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Functional Characterization of Four Known Cav2.1 Variants Associated with Neurodevelopmental Disorders.

ABSTRACT: Cav2.1 channels are expressed throughout the brain and are the predominant Ca²⁺ channels in the Purkinje cells. These cerebellar neurons fire spontaneously, and Cav2.1 channels are involved in the regular pacemaking activity. The loss of precision of the firing pattern of Purkinje cells leads to ataxia, a disorder characterized by poor balance and difficulties in performing coordinated movements. In this study, we aimed at characterizing functional and structural consequences of four variations (p.A405T in I-II loop and p.R1359W, p.R1667W and p.S1799L in IIIS4, IVS4, and IVS6 helices, respectively) identified in patients exhibiting a wide spectrum of disorders including ataxia symptoms. Functional analysis using two major Cav2.1 splice variants (Cav2.1+e47 and Cav2.1-e47) in Xenopus laevis oocytes, revealed a lack of effect upon A405T substitution and a significant loss-of-function caused by R1359W, whereas R1667W and S1799L caused both channel gain-of-function and loss-of-function, in a splice variant-dependent manner. Structural analysis revealed the loss of interactions with S1, S2, and S3 helices upon R1359W and R1667W substitutions, but a lack of obvious structural changes with S1799L. Computational modeling suggests that biophysical changes induced by Cav2.1 pathogenic mutations might affect action potential frequency in Purkinje cells.

SUBMITTER: Folacci M

PROVIDER: S-EPMC9864995 | biostudies-literature | 2023 Jan

REPOSITORIES: biostudies-literature

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Functional Characterization of Four Known Cav2.1 Variants Associated with Neurodevelopmental Disorders.

Folacci Mathilde M Estaran Sébastien S Ménard Claudine C Bertaud Anaïs A Rousset Matthieu M Roussel Julien J Thibaud Jean-Baptiste JB Vignes Michel M Chavanieu Alain A Charnet Pierre P Cens Thierry T

Membranes 20230111 1

Cav2.1 channels are expressed throughout the brain and are the predominant Ca<sup>2+</sup> channels in the Purkinje cells. These cerebellar neurons fire spontaneously, and Cav2.1 channels are involved in the regular pacemaking activity. The loss of precision of the firing pattern of Purkinje cells leads to ataxia, a disorder characterized by poor balance and difficulties in performing coordinated movements. In this study, we aimed at characterizing functional and structural consequences of four ...[more]

PMID: 36676903

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Project description:BackgroundHeterozygous disruption of FOXP2 causes a rare form of speech and language impairment. Screens of the FOXP2 sequence in individuals with speech/language-related disorders have identified several rare protein-altering variants, but their phenotypic relevance is often unclear. FOXP2 encodes a transcription factor with a forkhead box DNA-binding domain, but little is known about the functions of protein regions outside this domain.MethodsWe performed detailed functional analyses of seven rare FOXP2 variants found in affected cases, including three which have not been previously characterized, testing intracellular localization, transcriptional regulation, dimerization, and interaction with other proteins. To shed further light on molecular functions of FOXP2, we characterized the interaction between this transcription factor and co-repressor proteins of the C-terminal binding protein (CTBP) family. Finally, we analysed the functional significance of the polyglutamine tracts in FOXP2, since tract length variations have been reported in cases of neurodevelopmental disorder.ResultsWe confirmed etiological roles of multiple FOXP2 variants. Of three variants that have been suggested to cause speech/language disorder, but never before been characterized, only one showed functional effects. For the other two, we found no effects on protein function in any assays, suggesting that they are incidental to the phenotype. We identified a CTBP-binding region within the N-terminal portion of FOXP2. This region includes two amino acid substitutions that occurred on the human lineage following the split from chimpanzees. However, we did not observe any effects of these amino acid changes on CTBP binding or other core aspects of FOXP2 function. Finally, we found that FOXP2 variants with reduced polyglutamine tracts did not exhibit altered behaviour in cellular assays, indicating that such tracts are non-essential for core aspects of FOXP2 function, and that tract variation is unlikely to be a highly penetrant cause of speech/language disorder.ConclusionsOur findings highlight the importance of functional characterization of novel rare variants in FOXP2 in assessing the contribution of such variants to speech/language disorder and provide further insights into the molecular function of the FOXP2 protein.

Project description:ImportancePast studies identified rare copy number variants (CNVs) as risk factors for neurodevelopmental disorders (NDDs), including autism spectrum disorder and schizophrenia. However, the clinical characterization of NDD CNVs is understudied in population cohorts unselected for neuropsychiatric disorders and in cohorts of diverse ancestry.ObjectiveTo identify individuals harboring NDD CNVs in a multiancestry biobank and to query their enrichment for select neuropsychiatric disorders as well as association with multiple medical disorders.Design, settings, and participantsIn a series of phenotypic enrichment and association analyses, NDD CNVs were clinically characterized among 24 877 participants in the BioMe biobank, an electronic health record-linked biobank derived from the Mount Sinai Health System, New York, New York. Participants were recruited into the biobank since September 2007 across diverse ancestry and medical and neuropsychiatric specialties. For the current analyses, electronic health record data were analyzed from May 2004 through May 2019.Main outcomes and measuresNDD CNVs were identified using a consensus of 2 CNV calling algorithms, based on whole-exome sequencing and genotype array data, followed by novel in-silico clinical assessments.ResultsOf 24 877 participants, 14 586 (58.7%) were female; self-reported ancestry categories included 5965 (24.0%) who were of African ancestry, 7892 (31.7%) who were of European ancestry, and 8536 (34.3%) who were of Hispanic ancestry; and the mean (SD) age was 50.5 (17.3) years. Among 24 877 individuals, the prevalence of 64 NDD CNVs was 2.5% (n = 627), with prevalence varying by locus, corroborating the presence of some relatively highly prevalent NDD CNVs (eg, 15q11.2 deletion/duplication). An aggregate set of NDD CNVs were enriched for congenital disorders (odds ratio, 2.0; 95% CI, 1.1-3.5; P = .01) and major depressive disorder (odds ratio, 1.5; 95% CI, 1.1-2.0; P = .01). In a meta-analysis of medical diagnoses (n = 195 hierarchically clustered diagnostic codes), NDD CNVs were significantly associated with several medical outcomes, including essential hypertension (z score = 3.6; P = 2.8 × 10-4), kidney failure (z score = 3.3; P = 1.1 × 10-3), and obstructive sleep apnea (z score = 3.4; P = 8.1 × 10-4) and, in another analysis, morbid obesity (z score = 3.8; P = 1.3 × 10-4). Further, NDD CNVs were associated with increased body mass index in a multiancestry analysis (β = 0.19; 95% CI, 0.10-0.31; P = .003). For 36 common serum tests, there was no association with NDD CNVs.Conclusions and relevanceClinical features of individuals harboring NDD CNVs were elucidated in a large-scale, multiancestry biobank, identifying enrichments for congenital disorders and major depressive disorder as well as associations with several medical outcomes, including hypertension, kidney failure, and obesity and obesity-related phenotypes, specifically obstructive sleep apnea and increased body mass index. The association between NDD CNVs and obesity outcomes indicate further potential pleiotropy of NDD CNVs beyond neurodevelopmental outcomes previously reported. Future clinical genetic investigations may lead to insights of at-risk individuals and therapeutic strategies targeting specific genetic variants. The importance of diverse inclusion within biobanks and considering the effect of rare genetic variants in a multiancestry context is evident.

Project description:ObjectiveN-methyl-d-aspartate (NMDA) receptors are expressed at synaptic sites, where they mediate fast excitatory neurotransmission. NMDA receptors are critical to brain development and cognitive function. Natural variants to the GRIN1 gene, which encodes the obligatory GluN1 subunit of the NMDA receptor, are associated with severe neurological disorders that include epilepsy, intellectual disability, and developmental delay. Here, we investigated the pathogenicity of three missense variants to the GRIN1 gene, p. Ile148Val (GluN1-3b[I481V]), p.Ala666Ser (GluN1-3b[A666S]), and p.Tyr668His (GluN1-3b[Y668H]).MethodsWild-type and variant-containing NMDA receptors were expressed in HEK293 cells and primary hippocampal neurons. Patch-clamp electrophysiology and pharmacology were used to profile the functional properties of the receptors. Receptor surface expression was evaluated using fluorescently tagged receptors and microscopy.ResultsOur data demonstrate that the GluN1(I481V) variant is inhibited by the open pore blockers ketamine and memantine with reduce potency but otherwise has little effect on receptor function. By contrast, the other two variants exhibit gain-of-function molecular phenotypes. Glycine sensitivity was enhanced in receptors containing the GluN1(A666S) variant and the potency of pore block by memantine and ketamine was reduced, whereas that for MK-801 was increased. The most pronounced functional deficits, however, were found in receptors containing the GluN1(Y668H) variant. GluN1(Y668H)/2A receptors showed impaired surface expression, were more sensitive to glycine and glutamate by an order of magnitude, and exhibited impaired block by extracellular magnesium ions, memantine, ketamine, and MK-801. These variant receptors were also activated by either glutamate or glycine alone. Single-receptor recordings revealed that this receptor variant opened to several conductance levels and activated more frequently than wild-type GluN1/2A receptors.SignificanceOur study reveals a critical functional locus of the receptor (GluN1[Y668]) that couples receptor gating to ion channel conductance, which when mutated may be associated with neurological disorder.

Dataset Information

Functional Characterization of Four Known Cav2.1 Variants Associated with Neurodevelopmental Disorders.

Publications

Functional Characterization of Four Known Cav2.1 Variants Associated with Neurodevelopmental Disorders.

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