SLC6A1 variant pathogenicity, molecular function, and phenotype: a genetic and clinical analysis

in Brain: a Journal of Neurology (2023)

Genetic variants in the SLC6A1 gene can cause a broad phenotypic disease spectrum by altering the protein function. Thus, systematically curated clinically relevant genotype-phenotype associations are ... [more ▼]

Genetic variants in the SLC6A1 gene can cause a broad phenotypic disease spectrum by altering the protein function. Thus, systematically curated clinically relevant genotype-phenotype associations are needed to understand the disease mechanism and improve therapeutic decision-making. We aggregated genetic and clinical data from 172 individuals with likely pathogenic/pathogenic (lp/p) SLC6A1 variants and functional data for 184 variants (14.1% lp/p). Clinical and functional data were available for a subset of 126 individuals. We explored the potential associations of variant positions on the GAT1 3D structure with variant pathogenicity, altered molecular function, and phenotype severity using bioinformatic approaches. The GAT1 transmembrane domains 1, 6, and extracellular loop 4 (EL4) were enriched for patient over population variants. Across functionally tested missense variants (n = 156), the spatial proximity from the ligand was associated with loss-of-function in the GAT1 transporter activity. For variants with complete loss of in vitro GABA uptake, we found a 4.6-fold enrichment in patients having severe disease vs. non-severe disease (P = 2.9e-3, 95% CI: 1.5 - 15.3). In summary, we delineated associations between the 3D structure and variant pathogenicity, variant function, and phenotype in SLC6A1-related disorders. This knowledge supports biology-informed variant interpretation and research on GAT1 function. All our data can be interactively explored in the SLC6A1 Portal (https://slc6a1-portal.broadinstitute.org/). [less ▲]

Delineation of functionally essential protein regions for 242 neurodevelopmental genes

in Brain: a Journal of Neurology (2023), 146(2), 519-533

Neurodevelopmental disorders (NDDs), including severe pediatric epilepsy, autism, and intellectual disabilities are heterogeneous conditions in which clinical genetic testing can often identify a ... [more ▼]

Neurodevelopmental disorders (NDDs), including severe pediatric epilepsy, autism, and intellectual disabilities are heterogeneous conditions in which clinical genetic testing can often identify a pathogenic variant. For many of them, genetic therapies will be tested in this or the coming years in clinical trials. In contrast to first-generation symptomatic treatments, the new disease-modifying precision medicines require a genetic test-informed diagnosis before a patient can be enrolled in a clinical trial. However, even in 2022, most identified genetic variants in NDD genes are ‘Variants of Uncertain Significance’. To safely enroll patients in precision medicine clinical trials, it is important to increase our knowledge about which regions in NDD-associated proteins can ‘tolerate’ missense variants and which ones are ‘essential’ and will cause a NDD when mutated. In addition, knowledge about functionally indispensable regions in the three-dimensional (3D) structure context of proteins can also provide insights into the molecular mechanisms of disease variants. We developed a novel consensus approach that overlays evolutionary, and population based genomic scores to identify 3D essential sites (Essential3D) on protein structures. After extensive benchmarking of AlphaFold predicted and experimentally solved protein structures, we generated the currently largest expert curated protein structure set for 242 NDDs and identified 14,377 Essential3D sites across 189 gene disorders associated proteins. We demonstrate that the consensus annotation of Essential3D sites improves prioritization of disease mutations over single annotations. The identified Essential3D sites were enriched for functional features such as intermembrane regions or active sites and discovered key inter-molecule interactions in protein complexes that were otherwise not annotated. Using the currently largest autism, developmental disorders, and epilepsies exome sequencing studies including >360,000 NDD patients and population controls, we found that missense variants at Essential3D sites are 8-fold enriched in patients. In summary, we developed a comprehensive protein structure set for 242 neurodevelopmental disorders and identified 14,377 Essential3D sites in these. All data are available at https://es-ndd.broadinstitute.org for interactive visual inspection to enhance variant interpretation and development of mechanistic hypotheses for 242 NDDs genes. The provided resources will enhance clinical variant interpretation and in silico drug target development for NDD-associated genes and encoded proteins. [less ▲]

Mitochondrial damage-associated inflammation highlights biomarkers in PRKN/PINK1 parkinsonism

in Brain: a Journal of Neurology (2020)

There is increasing evidence for a role of inflammation in Parkinson’s disease. Recent research in murine models suggests that parkin and PINK1 deficiency leads to impaired mitophagy, which causes the ... [more ▼]

There is increasing evidence for a role of inflammation in Parkinson’s disease. Recent research in murine models suggests that parkin and PINK1 deficiency leads to impaired mitophagy, which causes the release of mitochondrial DNA (mtDNA), thereby triggering inflammation. Specifically, the CGAS (cyclic GMP-AMP synthase)-STING (stimulator of interferon genes) pathway mitigates activation of the innate immune system, quantifiable as increased interleukin-6 (IL6) levels. However, the role of IL6 and circulating cell-free mtDNA in unaffected and affected individuals harbouring mutations in PRKN/PINK1 and idiopathic Parkinson’s disease patients remain elusive. We investigated IL6, C-reactive protein, and circulating cell-free mtDNA in serum of 245 participants in two cohorts from tertiary movement disorder centres. We performed a hypothesis-driven rank-based statistical approach adjusting for multiple testing. We detected (i) elevated IL6 levels in patients with biallelic PRKN/PINK1 mutations compared to healthy control subjects in a German cohort, supporting the concept of a role for inflammation in PRKN/PINK1-linked Parkinson’s disease. In addition, the comparison of patients with biallelic and heterozygous mutations in PRKN/PINK1 suggests a gene dosage effect. The differences in IL6 levels were validated in a second independent Italian cohort; (ii) a correlation between IL6 levels and disease duration in carriers of PRKN/PINK1 mutations, while no such association was observed for idiopathic Parkinson’s disease patients. These results highlight the potential of IL6 as progression marker in Parkinson’s disease due to PRKN/PINK1 mutations; (iii) increased circulating cell-free mtDNA serum levels in both patients with biallelic or with heterozygous PRKN/PINK1 mutations compared to idiopathic Parkinson’s disease, which is in line with previous findings in murine models. By contrast, circulating cell-free mtDNA concentrations in unaffected heterozygous carriers of PRKN/PINK1 mutations were comparable to control levels; and (iv) that circulating cell-free mtDNA levels have good predictive potential to discriminate between idiopathic Parkinson’s disease and Parkinson’s disease linked to heterozygous PRKN/PINK1 mutations, providing functional evidence for a role of heterozygous mutations in PRKN or PINK1 as Parkinson’s disease risk factor. Taken together, our study further implicates inflammation due to impaired mitophagy and subsequent mtDNA release in the pathogenesis of PRKN/PINK1-linked Parkinson’s disease. In individuals carrying mutations in PRKN/PINK1, IL6 and circulating cell-free mtDNA levels may serve as markers of Parkinson’s disease state and progression, respectively. Finally, our study suggests that targeting the immune system with anti-inflammatory medication holds the potential to influence the disease course of Parkinson’s disease, at least in this subset of patients. [less ▲]

Bi-allelic GAD1 variants cause a neonatal onset syndromic developmental and epileptic encephalopathy

in Brain: a Journal of Neurology (2020)

Developmental and Epileptic Encephalopathies are a heterogeneous group of early-onset epilepsy syndromes dramatically impairing neurodevelopment. Modern genomic technologies have revealed a number of ... [more ▼]

Developmental and Epileptic Encephalopathies are a heterogeneous group of early-onset epilepsy syndromes dramatically impairing neurodevelopment. Modern genomic technologies have revealed a number of monogenic origins and opened the door to therapeutic hopes. Here we describe a new syndromic developmental and epileptic encephalopathies caused by bi-allelic loss of function variants in GAD1, as presented by eleven patients from 6 independent consanguineous families. Seizure onset occurred in the two first months of life in all patients. All 10 patients from whom early disease history was available, presented seizure onset in the first month of life, mainly consisting of epileptic spasms or myoclonic seizures. Early electroencephalography showed suppression-burst or pattern of burst attenuation or hypsarrhythmia if only recorded in the post-neonatal period. Eight patients had joint contractures and/or pes equinovarus. Seven patients presented a cleft palate and two also had an omphalocele, reproducing the phenotype of the knockout Gad1-/- mouse model. Four patients died before four years of age. GAD1 encodes the glutamate decarboxylase enzyme GAD67, a critical actor of the γ-aminobutyric acid (GABA) metabolism as it catalyzes the decarboxylation of glutamic acid to form GABA. Our findings evoke a novel syndrome related to GAD67 deficiency, characterized by the unique association of developmental and epileptic encephalopathies, cleft palate, joint contractures and/or omphalocele. [less ▲]

CHD2 variants are a risk factor for photosensitivity in epilepsy

in Brain: a Journal of Neurology (2015)