POLG2-Linked Mitochondrial Disease: Functional Insights from New Mutation Carriers and Review of the Literature.

Different pathogenic variants in the DNA polymerase-gamma2 (POLG2) gene cause a rare, clinically heterogeneous mitochondrial disease. We detected a novel POLG2 variant (c.1270 T > C, p.Ser424Pro) in a family with adult-onset cerebellar ataxia and progressive ophthalmoplegia. We demonstrated altered mitochondrial integrity in patients' fibroblast cultures but no changes of the mitochondrial DNA were found when compared to controls. We consider this novel, segregating POLG2 variant as disease-causing in this family. Moreover, we systematically screened the literature for POLG2-linked phenotypes and re-evaluated all mutations published to date for pathogenicity according to current knowledge. Thereby, we identified twelve published, likely disease-causing variants in 19 patients only. The core features included progressive ophthalmoplegia and cerebellar ataxia; parkinsonism, neuropathy, cognitive decline, and seizures were also repeatedly found in adult-onset heterozygous POLG2-related disease. A severe phenotype relates to biallelic pathogenic variants in POLG2, i.e., newborn-onset liver failure, referred to as mitochondrial depletion syndrome. Our work underlines the broad clinical spectrum of POLG2-related disease and highlights the importance of functional characterization of variants of uncertain significance to enable meaningful genetic counseling.

De Novo Variants in TAOK1 Cause Neurodevelopmental Disorders.

De novo variants represent a significant cause of neurodevelopmental delay and intellectual disability. A genetic basis can be identified in only half of individuals who have neurodevelopmental disorders (NDDs); this indicates that additional causes need to be elucidated. We compared the frequency of de novo variants in patient-parent trios with (n = 2,030) versus without (n = 2,755) NDDs. We identified de novo variants in TAOK1 (thousand and one [TAO] amino acid kinase 1), which encodes the serine/threonine-protein kinase TAO1, in three individuals with NDDs but not in persons who did not have NDDs. Through further screening and the use of GeneMatcher, five additional individuals with NDDs were found to have de novo variants. All eight variants were absent from gnomAD (Genome Aggregation Database). The variant carriers shared a non-specific phenotype of developmental delay, and six individuals had additional muscular hypotonia. We established a fibroblast line of one mutation carrier, and we demonstrated that reduced mRNA levels of TAOK1 could be increased upon cycloheximide treatment. These results indicate nonsense-mediated mRNA decay. Further, there was neither detectable phosphorylated TAO1 kinase nor phosphorylated tau in these cells, and mitochondrial morphology was altered. Knockdown of the ortholog gene Tao1 (Tao, CG14217) in Drosophila resulted in delayed early development. The majority of the Tao1-knockdown flies did not survive beyond the third instar larval stage. When compared to control flies, Tao1 knockdown flies revealed changed morphology of the ventral nerve cord and the neuromuscular junctions as well as a decreased number of endings (boutons). Furthermore, mitochondria in mutant flies showed altered distribution and decreased size in axons of motor neurons. Thus, we provide compelling evidence that de novo variants in TAOK1 cause NDDs.

Linking Penetrance and Transcription in DYT-THAP1: Insights From a Human iPSC-Derived Cortical Model.

BACKGROUND: The THAP1 gene encodes a transcription factor, and pathogenic variants cause a form of autosomal dominant, isolated dystonia (DYT-THAP1) with reduced penetrance. Factors underlying both reduced penetrance and the disease mechanism of DYT-THAP1 are largely unknown. METHODS: We performed transcriptome analysis on 29 cortical neuronal precursors derived from human-induced pluripotent stem cell lines generated from manifesting and nonmanifesting THAP1 mutation carriers and control individuals. RESULTS: Whole transcriptome analysis showed a penetrance-linked signature with expressional changes more pronounced in the group of manifesting (MMCs) than in nonmanifesting mutation carriers (NMCs) when compared to controls. A direct comparison of the transcriptomes in MMCs versus NMCs showed significant upregulation of the DRD4 gene in MMCs. A gene set enrichment analysis demonstrated alterations in various neurotransmitter release cycle pathways, extracellular matrix organization, and deoxyribonucleic acid methylation between MMCs and NMCs. When specifically considering transcription factors, the expression of YY1 and SIX2 differed in MMCs versus NMCs. Further, THAP1 was upregulated in the group of MMCs. CONCLUSIONS: To our knowledge, this is the first report systematically analyzing reduced penetrance in DYT-THAP1 in a human model using transcriptomes. Our findings indicate that transcriptional alterations during cortical development influence DYT-THAP1 pathogenesis and penetrance. We reinforce previously linked pathways including dopamine and eukaryotic translation initiation factor 2 alpha signaling in the pathogenesis of dystonia including DYT-THAP1 and suggest extracellular matrix organization and deoxyribonucleic acid methylation as mediators of disease protection. © 2021 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Genotype-Phenotype Relations for Isolated Dystonia Genes: MDSGene Systematic Review.

This comprehensive MDSGene review is devoted to 7 genes - TOR1A, THAP1, GNAL, ANO3, PRKRA, KMT2B, and HPCA - mutations in which may cause isolated dystonia. It followed MDSGene's standardized data extraction protocol and screened a total of ~1200 citations. Phenotypic and genotypic data on ~1200 patients with 254 different mutations were curated and analyzed. There were differences regarding age at onset, site of onset, and distribution of symptoms across mutation carriers in all 7 genes. Although carriers of TOR1A, THAP1, PRKRA, KMT2B, or HPCA mutations mostly showed childhood and adolescent onset, patients with GNAL and ANO3 mutations often developed first symptoms in adulthood. GNAL and KMT2B mutation carriers frequently have 1 predominant site of onset, that is, the neck (GNAL) or the lower limbs (KMT2B), whereas site of onset in DYT-TOR1A, DYT-THAP1, DYT-ANO3, DYT-PRKRA, and DYT-HPCA was broader. However, in most DYT-THAP1 and DYT-ANO3 patients, dystonia first manifested in the upper half of the body (upper limb, neck, and craniofacial/laryngeal), whereas onset in DYT-TOR1A, DYT-PRKRA and DYT-HPCA was frequently observed in an extremity, including both upper and lower ones. For ANO3, a segmental/multifocal distribution was typical, whereas TOR1A, PRKRA, KMT2B, and HPCA mutation carriers commonly developed generalized dystonia. THAP1 mutation carriers presented with focal, segmental/multifocal, or generalized dystonia in almost equal proportions. GNAL mutation carriers rarely showed generalization. This review provides a comprehensive overview of the current knowledge of hereditary isolated dystonia. The data are also available in an online database (http://www.mdsgene.org), which additionally offers descriptive summary statistics. © 2021 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Novel GNB1 mutations disrupt assembly and function of G protein heterotrimers and cause global developmental delay in humans.

Global developmental delay (GDD), often accompanied by intellectual disability, seizures and other features is a severe, clinically and genetically highly heterogeneous childhood-onset disorder. In cases where genetic causes have been identified, de novo mutations in neuronally expressed genes are a common scenario. These mutations can be best identified by exome sequencing of parent-offspring trios. De novo mutations in the guanine nucleotide-binding protein, beta 1 (GNB1) gene, encoding the Gß1 subunit of heterotrimeric G proteins, have recently been identified as a novel genetic cause of GDD. Using exome sequencing, we identified 14 different novel variants (2 splice site, 2 frameshift and 10 missense changes) in GNB1 in 16 pediatric patients. One mutation (R96L) was recurrently found in three ethnically diverse families with an autosomal dominant mode of inheritance. Ten variants occurred de novo in the patients. Missense changes were functionally tested for their pathogenicity by assaying the impact on complex formation with Gγ and resultant mutant Gßγ with Gα. Signaling properties of G protein complexes carrying mutant Gß1 subunits were further analyzed by their ability to couple to dopamine D1R receptors by real-time bioluminescence resonance energy transfer (BRET) assays. These studies revealed altered functionality of the missense mutations R52G, G64V, A92T, P94S, P96L, A106T and D118G but not for L30F, H91R and K337Q. In conclusion, we demonstrate a pathogenic role of de novo and autosomal dominant mutations in GNB1 as a cause of GDD and provide insights how perturbation in heterotrimeric G protein function contributes to the disease.

Utility and implications of exome sequencing in early-onset Parkinson's disease.

BACKGROUND: Although the genetic load is high in early-onset Parkinson's disease, thorough investigation of the genetic diagnostic yield has yet to be established. The objectives of this study were to assess variants in known genes for PD and other movement disorders and to find new candidates in 50 patients with early-onset PD. METHODS: We searched for variants either within genes listed by the International Parkinson and Movement Disorder Society Task Force on Genetic Nomenclature or rare homozygous variants in novel candidate genes. Further, exome data from 1148 European PD patients (International Parkinson Disease Genomics Consortium) were used for association testing. RESULTS: Seven patients (14%) carried pathogenic or likely pathogenic variants in Parkin, PLA2G6, or GBA. In addition, rare missense variants in DNAJC13:p.R1830C and in PPM1K:p.Y352C were detected. SPG7:p.A510V and PPM1K:p.Y352C revealed significant association with PD risk (P < 0.05). CONCLUSIONS: Although we identified pathogenic variants in 14% of our early-onset PD patients, the majority remain unexplained, and novel candidates need to be validated independently to better further evaluate their role in PD. © 2018 International Parkinson and Movement Disorder Society.

Spinocerebellar Ataxia Type 28-Phenotypic and Molecular Characterization of a Family with Heterozygous and Compound-Heterozygous Mutations in AFG3L2.

While heterozygous mutations in the AFG3L2 gene have been linked to spinocerebellar ataxia 28 (SCA28), homozygous mutations in the same gene can cause spastic ataxia 5 (SPAX5). AFG3L2 encodes a mitochondrial ATP-dependent metalloprotease. We here report a SCA28 patient with biallelic AFG3L2 variants and his heterozygous mother. The patient and his mother underwent a detailed neurological examination and fibroblast lines were established. The effect of the two missense variants on mitochondria was assessed by form factor analysis and quantification of mitochondrial proteins (TOMM70, complex V). The 39-year-old index patient presented with a slowly progressive cerebellar gait disorder for 19 years, bilateral ptosis, and dysarthria. A cranial MRI showed mild cerebellar atrophy. He carried two compound-heterozygous, rare, missense variants (c.1847A>G [p.Y616C], c.2167G>A [p.V723M]) in AFG3L2, while his mother was heterozygous for the first change that had previously been described in SPAX5. Altered mitochondrial morphology and interconnectivity, together with reduced protein levels of TOMM70 and complex V (ATPase), suggest mitochondrial structural defects in the patient's fibroblasts. No significant abnormalities were found in his mother's fibroblast cultures albeit all measurements were slightly below the control level. We here present a SCA28 patient with compound-heterozygous AFG3L2 variants and demonstrate mitochondrial abnormalities in skin fibroblast cultures from this patient. Thus, AFG3L2 variants should be considered in both slowly progressive ataxias and phenotypes with clinical features reminiscent of mitochondrial disease. Of note, ptosis was present in both mutation carriers and may serve as a red flag in the diagnosis of SCA28.

Reduced Expression of GABA A Receptor Alpha2 Subunit Is Associated With Disinhibition of DYT-THAP1 Dystonia Patient-Derived Striatal Medium Spiny Neurons.

DYT-THAP1 dystonia (formerly DYT6) is an adolescent-onset dystonia characterized by involuntary muscle contractions usually involving the upper body. It is caused by mutations in the gene THAP1 encoding for the transcription factor Thanatos-associated protein (THAP) domain containing apoptosis-associated protein 1 and inherited in an autosomal-dominant manner with reduced penetrance. Alterations in the development of striatal neuronal projections and synaptic function are known from transgenic mice models. To investigate pathogenetic mechanisms, human induced pluripotent stem cell (iPSC)-derived medium spiny neurons (MSNs) from two patients and one family member with reduced penetrance carrying a mutation in the gene THAP1 (c.474delA and c.38G > A) were functionally characterized in comparison to healthy controls. Calcium imaging and quantitative PCR analysis revealed significantly lower Ca2+ amplitudes upon GABA applications and a marked downregulation of the gene encoding the GABA A receptor alpha2 subunit in THAP1 MSNs indicating a decreased GABAergic transmission. Whole-cell patch-clamp recordings showed a significantly lower frequency of miniature postsynaptic currents (mPSCs), whereas the frequency of spontaneous action potentials (APs) was elevated in THAP1 MSNs suggesting that decreased synaptic activity might have resulted in enhanced generation of APs. Our molecular and functional data indicate that a reduced expression of GABA A receptor alpha2 subunit could eventually lead to limited GABAergic synaptic transmission, neuronal disinhibition, and hyperexcitability of THAP1 MSNs. These data give pathophysiological insight and may contribute to the development of novel treatment strategies for DYT-THAP1 dystonia.

Altered homodimer formation and increased iron accumulation in VAC14-related disease: Case report and review of the literature.

BACKGROUND: Pathogenic variants in the VAC14 component of PIKFYVE complex (VAC14) gene have been identified as a cause of a childhood-onset complex dystonia with striato-nigral degeneration. VAC14 is a scaffold protein relevant for the regulation of phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) and is known to form homodimers. METHODS: Whole exome sequencing was performed in a 32-year-old patient with adolescence-onset complex dystonia and his unaffected mother. We established primary fibroblast cultures from the patient and used stably transfected SH-SY5Y cells overexpressing wildtype or mutant VAC14 to investigate the influence of VAC14 variants on the homodimer formation. Furthermore, the current literature on VAC14-related disorders was reviewed. RESULTS: Our patient presented with progressive, complex dystonia with anarthria, dysphagia, sensorineural deafness, spasticity and nigral and pallidal iron deposition and striatal hyperintensities upon MRI. We identified two rare compound-heterozygous VAC14 variants (p.Leu648Phe and p.Arg623His), both located at the C-terminus in the predicted homodimerization domain. Enhanced VAC14 homodimer formation was observed for two missense variants (p.Leu648Phe and p.Ala562Val, a published mutation), but not for p.Arg623His, compared to wildtype VAC14. In contrast to previous reports, no enlarged vacuoles were detected in fibroblasts of our patient. CONCLUSIONS: We report a novel patient with a VAC14-related disorder and provide first evidence of an enhanced VAC14 homodimerization as a possible disease mechanism. Due to the increased iron deposition and the clinical overlap, this disorder should be discussed as a new form of neurodegeneration with brain iron accumulation (NBIA). We suggest that VAC14 should be implemented in NBIA gene panels.

Generation and characterization of eight human-derived iPSC lines from affected and unaffected THAP1 mutation carriers.

Mutations in THAP1 (THAP domain-containing apoptosis-associated protein 1) cause a form of early-onset, isolated dystonia (DYT-THAP1, aka DYT6). Here, we describe the generation of eight human induced pluripotent stem cell (iPSC) lines of manifesting and non-manifesting carriers of the THAP1 mutations p.Lys158Asnfs*23 or p.Arg13His (each 4 lines). Dermal fibroblasts were reprogrammed using non-integrating Sendai virus. The iPSC lines were comprehensively characterized including expression analyses of pluripotency markers, the potential to differentiate into cells of all three germ layers, and stable karyotypes. These lines provide a valuable resource for studying the impact of THAP1 mutations on the pathology of dystonia.