools indicated that p.Met544Arg is deleterious for GSN functionality or stability. Conclusions: The results expand the molecular spectrum of GSN-linked systemic amyloidosis. The novel p.Met544Arg pathogenic variant is predicted to affect gelsolin function, presumably by impairing a potential calcium-sensitive, actin-binding region.

Identification of novel pathogenic variants and novel gene-phenotype correlations in Mexican subjects with microphthalmia and/or anophthalmia by next-generation sequencing.

Severe congenital eye malformations, particularly microphthalmia and anophthalmia, are one of the main causes of visual handicap worldwide. They can arise from multifactorial, chromosomal, or monogenic factors and can be associated with extensive clinical variability. Genetic analysis of individuals with these defects has allowed the recognition of dozens of genes whose mutations lead to disruption of normal ocular embryonic development. Recent application of next generation sequencing (NGS) techniques for genetic screening of patients with congenital eye defects has greatly improved the recognition of monogenic cases. In this study, we applied clinical exome NGS to a group of 14 Mexican patients (including 7 familial and 7 sporadic cases) with microphthalmia and/or anophthalmia. Causal or likely causal pathogenic variants were demonstrated in ~60% (8 out of 14 patients) individuals. Seven out of 8 different identified mutations occurred in well-known microphthalmia/anophthalmia genes (OTX2, VSX2, MFRP, VSX1) or in genes associated with syndromes that include ocular defects (CHD7, COL4A1) (including two instances of CHD7 pathogenic variants). A single pathogenic variant was identified in PIEZO2, a gene that was not previously associated with isolated ocular defects. NGS efficiently identified the genetic etiology of microphthalmia/anophthalmia in ~60% of cases included in this cohort, the first from Mexican origin analyzed to date. The molecular defects identified through clinical exome sequencing in this study expands the phenotypic spectra of CHD7-associated disorders and implicate PIEZO2 as a candidate gene for major eye developmental defects.

Next generation sequencing-based molecular diagnosis in familial congenital cataract expands the mutational spectrum in known congenital cataract genes.

Congenital cataract (CC) is a significant cause of childhood blindness worldwide. CC is a genetically heterogeneous disease because mutations in over 40 genes have been demonstrated to cause the disorder and up to 40% of cases arise from single-gene mutations. Hence, next generation sequencing (NGS) of deoxyribonucleic acid is a suitable approach for CC molecular diagnosis. In this study, we used commercially available inherited disease NGS panels including 50 CC genes for the genetic diagnosis of 11 probands with hereditary CC. Causal variants were recognized in six families. A novel CRYGC variant, p.(Phe6Ser), was identified in two apparently unrelated families. Two additional novel variants in the crystallin genes CRYBB2 (p.[Gly149Asp]) and CRYGA (p.[Arg48Cys]) were also identified. One family carried the novel p.[Gly8_Leu11del] variant in GJA8, while another family exhibited the previously reported c.2826-9G>A pathogenic change in EPHA2. Our results illustrate the utility of NGS for diagnosing CC in our population, and our results contribute to expand the mutational spectrum with four novel pathogenic variants in known CC genes.

Dissecting Intra-tumor Heterogeneity in the Glioblastoma Microenvironment Using Fluorescence-Guided Multiple Sampling.

The treatment of the most aggressive primary brain tumor in adults, glioblastoma (GBM), is challenging due to its heterogeneous nature, invasive potential, and poor response to chemo- and radiotherapy. As a result, GBM inevitably recurs and only a few patients survive 5 years post-diagnosis. GBM is characterized by extensive phenotypic and genetic heterogeneity, creating a diversified genetic landscape and a network of biological interactions between subclones, ultimately promoting tumor growth and therapeutic resistance. This includes spatial and temporal changes in the tumor microenvironment, which influence cellular and molecular programs in GBM and therapeutic responses. However, dissecting phenotypic and genetic heterogeneity at spatial and temporal levels is extremely challenging, and the dynamics of the GBM microenvironment cannot be captured by analysis of a single tumor sample. In this review, we discuss the current research on GBM heterogeneity, in particular, the utility and potential applications of fluorescence-guided multiple sampling to dissect phenotypic and genetic intra-tumor heterogeneity in the GBM microenvironment, identify tumor and non-tumor cell interactions and novel therapeutic targets in areas that are key for tumor growth and recurrence, and improve the molecular classification of GBM.

Dissecting intra-tumor heterogeneity in the glioblastoma microenvironment using fluorescence-guided multiple sampling.

The treatment of the most aggressive primary brain tumor in adults, glioblastoma (GBM), is challenging due to its heterogeneous nature, invasive potential, and poor response to chemo- and radiotherapy. As a result, GBM inevitably recurs and only a few patients survive 5 years post-diagnosis. GBM is characterized by extensive phenotypic and genetic heterogeneity, creating a diversified genetic landscape and a network of biological interactions between subclones, ultimately promoting tumor growth and therapeutic resistance. This includes spatial and temporal changes in the tumor microenvironment, which influence cellular and molecular programs in GBM and therapeutic responses. However, dissecting phenotypic and genetic heterogeneity at spatial and temporal levels is extremely challenging, and the dynamics of the GBM microenvironment cannot be captured by analysis of a single tumor sample. In this review, we discuss the current research on GBM heterogeneity, in particular, the utility and potential applications of fluorescence-guided multiple sampling to dissect phenotypic and genetic intra-tumor heterogeneity in the GBM microenvironment, identify tumor and non-tumor cell interactions and novel therapeutic targets in areas that are key for tumor growth and the recurrence, and improve the molecular classification of GBM.

Extensive genic and allelic heterogeneity underlying inherited retinal dystrophies in Mexican patients molecularly analyzed by next-generation sequencing.

BACKGROUND: Retinal dystrophies (RDs) are one of the most genetically heterogeneous monogenic disorders with ~270 associated loci identified by early 2019. The recent application of next-generation sequencing (NGS) has greatly improved the molecular diagnosis of RD patients. Genetic characterization of RD cohorts from different ethnic groups is justified, as it would improve the knowledge of molecular basis of the disease. Here, we present the results of genetic analysis in a large cohort of 143 unrelated Mexican subjects with a variety of RDs. METHODS: A targeted NGS approach covering 199 RD genes was employed for molecular screening of 143 unrelated patients. In addition to probands, 258 relatives were genotyped by Sanger sequencing for familial segregation of pathogenic variants. RESULTS: A solving rate of 66% (95/143) was achieved, with evidence of extensive loci (44 genes) and allelic (110 pathogenic variants) heterogeneity. Forty-eight percent of the identified pathogenic variants were novel while ABCA4, CRB1, USH2A, and RPE65 carried the greatest number of alterations. Novel deleterious variants in IDH3B and ARL6 were identified, supporting their involvement in RD. Familial segregation of causal variants allowed the recognition of 124 autosomal or X-linked carriers. CONCLUSION: Our results illustrate the utility of NGS for genetic diagnosis of RDs of different populations for a better knowledge of the mutational landscape associated with the disease.

A recurrent de novo mutation in ATP1A3 gene in a Mexican patient with alternating hemiplegia of childhood detected by massively parallel sequencing.

Background: Pediatric movement disorders represent a diagnostic challenge for pediatricians and pediatric neurologists due to their high clinical heterogeneity and shared common features. Therefore, specific diagnoses require different approaches including metabolic work-up and specific tests for frequent genetic conditions. Alternating hemiplegia of childhood (AHC) is an ultra-rare pediatric movement disorder, characterized by paroxysmal alternating hemiplegia, dystonia, and seizure-like episodes that can be misleading during the evaluation of a child with a movement disorder. Case report: We present a Mexican patient with abnormal movements referred to the Genetics clinic because of hyperammonemia and a possible organic acidemia. Our assessment did not find clinical features compatible with an inborn error of metabolism. A massively parallel sequencing approach with targeted panel sequencing was used to get a final diagnosis. A missense variant c.2839G>A (p.Gly947Arg) located at exon 21 of ATP1A3 gene was demonstrated. This variant (rs398122887) has been previously reported as de novo producing alternating hemiplegia of childhood (AHC). Conclusions: AHC is an ultra-rare syndrome presented as a movement disorder with seizure-like episodes and a unique facial phenotype. Clinicians should be aware of this combination in order to diagnose this condition in a timely manner. Massive parallel sequencing panels are emerging as the best approach to diagnose rare movement disorders and simultaneously rule out metabolic disorders and common epileptic syndromes.

Introducción: Los trastornos pediátricos del movimiento representan un reto diagnóstico para pediatras y neurólogos pediatras debido a su gran heterogeneidad clínica y características comunes compartidas. Por lo tanto, los diagnósticos específicos requieren de diferentes abordajes que incluyen la búsqueda de desórdenes metabólicos y pruebas específicas para condiciones genéticas frecuentes. La hemiplejia alternante de la infancia (AHC) es un trastorno pediátrico del movimiento poco común, caracterizado por cuadros paroxísticos de hemiplejia alternante, distonía y episodios semejantes a crisis epilépticas, que pueden resultar desorientadores durante el abordaje diagnóstico de un infante con un desorden del movimiento. Caso clínico: Presentamos una paciente mexicana con movimientos anormales referida a la Clínica de Genética por hiperamonemia y una posible acidemia orgánica. Nuestro abordaje no identificó características clínicas compatibles con un error innato del metabolismo. Se utilizó un abordaje basado en secuenciación masiva en paralelo para obtener un diagnóstico final. Se demostró una variante de sentido equivocado c.2839G>A (p.Gly947Arg) localizada en el exón 21 del gen ATP1A3. Esta variante (rs398122887) ha sido previamente reportada como de novo, ocasionando AHC. Conclusiones: La AHC es un síndrome excepcionalmente raro que se presenta con un trastorno del movimiento con cuadros semejantes a crisis epilépticas y un fenotipo facial particular. Los médicos deben ser conscientes de esta combinación con el fin de diagnosticar oportunamente esta condición. Los paneles de secuenciación masiva están emergiendo como el mejor abordaje para diagnosticar trastornos del movimiento raros y, simultáneamente, descartar trastornos metabólicos y síndromes epilépticos comunes.

The clinical implications of molecular monitoring and analyses of inherited retinal diseases.

INTRODUCTION: Retinal dystrophies (RDs) are the most common cause of inherited blindness and one of the most genetically heterogeneous human diseases. RDs arise from mutations in genes involved in development and function of photoreceptors or other retinal cells. Identification of the genetic defect causing RD allows accurate diagnosis, prognosis, and counseling in affected patients. Molecular diagnosis is a tremendous challenge in RDs due to their locus and phenotypic heterogeneity. As conventional DNA sequencing approaches are impractical in such situation, Next Generation Sequencing (NGS)-based protocols are needed to identify RD-causing mutations. This is being accomplished by sequencing RD gene panels or by whole exome or whole genome sequencing approaches. Areas covered: This review discusses the current strategies for molecular diagnosis in RDs including their advantages and limitations, as well as their utility in diagnosis of non-syndromic versus syndromic RDs. Results of ongoing gene therapy protocols in RDs are also presented. Expert commentary: Molecular diagnosis in RD improves the medical management of patients. Importantly, demand for molecular screening for RDs is greatly expanding not only as a result of increasing development and availability of NGS technologies, but also of the growing number of gene-based clinical trials offering a potential treatment to patients.

A recurrent de novo mutation in ATP1A3 gene in a Mexican patient with alternating hemiplegia of childhood detected by massively parallel sequencing / Mutación de novo recurrente en el gen ATP1A3 en una paciente mexicana con hemiplejia alternante de la infancia detectada por secuenciación masiva en paralelo

Abstract Background: Pediatric movement disorders represent a diagnostic challenge for pediatricians and pediatric neurologists due to their high clinical heterogeneity and shared common features. Therefore, specific diagnoses require different approaches including metabolic work-up and specific tests for frequent genetic conditions. Alternating hemiplegia of childhood (AHC) is an ultra-rare pediatric movement disorder, characterized by paroxysmal alternating hemiplegia, dystonia, and seizure-like episodes that can be misleading during the evaluation of a child with a movement disorder. Case report: We present a Mexican patient with abnormal movements referred to the Genetics clinic because of hyperammonemia and a possible organic acidemia. Our assessment did not find clinical features compatible with an inborn error of metabolism. A massively parallel sequencing approach with targeted panel sequencing was used to get a final diagnosis. A missense variant c.2839G>A (p.Gly947Arg) located at exon 21 of ATP1A3 gene was demonstrated. This variant (rs398122887) has been previously reported as de novo producing alternating hemiplegia of childhood (AHC). Conclusions: AHC is an ultra-rare syndrome presented as a movement disorder with seizure-like episodes and a unique facial phenotype. Clinicians should be aware of this combination in order to diagnose this condition in a timely manner. Massive parallel sequencing panels are emerging as the best approach to diagnose rare movement disorders and simultaneously rule out metabolic disorders and common epileptic syndromes.

Resumen Introducción: Los trastornos pediátricos del movimiento representan un reto diagnóstico para pediatras y neurólogos pediatras debido a su gran heterogeneidad clínica y características comunes compartidas. Por lo tanto, los diagnósticos específicos requieren de diferentes abordajes que incluyen la búsqueda de desórdenes metabólicos y pruebas específicas para condiciones genéticas frecuentes. La hemiplejia alternante de la infancia (AHC) es un trastorno pediátrico del movimiento poco común, caracterizado por cuadros paroxísticos de hemiplejia alternante, distonía y episodios semejantes a crisis epilépticas, que pueden resultar desorientadores durante el abordaje diagnóstico de un infante con un desorden del movimiento. Caso clínico: Presentamos una paciente mexicana con movimientos anormales referida a la Clínica de Genética por hiperamonemia y una posible acidemia orgánica. Nuestro abordaje no identificó características clínicas compatibles con un error innato del metabolismo. Se utilizó un abordaje basado en secuenciación masiva en paralelo para obtener un diagnóstico final. Se demostró una variante de sentido equivocado c.2839G>A (p.Gly947Arg) localizada en el exón 21 del gen ATP1A3. Esta variante (rs398122887) ha sido previamente reportada como de novo, ocasionando AHC. Conclusiones: La AHC es un síndrome excepcionalmente raro que se presenta con un trastorno del movimiento con cuadros semejantes a crisis epilépticas y un fenotipo facial particular. Los médicos deben ser conscientes de esta combinación con el fin de diagnosticar oportunamente esta condición. Los paneles de secuenciación masiva están emergiendo como el mejor abordaje para diagnosticar trastornos del movimiento raros y, simultáneamente, descartar trastornos metabólicos y síndromes epilépticos comunes.