RESUMO
Inherited retinal diseases are clinically heterogeneous and are associated with nearly 300 different genes. In this retrospective, observational study of a consecutive cohort of 159 patients (134 families) with childhood-onset (<16 years of age) retinal dystrophy, molecular investigations, and in-depth phenotyping were performed to determine key clinical and molecular characteristics. The most common ocular phenotype was rod-cone dystrophy in 40 patients. Leber Congenital Amaurosis, the most severe form of retinal dystrophy, was present in 10 patients, and early onset severe retinal dystrophy in 22 patients. Analysis has so far identified 131 pathogenic or likely pathogenic variants including 22 novel variants. Molecular diagnosis was achieved in 112 of 134 families (83.6%) by NGS gene panel investigation in 60 families, Sanger sequencing in 27 families, and Asper microarray in 25 families. An additional nine variants of uncertain significance were also found including three novel variants. Variants in 36 genes have been identified with the most common being ABCA4 retinopathy in 36 families. Five sporadic retinal dystrophy patients were found to have variants in dominant and X-linked genes (CRX, RHO, RP2, and RPGR) resulting in more accurate genetic counseling of inheritance for these families. Variants in syndromic associated genes including ALMS1, SDCCAG8, and PPT1 were identified in eight families enabling directed systemic care.
Assuntos
Proteínas de Ciclo Celular/genética , Distrofias de Cones e Bastonetes/genética , Amaurose Congênita de Leber/genética , Distrofias Retinianas/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Distrofias de Cones e Bastonetes/diagnóstico por imagem , Distrofias de Cones e Bastonetes/epidemiologia , Distrofias de Cones e Bastonetes/patologia , Feminino , Testes Genéticos , Proteínas de Homeodomínio/genética , Humanos , Amaurose Congênita de Leber/diagnóstico por imagem , Amaurose Congênita de Leber/epidemiologia , Amaurose Congênita de Leber/patologia , Masculino , Pessoa de Meia-Idade , Mutação/genética , Nova Zelândia/epidemiologia , Linhagem , Fenótipo , Distrofias Retinianas/diagnóstico por imagem , Distrofias Retinianas/epidemiologia , Distrofias Retinianas/patologia , Adulto JovemRESUMO
Inherited retinal diseases (IRDs) are genetically and phenotypically diverse, and they cause significant morbidity worldwide. Importantly, IRDs may be amenable to precision medicine strategies, and thus the molecular characterisation of causative variants is becoming increasingly important with the promise of personalised therapies on the horizon. ABCA4, involved in the translocation of visual cycle derivatives, is a well-established, frequent cause of IRDs worldwide, with pathogenic variants implicated in phenotypically diverse diseases. Identification of causative ABCA4 variants in some individuals, however, has been enigmatic, and resolution of this issue is currently a hotbed of research. Recent evidence has indicated that hypomorphic alleles, which cause disease under certain conditions, may account for some of the missing causal variants. It has been postulated that the ABCA4 c.5603A>T (p.Asn1868Ile) variant, previously considered benign, be reclassified as hypomorphic when in cis configuration with c.2588G>C (p.Gly863Ala/Gly863del), a variant previously considered to be pathogenic in its own right. We are exploring this relationship within an Australian cohort to test this theory.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Doenças Retinianas/genética , Alelos , Austrália , Humanos , Mutação , Linhagem , Retina/patologiaRESUMO
Purpose: The aim of this study was to analyze and report pathogenic variants in the ABCA4 gene in Brazilian patients with a clinical diagnosis of Stargardt disease. Methods: This retrospective study evaluated variants in the ABCA4 gene in Brazilian patients with Stargardt disease. The patients' visual acuity and age of symptom onset were obtained from previous medical records. The patients were classified according to the autofluorescence patterns. Results: Fifty patients aged between 10 and 65 years from 44 families were included in the study. Among these cases, the mean age of symptom onset was 14 years (range, 5-40 years). ABCA4 gene sequencing was conclusive in 40 patients (80%), negative in two patients (4%), and inconclusive in eight patients (16%). Four families carried homozygous pathogenic variants. Segregation analysis results were available for 23 families. One novel variant was found: p.Ala2084Pro. The most frequent pathogenic variant in this group was p.Arg602Trp (12/100 alleles). Based on the phenotypic characteristics assessed with fundus autofluorescence imaging, 12 patients were classified as having type I phenotype, 16 as having type II, and 18 patients as having type III. The cases classified as type III phenotype included patients who were homozygous for the p.Asn96Asp and p.Arg2030* variants. One patient with a type I phenotype carried the homozygous intronic variant c.3862+1G>A. Conclusions: Next-generation sequencing was effective for the molecular diagnosis of genetic diseases and specifically allowed a conclusive diagnosis in 80% (40/50) of the patients. As the ABCA4 gene does not show a preferential region for pathogenic variants, the diagnosis of Stargardt disease depends on broader analysis of the gene. The most common pathogenic variants in the ABCA4 gene described in the literature were also found in these Brazilian patients. Although some genotype-phenotype correlations were found, more studies regarding the progression of Stargardt disease will help increase our understanding of the pathogenicity of these gene variants.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Degeneração Macular/congênito , Mutação , Polimorfismo de Nucleotídeo Único , Adolescente , Adulto , Idoso , Brasil/etnologia , Criança , Consanguinidade , Análise Mutacional de DNA , Eletrorretinografia , Feminino , Angiofluoresceinografia , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Degeneração Macular/diagnóstico , Degeneração Macular/etnologia , Degeneração Macular/genética , Masculino , Pessoa de Meia-Idade , Linhagem , Fenótipo , Estudos Retrospectivos , Doença de Stargardt , Tomografia de Coerência Óptica , Acuidade Visual , Adulto JovemRESUMO
PURPOSE OF REVIEW: We are witnessing lightning-fast advances in the molecular diagnosis of inherited retinal dystrophies, mainly due to the widespread use of next-generation sequencing technologies. The purpose of this review is to highlight the breadth of findings from this in-depth testing approach, and to propose changes to our traditional testing and diagnostic paradigms. Lessons learned from modern molecular testing suggest that the previous concept of inherited retinal dystrophies as a group of 'single gene diseases' may require a significant update. RECENT FINDINGS: All of the known retinal dystrophies genes can now be sequenced. In many cases, this nonhypothesis driven testing strategy is uncovering mutations in unsuspected genes, generating data that challenges established concepts of genetic mechanisms and provides insights regarding genes previously thought to be exclusively related to syndromic disease. Recent advances in testing have improved not only the breadth, but also the depth of genetic data. For example, deep intronic sequencing has uncovered many novel intronic mutations/variations in the ABCA4 gene. SUMMARY: Currently, in approximately 50-60% of patients with nonsyndromic retinal dystrophy, the disease mechanism can be identified. The presence of pathogenic alleles in more than one gene is not uncommon. Retinal dystrophy, with relatively defined clinical presentations and a large but limited number of genes involved, is becoming a model for the next-generation study of molecular disease mechanisms.
Assuntos
Distrofias Retinianas/diagnóstico , Distrofias Retinianas/genética , Transportadores de Cassetes de Ligação de ATP/genética , Bases de Dados de Compostos Químicos , Predisposição Genética para Doença , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Íntrons , MutaçãoRESUMO
Cerebrotendinous xanthomatosis (CTX) is a rare, difficult-to-diagnose genetic disorder of bile acid (BA) synthesis that can cause progressive neurological damage and premature death. Detection of CTX in the newborn period would be beneficial because an effective oral therapy for CTX is available to prevent disease progression. There is no suitable test to screen newborn dried bloodspots (DBS) for CTX. Blood screening for CTX is currently performed by GC-MS measurement of elevated 5α-cholestanol. We present here LC-ESI/MS/MS methodology utilizing keto derivatization with (O-(3-trimethylammonium-propyl) hydroxylamine) reagent to enable sensitive detection of ketosterol BA precursors that accumulate in CTX. The availability of isotopically enriched derivatization reagent allowed ready tagging of ketosterols to generate internal standards for isotope dilution quantification. Ketosterols were quantified and their utility as markers for CTX was compared with 5α-cholestanol. 7α,12α-Dihydroxy-4-cholesten-3-one provided the best discrimination between CTX and unaffected samples. In two CTX, newborn DBS concentrations of this ketosterol (120-214 ng/ml) were â¼10-fold higher than in unaffected newborn DBS (16.4 ± 6.0 ng/ml), such that quantification of this ketosterol provides a test with potential to screen newborn DBS for CTX. Early detection and intervention through newborn screening would greatly benefit those affected with CTX by preventing morbidity and mortality.
Assuntos
Colestenonas/sangue , Xantomatose Cerebrotendinosa/diagnóstico , Adulto , Calibragem , Estudos de Casos e Controles , Teste em Amostras de Sangue Seco , Humanos , Recém-Nascido , Triagem Neonatal , Padrões de Referência , Espectrometria de Massas por Ionização por Electrospray/normas , Espectrometria de Massas em Tandem/normas , Xantomatose Cerebrotendinosa/sangueAssuntos
Tendão do Calcâneo/efeitos dos fármacos , Ácido Quenodesoxicólico/uso terapêutico , Paraplegia/tratamento farmacológico , Xantomatose Cerebrotendinosa/tratamento farmacológico , Tendão do Calcâneo/patologia , Adulto , Colestanotriol 26-Mono-Oxigenase/genética , Predisposição Genética para Doença , Humanos , Imageamento por Ressonância Magnética , Masculino , Mutação de Sentido Incorreto , Paraplegia/diagnóstico , Paraplegia/genética , Fenótipo , Resultado do Tratamento , Xantomatose Cerebrotendinosa/complicações , Xantomatose Cerebrotendinosa/diagnóstico , Xantomatose Cerebrotendinosa/genéticaRESUMO
Congenital stationary night blindness (CSNB) is a disease affecting the night vision of individuals. Previous studies identified TRPM1 as a gene involved in reduced night vision. Homozygous deletion of TRPM1 was the cause of CSNB in several children in 6 Ashkenazi Jewish families, thereby prompting further investigation of the carrier status within the families as well as in large cohorts of unrelated Ashkenazi and Sephardi individuals. Affected children were tested with a CSNB next-generation (NextGen) sequencing panel. A deletion of TRPM1 exons 2 through 7 was detected and confirmed by PCR and sequence analysis. A TaqMan-based assay was used to assess the frequency of this deletion in 18266 individuals of Jewish descent. High-throughput amplicon sequencing was performed on 380 samples to determine the putative deletion-flanking founder haplotype. Heterozygous TRPM1 deletions were found in 2.75% (1/36) of Ashkenazi subjects and in 1.22% (1/82) individuals of mixed Ashkenazi/Sephardic origin. The homozygous deletion frequency in our data was 0.03% (1/4025) and was only found in Ashkenazi Jewish individuals. Homozygous deletion of exons 2-7 in TRPM1 is a common cause of CSNB and myopia in many Ashkenazi Jewish patients. This deletion is a founder Ashkenazi Jewish deletion.
RESUMO
BACKGROUND/AIMS: To describe the genetic characteristics of the cohort enrolled in the international multicentre progression of Stargardt disease 1 (STGD1) studies (ProgStar) and to determine geographic differences based on the allele frequency. METHODS: 345 participants with a clinical diagnosis of STGD1 and harbouring at least one disease-causing ABCA4 variant were enrolled from 9 centres in the USA and Europe. All variants were reviewed and in silico analysis was performed including allele frequency in public databases and pathogenicity predictions. Participants with multiple likely pathogenic variants were classified into four national subgroups (USA, UK, France, Germany), with subsequent comparison analysis of the allele frequency for each prevalent allele. RESULTS: 211 likely pathogenic variants were identified in the total cohort, including missense (63%), splice site alteration (18%), stop (9%) and others. 50 variants were novel. Exclusively missense variants were detected in 139 (50%) of 279 patients with multiple pathogenic variants. The three most prevalent variants of these patients with multiple pathogenic variants were p.G1961E (15%), p.G863A (7%) and c.5461-10 T>C (5%). Subgroup analysis revealed a statistically significant difference between the four recruiting nations in the allele frequency of nine variants. CONCLUSIONS: There is a large spectrum of ABCA4 sequence variants, including 50 novel variants, in a well-characterised cohort thereby further adding to the unique allelic heterogeneity in STGD1. Approximately half of the cohort harbours missense variants only, indicating a relatively mild phenotype of the ProgStar cohort. There are significant differences in allele frequencies between nations, although the three most prevalent variants are shared as frequent variants.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Degeneração Macular/congênito , Mutação , Adolescente , Adulto , Idade de Início , Idoso , Criança , Pré-Escolar , Estudos de Coortes , Análise Mutacional de DNA , Bases de Dados Factuais , Feminino , Frequência do Gene , Técnicas de Genotipagem , Geografia , Humanos , Internacionalidade , Degeneração Macular/diagnóstico , Degeneração Macular/genética , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Doença de StargardtRESUMO
Purpose: To analyze the presence of complex alleles of the ABCA4 gene in Brazilian patients with Stargardt disease and to assess the correlation with clinical features. Methods: This was an observational cross-sectional study. Patients with a diagnosis of Stargardt disease who presented three pathogenic variants of the ABCA4 gene or who had variants previously described as complex alleles were included. The relatives of these probands were evaluated in the segregation analysis. The patients were evaluated based on age at symptom onset and visual acuity, and the clinical characteristics were classified according to the findings observed on autofluorescence examination. Results: Among the 47 families analyzed, approximately 30% (14/47) presented complex alleles. The segregation analysis in 14 families with cases of Stargardt disease identified three novel complex alleles and one previously described complex allele. The known complex allele p.[Leu541Pro; Ala1038Val] was identified in two families. The novel complex alleles identified were p.[Leu541Pro; Arg1443His] in five families, p.[Ser1642Arg; Val1682_Val1686del] in seven families, and p.[Pro1761Arg; Arg2106Cys] in one family. Furthermore, four new variants (p.Lys22Asn, p.Asp915Asn, p.Glu1447Val, and p.Pro1761Arg) were identified in the second allele of the ABCA4 gene. Conclusions: Segregation analysis is important in order to confirm the molecular diagnosis of patients with Stargardt disease, given the frequency of complex alleles in the ABCA4 gene. The various pathogenic variation combinations observed in this study were associated with different phenotypes.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Alelos , Degeneração Macular/congênito , Mutação , Adolescente , Adulto , Idoso , Brasil , Criança , Estudos Transversais , Análise Mutacional de DNA , Eletrorretinografia , Feminino , Estudos de Associação Genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Degeneração Macular/diagnóstico , Degeneração Macular/genética , Degeneração Macular/fisiopatologia , Masculino , Pessoa de Meia-Idade , Linhagem , Retina/fisiologia , Doença de Stargardt , Acuidade Visual/fisiologia , Adulto JovemRESUMO
PURPOSE: To determine the effect of 15 individual ABCA4 mutations on disease severity. METHODS: Eighty-two patients harboring 15 distinct ABCA4 mutations in trans with null (hemizygous), 10 homozygous, and 20 nullizygous patients were recruited. Age of onset was determined from medical histories. Electroretinography (ERG) responses were classified into three groups (normal; cone dysfunction; cone and rod dysfunction). The dark-adapted bright-flash (DA 10.0) a-wave amplitudes and the light-adapted flicker ERG (LA 3.0 30 Hz) amplitudes were plotted against age and compared with the nullizygous patients. Fundus autofluorescence imaging (FAF) was assessed when available. RESULTS: Patients hemizygous for p.G1961E and p.R2030Q had normal ERGs. Patients harboring p.R24H, p.R212C, p.G863A/delG, p.R1108C, p.P1380L, p.L2027F, and c.5714+5G>A had abnormal ERGs (ERG group 2 or 3) at older ages, in most cases with significantly higher amplitudes than nullizygous patients. Mutations p.L541P+A1038V, p.E1022K, p.C1490Y, p.E1087K, p.T1526M, and p.C2150Y were associated with abnormal ERGs (group 2 or 3) and amplitudes comparable to those of nullizygous patients. The majority of patients, including those harboring p.G1961E, had foveal atrophy; while both patients harboring p.R2030Q had foveal sparing. Most patients harboring intermediate and null-like mutations displayed FAF abnormalities extending beyond the vascular arcades. CONCLUSIONS: In the hemizygous state, 2/15 ABCA4 alleles retain preserved peripheral retinal function; 7/15 are associated with either preserved or only mildly abnormal retinal function, worse in older patients; 6/15 behave like null mutations. These data help characterize the degree of dysfunction conferred by specific mutant ABCA4 proteins in the human retina.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Mutação , Retina/fisiopatologia , Adolescente , Adulto , Idade de Início , Idoso , Criança , Eletrorretinografia , Feminino , Angiofluoresceinografia , Fundo de Olho , Humanos , Masculino , Pessoa de Meia-Idade , Células Fotorreceptoras Retinianas Cones/fisiologia , Degeneração Retiniana/genética , Degeneração Retiniana/fisiopatologia , Células Fotorreceptoras Retinianas Bastonetes/fisiologia , Tomografia de Coerência Óptica , Adulto JovemRESUMO
Next-generation sequencing, also known as massively paralleled sequencing, offers an unprecedented opportunity to study disease mechanisms of inherited retinal dystrophies: a dramatic change from a few years ago. The specific involvement of the retina and the manageable number of genes to sequence make inherited retinal dystrophies an attractive model to study genotype-phenotype correlations. Costs are reducing rapidly and the current overall mutation detection rate of approximately 60% offers real potential for personalized medicine and treatments. This report addresses the challenges ahead, which include: better understanding of the mutation mechanisms of syndromic genes in apparent non-syndromic patients; finding mutations in patients who have tested negative or inconclusive; better variant calling, especially for intronic and synonymous variants; more precise genotype-phenotype correlations and making genetic testing more broadly accessible.
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Sequenciamento de Nucleotídeos em Larga Escala , Distrofias Retinianas/diagnóstico , Análise Mutacional de DNA , Exoma , Humanos , Técnicas de Diagnóstico Molecular , Distrofias Retinianas/genéticaRESUMO
OBJECTIVES: Cerebrotendinous xanthomatosis (CTX) is a rare genetic disorder of bile acid (BA) synthesis that can cause progressive neurological damage and premature death. Blood (normally serum or plasma) testing for CTX is performed by a small number of specialized laboratories, routinely by gas chromatography-mass spectrometry (GC-MS) measurement of elevated 5α-cholestanol. We report here on a more sensitive biochemical approach to test for CTX particularly useful for confirmation of CTX in the case of a challenging diagnostic sample with 5α-cholestanol that, although elevated, was below the cut-off used for diagnosis of CTX (10 µg/mL or 1.0 mg/dL). DESIGN AND METHODS: We have previously described liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) methodology utilizing keto derivatization to enable the sensitive quantification of plasma ketosterol BA precursors that accumulate in CTX. We have expanded this methodology to perform isotope dilution LC-ESI-MS/MS quantification of a panel of plasma ketosterol BA precursors, with internal standards readily generated using isotopically-enriched derivatization reagent. RESULTS: Quantification of plasma ketosterol BA precursors (7α-hydroxy-4-cholesten-3-one, 7α,12α-dihydroxy-4-cholesten-3-one and 7α,12α-dihydroxy-5ß-cholestan-3-one) in a single LC-ESI/MS/MS test provided better discrimination between a CTX-positive and negative samples analyzed (n=20) than measurement of 5α-cholestanol alone. CONCLUSIONS: Quantification of plasma ketosterol BA precursors provides a more sensitive biochemical approach to discriminate between CTX negative and positive samples. A multiplexed LC-ESI-MS/MS test quantifying a panel of plasma ketosterols, with simple sample preparation, rapid analysis time and readily available internal standards, can be performed by most clinical laboratories. Wider availability of testing will benefit those affected with CTX.