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1.
J Med Genet ; 2019 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-31439720

RESUMO

BACKGROUND: Autistic spectrum disorders (ASDs) with developmental delay and seizures are a genetically heterogeneous group of diseases caused by at least 700 different genes. Still, a number of cases remain genetically undiagnosed. OBJECTIVE: The objective of this study was to identify and characterise pathogenic variants in two individuals from unrelated families, both of whom presented a similar clinical phenotype that included an ASD, intellectual disability (ID) and seizures. METHODS: Whole-exome sequencing was used to identify pathogenic variants in the two individuals. Functional studies performed in the Drosophila melanogaster model was used to assess the protein function in vivo. RESULTS: Probands shared a heterozygous de novo secretory carrier membrane protein (SCAMP5) variant (NM_001178111.1:c.538G>T) resulting in a p.Gly180Trp missense variant. SCAMP5 belongs to a family of tetraspanin membrane proteins found in secretory and endocytic compartments of neuronal synapses. In the fly SCAMP orthologue, the p.Gly302Trp genotype corresponds to human p.Gly180Trp. Western blot analysis of proteins overexpressed in the Drosophila fat body showed strongly reduced levels of the SCAMP p.Gly302Trp protein compared with the wild-type protein, indicating that the mutant either reduced expression or increased turnover of the protein. The expression of the fly homologue of the human SCAMP5 p.Gly180Trp mutation caused similar eye and neuronal phenotypes as the expression of SCAMP RNAi, suggesting a dominant-negative effect. CONCLUSION: Our study identifies SCAMP5 deficiency as a cause for ASD and ID and underscores the importance of synaptic vesicular trafficking in neurodevelopmental disorders.

2.
Mol Autism ; 10: 33, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31406558

RESUMO

Background: Neurogenetics investigations and diagnostic yield in patients with autism spectrum disorder (ASD) have significantly improved over the last few years. Yet, many patients still fail to be systematically investigated. Methods: To improve access to services, an ambulatory team has been established since 1998, delivering on-site clinical genetics consultations and gradually upgrading services to 502 children and young adults with ASD in their standard environment across 26 day-care hospitals and specialized institutions within the Greater Paris region. The evaluation included a clinical genetics consultation, screening for fragile X syndrome, metabolic workup, chromosomal microarray analysis, and, in a proportion of patients, next-generation sequencing of genes reported in ASD and other neurodevelopmental disorders. Results: Fragile X syndrome and pathogenic copy number variants (CNVs) accounted for the disease in 10% of cases, including 4/312 (1.3%) with fragile X syndrome and 34/388 (8.8%) with pathogenic CNVs (19 de novo and 4 inherited). Importantly, adding high-throughput resequencing of reported intellectual disability/ASD genes to the screening procedure had a major impact on diagnostic yield in the 141 patients examined most recently. Pathogenic or likely pathogenic sequence variants in 27 disease genes were identified in 33/141 patients (23.4%; 23 were de novo and 10 inherited, including five X-linked and five recessive compound heterozygous variants). Diagnosed cases presented atypical and/or syndromic ASD with moderate to severe intellectual disability. The diagnostic yield of fragile X syndrome and array CGH testing combined with next-generation sequencing was significantly higher than fragile X syndrome and array CGH alone (p value 0.009). No inborn errors of metabolism were detected with the metabolic screening. Conclusion: Based on the diagnostic rate observed in this cohort, we suggest that a stepwise procedure be considered, first screening pathogenic CNVs and a limited number of disease genes in a much larger number of patients, especially those with syndromic ASD and intellectual disability.

3.
Psychiatr Genet ; 29(6): 237-242, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30933046

RESUMO

ARID1B mutations in Coffin-Siris syndrome are a cause of intellectual disability (0.5-1%), with various degrees of autism and agenesis of the corpus callosum (10%). Little is known regarding the cognitive and motor consequences of ARID1B mutations in humans and no link has been made between corpus callosum anomalies and visuospatial and neuromotor dysfunctions. We have investigated the visuospatial and neuromotor phenotype in eight patients with ARID1B mutations. A paramedian sagittal section of the brain MRI was selected, and corpus callosum was measured in anteroposterior length, genu and trunk width. Spearman's rank order coefficients were used to explore correlations between visuospatial and social cognitive variables and dimensions of the corpus callosum. A significant correlation between genu width size and visual cognition was observed. Retrocerebellar cysts were associated with corpus callosum anomalies. Here, we show that corpus callosum anomalies caused in ARID1B mutations may be predictive of the visuospatial and motor phenotype in Coffin-Siris syndrome.

5.
EMBO Mol Med ; 2018 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-30446499

RESUMO

The genetic causes of congenital hypothyroidism due to thyroid dysgenesis (TD) remain largely unknown. We identified three novel TUBB1 gene mutations that co-segregated with TD in three distinct families leading to 1.1% of TUBB1 mutations in TD study cohort. TUBB1 (Tubulin, Beta 1 Class VI) encodes for a member of the ß-tubulin protein family. TUBB1 gene is expressed in the developing and adult thyroid in humans and mice. All three TUBB1 mutations lead to non-functional α/ß-tubulin dimers that cannot be incorporated into microtubules. In mice, Tubb1 knock-out disrupted microtubule integrity by preventing ß1-tubulin incorporation and impaired thyroid migration and thyroid hormone secretion. In addition, TUBB1 mutations caused the formation of macroplatelets and hyperaggregation of human platelets after stimulation by low doses of agonists. Our data highlight unexpected roles for ß1-tubulin in thyroid development and in platelet physiology. Finally, these findings expand the spectrum of the rare paediatric diseases related to mutations in tubulin-coding genes and provide new insights into the genetic background and mechanisms involved in congenital hypothyroidism and thyroid dysgenesis.

6.
Eur J Med Genet ; 61(12): 759-764, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30268909

RESUMO

The advent of next generation sequencing has improved gene discovery in neurodevelopmental disorders. A greater understanding of the genetic basis of these disorders has expanded the spectrum of pathogenic genes, thus enhancing diagnosis and therapeutic management. Genetic overlap between distinct neurodevelopmental disorders has also been revealed, which can make determining a strict genotype-phenotype correlation more difficult. Intellectual disability and cortical malformations are two neurodevelopmental disorders particularly confronted by this difficulty. Indeed, for a given pathogenic gene, intellectual disability can be associated, or not, with cortical malformations. Here, we report for the first time, two individuals with the same de novo mutation in TBR1, leading to a frameshift starting at codon Thr532, and resulting in a premature stop codon 143 amino acids downstream (c.1588_1594dup, p.(Thr532Argfs*144)). These individuals presented with a developmental encephalopathy characterized by frontal pachygyria and severe intellectual disability. Remarkably, 11 TBR1 gene mutations were previously reported in intellectual disability and autism spectrum disorders. Our study supports the observation that TBR1-related disorders range from intellectual disability to frontal pachygyria. We also highlight the need for first-line, good quality neuroimaging for patients with intellectual disability.

8.
Genet Med ; 2018 Sep 12.
Artigo em Inglês | MEDLINE | ID: mdl-30206421

RESUMO

PURPOSE: Variants in IQSEC2, escaping X inactivation, cause X-linked intellectual disability with frequent epilepsy in males and females. We aimed to investigate sex-specific differences. METHODS: We collected the data of 37 unpublished patients (18 males and 19 females) with IQSEC2 pathogenic variants and 5 individuals with variants of unknown significance and reviewed published variants. We compared variant types and phenotypes in males and females and performed an analysis of IQSEC2 isoforms. RESULTS: IQSEC2 pathogenic variants mainly led to premature truncation and were scattered throughout the longest brain-specific isoform, encoding the synaptic IQSEC2/BRAG1 protein. Variants occurred de novo in females but were either de novo (2/3) or inherited (1/3) in males, with missense variants being predominantly inherited. Developmental delay and intellectual disability were overall more severe in males than in females. Likewise, seizures were more frequently observed and intractable, and started earlier in males than in females. No correlation was observed between the age at seizure onset and severity of intellectual disability or resistance to antiepileptic treatments. CONCLUSION: This study provides a comprehensive overview of IQSEC2-related encephalopathy in males and females, and suggests that an accurate dosage of IQSEC2 at the synapse is crucial during normal brain development.

9.
J Med Genet ; 55(6): 378-383, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29358270

RESUMO

BACKGROUND: Because the mitochondrial respiratory chain (RC) is ubiquitous, its deficiency can theoretically give rise to any symptom in any organ or tissue at any age with any mode of inheritance, owing to the twofold genetic origin of respiratory enzyme machinery, that is, nuclear and mitochondrial. Not all respiratory enzyme deficiencies are primary and secondary or artefactual deficiency is frequently observed, leading to a number of misleading conclusions and inappropriate investigations in clinical practice. This study is aimed at investigating the potential role of brain MRI in distinguishing primary RC deficiency from phenocopies and other aetiologies. METHODS: Starting from a large series of 189 patients (median age: 3.5 years (8 days-56 years), 58% males) showing signs of RC enzyme deficiency, for whom both brain MRIs and disease-causing mutations were available, we retrospectively studied the positive predictive value (PPV) and the positive likelihood ratio (LR+) of brain MRI imaging and its ability to discriminate between two groups: primary deficiency of the mitochondrial RC machinery and phenocopies. RESULTS: Detection of (1) brainstem hyperintensity with basal ganglia involvement (P≤0.001) and (2) lactate peak with either brainstem or basal ganglia hyperintensity was highly suggestive of primary RC deficiency (P≤0.01). Fourteen items had a PPV>95% and LR+ was greater than 9 for seven signs. Biallelic SLC19A3 mutations represented the main differential diagnosis. Non-significant differences between the two groups were found for cortical/subcortical atrophy, leucoencephalopathy and involvement of caudate nuclei, spinothalamic tract and corpus callosum. CONCLUSION: Based on these results and owing to invasiveness of skeletal muscle biopsies and cost of high-throughput DNA sequencing, we suggest giving consideration to brain MRI imaging as a diagnostic marker and an informative investigation to be performed in patients showing signs of RC enzyme deficiency.

10.
Liver Int ; 38(2): 358-364, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-28792652

RESUMO

BACKGROUND & AIMS: Obliterative portal venopathy (OPV) is characterized by lesions of portal vein intrahepatic branches and is thought to be responsible for many cases of portal hypertension in the absence of cirrhosis or obstruction of large portal or hepatic veins. In most cases the cause of OPV remains unknown. The aim was to identify a candidate gene of OPV. METHODS: Whole exome sequencing was performed in two families, including 6 patients with OPV. Identified mutations were confirmed by Sanger sequencing and expression of candidate gene transcript was studied by real time qPCR in human tissues. RESULTS: In both families, no mutations were identified in genes previously reported to be associated with OPV. In each family, we identified a heterozygous mutation (c.1783G>A, p.Gly595Arg and c.4895C>T, p.Thr1632Ile) in a novel gene located on chromosome 4, that we called FOPV (Familial Obliterative Portal Venopathy), and having a cDNA coding for 1793 amino acids. The FOPV mutations segregated with the disease in families and the pattern of inheritance was suggestive of autosomal dominant inherited OPV, with incomplete penetrance and variable expressivity. In silico analysis predicted a deleterious effect of each mutant and mutations concerned highly conserved amino acids in mammals. A deleterious heterozygous FOPV missense mutation (c.4244T>C, p.Phe1415Ser) was also identified in a patient with non-familial OPV. Expression study in liver veins showed that FOPV transcript was mainly expressed in intrahepatic portal vein. CONCLUSIONS: This report suggests that FOPV mutations may have a pathogenic role in some cases of familial and non-familial OPV.

12.
Am J Hum Genet ; 101(6): 995-1005, 2017 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-29198722

RESUMO

A recurrent de novo missense variant within the C-terminal Sin3-like domain of ZSWIM6 was previously reported to cause acromelic frontonasal dysostosis (AFND), an autosomal-dominant severe frontonasal and limb malformation syndrome, associated with neurocognitive and motor delay, via a proposed gain-of-function effect. We present detailed phenotypic information on seven unrelated individuals with a recurrent de novo nonsense variant (c.2737C>T [p.Arg913Ter]) in the penultimate exon of ZSWIM6 who have severe-profound intellectual disability and additional central and peripheral nervous system symptoms but an absence of frontonasal or limb malformations. We show that the c.2737C>T variant does not trigger nonsense-mediated decay of the ZSWIM6 mRNA in affected individual-derived cells. This finding supports the existence of a truncated ZSWIM6 protein lacking the Sin3-like domain, which could have a dominant-negative effect. This study builds support for a key role for ZSWIM6 in neuronal development and function, in addition to its putative roles in limb and craniofacial development, and provides a striking example of different variants in the same gene leading to distinct phenotypes.


Assuntos
Proteínas de Ligação a DNA/genética , Deficiência Intelectual/genética , Transtornos Neurocognitivos/genética , Sistema Nervoso Central/anormalidades , Sistema Nervoso Central/embriologia , Códon sem Sentido/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Deformidades Congênitas dos Membros/genética , Disostose Mandibulofacial/genética , Sistema Nervoso Periférico/anormalidades , Sistema Nervoso Periférico/enzimologia
13.
Hum Mutat ; 38(8): 932-941, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28585349

RESUMO

De novo mutations are a frequent cause of disorders related to brain development. We report the results from the screening of two patients diagnosed with intellectual disability (ID) using exome sequencing to identify new causative de novo mutations. Exome sequencing was conducted in two patient-parent trios to identify de novo variants. In silico and expression studies were also performed to evaluate the functional consequences of these variants. The two patients presented developmental delay with minor facial dysmorphy. One of them presented pharmacoresistant myoclonic epilepsy. We identified two de novo splice variants (c.175+2T>G; c.367+2T>C) in the CSNK2B gene encoding the ß subunit of the Caseine kinase 2 (CK2). CK2 is a ubiquitously expressed kinase that is present in high levels in brain and it appears to be constitutively active. The mRNA transcripts were abnormal and significantly reduced in affected fibroblasts and most likely produced truncated proteins. Taking into account that mutations in CSNK2A1, encoding the α subunit of CK2, were previously identified in patients with neurodevelopmental disorders and dysmorphic features, our study confirmed that the protein kinase CK2 plays a major role in brain, and showed that CSNK2, encoding the ß subunit, is a novel ID gene. This study adds knowledge to the increasingly growing list of causative and candidate genes in ID and epilepsy, and highlights CSNK2B as a new gene for neurodevelopmental disorders.


Assuntos
Caseína Quinase II/genética , Deficiências do Desenvolvimento/genética , Epilepsias Mioclônicas/genética , Deficiência Intelectual/genética , Caseína Quinase II/metabolismo , Pré-Escolar , Hibridização Genômica Comparativa , Exoma/genética , Feminino , Humanos , Lactente , Masculino , Mutação/genética , Transtornos do Neurodesenvolvimento/genética , Sequenciamento Completo do Exoma/métodos
14.
Am J Hum Genet ; 100(4): 592-604, 2017 Apr 06.
Artigo em Inglês | MEDLINE | ID: mdl-28285769

RESUMO

Pre-mRNA splicing factors play a fundamental role in regulating transcript diversity both temporally and spatially. Genetic defects in several spliceosome components have been linked to a set of non-overlapping spliceosomopathy phenotypes in humans, among which skeletal developmental defects and non-syndromic retinitis pigmentosa (RP) are frequent findings. Here we report that defects in spliceosome-associated protein CWC27 are associated with a spectrum of disease phenotypes ranging from isolated RP to severe syndromic forms. By whole-exome sequencing, recessive protein-truncating mutations in CWC27 were found in seven unrelated families that show a range of clinical phenotypes, including retinal degeneration, brachydactyly, craniofacial abnormalities, short stature, and neurological defects. Remarkably, variable expressivity of the human phenotype can be recapitulated in Cwc27 mutant mouse models, with significant embryonic lethality and severe phenotypes in the complete knockout mice while mice with a partial loss-of-function allele mimic the isolated retinal degeneration phenotype. Our study describes a retinal dystrophy-related phenotype spectrum as well as its genetic etiology and highlights the complexity of the spliceosomal gene network.


Assuntos
Anormalidades Múltiplas/genética , Ciclofilinas/genética , Mutação , Peptidilprolil Isomerase/genética , Degeneração Retiniana/genética , Adolescente , Animais , Criança , Pré-Escolar , Ciclofilinas/metabolismo , Feminino , Humanos , Masculino , Camundongos , Linhagem , Peptidilprolil Isomerase/metabolismo , Adulto Jovem
15.
Neurogenetics ; 18(1): 23-28, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27726050

RESUMO

Mutations in ATP1A3 are involved in a large spectrum of neurological disorders, including rapid onset dystonia parkinsonism (RDP), alternating hemiplegia of childhood (AHC), and cerebellar ataxia, pes cavus, optic atrophy, and sensorineural hearing loss (CAPOS), with recent descriptions of overlapping phenotypes. In AHC, a few familial cases of autosomal dominant inheritance have been reported, along with cases of de novo sporadic mutations. In contrast, autosomal dominant inheritance has frequently been associated with RDP and CAPOS. Here, we report on two unrelated sets of full siblings with ATP1A3 mutations, (c.2116G>A) p. Gly706Arg in the first family, and (c.2266C>T) p. Arg756Cys in the second family, presenting with familial recurrence of the disease. Both families displayed parental germline mosaicism. In the first family, the brother and sister presented with severe intellectual deficiency, early onset pharmacoresistant epilepsy, ataxia, and autistic features. In the second family, both sisters demonstrated severe encephalopathy with ataxia and dystonia following a regression episode during a febrile episode during infancy. To our knowledge, mosaicism has not previously been reported in ATP1A3-related disorders. This report, therefore, provides evidence that germline mosaicism for ATP1A3 mutations is a likely explanation for familial recurrence and should be considered during recurrence risk counseling for families of children with ATP1A3-related disorders.


Assuntos
Transtornos Cognitivos/genética , Distonia/genética , Mosaicismo , Mutação de Sentido Incorreto , Convulsões/genética , ATPase Trocadora de Sódio-Potássio/genética , Transtorno Autístico/complicações , Transtorno Autístico/genética , Transtorno Autístico/patologia , Criança , Transtornos Cognitivos/complicações , Transtornos Cognitivos/patologia , Distonia/complicações , Distonia/patologia , Feminino , Mutação em Linhagem Germinativa , Humanos , Masculino , Linhagem , Fenótipo , Recidiva , Convulsões/complicações , Convulsões/patologia , Irmãos , Estrabismo/complicações , Estrabismo/genética , Estrabismo/patologia
16.
Hum Mol Genet ; 26(3): 599-610, 2017 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-28025328

RESUMO

Congenital hypothyroidism is the most common neonatal endocrine disorder and is primarily caused by developmental abnormalities otherwise known as thyroid dysgenesis (TD). We performed whole exome sequencing (WES) in a consanguineous family with TD and subsequently sequenced a cohort of 134 probands with TD to identify genetic factors predisposing to the disease. We identified the novel missense mutations p.S148F, p.R114Q and p.L177W in the BOREALIN gene in TD-affected families. Borealin is a major component of the Chromosomal Passenger Complex (CPC) with well-known functions in mitosis. Further analysis of the missense mutations showed no apparent effects on mitosis. In contrast, expression of the mutants in human thyrocytes resulted in defects in adhesion and migration with corresponding changes in gene expression suggesting others functions for this mitotic protein. These results were well correlated with the same gene expression pattern analysed in the thyroid tissue of the patient with BOREALIN-p.R114W. These studies open new avenues in the genetics of TD in humans.


Assuntos
Proteínas de Ciclo Celular/genética , Predisposição Genética para Doença , Mutação de Sentido Incorreto/genética , Disgenesia da Tireoide/genética , Proteínas de Ciclo Celular/biossíntese , Movimento Celular/genética , Exoma/genética , Feminino , Regulação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Mitose/genética , Linhagem , Disgenesia da Tireoide/patologia
18.
J Med Genet ; 51(12): 834-8, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25351951

RESUMO

BACKGROUND: Inherited optic neuropathy has been ascribed to mutations in mitochondrial fusion/fission dynamics genes, nuclear and mitochondrial DNA-encoded respiratory enzyme genes or nuclear genes of poorly known mitochondrial function. However, the disease causing gene remains unknown in many families. METHODS: We used exome sequencing in order to identify the gene responsible for isolated or syndromic optic atrophy in five patients from three independent families. RESULTS: We found homozygous or compound heterozygous missense and frameshift mutations in the gene encoding mitochondrial aconitase (ACO2), a tricarboxylic acid cycle enzyme, catalysing interconversion of citrate into isocitrate. Unlike wild type ACO2, all mutant ACO2 proteins failed to complement the respiratory growth of a yeast aco1-deletion strain. Retrospective studies using patient-derived cultured skin fibroblasts revealed various degrees of deficiency in ACO2 activity, but also in ACO1 cytosolic activity. CONCLUSIONS: Our study shows that autosomal recessive ACO2 mutations can cause either isolated or syndromic optic neuropathy. This observation identifies ACO2 as the second gene responsible for non-syndromic autosomal recessive optic neuropathies and provides evidence for a genetic overlap between isolated and syndromic forms, giving further support to the view that optic atrophy is a hallmark of defective mitochondrial energy supply.


Assuntos
Aconitato Hidratase/genética , Mutação , Doenças do Nervo Óptico/genética , Aconitato Hidratase/metabolismo , Adulto , Encéfalo/patologia , Pré-Escolar , Ciclo do Ácido Cítrico , Ativação Enzimática , Exoma , Evolução Fatal , Feminino , Expressão Gênica , Genes Recessivos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Imagem por Ressonância Magnética , Masculino , Oftalmoscópios , Atrofia Óptica/diagnóstico , Atrofia Óptica/genética , Doenças do Nervo Óptico/diagnóstico , Doenças do Nervo Óptico/metabolismo , Avaliação de Resultados da Assistência ao Paciente , Irmãos
19.
Orphanet J Rare Dis ; 8: 187, 2013 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-24289864

RESUMO

Despite it has been reported that several loci are involved in Hirschsprung's disease, the molecular basis of the disease remains yet essentially unknown. The study of collective properties of modules of functionally-related genes provides an efficient and sensitive statistical framework that can overcome sample size limitations in the study of rare diseases. Here, we present the extension of a previous study of a Spanish series of HSCR trios to an international cohort of 162 HSCR trios to validate the generality of the underlying functional basis of the Hirschsprung's disease mechanisms previously found. The Pathway-Based Analysis (PBA) confirms a strong association of gene ontology (GO) modules related to signal transduction and its regulation, enteric nervous system (ENS) formation and other processes related to the disease. In addition, network analysis recovers sub-networks significantly associated to the disease, which contain genes related to the same functionalities, thus providing an independent validation of these findings. The functional profiles of association obtained for patients populations from different countries were compared to each other. While gene associations were different at each series, the main functional associations were identical in all the five populations. These observations would also explain the reported low reproducibility of associations of individual disease genes across populations.


Assuntos
Doença de Hirschsprung/metabolismo , Feminino , Predisposição Genética para Doença , Genótipo , Doença de Hirschsprung/genética , Humanos , Masculino , Polimorfismo de Nucleotídeo Único/genética
20.
BMC Genomics ; 10: 246, 2009 May 26.
Artigo em Inglês | MEDLINE | ID: mdl-19470167

RESUMO

BACKGROUND: For more than a decade, microarrays have been a powerful and widely used tool to explore the transcriptome of biological systems. However, the amount of biological material from cell sorting or laser capture microdissection is much too small to perform microarray studies. To address this issue, RNA amplification methods have been developed to generate sufficient targets from picogram amounts of total RNA to perform microarray hybridisation. RESULTS: In this study, four commercial protocols for amplification of picograms amounts of input RNA for microarray expression profiling were evaluated and compared. The quantitative and qualitative performances of the methods were assessed. Microarrays were hybridised with the amplified targets and the amplification protocols were compared with respect to the quality of expression profiles, reproducibility within a concentration range of input RNA, and sensitivity. The results demonstrate significant differences between these four methods. CONCLUSION: In our hands, the WT-Ovation pico system proposed by Nugen appears to be the most suitable for RNA amplification. This comparative study will be useful to scientists needing to choose an amplification method to carry out microarray experiments involving samples comprising only a few cells and generating picogram amounts of RNA.


Assuntos
Perfilação da Expressão Gênica , Técnicas de Amplificação de Ácido Nucleico/métodos , RNA/isolamento & purificação , Linhagem Celular , Humanos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
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