RESUMEN
Short-read next-generation sequencing has revolutionized our ability to identify variants underlying inherited diseases; however, it does not allow the phasing of variants to clarify their diagnostic interpretation. The advent of widespread, increasingly accurate long-read sequencing has opened up new applications not currently available through short-read next-generation sequencing. One such use is the ability to phase variants to clarify their diagnostic interpretation and to investigate the increasingly prevalent role of cis-acting variants in the pathogenesis of the inherited disease, so-called complex alleles. Complex alleles are becoming an increasingly prevalent part of the study of genes associated with inherited diseases, for example, in ABCA4-related diseases. We sought to establish a cost-effective method to phase contiguous segments of the 130-kb ABCA4 locus by long-read sequencing of overlapping amplification products. Using the comprehensively characterized CEPH sample, NA12878, we verified the accuracy and robustness of our assay. However, in-field assessment of its utility using clinical test cases was hampered by the paucity and distribution of identified variants and by PCR chimerism, particularly where the number of PCR cycles was high. Despite this, we were able to construct robust phase blocks of up to 94.9 kb, representing 73% of the ABCA4 locus. We conclude that, although haplotype analysis of variants located within discrete amplification products was robust and informative, the stitching together of larger phase blocks using overlapping single-molecule reads remained practically challenging.
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Secuenciación de Nanoporos , Haplotipos/genética , Alelos , Reacción en Cadena de la Polimerasa , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Análisis de Secuencia de ADN/métodosRESUMEN
PURPOSE: To characterize the phenotype observed in a case series with macular disease and determine the cause. DESIGN: Multicenter case series. PARTICIPANTS: Six families (7 patients) with sporadic or multiplex macular disease with onset at 20 to 78 years, and 1 patient with age-related macular degeneration. METHODS: Patients underwent ophthalmic examination; exome, genome, or targeted sequencing; and/or polymerase chain reaction (PCR) amplification of the breakpoint, followed by cloning and Sanger sequencing or direct Sanger sequencing. MAIN OUTCOME MEASURES: Clinical phenotypes, genomic findings, and a hypothesis explaining the mechanism underlying disease in these patients. RESULTS: All 8 cases carried the same deletion encompassing the genes TPRX1, CRX, and SULT2A1, which was absent from 382 control individuals screened by breakpoint PCR and 13 096 Clinical Genetics patients with a range of other inherited conditions screened by array comparative genomic hybridization. Microsatellite genotypes showed that these 7 families are not closely related, but genotypes immediately adjacent to the deletion breakpoints suggest they may share a distant common ancestor. CONCLUSIONS: Previous studies had found that carriers for a single defective CRX allele that was predicted to produce no functional CRX protein had a normal ocular phenotype. Here, we show that CRX whole-gene deletion in fact does cause a dominant late-onset macular disease.
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Degeneración Macular , Humanos , Hibridación Genómica Comparativa , Degeneración Macular/diagnóstico , Degeneración Macular/genética , Linaje , Fenotipo , Transactivadores/genética , Proteínas de Homeodominio/genéticaRESUMEN
BACKGROUND: Primary ciliopathies represent a group of inherited disorders due to defects in the primary cilium, the 'cell's antenna'. The 100,000 Genomes Project was launched in 2012 by Genomics England (GEL), recruiting National Health Service (NHS) patients with eligible rare diseases and cancer. Sequence data were linked to Human Phenotype Ontology (HPO) terms entered by recruiting clinicians. METHODS: Eighty-three prescreened probands were recruited to the 100,000 Genomes Project suspected to have congenital malformations caused by ciliopathies in the following disease categories: Bardet-Biedl syndrome (n=45), Joubert syndrome (n=14) and 'Rare Multisystem Ciliopathy Disorders' (n=24). We implemented a bespoke variant filtering and analysis strategy to improve molecular diagnostic rates for these participants. RESULTS: We determined a research molecular diagnosis for n=43/83 (51.8%) probands. This is 19.3% higher than previously reported by GEL (n=27/83 (32.5%)). A high proportion of diagnoses are due to variants in non-ciliopathy disease genes (n=19/43, 44.2%) which may reflect difficulties in clinical recognition of ciliopathies. n=11/83 probands (13.3%) had at least one causative variant outside the tiers 1 and 2 variant prioritisation categories (GEL's automated triaging procedure), which would not be reviewed in standard 100,000 Genomes Project diagnostic strategies. These include four structural variants and three predicted to cause non-canonical splicing defects. Two unrelated participants have biallelic likely pathogenic variants in LRRC45, a putative novel ciliopathy disease gene. CONCLUSION: These data illustrate the power of linking large-scale genome sequence to phenotype information. They demonstrate the value of research collaborations in order to maximise interpretation of genomic data.
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Anomalías Múltiples , Ciliopatías , Anomalías del Ojo , Enfermedades Renales Quísticas , Anomalías Múltiples/genética , Ciliopatías/diagnóstico , Ciliopatías/genética , Ciliopatías/patología , Anomalías del Ojo/genética , Humanos , Enfermedades Renales Quísticas/genética , Fenotipo , Medicina EstatalRESUMEN
BACKGROUND: The 100 000 Genomes Project (100K) recruited National Health Service patients with eligible rare diseases and cancer between 2016 and 2018. PanelApp virtual gene panels were applied to whole genome sequencing data according to Human Phenotyping Ontology (HPO) terms entered by recruiting clinicians to guide focused analysis. METHODS: We developed a reverse phenotyping strategy to identify 100K participants with pathogenic variants in nine prioritised disease genes (BBS1, BBS10, ALMS1, OFD1, DYNC2H1, WDR34, NPHP1, TMEM67, CEP290), representative of the full phenotypic spectrum of multisystemic primary ciliopathies. We mapped genotype data 'backwards' onto available clinical data to assess potential matches against phenotypes. Participants with novel molecular diagnoses and key clinical features compatible with the identified disease gene were reported to recruiting clinicians. RESULTS: We identified 62 reportable molecular diagnoses with variants in these nine ciliopathy genes. Forty-four have been reported by 100K, 5 were previously unreported and 13 are new diagnoses. We identified 11 participants with unreportable, novel molecular diagnoses, who lacked key clinical features to justify reporting to recruiting clinicians. Two participants had likely pathogenic structural variants and one a deep intronic predicted splice variant. These variants would not be prioritised for review by standard 100K diagnostic pipelines. CONCLUSION: Reverse phenotyping improves the rate of successful molecular diagnosis for unsolved 100K participants with primary ciliopathies. Previous analyses likely missed these diagnoses because incomplete HPO term entry led to incorrect gene panel choice, meaning that pathogenic variants were not prioritised. Better phenotyping data are therefore essential for accurate variant interpretation and improved patient benefit.
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Síndrome de Bardet-Biedl , Ciliopatías , Humanos , Antígenos de Neoplasias , Síndrome de Bardet-Biedl/genética , Proteínas Portadoras/genética , Proteínas de Ciclo Celular/genética , Ciliopatías/diagnóstico , Ciliopatías/genética , Proteínas del Citoesqueleto/genética , Genotipo , Proteínas Asociadas a Microtúbulos/genética , Fenotipo , Medicina Estatal , Genoma HumanoRESUMEN
Purpose: To investigate the molecular basis of recessively inherited congenital cataract, microcornea, and corneal opacification with or without coloboma and microphthalmia in two consanguineous families. Methods: Conventional autozygosity mapping was performed using single nucleotide polymorphism (SNP) microarrays. Whole-exome sequencing was completed on genomic DNA from one affected member of each family. Exome sequence data were also used for homozygosity mapping and copy number variation analysis. PCR and Sanger sequencing were used to confirm the identification of mutations and to screen further patients. Evolutionary conservation of protein sequences was assessed using CLUSTALW, and protein structures were modeled using PyMol. Results: In family MEP68, a novel homozygous nucleotide substitution in SIX6 was found, c.547G>C, that converts the evolutionarily conserved aspartic acid residue at the 183rd amino acid in the protein to a histidine, p.(Asp183His). This residue mapped to the third helix of the DNA-binding homeobox domain in SIX6, which interacts with the major groove of double-stranded DNA. This interaction is likely to be disrupted by the mutation. In family F1332, a novel homozygous 1034 bp deletion that encompasses the first exon of SIX6 was identified, chr14:g.60975890_60976923del. Both mutations segregated with the disease phenotype as expected for a recessive condition and were absent from publicly available variant databases. Conclusions: Our findings expand the mutation spectrum in this form of inherited eye disease and confirm that homozygous human SIX6 mutations cause a developmental spectrum of ocular phenotypes that includes not only the previously described features of microphthalmia, coloboma, and congenital cataract but also corneal abnormalities.
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Catarata , Coloboma , Enfermedades de la Córnea , Anomalías del Ojo , Microftalmía , Catarata/congénito , Catarata/genética , Coloboma/genética , Enfermedades de la Córnea/genética , ADN/genética , Variaciones en el Número de Copia de ADN , Análisis Mutacional de ADN , Anomalías del Ojo/genética , Proteínas de Homeodominio/genética , Humanos , Microftalmía/genética , Mutación , Linaje , Fenotipo , Transactivadores/genéticaRESUMEN
Purpose: To describe the clinical phenotype and genetic basis of non-syndromic retinitis pigmentosa (RP) in one family and two sporadic cases with biallelic mutations in the transcription factor neural retina leucine zipper (NRL). Methods: Exome sequencing was performed in one affected family member. Microsatellite genotyping was used for haplotype analysis. PCR and Sanger sequencing were used to confirm mutations in and screen other family members where they were available. The SMART tool for domain prediction helped us build the protein schematic diagram. Results: For family MM1 of Pakistani origin, whole-exome sequencing and microsatellite genotyping revealed homozygosity on chromosome 14 and identified a homozygous stop-loss mutation in NRL, NM_006177.5: c.713G>T, p.*238Lext57, which is predicted to add an extra 57 amino acids to the normal protein chain. The variant segregated with disease symptoms in the family. For case RP-3051 of Spanish ancestry, clinical exome sequencing focusing on the morbid genome highlighted a homozygous nonsense mutation in NRL, c.238C>T, p.Gln80*, as the most likely disease candidate. For case RP-1553 of Romanian ethnicity, targeted-exome sequencing of 73 RP/LCA genes identified a homozygous nonsense mutation in NRL, c.544C>T, p.Gln182*. The variants were either rare or absent in the gnomAD database. Conclusions: NRL mutations predominantly cause dominant retinal disease, but there have been five published reports of mutations causing recessive disease. Here, we present three further examples of recessive RP due to NRL mutations. The phenotypes observed are consistent with those in the previous reports, and the observed mutation types and distribution further confirm distinct patterns for variants in NRL causing recessive and dominant diseases.
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Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Proteínas del Ojo/genética , Retinitis Pigmentosa , Factores de Transcripción , Codón sin Sentido , Análisis Mutacional de ADN , Humanos , Mutación , Linaje , Retinitis Pigmentosa/diagnóstico , Retinitis Pigmentosa/genética , Factores de Transcripción/genéticaRESUMEN
Biallelic mutations in G-Protein coupled receptor kinase 1 (GRK1) cause Oguchi disease, a rare subtype of congenital stationary night blindness (CSNB). The purpose of this study was to identify disease causing GRK1 variants and use in-depth bioinformatic analyses to evaluate how their impact on protein structure could lead to pathogenicity. Patients' genomic DNA was sequenced by whole genome, whole exome or focused exome sequencing. Disease associated variants, published and novel, were compared to nondisease associated missense variants. The impact of GRK1 missense variants at the protein level were then predicted using a series of computational tools. We identified twelve previously unpublished cases with biallelic disease associated GRK1 variants, including eight novel variants, and reviewed all GRK1 disease associated variants. Further structure-based scoring revealed a hotspot for missense variants in the kinase domain. In addition, to aid future clinical interpretation, we identified the bioinformatics tools best able to differentiate disease associated from nondisease associated variants. We identified GRK1 variants in Oguchi disease patients and investigated how disease-causing variants may impede protein function in-silico.
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Enfermedades Hereditarias del Ojo , Quinasa 1 del Receptor Acoplado a Proteína-G , Ceguera Nocturna , Enfermedades Hereditarias del Ojo/genética , Quinasa 1 del Receptor Acoplado a Proteína-G/genética , Humanos , Ceguera Nocturna/genéticaRESUMEN
Familial exudative vitreoretinopathy (FEVR) is an inherited blinding disorder characterized by the abnormal development of the retinal vasculature. The majority of mutations identified in FEVR are found within four genes that encode the receptor complex (FZD4, LRP5, and TSPAN12) and ligand (NDP) of a molecular pathway that controls angiogenesis, the Norrin-ß-catenin signaling pathway. However, half of all FEVR-affected case subjects do not harbor mutations in these genes, indicating that further mutated genes remain to be identified. Here we report the identification of mutations in CTNNB1, the gene encoding ß-catenin, as a cause of FEVR. We describe heterozygous mutations (c.2142_2157dup [p.His720∗] and c.2128C>T [p.Arg710Cys]) in two dominant FEVR-affected families and a de novo mutation (c.1434_1435insC [p.Glu479Argfs∗18]) in a simplex case subject. Previous studies have reported heterozygous de novo CTNNB1 mutations as a cause of syndromic intellectual disability (ID) and autism spectrum disorder, and somatic mutations are linked to many cancers. However, in this study we show that Mendelian inherited CTNNB1 mutations can cause non-syndromic FEVR and that FEVR can be a part of the syndromic ID phenotype, further establishing the role that ß-catenin signaling plays in the development of the retinal vasculature.
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Enfermedades de la Retina/genética , Transducción de Señal , beta Catenina/metabolismo , Secuencia de Bases , Enfermedades Hereditarias del Ojo , Vitreorretinopatías Exudativas Familiares , Femenino , Heterocigoto , Humanos , Luciferasas/metabolismo , Masculino , Modelos Biológicos , Proteínas Mutantes/metabolismo , Mutación/genética , Linaje , Fenotipo , Transcripción GenéticaRESUMEN
PURPOSE: Determining the role of DYNC2H1 variants in nonsyndromic inherited retinal disease (IRD). METHODS: Genome and exome sequencing were performed for five unrelated cases of IRD with no identified variant. In vitro assays were developed to validate the variants identified (fibroblast assay, induced pluripotent stem cell [iPSC] derived retinal organoids, and a dynein motility assay). RESULTS: Four novel DYNC2H1 variants (V1, g.103327020_103327021dup; V2, g.103055779A>T; V3, g.103112272C>G; V4, g.103070104A>C) and one previously reported variant (V5, g.103339363T>G) were identified. In proband 1 (V1/V2), V1 was predicted to introduce a premature termination codon (PTC), whereas V2 disrupted the exon 41 splice donor site causing incomplete skipping of exon 41. V1 and V2 impaired dynein-2 motility in vitro and perturbed IFT88 distribution within cilia. V3, homozygous in probands 2-4, is predicted to cause a PTC in a retina-predominant transcript. Analysis of retinal organoids showed that this new transcript expression increased with organoid differentiation. V4, a novel missense variant, was in trans with V5, previously associated with Jeune asphyxiating thoracic dystrophy (JATD). CONCLUSION: The DYNC2H1 variants discussed herein were either hypomorphic or affecting a retina-predominant transcript and caused nonsyndromic IRD. Dynein variants, specifically DYNC2H1 variants are reported as a cause of non syndromic IRD.
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Síndrome de Ellis-Van Creveld , Degeneración Retiniana , Dineínas Citoplasmáticas/genética , Síndrome de Ellis-Van Creveld/genética , Exones , Humanos , Mutación , Linaje , Retina , Degeneración Retiniana/genéticaRESUMEN
PURPOSE: RAX2 encodes a homeobox-containing transcription factor, in which four monoallelic pathogenic variants have been described in autosomal dominant cone-dominated retinal disease. METHODS: Exome sequencing in a European cohort with inherited retinal disease (IRD) (n = 2086) was combined with protein structure modeling of RAX2 missense variants, bioinformatics analysis of deletion breakpoints, haplotyping of RAX2 variant c.335dup, and clinical assessment of biallelic RAX2-positive cases and carrier family members. RESULTS: Biallelic RAX2 sequence and structural variants were found in five unrelated European index cases, displaying nonsyndromic autosomal recessive retinitis pigmentosa (ARRP) with an age of onset ranging from childhood to the mid-40s (average mid-30s). Protein structure modeling points to loss of function of the novel recessive missense variants and to a dominant-negative effect of the reported dominant RAX2 alleles. Structural variants were fine-mapped to disentangle their underlying mechanisms. Haplotyping of c.335dup in two cases suggests a common ancestry. CONCLUSION: This study supports a role for RAX2 as a novel disease gene for recessive IRD, broadening the mutation spectrum from sequence to structural variants and revealing a founder effect. The identification of biallelic RAX2 pathogenic variants in five unrelated families shows that RAX2 loss of function may be a nonnegligible cause of IRD in unsolved ARRP cases.
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Proteínas del Ojo/genética , Proteínas de Homeodominio/genética , Retinitis Pigmentosa/genética , Factores de Transcripción/genética , Adulto , Análisis Mutacional de ADN/métodos , Proteínas del Ojo/metabolismo , Proteínas del Ojo/fisiología , Femenino , Genes Recesivos/genética , Estudios de Asociación Genética/métodos , Genotipo , Haplotipos/genética , Proteínas de Homeodominio/metabolismo , Proteínas de Homeodominio/fisiología , Humanos , Masculino , Persona de Mediana Edad , Mutación/genética , Mutación Missense/genética , Linaje , Fenotipo , Retina/metabolismo , Retina/patología , Enfermedades de la Retina/genética , Factores de Transcripción/metabolismo , Factores de Transcripción/fisiología , Población Blanca/genéticaRESUMEN
The original version of this Article contained an incorrect version of Fig. 3, which included two variants initially shown in black text in Fig. 3a that the authors removed from the final manuscript. The correct version of Fig. 3 without the two variants now appears in the PDF and HTML versions of the Article.
RESUMEN
Hereditary retinal degenerations encompass a group of genetic diseases characterized by extreme clinical variability. Following next-generation sequencing and autozygome-based screening of patients presenting with a peculiar, recessive form of cone-dominated retinopathy, we identified five homozygous variants [p.(Asp594fs), p.(Gln117*), p.(Met712fs), p.(Ile756Phe), and p.(Glu543Lys)] in the polyglutamylase-encoding gene TTLL5, in eight patients from six families. The two male patients carrying truncating TTLL5 variants also displayed a substantial reduction in sperm motility and infertility, whereas those carrying missense changes were fertile. Defects in this polyglutamylase in humans have recently been associated with cone photoreceptor dystrophy, while mouse models carrying truncating mutations in the same gene also display reduced fertility in male animals. We examined the expression levels of TTLL5 in various human tissues and determined that this gene has multiple viable isoforms, being highly expressed in testis and retina. In addition, antibodies against TTLL5 stained the basal body of photoreceptor cells in rat and the centrosome of the spermatozoon flagellum in humans, suggesting a common mechanism of action in these two cell types. Taken together, our data indicate that mutations in TTLL5 delineate a novel, allele-specific syndrome causing defects in two as yet pathogenically unrelated functions, reproduction and vision.
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Proteínas Portadoras/genética , Distrofias de Conos y Bastones/enzimología , Expresión Génica , Infertilidad Masculina/enzimología , Mutación , Adolescente , Adulto , Anciano , Animales , Distrofias de Conos y Bastones/genética , Análisis Mutacional de ADN , Modelos Animales de Enfermedad , Proteínas del Ojo/genética , Femenino , Homocigoto , Humanos , Infertilidad Masculina/genética , Masculino , Ratones , Persona de Mediana Edad , Especificidad de Órganos , Linaje , Células Fotorreceptoras de Vertebrados/enzimología , Ratas , Motilidad Espermática , Espermatozoides/enzimología , Testículo/enzimologíaRESUMEN
Retinal dystrophies are an overlapping group of genetically heterogeneous conditions resulting from mutations in more than 250 genes. Here we describe five families affected by an adult-onset retinal dystrophy with early macular involvement and associated central visual loss in the third or fourth decade of life. Affected individuals were found to harbor disease-causing variants in DRAM2 (DNA-damage regulated autophagy modulator protein 2). Homozygosity mapping and exome sequencing in a large, consanguineous British family of Pakistani origin revealed a homozygous frameshift variant (c.140delG [p.Gly47Valfs(∗)3]) in nine affected family members. Sanger sequencing of DRAM2 in 322 unrelated probands with retinal dystrophy revealed one European subject with compound heterozygous DRAM2 changes (c.494G>A [p.Trp165(∗)] and c.131G>A [p.Ser44Asn]). Inspection of previously generated exome sequencing data in unsolved retinal dystrophy cases identified a homozygous variant in an individual of Indian origin (c.64_66del [p.Ala22del]). Independently, a gene-based case-control association study was conducted via an exome sequencing dataset of 18 phenotypically similar case subjects and 1,917 control subjects. Using a recessive model and a binomial test for rare, presumed biallelic, variants, we found DRAM2 to be the most statistically enriched gene; one subject was a homozygote (c.362A>T [p.His121Leu]) and another a compound heterozygote (c.79T>C [p.Tyr27His] and c.217_225del [p.Val73_Tyr75del]). DRAM2 encodes a transmembrane lysosomal protein thought to play a role in the initiation of autophagy. Immunohistochemical analysis showed DRAM2 localization to photoreceptor inner segments and to the apical surface of retinal pigment epithelial cells where it might be involved in the process of photoreceptor renewal and recycling to preserve visual function.
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Degeneración Macular/genética , Degeneración Macular/patología , Proteínas de la Membrana/genética , Mutación/genética , Distrofias Retinianas/genética , Distrofias Retinianas/patología , Adulto , Secuencia de Bases , Exoma/genética , Homocigoto , Humanos , Inmunohistoquímica , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Pakistán/etnología , Linaje , Análisis de Secuencia de ADN , Reino UnidoRESUMEN
BACKGROUND: Albinism refers to a group of disorders primarily characterized by hypopigmentation. Affected individuals usually manifest both ocular and cutaneous features of the disease, but occasionally hair and skin pigmentation may appear normal. This is the case in ocular albinism, an X chromosome linked disorder resulting from mutation of GPR143. Female carriers may be recognized by a "mud-splatter" appearance in the peripheral retina. The macula is thought to be normal, however. METHODS: Obligate female carriers of pathogenic GPR143 alleles were recruited. Molecular confirmation of disease was performed only for atypical cases. Detailed retinal imaging was performed (colour fundus photography, optical coherence tomography, fundus autofluorescence. RESULTS: Eight individuals were ascertained. A novel GPR143 mutation was identified in one family (p.Gln328Ter). Foveal fundus autofluorescence was subjectively reduced in 6/6 patients imaged. A "tapetal-like" pattern of autofluorescence was visible at the macula in 3/6. Persistence of the inner retinal layers at the fovea was observed in 6/8 females. CONCLUSION: Female carriers of ocular albinism may manifest signs of retinal pigment epithelium mosaicism at the macula and the peripheral fundus. A tapetal-like reflex on fundus autofluorescence may be considered the macular correlate of "mud-splatter."
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Albinismo Ocular/patología , Retina/patología , Adulto , Albinismo Ocular/genética , Proteínas del Ojo/genética , Femenino , Heterocigoto , Humanos , Mácula Lútea/patología , Glicoproteínas de Membrana/genética , Persona de Mediana Edad , Estudios Prospectivos , Epitelio Pigmentado de la Retina/patologíaRESUMEN
Foveal hypoplasia and optic nerve misrouting are developmental defects of the visual pathway and only co-occur in connection with albinism; to date, they have only been associated with defects in the melanin-biosynthesis pathway. Here, we report that these defects can occur independently of albinism in people with recessive mutations in the putative glutamine transporter gene SLC38A8. Nine different mutations were identified in seven Asian and European families. Using morpholino-mediated ablation of Slc38a8 in medaka fish, we confirmed that pigmentation is unaffected by loss of SLC38A8. Furthermore, by undertaking an association study with SNPs at the SLC38A8 locus, we showed that common variants within this gene modestly affect foveal thickness in the general population. This study reveals a melanin-independent component underpinning the development of the visual pathway that requires a functional role for SLC38A8.
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Albinismo , Sistemas de Transporte de Aminoácidos Neutros/genética , Fóvea Central/anomalías , Genes Recesivos , Mutación , Nervio Óptico/fisiopatología , Animales , Niño , Consanguinidad , Análisis Mutacional de ADN , Femenino , Homocigoto , Humanos , Masculino , Linaje , Fenotipo , SíndromeRESUMEN
Familial exudative vitreoretinopathy (FEVR) is a genetically heterogeneous disorder characterized by abnormal vascularization of the peripheral retina, which can result in retinal detachment and severe visual impairment. In a large Dutch FEVR family, we performed linkage analysis, exome sequencing, and segregation analysis of DNA variants. We identified putative disease-causing DNA variants in proline-alanine-rich ste20-related kinase (c.791dup; p.Ser265ValfsX64) and zinc finger protein 408 (ZNF408) (c.1363C>T; p.His455Tyr), the latter of which was also present in an additional Dutch FEVR family that subsequently appeared to share a common ancestor with the original family. Sequence analysis of ZNF408 in 132 additional individuals with FEVR revealed another potentially pathogenic missense variant, p.Ser126Asn, in a Japanese family. Immunolocalization studies in COS-1 cells transfected with constructs encoding the WT and mutant ZNF408 proteins, revealed that the WT and the p.Ser126Asn mutant protein show complete nuclear localization, whereas the p.His455Tyr mutant protein was localized almost exclusively in the cytoplasm. Moreover, in a cotransfection assay, the p.His455Tyr mutant protein retains the WT ZNF408 protein in the cytoplasm, suggesting that this mutation acts in a dominant-negative fashion. Finally, morpholino-induced knockdown of znf408 in zebrafish revealed defects in developing retinal and trunk vasculature, that could be rescued by coinjection of RNA encoding human WT ZNF408 but not p.His455Tyr mutant ZNF408. Together, our data strongly suggest that mutant ZNF408 results in abnormal retinal vasculogenesis in humans and is associated with FEVR.
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Mutación , Vasos Retinianos/metabolismo , Vitreorretinopatía Proliferativa/genética , Proteínas de Pez Cebra/genética , Secuencia de Aminoácidos , Animales , Animales Modificados Genéticamente , Células COS , Núcleo Celular/metabolismo , Chlorocebus aethiops , Análisis Mutacional de ADN , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Salud de la Familia , Femenino , Perfilación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Masculino , Microscopía Fluorescente , Datos de Secuencia Molecular , Linaje , Vasos Retinianos/embriología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Vitreorretinopatía Proliferativa/metabolismo , Vitreorretinopatía Proliferativa/patología , Pez Cebra/embriología , Pez Cebra/genética , Pez Cebra/metabolismo , Proteínas de Pez Cebra/metabolismoRESUMEN
The ciliopathies are a group of heterogeneous diseases with considerable variations in phenotype for allelic conditions such as Meckel-Gruber syndrome (MKS) and Joubert syndrome (JBTS) even at the inter-individual level within families. In humans, mutations in TMEM67 (also known as MKS3) cause both MKS and JBTS, with TMEM67 encoding the orphan receptor meckelin (TMEM67) that localizes to the ciliary transition zone. We now describe the Tmem67(tm1(Dgen/H)) knockout mouse model that recapitulates the brain phenotypic variability of these human ciliopathies, with categorization of Tmem67 mutant animals into two phenotypic groups. An MKS-like incipient congenic group (F6 to F10) manifested very variable neurological features (including exencephaly, and frontal/occipital encephalocele) that were associated with the loss of primary cilia, diminished Shh signalling and dorsalization of the caudal neural tube. The 'MKS-like' group also had high de-regulated canonical Wnt/ß-catenin signalling associated with hyper-activated Dishevelled-1 (Dvl-1) localized to the basal body. Conversely, a second fully congenic group (F > 10) had less variable features pathognomonic for JBTS (including cerebellar hypoplasia), and retention of abnormal bulbous cilia associated with mild neural tube ventralization. The 'JBTS-like' group had de-regulated low levels of canonical Wnt signalling associated with the loss of Dvl-1 localization to the basal body. Our results suggest that modifier alleles partially determine the variation between MKS and JBTS, implicating the interaction between Dvl-1 and meckelin, or other components of the ciliary transition zone. The Tmem67(tm1(Dgen/H)) line is unique in modelling the variable expressivity of phenotypes in these two ciliopathies.
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Enfermedades Cerebelosas/metabolismo , Trastornos de la Motilidad Ciliar/metabolismo , Encefalocele/metabolismo , Anomalías del Ojo/metabolismo , Proteínas Hedgehog/metabolismo , Enfermedades Renales Quísticas/metabolismo , Proteínas de la Membrana/genética , Enfermedades Renales Poliquísticas/metabolismo , Vía de Señalización Wnt , Anomalías Múltiples , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Tipificación del Cuerpo/genética , Enfermedades Cerebelosas/genética , Enfermedades Cerebelosas/patología , Cerebelo/anomalías , Cilios/patología , Trastornos de la Motilidad Ciliar/genética , Trastornos de la Motilidad Ciliar/patología , Modelos Animales de Enfermedad , Proteínas Dishevelled , Encefalocele/genética , Encefalocele/patología , Anomalías del Ojo/genética , Anomalías del Ojo/patología , Regulación de la Expresión Génica , Genes Reporteros , Células HEK293 , Humanos , Enfermedades Renales Quísticas/genética , Enfermedades Renales Quísticas/patología , Luciferasas de Luciérnaga/biosíntesis , Luciferasas de Luciérnaga/genética , Proteínas de la Membrana/deficiencia , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Defectos del Tubo Neural/genética , Defectos del Tubo Neural/metabolismo , Defectos del Tubo Neural/patología , Fenotipo , Fosfoproteínas/metabolismo , Enfermedades Renales Poliquísticas/genética , Enfermedades Renales Poliquísticas/patología , Transporte de Proteínas , Retina/anomalías , Retina/metabolismo , Retina/patología , Retinitis PigmentosaRESUMEN
PURPOSE: To investigate the molecular basis of retinitis pigmentosa in two consanguineous families of Pakistani origin with multiple affected members. METHODS: Homozygosity mapping and Sanger sequencing of candidate genes were performed in one family while the other was analyzed with whole exome next-generation sequencing. A minigene splicing assay was used to confirm the splicing defects. RESULTS: In family MA48, a novel homozygous nucleotide substitution in C8orf37, c.244-2A>C, that disrupted the consensus splice acceptor site of exon 3 was found. The minigene splicing assay revealed that this mutation activated a cryptic splice site within exon 3, causing a 22 bp deletion in the transcript that is predicted to lead to a frameshift followed by premature protein truncation. In family MA13, a novel homozygous null mutation in C8orf37, c.555G>A, p.W185*, was identified. Both mutations segregated with the disease phenotype as expected in a recessive manner and were absent in 8,244 unrelated individuals of South Asian origin. CONCLUSIONS: In this report, we describe C8orf37 mutations that cause retinal dystrophy in two families of Pakistani origin, contributing further data on the phenotype and the spectrum of mutations in this form of retinitis pigmentosa.