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1.
Genet Med ; 2021 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-33442021

RESUMO

PURPOSE: A COVID-19 pandemic business continuity plan (BCP) was rapidly developed to protect the Victorian newborn screening (NBS) program. Here, we present the outcomes of our COVID-19 BCP and its impact on the Victorian NBS laboratory service. METHODS: Change management principles were used to develop a BCP that included mapping of NBS processes against staff resources, triaging priorities, technology solutions, supply chain continuity, gap analysis, and supporting maternity service providers. The effect was assessed quantitatively by review of key performance indicator data and qualitatively from staff feedback. RESULTS: A four-stage BCP was implemented. Stage 1 split teams into two, which rotated weekly, onsite (laboratory) and offsite (home). At 20 weeks post-implementation the BCP only progressed to stage 1 and the overall turnaround time was maintained. Staff experience indicated benefits from the review of workflow but noted some social impact associated with the change. CONCLUSION: The preparedness and agility of implementation was based on our focus on the newborn babies and their families, our production system, and a continuous improvement mindset. Both our people and technology infrastructure processes are crucial to this success as we continue to adapt to new challenges.

2.
Nat Commun ; 11(1): 6371, 2020 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-33311470

RESUMO

Genetic mutations predispose the serine protease inhibitor α1-antitrypsin to misfolding and polymerisation within hepatocytes, causing liver disease and chronic obstructive pulmonary disease. This misfolding occurs via a transiently populated intermediate state, but our structural understanding of this process is limited by the instability of recombinant α1-antitrypsin variants in solution. Here we apply NMR spectroscopy to patient-derived samples of α1-antitrypsin at natural isotopic abundance to investigate the consequences of disease-causing mutations, and observe widespread chemical shift perturbations for methyl groups in Z AAT (E342K). By comparison with perturbations induced by binding of a small-molecule inhibitor of misfolding we conclude that they arise from rapid exchange between the native conformation and a well-populated intermediate state. The observation that this intermediate is stabilised by inhibitor binding suggests a paradoxical approach to the targeted treatment of protein misfolding disorders, wherein the stabilisation of disease-associated states provides selectivity while inhibiting further transitions along misfolding pathways.


Assuntos
Espectroscopia de Ressonância Magnética/métodos , Deficiência de alfa 1-Antitripsina/genética , Deficiência de alfa 1-Antitripsina/metabolismo , alfa 1-Antitripsina/química , alfa 1-Antitripsina/genética , alfa 1-Antitripsina/metabolismo , Predisposição Genética para Doença/genética , Glicoproteínas , Humanos , Modelos Moleculares , Medicina Molecular , Mutação , Agregação Patológica de Proteínas , Conformação Proteica , Proteínas Recombinantes , Inibidores de Serino Proteinase/química
3.
Hum Mutat ; 2020 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-33169436

RESUMO

The diagnosis of Mendelian disorders following uninformative exome and genome sequencing remains a challenging and often unmet need. Following uninformative exome and genome sequencing of a family quartet including two siblings with suspected mitochondrial disorder, RNA sequencing (RNAseq) was pursued in one sibling. Long-read amplicon sequencing was used to determine and quantify transcript structure. Immunoblotting studies and quantitative proteomics were performed to demonstrate functional impact. Differential expression analysis of RNAseq data identified significantly decreased expression of the mitochondrial OXPHOS Complex I subunit NDUFB10 associated with a cryptic exon in intron 1 of NDUFB10, that included an in-frame stop codon. The cryptic exon contained a rare intronic variant that was homozygous in both affected siblings. Immunoblot and quantitative proteomic analysis of fibroblasts revealed decreased abundance of Complex I subunits, providing evidence of isolated Complex I deficiency. Through multiomic analysis we present data implicating a deep intronic variant in NDUFB10 as the cause of mitochondrial disease in two individuals, providing further support of the gene-disease association. This study highlights the importance of transcriptomic and proteomic analyses as complementary diagnostic tools in patients undergoing genome-wide diagnostic evaluation.

4.
Amyloid ; : 1-10, 2020 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-33026249

RESUMO

Accumulation of ubiquitin-positive, tau- and α-synuclein-negative intracellular inclusions of TDP-43 in the central nervous system represents the major hallmark correlated to amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U). Such inclusions have variably been described as amorphous aggregates or more structured deposits having amyloid properties. Here we have purified full-length TDP-43 (FL TDP-43) and its C-terminal domain (Ct TDP-43) to investigate the morphological, structural and tinctorial features of aggregates formed in vitro by them at pH 7.4 and 37 °C. AFM images indicate that both protein variants show a tendency to form filaments. Moreover, we show that both FL TDP-43 and Ct TDP-43 filaments possess a largely disordered secondary structure, as ascertained by far-UV circular dichroism and Fourier transform infra-red spectroscopy, do not bind Congo red and induce a very weak increase of thioflavin T fluorescence, indicating the absence of a clear amyloid-like signature.

6.
Hum Mutat ; 41(11): 1884-1891, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32906196

RESUMO

Rapid genomic diagnosis programs are transforming rare disease diagnosis in acute pediatrics. A ventilated newborn with cerebellar hypoplasia underwent rapid exome sequencing (75 h), identifying a novel homozygous ASNS splice-site variant (NM_133436.3:c.1476+1G>A) of uncertain significance. Rapid ASNS splicing studies using blood-derived messenger RNA from the family trio confirmed a consistent pattern of abnormal splicing induced by the variant (cryptic 5' splice-site or exon 12 skipping) with absence of normal ASNS splicing in the proband. Splicing studies reported within 10 days led to reclassification of c.1476+1G>A as pathogenic at age 27 days. Intensive care was redirected toward palliation. Cost analyses for the neonate and his undiagnosed, similarly affected deceased sibling, demonstrate that early diagnosis reduced hospitalization costs by AU$100,828. We highlight the diagnostic benefits of adjunct RNA testing to confirm the pathogenicity of splicing variants identified via rapid genomic testing pipelines for precision and preventative medicine.

7.
Genet Med ; 2020 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-32839572

RESUMO

PURPOSE: To estimate the value of genomic sequencing for complex pediatric neurological disorders of suspected genetic origin. METHODS: A discrete choice experiment (DCE) was undertaken to elicit societal preferences and values. A Bayesian D-efficient and explicit partial profile design was used. The design included 72 choice tasks, split across six blocks, with eight attributes (three overlapping per choice task) and three alternatives. Choice data were analyzed using a panel error component mixed logit model and a latent class model. Preference heterogeneity according to personal socioeconomic, demographic, and attitudinal characteristics was explored using linear and fractional logistic regressions. RESULTS: In total, 820 members of the Australian public were recruited. Statistically significant preferences were identified across all eight DCE attributes. We estimated that society on average would be willing to pay AU$5650 more (95% confidence interval [CI]: AU$5500 to $5800) (US$3955 [95% CI: US$3850 to $4060]) for genomic sequencing relative to standard care. Preference heterogeneity was identified for some personal characteristics. CONCLUSION: On average, society highly values all diagnostic, process, clinical, and nonclinical components of personal utility. To ensure fair prioritization of genomics, decision makers need to consider the wide range of risks and benefits associated with genomic information.

8.
Methods Mol Biol ; 2141: 477-504, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32696373

RESUMO

Interactions of intrinsically disordered proteins are central to their cellular functions, and solution-state NMR spectroscopy provides a powerful tool for characterizing both structural and mechanistic aspects of such interactions. Here we focus on the analysis of IDP interactions using NMR titration measurements. Changes in resonance lineshapes in two-dimensional NMR spectra upon titration with a ligand contain rich information on structural changes in the protein and the thermodynamics and kinetics of the interaction, as well as on the microscopic association mechanism. Here we present protocols for the optimal design of titration experiments, data acquisition, and data analysis by two-dimensional lineshape fitting using the TITAN software package.

9.
Hum Mutat ; 2020 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-32652677

RESUMO

Defects in the motor domain of kinesin family member 1A (KIF1A), a neuron-specific ATP-dependent anterograde axonal transporter of synaptic cargo, are well-recognized to cause a spectrum of neurological conditions, commonly known as KIF1A-associated neurological disorders (KAND). Here, we report one mutation-negative female with classic Rett syndrome (RTT) harboring a de novo heterozygous novel variant [NP_001230937.1:p.(Asp248Glu)] in the highly conserved motor domain of KIF1A. In addition, three individuals with severe neurodevelopmental disorder along with clinical features overlapping with KAND are also reported carrying de novo heterozygous novel [NP_001230937.1:p.(Cys92Arg) and p.(Pro305Leu)] or previously reported [NP_001230937.1:p.(Thr99Met)] variants in KIF1A. In silico tools predicted these variants to be likely pathogenic, and 3D molecular modeling predicted defective ATP hydrolysis and/or microtubule binding. Using the neurite tip accumulation assay, we demonstrated that all novel KIF1A variants significantly reduced the ability of the motor domain of KIF1A to accumulate along the neurite lengths of differentiated SH-SY5Y cells. In vitro microtubule gliding assays showed significantly reduced velocities for the variant p.(Asp248Glu) and reduced microtubule binding for the p.(Cys92Arg) and p.(Pro305Leu) variants, suggesting a decreased ability of KIF1A to move along microtubules. Thus, this study further expanded the phenotypic characteristics of KAND individuals with pathogenic variants in the KIF1A motor domain to include clinical features commonly seen in RTT individuals.

10.
Am J Hum Genet ; 107(2): 234-250, 2020 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-32668217

RESUMO

Phenylketonuria (PKU), caused by variants in the phenylalanine hydroxylase (PAH) gene, is the most common autosomal-recessive Mendelian phenotype of amino acid metabolism. We estimated that globally 0.45 million individuals have PKU, with global prevalence 1:23,930 live births (range 1:4,500 [Italy]-1:125,000 [Japan]). Comparing genotypes and metabolic phenotypes from 16,092 affected subjects revealed differences in disease severity in 51 countries from 17 world regions, with the global phenotype distribution of 62% classic PKU, 22% mild PKU, and 16% mild hyperphenylalaninemia. A gradient in genotype and phenotype distribution exists across Europe, from classic PKU in the east to mild PKU in the southwest and mild hyperphenylalaninemia in the south. The c.1241A>G (p.Tyr414Cys)-associated genotype can be traced from Northern to Western Europe, from Sweden via Norway, to Denmark, to the Netherlands. The frequency of classic PKU increases from Europe (56%) via Middle East (71%) to Australia (80%). Of 758 PAH variants, c.1222C>T (p.Arg408Trp) (22.2%), c.1066-11G>A (IVS10-11G>A) (6.4%), and c.782G>A (p.Arg261Gln) (5.5%) were most common and responsible for two prevalent genotypes: p.[Arg408Trp];[Arg408Trp] (11.4%) and c.[1066-11G>A];[1066-11G>A] (2.6%). Most genotypes (73%) were compound heterozygous, 27% were homozygous, and 55% of 3,659 different genotypes occurred in only a single individual. PAH variants were scored using an allelic phenotype value and correlated with pre-treatment blood phenylalanine concentrations (n = 6,115) and tetrahydrobiopterin loading test results (n = 4,381), enabling prediction of both a genotype-based phenotype (88%) and tetrahydrobiopterin responsiveness (83%). This study shows that large genotype databases enable accurate phenotype prediction, allowing appropriate targeting of therapies to optimize clinical outcome.


Assuntos
Predisposição Genética para Doença/genética , Fenilcetonúrias/epidemiologia , Fenilcetonúrias/genética , Alelos , Biopterina/análogos & derivados , Biopterina/genética , Europa (Continente) , Frequência do Gene/genética , Estudos de Associação Genética/métodos , Genótipo , Homozigoto , Humanos , Mutação/genética , Fenótipo , Fenilalanina/sangue , Fenilalanina Hidroxilase/genética , Fenilcetonúrias/sangue
11.
J Inherit Metab Dis ; 43(6): 1333-1348, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32681751

RESUMO

Asparagine-linked glycosylation 13 homolog (ALG13) encodes a nonredundant, highly conserved, X-linked uridine diphosphate (UDP)-N-acetylglucosaminyltransferase required for the synthesis of lipid linked oligosaccharide precursor and proper N-linked glycosylation. De novo variants in ALG13 underlie a form of early infantile epileptic encephalopathy known as EIEE36, but given its essential role in glycosylation, it is also considered a congenital disorder of glycosylation (CDG), ALG13-CDG. Twenty-four previously reported ALG13-CDG cases had de novo variants, but surprisingly, unlike most forms of CDG, ALG13-CDG did not show the anticipated glycosylation defects, typically detected by altered transferrin glycosylation. Structural homology modeling of two recurrent de novo variants, p.A81T and p.N107S, suggests both are likely to impact the function of ALG13. Using a corresponding ALG13-deficient yeast strain, we show that expressing yeast ALG13 with either of the highly conserved hotspot variants rescues the observed growth defect, but not its glycosylation abnormality. We present molecular and clinical data on 29 previously unreported individuals with de novo variants in ALG13. This more than doubles the number of known cases. A key finding is that a vast majority of the individuals presents with West syndrome, a feature shared with other CDG types. Among these, the initial epileptic spasms best responded to adrenocorticotropic hormone or prednisolone, while clobazam and felbamate showed promise for continued epilepsy treatment. A ketogenic diet seems to play an important role in the treatment of these individuals.

12.
J Exp Med ; 217(10)2020 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-32639540

RESUMO

The exocyst, an octameric protein complex, is an essential component of the membrane transport machinery required for tethering and fusion of vesicles at the plasma membrane. We report pathogenic variants in an exocyst subunit, EXOC2 (Sec5). Affected individuals have severe developmental delay, dysmorphism, and brain abnormalities; variability associated with epilepsy; and poor motor skills. Family 1 had two offspring with a homozygous truncating variant in EXOC2 that leads to nonsense-mediated decay of EXOC2 transcript, a severe reduction in exocytosis and vesicle fusion, and undetectable levels of EXOC2 protein. The patient from Family 2 had a milder clinical phenotype and reduced exocytosis. Cells from both patients showed defective Arl13b localization to the primary cilium. The discovery of mutations that partially disable exocyst function provides valuable insight into this essential protein complex in neural development. Since EXOC2 and other exocyst complex subunits are critical to neuronal function, our findings suggest that EXOC2 variants are the cause of the patients' neurological disorders.

13.
J Vis Exp ; (159)2020 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-32510486

RESUMO

The ECLIA is a versatile method which is able to quantify endogenous and recombinant protein amounts in a 96-well format. To demonstrate ECLIA efficiency, this assay was used to analyze intrinsic levels of MeCP2 in mouse brain tissue and the uptake of TAT-MeCP2 in human dermal fibroblasts. The MeCP2-ECLIA produces highly accurate and reproducible measurements with low intra- and inter-assay error. In summary, we developed a quantitative method for the evaluation of MeCP2 protein variants that can be utilized in high-throughput screens.


Assuntos
Encéfalo/metabolismo , Medições Luminescentes , Proteína 2 de Ligação a Metil-CpG/metabolismo , Animais , Eletroquímica , Fibroblastos/metabolismo , Proteína 2 de Ligação a Metil-CpG/genética , Camundongos , Transporte Proteico
14.
Annu Rev Biochem ; 89: 389-415, 2020 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-32569518

RESUMO

Folding of polypeptides begins during their synthesis on ribosomes. This process has evolved as a means for the cell to maintain proteostasis, by mitigating the risk of protein misfolding and aggregation. The capacity to now depict this cellular feat at increasingly higher resolution is providing insight into the mechanistic determinants that promote successful folding. Emerging from these studies is the intimate interplay between protein translation and folding, and within this the ribosome particle is the key player. Its unique structural properties provide a specialized scaffold against which nascent polypeptides can begin to form structure in a highly coordinated, co-translational manner. Here, we examine how, as a macromolecular machine, the ribosome modulates the intrinsic dynamic properties of emerging nascent polypeptide chains and guides them toward their biologically active structures.

15.
Am J Med Genet A ; 182(7): 1576-1591, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32500973

RESUMO

More than 50 individuals with activating variants in the receptor tyrosine kinase PDGFRB have been reported, separated based on clinical features into solitary myofibromas, infantile myofibromatosis, Penttinen syndrome with premature aging and osteopenia, Kosaki overgrowth syndrome, and fusiform aneurysms. Despite their descriptions as distinct clinical entities, review of previous reports demonstrates substantial phenotypic overlap. We present a case series of 12 patients with activating variants in PDGFRB and review of the literature. We describe five patients with PDGFRB activating variants whose clinical features overlap multiple diagnostic entities. Seven additional patients from a large family had variable expressivity and late-onset disease, including adult onset features and two individuals with sudden death. Three patients were treated with imatinib and had robust and rapid response, including the first two reported infants with multicentric myofibromas treated with imatinib monotherapy and one with a recurrent p.Val665Ala (Penttinen) variant. Along with previously reported individuals, our cohort suggests infants and young children had few abnormal features, while older individuals had multiple additional features, several of which appeared to worsen with advancing age. Our analysis supports a diagnostic entity of a spectrum disorders due to activating variants in PDGFRB. Differences in reported phenotypes can be dramatic and correlate with advancing age, genotype, and to mosaicism in some individuals.

16.
Am J Hum Genet ; 106(6): 779-792, 2020 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-32413283

RESUMO

The evolutionarily conserved hedgehog (Hh) pathway is essential for organogenesis and plays critical roles in postnatal tissue maintenance and renewal. A unique feature of the vertebrate Hh pathway is that signal transduction requires the primary cilium (PC) where major pathway components are dynamically enriched. These factors include smoothened (SMO) and patched, which constitute the core reception system for sonic hedgehog (SHH) as well as GLI transcription factors, the key mediators of the pathway. Here, we report bi-allelic loss-of-function variations in SMO in seven individuals from five independent families; these variations cause a wide phenotypic spectrum of developmental anomalies affecting the brain (hypothalamic hamartoma and microcephaly), heart (atrioventricular septal defect), skeleton (postaxial polydactyly, narrow chest, and shortening of long bones), and enteric nervous system (aganglionosis). Cells derived from affected individuals showed normal ciliogenesis but severely altered Hh-signal transduction as a result of either altered PC trafficking or abnormal activation of the pathway downstream of SMO. In addition, Hh-independent GLI2 accumulation at the PC tip in cells from the affected individuals suggests a potential function of SMO in regulating basal ciliary trafficking of GLI2 when the pathway is off. Thus, loss of SMO function results in abnormal PC dynamics of key components of the Hh signaling pathway and leads to a large continuum of malformations in humans.


Assuntos
Alelos , Deficiências do Desenvolvimento/genética , Proteínas Hedgehog/metabolismo , Transdução de Sinais , Receptor Smoothened/genética , Sequência de Bases , Criança , Pré-Escolar , Cílios/fisiologia , Feminino , Humanos , Lactente , Masculino , Modelos Moleculares , Neoplasias/genética , Proteínas do Tecido Nervoso , Proteínas Nucleares , Linhagem , Proteína Gli2 com Dedos de Zinco , Proteína Gli3 com Dedos de Zinco
17.
Mov Disord ; 35(9): 1675-1679, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32407596

RESUMO

BACKGROUND: Spinocerebellar ataxias are often caused by expansions of short tandem repeats. Recent methodological advances have made repeat expansion (RE) detection with whole-genome sequencing (WGS) feasible. OBJECTIVES: The objective of this study was to determine the genetic basis of ataxia in a multigenerational Australian pedigree with autosomal-dominant inheritance. METHODS AND RESULTS: WGS was performed on 3 affected relatives. The sequence data were screened for known pathogenic REs using 2 RE detection tools: exSTRa and ExpansionHunter. This screen provided a clear and rapid diagnosis (<5 days from receiving the sequencing data) of spinocerebellar ataxia 36, a rare form of ataxia caused by an intronic GGCCTG RE in NOP56. CONCLUSIONS: The diagnosis of rare ataxias caused by REs is highly feasible and cost-effective with WGS. We propose that WGS could potentially be implemented as the frontline, cost-effective methodology for the molecular testing of individuals with a clinical diagnosis of ataxia. © 2020 International Parkinson and Movement Disorder Society.

18.
Genet Med ; 22(8): 1311-1319, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32371919

RESUMO

PURPOSE: To estimate the personal utility and uptake of genomic sequencing (GS) across pediatric and adult-onset genetic conditions. METHODS: Three discrete choice experiment (DCE) surveys were designed and administered to separate representative samples of the Australian public. Bayesian D-efficient explicit partial profile designs were used. Choice data were analyzed using a panel error component random parameter logit model. RESULTS: Overall, 1913 participants completed the pediatric (n = 533), symptomatic adult (n = 700) and at-risk adult (n = 680) surveys. The willingness-to-pay for GS information in pediatric conditions was estimated at $5470-$15,250 (US$3830-$10,675) depending on the benefits of genomic information. Uptake ranged between 60% and 81%. For symptomatic adults, the value of GS was estimated at $1573-$8102 (US$1100-$5671) and uptake at 34-82%. For at-risk adults, GS was valued at $2036-$5004 (US$1425-$3503) and uptake was predicted at 35-61%. CONCLUSION: There is substantial personal utility in GS, particularly for pediatric conditions. Personal utility increased as the perceived benefits of genomic information increased. The clinical and regulatory context, and individuals' sociodemographic and attitudinal characteristics influenced the value and uptake of GS. Society values highly the diagnostic, clinical, and nonclinical benefits of GS. The personal utility of GS should be considered in health-care decision-making.

20.
Hum Mutat ; 41(8): 1425-1434, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32442335

RESUMO

LARS2 variants are associated with Perrault syndrome, characterized by premature ovarian failure and hearing loss, and with an infantile lethal multisystem disorder: Hydrops, lactic acidosis, sideroblastic anemia (HLASA) in one individual. Recently we reported LARS2 deafness with (ovario) leukodystrophy. Here we describe five patients with a range of phenotypes, in whom we identified biallelic LARS2 variants: three patients with a HLASA-like phenotype, an individual with Perrault syndrome whose affected siblings also had leukodystrophy, and an individual with a reversible mitochondrial myopathy, lactic acidosis, and developmental delay. Three HLASA cases from two unrelated families were identified. All were males with genital anomalies. Two survived multisystem disease in the neonatal period; both have developmental delay and hearing loss. A 55-year old male with deafness has not displayed neurological symptoms while his female siblings with Perrault syndrome developed leukodystrophy and died in their 30s. Analysis of muscle from a child with a reversible myopathy showed reduced LARS2 and mitochondrial complex I levels, and an unusual form of degeneration. Analysis of recombinant LARS2 variant proteins showed they had reduced aminoacylation efficiency, with HLASA-associated variants having the most severe effect. A broad phenotypic spectrum should be considered in association with LARS2 variants.

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