Bi-allelic variants in TSPOAP1, encoding the active zone protein RIMBP1, cause autosomal recessive dystonia

Dystonia is a debilitating hyperkinetic movement disorder, which can be transmitted as a monogenic trait. Here, we describe homozygous frameshift, nonsense and missense variants in TSPOAP1, encoding the active zone RIM-binding protein 1 (RIMBP1), as a novel genetic cause of autosomal recessive dystonia in seven subjects from three unrelated families. Subjects carrying loss-of-function variants presented with juvenile-onset progressive generalized dystonia, associated with intellectual disability and cerebellar atrophy. Conversely, subjects carrying a pathogenic missense variant (p.Gly1808Ser) presented with isolated adult-onset focal dystonia. In mice, complete loss of RIMBP1, known to reduce neurotransmission, led to motor abnormalities reminiscent of dystonia, decreased Purkinje cell dendritic arborization, and reduced numbers of cerebellar synapses. In vitro analysis of the p.Gly1808Ser variant showed larger spike-evoked calcium transients and enhanced neurotransmission, suggesting that RIMBP1-linked dystonia can be caused by either reduced or enhanced rates of spike-evoked release in relevant neural networks. Our findings establish a direct link between dysfunction of the presynaptic active zone and dystonia and highlight the critical role played by well-balanced neurotransmission in motor control and disease pathogenesis

Exploring next generation sequencing in the diagnosis of inherited ataxia and other neurological diseases

Neurodegenerative diseases are a complex and heterogeneous group of disorders characterised by progressive loss of cells from the central nervous system which can result in a broad range of clinical phenotypes including cognitive impairment, motor dysfunction, epilepsy and visual loss. Next generation sequencing technologies have greatly facilitated the search for novel genes underlying neurodegenerative disorders. In this thesis, I present the results of my efforts to understand and clarify the genetic bases for a range of neurological disorders with a focus on cerebellar ataxia. I have used a range of techniques including whole exome sequencing, together with traditional genetic approaches to investigate several families with hitherto genetically undiagnosed neurological syndromes. The first project I present is the identification of a novel gene (TUBB4A) underlying a form of dominantly inherited dystonia (DYT4). With the understanding I gained in the application of exome sequencing and mapping strategies, I go on to present my findings of a further novel gene underlying a recessive cerebellar ataxia (CAPN1). I describe the identification of the genetic cause in several families with recessive ataxia and explore functional studies to confirm the pathogenicity of novel mutations in a rare form of neuronal ceroid lipofuscinosis (CLN10). I present a study in which I developed a next-generation sequencing panel for large scale sequencing of a cohort of autosomal dominant ataxia patients. Finally, I present data on the identification of PNPLA6 mutations as the genetic cause of a rare form of spastic ataxia associated with hypogonadrotropic hypogonadism – Oliver McFarlane syndrome

Combined genomics and proteomics unveils elusive variants and vast aetiologic heterogeneity in dystonia

Dystonia is a rare-disease trait for which large-scale genomic investigations are still underrepresented. Genetic heterogeneity among patients with unexplained dystonia warrants interrogation of entire genome sequences, but this has not yet been systematically evaluated. To significantly enhance our understanding of the genetic contribution to dystonia, we (re)analyzed 2,874 whole-exome sequencing (WES), 564 whole-genome sequencing (WGS), as well as 80 fibroblast-derived proteomics datasets, representing the output of high-throughput analyses in 1,990 patients and 973 unaffected relatives from 1,877 families. Recruitment and precision-phenotyping procedures were driven by long-term collaborations of international experts with access to overlooked populations. By exploring WES data, we found that continuous scaling of sample sizes resulted in steady gains in the number of associated disease genes without plateauing. On average, every second diagnosis involved a gene not previously implicated in our cohort. Second-line WGS focused on a subcohort of undiagnosed individuals with high likelihood of having monogenic forms of dystonia, comprising large proportions of patients with early onset (81.3%), generalized symptom distribution (50.8%) and/or coexisting features (68.9%). We undertook extensive searches for variants in nuclear and mitochondrial genomes to uncover 38 (ultra)rare diagnostic-grade findings in 37 of 305 index patients (12.1%), many of which had remained undetected due to methodological inferiority of WES or pipeline limitations. WGS-identified elusive variations included alterations in exons poorly covered by WES, RNA-gene variants, mitochondrial-DNA mutations, small copy-number variants, complex rearranged genome structure, and short tandem repeats. For improved variant interpretation in WGS-inconclusive cases, we employed systematic integration of quantitative proteomics. This aided in verifying diagnoses related to technically challenging variants and in upgrading a variant of uncertain significance (3 of 70 WGS-inconclusive index patients, 4.3%). Further, unsupervised proteomic outlier-analysis supplemented with transcriptome sequencing revealed pathological gene underexpression induced by transcript disruptions in three more index patients with underlying (deep) intronic variants (3/70, 4.3%), highlighting the potential for targeted antisense-oligonucleotide therapy development. Finally, trio-WGS prioritized a de-novo missense change in the candidate PRMT1, encoding a histone-methyltransferase. Data-sharing strategies supported the discovery of three distinct PRMT1 de-novo variants in four phenotypically similar patients, associated with loss-of-function effects in in-vitro assays. This work underscores the importance of continually expanding sequencing cohorts to characterize the extensive spectrum of gene aberrations in dystonia. We show that a pool of unresolved cases is amenable to WGS and complementary multi-omic studies, directing advanced etiopathological concepts and future diagnostic-practice workflows for dystonia

Challenges in clinicogenetic correlations: one gene - many phenotypes

Background: Progress in genetics - particularly the advent of next-generation sequencing (NGS) - has enabled an unparalleled gene discovery and revealed unmatched complexity of genotype-phenotype correlations in movement disorders. Among other things, it has emerged that mutations in one and the same gene can cause multiple, often markedly different phenotypes. Consequently, movement disorder specialists have increasingly experienced challenges in clinicogenetic correlations. Objectives: To deconstruct biological phenomena and mechanistic bases of phenotypic heterogeneity in monogenic movement disorders and neurodegenerative diseases. To discuss the evolving role of movement disorder specialists in reshaping disease phenotypes in the NGS era. Methods: This scoping review details phenomena contributing to phenotypic heterogeneity and their underlying mechanisms. Results: Three phenomena contribute to phenotypic heterogeneity, namely incomplete penetrance, variable expressivity and pleiotropy. Their underlying mechanisms, which are often shared across phenomena and non-mutually exclusive, are not fully elucidated. They involve genetic factors (ie, different mutation types, dynamic mutations, somatic mosaicism, intragenic intra- and inter-allelic interactions, modifiers and epistatic genes, mitochondrial heteroplasmy), epigenetic factors (ie, genomic imprinting, X-chromosome inactivation, modulation of genetic and chromosomal defects), and environmental factors. Conclusion: Movement disorders is unique in its reliance on clinical judgment to accurately define disease phenotypes. This has been reaffirmed by the NGS revolution, which provides ever-growing sequencing data and fuels challenges in variant pathogenicity assertions for such clinically heterogeneous disorders. Deep phenotyping, with characterization and continual updating of "core" phenotypes, and comprehension of determinants of genotype-phenotype complex relationships are crucial for clinicogenetic correlations and have implications for the diagnosis, treatment and counseling

Atypical Ataxia Presentation in Variant Ataxia Telangiectasia: Iranian Case-Series and Review of the Literature

Ataxia-telangiectasia (AT) is a rare autosomal recessive neurodegenerative multisystem disorder. A minority of AT patients can present late-onset atypical presentations due to unknown mechanisms. The demographic, clinical, immunological and genetic data were collected by direct interview and examining the Iranian AT patients with late-onset manifestations. We also conducted a systematic literature review for reported atypical AT patients. We identified three Iranian AT patients (3/249, 1.2% of total registry) with later age at ataxia onset and slower neurologic progression despite elevated alpha-fetoprotein levels, history of respiratory infections, and immunological features of the syndrome. Of note, all patients developed autoimmunity in which a decrease of naïve T cells and regulatory T cells were observed. The literature searches also summarized data from 73 variant AT patients with atypical presentation indicating biallelic mild mutations mainly lead to an atypical phenotype with an increased risk of cancer. Variant AT patients present with milder phenotype or atypical form of classical symptoms causing under- or mis- diagnosis. Although missense mutations are more frequent, an atypical presentation can be associated with deleterious mutations due to unknown modifying factors

The Classification of Autosomal Recessive Cerebellar Ataxias : a Consensus Statement from the Society for Research on the Cerebellum and Ataxias Task Force

There is currently no accepted classification of autosomal recessive cerebellar ataxias, a group of disorders characterized by important genetic heterogeneity and complex phenotypes. The objective of this task force was to build a consensus on the classification of autosomal recessive ataxias in order to develop a general approach to a patient presenting with ataxia, organize disorders according to clinical presentation, and define this field of research by identifying common pathogenic molecular mechanisms in these disorders. The work of this task force was based on a previously published systematic scoping review of the literature that identified autosomal recessive disorders characterized primarily by cerebellar motor dysfunction and cerebellar degeneration. The task force regrouped 12 international ataxia experts who decided on general orientation and specific issues. We identified 59 disorders that are classified as primary autosomal recessive cerebellar ataxias. For each of these disorders, we present geographical and ethnical specificities along with distinctive clinical and imagery features. These primary recessive ataxias were organized in a clinical and a pathophysiological classification, and we present a general clinical approach to the patient presenting with ataxia. We also identified a list of 48 complex multisystem disorders that are associated with ataxia and should be included in the differential diagnosis of autosomal recessive ataxias. This classification is the result of a consensus among a panel of international experts, and it promotes a unified understanding of autosomal recessive cerebellar disorders for clinicians and researchers

De novo and biallelic DEAF1 variants cause a phenotypic spectrum.

PURPOSE: To investigate the effect of different DEAF1 variants on the phenotype of patients with autosomal dominant and recessive inheritance patterns and on DEAF1 activity in vitro. METHODS: We assembled a cohort of 23 patients with de novo and biallelic DEAF1 variants, described the genotype-phenotype correlation, and investigated the differential effect of de novo and recessive variants on transcription assays using DEAF1 and Eif4g3 promoter luciferase constructs. RESULTS: The proportion of the most prevalent phenotypic features, including intellectual disability, speech delay, motor delay, autism, sleep disturbances, and a high pain threshold, were not significantly different in patients with biallelic and pathogenic de novo DEAF1 variants. However, microcephaly was exclusively observed in patients with recessive variants (p < 0.0001). CONCLUSION: We propose that different variants in the DEAF1 gene result in a phenotypic spectrum centered around neurodevelopmental delay. While a pathogenic de novo dominant variant would also incapacitate the product of the wild-type allele and result in a dominant-negative effect, a combination of two recessive variants would result in a partial loss of function. Because the clinical picture can be nonspecific, detailed phenotype information, segregation, and functional analysis are fundamental to determine the pathogenicity of novel variants and to improve the care of these patients

Using next-generation sequencing to understand the aetiology of dystonia and other neurological diseases

This thesis presents my work using both next-generation and traditional genetic techniques aimed at further clarifying the aetiology of hereditary neurological disorders, with a particular focus on dystonia. A large part of this was focused on the identification, clinical phenotyping, and genetic analysis of kindreds with neurological disease inherited in a Mendelian fashion but for which no causal mutation had yet been identified. My work led directly to the discovery of two new dystonia genes, ANO3 and HPCA, and the identification of two novel phenotypes for the known disease-associated genes, ATM and NUBPL. Mutations in a third novel gene, SLC25A46, was identified as the most likely cause of disease in a kindred with a complex neurological disorder consisting of optic atrophy, severe action myoclonus and peripheral neuropathy, but could not be confirmed due to lack of a second segregating kindred – it was published, a year and a half after we had first identified it, by another group, just as I was in the process of submitting thesis. Taken together, these results confirm that whole exome sequencing in combination with linkage analysis or homozygosity mapping represents a powerful means of dissecting out the genetic aetiology of Mendelian disease. In addition, this thesis summarises my foray in the world of association analyses, a technique which underpins the recent explosion in knowledge regarding the genetic architecture of so-called ‘complex’ disease. I use this technique to show that the association signal over MAPT in Parkinson’s disease survives when affectation status is defined neuropathologically. Finally, I present my work using traditional Sanger sequencing to better understand the prevalence of already published Mendelian disease genes for both dystonia and Parkinson’s disease