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1.
Hum Mutat ; 2019 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-31696996

RESUMO

Hereditary spastic paraplegia (HSP) is a group of disorders with predominant symptoms of lower-extremity weakness and spasticity. Despite the delineation of numerous genetic causes of HSP, a significant portion of individuals with HSP remain molecularly undiagnosed. Through exome sequencing, we identified five unrelated families with childhood-onset nonsyndromic HSP, all presenting with progressive spastic gait, leg clonus, and toe walking starting from 7-8 years old. A recurrent two-base pair deletion (c.426_427delGA, p.K143Sfs*15) in the UBAP1 gene was found in four families, and a similar variant (c.475_476delTT, p.F159*) was detected in a fifth family. The variant was confirmed to be de novo in two families and inherited from affected parent in two other families. RNA studies performed in lymphocytes from one patient with the de novo c.426_427delGA variant demonstrated escape of nonsense-mediated decay (NMD) of the UBAP1 mutant transcript, suggesting the generation of a truncated protein. Both variants identified in this study are predicted to result in truncated proteins losing the capacity of binding to ubiquitinated proteins, hence appearing to exhibit a dominant-negative effect on the normal function of the endosome-specific ESCRT-I (endosomal sorting complexes required for transport-I) complex. This article is protected by copyright. All rights reserved.

2.
Genome Med ; 11(1): 48, 2019 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-31349857

RESUMO

BACKGROUND: Although mosaic variation has been known to cause disease for decades, high-throughput sequencing technologies with the analytical sensitivity to consistently detect variants at reduced allelic fractions have only recently emerged as routine clinical diagnostic tests. To date, few systematic analyses of mosaic variants detected by diagnostic exome sequencing for diverse clinical indications have been performed. METHODS: To investigate the frequency, type, allelic fraction, and phenotypic consequences of clinically relevant somatic mosaic single nucleotide variants (SNVs) and characteristics of the corresponding genes, we retrospectively queried reported mosaic variants from a cohort of ~ 12,000 samples submitted for clinical exome sequencing (ES) at Baylor Genetics. RESULTS: We found 120 mosaic variants involving 107 genes, including 80 mosaic SNVs in proband samples and 40 in parental/grandparental samples. Average mosaic alternate allele fraction (AAF) detected in autosomes and in X-linked disease genes in females was 18.2% compared with 34.8% in X-linked disease genes in males. Of these mosaic variants, 74 variants (61.7%) were classified as pathogenic or likely pathogenic and 46 (38.3%) as variants of uncertain significance. Mosaic variants occurred in disease genes associated with autosomal dominant (AD) or AD/autosomal recessive (AR) (67/120, 55.8%), X-linked (33/120, 27.5%), AD/somatic (10/120, 8.3%), and AR (8/120, 6.7%) inheritance. Of note, 1.7% (2/120) of variants were found in genes in which only somatic events have been described. Nine genes had recurrent mosaic events in unrelated individuals which accounted for 18.3% (22/120) of all detected mosaic variants in this study. The proband group was enriched for mosaicism affecting Ras signaling pathway genes. CONCLUSIONS: In sum, an estimated 1.5% of all molecular diagnoses made in this cohort could be attributed to a mosaic variant detected in the proband, while parental mosaicism was identified in 0.3% of families analyzed. As ES design favors breadth over depth of coverage, this estimate of the prevalence of mosaic variants likely represents an underestimate of the total number of clinically relevant mosaic variants in our cohort.

4.
Hum Mol Genet ; 28(17): 2900-2919, 2019 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-31127942

RESUMO

N-alpha-acetylation is one of the most common co-translational protein modifications in humans and is essential for normal cell function. NAA10 encodes for the enzyme NAA10, which is the catalytic subunit in the N-terminal acetyltransferase A (NatA) complex. The auxiliary and regulatory subunits of the NatA complex are NAA15 and Huntington-interacting protein (HYPK), respectively. Through a genotype-first approach with exome sequencing, we identified and phenotypically characterized 30 individuals from 30 unrelated families with 17 different de novo or inherited, dominantly acting missense variants in NAA10 or NAA15. Clinical features of affected individuals include variable levels of intellectual disability, delayed speech and motor milestones and autism spectrum disorder. Additionally, some subjects present with mild craniofacial dysmorphology, congenital cardiac anomalies and seizures. One of the individuals is an 11-year-old boy with a frameshift variant in exon 7 of NAA10, who presents most notably with microphthalmia, which confirms a prior finding with a single family with Lenz microphthalmia syndrome. Biochemical analyses of variants as part of the human NatA complex, as well as enzymatic analyses with and without the HYPK regulatory subunit, help to explain some of the phenotypic differences seen among the different variants.

5.
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.

6.
Genet Med ; 21(9): 2135-2144, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-30890783

RESUMO

PURPOSE: To provide a validated method to confidently identify exon-containing copy-number variants (CNVs), with a low false discovery rate (FDR), in targeted sequencing data from a clinical laboratory with particular focus on single-exon CNVs. METHODS: DNA sequence coverage data are normalized within each sample and subsequently exonic CNVs are identified in a batch of samples, when the target log2 ratio of the sample to the batch median exceeds defined thresholds. The quality of exonic CNV calls is assessed by C-scores (Z-like scores) using thresholds derived from gold standard samples and simulation studies. We integrate an ExonQC threshold to lower FDR and compare performance with alternate software (VisCap). RESULTS: Thirteen CNVs were used as a truth set to validate Atlas-CNV and compared with VisCap. We demonstrated FDR reduction in validation, simulation, and 10,926 eMERGESeq samples without sensitivity loss. Sixty-four multiexon and 29 single-exon CNVs with high C-scores were assessed by Multiplex Ligation-dependent Probe Amplification (MLPA). CONCLUSION: Atlas-CNV is validated as a method to identify exonic CNVs in targeted sequencing data generated in the clinical laboratory. The ExonQC and C-score assignment can reduce FDR (identification of targets with high variance) and improve calling accuracy of single-exon CNVs respectively. We propose guidelines and criteria to identify high confidence single-exon CNVs.

7.
Am J Hum Genet ; 104(2): 319-330, 2019 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-30639322

RESUMO

ZMIZ1 is a coactivator of several transcription factors, including p53, the androgen receptor, and NOTCH1. Here, we report 19 subjects with intellectual disability and developmental delay carrying variants in ZMIZ1. The associated features include growth failure, feeding difficulties, microcephaly, facial dysmorphism, and various other congenital malformations. Of these 19, 14 unrelated subjects carried de novo heterozygous single-nucleotide variants (SNVs) or single-base insertions/deletions, 3 siblings harbored a heterozygous single-base insertion, and 2 subjects had a balanced translocation disrupting ZMIZ1 or involving a regulatory region of ZMIZ1. In total, we identified 13 point mutations that affect key protein regions, including a SUMO acceptor site, a central disordered alanine-rich motif, a proline-rich domain, and a transactivation domain. All identified variants were absent from all available exome and genome databases. In vitro, ZMIZ1 showed impaired coactivation of the androgen receptor. In vivo, overexpression of ZMIZ1 mutant alleles in developing mouse brains using in utero electroporation resulted in abnormal pyramidal neuron morphology, polarization, and positioning, underscoring the importance of ZMIZ1 in neural development and supporting mutations in ZMIZ1 as the cause of a rare neurodevelopmental syndrome.

8.
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.

9.
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
10.
Genet Med ; 19(8): 936-944, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28125085

RESUMO

PURPOSE: To investigate pan-ethnic SMN1 copy-number and sequence variation by hybridization-based target enrichment coupled with massively parallel sequencing or next-generation sequencing (NGS). METHODS: NGS reads aligned to SMN1 and SMN2 exon 7 were quantified to determine the total combined copy number of SMN1 and SMN2. The ratio of SMN1 to SMN2 was calculated based on a single-nucleotide difference that distinguishes the two genes. SMN1 copy-number results were compared between the NGS and quantitative polymerase chain reaction and/or multiplex ligation-dependent probe amplification. The NGS data set was also queried for the g.27134T>G single-nucleotide polymorphism (SNP) and other SMN1 sequence pathogenic variants. RESULTS: The sensitivity of the test to detect spinal muscular atrophy (SMA) carriers with one copy of SMN1 was 100% (95% confidence interval (CI): 95.9-100%; n = 90) and specificity was 99.6% (95% CI: 99.4-99.7%; n = 6,648). Detection of the g.27134T>G SNP by NGS was 100% concordant with an restriction fragment-length polymorphism method (n = 493). Ten single-nucleotide variants in SMN1 were detectable by NGS and confirmed by gene-specific amplicon-based sequencing. This comprehensive approach yielded SMA carrier detection rates of 90.3-95.0% in five ethnic groups studied. CONCLUSION: We have developed a novel, comprehensive SMN1 copy-number and sequence variant analysis method by NGS that demonstrated improved SMA carrier detection rates across the entire population examined.Genet Med advance online publication 19 January 2017.


Assuntos
Triagem de Portadores Genéticos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Atrofia Muscular Espinal/genética , Proteína 1 de Sobrevivência do Neurônio Motor/genética , Dosagem de Genes , Humanos , Atrofia Muscular Espinal/etnologia , Polimorfismo de Nucleotídeo Único , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Proteína 2 de Sobrevivência do Neurônio Motor/genética
11.
Am J Hum Genet ; 100(2): 343-351, 2017 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-28132692

RESUMO

Whole-exome sequencing (WES) has increasingly enabled new pathogenic gene variant identification for undiagnosed neurodevelopmental disorders and provided insights into both gene function and disease biology. Here, we describe seven children with a neurodevelopmental disorder characterized by microcephaly, profound developmental delays and/or intellectual disability, cataracts, severe epilepsy including infantile spasms, irritability, failure to thrive, and stereotypic hand movements. Brain imaging in these individuals reveals delay in myelination and cerebral atrophy. We observe an identical recurrent de novo heterozygous c.892C>T (p.Arg298Trp) variant in the nucleus accumbens associated 1 (NACC1) gene in seven affected individuals. One of the seven individuals is mosaic for this variant. NACC1 encodes a transcriptional repressor implicated in gene expression and has not previously been associated with germline disorders. The probability of finding the same missense NACC1 variant by chance in 7 out of 17,228 individuals who underwent WES for diagnoses of neurodevelopmental phenotypes is extremely small and achieves genome-wide significance (p = 1.25 × 10-14). Selective constraint against missense variants in NACC1 makes this excess of an identical missense variant in all seven individuals more remarkable. Our findings are consistent with a germline recurrent mutational hotspot associated with an allele-specific neurodevelopmental phenotype in NACC1.


Assuntos
Catarata/genética , Variação Genética , Deficiência Intelectual/genética , Proteínas de Neoplasias/genética , Proteínas Repressoras/genética , Espasmos Infantis/genética , Alelos , Sequência de Aminoácidos , Encéfalo/diagnóstico por imagem , Catarata/diagnóstico por imagem , Criança , Pré-Escolar , Feminino , Estudo de Associação Genômica Ampla , Humanos , Lactente , Deficiência Intelectual/diagnóstico por imagem , Imagem por Ressonância Magnética , Masculino , Microcefalia/genética , Mutação de Sentido Incorreto , Linhagem , Fenótipo , Espasmos Infantis/diagnóstico por imagem
12.
Am J Med Genet A ; 173(2): 460-470, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-27860360

RESUMO

Pyrroline-5-carboxylate reductase 2, encoded by PYCR2, is one of the three homologous enzymes that catalyze the last step of proline synthesis. Homozygous variants in PYCR2 have been reported in patients from multiple consanguineous families with hypomyelinating leukodystrophy 10 (HLD10) (MIM: 616420). Here, we report five additional patients from three families with homozygous nonsense or missense variants in PYCR2, identified through clinical exome sequencing. All patients presented with postnatally acquired microcephaly, moderate to profound global developmental delay, and failure to thrive. Brain MRI in these patients showed thin corpus callosum, delayed myelination, and generalized white-matter volume loss. Additional phenotypes that were less consistent among patients included seizures or seizure-like movements, spasticity and ataxic gait, recurrent vomiting, cortical blindness, dysmorphic features, joint contractures, and irritability. Exome sequencing identified homozygous variants in PYCR2 in the proband from each family: c.28C>T (p.(Glu10Ter)), c.796C>T (p.(Arg266Ter)), and c.577G>A (p.(Val193Met)). Subsequent targeted analyses demonstrated co-segregation of the disease with the variant in the family. Despite the metabolic role of PYCR2, routine serum metabolic test in these patients were normal. To further understand the disease etiology and functions of PYCR2, small molecule metabolomics profiling was performed in plasma from three severely affected patients. No significant changes were identified in proline biosynthesis pathway or related metabolites. Studying the clinical features and the metabolic profiles of the PYCR2-deficient patients provides a more comprehensive picture for this newly identified disorder and facilitates further research on the gene function and disease etiology. © 2016 Wiley Periodicals, Inc.


Assuntos
Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/diagnóstico , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/genética , Homozigoto , Microcefalia/diagnóstico , Microcefalia/genética , Mutação , Pirrolina Carboxilato Redutases/genética , Adolescente , Alelos , Substituição de Aminoácidos , Encéfalo/anormalidades , Criança , Pré-Escolar , Códon , Análise Mutacional de DNA , Exoma , Feminino , Estudos de Associação Genética , Gráficos de Crescimento , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Metabolômica/métodos , Linhagem , Fenótipo
14.
Diabetes Care ; 39(4): 555-62, 2016 04.
Artigo em Inglês | MEDLINE | ID: mdl-26884472

RESUMO

OBJECTIVE: Treatment of severe hypoglycemia outside of the hospital setting is limited to intramuscular glucagon requiring reconstitution prior to injection. The current study examined the safety and dose-response relationships of a needle-free intranasal glucagon preparation in youth aged 4 to <17 years. RESEARCH DESIGN AND METHODS: A total of 48 youth with type 1 diabetes completed the study at seven clinical centers. Participants in the two youngest cohorts (4 to <8 and 8 to <12 years old) were randomly assigned to receive either 2 or 3 mg intranasal glucagon in two separate sessions or to receive a single, weight-based dose of intramuscular glucagon. Participants aged 12 to <17 years received 1 mg intramuscular glucagon in one session and 3 mg intranasal glucagon in the other session. Glucagon was given after glucose was lowered to <80 mg/dL (mean nadir ranged between 67 and 75 mg/dL). RESULTS: All 24 intramuscular and 58 of the 59 intranasal doses produced a ≥25 mg/dL rise in glucose from nadir within 20 min of dosing. Times to peak plasma glucose and glucagon levels were similar under both intramuscular and intranasal conditions. Transient nausea occurred in 67% of intramuscular sessions versus 42% of intranasal sessions (P = 0.05); the efficacy and safety of the 2- and 3-mg intranasal doses were similar in the youngest cohorts. CONCLUSIONS: Results of this phase 1, pharmacokinetic, and pharmacodynamic study support the potential efficacy of a needle-free glucagon nasal powder delivery system for treatment of hypoglycemia in youth with type 1 diabetes. Given the similar frequency and transient nature of adverse effects of the 2- and 3-mg intranasal doses in the two youngest cohorts, a single 3-mg intranasal dose appears to be appropriate for use across the entire 4- to <17-year age range.


Assuntos
Administração Intranasal , Diabetes Mellitus Tipo 1/tratamento farmacológico , Glucagon/administração & dosagem , Adolescente , Glicemia/metabolismo , Criança , Pré-Escolar , Estudos de Coortes , Estudos Cross-Over , Relação Dose-Resposta a Droga , Método Duplo-Cego , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/prevenção & controle , Feminino , Glucagon/efeitos adversos , Humanos , Hipoglicemia/sangue , Hipoglicemia/tratamento farmacológico , Injeções Intramusculares , Insulina/sangue , Insulina/uso terapêutico , Masculino , Náusea/etiologia , Náusea/prevenção & controle , Pós
15.
Diabetes Care ; 39(2): 264-70, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26681725

RESUMO

OBJECTIVE: Treatment of severe hypoglycemia with loss of consciousness or seizure outside of the hospital setting is presently limited to intramuscular glucagon requiring reconstitution immediately prior to injection, a process prone to error or omission. A needle-free intranasal glucagon preparation was compared with intramuscular glucagon for treatment of insulin-induced hypoglycemia. RESEARCH DESIGN AND METHODS: At eight clinical centers, a randomized crossover noninferiority trial was conducted involving 75 adults with type 1 diabetes (mean age, 33 ± 12 years; median diabetes duration, 18 years) to compare intranasal (3 mg) versus intramuscular (1 mg) glucagon for treatment of hypoglycemia induced by intravenous insulin. Success was defined as an increase in plasma glucose to ≥70 mg/dL or ≥20 mg/dL from the glucose nadir within 30 min after receiving glucagon. RESULTS: Mean plasma glucose at time of glucagon administration was 48 ± 8 and 49 ± 8 mg/dL at the intranasal and intramuscular visits, respectively. Success criteria were met at all but one intranasal visit and at all intramuscular visits (98.7% vs. 100%; difference 1.3%, upper end of 1-sided 97.5% CI 4.0%). Mean time to success was 16 min for intranasal and 13 min for intramuscular (P < 0.001). Head/facial discomfort was reported during 25% of intranasal and 9% of intramuscular dosing visits; nausea (with or without vomiting) occurred with 35% and 38% of visits, respectively. CONCLUSIONS: Intranasal glucagon was highly effective in treating insulin-induced hypoglycemia in adults with type 1 diabetes. Although the trial was conducted in a controlled setting, the results are applicable to real-world management of severe hypoglycemia, which occurs owing to excessive therapeutic insulin relative to the impaired or absent endogenous glucagon response.


Assuntos
Diabetes Mellitus Tipo 1/tratamento farmacológico , Glucagon/administração & dosagem , Hormônios/uso terapêutico , Administração Intranasal , Adulto , Glicemia/efeitos dos fármacos , Estudos Cross-Over , Diabetes Mellitus Tipo 1/sangue , Feminino , Humanos , Hipoglicemia/induzido quimicamente , Insulina/uso terapêutico , Masculino , Pessoa de Meia-Idade , Adulto Jovem
16.
Hum Mol Genet ; 25(R1): R18-26, 2016 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-26628634

RESUMO

The concept of orphan drugs for treatment of orphan genetic diseases is perceived enthusiastically at present, and this is leading to research investment on the part of governments, disease-specific foundations and industry. This review attempts to survey the potential to use traditional pharmaceuticals as opposed to biopharmaceuticals to treat single-gene disorders. The available strategies include the use of antisense oligonucleotides (ASOs) to alter splicing or knock-down expression of a transcript, siRNAs to knock-down gene expression and drugs for nonsense mutation read-through. There is an approved drug for biallelic knock-down of the APOB gene as treatment for familial hypercholesterolemia. Both ASOs and siRNAs are being explored to knock-down the transthyretin gene to prevent the related form of amyloidosis. The use of ASOs to alter gene-splicing to treat spinal muscular atrophy is in phase 3 clinical trials. Work is progressing on the use of ASOs to activate the normally silent paternal copy of the imprinted UBE3A gene in neurons as a treatment for Angelman syndrome. A gene-activation or gene-specific ramp-up strategy would be generally helpful if such could be developed. There is exciting theoretical potential for converting biopharmaceutical strategies such gene correction and CRISPR-Cas9 editing to a synthetic pharmaceutical approach.


Assuntos
Marcação de Genes , Doenças Genéticas Inatas/tratamento farmacológico , Doenças Raras/tratamento farmacológico , Humanos
17.
Nature ; 518(7539): 409-12, 2015 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-25470045

RESUMO

Angelman syndrome is a single-gene disorder characterized by intellectual disability, developmental delay, behavioural uniqueness, speech impairment, seizures and ataxia. It is caused by maternal deficiency of the imprinted gene UBE3A, encoding an E3 ubiquitin ligase. All patients carry at least one copy of paternal UBE3A, which is intact but silenced by a nuclear-localized long non-coding RNA, UBE3A antisense transcript (UBE3A-ATS). Murine Ube3a-ATS reduction by either transcription termination or topoisomerase I inhibition has been shown to increase paternal Ube3a expression. Despite a clear understanding of the disease-causing event in Angelman syndrome and the potential to harness the intact paternal allele to correct the disease, no gene-specific treatment exists for patients. Here we developed a potential therapeutic intervention for Angelman syndrome by reducing Ube3a-ATS with antisense oligonucleotides (ASOs). ASO treatment achieved specific reduction of Ube3a-ATS and sustained unsilencing of paternal Ube3a in neurons in vitro and in vivo. Partial restoration of UBE3A protein in an Angelman syndrome mouse model ameliorated some cognitive deficits associated with the disease. Although additional studies of phenotypic correction are needed, we have developed a sequence-specific and clinically feasible method to activate expression of the paternal Ube3a allele.


Assuntos
Síndrome de Angelman/genética , Síndrome de Angelman/terapia , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/uso terapêutico , RNA Longo não Codificante/antagonistas & inibidores , RNA Longo não Codificante/genética , Alelos , Síndrome de Angelman/complicações , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Células Cultivadas , Modelos Animais de Doenças , Pai , Feminino , Inativação Gênica/efeitos dos fármacos , Impressão Genômica/genética , Masculino , Transtornos da Memória/complicações , Transtornos da Memória/genética , Transtornos da Memória/terapia , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Obesidade/complicações , Obesidade/genética , Obesidade/terapia , Oligonucleotídeos Antissenso/farmacologia , Fenótipo , RNA Antissenso/antagonistas & inibidores , RNA Antissenso/deficiência , RNA Antissenso/genética , Fatores de Tempo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo
18.
PLoS Genet ; 9(12): e1004039, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24385930

RESUMO

Angelman syndrome (AS) is a severe neurodevelopmental disorder caused by maternal deficiency of the imprinted gene UBE3A. Individuals with AS suffer from intellectual disability, speech impairment, and motor dysfunction. Currently there is no cure for the disease. Here, we evaluated the phenotypic effect of activating the silenced paternal allele of Ube3a by depleting its antisense RNA Ube3a-ATS in mice. Premature termination of Ube3a-ATS by poly(A) cassette insertion activates expression of Ube3a from the paternal chromosome, and ameliorates many disease-related symptoms in the AS mouse model, including motor coordination defects, cognitive deficit, and impaired long-term potentiation. Studies on the imprinting mechanism of Ube3a revealed a pattern of biallelic transcription initiation with suppressed elongation of paternal Ube3a, implicating transcriptional collision between sense and antisense polymerases. These studies demonstrate the feasibility and utility of unsilencing the paternal copy of Ube3a via targeting Ube3a-ATS as a treatment for Angelman syndrome.


Assuntos
Síndrome de Angelman/genética , Deficiência Intelectual/patologia , Distúrbios da Fala/genética , Ubiquitina-Proteína Ligases/genética , Síndrome de Angelman/complicações , Síndrome de Angelman/patologia , Animais , Modelos Animais de Doenças , Inativação Gênica , Impressão Genômica , Humanos , Deficiência Intelectual/complicações , Deficiência Intelectual/genética , Camundongos , Terapia de Alvo Molecular , Neurônios/metabolismo , RNA Antissenso/genética , Distúrbios da Fala/complicações , Distúrbios da Fala/patologia , Transcrição Genética , Ubiquitina-Proteína Ligases/biossíntese , Ubiquitina-Proteína Ligases/metabolismo
19.
Hum Mol Genet ; 21(13): 3001-12, 2012 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-22493002

RESUMO

The Angelman syndrome gene, UBE3A, is subject to genomic imprinting controlled by mechanisms that are only partially understood. Its antisense transcript, UBE3A-ATS, is also imprinted and hypothesized to suppress UBE3A in cis. In this research, we showed that the mouse antisense ortholog, Ube3a-ATS, was transcribed by RNA polymerase (RNAP) II. However, unlike typical protein-coding transcripts, Ube3a-ATS was not poly-adenylated and was localized exclusively in the nucleus. It was relatively unstable with a half-life of 4 h, shorter than most protein-coding RNAs tested. To understand the role of Ube3a-ATS in vivo, a mouse model with a 0.9-kb genomic deletion over the paternal Snrpn major promoter was studied. The mice showed partial activation of paternal Ube3a, with decreased expression of Ube3a-ATS but not any imprinting defects in the Prader-Willi syndrome/Angelman syndrome region. A novel cell culture model was also generated with a transcriptional termination cassette inserted downstream of Ube3a on the paternal chromosome to reduce Ube3a-ATS transcription. In neuronally differentiated embryonic stem (ES) cells, paternal Ube3a was found to be expressed at a high level, comparable with that of the maternal allele. To further characterize the antisense RNA, a strand-specific microarray was performed. Ube3a-ATS was detectable across the entire locus of Ube3a and extended beyond the transcriptional start site of Ube3a. In summary, we conclude that Ube3a-ATS is an atypical RNAPII transcript that represses Ube3a on the paternal chromosome. These results suggest that the repression of human UBE3A-ATS may activate the expression of UBE3A from the paternal chromosome, providing a potential therapeutic strategy for patients with Angelman syndrome.


Assuntos
RNA Antissenso/genética , Ubiquitina-Proteína Ligases/genética , Síndrome de Angelman/genética , Síndrome de Angelman/patologia , Síndrome de Angelman/terapia , Animais , Células Cultivadas , Células-Tronco Embrionárias/metabolismo , Feminino , Impressão Genômica , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/metabolismo , Poliadenilação , Regiões Promotoras Genéticas , RNA Polimerase II , RNA Antissenso/metabolismo , Transcrição Genética , Ubiquitina-Proteína Ligases/biossíntese , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Centrais de snRNP/genética
20.
Virus Res ; 163(2): 512-9, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22142475

RESUMO

RNA-dependent RNA polymerases (RDRs) from fungi, plants and some invertebrate animals play fundamental roles in antiviral defense. Here, we investigated the role of RDR6 in the defense of economically important rice plants against a negative-strand RNA virus (Rice stripe virus, RSV) that causes enormous crop damage. In three independent transgenic lines (OsRDR6AS line A, B and C) in which OsRDR6 transcription levels were reduced by 70-80% through antisense silencing, the infection and disease symptoms of RSV were shown to be significantly enhanced. The hypersusceptibilities of the OsRDR6AS plants were attributed not to enhanced insect infestation but to enhanced virus infection. The rise in symptoms was associated with the increased accumulation of RSV genomic RNA in the OsRDR6AS plants. The deep sequencing data showed reduced RSV-derived siRNA accumulation in the OsRDR6AS plants compared with the wild type plants. This is the first report of the antiviral role of a RDR in a monocot crop plant in the defense against a negative-strand RNA virus and significantly expands upon the current knowledge of the antiviral roles of RDRs in the defense against different types of viral genomes in numerous groups of plants.


Assuntos
Oryza/enzimologia , Oryza/imunologia , RNA Replicase/imunologia , RNA Replicase/metabolismo , Tenuivirus/imunologia , Tenuivirus/patogenicidade , Oryza/virologia , Doenças das Plantas/imunologia , Doenças das Plantas/virologia , Proteínas de Plantas/imunologia , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Tenuivirus/crescimento & desenvolvimento , Carga Viral
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