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1.
Parasit Vectors ; 13(1): 276, 2020 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-32487217

RESUMO

BACKGROUND: In eco-epidemiological studies, Leishmania detection in vectors and reservoirs is frequently accomplished by high-throughput and sensitive molecular methods that target minicircle kinetoplast DNA (kDNA). A pan-Leishmania SYBR green quantitative PCR (qPCR) assay which detects the conserved spliced-leader RNA (SL RNA) sequence was developed recently. This study assessed the SL RNA assay performance combined with a crude extraction method for the detection of Leishmania in field-collected and laboratory-reared sand flies and in tissue samples from hyraxes as reservoir hosts. METHODS: Field-collected and laboratory-infected sand fly and hyrax extracts were subjected to three different qPCR approaches to assess the suitability of the SL RNA target for Leishmania detection. Nucleic acids of experimentally infected sand flies were isolated with a crude extraction buffer with ethanol precipitation and a commercial kit and tested for downstream DNA and RNA detection. Promastigotes were isolated from culture and sand fly midguts to assess whether there was difference in SL RNA and kDNA copy numbers. Naive sand flies were spiked with a serial dilution of promastigotes to make a standard curve. RESULTS: The qPCR targeting SL RNA performed well on infected sand fly samples, despite preservation and extraction under presumed unfavorable conditions for downstream RNA detection. Nucleic acid extraction by a crude extraction buffer combined with a precipitation step was highly compatible with downstream SL RNA and kDNA detection. Copy numbers of kDNA were found to be identical in culture-derived parasites and promastigotes isolated from sand fly midguts. SL RNA levels were slightly lower in sand fly promastigotes (ΔCq 1.7). The theoretical limit of detection and quantification of the SL RNA qPCR respectively reached down to 10-3 and 10 parasite equivalents. SL RNA detection in stored hyrax samples was less efficient with some false-negative assay results, most likely due to the long-term tissue storage in absence of RNA stabilizing reagents. CONCLUSIONS: This study shows that a crude extraction method in combination with the SL RNA qPCR assay is suitable for the detection and quantification of Leishmania in sand flies. The assay is inexpensive, sensitive and pan-Leishmania specific, and accordingly an excellent assay for high-throughput screening in entomological research.

2.
J Microbiol Methods ; 173: 105935, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32376283

RESUMO

BACKGROUND: Molecular detection techniques using peripheral blood are preferred over invasive tissue aspiration for the diagnosis and post-treatment follow-up of visceral leishmaniasis (VL) patients. This study aims to identify suitable stabilizing reagents to prevent DNA and RNA degradation during storage and transport to specialized laboratories where molecular diagnosis is performed. METHODOLOGY: The stabilizing capacities of different commercially available reagents were compared using promastigote-spiked human blood and peripheral blood of Syrian golden hamsters subjected to experimental infection, treatment (miltefosine or aminopyrazole DNDi-1044) and immunosuppression. The impact of various storage temperature conditions was tested in combination with an established kinetoplast DNA (kDNA) qPCR and a recently developed spliced leader RNA (SL-RNA) assay for Leishmania detection. PRINCIPAL FINDINGS: Irrespective of the blood type and stabilizer used, threshold (cT) values obtained with the SL-RNA qPCR were systematically lower than those obtained with the kDNA assay, confirming the advantage of the SL-RNA assay over the widely used kDNA assay for low-level Leishmania detection. Peripheral blood parasite levels correlated relatively well with hepatic burdens. RNA protect cell reagent provided the most optimal simultaneous DNA and RNA stabilization in both human and hamster blood. However, this stabilizer requires an erythrocyte lysis step, which can be challenging under field conditions. DNA/RNA shield provides a good alternative for downstream kDNA and SL-RNA assays, especially if sample storage capacity at 4 °C can be guaranteed. CONCLUSIONS/SIGNIFICANCE: The recommended stabilizing reagents are compatible with RNA- and DNA-based Leishmania detection in peripheral blood in the VL hamster model and spiked human blood. Since molecular detection techniques using peripheral blood are less invasive than microscopic assessment of tissue aspirates, the findings of this study may be applied to human VL clinical studies.

3.
Mol Genet Genomic Med ; 4(5): 568-80, 2016 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-27652284

RESUMO

BACKGROUND: Many genes are candidates for involvement in epileptic encephalopathy (EE) because one or a few possibly pathogenic variants have been found in patients, but insufficient genetic or functional evidence exists for a definite annotation. METHODS: To increase the number of validated EE genes, we sequenced 26 known and 351 candidate genes for EE in 360 patients. Variants in 25 genes known to be involved in EE or related phenotypes were followed up in 41 patients. We prioritized the candidate genes, and followed up 31 variants in this prioritized subset of candidate genes. RESULTS: Twenty-nine genotypes in known genes for EE (19) or related diseases (10), dominant as well as recessive or X-linked, were classified as likely pathogenic variants. Among those, likely pathogenic de novo variants were found in EE genes that act dominantly, including the recently identified genes EEF1A2, KCNB1 and the X-linked gene IQSEC2. A de novo frameshift variant in candidate gene HNRNPU was the only de novo variant found among the followed-up candidate genes, and the patient's phenotype was similar to a few recent publications. CONCLUSION: Mutations in genes described in OMIM as, for example, intellectual disability gene can lead to phenotypes that get classified as EE in the clinic. We confirmed existing literature reports that de novo loss-of-function HNRNPUmutations lead to severe developmental delay and febrile seizures in the first year of life.

4.
Sci Rep ; 5: 17816, 2015 Dec 09.
Artigo em Inglês | MEDLINE | ID: mdl-26647834

RESUMO

Febrile seizures (FS) are the most common seizure syndrome and are potentially a prelude to more severe epilepsy. Although zinc (Zn(2+)) metabolism has previously been implicated in FS, whether or not variation in proteins essential for Zn(2+) homeostasis contributes to susceptibility is unknown. Synaptic Zn(2+) is co-released with glutamate and modulates neuronal excitability. SLC30A3 encodes the zinc transporter 3 (ZNT3), which is primarily responsible for moving Zn(2+) into synaptic vesicles. Here we sequenced SLC30A3 and discovered a rare variant (c.892C > T; p.R298C) enriched in FS populations but absent in population-matched controls. Functional analysis revealed a significant loss-of-function of the mutated protein resulting from a trafficking deficit. Furthermore, mice null for ZnT3 were more sensitive than wild-type to hyperthermia-induced seizures that model FS. Together our data suggest that reduced synaptic Zn(2+) increases the risk of FS and more broadly support the idea that impaired synaptic Zn(2+) homeostasis can contribute to neuronal hyperexcitability.


Assuntos
Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Convulsões Febris/genética , Convulsões Febris/metabolismo , Zinco/metabolismo , Sequência de Aminoácidos , Animais , Estudos de Casos e Controles , Proteínas de Transporte de Cátions/química , Linhagem Celular , Análise Mutacional de DNA , Predisposição Genética para Doença , Humanos , Padrões de Herança , Estimativa de Kaplan-Meier , Camundongos Knockout , Dados de Sequência Molecular , Mutação , Linhagem , Ratos , Risco , Convulsões Febris/mortalidade , Alinhamento de Sequência , Análise de Sequência de DNA
5.
J Neurol Neurosurg Psychiatry ; 85(4): 462-5, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24101679

RESUMO

BACKGROUND: Mutations in the proline-rich transmembrane protein 2 (PRRT2) gene have been identified in patients with benign (familial) infantile convulsions (B(F)IC), infantile convulsions with choreoathetosis (ICCA) and paroxysmal dyskinesias (PDs). However it remains unknown whether PRRT2 mutations are causal in other epilepsy syndromes. After we discovered a PRRT2 mutation in a large family with ICCA containing one individual with febrile seizures (FS) and one individual with West syndrome, we analysed PRRT2 in a heterogeneous cohort of patients with different types of infantile epilepsy. METHODS: We screened a cohort of 460 patients with B(F)IC or ICCA, fever related seizures or infantile epileptic encephalopathies. All patients were tested for point mutations using direct sequencing. RESULTS: We identified heterozygous mutations in 16 individuals: 10 familial and 6 sporadic cases. All patients were diagnosed with B(F)IC, ICCA or PD. We were not able to detect mutations in any of the other epilepsy syndromes. Several mutation carriers had learning disabilities and/or impaired fine motor skills later in life. CONCLUSIONS: PRRT2 mutations do not seem to be involved in the aetiology of FS or infantile epileptic encephalopathies. Therefore B(F)IC, ICCA and PD remain the core phenotypes associated with PRRT2 mutations. The presence of learning disabilities or neuropsychiatric problems in several mutation carriers calls for additional clinical studies addressing this developmental aspect in more detail.


Assuntos
Epilepsia/genética , Proteínas de Membrana/genética , Proteínas do Tecido Nervoso/genética , Mutação Puntual/genética , Epilepsia/complicações , Epilepsia/diagnóstico , Feminino , Humanos , Deficiências da Aprendizagem/complicações , Deficiências da Aprendizagem/genética , Masculino , Transtornos das Habilidades Motoras/complicações , Transtornos das Habilidades Motoras/genética , Linhagem , Fenótipo
6.
Ann Neurol ; 75(1): 147-54, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24272827

RESUMO

OBJECTIVE: To identify novel epilepsy genes using a panel approach and describe the functional consequences of mutations. METHODS: Using a panel approach, we screened 357 patients comprising a vast spectrum of epileptic disorders for defects in genes known to contribute to epilepsy and/or intellectual disability (ID). After detection of mutations in a novel epilepsy gene, we investigated functional effects in Xenopus laevis oocytes and screened a follow-up cohort. RESULTS: We revealed de novo mutations in GRIN2B encoding the NR2B subunit of the N-methyl-D-aspartate (NMDA) receptor in 2 individuals with West syndrome and severe developmental delay as well as 1 individual with ID and focal epilepsy. The patient with ID and focal epilepsy had a missense mutation in the extracellular glutamate-binding domain (p.Arg540His), whereas both West syndrome patients carried missense mutations within the NR2B ion channel-forming re-entrant loop (p.Asn615Ile, p.Val618Gly). Subsequent screening of 47 patients with unexplained infantile spasms did not reveal additional de novo mutations, but detected a carrier of a novel inherited GRIN2B splice site variant in close proximity (c.2011-5_2011-4delTC). Mutations p.Asn615Ile and p.Val618Gly cause a significantly reduced Mg(2+) block and higher Ca(2+) permeability, leading to a dramatically increased Ca(2+) influx, whereas p.Arg540His caused less severe disturbance of channel function, corresponding to the milder patient phenotype. INTERPRETATION: We identified GRIN2B gain-of-function mutations as a cause of West syndrome with severe developmental delay as well as of ID with childhood onset focal epilepsy. Severely disturbed channel function corresponded to severe clinical phenotypes, underlining the important role of facilitated NMDA receptor signaling in epileptogenesis.


Assuntos
Epilepsias Parciais/genética , Deficiência Intelectual/genética , Mutação/genética , Receptores de N-Metil-D-Aspartato/genética , Espasmos Infantis/genética , Animais , Criança , Pré-Escolar , Cristalografia por Raios X , Epilepsias Parciais/complicações , Epilepsias Parciais/diagnóstico , Feminino , Humanos , Recém-Nascido , Deficiência Intelectual/complicações , Deficiência Intelectual/diagnóstico , Ratos , Receptores de N-Metil-D-Aspartato/química , Espasmos Infantis/complicações , Espasmos Infantis/diagnóstico , Xenopus laevis
7.
Neurology ; 81(19): 1697-703, 2013 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-24107868

RESUMO

OBJECTIVES: To determine the frequency of KCNQ2 mutations in patients with neonatal epileptic encephalopathy (NEE), and to expand the phenotypic spectrum of KCNQ2 epileptic encephalopathy. METHODS: Eighty-four patients with unexplained NEE were screened for KCNQ2 mutations using classic Sanger sequencing. Clinical data of 6 additional patients with KCNQ2 mutations detected by gene panel were collected. Detailed phenotyping was performed with particular attention to seizure frequency, cognitive outcome, and video-EEG. RESULTS: In the cohort, we identified 9 different heterozygous de novo KCNQ2 missense mutations in 11 of 84 patients (13%). Two of 6 missense mutations detected by gene panel were recurrent and present in patients of the cohort. Seizures at onset typically consisted of tonic posturing often associated with focal clonic jerking, and were accompanied by apnea with desaturation. One patient diagnosed by gene panel had seizure onset at the age of 5 months. Based on seizure frequency at onset and cognitive outcome, we delineated 3 clinical subgroups, expanding the spectrum of KCNQ2 encephalopathy to patients with moderate intellectual disability and/or infrequent seizures at onset. Recurrent mutations lead to relatively homogenous phenotypes. One patient responded favorably to retigabine; 5 patients had a good response to carbamazepine. In 6 patients, seizures with bradycardia were recorded. One patient died of probable sudden unexpected death in epilepsy. CONCLUSION: KCNQ2 mutations cause approximately 13% of unexplained NEE. Patients present with a wide spectrum of severity and, although rare, infantile epilepsy onset is possible.


Assuntos
Predisposição Genética para Doença/genética , Canal de Potássio KCNQ2/genética , Mutação/genética , Espasmos Infantis/genética , Estudos de Coortes , Análise Mutacional de DNA , Eletroencefalografia , Feminino , Humanos , Lactente , Masculino , Gravação em Vídeo
8.
Epilepsia ; 54(5): e74-80, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23409955

RESUMO

Mutations in STXBP1 have been identified in a subset of patients with early onset epileptic encephalopathy (EE), but the full phenotypic spectrum remains to be delineated. Therefore, we screened a cohort of 160 patients with an unexplained EE, including patients with early myoclonic encephalopathy (EME), Ohtahara syndrome, West syndrome, nonsyndromic EE with onset in the first year, and Lennox-Gastaut syndrome (LGS). We found six de novo mutations in six patients presenting as Ohtahara syndrome (2/6, 33%), West syndrome (1/65, 2%), and nonsyndromic early onset EE (3/64, 5%). No mutations were found in LGS or EME. Only two of four mutation carriers with neonatal seizures had Ohtahara syndrome. Epileptic spasms were present in five of six patients. One patient with normal magnetic resonance imaging (MRI) but focal seizures underwent epilepsy surgery and seizure frequency dropped drastically. Neuropathology showed a focal cortical dysplasia type 1a. There is a need for additional neuropathologic studies to explore whether STXBP1 mutations can lead to structural brain abnormalities.


Assuntos
Predisposição Genética para Doença/genética , Proteínas Munc18/genética , Mutação/genética , Convulsões/genética , Convulsões/cirurgia , Espasmos Infantis/genética , Encéfalo/metabolismo , Encéfalo/patologia , Criança , Pré-Escolar , Eletroencefalografia , Feminino , Humanos , Lactente , Masculino , Fosfopiruvato Hidratase/metabolismo , Convulsões/etiologia , Convulsões/patologia , Espasmos Infantis/complicações , Adulto Jovem
9.
Endocrine ; 44(2): 386-90, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23264184

RESUMO

The role of mutations in the melanocortin-3 receptor (MC3R) gene, which is implicated in the regulation of energy homeostasis, is still under debate. Animal studies have clearly proven that, together with the melanocortin-4 receptor (MC4R), the MC3R is a critical receptor for melanocortin peptides within the leptin-melanocortin signaling cascade. However, as several mutations have been found in lean individuals and not all mutations seem to cause receptor dysfunction, results from mutation screens in obese humans remain controversial. In the present study, we screened for rare variants in the MC3R gene of obese children and lean controls to assess the prevalence of MC3R mutations in the Belgian population. We screened 249 severely overweight and obese children and adolescents and 239 lean adults for mutations in the coding region of MC3R. Mutation screening was performed by high resolution melting curve analysis and direct sequencing. We identified four non-synonymous coding variations in the obese population, all of which had been reported previously. In addition, we also found four novel rare MC3R variants in the lean control population, suggesting that not all MC3R mutations are disease-causing. Overall, the total prevalence of rare MC3R variants was 1 % in Belgian obese children and adolescents compared to 1.02 % in lean controls. Ultimately, cosegregation studies combined with comprehensive functional analysis is required to determine the potential pathogenic role of rare MC3R variants in causing human obesity.


Assuntos
Obesidade/genética , Polimorfismo de Nucleotídeo Único , Receptor Tipo 3 de Melanocortina/genética , Magreza/genética , Adolescente , Adulto , Substituição de Aminoácidos , Bélgica/epidemiologia , Estudos de Casos e Controles , Criança , Análise Mutacional de DNA , Feminino , Frequência do Gene , Predisposição Genética para Doença , Humanos , Masculino , Obesidade/epidemiologia , Prevalência , Magreza/epidemiologia
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