Two newly defined inherited disorders due to cholinergic transporter dysfunction with distinct clinical outcomes, disease mechanisms and modes of inheritance.

Neurodegenerative diseases are becoming increasingly prevalent due to the ageing population, and are among the major contributors to disability and disease worldwide. The identification of the gene defects responsible for many of these conditions has played a major role in our understanding of the pathogenic processes involved, and provided opportunity to develop targeted treatment strategies. Cholinergic neurotransmission supports a wide range of physiological and behavioural processes and its dysfunction of cholinergic signalling has been associated with a number of disorders, including myasthenias, cardiovascular disease(1), attention-deficit hyperactivity disorder (ADHD) (2), Alzheimer’s disease (ADi), schizophrenia, addiction(3), and depression(4). SLC5A7 encodes the Na+/Cl- dependent, high-affinity choline transporter (CHT) which represents the rate limiting step in acetylcholine (Ach) synthesis and is critical for normal cholinergic signalling. The work in this thesis details two new inherited disorders, caused by distinct pathogenic disease mechanisms, associated with novel SLC5A7 mutations. Chapter three documents the discovery of two autosomal-dominantly acting SLC5A7/CHT mutations associated with adult onset motor neurone disorders. Initially we identified a frameshift mutation that results in premature truncation of the transporter protein in a large Welsh kindred affected with distal hereditary motor neuropathy type VII (dHMN-VII), in which neurodegeneration and muscle paresis is largely restricted to the distal limb muscles and vocal cords. The mutation responsible results in the dominant-negative interference of the mutant molecule with function of the wild type choline transporter, resulting in significantly reduced (although not completely abolished) transporter activity. This finding is further evidenced by the discovery of a second dHMN family associated with a distinct frameshift SLC5A7 mutation indicative of a similar dominant-negative disease mechanism. Together these findings corroborate a dominant-negative disease mechanism arising from C-terminal truncating SLC5A7 mutations associated with dHMN, and provide further insight into the role of aberrant choline transporter function in neurological disease. Chapter four describes N-terminal missense mutations located in the transmembrane spanning region of SLC5A7/CHT, associated with a severe infantile neuromuscular disorder characterised by predominantly central hypotonia and developmental delay. The phenotypic effects of these mutations are likely to result from the near abolition of CHT-mediated choline transport in homozygous individuals, and are in keeping with those observed in CHT knock-out mouse models(5). The development of a mouse model of the human motor neurone disease arising from SLC5A7 frameshift mutations should allow for further investigation of the mechanism by which truncated CHT leads to the dHMN phenotype. Chapter 5 details treatment hypotheses for dHMN, as well as the generation of a patient-specific knock-in mouse model carrying an Slc5a7 mutation orthologous to that identified in dHMN-VII families in chapter 3, and results from preliminary neurological phenotyping of the mouse model. This model will be crucially important for the exploration of treatment options in dHMN-VII motor neurone disease as a prelude to clinical trials in humans.Neurosciences Research FoundationJohn McDowall Bursary (for MND research

Loss-of-Function Variants in DRD1 in Infantile Parkinsonism-Dystonia

The human dopaminergic system is vital for a broad range of neurological processes, including the control of voluntary movement. Here we report a proband presenting with clinical features of dopamine deficiency: severe infantile parkinsonism-dystonia, characterised by frequent oculogyric crises, dysautonomia and global neurodevelopmental impairment. CSF neurotransmitter analysis was unexpectedly normal. Triome whole-genome sequencing revealed a homozygous variant (c.110C>A, (p.T37K)) in DRD1, encoding the most abundant dopamine receptor (D1) in the central nervous system, most highly expressed in the striatum. This variant was absent from gnomAD, with a CADD score of 27.5. Using an in vitro heterologous expression system, we determined that DRD1-T37K results in loss of protein function. Structure-function modelling studies predicted reduced substrate binding, which was confirmed in vitro. Exposure of mutant protein to the selective D1 agonist Chloro APB resulted in significantly reduced cyclic AMP levels. Numerous D1 agonists failed to rescue the cellular defect, reflected clinically in the patient, who had no benefit from dopaminergic therapy. Our study identifies DRD1 as a new disease-associated gene, suggesting a crucial role for the D1 receptor in motor control

Defective Presynaptic Choline Transport Underlies Hereditary Motor Neuropathy

The neuromuscular junction (NMJ) is a specialized synapse with a complex molecular architecture that provides for reliable transmission between the nerve terminal and muscle fiber. Using linkage analysis and whole-exome sequencing of DNA samples from subjects with distal hereditary motor neuropathy type VII, we identified a mutation in SLC5A7, which encodes the presynaptic choline transporter (CHT), a critical determinant of synaptic acetylcholine synthesis and release at the NMJ. This dominantly segregating SLC5A7 mutation truncates the encoded product just beyond the final transmembrane domain, eliminating cytosolic-C-terminus sequences known to regulate surface transporter trafficking. Choline-transport assays in both transfected cells and monocytes from affected individuals revealed significant reductions in hemicholinium-3-sensitive choline uptake, a finding consistent with a dominant-negative mode of action. The discovery of CHT dysfunction underlying motor neuropathy identifies a biological basis for this group of conditions and widens the spectrum of disorders that derive from impaired NMJ transmission. Our findings compel consideration of mutations in SLC5A7 or its functional partners in relation to unexplained motor neuronopathies

The Genetic Landscape of Complex Childhood-Onset Hyperkinetic Movement Disorders

Acord transformatiu CRUE-CSICThis work was supported by an NIHR Professorship (to M.A.K.). M.A.K. has received funding from the Sir Jules Thorn Award for Biomedical Research and Wellcome Trust. B.P.-D. was supported by Instituto de Salud Carlos III, PI 18/01319 and PI21/00248, and has received funding from Beca José Castillejos (CAS14/00328). K.J.P. was supported by an MRC Clinician-Scientist Fellowship (511015) and was supported by the Dystonia Medical Research Foundation and Fight for Sight. S.S.M. has received funding from the Winston Churchill Memorial trust and Cerebral Palsy Alliance.Background and Objective: The objective of this study was to better delineate the genetic landscape and key clinical characteristics of complex, early-onset, monogenic hyperkinetic movement disorders. Methods: Patients were recruited from 14 international centers. Participating clinicians completed standardized proformas capturing demographic, clinical, and genetic data. Two pediatric movement disorder experts reviewed available video footage, classifying hyperkinetic movements according to published criteria. Results: One hundred forty patients with pathogenic variants in 17 different genes (ADCY5, ATP1A3, DDC, DHPR, FOXG1, GCH1, GNAO1, KMT2B, MICU1, NKX2.1, PDE10A, PTPS, SGCE, SLC2A1, SLC6A3, SPR, and TH) were identified. In the majority, hyperkinetic movements were generalized (77%), with most patients (69%) manifesting combined motor semiologies. Parkinsonism-dystonia was characteristic of primary neurotransmitter disorders (DDC, DHPR, PTPS, SLC6A3, SPR, TH); chorea predominated in ADCY5-, ATP1A3-, FOXG1-, NKX2.1-, SLC2A1-, GNAO1-, and PDE10A-related disorders; and stereotypies were a prominent feature in FOXG1- and GNAO1-related disease. Those with generalized hyperkinetic movements had an earlier disease onset than those with focal/segmental distribution (2.5 ± 0.3 vs. 4.7 ± 0.7 years; P = 0.007). Patients with developmental delay also presented with hyperkinetic movements earlier than those with normal neurodevelopment (1.5 ± 2.9 vs. 4.7 ± 3.8 years; P < 0.001). Effective disease-specific therapies included dopaminergic agents for neurotransmitters disorders, ketogenic diet for glucose transporter deficiency, and deep brain stimulation for SGCE-, KMT2B-, and GNAO1-related hyperkinesia. Conclusions: This study highlights the complex phenotypes observed in children with genetic hyperkinetic movement disorders that can lead to diagnostic difficulty. We provide a comprehensive analysis of motor semiology to guide physicians in the genetic investigation of these patients, to facilitate early diagnosis, precision medicine treatments, and genetic counseling. © 2022 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society

Novel homozygous missense mutation in GAN associated with Charcot-Marie-Tooth disease type 2 in a large consanguineous family from Israel.

BACKGROUND: CMT-2 is a clinically and genetically heterogeneous group of peripheral axonal neuropathies characterized by slowly progressive weakness and atrophy of distal limb muscles resulting from length-dependent motor and sensory neurodegeneration. Classical giant axonal neuropathy (GAN) is an autosomal recessively inherited progressive neurodegenerative disorder of the peripheral and central nervous systems, typically diagnosed in early childhood and resulting in death by the end of the third decade. Distinctive phenotypic features are the presence of "kinky" hair and long eyelashes. The genetic basis of the disease has been well established, with over 40 associated mutations identified in the gene GAN, encoding the BTB-KELCH protein gigaxonin, involved in intermediate filament regulation. METHODS: An Illumina Human CytoSNP-12 array followed by whole exome sequence analysis was used to identify the disease associated gene mutation in a large consanguineous family diagnosed with Charcot-Marie-Tooth disease type 2 (CMT-2) from which all but one affected member had straight hair. RESULTS: Here we report the identification of a novel GAN missense mutation underlying the CMT-2 phenotype observed in this family. Although milder forms of GAN, with and without the presence of kinky hair have been reported previously, a phenotype distinct from that was investigated in this study. All family members lacked common features of GAN, including ataxia, nystagmus, intellectual disability, seizures, and central nervous system involvement. CONCLUSIONS: Our findings broaden the spectrum of phenotypes associated with GAN mutations and emphasize a need to proceed with caution when providing families with diagnostic or prognostic information based on either clinical or genetic findings alone

TNPO2 variants associate with human developmental delays, neurologic deficits, and dysmorphic features and alter TNPO2 activity in Drosophila

Transportin-2 (TNPO2) mediates multiple pathways including non-classical nucleocytoplasmic shuttling of >60 cargoes, such as developmental and neuronal proteins. We identified 15 individuals carrying de novo coding variants in TNPO2 who presented with global developmental delay (GDD), dysmorphic features, ophthalmologic abnormalities, and neurological features. To assess the nature of these variants, functional studies were performed in Drosophila. We found that fly dTnpo (orthologous to TNPO2) is expressed in a subset of neurons. dTnpo is critical for neuronal maintenance and function as downregulating dTnpo in mature neurons using RNAi disrupts neuronal activity and survival. Altering the activity and expression of dTnpo using mutant alleles or RNAi causes developmental defects, including eye and wing deformities and lethality. These effects are dosage dependent as more severe phenotypes are associated with stronger dTnpo loss. Interestingly, similar phenotypes are observed with dTnpo upregulation and ectopic expression of TNPO2, showing that loss and gain of Transportin activity causes developmental defects. Further, proband-associated variants can cause more or less severe developmental abnormalities compared to wild-type TNPO2 when ectopically expressed. The impact of the variants tested seems to correlate with their position within the protein. Specifically, those that fall within the RAN binding domain cause more severe toxicity and those in the acidic loop are less toxic. Variants within the cargo binding domain show tissue-dependent effects. In summary, dTnpo is an essential gene in flies during development and in neurons. Further, proband-associated de novo variants within TNPO2 disrupt the function of the encoded protein. Hence, TNPO2 variants are causative for neurodevelopmental abnormalities

The Genetic Landscape of Complex Childhood-Onset Hyperkinetic Movement Disorders

Background and Objective: The objective of this study was to better delineate the genetic landscape and key clinical characteristics of complex, early-onset, monogenic hyperkinetic movement disorders. Methods: Patients were recruited from 14 international centers. Participating clinicians completed standardized proformas capturing demographic, clinical, and genetic data. Two pediatric movement disorder experts reviewed available video footage, classifying hyperkinetic movements according to published criteria. Results: One hundred forty patients with pathogenic variants in 17 different genes (ADCY5, ATP1A3, DDC, DHPR, FOXG1, GCH1, GNAO1, KMT2B, MICU1, NKX2.1, PDE10A, PTPS, SGCE, SLC2A1, SLC6A3, SPR, and TH) were identified. In the majority, hyperkinetic movements were generalized (77%), with most patients (69%) manifesting combined motor semiologies. Parkinsonism-dystonia was characteristic of primary neurotransmitter disorders (DDC, DHPR, PTPS, SLC6A3, SPR, TH); chorea predominated in ADCY5-, ATP1A3-, FOXG1-, NKX2.1-, SLC2A1-, GNAO1-, and PDE10A-related disorders; and stereotypies were a prominent feature in FOXG1- and GNAO1-related disease. Those with generalized hyperkinetic movements had an earlier disease onset than those with focal/segmental distribution (2.5 +/- 0.3 vs. 4.7 +/- 0.7 years; P = 0.007). Patients with developmental delay also presented with hyperkinetic movements earlier than those with normal neurodevelopment (1.5 +/- 2.9 vs. 4.7 +/- 3.8 years; P < 0.001). Effective disease-specific therapies included dopaminergic agents for neurotransmitters disorders, ketogenic diet for glucose transporter deficiency, and deep brain stimulation for SGCE-, KMT2B-, and GNAO1-related hyperkinesia. Conclusions: This study highlights the complex phenotypes observed in children with genetic hyperkinetic movement disorders that can lead to diagnostic difficulty. We provide a comprehensive analysis of motor semiology to guide physicians in the genetic investigation of these patients, to facilitate early diagnosis, precision medicine treatments, and genetic counseling. (C) 2022 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder SocietyNIHR Professorship; Sir Jules Thorn Award for Biomedical Research and Wellcome Trust; Instituto de Salud Carlos III [PI 18/01319, PI21/00248]; MRC Clinician-Scientist Fellowship [511015]; Dystonia Medical Research Foundation; Fight for Sight; Winston Churchill Memorial trust and Cerebral Palsy Alliance; Beca Jose Castillejos [CAS14/00328]This work was supported by an NIHR Professorship (to M.A.K.). M.A.K. has received funding from the Sir Jules Thorn Award for Biomedical Research and Wellcome Trust. B.P.-D. was supported by Instituto de Salud Carlos III, PI 18/01319 and PI21/00248, and has received funding from Beca Jose Castillejos (CAS14/00328). K.J.P. was supported by an MRC Clinician-Scientist Fellowship (511015) and was supported by the Dystonia Medical Research Foundation and Fight for Sight. S.S.M. has received funding from the Winston Churchill Memorial trust and Cerebral Palsy Alliance

Comparison of methylation episignatures in KMT2B- and KMT2D-related human disorders.

Funder: Rosetrees TrustAim & methods: To investigate peripheral blood methylation episignatures in KMT2B-related dystonia (DYT-KMT2B), the authors undertook genome-wide methylation profiling of ∼2 M CpGs using a next-generation sequencing-based assay and compared the findings with those in controls and patients with KMT2D-related Kabuki syndrome type 1 (KS1). Results: A total of 1812 significantly differentially methylated CpG positions (false discovery rate < 0.05) were detected in DYT-KMT2B samples compared with controls. Multi-dimensional scaling analysis showed that the 10 DYT-KMT2B samples clustered together and separately from 29 controls and 10 with pathogenic variants in KMT2D. The authors found that most differentially methylated CpG positions were specific to one disorder and that all (DYT-KMT2B) and most (Kabuki syndrome type 1) methylation alterations in CpG islands were gain of methylation events. Conclusion: Using sensitive methylation profiling methodology, the authors replicated recent reports of a methylation episignature for DYT-KMT2B. These findings will facilitate the development of episignature-based assays to improve diagnostic accuracy