Display omitted
•Use of napkin paper for production of low-cost analytical devices is reported.•New stamping method for creation of paraffin hydrophobic barriers is described.•Novel method for ...colorimetric quantification of ethanol in beverages is presented.•Analytical application for analysis of real samples is successfully demonstrated.•Results showed great concordance when compared to UV–vis spectrophotometry.
In this work we report for the first time the use of napkin paper as substrate for production of paper analytical devices (PADs). Napkin paper is a more accessible and cheaper option compared to the most commonly used chromatographic and filter papers. The PADs were easily fabricated by depositing hydrophobic paraffin barriers using a simple stamping contact method. The stamps used here were fabricated by 3D printing. To the best of our knowledge, this is the first report of this stamping approach aiming production of paraffin barriers in PADs. The PADs proposed here were utilized for colorimetric determination of alcohol content in beverages. This method was based on the redox reaction between ethanol and dichromate in acidic medium. As far as we know, this is the first PAD-based method exploring this principle. The image-based method showed a linear range between 0 and 50 % (v/v) ethanol, with a limit of detection of 1.5 %. This concentration range allows the analysis of various alcoholic beverages. The results obtained during the analysis of real samples presented great concordance with the values found by UV–vis spectrophotometry, used for validation (relative error < 5 %). Thus, the new PAD-based colorimetric method reported here shows great potential for applications in areas such as food control and forensics. Moreover, the use of napkin paper for fabrication of PADs appears to be a promising, easier and less expensive option, being specially interesting for places with limited resources.
Hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels contribute to cationic Ih current in neurons and regulate the excitability of neuronal networks. Studies in rat models have shown ...that the Hcn1 gene has a key role in epilepsy, but clinical evidence implicating HCN1 mutations in human epilepsy is lacking. We carried out exome sequencing for parent-offspring trios with fever-sensitive, intractable epileptic encephalopathy, leading to the discovery of two de novo missense HCN1 mutations. Screening of follow-up cohorts comprising 157 cases in total identified 4 additional amino acid substitutions. Patch-clamp recordings of Ih currents in cells expressing wild-type or mutant human HCN1 channels showed that the mutations had striking but divergent effects on homomeric channels. Individuals with mutations had clinical features resembling those of Dravet syndrome with progression toward atypical absences, intellectual disability and autistic traits. These findings provide clear evidence that de novo HCN1 point mutations cause a recognizable early-onset epileptic encephalopathy in humans.
Disease gene discovery on chromosome (chr) X is challenging owing to its unique modes of inheritance. We undertook a systematic analysis of human chrX genes. We observe a higher proportion of ...disorder-associated genes and an enrichment of genes involved in cognition, language, and seizures on chrX compared to autosomes. We analyze gene constraints, exon and promoter conservation, expression, and paralogues, and report 127 genes sharing one or more attributes with known chrX disorder genes. Using machine learning classifiers trained to distinguish disease-associated from dispensable genes, we classify 247 genes, including 115 of the 127, as having high probability of being disease-associated. We provide evidence of an excess of variants in predicted genes in existing databases. Finally, we report damaging variants in CDK16 and TRPC5 in patients with intellectual disability or autism spectrum disorders. This study predicts large-scale gene-disease associations that could be used for prioritization of X-linked pathogenic variants.
KIF21B is a kinesin protein that promotes intracellular transport and controls microtubule dynamics. We report three missense variants and one duplication in KIF21B in individuals with ...neurodevelopmental disorders associated with brain malformations, including corpus callosum agenesis (ACC) and microcephaly. We demonstrate, in vivo, that the expression of KIF21B missense variants specifically recapitulates patients' neurodevelopmental abnormalities, including microcephaly and reduced intra- and inter-hemispheric connectivity. We establish that missense KIF21B variants impede neuronal migration through attenuation of kinesin autoinhibition leading to aberrant KIF21B motility activity. We also show that the ACC-related KIF21B variant independently perturbs axonal growth and ipsilateral axon branching through two distinct mechanisms, both leading to deregulation of canonical kinesin motor activity. The duplication introduces a premature termination codon leading to nonsense-mediated mRNA decay. Although we demonstrate that Kif21b haploinsufficiency leads to an impaired neuronal positioning, the duplication variant might not be pathogenic. Altogether, our data indicate that impaired KIF21B autoregulation and function play a critical role in the pathogenicity of human neurodevelopmental disorder.
Massively parallel sequencing has revealed many de novo mutations in the etiology of developmental and epileptic encephalopathies (EEs), highlighting their genetic heterogeneity. Additional candidate ...genes have been prioritized in silico by their co-expression in the brain. Here, we evaluate rare coding variability in 20 candidates nominated with the use of a reference gene set of 51 established EE-associated genes. Variants within the 20 candidate genes were extracted from exome-sequencing data of 42 subjects with EE and no previous genetic diagnosis. We identified 7 rare non-synonymous variants in 7 of 20 genes and performed Sanger sequence validation in affected probands and parental samples. De novo variants were found only in SLC1A2 (aka EAAT2 or GLT1) (c.244G>A p.Gly82Arg) and YWHAG (aka 14-3-3γ) (c.394C>T p.Arg132Cys), highlighting the potential cause of EE in 5% (2/42) of subjects. Seven additional subjects with de novo variants in SLC1A2 (n = 1) and YWHAG (n = 6) were subsequently identified through online tools. We identified a highly significant enrichment of de novo variants in YWHAG, establishing their role in early-onset epilepsy, and we provide additional support for the prior assignment of SLC1A2. Hence, in silico modeling of brain co-expression is an efficient method for nominating EE-associated genes to further elucidate the disorder’s etiology and genotype-phenotype correlations.
The leucine-rich glioma-inactivated (LGI) family consists of four highly conserved paralogous genes, LGI1-4, that are highly expressed in mammalian central and/or peripheral nervous systems. LGI1 ...antibodies are detected in subjects with autoimmune limbic encephalitis and peripheral nerve hyperexcitability syndromes (PNHSs) such as Isaacs and Morvan syndromes. Pathogenic variations of LGI1 and LGI4 are associated with neurological disorders as disease traits including familial temporal lobe epilepsy and neurogenic arthrogryposis multiplex congenita 1 with myelin defects, respectively. No human disease has been reported associated with either LGI2 or LGI3. We implemented exome sequencing and family-based genomics to identify individuals with deleterious variants in LGI3 and utilized GeneMatcher to connect practitioners and researchers worldwide to investigate the clinical and electrophysiological phenotype in affected subjects. We also generated Lgi3-null mice and performed peripheral nerve dissection and immunohistochemistry to examine the juxtaparanode LGI3 microarchitecture. As a result, we identified 16 individuals from eight unrelated families with loss-of-function (LoF) bi-allelic variants in LGI3. Deep phenotypic characterization showed LGI3 LoF causes a potentially clinically recognizable PNHS trait characterized by global developmental delay, intellectual disability, distal deformities with diminished reflexes, visible facial myokymia, and distinctive electromyographic features suggestive of motor nerve instability. Lgi3-null mice showed reduced and mis-localized Kv1 channel complexes in myelinated peripheral axons. Our data demonstrate bi-allelic LoF variants in LGI3 cause a clinically distinguishable disease trait of PNHS, most likely caused by disturbed Kv1 channel distribution in the absence of LGI3.
Display omitted
Through the power of human paralog gene studies, worldwide family-based genomics analyses, and mouse studies, we define a potentially-recognizable peripheral hyperexcitability syndrome in 16 individuals with bi-allelic LGI3 variants and show that LGI3 co-localizes with juxta-paranodal voltage-gated potassium channels and its loss results in mis-localization of potassium channel complexes.
Corpus callosum agenesis (CCA) is a brain malformation associated with a wide clinical spectrum including intellectual disability (ID) and an etiopathological complexity. We identified a novel ...missense G424R mutation in the X-linked p21-activated kinase 3 (PAK3) gene in a boy presenting with severe ID, microcephaly and CCA and his fetal sibling with CCA and severe hydrocephaly. PAK3 kinase is known to control synaptic plasticity and dendritic spine dynamics but its implication is less characterized in brain ontogenesis. In order to identify developmental functions of PAK3 impacted by mutations responsible for CCA, we compared the biochemical and biological effects of three PAK3 mutations localized in the catalytic domain. These mutations include two “severe” G424R and K389N variants (responsible for severe ID and CCA) and the “mild” A365E variant (responsible for nonsyndromic mild ID). Whereas they suppressed kinase activity, only the two severe variants displayed normal protein stability. Furthermore, they increased interactions between PAK3 and the guanine exchange factor αPIX/ARHGEF6, disturbed adhesion point dynamics and cell spreading, and severely impacted cell migration. Our findings highlight new molecular defects associated with mutations responsible for severe clinical phenotypes with developmental brain defects.
•A novel PAK3 variant (p.Gly424Arg) with severe phenotype has been identified.•Mutation impairs kinase activity and tends to increase PIX binding.•Variant expression affects cell morphology and migration.•Biological impairments may be correlated with clinical severity.
Biallelic loss-of-function variants in ST3GAL5 cause GM3 synthase deficiency (GM3SD) responsible for Amish infantile epilepsy syndrome. All Amish patients carry the homozygous p.(Arg288Ter) variant ...arising from a founder effect. To date only 10 patients from 4 non-Amish families have been reported. Thus, the phenotypical spectrum of GM3SD due to other variants and other genetic backgrounds is still poorly known.
We collected clinical and molecular data from 16 non-Amish patients with pathogenic ST3GAL5 variants resulting in GM3SD.
We identified 12 families originating from Reunion Island, Ivory Coast, Italy, and Algeria and carrying 6 ST3GAL5 variants, 5 of which were novel. Genealogical investigations and/or haplotype analyses showed that 3 of these variants were founder alleles. Glycosphingolipids quantification in patients' plasma confirmed the pathogenicity of 4 novel variants. All patients (N = 16), aged 2 to 12 years, had severe to profound intellectual disability, 14 of 16 had a hyperkinetic movement disorder, 11 of 16 had epilepsy and 9 of 16 had microcephaly. Other main features were progressive skin pigmentation anomalies, optic atrophy or pale papillae, and hearing loss.
The phenotype of non-Amish patients with GM3SD is similar to the Amish infantile epilepsy syndrome, which suggests that GM3SD is associated with a narrow and severe clinical spectrum.
Autism spectrum disorders affect millions of individuals worldwide, but their heterogeneity complicates therapeutic intervention that is essentially symptomatic. A versatile yet relevant model to ...rationally screen among hundreds of therapeutic options would help improving clinical practice. Here we investigated whether neurons differentiated from pluripotent stem cells can provide such a tool using SHANK3 haploinsufficiency as a proof of principle. A library of compounds was screened for potential to increase SHANK3 mRNA content in neurons differentiated from control human embryonic stem cells. Using induced pluripotent stem cell technology, active compounds were then evaluated for efficacy in correcting dysfunctional networks of neurons differentiated from individuals with deleterious point mutations of SHANK3. Among 202 compounds tested, lithium and valproic acid showed the best efficacy at corrected SHANK3 haploinsufficiency associated phenotypes in cellulo. Lithium pharmacotherapy was subsequently provided to one patient and, after one year, an encouraging decrease in autism severity was observed. This demonstrated that pluripotent stem cell-derived neurons provide a novel cellular paradigm exploitable in the search for specific disease-modifying treatments.
•Human neurons were used to screen for compounds correcting symptoms associated with SHANK3 haploinsufficiency syndrome.•Screening criteria were the ability to increase SHANK3 expression and to increase glutamatergic transmission.•Selected hit compounds were then validated using neurons differentiated from individuals with SHANK3 disrupting mutations.•Lithium was selected and delivered to one of SHANK3 patient showing encouraging positive clinical outcomes after one year.
The clinical heterogeneity between individuals affected by autism makes it difficult to anticipate the effectiveness of a treatment. Furthermore, clinical practice lacks biological tools to help make such decisions. Here we use neurons, produced from pluripotent stem cells derived from patients affected by SHANK3 haploinsufficiency syndrome, to test the efficiency of therapeutic compounds. We screened the biological activity of more than 200 compounds on SHANK3 expression. Lithium was ultimately selected and delivered to one patient with a SHANK3-disruptive mutation. This resulted in a positive outcome, as determined by improved autistic core symptoms, thus supporting the usefulness of this type of predictive approach.
Objective To evaluate the role that chromosomal micro-rearrangements play in patients with both corpus callosum abnormality and intellectual disability, we analyzed copy number variations (CNVs) in ...patients with corpus callosum abnormality/intellectual disability Study design We screened 149 patients with corpus callosum abnormality/intellectual disability using Illumina SNP arrays. Results In 20 patients (13%), we have identified at least 1 CNV that likely contributes to corpus callosum abnormality/intellectual disability phenotype. We confirmed that the most common rearrangement in corpus callosum abnormality/intellectual disability is inverted duplication with terminal deletion of the 8p chromosome (3.2%). In addition to the identification of known recurrent CNVs, such as deletions 6qter, 18q21 (including TCF4 ), 1q43q44, 17p13.3, 14q12, 3q13, 3p26, and 3q26 (including SOX2 ), our analysis allowed us to refine the 2 known critical regions associated with 8q21.1 deletion and 19p13.1 duplication relevant for corpus callosum abnormality; report a novel 10p12 deletion including ZEB1 recently implicated in corpus callosum abnormality with corneal dystrophy; and) report a novel pathogenic 7q36 duplication encompassing SHH . In addition, 66 variants of unknown significance were identified in 57 patients encompassed candidate genes. Conclusions Our results confirm the relevance of using microarray analysis as first line test in patients with corpus callosum abnormality/intellectual disability.
PDF
