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1.
Int J Mol Sci ; 20(16)2019 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-31405222

RESUMO

Although fragile X syndrome (FXS) is caused by a hypermethylated full mutation (FM) expansion with ≥200 cytosine-guanine-guanine (CGG) repeats, and a decrease in FMR1 mRNA and its protein (FMRP), incomplete silencing has been associated with more severe autism features in FXS males. This study reports on brothers (B1 and B2), aged 5 and 2 years, with autistic features and language delay, but a higher non-verbal IQ in comparison to typical FXS. CGG sizing using AmplideX PCR only identified premutation (PM: 55-199 CGGs) alleles in blood. Similarly, follow-up in B1 only revealed PM alleles in saliva and skin fibroblasts; whereas, an FM expansion was detected in both saliva and buccal DNA of B2. While Southern blot analysis of blood detected an unmethylated FM, methylation analysis with a more sensitive methodology showed that B1 had partially methylated PM alleles in blood and fibroblasts, which were completely unmethylated in buccal and saliva cells. In contrast, B2 was partially methylated in all tested tissues. Moreover, both brothers had FMR1 mRNA ~5 fold higher values than those of controls, FXS and PM cohorts. In conclusion, the presence of unmethylated FM and/or PM in both brothers may lead to an overexpression of toxic expanded mRNA in some cells, which may contribute to neurodevelopmental problems, including elevated autism features.

2.
Am J Hum Genet ; 104(5): 914-924, 2019 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-30982611

RESUMO

Glypicans are a family of cell-surface heparan sulfate proteoglycans that regulate growth-factor signaling during development and are thought to play a role in the regulation of morphogenesis. Whole-exome sequencing of the Australian family that defined Keipert syndrome (nasodigitoacoustic syndrome) identified a hemizygous truncating variant in the gene encoding glypican 4 (GPC4). This variant, located in the final exon of GPC4, results in premature termination of the protein 51 amino acid residues prior to the stop codon, and in concomitant loss of functionally important N-linked glycosylation (Asn514) and glycosylphosphatidylinositol (GPI) anchor (Ser529) sites. We subsequently identified seven affected males from five additional kindreds with novel and predicted pathogenic variants in GPC4. Segregation analysis and X-inactivation studies in carrier females provided supportive evidence that the GPC4 variants caused the condition. Furthermore, functional studies of recombinant protein suggested that the truncated proteins p.Gln506∗ and p.Glu496∗ were less stable than the wild type. Clinical features of Keipert syndrome included a prominent forehead, a flat midface, hypertelorism, a broad nose, downturned corners of mouth, and digital abnormalities, whereas cognitive impairment and deafness were variable features. Studies of Gpc4 knockout mice showed evidence of the two primary features of Keipert syndrome: craniofacial abnormalities and digital abnormalities. Phylogenetic analysis demonstrated that GPC4 is most closely related to GPC6, which is associated with a bone dysplasia that has a phenotypic overlap with Keipert syndrome. Overall, we have shown that pathogenic variants in GPC4 cause a loss of function that results in Keipert syndrome, making GPC4 the third human glypican to be linked to a genetic syndrome.

3.
Hum Mutat ; 39(12): 1995-2007, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30178502

RESUMO

Recessive SLC25A46 mutations cause a spectrum of neurodegenerative disorders with optic atrophy as a core feature. We report a patient with optic atrophy, peripheral neuropathy, ataxia, but not cerebellar atrophy, who is on the mildest end of the phenotypic spectrum. By studying seven different nontruncating mutations, we found that the stability of the SLC25A46 protein inversely correlates with the severity of the disease and the patient's variant does not markedly destabilize the protein. SLC25A46 belongs to the mitochondrial transporter family, but it is not known to have transport function. Apart from this possible function, SLC25A46 forms molecular complexes with proteins involved in mitochondrial dynamics and cristae remodeling. We demonstrate that the patient's mutation directly affects the SLC25A46 interaction with MIC60. Furthermore, we mapped all of the reported substitutions in the protein onto a 3D model and found that half of them fall outside of the signature carrier motifs associated with transport function. We thus suggest that there are two distinct molecular mechanisms in SLC25A46-associated pathogenesis, one that destabilizes the protein while the other alters the molecular interactions of the protein. These results have the potential to inform clinical prognosis of such patients and indicate a pathway to drug target development.

4.
Semin Pediatr Neurol ; 26: 2-9, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29961509

RESUMO

Brown-Vialetto-van Laere syndrome is characterized by a progressive sensorimotor neuropathy, optic atrophy, hearing loss, bulbar dysfunction, and respiratory insufficiency. Mutations in SLC52A2 and SLC52A3, encoding riboflavin transporters RFVT2 and RFVT3, respectively, are the genetic basis of this disorder, often referred to as riboflavin transporter deficiency types 2 and 3, respectively. We present cases of both types of riboflavin transporter deficiency, highlighting the distinguishing clinical features of a rapidly progressive motor or sensorimotor axonal neuropathy, optic atrophy, sensorineural hearing loss, and bulbar dysfunction. One child presented with isolated central apnea and hypoventilation, not previously described in genetically confirmed Brown-Vialetto-van Laere, later complicated by diaphragmatic paralysis secondary to phrenic nerve palsy. Magnetic resonance imaging showed T2 hyperintensity in the dorsal spinal cord in 2 children, as well as previously unreported cervical nerve root enlargement and cauda equina ventral nerve root enhancement in 1 child. Novel homozygous mutations were identified in each gene-a NM_024531.4(SLC52A2):c.505C > T, NP_078807.1(SLC52A2):p.(Arg169Cys) variant in SLC52A2 and NM_033409.3(SLC52A3):c.1316G > A, NP_212134.3(SLC52A3):p.(Gly439Asp) variant in SLC52A3. Both treated children showed improvement on high-dose riboflavin supplementation, highlighting the importance of early recognition of this treatable clinical entity.


Assuntos
Paralisia Bulbar Progressiva/diagnóstico por imagem , Paralisia Bulbar Progressiva/genética , Perda Auditiva Neurossensorial/diagnóstico por imagem , Perda Auditiva Neurossensorial/genética , Encéfalo/diagnóstico por imagem , Paralisia Bulbar Progressiva/fisiopatologia , Paralisia Bulbar Progressiva/terapia , Pré-Escolar , Consanguinidade , Feminino , Perda Auditiva Neurossensorial/fisiopatologia , Perda Auditiva Neurossensorial/terapia , Humanos , Lactente , Masculino , Proteínas de Membrana Transportadoras/genética , Receptores Acoplados a Proteínas-G/genética , Medula Espinal/diagnóstico por imagem
5.
Nat Commun ; 9(1): 1373, 2018 04 10.
Artigo em Inglês | MEDLINE | ID: mdl-29636455

RESUMO

Congenital heart defects can be caused by mutations in genes that guide cardiac lineage formation. Here, we show deletion of NKX2-5, a critical component of the cardiac gene regulatory network, in human embryonic stem cells (hESCs), results in impaired cardiomyogenesis, failure to activate VCAM1 and to downregulate the progenitor marker PDGFRα. Furthermore, NKX2-5 null cardiomyocytes have abnormal physiology, with asynchronous contractions and altered action potentials. Molecular profiling and genetic rescue experiments demonstrate that the bHLH protein HEY2 is a key mediator of NKX2-5 function during human cardiomyogenesis. These findings identify HEY2 as a novel component of the NKX2-5 cardiac transcriptional network, providing tangible evidence that hESC models can decipher the complex pathways that regulate early stage human heart development. These data provide a human context for the evaluation of pathogenic mutations in congenital heart disease.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Redes Reguladoras de Genes , Proteína Homeobox Nkx-2.5/genética , Células-Tronco Embrionárias Humanas/metabolismo , Miócitos Cardíacos/metabolismo , Organogênese/genética , Proteínas Repressoras/genética , Potenciais de Ação/fisiologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Diferenciação Celular , Linhagem Celular , Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento , Proteína Homeobox Nkx-2.5/deficiência , Células-Tronco Embrionárias Humanas/citologia , Humanos , Miocárdio/citologia , Miocárdio/metabolismo , Miócitos Cardíacos/citologia , Técnicas de Patch-Clamp , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Proteínas Repressoras/metabolismo , Transcrição Genética , Molécula 1 de Adesão de Célula Vascular/genética , Molécula 1 de Adesão de Célula Vascular/metabolismo
6.
Genet Med ; 2018 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-29543227

RESUMO

PurposeThe purpose of the study was to implement and prospectively evaluate the outcomes of a rapid genomic diagnosis program at two pediatric tertiary centers.MethodsRapid singleton whole-exome sequencing (rWES) was performed in acutely unwell pediatric patients with suspected monogenic disorders. Laboratory and clinical barriers to implementation were addressed through continuous multidisciplinary review of process parameters. Diagnostic and clinical utility and cost-effectiveness of rWES were assessed.ResultsOf 40 enrolled patients, 21 (52.5%) received a diagnosis, with median time to report of 16 days (range 9-109 days). A result was provided during the first hospital admission in 28 of 36 inpatients (78%). Clinical management changed in 12 of the 21 diagnosed patients (57%), including the provision of lifesaving treatment, avoidance of invasive biopsies, and palliative care guidance. The cost per diagnosis was AU$13,388 (US$10,453). Additional cost savings from avoidance of planned tests and procedures and reduced length of stay are estimated to be around AU$543,178 (US$424,101). The clear relative advantage of rWES, joint clinical and laboratory leadership, and the creation of a multidisciplinary "rapid team" were key to successful implementation.ConclusionRapid genomic testing in acute pediatrics is not only feasible but also cost-effective, and has high diagnostic and clinical utility. It requires a whole-of-system approach for successful implementation.GENETICS in MEDICINE advance online publication, 15 March 2018; doi:10.1038/gim.2018.37.

7.
Ann Clin Transl Neurol ; 4(5): 318-325, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28491899

RESUMO

OBJECTIVE: To explore the diagnostic utility and cost effectiveness of whole exome sequencing (WES) in a cohort of individuals with peripheral neuropathy. METHODS: Singleton WES was performed in individuals recruited though one pediatric and one adult tertiary center between February 2014 and December 2015. Initial analysis was restricted to a virtual panel of 55 genes associated with peripheral neuropathies. Patients with uninformative results underwent expanded analysis of the WES data. Data on the cost of prior investigations and assessments performed for diagnostic purposes in each patient was collected. RESULTS: Fifty patients with a peripheral neuropathy were recruited (median age 18 years; range 2-68 years). The median time from initial presentation to study enrollment was 6 years 9 months (range 2 months-62 years), and the average cost of prior investigations and assessments for diagnostic purposes AU$4013 per patient. Eleven individuals received a diagnosis from the virtual panel. Eight individuals received a diagnosis following expanded analysis of the WES data, increasing the overall diagnostic yield to 38%. Two additional individuals were diagnosed with pathogenic copy number variants through SNP microarray. CONCLUSIONS: This study provides evidence that WES has a high diagnostic utility and is cost effective in patients with a peripheral neuropathy. Expanded analysis of WES data significantly improves the diagnostic yield in patients in whom a diagnosis is not found on the initial targeted analysis. This is primarily due to diagnosis of conditions caused by newly discovered genes and the resolution of complex and atypical phenotypes.

8.
Eur Heart J ; 37(33): 2586-90, 2016 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-27106955

RESUMO

AIMS: We identified a novel homozygous truncating mutation in the gene encoding alpha kinase 3 (ALPK3) in a family presenting with paediatric cardiomyopathy. A recent study identified biallelic truncating mutations of ALPK3 in three unrelated families; therefore, there is strong genetic evidence that ALPK3 mutation causes cardiomyopathy. This study aimed to clarify the mutation mechanism and investigate the molecular and cellular pathogenesis underlying ALPK3-mediated cardiomyopathy. METHODS AND RESULTS: We performed detailed clinical and genetic analyses of a consanguineous family, identifying a new ALPK3 mutation (c.3792G>A, p.W1264X) which undergoes nonsense-mediated decay in ex vivo and in vivo tissues. Ultra-structural analysis of cardiomyocytes derived from patient-specific and human ESC-derived stem cell lines lacking ALPK3 revealed disordered sarcomeres and intercalated discs. Multi-electrode array analysis and calcium imaging demonstrated an extended field potential duration and abnormal calcium handling in mutant contractile cultures. CONCLUSIONS: This study validates the genetic evidence, suggesting that mutations in ALPK3 can cause familial cardiomyopathy and demonstrates loss of function as the underlying genetic mechanism. We show that ALPK3-deficient cardiomyocytes derived from pluripotent stem cell models recapitulate the ultrastructural and electrophysiological defects observed in vivo. Analysis of differentiated contractile cultures identified abnormal calcium handling as a potential feature of cardiomyocytes lacking ALPK3, providing functional insights into the molecular mechanisms underlying ALPK3-mediated cardiomyopathy.


Assuntos
Miócitos Cardíacos , Cálcio , Cardiomiopatias , Células-Tronco Embrionárias Humanas , Humanos , Células-Tronco Pluripotentes Induzidas , Proteínas Musculares , Proteínas Quinases
9.
Am J Hum Genet ; 95(6): 729-35, 2014 Dec 04.
Artigo em Inglês | MEDLINE | ID: mdl-25434005

RESUMO

Advances in understanding the etiology of Parkinson disease have been driven by the identification of causative mutations in families. Genetic analysis of an Australian family with three males displaying clinical features of early-onset parkinsonism and intellectual disability identified a ∼45 kb deletion resulting in the complete loss of RAB39B. We subsequently identified a missense mutation (c.503C>A [p.Thr168Lys]) in RAB39B in an unrelated Wisconsin kindred affected by a similar clinical phenotype. In silico and in vitro studies demonstrated that the mutation destabilized the protein, consistent with loss of function. In vitro small-hairpin-RNA-mediated knockdown of Rab39b resulted in a reduction in the density of α-synuclein immunoreactive puncta in dendritic processes of cultured neurons. In addition, in multiple cell models, we demonstrated that knockdown of Rab39b was associated with reduced steady-state levels of α-synuclein. Post mortem studies demonstrated that loss of RAB39B resulted in pathologically confirmed Parkinson disease. There was extensive dopaminergic neuron loss in the substantia nigra and widespread classic Lewy body pathology. Additional pathological features included cortical Lewy bodies, brain iron accumulation, tau immunoreactivity, and axonal spheroids. Overall, we have shown that loss-of-function mutations in RAB39B cause intellectual disability and pathologically confirmed early-onset Parkinson disease. The loss of RAB39B results in dysregulation of α-synuclein homeostasis and a spectrum of neuropathological features that implicate RAB39B in the pathogenesis of Parkinson disease and potentially other neurodegenerative disorders.


Assuntos
Genes Ligados ao Cromossomo X , Deficiência Intelectual/genética , Degeneração Neural/genética , Doença de Parkinson/genética , alfa-Sinucleína/metabolismo , Proteínas rab de Ligação ao GTP/genética , Substituição de Aminoácidos , Austrália , Sequência de Bases , Dopamina/metabolismo , Feminino , Regulação da Expressão Gênica , Humanos , Deficiência Intelectual/fisiopatologia , Corpos de Lewy/metabolismo , Masculino , Pessoa de Meia-Idade , Modelos Moleculares , Dados de Sequência Molecular , Mutação de Sentido Incorreto , Degeneração Neural/fisiopatologia , Doença de Parkinson/fisiopatologia , Linhagem , Análise de Sequência de DNA , Deleção de Sequência , Substância Negra/fisiopatologia , Proteínas rab de Ligação ao GTP/metabolismo
10.
J Cardiovasc Electrophysiol ; 22(9): 1073-6, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21288276

RESUMO

A 14-year-old boy presented with atrial flutter. His ECG showed Brugada changes, first-degree AV block and major sinus pauses. Polymorphic VT was inducible at electrophysiology study. A pacemaker defibrillator was placed. Classic sequencing for SCN5A was normal. Multiplex ligation-dependent probe amplification, however, detected a major deletion in SCN5A. It is predicted that this deletion would result in haploinsufficiency. The report is the first description of a large-scale rearrangement of the SCN5A gene and supports the association between the molecular pathology and the phenotypic expression.


Assuntos
Síndrome de Brugada/genética , Deleção de Genes , Canais de Sódio/genética , Adolescente , Síndrome de Brugada/diagnóstico , Síndrome de Brugada/patologia , Estudos de Associação Genética/métodos , Humanos , Masculino , Canal de Sódio Disparado por Voltagem NAV1.5 , Técnicas de Amplificação de Ácido Nucleico/métodos
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