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1.
J Inherit Metab Dis ; 42(5): 809-817, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31177572

RESUMO

The first step in branched-chain amino acid (BCAA) catabolism is catalyzed by the two BCAA transferase isoenzymes, cytoplasmic branched-chain amino acid transferase (BCAT) 1, and mitochondrial BCAT2. Defects in the second step of BCAA catabolism cause maple syrup urine disease (MSUD), a condition which has been far more extensively investigated. Here, we studied the consequences of BCAT2 deficiency, an ultra-rare condition in humans. We present genetic, clinical, and functional data in five individuals from four different families with homozygous or compound heterozygous BCAT2 mutations which were all detected following abnormal biochemical profile results or familial mutation segregation studies. We demonstrate that BCAT2 deficiency has a recognizable biochemical profile with raised plasma BCAAs and, in contrast with MSUD, low-normal branched-chain keto acids (BCKAs) with undetectable l-allo-isoleucine. Interestingly, unlike in MSUD, none of the individuals with BCAT2 deficiency developed acute encephalopathy even with exceptionally high BCAA levels. We observed wide-ranging clinical phenotypes in individuals with BCAT2 deficiency. While one adult was apparently asymptomatic, three individuals had presented with developmental delay and autistic features. We show that the biochemical characteristics of BCAT2 deficiency may be amenable to protein-restricted diet and that early treatment may improve outcome in affected individuals. BCAT2 deficiency is an inborn error of BCAA catabolism. At present, it is unclear whether developmental delay and autism are parts of the variable phenotypic spectrum of this condition or coincidental. Further studies will be required to explore this.

2.
Sci Rep ; 8(1): 1643, 2018 01 26.
Artigo em Inglês | MEDLINE | ID: mdl-29374201

RESUMO

Retinal inflammation plays a key role in the progression of age-related macular degeneration (AMD), a condition that leads to loss of central vision. The deposition of the acute phase pentraxin C-reactive protein (CRP) in the macula activates the complement system, thereby contributing to dysregulated inflammation. The complement protein factor H (FH) can bind CRP and down-regulate an inflammatory response. However, it is not known whether a truncated form of FH, called factor H-like protein 1 (FHL-1), which plays a significant regulatory role in the eye, also interacts with CRP. Here, we compare the binding properties of FHL-1 and FH to both CRP and the related protein pentraxin-3 (PTX3). We find that, unlike FH, FHL-1 can bind pro-inflammatory monomeric CRP (mCRP) as well as the circulating pentameric form. Furthermore, the four-amino acid C-terminal tail of FHL-1 (not present in FH) plays a role in mediating its binding to mCRP. PTX3 was found to be present in the macula of donor eyes and the AMD-associated Y402H polymorphism altered the binding of FHL-1 to PTX3. Our findings reveal that the binding characteristics of FHL-1 differ from those of FH, likely underpinning independent immune regulatory functions in the context of the human retina.


Assuntos
Proteína C-Reativa/metabolismo , Proteínas Inativadoras do Complemento C3b/metabolismo , Fator H do Complemento/metabolismo , Retinite/patologia , Componente Amiloide P Sérico/metabolismo , Humanos , Ligação Proteica
3.
Eur J Med Genet ; 60(10): 527-532, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28711741

RESUMO

We report three probands from two unrelated consanguineous families of South Asian origin who all carry the same rare novel homozygous variant within the dead box helicase gene DDX59 in association with features of oral-facial-digital syndrome (OFDS). DDX59 variants have been reported previously in an unclassified, autosomal recessive form of OFDS; clinically associated with features including tongue lobulation, cleft palate, frontal bossing, hypertelorism and postaxial polydactyly. All three probands had lobulated tongues with tongue hamartomas, abnormal tongue tip, developmental delay and microcephaly, with just one proband demonstrating polydactlyly. The novel DDX59 variant was identified through autozygosity studies followed by sequencing of homozygous regions identified. It affects a stop codon, extending the protein product and is therefore predicted to be pathogenic. It is only the third reported DDX59 mutation associated with OFDS reported so far.


Assuntos
Mutação , Síndromes Orofaciodigitais/genética , Fenótipo , RNA Helicases/genética , Adulto , Criança , Pré-Escolar , Códon de Terminação/genética , Feminino , Genes Recessivos , Humanos , Lactente , Masculino , Síndromes Orofaciodigitais/diagnóstico , Linhagem
4.
Orphanet J Rare Dis ; 11(1): 125, 2016 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-27628848

RESUMO

BACKGROUND: Although the majority of small in-frame insertions/deletions (indels) has no/little affect on protein function, a small subset of these changes has been causally associated with genetic disorders. Notably, the molecular mechanisms and frequency by which they give rise to disease phenotypes remain largely unknown. The aim of this study is to provide insights into the role of in-frame indels (≤21 nucleotides) in two genetically heterogeneous eye disorders. RESULTS: One hundred eighty-one probands with childhood cataracts and 486 probands with retinal dystrophy underwent multigene panel testing in a clinical diagnostic laboratory. In-frame indels were collected and evaluated both clinically and in silico. Variants that could be modeled in the context of protein structure were identified and analysed using integrative structural modeling. Overall, 55 small in-frame indels were detected in 112 of 667 probands (16.8 %); 17 of these changes were novel to this study and 18 variants were reported clinically. A reliable model of the corresponding protein sequence could be generated for 8 variants. Structural modeling indicated a diverse range of molecular mechanisms of disease including disruption of secondary and tertiary protein structure and alteration of protein-DNA binding sites. CONCLUSIONS: In childhood cataract and retinal dystrophy subjects, one small in-frame indel is clinically reported in every ~37 individuals tested. The clinical utility of computational tools evaluating these changes increases when the full complexity of the involved molecular mechanisms is embraced.


Assuntos
Oftalmopatias/genética , Mutação INDEL/genética , Fases de Leitura/genética , Catarata/genética , Biologia Computacional , Humanos , Distrofias Retinianas/genética
5.
Ophthalmology ; 123(5): 1143-50, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26872967

RESUMO

PURPOSE: To compare the efficacy of whole genome sequencing (WGS) with targeted next-generation sequencing (NGS) in the diagnosis of inherited retinal disease (IRD). DESIGN: Case series. PARTICIPANTS: A total of 562 patients diagnosed with IRD. METHODS: We performed a direct comparative analysis of current molecular diagnostics with WGS. We retrospectively reviewed the findings from a diagnostic NGS DNA test for 562 patients with IRD. A subset of 46 of 562 patients (encompassing potential clinical outcomes of diagnostic analysis) also underwent WGS, and we compared mutation detection rates and molecular diagnostic yields. In addition, we compared the sensitivity and specificity of the 2 techniques to identify known single nucleotide variants (SNVs) using 6 control samples with publically available genotype data. MAIN OUTCOME MEASURES: Diagnostic yield of genomic testing. RESULTS: Across known disease-causing genes, targeted NGS and WGS achieved similar levels of sensitivity and specificity for SNV detection. However, WGS also identified 14 clinically relevant genetic variants through WGS that had not been identified by NGS diagnostic testing for the 46 individuals with IRD. These variants included large deletions and variants in noncoding regions of the genome. Identification of these variants confirmed a molecular diagnosis of IRD for 11 of the 33 individuals referred for WGS who had not obtained a molecular diagnosis through targeted NGS testing. Weighted estimates, accounting for population structure, suggest that WGS methods could result in an overall 29% (95% confidence interval, 15-45) uplift in diagnostic yield. CONCLUSIONS: We show that WGS methods can detect disease-causing genetic variants missed by current NGS diagnostic methodologies for IRD and thereby demonstrate the clinical utility and additional value of WGS.


Assuntos
Oftalmopatias Hereditárias/genética , Genoma , Técnicas de Diagnóstico Molecular , Doenças Retinianas/genética , Análise de Sequência de DNA , Feminino , Genótipo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Polimorfismo de Nucleotídeo Único , Estudos Retrospectivos , Sensibilidade e Especificidade
6.
Hum Mol Genet ; 24(20): 5789-804, 2015 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-26231217

RESUMO

Correct morphogenesis and differentiation are critical in development and maintenance of the lens, which is a classic model system for epithelial development and disease. Through germline genomic analyses in patients with lens and eye abnormalities, we discovered functional mutations in the Signal Induced Proliferation Associated 1 Like 3 (SIPA1L3) gene, which encodes a previously uncharacterized member of the Signal Induced Proliferation Associated 1 (SIPA1 or SPA1) family, with a role in Rap1 signalling. Patient 1, with a de novo balanced translocation, 46,XY,t(2;19)(q37.3;q13.1), had lens and ocular anterior segment abnormalities. Breakpoint mapping revealed transection of SIPA1L3 at 19q13.1 and reduced SIPA1L3 expression in patient lymphoblasts. SIPA1L3 downregulation in 3D cell culture revealed morphogenetic and cell polarity abnormalities. Decreased expression of Sipa1l3 in zebrafish and mouse caused severe lens and eye abnormalities. Sipa1l3(-/-) mice showed disrupted epithelial cell organization and polarity and, notably, abnormal epithelial to mesenchymal transition in the lens. Patient 2 with cataracts was heterozygous for a missense variant in SIPA1L3, c.442G>T, p.Asp148Tyr. Examination of the p.Asp148Tyr mutation in an epithelial cell line showed abnormal clustering of actin stress fibres and decreased formation of adherens junctions. Our findings show that abnormalities of SIPA1L3 in human, zebrafish and mouse contribute to lens and eye defects, and we identify a critical role for SIPA1L3 in epithelial cell morphogenesis, polarity, adhesion and cytoskeletal organization.


Assuntos
Catarata/fisiopatologia , Polaridade Celular , Citoesqueleto/ultraestrutura , Anormalidades do Olho/fisiopatologia , Proteínas Ativadoras de GTPase/genética , Mutação , Proteínas de Peixe-Zebra/genética , Sequência de Aminoácidos , Animais , Catarata/genética , Catarata/metabolismo , Polaridade Celular/genética , Análise Mutacional de DNA , Transição Epitelial-Mesenquimal/genética , Anormalidades do Olho/genética , Anormalidades do Olho/metabolismo , Humanos , Camundongos , Dados de Sequência Molecular , Transdução de Sinais , Peixe-Zebra/genética , Proteínas rap1 de Ligação ao GTP/metabolismo
7.
Neurogenetics ; 16(1): 69-75, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25159689

RESUMO

Leber hereditary optic neuropathy and autosomal dominant optic atrophy are the two most common inherited optic neuropathies. The latter has been associated with mutations in the OPA1 and OPA3 genes. To date, only six families with OPA3-associated dominant optic atrophy have been reported. In order to identify additional families, we performed Sanger sequencing of the OPA3 gene in 75 unrelated optic neuropathy patients. Affected individuals from two families were found to harbour the c.313C > G, p.(Gln105Glu) change in heterozygous state; this genetic defect has been previously reported in four dominant optic atrophy families. Intra- and interfamilial variability in age of onset and presenting symptoms was observed. Although dominant OPA3 mutations are typically associated with optic atrophy and cataracts, the former can be observed in isolation; we report a case with no lens opacities at age 38. Conversely, it is important to consider OPA3-related disease in individuals with bilateral infantile-onset cataracts and to assess optic nerve health in those whose vision fail to improve following lens surgery. The papillomacular bundle is primarily affected and vision is typically worse than 20/40. Notably, we describe one subject who retained normal acuities into the fifth decade of life. The condition can be associated with extraocular clinical features: two affected individuals in the present study had sensorineural hearing loss. The clinical heterogeneity observed in the individuals reported here (all having the same genetic defect in OPA3) suggests that the molecular pathology of the disorder is likely to be complex.


Assuntos
Mutação , Atrofia Óptica Autossômica Dominante/diagnóstico , Atrofia Óptica Autossômica Dominante/genética , Proteínas/genética , Adulto , Idoso , Análise Mutacional de DNA , Feminino , Genes Dominantes , Humanos , Masculino , Pessoa de Meia-Idade , Disco Óptico/patologia , Linhagem , Acuidade Visual/genética , Adulto Jovem
8.
Eur J Hum Genet ; 23(9): 1165-70, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25424711

RESUMO

KAT6B sequence variants have been identified previously in both patients with the Say-Barber-Biesecker type of blepharophimosis mental retardation syndromes (SBBS) and in the more severe genitopatellar syndrome (GPS). We report on the findings in a previously unreported group of 57 individuals with suggestive features of SBBS or GPS. Likely causative variants have been identified in 34/57 patients and were commonly located in the terminal exons of KAT6B. Of those where parental samples could be tested, all occurred de novo. Thirty out of thirty-four had truncating variants, one had a missense variant and the remaining three had the same synonymous change predicted to affect splicing. Variants in GPS tended to occur more proximally to those in SBBS patients, and genotype/phenotype analysis demonstrated significant clinical overlap between SBBS and GPS. The de novo synonymous change seen in three patients with features of SBBS occurred more proximally in exon 16. Statistical analysis of clinical features demonstrated that KAT6B variant-positive patients were more likely to display hypotonia, feeding difficulties, long thumbs/great toes and dental, thyroid and patella abnormalities than KAT6B variant-negative patients. The few reported patients with KAT6B haploinsufficiency had a much milder phenotype, though with some features overlapping those of SBBS. We report the findings in a previously unreported patient with a deletion of the KAT6B gene to further delineate the haploinsufficiency phenotype. The molecular mechanisms giving rise to the SBBS and GPS phenotypes are discussed.


Assuntos
Blefarofimose/genética , Hipotireoidismo Congênito/genética , Anormalidades Craniofaciais/genética , Éxons , Cardiopatias Congênitas/genética , Histona Acetiltransferases/genética , Deficiência Intelectual/genética , Instabilidade Articular/genética , Rim/anormalidades , Mutação , Patela/anormalidades , Transtornos Psicomotores/genética , Escroto/anormalidades , Anormalidades Urogenitais/genética , Blefarofimose/diagnóstico , Blefarofimose/patologia , Pré-Escolar , Hipotireoidismo Congênito/diagnóstico , Hipotireoidismo Congênito/patologia , Anormalidades Craniofaciais/diagnóstico , Anormalidades Craniofaciais/patologia , Análise Mutacional de DNA , Diagnóstico Diferencial , Exoma , Facies , Feminino , Expressão Gênica , Estudos de Associação Genética , Genótipo , Cardiopatias Congênitas/diagnóstico , Cardiopatias Congênitas/patologia , Humanos , Deficiência Intelectual/diagnóstico , Deficiência Intelectual/patologia , Instabilidade Articular/diagnóstico , Instabilidade Articular/patologia , Rim/patologia , Masculino , Patela/patologia , Fenótipo , Transtornos Psicomotores/diagnóstico , Transtornos Psicomotores/patologia , Escroto/patologia , Índice de Gravidade de Doença , Anormalidades Urogenitais/diagnóstico , Anormalidades Urogenitais/patologia
9.
Clin Exp Ophthalmol ; 40(5): 476-83, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22574936

RESUMO

BACKGROUND: To characterize the clinical and genetic abnormalities within two Australian pedigrees with high incidences of retinal detachment and visual disability. DESIGN: Prospective review of two extended Australian pedigrees with high rates of retinal detachment. PARTICIPANTS: Twenty-two family members from two extended Australian pedigrees with high rates of retinal detachment were examined. METHODS: A full ophthalmic history and examination were performed, and DNA was analysed by linkage analysis and mutation screening. MAIN OUTCOME MEASURES: Characterization of a causative hereditary gene mutation in each family. RESULTS: All affected family members of one pedigree carried a C192A COL2A1 exon 2 mutation. None of the affected family members had early-onset arthritis, hearing abnormalities, abnormal clefting or facial features characteristic of classical Stickler syndrome. All affected members of the familial exudative vitreoretinopathy pedigree carried a 957delG FZD4 mutation. CONCLUSIONS: Patients with retinal detachment and a positive family history should be investigated for heritable conditions associated with retinal detachment such as Stickler syndrome and familial exudative vitreoretinopathy. The absence of non-ocular features of Stickler syndrome should raise the possibility of mutations in exon 2 of COL2A1. Similarly, late-onset familial exudative vitreoretinopathy may appear more like a rhegmatogenous detachment and not be correctly diagnosed. When a causative gene mutation is identified, cascade genetic screening of the family will facilitate genetic counselling and screening of high-risk relatives, allowing targeted management of the pre-detachment changes in affected patients.


Assuntos
Colágeno Tipo II/genética , Éxons/genética , Receptores Frizzled/genética , Mutação , Descolamento Retiniano/genética , Adolescente , Adulto , Idoso , Artrite/diagnóstico , Artrite/genética , Doenças do Tecido Conjuntivo/diagnóstico , Doenças do Tecido Conjuntivo/genética , Análise Mutacional de DNA , Feminino , Perda Auditiva Neurossensorial/diagnóstico , Perda Auditiva Neurossensorial/genética , Humanos , Masculino , Pessoa de Meia-Idade , Linhagem , Reação em Cadeia da Polimerase , Estudos Prospectivos , Descolamento Retiniano/diagnóstico , Acuidade Visual/fisiologia , Vitreorretinopatia Proliferativa/diagnóstico , Vitreorretinopatia Proliferativa/genética
10.
J Biol Chem ; 285(39): 30192-202, 2010 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-20660596

RESUMO

Age-related macular degeneration (AMD) is the predominant cause of blindness in the industrialized world where destruction of the macula, i.e. the central region of the retina, results in loss of vision. AMD is preceded by the formation of deposits in the macula, which accumulate between the Bruch's membrane and the retinal pigment epithelium (RPE). These deposits are associated with complement-mediated inflammation and perturb retinal function. Recent genetic association studies have demonstrated that a common allele (402H) of the complement factor H (CFH) gene is a major risk factor for the development of AMD; CFH suppresses complement activation on host tissues where it is believed to bind via its interaction with polyanionic structures. We have shown previously that this coding change (Y402H; from a tyrosine to histidine residue) alters the binding of the CFH protein to sulfated polysaccharides. Here we demonstrate that the AMD-associated polymorphism profoundly affects CFH binding to sites within human macula. Notably, the AMD-associated 402H variant binds less well to heparan sulfate and dermatan sulfate glycosaminoglycans within Bruch's membrane when compared with the 402Y form; both allotypes exhibit a similar level of binding to the RPE. We propose that the impaired binding of the 402H variant to Bruch's membrane results in an overactivation of the complement pathway leading to local chronic inflammation and thus contributes directly to the development and/or progression of AMD. These studies therefore provide a putative disease mechanism and add weight to the genetic association studies that implicate the 402H allele as an important risk factor in AMD.


Assuntos
Lâmina Basilar da Corioide/metabolismo , Ativação do Complemento , Heparitina Sulfato/metabolismo , Degeneração Macular/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Alelos , Substituição de Aminoácidos , Fator H do Complemento/genética , Fator H do Complemento/metabolismo , Heparitina Sulfato/genética , Humanos , Macula Lutea/metabolismo , Degeneração Macular/genética , Mutação de Sentido Incorreto , Ligação Proteica , Fatores de Risco
11.
Am J Hum Genet ; 86(5): 686-95, 2010 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-20398886

RESUMO

Retinitis pigmentosa is a genetically heterogeneous group of inherited ocular disorders characterized by progressive photoreceptor cell loss, night blindness, constriction of the visual field, and progressive visual disability. Homozygosity mapping and gene expression studies identified a 2 exon gene, C2ORF71. The encoded protein has no homologs and is highly expressed in the eye, where it is specifically expressed in photoreceptor cells. Two mutations were found in C2ORF71 in human RP patients: A nonsense mutation (p.W253X) in the first exon is likely to be a null allele; the second, a missense mutation (p.I201F) within a highly conserved region of the protein, leads to proteosomal degradation. Bioinformatic and functional studies identified and validated sites of lipid modification within the first three amino acids of the C2ORF71 protein. Using morpholino oligonucleotides to knockdown c2orf71 expression in zebrafish results in visual defects, confirming that C2ORF71 plays an important role in the development of normal vision. Finally, localization of C2ORF71 to primary cilia in cultured cells suggests that the protein is likely to localize to the connecting cilium or outer segment of photoreceptor cells.


Assuntos
Olho/metabolismo , Mutação , Células Fotorreceptoras de Vertebrados/metabolismo , Proteínas/genética , Retinite Pigmentosa/genética , Cegueira/genética , Cílios/genética , Cílios/metabolismo , Éxons , Proteínas do Olho/genética , Homozigoto , Humanos , Mutação de Sentido Incorreto , Retinite Pigmentosa/metabolismo
14.
Eur J Hum Genet ; 17(10): 1325-35, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19367324

RESUMO

Oculofaciocardiodental (OFCD) and Lenz microphthalmia syndromes form part of a spectrum of X-linked microphthalmia disorders characterized by ocular, dental, cardiac and skeletal anomalies and mental retardation. The two syndromes are allelic, caused by mutations in the BCL-6 corepressor gene (BCOR). To extend the series of phenotypes associated with pathogenic mutations in BCOR, we sequenced the BCOR gene in patients with (1) OFCD syndrome, (2) putative X-linked ('Lenz') microphthalmia syndrome, (3) isolated ocular defects and (4) laterality phenotypes. We present a new cohort of females with OFCD syndrome and null mutations in BCOR, supporting the hypothesis that BCOR is the sole molecular cause of this syndrome. We identify for the first time mosaic BCOR mutations in two females with OFCD syndrome and one apparently asymptomatic female. We present a female diagnosed with isolated ocular defects and identify minor features of OFCD syndrome, suggesting that OFCD syndrome may be mild and underdiagnosed. We have sequenced a cohort of males diagnosed with putative X-linked microphthalmia and found a mutation, p.P85L, in a single case, suggesting that BCOR mutations are not a major cause of X-linked microphthalmia in males. The absence of BCOR mutations in a panel of patients with non-specific laterality defects suggests that mutations in BCOR are not a major cause of isolated heart and laterality defects. Phenotypic analysis of OFCD and Lenz microphthalmia syndromes shows that in addition to the standard diagnostic criteria of congenital cataract, microphthalmia and radiculomegaly, patients should be examined for skeletal defects, particularly radioulnar synostosis, and cardiac/laterality defects.


Assuntos
Anormalidades do Olho/genética , Cardiopatias/genética , Deficiência Intelectual/genética , Microftalmia/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Repressoras/genética , Adolescente , Adulto , Idoso , Alelos , Animais , Criança , Pré-Escolar , Estudos de Coortes , Anormalidades do Olho/complicações , Feminino , Doenças Genéticas Ligadas ao Cromossomo X/complicações , Doenças Genéticas Ligadas ao Cromossomo X/genética , Cardiopatias/complicações , Humanos , Recém-Nascido , Deficiência Intelectual/complicações , Masculino , Microftalmia/complicações , Pessoa de Meia-Idade , Síndrome
15.
Hum Mutat ; 29(1): 83-90, 2008 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-17721977

RESUMO

Stickler syndrome type I (STL1) is a phenotypically heterogeneous disorder characterized by ocular and extraocular features. It is caused by null-allele mutations in the COL2A1 gene that codes for procollagen II. COL2A1 precursor mRNA undergoes alternative splicing, resulting in two isoforms, a long form including exon 2 (type IIA isoform) and a short form excluding exon 2 (type IIB isoform). The short form is predominantly expressed by differentiated chondrocytes in adult cartilage, and the long form in chondroprogenitor cells during early development and in the vitreous of the eye, which is the only adult tissue containing procollagen IIA. Recent evidence indicates that due to the tissue-specific expression of these two isoforms, premature termination codon mutations in exon 2 cause Stickler syndrome with minimal or no extraocular manifestations. We describe here two mutations in exon 2 of COL2A1 in three patients with predominantly ocular Stickler syndrome: Cys64Stop in two patients, and a novel structural mutation, Cys57Tyr, in one patient. RT-PCR of total lymphoblast RNA from one patient with the Cys64Stop mutation revealed that only the normal allele of the IIA form was present, indicating that the mutation resulted either in complete loss of the allele by nonsense-mediated mRNA decay or by skipping of exon 2 via nonsense-mediated altered splicing, resulting in production of the type IIB isoform. The results of COL2A1 minigene expression studies suggest that both Cys64Stop and Cys57Tyr alter positive cis regulatory elements for splicing, resulting in a lower IIA:IIB ratio.


Assuntos
Processamento Alternativo/genética , Códon sem Sentido , Colágeno Tipo II/genética , Doenças do Tecido Conjuntivo/genética , Éxons , Oftalmopatias Hereditárias/genética , Mutação de Sentido Incorreto , Adulto , Sequência de Bases , Criança , Colágeno Tipo II/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/metabolismo , Síndrome
16.
Hum Mutat ; 28(10): 968-77, 2007 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-17492639

RESUMO

Molecular characterization of chromosomal rearrangements is a powerful resource in identification of genes associated with monogenic disorders. We describe the molecular characterization of a balanced familial chromosomal translocation, t(16;22)(p13.3;q11.2), segregating with congenital lamellar cataract. This led to the discovery of a cluster of lens-derived expressed sequence tags (ESTs) close to the 16p13.3 breakpoint. This region harbors a locus associated with cataract and microphthalmia. Long-range PCR and 16p13.3 breakpoint sequencing identified genomic sequence in a human genome sequence gap, and allowed identification of a novel four-exon gene, designated TMEM114, which encodes a predicted protein of 223 amino acids. The breakpoint lies in the promoter region of TMEM114 and separates the gene from predicted eye-specific upstream transcription factor binding sites. There is sequence conservation among orthologs down to zebrafish. The protein is predicted to contain four transmembrane domains with homology to the lens intrinsic membrane protein, LIM2 (also known as MP20), in the PMP-22/EMP/MP20 family. TMEM114 mutation screening in 130 congenital cataract patients revealed missense mutations leading to the exchange of highly-conserved amino acids in the first extracellular domain of the protein (p.I35T, p.F106L) in two separate patients and their reportedly healthy sibling and mother, respectively. In the lens, Tmem114 shows expression in the lens epithelial cells extending into the transitional zone where early fiber differentiation occurs. Our findings implicate dysregulation of expression of this novel human gene, TMEM114, in mammalian cataract formation.


Assuntos
Catarata/genética , Cromossomos Humanos Par 16 , Cromossomos Humanos Par 22 , Cromossomos/ultraestrutura , Proteínas de Membrana/genética , Translocação Genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Etiquetas de Sequências Expressas , Feminino , Humanos , Cristalino/metabolismo , Masculino , Proteínas de Membrana/química , Camundongos , Dados de Sequência Molecular , Linhagem
17.
Invest Ophthalmol Vis Sci ; 47(8): 3565-72, 2006 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16877430

RESUMO

PURPOSE: Linkage intervals for erosive vitreoretinopathy (ERVR) and Wagner disease previously were found to overlap at 5q14.3. In a Japanese family with Wagner disease, a CSPG2/Versican splice site mutation (c.4004-2A-->G) was recently reported that resulted in a 39-nucleotide exon 8 in-frame deletion. We investigated whether CSPG2/Versican was mutated in six Dutch families and one Chinese family with Wagner disease and in a family with ERVR. METHODS: In all families, extensive ophthalmic examinations, haplotype analysis of the 5q14.3 region, and sequence analysis of CSPG2/Versican were performed. The effects of splice site mutations were assessed by reverse transcription-polymerase chain reaction (RT-PCR) and real-time quantitative RT-PCR (QPCR). RESULTS: Three novel intron 7 sequence variants (c.4004-5T-->C, c.4004-5T-->A, c.4004-1G-->A) were identified in seven families. The c.4004-5T-->C variant was identified in four families with Wagner disease and a family with ERVR. The families were shown to carry the same 5q14.3 haplotype, strongly suggesting that this is a common Dutch founder variant. All three changes segregated with the disease in the respective families and were absent in 250 healthy individuals. In patients with the c.4004-5T-->A and c.4004-1G-->A variants, RT-PCR analysis of CSPG2/Versican showed activation of a cryptic splice site resulting in a 39-nt exon 8 in-frame deletion in splice variant V0. QPCR revealed a highly significant (P < 0.0001) and consistent increase of the V2 (>38-fold) and V3 (>12-fold) splice variants in all patients with intron 7 nucleotide changes and in a Chinese Wagner disease family, in which the genetic defect remains to be found. CONCLUSIONS: Wagner disease and ERVR are allelic disorders. Seven of the eight families exhibit a variant in intron 7 of CSPG2/Versican. The conspicuous clustering of sequence variants in the splice acceptor site of intron 7 and the consistent upregulation of the V2 and V3 isoforms strongly suggest that Wagner disease and ERVR may belong to a largely overlooked group of diseases that are caused by mRNA isoform balance shifts, representing a novel disease mechanism.


Assuntos
Proteoglicanas de Sulfatos de Condroitina/genética , Oftalmopatias/genética , Íntrons/genética , Lectinas Tipo C/genética , Mutação , Sítios de Splice de RNA/genética , Doenças Retinianas/genética , Corpo Vítreo , Adolescente , Adulto , Idoso , Criança , Pré-Escolar , Cromossomos Humanos Par 5/genética , Feminino , Ligação Genética , Variação Genética , Genótipo , Haplótipos , Humanos , Masculino , Linhagem , Processamento de RNA , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Versicanas
18.
Invest Ophthalmol Vis Sci ; 45(10): 3683-9, 2004 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-15452077

RESUMO

PURPOSE: To investigate the genetic basis of autosomal dominant vitreoretinochoroidopathy (ADVIRC), a rare, inherited retinal dystrophy that may be associated with defects of ocular development, including nanophthalmos. METHODS: A combination of linkage analysis and DNA sequencing in five families was used to identify disease-causing mutations in VMD2. The effect of these mutations on splicing was assessed using a minigene system. RESULTS: Three pathogenic sequence alterations in VMD2 were identified in five families with nanophthalmos associated with ADVIRC. All sequences showed simultaneous missense substitutions and exon skipping. CONCLUSIONS: VMD2 encodes bestrophin, a transmembrane protein located at the basolateral membrane of the RPE, that is also mutated in Best macular dystrophy. We support that each heterozygous affected individual produces three bestrophin isoforms consisting of the wild type and two abnormal forms: one containing a missense substitution and the other an in-frame deletion. The data showed that VMD2 mutations caused defects of ocular patterning, supporting the hypothesized role for the RPE, and specifically VMD2, in the normal growth and development of the eye.


Assuntos
Doenças da Coroide/genética , Oftalmopatias/genética , Proteínas do Olho/genética , Microftalmia/genética , Mutação , Processamento de RNA/genética , Doenças Retinianas/genética , Corpo Vítreo , Bestrofinas , Canais de Cloreto , Mapeamento Cromossômico , Análise Mutacional de DNA , Feminino , Genes Dominantes , Humanos , Masculino , Linhagem , Reação em Cadeia da Polimerase , Análise de Sequência de DNA
19.
Nat Genet ; 36(4): 411-6, 2004 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-15004558

RESUMO

Lenz microphthalmia is inherited in an X-linked recessive pattern and comprises microphthalmia, mental retardation, and skeletal and other anomalies. Two loci associated with this syndrome, MAA (microphthalmia with associated anomalies) and MAA2, are situated respectively at Xq27-q28 (refs. 1,2) and Xp11.4-p21.2 (ref. 3). We identified a substitution, nt 254C-->T; P85L, in BCOR (encoding BCL-6-interacting corepressor, BCOR) in affected males from the family with Lenz syndrome previously used to identify the MAA2 locus. Oculofaciocardiodental syndrome (OFCD; OMIM 300166) is inherited in an X-linked dominant pattern with presumed male lethality and comprises microphthalmia, congenital cataracts, radiculomegaly, and cardiac and digital abnormalities. Given their phenotypic overlap, we proposed that OFCD and MAA2-associated Lenz microphthalmia were allelic, and we found different frameshift, deletion and nonsense mutations in BCOR in seven families affected with OFCD. Like wild-type BCOR, BCOR P85L and an OFCD-mutant form of BCOR can interact with BCL-6 and efficiently repress transcription. This indicates that these syndromes are likely to result from defects in alternative functions of BCOR, such as interactions with transcriptional partners other than BCL-6. We cloned the zebrafish (Danio rerio) ortholog of BCOR and found that knock-down of this ortholog caused developmental perturbations of the eye, skeleton and central nervous system consistent with the human syndromes, confirming that BCOR is a key transcriptional regulator during early embryogenesis.


Assuntos
Anormalidades Múltiplas/genética , Anormalidades do Olho/genética , Cardiopatias Congênitas/genética , Mutação , Proteínas Proto-Oncogênicas/genética , Proteínas Repressoras/genética , Sequência de Aminoácidos , Animais , Compensação de Dosagem (Genética) , Face/anormalidades , Humanos , Dados de Sequência Molecular , Proteínas Proto-Oncogênicas/química , Proteínas Repressoras/química , Homologia de Sequência de Aminoácidos , Síndrome , Anormalidades Dentárias/genética , Cromossomo X , Peixe-Zebra
20.
Am J Hum Genet ; 72(6): 1359-69, 2003 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-12730828

RESUMO

Cohen syndrome is an uncommon autosomal recessive disorder whose diagnosis is based on the clinical picture of nonprogressive psychomotor retardation and microcephaly, characteristic facial features, retinal dystrophy, and intermittent neutropenia. We have refined the critical region on chromosome 8q22 by haplotype analysis, and we report the characterization of a novel gene, COH1, that is mutated in patients with Cohen syndrome. The longest transcript (14,093 bp) is widely expressed and is transcribed from 62 exons that span a genomic region of approximately 864 kb. COH1 encodes a putative transmembrane protein of 4,022 amino acids, with a complex domain structure. Homology to the Saccharomyces cerevisiae VPS13 protein suggests a role for COH1 in vesicle-mediated sorting and transport of proteins within the cell.


Assuntos
Anormalidades Múltiplas/genética , Mutação , Anormalidades Múltiplas/patologia , Adulto , Criança , Cromossomos Humanos Par 8 , Estudos de Coortes , Anormalidades Craniofaciais/genética , Anormalidades Craniofaciais/patologia , Deficiências do Desenvolvimento/genética , Deficiências do Desenvolvimento/patologia , Feminino , Humanos , Deficiência Intelectual/genética , Deficiência Intelectual/patologia , Masculino , Proteínas de Membrana/genética , Microcefalia/genética , Microcefalia/patologia , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos , Síndrome , Proteínas de Transporte Vesicular
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