Dissecting the genetic basis of neurodevelopmental disorders and demyelinating neuropathies

The understanding of the pathophysiology of most rare, complex neurological disorders has been elusive, especially in the case of complex demyelinating neuropathies and neurodevelopmental disorders. In my work, I learnt to employ two main techniques that will help advance the search for better understanding of neurodevelopmental disorders: next generation sequencing and functional validation of rare genetic variants. The main aim of my research was to establish the genetic diagnosis in several patients affected by complex syndromes such as peripheral neuropathy with central nervous system involvement (Chapter 3), neurodevelopmental disorders (Chapter 4) and epilepsy (Chapter 5). The phenotypic and genotypic correlations of identified gene variants were investigated in these chapters and is a profound theme in my project. To achieve this, an integrated approach combining next generation sequencing (NGS) technology, homozygosity mapping, array genotyping, traditional Sanger sequencing and functional experiments was undertaken. Firstly, I describe the work performed in an attempt to identify the causative gene in a cohort of young children presented with an early-onset hereditary form of chronic inflammatory demyelinating polyneuropathy with a central and peripheral involvement. My key findings were that: i) neurofascin is the first gene causally responsible for an inherited disorder that resembles CIDP, ii) this is the largest clinical cohort to date of patients with NFASC mutations with 10 individuals, and iii) the functional evidence implicate the major protein isoforms, which were also shown to be the main targets for the autoantibodies in CIDP pathogenesis. Secondly, I describe the work done on various neurodevelopmental disorder (NDD) genes, with particular focus on a newly identified gene presenting with a complex neurodevelopmental phenotype comprised of developmental delay, epilepsy, and/or a demyelinating neuropathy. My key findings were that: i) NARS1, a cytoplasmic aminoacyl-tRNA synthetase enzyme can be causative for this disorder by either a de-novo heterozygous or a biallelic inheritance mode, ii) functional investigations showed reduced aminoacylation activity in the disease-associated biallelic mutations using fibroblasts and iNPCs transcriptomics, suggesting that the majority of NARS1 mutations cause a loss of function of the protein by reduced expression and disruption of dimer formation suggesting a loss-of-function mechanism, and iii) increased yeast growth in the disease-associated heterozygous mutations showing near normal protein expression are suggestive of a gain-of-function mechanism. Finally, I describe the work done on two relative new genes (PIGS and TARS1) in an attempt to expand the patient phenotypic spectrum, as well as an interesting candidate gene (SLITRK3) linked with epilepsy. I present my understanding for disease-gene discovery that will enable me and other members of the neurogenetics field to identify disease-mechanisms and address important gaps of translational research into rare neurological diseases such as those described in this thesis

Suppression of the activity of arbuscular mycorrhizal fungi by the soil microbiota

Arbuscular mycorrhizal fungi (AMF) colonise roots of most plants; their extra-radical mycelium (ERM) extends into the soil and acquires nutrients for the plant. The ERM coexists with soil microbial communities and it is unresolved whether these communities stimulate or suppress the ERM activity. This work studied the prevalence of suppressed ERM activity and identified main components behind the suppression. ERM activity was determined by quantifying ERM-mediated P uptake from radioisotope-labelled unsterile soil into plants, and compared to soil physicochemical characteristics and soil microbiome composition. ERM activity varied considerably and was greatly suppressed in 4 of 21 soils. Suppression was mitigated by soil pasteurisation and had a dominating biotic component. AMF-suppressive soils had high abundances of Acidobacteria, and other bacterial taxa being putative fungal antagonists. Suppression was also associated with low soil pH, but this effect was likely indirect, as the relative abundance of, e.g., Acidobacteria decreased after liming. Suppression could not be transferred by adding small amounts of suppressive soil to conducive soil, and thus appeared to involve the common action of several taxa. The presence of AMF antagonists resembles the phenomenon of disease-suppressive soils and implies that ecosystem services of AMF will depend strongly on the specific soil microbiome.publishedVersio

AMPA receptor GluA2 subunit defects are a cause of neurodevelopmental disorders

AMPA receptors (AMPARs) are tetrameric ligand-gated channels made up of combinations of GluA1-4 subunits encoded by GRIA1-4 genes. GluA2 has an especially important role because, following post-transcriptional editing at the Q607 site, it renders heteromultimeric AMPARs Ca2+-impermeable, with a linear relationship between current and trans-membrane voltage. Here, we report heterozygous de novo GRIA2 mutations in 28 unrelated patients with intellectual disability (ID) and neurodevelopmental abnormalities including autism spectrum disorder (ASD), Rett syndrome-like features, and seizures or developmental epileptic encephalopathy (DEE). In functional expression studies, mutations lead to a decrease in agonist-evoked current mediated by mutant subunits compared to wild-type channels. When GluA2 subunits are co-expressed with GluA1, most GRIA2 mutations cause a decreased current amplitude and some also affect voltage rectification. Our results show that de-novo variants in GRIA2 can cause neurodevelopmental disorders, complementing evidence that other genetic causes of ID, ASD and DEE also disrupt glutamatergic synaptic transmission

Neuroinflammation and Lysosomal Abnormalities Characterise the Essential Role for Oxidation Resistance 1 in the Developing and Adult Cerebellum

Loss-of-function mutations in the TLDc family of proteins cause a range of severe childhood-onset neurological disorders with common clinical features that include cerebellar neurodegeneration, ataxia and epilepsy. Of these proteins, oxidation resistance 1 (OXR1) has been implicated in multiple cellular pathways related to antioxidant function, transcriptional regulation and cellular survival; yet how this relates to the specific neuropathological features in disease remains unclear. Here, we investigate a range of loss-of-function mouse model systems and reveal that constitutive deletion of Oxr1 leads to a rapid and striking neuroinflammatory response prior to neurodegeneration that is associated with lysosomal pathology. We go on to show that neuroinflammation and cell death in Oxr1 knockouts can be completely rescued by the neuronal expression of Oxr1, suggesting that the phenotype is driven by the cell-intrinsic defects of neuronal cells lacking the gene. Next, we generate a ubiquitous, adult inducible knockout of Oxr1 that surprisingly displays rapid-onset ataxia and cerebellar neurodegeneration, establishing for the first time that the distinctive pathology associated with the loss of Oxr1 occurs irrespective of developmental stage. Finally, we describe two new homozygous human pathogenic variants in OXR1 that cause neurodevelopmental delay, including a novel stop-gain mutation. We also compare functionally two missense human pathogenic mutations in OXR1, including one newly described here, that cause different clinical phenotypes but demonstrate partially retained neuroprotective activity against oxidative stress. Together, these data highlight the essential role of Oxr1 in modulating neuroinflammatory and lysosomal pathways in the mammalian brain and support the hypothesis that OXR1 protein dosage may be critical for pathological outcomes in disease

Not to Miss: Intronic Variants, Treatment, and Review of the Phenotypic Spectrum in VPS13D-Related Disorder

VPS13D is one of four human homologs of the vacuolar sorting protein 13 gene (VPS13). Biallelic pathogenic variants in the gene are associated with spastic ataxia or spastic paraplegia. Here, we report two patients with intronic pathogenic variants: one patient with early onset severe spastic ataxia and debilitating tremor, which is compound-heterozygous for a canonical (NM_018156.4: c.2237−1G > A) and a non-canonical (NM_018156.4: c.941+3G>A) splice site variant. The second patient carries the same non-canonical splice site variant in the homozygous state and is affected by late-onset spastic paraplegia. We confirmed altered splicing as a result of the intronic variants and demonstrated disturbed mitochondrial integrity. Notably, tremor in the first patient improved significantly by bilateral deep brain stimulation (DBS) in the ventralis intermedius (VIM) nucleus of the thalamus. We also conducted a literature review and summarized the phenotypical spectrum of reported VPS13D-related disorders. Our study underscores that looking for mutations outside the canonical splice sites is important not to miss a genetic diagnosis, especially in disorders with a highly heterogeneous presentation without specific red flags

Post-transcriptional microRNA repression of PMP22 dose in severe Charcot-Marie-Tooth disease type 1

Copy number variation (CNV) may lead to pathological traits, and Charcot-Marie-Tooth disease type 1A (CMT1A), the commonest inherited peripheral neuropathy, is due to a genomic duplication encompassing the dosage-sensitive PMP22 gene. MicroRNAs act as repressors on post-transcriptional regulation of gene expression and in rodent models of CMT1A, overexpression of one such microRNA (miR-29a) has been shown to reduce the PMP22 transcript and protein level. Here we present genomic and functional evidence, for the first time in a human CNV-associated phenotype, of the 3' untranslated region (3'-UTR)-mediated role of microRNA repression on gene expression. The proband of the family presented with an early-onset, severe sensorimotor demyelinating neuropathy and harboured a novel de novo deletion in the PMP22 3'-UTR. The deletion is predicted to include the miR-29a seed binding site and transcript analysis of dermal myelinated nerve fibres using a novel platform, revealed a marked increase in PMP22 transcript levels. Functional evidence from Schwann cell lines harbouring the wildtype and mutant 3'-UTR showed significantly increased reporter assay activity in the latter which was not ameliorated by overexpression of a miR-29a mimic. This shows the importance of miR-29a in regulating PMP22 expression and opens an avenue for therapeutic drug development

Consolidating the association of biallelic MAPKAPK5 pathogenic variants with a distinct syndromic neurodevelopmental disorder

[Background]: MAPK-activated protein kinase 5 (MAPKAPK5) is an essential enzyme for diverse cellular processes. Dysregulation of the pathways regulated by MAPKAPK enzymes can lead to the development of variable diseases. Recently, homozygous loss-of-function variants in MAPKAPK5 were reported in four patients from three families presenting with a recognisable neurodevelopmental disorder, so-called ‘neurocardiofaciodigital’ syndrome. [Objective and methods]: In order to improve characterisation of the clinical features associated with biallelic MAPKAPK5 variants, we employed a genotype-first approach combined with reverse deep-phenotyping of three affected individuals. [Results]: In the present study, we identified biallelic loss-of-function and missense MAPKAPK5 variants in three unrelated individuals from consanguineous families. All affected individuals exhibited a syndromic neurodevelopmental disorder characterised by severe global developmental delay, intellectual disability, characteristic facial morphology, brachycephaly, digital anomalies, hair and nail defects and neuroradiological findings, including cerebellar hypoplasia and hypomyelination, as well as variable vision and hearing impairment. Additional features include failure to thrive, hypotonia, microcephaly and genitourinary anomalies without any reported congenital heart disease. [Conclusion]: In this study, we consolidate the causality of loss of MAPKAPK5 function and further delineate the molecular and phenotypic spectrum associated with this new ultra-rare neurodevelopmental syndrome.HH is funded by the MRC (MR/S01165X/1, MR/S005021/1, G0601943), the National Institute for Health Research University College London Hospitals Biomedical Research Centre, Rosetree Trust, Ataxia UK, MSA Trust, Brain Research UK, Sparks GOSH Charity, Muscular Dystrophy UK (MDUK), Muscular Dystrophy Association (MDA USA). SE is supported by an MRC strategic award to establish an International Centre for Genomic Medicine in Neuromuscular Diseases (ICGNMD) MR/S005021/1’

Consolidating the association of biallelic MAPKAPK5 pathogenic variants with a distinct syndromic neurodevelopmental disorder

BACKGROUND: MAPK-activated protein kinase 5 (MAPKAPK5) is an essential enzyme for diverse cellular processes. Dysregulation of the pathways regulated by MAPKAPK enzymes can lead to the development of variable diseases. Recently, homozygous loss-of-function variants in MAPKAPK5 were reported in four patients from three families presenting with a recognisable neurodevelopmental disorder, so-called 'neurocardiofaciodigital' syndrome. OBJECTIVE AND METHODS: In order to improve characterisation of the clinical features associated with biallelic MAPKAPK5 variants, we employed a genotype-first approach combined with reverse deep-phenotyping of three affected individuals. RESULTS: In the present study, we identified biallelic loss-of-function and missense MAPKAPK5 variants in three unrelated individuals from consanguineous families. All affected individuals exhibited a syndromic neurodevelopmental disorder characterised by severe global developmental delay, intellectual disability, characteristic facial morphology, brachycephaly, digital anomalies, hair and nail defects and neuroradiological findings, including cerebellar hypoplasia and hypomyelination, as well as variable vision and hearing impairment. Additional features include failure to thrive, hypotonia, microcephaly and genitourinary anomalies without any reported congenital heart disease. CONCLUSION: In this study, we consolidate the causality of loss of MAPKAPK5 function and further delineate the molecular and phenotypic spectrum associated with this new ultra-rare neurodevelopmental syndrome

Pure cerebellar ataxia due to bi-allelic PRDX3 variants including recurring p.Asp202Asn

Bi-allelic variants in peroxiredoxin 3 (PRDX3) have only recently been associated with autosomal recessive spinocerebellar ataxia characterized by early onset slowly progressive cerebellar ataxia, variably associated with hyperkinetic and hypokinetic features, accompanied by cerebellar atrophy and occasional olivary and brainstem involvement. Herein, we describe a further simplex case carrying a reported PRDX3 variant as well as two additional cases with novel variants. We report the first Brazilian patient with SCAR32, replicating the pathogenic status of a known variant. All presented cases from the Brazilian and Indian populations expand the phenotypic spectrum of the disease by displaying prominent neuroradiological findings. SCAR32, although rare, should be included in the differential diagnosis of sporadic or recessive childhood and adolescent-onset pure and complex cerebellar ataxia

Mutations in NKX6-2 Cause Progressive Spastic Ataxia and Hypomyelination

Progressive limb spasticity and cerebellar ataxia are frequently found together in clinical practice and form a heterogeneous group of degenerative disorders that are classified either as pure spastic ataxia or as complex spastic ataxia with additional neurological signs. Inheritance is either autosomal dominant or autosomal recessive. Hypomyelinating features on MRI are sometimes seen with spastic ataxia, but this is usually mild in adults and severe and life limiting in children. We report seven individuals with an early-onset spastic-ataxia phenotype. The individuals come from three families of different ethnic backgrounds. Affected members of two families had childhood onset disease with very slow progression. They are still alive in their 30s and 40s and show predominant ataxia and cerebellar atrophy features on imaging. Affected members of the third family had a similar but earlier-onset presentation associated with brain hypomyelination. Using a combination of homozygozity mapping and exome sequencing, we mapped this phenotype to deleterious nonsense or homeobox domain missense mutations in NKX6-2. NKX6-2 encodes a transcriptional repressor with early high general and late focused CNS expression. Deficiency of its mouse ortholog results in widespread hypomyelination in the brain and optic nerve, as well as in poor motor coordination in a pattern consistent with the observed human phenotype. In-silico analysis of human brain expression and network data provides evidence that NKX6-2 is involved in oligodendrocyte maturation and might act within the same pathways of genes already associated with central hypomyelination. Our results support a non-redundant developmental role of NKX6-2 in humans and imply that NKX6-2 mutations should be considered in the differential diagnosis of spastic ataxia and hypomyelination.Fil: Chelban, Viorica. University College London; Estados Unidos. Institute of Emergency Medicine; MoldaviaFil: Patel, Nisha. King Faisal Specialist Hospital and Research Center; Arabia SauditaFil: Vandrovcova, Jana. University College London; Estados UnidosFil: Zanetti, Maria Natalia. University College London; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Lynch, David S.. University College London; Estados UnidosFil: Ryten, Mina. University College London; Estados Unidos. King’s College London; Reino UnidoFil: Botía, Juan A.. University College London; Estados Unidos. Universidad de Murcia; EspañaFil: Bello, Oscar Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; Argentina. University College London; Estados UnidosFil: Tribollet, Eloise. University College London; Estados UnidosFil: Efthymiou, Stephanie. University College London; Estados UnidosFil: Davagnanam, Indran. University College London; Estados UnidosFil: Bashiri, Fahad A.. King Saud University; Arabia SauditaFil: Wood, Nicholas W.. University College London; Estados Unidos. The National Hospital for Neurology and Neurosurgery; Reino UnidoFil: Rothman, James E.. University of Yale. School of Medicine; Estados Unidos. University College London; Estados UnidosFil: Alkuraya, Fowzan S.. King Faisal Specialist Hospital and Research Center; Arabia Saudita. Alfaisal University; Arabia Saudita. King Abdulaziz City for Science and Technology; Arabia SauditaFil: Houlden, Henry. The National Hospital for Neurology and Neurosurgery; Reino Unido. University College London; Estados Unido