Biology subjects

Houlden, H.

Publications and source records attributed to Houlden, H..

At least 37 records · Page 2Linked to original sources

bioRxiv2022

Human mutations in SLITRK3 implicated in GABAergic synapse development in mice

We report on biallelic homozygous and monoallelic de-novo variants in SLITRK3 in 3 unrelated families presenting with epileptic encephalopathy associated with a broad neurological involvement characterized by microcephaly, intellectual disability, seizures, and global developmental delay. SLITRK3 encodes for a transmembrane protein that is involved in controlling neurite outgrowth and inhibitory synapse development and that has an important role in brain function and neurological diseases. Using primary cultures of hippocampal neurons carrying patients SLITRK3 variants and in combination with electrophysiology, we demonstrate that recessive variants are loss-of-function alleles. By analyzing the development and phenotype of SLITRK3 KO (SLITRK3-/-) mice, we bring additional evidence of enhanced susceptibility to pentylenetetrazole-induced seizure with the appearance of spontaneous epileptiform EEG, as well as developmental deficits such as higher motor activities and reduced parvalbumin interneurons. Taken together, our results exhibit impaired development of peripheral and central nervous system and support a conserved role of this transmembrane protein in neurological function. Our study delineates an emerging spectrum of human core synaptopathies caused by variants in genes that encode SLITRK proteins and essential regulatory components of the synaptic machinery. The hallmark of these disorders is impaired postsynaptic neurotransmission at nerve terminals; an impaired neurotransmission resulting in a wide array of (often overlapping) clinical features, including neurodevelopmental impairment, weakness, seizures, and abnormal movements. The genetic synaptopathy caused by SLITRK3 mutations highlights the key roles of this gene in human brain development and function.

genetics↗

bioRxiv2022

Upregulated extracellular matrix-related genes and impaired synaptic activity in dopaminergic and hippocampal neurons derived from Parkinson's disease patients with PINK1 and PARK2 mutations

Parkinsons disease (PD) is the second most prevalent neurodegenerative disease. Primary symptoms of PD arise with the loss of dopaminergic (DA) neurons in the Substantia Nigra Pars Compacta, but it affects the hippocampus and cortex also, usually in its later stage. Approximately 15% of PD cases familial with a genetic mutation. Two of the most associated genes with autosomal recessive (AR) early-onset familial PD are PINK1 and PARK2. There is a need for in-vitro studies of these genetic mutations in order to understand the neurophysiological changes in patients neurons that may contribute to neurodegeneration. In this work, we generated and differentiated DA and hippocampal neurons from iPSCs derived from two patients with a double mutation in their PINK1 and PARK2 (one homozygous and one heterozygous) genes and assessed their neurophysiology compared to two healthy controls. We showed that the synaptic activity of PD neurons generated from patients with the PINK1 and PARK2 mutations is impaired in the hippocampus and dopaminergic neurons. Mutant dopaminergic neurons had enhanced excitatory post-synaptic activity. In addition, DA neurons with the homozygous mutation of PINK1 exhibited more pronounced electrophysiological differences compared to the control neurons. Signaling network analysis of RNA sequencing results revealed that Focal adhesion and ECM receptor pathway were the top 2 upregulated pathways in the mutant PD neurons. These phenotypes are reversed to PD phenotypes of other mutations, suggesting that the interaction of the two mutations may yield different mechanisms of PD.

neuroscience↗

bioRxiv2022

Pseudogenes limit the identification of novel common transcripts generated by their parent genes

The human genome contains numerous duplicated regions, such as parent-pseudogene pairs, causing sequencing reads to align equally well to either gene. The extent to which this ambiguity complicates transcriptomic analyses is currently unknown. This is concerning as many parent genes have been linked to disease, including GBA1, causally linked to both Parkinsons and Gaucher disease. We find that most of the short sequencing reads that map to GBA1, also map to its pseudogene, GBAP1. Using long-read RNA-sequencing in human brain, where all reads mapped uniquely, we demonstrate significant differences in expression compared to short-read data. We identify novel transcripts from both GBA1 and GBAP1, including protein-coding transcripts that are translated in vitro and detected in proteomic data, but that lack GCase activity. By combining long-read with single-nuclear RNA-sequencing to analyse brain-relevant cell types we demonstrate that transcript expression varies by brain region with cell-type-selectivity. Taken together, these results suggest a non-lysosomal function for both GBA1 and GBAP1 in brain. Finally, we demonstrate that inaccuracies in annotation are widespread among parent genes, with implications for many human diseases.

genetics↗

bioRxiv2022

Molecular basis of FAAH-OUT-associated human pain insensitivity

Chronic pain affects millions of people worldwide. Studying pain insensitive individuals helps to identify novel analgesic strategies. Here we report how the recently discovered FAAH-OUT lncRNA-encoding gene, which was found from studying a pain insensitive patient with reduced anxiety and fast wound healing, regulates the adjacent key endocannabinoid system gene FAAH, which encodes the anandamide-degrading fatty acid amide hydrolase enzyme. We demonstrate that the disruption in FAAH-OUT lncRNA transcription leads to DNMT1-dependent DNA methylation within the FAAH promoter. In addition, FAAH-OUT contains a conserved regulatory element, FAAH-AMP, that acts as an enhancer for FAAH expression. Furthermore, using transcriptomic analyses we have uncovered a network of genes that are dysregulated from disruption of the FAAH-FAAH-OUT axis, thus providing a coherent mechanistic basis to understand the human phenotype observed and a platform for development of future gene and small molecule therapies.

neuroscience↗

bioRxiv2022

The contribution of Neanderthal introgression and natural selection to neurodegenerative diseases

Humans are thought to be more susceptible to neurodegeneration than equivalently-aged primates. It is not known whether this vulnerability is specific to anatomically-modern humans or shared with other hominids. The contribution of introgressed Neanderthal DNA to neurodegenerative disorders remains uncertain. It is also unclear how common variants associated with neurodegenerative disease risk are maintained by natural selection in the population despite their deleterious effects. In this study, we aimed to quantify the genome-wide contribution of Neanderthal introgression and positive selection to the heritability of complex neurodegenerative disorders to address these questions. We used stratified-linkage disequilibrium score regression to investigate the relationship between five SNP-based signatures of natural selection, reflecting different timepoints of evolution, and genome-wide associated variants of the three most prevalent neurodegenerative disorders: Alzheimers disease, amyotrophic lateral sclerosis and Parkinsons disease. We found no evidence for enrichment of positively-selected SNPs in the heritability of Alzheimers disease, amyotrophic lateral sclerosis and Parkinsons disease,, suggesting that common deleterious disease variants are unlikely to be maintained by positive selection. There was no enrichment of Neanderthal introgression in the SNP-heritability of these disorders, suggesting that Neanderthal admixture is unlikely to have contributed to disease risk. These findings provide insight into the origins of neurodegenerative disorders within the evolution of Homo sapiens and addresses a long-standing debate, showing that Neanderthal admixture is unlikely to have contributed to common genetic risk of neurodegeneration in anatomically-modern humans.

neuroscience↗

bioRxiv2021

Biallelic loss-of-function OBSCN variants predispose individuals to severe, recurrent rhabdomyolysis

Rhabdomyolysis is the acute breakdown of skeletal myofibres in response to an initiating factor, most commonly toxins and over exertion. A variety of genetic disorders predispose to rhabdomyolysis through different pathogenic mechanisms, particularly in patients with recurrent episodes. However, the majority of cases remain without a genetic diagnosis. Here we present six patients who presented with severe and recurrent rhabdomyolysis, usually with onset in the teenage years; other features included a history of myalgia and muscle cramps. We identified ten bi-allelic loss-of-function variants in the gene encoding obscurin (OBSCN) co-segregating with disease. We show reduced expression of OBSCN and loss of obscurin protein in patient muscle. Obscurin is proposed to be involved in SR function and Ca2+ handling. Patient cultured myoblasts appear more susceptible to starvation as evidenced by a greater decreased in SR Ca2+ content compared to control myoblasts. This likely reflects a lower efficiency when pumping Ca2+ back into the SR and/or a decrease in Ca2+ SR storage ability when metabolism is diminished. OSBCN variants have previously been associated with cardiomyopathies. None of the patients presented with a cardiomyopathy and cardiac examinations were normal in all cases in which cardiac function was assessed. There was also no history of cardiomyopathy in first degree relatives, in particular in any of the carrier parents. This cohort is relatively young, thus follow-up studies and the identification of additional cases with bi-allelic null OBSCN variants will further delineate OBSCN-related disease and the clinical course of disease.

genetics↗

bioRxiv2021

Delineating the molecular and phenotypic spectrum of the SETD1B-related syndrome

Pathogenic variants in SETD1B have been associated with a syndromic neurodevelopmental disorder including intellectual disability, language delay and seizures. To date, clinical features have been described for eleven patients with (likely) pathogenic SETD1B sequence variants. We perform an in-depth clinical characterization of a cohort of 36 unpublished individuals with SETD1B sequence variants, describing their molecular and phenotypic spectrum. Selected variants were functionally tested using in vitro and genome-wide methylation assays. Our data present evidence for a loss-of-function mechanism of SETD1B variants, resulting in a core clinical phenotype of global developmental delay, language delay including regression, intellectual disability, autism and other behavioral issues, and variable epilepsy phenotypes. Developmental delay appeared to precede seizure onset, suggesting SETD1B dysfunction impacts physiological neurodevelopment even in the absence of epileptic activity. Interestingly, males are significantly overrepresented and more severely affected, and we speculate that sex-linked traits could affect susceptibility to penetrance and the clinical spectrum of SETD1B variants. Finally, despite the possibility of non-redundant contributions of SETD1B and its paralogue SETD1A to epigenetic control, the clinical phenotypes of the related disorders share many similarities, indicating that elucidating shared and divergent downstream targets of both genes will help to understand the mechanism leading to the neurobehavioral phenotypes. Insights from this extensive cohort will facilitate the counseling regarding the molecular and phenotypic landscape of newly diagnosed patients with the SETD1B-related syndrome.

genetics↗

bioRxiv2021

Integrating protein networks and machine learning for disease stratification in the Hereditary Spastic Paraplegias

The Hereditary Spastic Paraplegias are a group of neurodegenerative diseases characterized by spasticity and weakness in the lower body. Despite the identification of causative mutations in over 70 genes, the molecular aetiology remains unclear. Due to the combination of genetic diversity and variable clinical presentation, the Hereditary Spastic Paraplegias are a strong candidate for protein-protein interaction network analysis as a tool to understand disease mechanism(s) and to aid functional stratification of phenotypes. In this study, experimentally validated human protein-protein interactions were used to create a protein-protein interaction network based on the causative Hereditary Spastic Paraplegia genes. Network evaluation as a combination of both topological analysis and functional annotation led to the identification of core proteins in putative shared biological processes such as intracellular transport and vesicle trafficking. The application of machine learning techniques suggested a functional dichotomy linked with distinct sets of clinical presentations, suggesting there is scope to further classify conditions currently described under the same umbrella term of Hereditary Spastic Paraplegias based on specific molecular mechanisms of disease.

systems biology↗

bioRxiv2020

Biallelic mutation of CLRN2 causes non-syndromic hearing loss in humans

Deafness, the most frequent sensory deficit in humans, is extremely heterogenous with hundreds of genes probably involved. Clinical and genetic analyses of an extended consanguineous family with pre-lingual, moderate-to-profound autosomal recessive sensorineural hearing loss, allowed us to identify CLRN2, encoding a tetraspan protein as a new deafness gene. Homozygosity mapping followed by exome sequencing identified a 15.2 Mb locus on chromosome 4p15.32p15.1 containing a missense pathogenic variant in CLRN2 (c.494C>A, NM_001079827.2) segregating with the disease. Using in vitro RNA splicing analysis, we show that the CLRN2 c.494C>A mutation leads to two events: 1) the substitution of a highly conserved threonine (uncharged amino acid) to lysine (charged amino acid) at position 165, p.(Thr165Lys), and 2) aberrant splicing, with the retention of intron 2 resulting in a stop codon after 26 additional amino acids, p.(Gly146Lysfs*26). Expression studies and phenotyping of newly produced zebrafish and mouse models deficient for clarin 2 further confirm that clarin 2, expressed in the inner ear hair cells, is essential for normal organization and maintenance of the auditory hair bundles, and for hearing function. Together, our findings identify CLRN2 as a new deafness gene, which will impact future diagnosis and treatment for deaf patients.

genetics↗

bioRxiv2020

Negative screening for 12 rare LRRK2 pathogenic variants in a cohort of Nigerians with Parkinson disease

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most commonly identified genetic variants in familial and sporadic Parkinson’s disease (PD). Over three hundred LRRK2 variants have been described in the literature, of which at least 17 have a confirmed or probable pathogenic role in PD. The distribution of these rare pathogenic variants has been shown to be different among ethnic groups including Caucasians, Latin Americans and East and South Asians. However, to date no PD-related LRRK2 pathogenic variant has been described in persons of black African ancestry within or outside Africa. We previously reported that the LRRK2 p.gly2019ser mutation was not found in 126 PD patients and 55 controls from Nigeria. Using Kompetitive Allele-Specific Polymerase chain reaction (KASP), we screened a new cohort of 92 Nigerians with PD and 210 healthy ethnically matched controls for 12 rare LRRK2 variants (which have been shown to be pathogenic in other ethnic populations) including: p.gly2019ser, p.Arg1441His, p.Gly2385Arg, p.Ala419Val, p.Arg1628Pro, p.Pro755Leu, p.Ile2020Thr and Tyr1699Cys. All 12 rare variants were absent in PD patients and controls from this cohort. These results endorse our previous findings and confirm that rare LRRK2 pathogenic variants reported in Caucasians, Asians and persons of mixed ancestry are absent in West Africans. Applying next generation sequencing technologies in future studies is necessary to explore possible novel LRRK2 variants indigenous to black Africans.Competing Interest StatementThe authors have declared no competing interest.View Full Text

genetics↗

bioRxiv2020

Human-lineage-specific genomic elements: relevance to neurodegenerative disease and APOE transcript usage

Knowledge of genomic features specific to the human lineage may provide insights into brain-related diseases. We leverage high-depth whole genome sequencing data to generate a combined annotation identifying regions simultaneously depleted for genetic variation (constrained regions) and poorly conserved across primates. We propose that these constrained, non-conserved regions (CNCRs) have been subject to human-specific purifying selection and are enriched for brain-specific elements. We find that CNCRs are depleted from protein-coding genes but enriched within lncRNAs. We demonstrate that per-SNP heritability of a range of brain-relevant phenotypes are enriched within CNCRs. We find that genes implicated in neurological diseases have high CNCR density, including APOE, highlighting an unannotated intron-3 retention event. Using human brain RNA-sequencing data, we show the intron-3-retaining transcript/s to be more abundant in Alzheimers disease with more severe tau and amyloid pathological burden. Thus, we demonstrate the importance of human-lineage-specific sequences in brain development and neurological disease. We release our annotation through vizER (https://snca.atica.um.es/browser/app/vizER).

neuroscience↗

bioRxiv2020

Human patient SFPQ homozygous mutation is found deleterious for brain and motor development in a zebrafish model

SFPQ (Splicing factor proline- and glutamine-rich) is a DNA and RNA binding protein involved in transcription, pre-mRNA splicing, and DNA damage repair and it has been previously implicated in neurodegenerative disorders. A homozygous p.Ser660Asn variant in SFPQ was identified through whole exome sequencing (WES) in an Italian woman presented a complex neurological phenotype with intellectual disability, peripheral neuropathy, bradykinesia, extrapyramidal rigidity and rest (heads) tremor and neuroradiological anomalies including thin dysplastic corpus callosum, hypomyelination and hypointensity of the globus pallidus and of the mesencephalic substantia nigra (resembling neurodegeneration with brain iron accumulation; NBIA). Using a zebrafish SFPQ genetic model we have showed that a rescue with this SFPQS660N mutant revealed robust defects in the developing central nervous system (CNS) of the embryos, including abnormal branching of the motor axons innervating body muscles and misfolding of the posterior brain neuroepithelium. The defects hereby identified in the model organism indicate a potential contribution of the homozygous SFPQ p.Ser660Asn variant in some of the patient's neurodegenerative features, including the clinical parkinsonism and the NBIA-like pattern on brain imaging.

neuroscience↗

bioRxiv2020

Cell-based analysis of CAD variants identifies individuals likely to benefit from uridine therapy

PurposePathogenic autosomal recessive variants in CAD, encoding the multienzymatic protein initiating pyrimidine de novo biosynthesis, cause a severe inborn metabolic disorder treatable with a dietary supplement of uridine. This condition is difficult to diagnose given the large size of CAD with over 1000 missense variants and the non-specific clinical presentation. We aimed to develop a reliable and discerning assay to assess the pathogenicity of CAD variants and to select affected individuals that might benefit from uridine therapy. MethodsUsing CRISPR/Cas9, we generated a human CAD-knockout cell line that requires uridine supplements for survival. Transient transfection of the knockout cells with recombinant CAD restores growth in absence of uridine. This system determines missense variants that inactivate CAD and do not rescue the growth phenotype. ResultsWe identified 25 individuals with biallelic variants in CAD and a phenotype consistent with a CAD deficit. We used the CAD-knockout complementation assay to test a total of 34 variants, identifying 16 as deleterious for CAD activity. Combination of these pathogenic variants confirmed 11 subjects with a CAD deficit, for whom we describe the clinical phenotype. ConclusionsWe designed a cell-based assay to test the pathogenicity of CAD variants, identifying 11 CAD deficient individuals, who could benefit from uridine therapy.

genetics↗

bioRxiv2020

Regulation of mitophagy by the NSL complex underlies genetic risk for Parkinson's disease at Chr16q11.2 and on the MAPT H1 allele.

Parkinsons disease (PD) is a common incurable neurodegenerative disease. The identification of genetic variants via genome-wide association studies (GWAS) has considerably advanced our understanding of the PD genetic risk. Understanding the functional significance of the risk loci is now a critical step towards translating these genetic advances into an enhanced biological understanding of the disease. Impaired mitophagy is a key causative pathway in familial PD, but its relevance to idiopathic PD is unclear. We used a mitophagy screening assay to evaluate the functional significance of risk genes identified through GWAS. We identified two new regulators of PINK1-mitophagy, KAT8 and KANSL1, previously shown to modulate lysine acetylation. We show that KAT8 and KANSL1 modulate PINK1 gene expression and subsequent PINK1-mitophagy. These findings suggest PINK1-mitophagy is a contributing factor to idiopathic PD. KANSL1 is located on chromosome 17q21 where the risk associated gene has long been considered to be MAPT. While our data does not exclude a possible association between the MAPT gene and PD, it provides strong evidence that KANSL1 plays a crucial role in the disease. Finally, these results enrich our understanding of physiological events regulating mitophagy and establish a novel pathway for drug targeting in neurodegeneration.

neuroscience↗

bioRxiv2019

A genome-wide genetic pleiotropy approach identified shared loci between multiple system atrophy and inflammatory bowel disease

We aimed to identify shared genetic background between multiple system atrophy (MSA) and autoimmune diseases by using the conjFDR approach. Our study showed significant genetic overlap between MSA and inflammatory bowel disease and identified DENND1B, C7, and RSP04 loci, which are linked to significant changes in methylation or expression levels of adjacent genes. We obtained evidence of enriched heritability involving immune/digestive categories. Finally, an MSA mouse model showed dysregulation of the C7 gene in the degenerating midbrain compared to wildtype mice. The results identify novel molecular mechanisms and implicate immune and gut dysfunction in MSA pathophysiology.

genetics↗

bioRxiv2018

Leucine Rich Repeat Kinase 2 (LRRK2) Gly2019Ser Mutation Is Absent In A Second Cohort Of Nigerian Africans With Parkinson Disease.

To date the LRRK2 p.G2019S mutation remains the most common genetic cause of Parkinson disease (PD) worldwide. It accounts for up to 6% of familial and approximately 1.5% of sporadic cases. LRRK2 has a kinase enzymatic domain which provides an attractive potential target for drug therapies and LRRK2 kinase inhibitors are in development. Prevalence of the p.G2019S has a variable ethnic and geographic distribution, the highest was reported among Ashkenazi Jews (30% in patients with familial PD, 14% in sporadic PD, 2.0% in controls) and North African Berbers (37% in patients with familial PD, 41% in sporadic PD, and 1% in controls). Little is known about the frequency of the LRRK2 p.G2019S among populations in Sub Saharan Africa. Our group and others previously reported that the p.G2019S is absent in a small cohort from Nigerian PD patients and controls. Here we used Kompetitive Allele Specific PCR (KASP) assay to screen for the p.G2019S in a larger cohort of Black African PD patients (n =126) and healthy controls (n = 55) from Nigeria. Our analysis confirmed that all patients and controls are negative for the p.G2019S mutation. This report provides further evidence that the LRRK2 p.G2019S is not implicated in PD in black populations from Nigeria and support the notion that p.G2019S mutation originated after the early human dispersal from sub-Saharan Africa. Further studies using larger cohorts and advance sequencing technology are required to underpin the genetic causes of PD in this region.

genetics↗

bioRxiv2018

G2P: Using machine learning to understand and predict genes causing rare neurological disorders

To facilitate precision medicine and neuroscience research, we developed a machine-learning technique that scores the likelihood that a gene, when mutated, will cause a neurological phenotype. We analysed 1126 genes relating to 25 subtypes of Mendelian neurological disease defined by Genomics England (March 2017) together with 154 gene-specific features capturing genetic variation, gene structure and tissue-specific expression and co-expression. We randomly re-sampled genes with no known disease association to develop bootstrapped decision-tree models, which were integrated to generate a decision tree-based ensemble for each disease subtype. Genes generating larger numbers of distinct transcripts and with higher probability of having missense mutations in normal individuals were significantly more likely to cause neurological diseases. Using mouse-mutant phenotypic data we tested the accuracy of gene-phenotype predictions and found that for 88% of all disease subtypes there was a significant enrichment of relevant phenotypic abnormalities when predicted genes were mutated in mice and in many cases mutations produced specific and matching phenotypes. Furthermore, using only newly identified genes included in the Genomics England November 2017 release, we assessed our gene-phenotype predictions and showed an 8.3 fold enrichment relative to chance for correct predictions. Thus, we demonstrate both the explanatory and predictive power of machine-learning-based models in neurological disease.

genetics↗

bioRxiv2017

DNA isolation protocol effects on nuclear DNA analysis by microarrays, droplet digital PCR, and whole genome sequencing, and on mitochondrial DNA copy number estimation

Potential bias introduced during DNA isolation is inadequately explored, although it could have significant impact on downstream analysis. To investigate this in human brain, we isolated DNA from cerebellum and frontal cortex using spin columns under different conditions, and salting-out. We first analysed DNA using array CGH, which revealed a striking wave pattern suggesting primarily GC-rich cerebellar losses, even against matched frontal cortex DNA, with a similar pattern on a SNP array. The aCGH changes varied with the isolation protocol. Droplet digital PCR of two genes also showed protocol-dependent losses. Whole genome sequencing showed GC-dependent variation in coverage with spin column isolation from cerebellum. We also extracted and sequenced DNA from substantia nigra using salting-out and phenol / chloroform. The mtDNA copy number, assessed by reads mapping to the mitochondrial genome, was higher in substantia nigra when using phenol / chloroform. We thus provide evidence for significant method-dependent bias in DNA isolation from human brain, as reported in rat tissues. This may contribute to array \"waves\", and could affect copy number determination, particularly if mosaicism is being sought, and sequencing coverage. Variations in isolation protocol may also affect apparent mtDNA abundance.

genomics↗