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1.
Am J Med Genet A ; 2019 Sep 12.
Artigo em Inglês | MEDLINE | ID: mdl-31512387

RESUMO

Coffin-Lowry syndrome (CLS) is a rare X-linked disorder characterized by moderate to severe intellectual disability, hypotonia, craniofacial features, tapering digits, short stature, and skeletal deformities. Using whole exome sequencing and high-resolution targeted comparative genomic hybridization array analysis, we identified a novel microduplication encompassing exons five through nine of RPS6KA3 in three full brothers. Each brother presented with intellectual disability and clinical and radiographic features consistent with CLS. qRT-PCR analyses performed on mRNA from the peripheral blood of the three siblings revealed a marked reduction of RPS6KA3 levels suggesting a loss-of-function mechanism. PCR analysis of the patients' cDNA detected a band greater than expected for an exon 4-10 amplicon, suggesting this was likely a direct duplication that lies between exons 4 through 10, which was later confirmed by Sanger sequencing. This microduplication is only the third intragenic duplication of RPS6KA3, and the second and smallest reported to date thought to cause CLS. Our study further supports the clinical utility of methods such as next-generation sequencing and high-resolution genomic arrays to detect small intragenic duplications. These methods, coupled with expression studies and cDNA structural analysis have the capacity to confirm the diagnosis of CLS in these rare cases.

3.
Genome Med ; 11(1): 30, 2019 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-31101064

RESUMO

BACKGROUND: Exome sequencing (ES) has been successfully applied in clinical detection of single nucleotide variants (SNVs) and small indels. However, identification of copy number variants (CNVs) using ES data remains challenging. The purpose of this study is to understand the contribution of CNVs and copy neutral runs of homozygosity (ROH) in molecular diagnosis of patients referred for ES. METHODS: In a cohort of 11,020 consecutive ES patients, an Illumina SNP array analysis interrogating mostly coding SNPs was performed as a quality control (QC) measurement and for CNV/ROH detection. Among these patients, clinical chromosomal microarray analysis (CMA) was performed at Baylor Genetics (BG) on 3229 patients, either before, concurrently, or after ES. We retrospectively analyzed the findings from CMA and the QC array. RESULTS: The QC array can detect ~ 70% of pathogenic/likely pathogenic CNVs (PCNVs) detectable by CMA. Out of the 11,020 ES cases, the QC array identified PCNVs in 327 patients and uniparental disomy (UPD) disorder-related ROH in 10 patients. The overall PCNV/UPD detection rate was 5.9% in the 3229 ES patients who also had CMA at BG; PCNV/UPD detection rate was higher in concurrent ES and CMA than in ES with prior CMA (7.2% vs 4.6%). The PCNVs/UPD contributed to the molecular diagnoses in 17.4% (189/1089) of molecularly diagnosed ES cases with CMA and were estimated to contribute in 10.6% of all molecularly diagnosed ES cases. Dual diagnoses with both PCNVs and SNVs were detected in 38 patients. PCNVs affecting single recessive disorder genes in a compound heterozygous state with SNVs were detected in 4 patients, and homozygous deletions (mostly exonic deletions) were detected in 17 patients. A higher PCNV detection rate was observed for patients with syndromic phenotypes and/or cardiovascular abnormalities. CONCLUSIONS: Our clinical genomics study demonstrates that detection of PCNV/UPD through the QC array or CMA increases ES diagnostic rate, provides more precise molecular diagnosis for dominant as well as recessive traits, and enables more complete genetic diagnoses in patients with dual or multiple molecular diagnoses. Concurrent ES and CMA using an array with exonic coverage for disease genes enables most effective detection of both CNVs and SNVs and therefore is recommended especially in time-sensitive clinical situations.

4.
Genome Med ; 11(1): 12, 2019 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-30819258

RESUMO

BACKGROUND: Neurodevelopmental disorders are genetically and phenotypically heterogeneous encompassing developmental delay (DD), intellectual disability (ID), autism spectrum disorders (ASDs), structural brain abnormalities, and neurological manifestations with variants in a large number of genes (hundreds) associated. To date, a few de novo mutations potentially disrupting TCF20 function in patients with ID, ASD, and hypotonia have been reported. TCF20 encodes a transcriptional co-regulator structurally related to RAI1, the dosage-sensitive gene responsible for Smith-Magenis syndrome (deletion/haploinsufficiency) and Potocki-Lupski syndrome (duplication/triplosensitivity). METHODS: Genome-wide analyses by exome sequencing (ES) and chromosomal microarray analysis (CMA) identified individuals with heterozygous, likely damaging, loss-of-function alleles in TCF20. We implemented further molecular and clinical analyses to determine the inheritance of the pathogenic variant alleles and studied the spectrum of phenotypes. RESULTS: We report 25 unique inactivating single nucleotide variants/indels (1 missense, 1 canonical splice-site variant, 18 frameshift, and 5 nonsense) and 4 deletions of TCF20. The pathogenic variants were detected in 32 patients and 4 affected parents from 31 unrelated families. Among cases with available parental samples, the variants were de novo in 20 instances and inherited from 4 symptomatic parents in 5, including in one set of monozygotic twins. Two pathogenic loss-of-function variants were recurrent in unrelated families. Patients presented with a phenotype characterized by developmental delay, intellectual disability, hypotonia, variable dysmorphic features, movement disorders, and sleep disturbances. CONCLUSIONS: TCF20 pathogenic variants are associated with a novel syndrome manifesting clinical characteristics similar to those observed in Smith-Magenis syndrome. Together with previously described cases, the clinical entity of TCF20-associated neurodevelopmental disorders (TAND) emerges from a genotype-driven perspective.


Assuntos
Anormalidades Craniofaciais/genética , Deficiências do Desenvolvimento/genética , Mutação INDEL , Deficiência Intelectual/genética , Hipotonia Muscular/genética , Síndrome de Smith-Magenis/genética , Fatores de Transcrição/genética , Adolescente , Criança , Pré-Escolar , Anormalidades Craniofaciais/patologia , Deficiências do Desenvolvimento/patologia , Feminino , Humanos , Lactente , Deficiência Intelectual/patologia , Masculino , Hipotonia Muscular/patologia , Síndrome de Smith-Magenis/patologia , Fatores de Transcrição/metabolismo , Adulto Jovem
6.
Am J Hum Genet ; 104(2): 213-228, 2019 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-30639323

RESUMO

Primary defects in lung branching morphogenesis, resulting in neonatal lethal pulmonary hypoplasias, are incompletely understood. To elucidate the pathogenetics of human lung development, we studied a unique collection of samples obtained from deceased individuals with clinically and histopathologically diagnosed interstitial neonatal lung disorders: acinar dysplasia (n = 14), congenital alveolar dysplasia (n = 2), and other lethal lung hypoplasias (n = 10). We identified rare heterozygous copy-number variant deletions or single-nucleotide variants (SNVs) involving TBX4 (n = 8 and n = 2, respectively) or FGF10 (n = 2 and n = 2, respectively) in 16/26 (61%) individuals. In addition to TBX4, the overlapping ∼2 Mb recurrent and nonrecurrent deletions at 17q23.1q23.2 identified in seven individuals with lung hypoplasia also remove a lung-specific enhancer region. Individuals with coding variants involving either TBX4 or FGF10 also harbored at least one non-coding SNV in the predicted lung-specific enhancer region, which was absent in 13 control individuals with the overlapping deletions but without any structural lung anomalies. The occurrence of rare coding variants involving TBX4 or FGF10 with the putative hypomorphic non-coding SNVs implies a complex compound inheritance of these pulmonary hypoplasias. Moreover, they support the importance of TBX4-FGF10-FGFR2 epithelial-mesenchymal signaling in human lung organogenesis and help to explain the histopathological continuum observed in these rare lethal developmental disorders of the lung.

7.
Hum Mutat ; 2018 Dec 06.
Artigo em Inglês | MEDLINE | ID: mdl-30520571

RESUMO

Next-generation sequencing has been instrumental in solving the genetic basis of rare inherited diseases, especially neurodevelopmental syndromes. However, functional workup is essential for precise phenotype definition and to understand the underlying disease mechanisms. Using whole exome and whole genome sequencing in 4 independent families with hypotonia, neurodevelopmental delay, facial dysmorphism, loss of white matter and thinning of the corpus callosum, we identified 4 previously unreported homozygous truncating PPP1R21 alleles: c.347delT p.(Ile116Lysfs*25), c.2170_2171insGGTA p.(Ile724Argfs*8), c.1607dupT p.(Leu536Phefs*7), c.2063delA p.(Lys688Serfs*26) and found that PPP1R21 was absent in fibroblasts of an affected individual, supporting the allele's loss of function effect. PPP1R21 function had not been studied except that a large scale affinity proteomics approach suggested an interaction with PIBF1 defective in Joubert syndrome. Our co-Immunoprecipitation studies did not confirm this but in contrast defined the localization of PPP1R21 to the early endosome. Consistent with the subcellular expression pattern and the clinical phenotype exhibiting features of storage diseases, we found patient fibroblasts exhibited a delay in clearance of transferrin-488 while uptake was normal. In summary, we delineate a novel neurodevelopmental syndrome caused by bi-allelic PPP1R21 loss of function variants, and suggest a role of PPP1R21 within the endosomal sorting process or endosome maturation pathway. This article is protected by copyright. All rights reserved.

8.
Ann Clin Transl Neurol ; 5(10): 1277-1285, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30349862

RESUMO

De novo variants in DDX3X account for 1-3% of unexplained intellectual disability (ID) cases and are amongst the most common causes of ID especially in females. Forty-seven patients (44 females, 3 males) have been described. We identified 31 additional individuals carrying 29 unique DDX3X variants, including 30 postnatal individuals with complex clinical presentations of developmental delay or ID, and one fetus with abnormal ultrasound findings. Rare or novel phenotypes observed include respiratory problems, congenital heart disease, skeletal muscle mitochondrial DNA depletion, and late-onset neurologic decline. Our findings expand the spectrum of DNA variants and phenotypes associated with DDX3X disorders.

9.
Genome Med ; 10(1): 74, 2018 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-30266093

RESUMO

BACKGROUND: Exome sequencing is now being incorporated into clinical care for pediatric and adult populations, but its integration into prenatal diagnosis has been more limited. One reason for this is the paucity of information about the clinical utility of exome sequencing in the prenatal setting. METHODS: We retrospectively reviewed indications, results, time to results (turnaround time, TAT), and impact of exome results for 146 consecutive "fetal exomes" performed in a clinical diagnostic laboratory between March 2012 and November 2017. We define a fetal exome as one performed on a sample obtained from a fetus or a product of conception with at least one structural anomaly detected by prenatal imaging or autopsy. Statistical comparisons were performed using Fisher's exact test. RESULTS: Prenatal exome yielded an overall molecular diagnostic rate of 32% (n = 46/146). Of the 46 molecular diagnoses, 50% were autosomal dominant disorders (n = 23/46), 41% were autosomal recessive disorders (n = 19/46), and 9% were X-linked disorders (n = 4/46). The molecular diagnostic rate was highest for fetuses with anomalies affecting multiple organ systems and for fetuses with craniofacial anomalies. Out of 146 cases, a prenatal trio exome option designed for ongoing pregnancies was performed on 62 fetal specimens, resulting in a diagnostic yield of 35% with an average TAT of 14 days for initial reporting (excluding tissue culture time). The molecular diagnoses led to refined recurrence risk estimates, altered medical management, and informed reproductive planning for families. CONCLUSION: Exome sequencing is a useful diagnostic tool when fetal structural anomalies suggest a genetic etiology, but other standard prenatal genetic tests did not provide a diagnosis.

10.
Am J Med Genet A ; 176(4): 973-979, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29423971

RESUMO

SHANK3 encodes for a scaffolding protein that links neurotransmitter receptors to the cytoskeleton and is enriched in postsynaptic densities of excitatory synapses. Deletions or mutations in one copy of the SHANK3 gene cause Phelan-McDermid syndrome, also called 22q13.3 deletion syndrome, a neurodevelopmental disorder with common features including global developmental delay, absent to severely impaired language, autistic behavior, and minor dysmorphic features. By whole exome sequencing, we identified two de novo novel variants including one frameshift pathogenic variant and one missense variant of unknown significance in a 14-year-old boy with delayed motor milestones, delayed language acquisition, autism, intellectual disability, ataxia, progressively worsening spasticity of the lower extremities, dysmorphic features, short stature, microcephaly, failure to thrive, chronic constipation, intrauterine growth restriction, and bilateral inguinal hernias. Both changes are within the CpG island in exon 21, separated by a 375 bp sequence. Next generation sequencing of PCR products revealed that the two variants are most frequently associated with each other. Sanger sequencing of the cloned PCR products further confirmed that both changes were on a single allele. The clinical presentation in this individual is consistent with other patients with a truncating mutation in exon 21, suggesting that the missense change contributes none or minimally to the phenotypes. This is the first report of two de novo mutations in one SHANK3 allele.

11.
JAMA Pediatr ; 171(12): e173438, 2017 Dec 04.
Artigo em Inglês | MEDLINE | ID: mdl-28973083

RESUMO

Importance: While congenital malformations and genetic diseases are a leading cause of early infant death, to our knowledge, the contribution of single-gene disorders in this group is undetermined. Objective: To determine the diagnostic yield and use of clinical exome sequencing in critically ill infants. Design, Setting, and Participants: Clinical exome sequencing was performed for 278 unrelated infants within the first 100 days of life who were admitted to Texas Children's Hospital in Houston, Texas, during a 5-year period between December 2011 and January 2017. Exome sequencing types included proband exome, trio exome, and critical trio exome, a rapid genomic assay for seriously ill infants. Main Outcomes and Measures: Indications for testing, diagnostic yield of clinical exome sequencing, turnaround time, molecular findings, patient age at diagnosis, and effect on medical management among a group of critically ill infants who were suspected to have genetic disorders. Results: The mean (SEM) age for infants participating in the study was 28.5 (1.7) days; of these, the mean (SEM) age was 29.0 (2.2) days for infants undergoing proband exome sequencing, 31.5 (3.9) days for trio exome, and 22.7 (3.9) days for critical trio exome. Clinical indications for exome sequencing included a range of medical concerns. Overall, a molecular diagnosis was achieved in 102 infants (36.7%) by clinical exome sequencing, with relatively low yield for cardiovascular abnormalities. The diagnosis affected medical management for 53 infants (52.0%) and had a substantial effect on informed redirection of care, initiation of new subspecialist care, medication/dietary modifications, and furthering life-saving procedures in select patients. Critical trio exome sequencing revealed a molecular diagnosis in 32 of 63 infants (50.8%) at a mean (SEM) of 33.1 (5.6) days of life with a mean (SEM) turnaround time of 13.0 (0.4) days. Clinical care was altered by the diagnosis in 23 of 32 patients (71.9%). The diagnostic yield, patient age at diagnosis, and medical effect in the group that underwent critical trio exome sequencing were significantly different compared with the group who underwent regular exome testing. For deceased infants (n = 81), genetic disorders were molecularly diagnosed in 39 (48.1%) by exome sequencing, with implications for recurrence risk counseling. Conclusions and Relevance: Exome sequencing is a powerful tool for the diagnostic evaluation of critically ill infants with suspected monogenic disorders in the neonatal and pediatric intensive care units and its use has a notable effect on clinical decision making.


Assuntos
Doenças Genéticas Inatas/diagnóstico , Unidades de Terapia Intensiva Pediátrica , Sequenciamento Completo do Exoma/métodos , Adulto , Cuidados Críticos/métodos , Gerenciamento Clínico , Exoma , Aconselhamento Genético/métodos , Doenças Genéticas Inatas/genética , Doenças Genéticas Inatas/terapia , Humanos , Lactente , Cuidado do Lactente/métodos , Recém-Nascido , Tempo de Internação/estatística & dados numéricos , Estudos Retrospectivos , Texas
12.
Am J Hum Genet ; 101(4): 503-515, 2017 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-28942966

RESUMO

Bromodomain PHD finger transcription factor (BPTF) is the largest subunit of nucleosome remodeling factor (NURF), a member of the ISWI chromatin-remodeling complex. However, the clinical consequences of disruption of this complex remain largely uncharacterized. BPTF is required for anterior-posterior axis formation of the mouse embryo and was shown to promote posterior neuroectodermal fate by enhancing Smad2-activated wnt8 expression in zebrafish. Here, we report eight loss-of-function and two missense variants (eight de novo and two of unknown origin) in BPTF on 17q24.2. The BPTF variants were found in unrelated individuals aged between 2.1 and 13 years, who manifest variable degrees of developmental delay/intellectual disability (10/10), speech delay (10/10), postnatal microcephaly (7/9), and dysmorphic features (9/10). Using CRISPR-Cas9 genome editing of bptf in zebrafish to induce a loss of gene function, we observed a significant reduction in head size of F0 mutants compared to control larvae. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and phospho-histone H3 (PH3) staining to assess apoptosis and cell proliferation, respectively, showed a significant increase in cell death in F0 mutants compared to controls. Additionally, we observed a substantial increase of the ceratohyal angle of the craniofacial skeleton in bptf F0 mutants, indicating abnormal craniofacial patterning. Taken together, our data demonstrate the pathogenic role of BPTF haploinsufficiency in syndromic neurodevelopmental anomalies and extend the clinical spectrum of human disorders caused by ablation of chromatin remodeling complexes.


Assuntos
Anormalidades Múltiplas/genética , Antígenos Nucleares/genética , Anormalidades Craniofaciais/genética , Regulação da Expressão Gênica no Desenvolvimento , Haploinsuficiência/genética , Transtornos do Desenvolvimento da Linguagem/genética , Microcefalia/genética , Proteínas do Tecido Nervoso/genética , Fatores de Transcrição/genética , Anormalidades Múltiplas/patologia , Adolescente , Animais , Antígenos Nucleares/metabolismo , Sistemas CRISPR-Cas , Proliferação de Células , Células Cultivadas , Criança , Pré-Escolar , Montagem e Desmontagem da Cromatina , Estudos de Coortes , Anormalidades Craniofaciais/patologia , Feminino , Edição de Genes , Haploinsuficiência/fisiologia , Humanos , Transtornos do Desenvolvimento da Linguagem/patologia , Larva/genética , Larva/crescimento & desenvolvimento , Masculino , Microcefalia/patologia , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Neurônios/patologia , Fenótipo , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimento
14.
Hum Genet ; 136(4): 377-386, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28251352

RESUMO

Impairment of ubiquitin-proteasome system activity involving ubiquitin ligase genes UBE3A, UBE3B, and HUWE1 and deubiquitinating enzyme genes USP7 and USP9X has been reported in patients with neurodevelopmental delays. To date, only a handful of single-nucleotide variants (SNVs) and copy-number variants (CNVs) involving TRIP12, encoding a member of the HECT domain E3 ubiquitin ligases family on chromosome 2q36.3 have been reported. Using chromosomal microarray analysis and whole-exome sequencing (WES), we have identified, respectively, five deletion CNVs and four inactivating SNVs (two frameshifts, one missense, and one splicing) in TRIP12. Seven of these variants were found to be de novo; parental studies could not be completed in two families. Quantitative PCR analyses of the splicing mutation showed a dramatically decreased level of TRIP12 mRNA in the proband compared to the family controls, indicating a loss-of-function mechanism. The shared clinical features include intellectual disability with or without autistic spectrum disorders, speech delay, and facial dysmorphism. Our findings demonstrate that E3 ubiquitin ligase TRIP12 plays an important role in nervous system development and function. The nine presented pathogenic variants further document that TRIP12 haploinsufficiency causes a childhood-onset neurodevelopmental disorder. Finally, our data enable expansion of the phenotypic spectrum of ubiquitin-proteasome dependent disorders.


Assuntos
Transtorno do Espectro Autista/genética , Proteínas de Transporte/genética , Facies , Haploinsuficiência , Deficiência Intelectual/genética , Transtornos do Desenvolvimento da Linguagem/genética , Ubiquitina-Proteína Ligases/genética , Adolescente , Transtorno do Espectro Autista/complicações , Criança , Pré-Escolar , Estudos de Coortes , Variações do Número de Cópias de DNA , Feminino , Humanos , Lactente , Deficiência Intelectual/complicações , Transtornos do Desenvolvimento da Linguagem/complicações , Masculino
15.
Am J Hum Genet ; 99(4): 886-893, 2016 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-27616478

RESUMO

Disruption of the establishment of left-right (L-R) asymmetry leads to situs anomalies ranging from situs inversus totalis (SIT) to situs ambiguus (heterotaxy). The genetic causes of laterality defects in humans are highly heterogeneous. Via whole-exome sequencing (WES), we identified homozygous mutations in PKD1L1 from three affected individuals in two unrelated families. PKD1L1 encodes a polycystin-1-like protein and its loss of function is known to cause laterality defects in mouse and medaka fish models. Family 1 had one fetus and one deceased child with heterotaxy and complex congenital heart malformations. WES identified a homozygous splicing mutation, c.6473+2_6473+3delTG, which disrupts the invariant splice donor site in intron 42, in both affected individuals. In the second family, a homozygous c.5072G>C (p.Cys1691Ser) missense mutation was detected in an individual with SIT and congenital heart disease. The p.Cys1691Ser substitution affects a highly conserved cysteine residue and is predicted by molecular modeling to disrupt a disulfide bridge essential for the proper folding of the G protein-coupled receptor proteolytic site (GPS) motif. Damaging effects associated with substitutions of this conserved cysteine residue in the GPS motif have also been reported in other genes, namely GPR56, BAI3, and PKD1 in human and lat-1 in C. elegans, further supporting the likely pathogenicity of p.Cys1691Ser in PKD1L1. The identification of bi-allelic PKD1L1 mutations recapitulates previous findings regarding phenotypic consequences of loss of function of the orthologous genes in mice and medaka fish and further expands our understanding of genetic contributions to laterality defects in humans.


Assuntos
Alelos , Lateralidade Funcional/genética , Proteínas de Membrana/genética , Mutação , Situs Inversus/genética , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Caenorhabditis elegans/genética , Cisteína/genética , Exoma/genética , Feminino , Doenças Fetais/genética , Cardiopatias Congênitas/genética , Síndrome de Heterotaxia , Homozigoto , Humanos , Recém-Nascido , Íntrons/genética , Masculino , Proteínas de Membrana/química , Camundongos , Pessoa de Meia-Idade , Modelos Moleculares , Mutação de Sentido Incorreto , Oryzias/genética , Linhagem , Processamento de RNA/genética
16.
Am J Hum Genet ; 98(2): 347-57, 2016 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-26805781

RESUMO

The underlying genetic etiology of rhabdomyolysis remains elusive in a significant fraction of individuals presenting with recurrent metabolic crises and muscle weakness. Using exome sequencing, we identified bi-allelic mutations in TANGO2 encoding transport and Golgi organization 2 homolog (Drosophila) in 12 subjects with episodic rhabdomyolysis, hypoglycemia, hyperammonemia, and susceptibility to life-threatening cardiac tachyarrhythmias. A recurrent homozygous c.460G>A (p.Gly154Arg) mutation was found in four unrelated individuals of Hispanic/Latino origin, and a homozygous ∼34 kb deletion affecting exons 3-9 was observed in two families of European ancestry. One individual of mixed Hispanic/European descent was found to be compound heterozygous for c.460G>A (p.Gly154Arg) and the deletion of exons 3-9. Additionally, a homozygous exons 4-6 deletion was identified in a consanguineous Middle Eastern Arab family. No homozygotes have been reported for these changes in control databases. Fibroblasts derived from a subject with the recurrent c.460G>A (p.Gly154Arg) mutation showed evidence of increased endoplasmic reticulum stress and a reduction in Golgi volume density in comparison to control. Our results show that the c.460G>A (p.Gly154Arg) mutation and the exons 3-9 heterozygous deletion in TANGO2 are recurrent pathogenic alleles present in the Latino/Hispanic and European populations, respectively, causing considerable morbidity in the homozygotes in these populations.


Assuntos
Arritmias Cardíacas/genética , Debilidade Muscular/genética , Rabdomiólise/genética , Alelos , Árabes/genética , Arritmias Cardíacas/diagnóstico , Sequência de Bases , Criança , Pré-Escolar , Estresse do Retículo Endoplasmático/genética , Grupo com Ancestrais do Continente Europeu/genética , Exoma , Éxons , Feminino , Deleção de Genes , Complexo de Golgi/genética , Complexo de Golgi/metabolismo , Hispano-Americanos/genética , Homozigoto , Humanos , Lactente , Masculino , Dados de Sequência Molecular , Debilidade Muscular/diagnóstico , Linhagem , Rabdomiólise/diagnóstico
17.
Hum Gene Ther Methods ; 24(5): 321-7, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23947957

RESUMO

Crigler-Najjar syndrome type I is caused by mutations of the uridine diphospho-glucuronosyl transferase 1A1 (UGT1A1) gene resulting in life-threatening increase of serum bilirubin. Life-long correction of hyperbilirubinemia was previously shown with intravenous injection of high doses of a helper-dependent adenoviral (HDAd) vector expressing UGT1A1 in the Gunn rat, the animal model of Crigler-Najjar syndrome. However, such high vector doses can activate an acute and potentially lethal inflammatory response with elevated serum interleukin-6 (IL-6). To overcome this obstacle, we investigated safety and efficacy of direct injections of low HDAd doses delivered directly into the liver parenchyma of Gunn rats. Direct hepatic injections performed by either laparotomy or ultrasound-guided percutaneous injections were compared with the same doses given by intravenous injections. A greater reduction of hyperbilirubinemia and increased conjugated bilirubin in bile were achieved with 1 × 10(11) vp/kg by direct liver injections compared with intravenous injections. In sharp contrast to intravenous injections, direct hepatic injections neither raised serum IL-6 nor resulted in thrombocytopenia. In conclusion, ultrasound-guided percutaneous injection of HDAd vectors into liver parenchyma resulted in improved hepatocyte transduction and reduced toxicity compared with systemic injections and is clinically attractive for liver-directed gene therapy of Crigler-Najjar syndrome.


Assuntos
Adenoviridae/genética , Síndrome de Crigler-Najjar/terapia , Terapia Genética , Vetores Genéticos/administração & dosagem , Glucuronosiltransferase/genética , Animais , Vetores Genéticos/toxicidade , Glucuronosiltransferase/metabolismo , Vírus Auxiliares/genética , Humanos , Injeções/métodos , Fígado/diagnóstico por imagem , Ratos , Ratos Gunn , Resultado do Tratamento , Ultrassonografia
18.
Nat Cell Biol ; 15(6): 647-58, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23604321

RESUMO

The lysosomal-autophagic pathway is activated by starvation and plays an important role in both cellular clearance and lipid catabolism. However, the transcriptional regulation of this pathway in response to metabolic cues is uncharacterized. Here we show that the transcription factor EB (TFEB), a master regulator of lysosomal biogenesis and autophagy, is induced by starvation through an autoregulatory feedback loop and exerts a global transcriptional control on lipid catabolism via Ppargc1α and Ppar1α. Thus, during starvation a transcriptional mechanism links the autophagic pathway to cellular energy metabolism. The conservation of this mechanism in Caenorhabditis elegans suggests a fundamental role for TFEB in the evolution of the adaptive response to food deprivation. Viral delivery of TFEB to the liver prevented weight gain and metabolic syndrome in both diet-induced and genetic mouse models of obesity, suggesting a new therapeutic strategy for disorders of lipid metabolism.


Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Metabolismo dos Lipídeos , Síndrome Metabólica/metabolismo , Obesidade/metabolismo , Inanição/metabolismo , Animais , Autofagia/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/farmacologia , Caenorhabditis elegans/metabolismo , Linhagem Celular Tumoral , Metabolismo Energético , Retroalimentação Fisiológica , Regulação da Expressão Gênica , Células HeLa , Homeostase , Humanos , Fígado/metabolismo , Lisossomos/genética , Masculino , Síndrome Metabólica/genética , Síndrome Metabólica/prevenção & controle , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Obesidade/genética , PPAR alfa/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Inanição/genética , Transativadores/metabolismo , Fatores de Transcrição , Transcrição Genética , Ganho de Peso
19.
EMBO Mol Med ; 5(3): 397-412, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23381957

RESUMO

Alpha-1-anti-trypsin deficiency is the most common genetic cause of liver disease in children and liver transplantation is currently the only available treatment. Enhancement of liver autophagy increases degradation of mutant, hepatotoxic alpha-1-anti-trypsin (ATZ). We investigated the therapeutic potential of liver-directed gene transfer of transcription factor EB (TFEB), a master gene that regulates lysosomal function and autophagy, in PiZ transgenic mice, recapitulating the human hepatic disease. Hepatocyte TFEB gene transfer resulted in dramatic reduction of hepatic ATZ, liver apoptosis and fibrosis, which are key features of alpha-1-anti-trypsin deficiency. Correction of the liver phenotype resulted from increased ATZ polymer degradation mediated by enhancement of autophagy flux and reduced ATZ monomer by decreased hepatic NFκB activation and IL-6 that drives ATZ gene expression. In conclusion, TFEB gene transfer is a novel strategy for treatment of liver disease of alpha-1-anti-trypsin deficiency. This study may pave the way towards applications of TFEB gene transfer for treatment of a wide spectrum of human disorders due to intracellular accumulation of toxic proteins.


Assuntos
Autofagia , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Técnicas de Transferência de Genes , Terapia Genética/métodos , Cirrose Hepática/terapia , Fígado/enzimologia , Deficiência de alfa 1-Antitripsina/terapia , alfa 1-Antitripsina/metabolismo , Animais , Apoptose , Autofagia/genética , Proteína 7 Relacionada à Autofagia , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Modelos Animais de Doenças , Predisposição Genética para Doença , Células HeLa , Humanos , Interleucina-6/metabolismo , Fígado/patologia , Cirrose Hepática/genética , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , Lisossomos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Proteínas Associadas aos Microtúbulos/deficiência , Proteínas Associadas aos Microtúbulos/genética , Mutação de Sentido Incorreto , NF-kappa B/metabolismo , Papio , Fenótipo , Fatores de Tempo , Transfecção , alfa 1-Antitripsina/genética , Deficiência de alfa 1-Antitripsina/complicações , Deficiência de alfa 1-Antitripsina/genética , Deficiência de alfa 1-Antitripsina/metabolismo
20.
Mol Ther ; 21(4): 767-74, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23358188

RESUMO

Helper-dependent adenoviral (HDAd) vectors can mediate long-term, high-level transgene expression from transduced hepatocytes with no chronic toxicity. However, a toxic acute response with potentially lethal consequences has hindered their clinical applications. Liver sinusoidal endothelial cells (LSECs) and Kupffer cells are major barriers to efficient hepatocyte transduction. Understanding the mechanisms of adenoviral vector uptake by non-parenchymal cells may allow the development of strategies aimed at overcoming these important barriers and to achieve preferential hepatocyte gene transfer with reduced toxicity. Scavenger receptors on Kupffer cells bind adenoviral particles and remove them from the circulation, thus preventing hepatocyte transduction. In the present study, we show that HDAd particles interact in vitro and in vivo with scavenger receptor-A (SR-A) and with scavenger receptor expressed on endothelial cells-I (SREC-I) and we exploited this knowledge to increase the efficiency of hepatocyte transduction by HDAd vectors in vivo through blocking of SR-A and SREC-I with specific fragments antigen-binding (Fabs).


Assuntos
Adenoviridae/genética , Receptor de Asialoglicoproteína/genética , Vetores Genéticos/genética , Receptores Imunológicos/genética , Receptores Depuradores Classe A/genética , Receptores Depuradores Classe F/genética , Animais , Linhagem Celular , Imunofluorescência , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase em Tempo Real
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