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1.
Genet Med ; 2021 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-33580225

RESUMO

PURPOSE: Genomic sequencing has become an increasingly powerful and relevant tool to be leveraged for the discovery of genetic aberrations underlying rare, Mendelian conditions. Although the computational tools incorporated into diagnostic workflows for this task are continually evolving and improving, we nevertheless sought to investigate commonalities across sequencing processing workflows to reveal consensus and standard practice tools and highlight exploratory analyses where technical and theoretical method improvements would be most impactful. METHODS: We collected details regarding the computational approaches used by a genetic testing laboratory and 11 clinical research sites in the United States participating in the Undiagnosed Diseases Network via meetings with bioinformaticians, online survey forms, and analyses of internal protocols. RESULTS: We found that tools for processing genomic sequencing data can be grouped into four distinct categories. Whereas well-established practices exist for initial variant calling and quality control steps, there is substantial divergence across sites in later stages for variant prioritization and multimodal data integration, demonstrating a diversity of approaches for solving the most mysterious undiagnosed cases. CONCLUSION: The largest differences across diagnostic workflows suggest that advances in structural variant detection, noncoding variant interpretation, and integration of additional biomedical data may be especially promising for solving chronically undiagnosed cases.

2.
Am J Hum Genet ; 107(6): 1096-1112, 2020 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-33232675

RESUMO

SWI/SNF-related intellectual disability disorders (SSRIDDs) are rare neurodevelopmental disorders characterized by developmental disability, coarse facial features, and fifth digit/nail hypoplasia that are caused by pathogenic variants in genes that encode for members of the SWI/SNF (or BAF) family of chromatin remodeling complexes. We have identified 12 individuals with rare variants (10 loss-of-function, 2 missense) in the BICRA (BRD4 interacting chromatin remodeling complex-associated protein) gene, also known as GLTSCR1, which encodes a subunit of the non-canonical BAF (ncBAF) complex. These individuals exhibited neurodevelopmental phenotypes that include developmental delay, intellectual disability, autism spectrum disorder, and behavioral abnormalities as well as dysmorphic features. Notably, the majority of individuals lack the fifth digit/nail hypoplasia phenotype, a hallmark of most SSRIDDs. To confirm the role of BICRA in the development of these phenotypes, we performed functional characterization of the zebrafish and Drosophila orthologs of BICRA. In zebrafish, a mutation of bicra that mimics one of the loss-of-function variants leads to craniofacial defects possibly akin to the dysmorphic facial features seen in individuals harboring putatively pathogenic BICRA variants. We further show that Bicra physically binds to other non-canonical ncBAF complex members, including the BRD9/7 ortholog, CG7154, and is the defining member of the ncBAF complex in flies. Like other SWI/SNF complex members, loss of Bicra function in flies acts as a dominant enhancer of position effect variegation but in a more context-specific manner. We conclude that haploinsufficiency of BICRA leads to a unique SSRIDD in humans whose phenotypes overlap with those previously reported.

3.
J Clin Invest ; 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-33001864

RESUMO

BACKGROUND: Transcriptome sequencing (RNA-seq) improves diagnostic rates in individuals with suspected Mendelian conditions to varying degrees, primarily by directing the prioritization of candidate DNA variants identified on exome or genome sequencing (ES/GS). Here we implemented an RNA-seq guided method to diagnose individuals across a wide range of ages and clinical phenotypes. METHODS: One hundred fifteen undiagnosed adult and pediatric patients with diverse phenotypes and 67 family members (182 total individuals) underwent RNA-seq from whole blood and fibroblasts at the Baylor College of Medicine (BCM) Undiagnosed Diseases Network (UDN) clinical site from 2014-2020. We implemented a workflow to detect outliers in gene expression and splicing for cases that remained undiagnosed despite standard genomic and transcriptomic analysis. RESULTS: The transcriptome-directed approach resulted in a diagnostic rate of 12% across the entire cohort, or 17% after excluding cases solved on ES/GS alone. Newly diagnosed conditions included Koolen-de Vries syndrome (KANSL1), Renpenning syndrome (PQBP1), TBCK-associated encephalopathy, NSD2- and CLTC-related intellectual disability, and others, all with negative conventional genomic testing, including ES and chromosomal microarray (CMA). Fibroblasts exhibited higher and more consistent expression of clinically relevant genes than whole blood. In solved cases with RNA-seq from both tissues, the causative defect was missed in blood in half the cases but none from fibroblasts. CONCLUSION: For our cohort of undiagnosed individuals with suspected Mendelian conditions, transcriptome-directed genomic analysis facilitated diagnoses, primarily through the identification of variants missed on ES and CMA.

5.
Clin Lab Med ; 40(2): 113-119, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32439063

RESUMO

The diagnostic rate of comprehensive genomic sequencing remains only 25% to 30% due to the difficulty in interpreting variants of uncertain significance and noncoding mutations and in elucidating downstream effects of these and other genetic changes. Unlike DNA sequencing, RNA sequencing (RNAseq) reveals the functional consequence of genetic variation through the detection of abnormal gene expression levels, differences in gene splicing, and allele-specific expression. RNAseq can provide nearly 40% improvement in diagnostic rates depending on disease and tissue source. In this burgeoning era of precision medicine, RNAseq offers a powerful tool to improve diagnostic rates and understand disease mechanisms.

6.
Am J Hum Genet ; 106(5): 717-725, 2020 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-32330417

RESUMO

We identified three unrelated individuals with de novo missense variants in CDK19, encoding a cyclin-dependent kinase protein family member that predominantly regulates gene transcription. These individuals presented with hypotonia, global developmental delay, epileptic encephalopathy, and dysmorphic features. CDK19 is conserved between vertebrate and invertebrate model organisms, but currently abnormalities in CDK19 are not known to be associated with a human disorder. Loss of Cdk8, the fly homolog of CDK19, causes larval lethality, which is suppressed by expression of human CDK19 reference cDNA. In contrast, the CDK19 p.Tyr32His and p.Thr196Ala variants identified in the affected individuals fail to rescue the loss of Cdk8 and behave as null alleles. Additionally, neuronal RNAi-mediated knockdown of Cdk8 in flies results in semi-lethality. The few eclosing flies exhibit severe seizures and a reduced lifespan. Both phenotypes are fully suppressed by moderate expression of the CDK19 reference cDNA but not by expression of the two variants. Finally, loss of Cdk8 causes an obvious loss of boutons and synapses at larval neuromuscular junctions (NMJs). Together, our findings demonstrate that human CDK19 fully replaces the function of Cdk8 in the fly, the human disease-associated CDK19 variants behave as strong loss-of-function variants, and deleterious CDK19 variants underlie a syndromic neurodevelopmental disorder.


Assuntos
Encefalopatias/genética , Quinases Ciclina-Dependentes/genética , Epilepsia Generalizada/genética , Deficiência Intelectual/genética , Mutação de Sentido Incorreto/genética , Adulto , Sequência de Aminoácidos , Animais , Pré-Escolar , Quinase 8 Dependente de Ciclina/deficiência , Quinase 8 Dependente de Ciclina/genética , Proteínas de Drosophila/deficiência , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Junção Neuromuscular , Doenças Raras/genética , Convulsões/genética , Síndrome , Adulto Jovem
7.
J Exp Med ; 217(6)2020 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-32207811

RESUMO

Autosomal dominant hyper-IgE syndrome (AD-HIES) is typically caused by dominant-negative (DN) STAT3 mutations. Patients suffer from cold staphylococcal lesions and mucocutaneous candidiasis, severe allergy, and skeletal abnormalities. We report 12 patients from 8 unrelated kindreds with AD-HIES due to DN IL6ST mutations. We identified seven different truncating mutations, one of which was recurrent. The mutant alleles encode GP130 receptors bearing the transmembrane domain but lacking both the recycling motif and all four STAT3-recruiting tyrosine residues. Upon overexpression, the mutant proteins accumulate at the cell surface and are loss of function and DN for cellular responses to IL-6, IL-11, LIF, and OSM. Moreover, the patients' heterozygous leukocytes and fibroblasts respond poorly to IL-6 and IL-11. Consistently, patients with STAT3 and IL6ST mutations display infectious and allergic manifestations of IL-6R deficiency, and some of the skeletal abnormalities of IL-11R deficiency. DN STAT3 and IL6ST mutations thus appear to underlie clinical phenocopies through impairment of the IL-6 and IL-11 response pathways.

8.
Brain ; 142(9): 2631-2643, 2019 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-31334757

RESUMO

Marked by incomplete division of the embryonic forebrain, holoprosencephaly is one of the most common human developmental disorders. Despite decades of phenotype-driven research, 80-90% of aneuploidy-negative holoprosencephaly individuals with a probable genetic aetiology do not have a genetic diagnosis. Here we report holoprosencephaly associated with variants in the two X-linked cohesin complex genes, STAG2 and SMC1A, with loss-of-function variants in 10 individuals and a missense variant in one. Additionally, we report four individuals with variants in the cohesin complex genes that are not X-linked, SMC3 and RAD21. Using whole mount in situ hybridization, we show that STAG2 and SMC1A are expressed in the prosencephalic neural folds during primary neurulation in the mouse, consistent with forebrain morphogenesis and holoprosencephaly pathogenesis. Finally, we found that shRNA knockdown of STAG2 and SMC1A causes aberrant expression of HPE-associated genes ZIC2, GLI2, SMAD3 and FGFR1 in human neural stem cells. These findings show the cohesin complex as an important regulator of median forebrain development and X-linked inheritance patterns in holoprosencephaly.


Assuntos
Proteínas de Ciclo Celular/genética , Proteínas Cromossômicas não Histona/genética , Holoprosencefalia/diagnóstico , Holoprosencefalia/genética , Adolescente , Animais , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Camundongos , Camundongos Endogâmicos C57BL
9.
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.


Assuntos
Variações do Número de Cópias de DNA/genética , Éxons/genética , Genoma Humano/genética , Software , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Análise de Sequência de DNA
10.
Artigo em Inglês | MEDLINE | ID: mdl-30622101

RESUMO

A 55-yr-old male with severe intellectual disability, behavioral problems, kyphoscoliosis, and dysmorphic features was referred for a genetic evaluation. Chromosomal microarray, RASopathy gene panel, mitochondrial sequencing, and fragile X testing were all negative. Subsequent whole-exome sequencing revealed a heterozygous, truncating variant in the AHDC1 gene, consistent with a diagnosis of Xia-Gibbs syndrome (XGS). Review of his clinical history showed many classic dysmorphic and clinical features of XGS, but no major health issues in adulthood other than intellectual disability. This individual is the oldest published XGS case to date, demonstrates the wide phenotypic spectrum of the disorder, and provides information on the condition's natural history. As more adults undergo genomic studies, we will continue to learn about the adult phenotypes of genetic conditions typically diagnosed in the pediatric setting.


Assuntos
Anormalidades Múltiplas/genética , Deficiência Intelectual/genética , Sequenciamento Completo do Exoma , Anormalidades Múltiplas/diagnóstico , Anormalidades Múltiplas/patologia , Heterozigoto , Humanos , Deficiência Intelectual/diagnóstico , Deficiência Intelectual/patologia , Masculino , Pessoa de Meia-Idade
11.
Am J Med Genet A ; 176(6): 1315-1326, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29696776

RESUMO

Xia-Gibbs syndrome (XGS: OMIM # 615829) results from de novo truncating mutations within the AT-Hook DNA Binding Motif Containing 1 gene (AHDC1). To further define the phenotypic and molecular spectrum of this disorder, we established an XGS Registry and recruited patients from a worldwide pool of approximately 60 probands. Additional de novo truncating mutations were observed among 25 individuals, extending both the known number of mutation sites and the range of positions within the coding region that were sensitive to alteration. Detailed phenotypic examination of 20 of these patients via clinical records review and data collection from additional surveys showed a wider age range than previously described. Data from developmental milestones showed evidence for delayed speech and that males were more severely affected. Neuroimaging from six available patients showed an associated thinning of the corpus callosum and posterior fossa cysts. An increased risk of both scoliosis and seizures relative to the population burden was also observed. Data from a modified autism screening tool revealed that XGS shares significant overlap with autism spectrum disorders. These details of the phenotypic heterogeneity of XGS implicate specific genotype/phenotype correlations and suggest potential clinical management guidelines.


Assuntos
Transtorno do Espectro Autista/etiologia , Proteínas de Ligação a DNA/genética , Deficiências do Desenvolvimento/etiologia , Mutação , Criança , Cognição/fisiologia , Corpo Caloso/diagnóstico por imagem , Corpo Caloso/patologia , Face/anormalidades , Feminino , Humanos , Masculino , Linhagem , Fenótipo , Sistema de Registros , Convulsões/etiologia , Síndrome , Adulto Jovem
12.
Congenit Anom (Kyoto) ; 58(1): 29-32, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-28670735

RESUMO

Holoprosencephaly (HPE) is failure of the forebrain to divide completely during embryogenesis. Incomplete penetrance has not been reported previously in SIX3 whole gene deletions, which are known to cause HPE. Both chromosomal microarray and whole exome sequencing (WES) were used to evaluate families with inherited HPE. Two families showed inherited deletions that contain SIX3 and were incompletely penetrant for HPE. Using WES, we ruled out parental mosaicism, a SIX3 hypomorph, and clinically significant variants in genes that are known to interact with SIX3 as causes of incomplete penetrance. We demonstrate the importance of molecular cascade testing in families with HPE and we answer important questions about incomplete penetrance.


Assuntos
Proteínas do Olho/genética , Deleção de Genes , Holoprosencefalia/genética , Proteínas de Homeodomínio/genética , Proteínas do Tecido Nervoso/genética , Prosencéfalo/anormalidades , Adulto , Pré-Escolar , Expressão Gênica , Holoprosencefalia/diagnóstico , Holoprosencefalia/metabolismo , Holoprosencefalia/patologia , Humanos , Lactente , Recém-Nascido , Análise em Microsséries , Proteínas do Tecido Nervoso/deficiência , Penetrância , Prosencéfalo/metabolismo , Sequenciamento Completo do Exoma
13.
J Clin Endocrinol Metab ; 102(5): 1529-1537, 2017 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-28324009

RESUMO

Context: Turner syndrome (TS) is due to a complete or partial loss of an X chromosome in female patients and is not currently part of newborn screening (NBS). Diagnosis is often delayed, resulting in missed crucial diagnostic and therapeutic opportunities. Objectives: This study sought to determine if whole-exome sequencing (WES) as part of a potential NBS program could be used to diagnose TS. Design, Setting, Patients: Karyotype, chromosomal microarray, and WES were performed on blood samples from women with TS (n = 27) enrolled in the Personalized Genomic Research study at the National Institutes of Health. Female control subjects (n = 37) and male subjects (n = 27) also underwent WES. Copy number variation was evaluated using EXCAVATOR2 and B allele frequency was calculated from informative single nucleotide polymorphisms. Simulated WES data were generated for detection of low-level mosaicism and complex structural chromosome abnormalities. Results: We detected monosomy for chromosome X in all 27 TS samples, including 1 mosaic for 45,X/46,XX and another with previously unreported material on chromosome Y. Sensitivity and specificity were both 100% for the diagnosis of TS with no false-positive or false-negative results. Using simulated WES data, we detected isochromosome Xq and low-level mosaicism as low as 5%. Conclusion: We present an accurate method of diagnosing TS using WES, including cases with low-level mosaicism, isochromosome Xq, and cryptic Y-chromosome material. Given the potential use of next-generation sequencing for NBS in many different diseases and syndromes, we propose WES can be used as a screening test for TS in newborns.


Assuntos
Cromossomos Humanos X/genética , Análise de Sequência de DNA/métodos , Síndrome de Turner/diagnóstico , Adulto , Idoso , Estudos de Casos e Controles , Exoma , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Recém-Nascido , Cariotipagem , Masculino , Análise em Microsséries , Pessoa de Meia-Idade , Mosaicismo , Triagem Neonatal , Polimorfismo de Nucleotídeo Único , Sensibilidade e Especificidade , Síndrome de Turner/genética , Adulto Jovem
14.
BMC Med Genet ; 15: 64, 2014 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-24898207

RESUMO

BACKGROUND: Shwachman-Diamond syndrome (SDS) is an autosomal recessive ribosomopathy caused mainly by compound heterozygous mutations in SBDS. Structural variation (SV) involving the SBDS locus has been rarely reported in association with the disease. We aimed to determine whether an SV contributed to the pathogenesis of a case lacking biallelic SBDS point mutations. CASE PRESENTATION: Whole exome sequencing was performed in a patient with SDS lacking biallelic SBDS point mutations. Array comparative genomic hybridization and Southern blotting were used to seek SVs across the SBDS locus. Locus-specific polymerase chain reaction (PCR) encompassing flanking intronic sequence was also performed to investigate mutation within the locus. RNA expression and Western blotting were performed to analyze allele and protein expression. We found the child harbored a single missense mutation in SBDS (c.98A > C; p.K33T), inherited from the mother, and an SV in the SBDS locus, inherited from the father. The missense allele and SV segregated in accordance with Mendelian expectations for autosomal recessive SDS. Complementary DNA and western blotting analysis and locus specific PCR support the contention that the SV perturbed SBDS protein expression in the father and child. CONCLUSION: Our findings implicate genomic rearrangements in the pathogenesis of some cases of SDS and support patients lacking biallelic SBDS point mutations be tested for SV within the SBDS locus.


Assuntos
Doenças da Medula Óssea/genética , Insuficiência Pancreática Exócrina/genética , Lipomatose/genética , Mutação de Sentido Incorreto , Proteínas/genética , Abdome/diagnóstico por imagem , Alelos , Doenças da Medula Óssea/diagnóstico , Linhagem Celular Transformada , Pré-Escolar , Hibridização Genômica Comparativa , Análise Mutacional de DNA , Insuficiência Pancreática Exócrina/diagnóstico , Feminino , Ordem dos Genes , Humanos , Lipomatose/diagnóstico , Mutagênese Insercional , Mutação , Linhagem , Radiografia Abdominal , Síndrome de Shwachman-Diamond , Ultrassonografia
15.
Clin Genet ; 83(5): 457-461, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-22901280

RESUMO

The advent of whole-exome next-generation sequencing (WES) has been pivotal for the molecular characterization of Mendelian disease; however, the clinical applicability of WES has remained relatively unexplored. We describe our exploration of WES as a diagnostic tool in a 3½-year old female patient with a 2-year history of episodic muscle weakness and paroxysmal dystonia who presented following a previous extensive but unrevealing diagnostic work-up. WES was performed on the proband and her two parents. Parental exome data was used to filter potential de novo genomic events in the proband and suspected variants were confirmed using di-deoxy sequencing. WES revealed a de novo non-synonymous mutation in exon 21 of the calcium channel gene CACNA1S that has been previously reported in a single patient as a rare cause of atypical hypokalemic periodic paralysis. This was unexpected, as the proband's original differential diagnosis had included hypokalemic periodic paralysis, but clinical and laboratory features were equivocal, and standard clinical molecular testing for hypokalemic periodic paralysis and related disorders was negative. This report highlights the potential diagnostic utility of WES in clinical practice, with implications for the approach to similar diagnostic dilemmas in the future.


Assuntos
Exoma , Sequenciamento de Nucleotídeos em Larga Escala , Debilidade Muscular/diagnóstico , Debilidade Muscular/genética , Sequência de Bases , Canais de Cálcio/genética , Canais de Cálcio Tipo L , Pré-Escolar , Éxons , Feminino , Genótipo , Humanos , Masculino , Mutação , Canal de Sódio Disparado por Voltagem NAV1.4/genética , Linhagem , Fenótipo
16.
Am J Med Genet A ; 155A(9): 2071-7, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21834044

RESUMO

Polymicrogyria is a disorder of neuronal development resulting in structurally abnormal cerebral hemispheres characterized by over-folding and abnormal lamination of the cerebral cortex. Polymicrogyria is frequently associated with severe neurologic deficits including intellectual disability, motor problems, and epilepsy. There are acquired and genetic causes of polymicrogyria, but most patients with a presumed genetic etiology lack a specific diagnosis. Here we report using whole-exome sequencing to identify compound heterozygous mutations in the WD repeat domain 62 (WDR62) gene as the cause of recurrent polymicrogyria in a sibling pair. Sanger sequencing confirmed that the siblings both inherited 1-bp (maternal allele) and 2-bp (paternal allele) frameshift deletions, which predict premature truncation of WDR62, a protein that has a role in early cortical development. The probands are from a non-consanguineous family of Northern European descent, suggesting that autosomal recessive PMG due to compound heterozygous mutation of WDR62 might be a relatively common cause of PMG in the population. Further studies to identify mutation frequency in the population are needed.


Assuntos
Anormalidades Múltiplas/genética , Exoma , Malformações do Desenvolvimento Cortical/genética , Proteínas do Tecido Nervoso/genética , Adulto , Sequência de Bases , Proteínas de Ciclo Celular , Criança , Anormalidades Craniofaciais/genética , Feminino , Mutação da Fase de Leitura , Testes Genéticos , Heterozigoto , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Imagem por Ressonância Magnética , Masculino , Mutação , Análise de Sequência de DNA , Deleção de Sequência , Irmãos
17.
Sci Transl Med ; 3(87): 87re3, 2011 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-21677200

RESUMO

Whole-genome sequencing of patient DNA can facilitate diagnosis of a disease, but its potential for guiding treatment has been under-realized. We interrogated the complete genome sequences of a 14-year-old fraternal twin pair diagnosed with dopa (3,4-dihydroxyphenylalanine)-responsive dystonia (DRD; Mendelian Inheritance in Man #128230). DRD is a genetically heterogeneous and clinically complex movement disorder that is usually treated with l-dopa, a precursor of the neurotransmitter dopamine. Whole-genome sequencing identified compound heterozygous mutations in the SPR gene encoding sepiapterin reductase. Disruption of SPR causes a decrease in tetrahydrobiopterin, a cofactor required for the hydroxylase enzymes that synthesize the neurotransmitters dopamine and serotonin. Supplementation of l-dopa therapy with 5-hydroxytryptophan, a serotonin precursor, resulted in clinical improvements in both twins.


Assuntos
Distúrbios Distônicos , Genoma Humano , Assistência ao Paciente , Análise de Sequência de DNA , Adolescente , Tomada de Decisões , Distúrbios Distônicos/tratamento farmacológico , Distúrbios Distônicos/genética , Feminino , Humanos , Levodopa/uso terapêutico , Masculino , Linhagem , Resultado do Tratamento , Gêmeos Dizigóticos/genética
18.
J Biomech ; 40(2): 476-80, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-16500663

RESUMO

We use optical tweezers in conjunction with an optical position-sensing system, which spectrally filters signals generated by a trapped fluorescent microsphere to study plasma membrane (PM) mechanics and its interactions with cytoskeleton. We dynamically measure the PM tethering force on human embryonic kidney cells that are a standard cultured cell line. Recorded tethering force vs. PM displacement profiles, revealed the tether formation process, initiated with linear deformation of the PM, followed by a nonlinear regime and terminated with the local separation of PM. Tethering force vs. displacement profiles were used to estimate tether formation force and stiffness parameter of the PM. Integration of the force-displacement profiles yielded the work of tether formation, including linear and nonlinear components. Our results demonstrate that spectral filtering of the optically trapped fluorescent microsphere image formed on the position-sensing system overcomes the artifacts introduced by the transillumination imaging and allows accurate measures of PM mechanics before and during the initial stages of tether formation.


Assuntos
Fenômenos Biomecânicos , Membrana Celular/fisiologia , Citoesqueleto/fisiologia , Diagnóstico por Imagem , Fluorescência , Pinças Ópticas , Linhagem Celular , Humanos
19.
Biophys J ; 89(6): 4090-5, 2005 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-16199506

RESUMO

An optical tweezers system was used to characterize the effects of chlorpromazine (CPZ) on the mechanical properties of the mammalian outer hair cell (OHC) through the formation of plasma membrane tethers. Such tethers exhibited force relaxation when held at a constant length for several minutes. We used a second-order generalized Kelvin body to model tether-force behavior from which several mechanical parameters were then calculated including stiffness, viscosity-associated measures, and force relaxation time constants. The results of the analysis portray a two-part relaxation process characterized by significantly different rates of force decay, which we propose is due to the local reorganization of lipids within the tether and the flow of external lipid into the tether. We found that CPZ's effect was limited to the latter phenomenon since only the second phase of relaxation was significantly affected by the drug. This finding coupled with an observed large reduction in overall tether forces implies a common basis for the drug's effects, the plasma membrane-cytoskeleton interaction. The CPZ-induced changes in tether viscoelastic behavior suggest that alterations in the mechanical properties of the OHC lateral wall could play a role in the modulation of OHC electromotility by CPZ.


Assuntos
Membrana Celular/fisiologia , Células Ciliadas Auditivas Externas/fisiologia , Mecanotransdução Celular/fisiologia , Modelos Biológicos , Animais , Fenômenos Biomecânicos/métodos , Células Cultivadas , Simulação por Computador , Elasticidade , Feminino , Fricção , Cobaias , Masculino , Estresse Mecânico
20.
J Neurophysiol ; 94(3): 2105-10, 2005 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-15958599

RESUMO

High concentrations of the amphipathic drug salicylate (Sal) block outer hair cell (OHC) electromotility resulting in reversible hearing loss. We used optical tweezers to study the effects of Sal on the mechanics of the cell plasma membrane. Membrane tethers were formed from guinea pig OHCs and cultured human embryonic kidney (HEK) cells as controls. HEK cells are commonly used in functional expression studies of electromotility. Effective tether viscosity (eta(eff)), steady-state tethering force extrapolated to zero pulling rate F(ss0), and time constant for tether growth (tau(tg)) were estimated from the measurements of the instantaneous tethering force at different tether pulling rates. Average values of eta(eff), F(ss0), and tau(tg) for the OHC lateral wall plasma membrane and control cell plasma membrane remained the same after Sal perfusion, which is consistent with the hypothesis that Sal-induced reversible hearing loss appears to be more the result of its competition with essential anions and less the result of a change in plasma membrane mechanics.


Assuntos
Membrana Celular/efeitos dos fármacos , Inibidores de Ciclo-Oxigenase/farmacologia , Células Ciliadas Auditivas Externas/citologia , Mecânica , Salicilatos/farmacologia , Animais , Células Cultivadas , Cóclea/citologia , Relação Dose-Resposta a Droga , Feminino , Cobaias , Humanos , Masculino , Estresse Mecânico , Fatores de Tempo
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