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

Regulatory sites for splicing in human basal ganglia are enriched for disease-relevant information

Genome-wide association studies have generated an increasing number of common genetic variants associated with neurological and psychiatric disease risk. An improved under- standing of the genetic control of gene expression in human brain is vital considering this is the likely modus operandum for many causal variants. However, human brain sampling complexities limit the explanatory power of brain-related expression quantitative trait loci (eQTL) and allele-specific expression (ASE) signals. We address this, using paired genomic and transcriptomic data from putamen and substantia nigra from 117 human brains, inter- rogating regulation at different RNA processing stages and uncovering novel transcripts. We identify disease-relevant regulatory loci, find that splicing eQTLs are enriched for regulatory information of neuron-specific genes, that ASEs provide cell-specific regulatory information with evidence for cellular specificity, and that incomplete annotation of the brain tran- scriptome limits interpretation of risk loci for neuropsychiatric disease. This resource of regulatory data is accessible through our web server, http://braineacv2.inf.um.es/

Analysis of subcellular RNA fractions demonstrates significant genetic regulation of gene expression in human brain post-transcriptionally

Gaining insight into the genetic regulation of gene expression in human brain is key to the interpretation of genome-wide association studies for major neurological and neuropsychiatric diseases. Expression quantitative trait loci (eQTL) analyses have largely been used to achieve this, providing valuable insights into the genetic regulation of steady-state RNA in human brain, but not distinguishing between molecular processes regulating transcription and stability. RNA quantification within cellular fractions can disentangle these processes in cell types and tissues which are challenging to model in vitro. We investigated the underlying molecular processes driving the genetic regulation of gene expression specific to a cellular fraction using allele-specific expression (ASE). Applying ASE analysis to genomic and transcriptomic data from paired nuclear and cytoplasmic fractions of anterior prefrontal cortex, cerebellar cortex and putamen tissues from 4 post-mortem neuropathologically-confirmed control human brains, we demonstrate that a significant proportion of genetic regulation of gene expression occurs post-transcriptionally in the cytoplasm, with genes undergoing this form of regulation more likely to be synaptic. These findings have implications for understanding the structure of gene expression regulation in human brain, and importantly the interpretation of rapidly growing single-nucleus brain RNA-sequencing and eQTL datasets, where cytoplasm-specific regulatory events could be missed

Targeted genetic testing for familial hypercholesterolaemia using next generation sequencing:a population-based study

Background<p></p> Familial hypercholesterolaemia (FH) is a common Mendelian condition which, untreated, results in premature coronary heart disease. An estimated 88% of FH cases are undiagnosed in the UK. We previously validated a method for FH mutation detection in a lipid clinic population using next generation sequencing (NGS), but this did not address the challenge of identifying index cases in primary care where most undiagnosed patients receive healthcare. Here, we evaluate the targeted use of NGS as a potential route to diagnosis of FH in a primary care population subset selected for hypercholesterolaemia.<p></p> Methods<p></p> We used microfluidics-based PCR amplification coupled with NGS and multiplex ligation-dependent probe amplification (MLPA) to detect mutations in LDLR, APOB and PCSK9 in three phenotypic groups within the Generation Scotland: Scottish Family Health Study including 193 individuals with high total cholesterol, 232 with moderately high total cholesterol despite cholesterol-lowering therapy, and 192 normocholesterolaemic controls.<p></p> Results<p></p> Pathogenic mutations were found in 2.1% of hypercholesterolaemic individuals, in 2.2% of subjects on cholesterol-lowering therapy and in 42% of their available first-degree relatives. In addition, variants of uncertain clinical significance (VUCS) were detected in 1.4% of the hypercholesterolaemic and cholesterol-lowering therapy groups. No pathogenic variants or VUCS were detected in controls.<p></p> Conclusions<p></p> We demonstrated that population-based genetic testing using these protocols is able to deliver definitive molecular diagnoses of FH in individuals with high cholesterol or on cholesterol-lowering therapy. The lower cost and labour associated with NGS-based testing may increase the attractiveness of a population-based approach to FH detection compared to genetic testing with conventional sequencing. This could provide one route to increasing the present low percentage of FH cases with a genetic diagnosis

Enhancement of biomimetic enzymatic mineralization of gellan gum polysaccharide hydrogels by plant-derived gallotannins

Mineralization of hydrogel biomaterials with calcium phosphate (CaP) is considered advantageous for bone regeneration. Mineralization can be both induced by the enzyme alkaline phosphatase (ALP) and promoted by calcium-binding biomolecules, such as plant-derived polyphenols. In this study, ALP-loaded gellan gum (GG) hydrogels were enriched with gallotannins, a subclass of polyphenols. Five preparations were compared, namely three tannic acids of differing molecular weight (MW), pentagalloyl glucose (PGG), and a gallotannin-rich extract from mango kernel (Mangifera indica L.). Certain gallotannin preparations promoted mineralization to a greater degree than others. The various gallotannin preparations bound differently to ALP and influenced the size of aggregates of ALP, which may be related to ability to promote mineralization. Human osteoblast-like Saos-2 cells grew in eluate from mineralized hydrogels. Gallotannin incorporation impeded cell growth on hydrogels and did not impart antibacterial activity. In conclusion, gallotannin incorporation aided mineralization but reduced cytocompatibility

Regulatory sites for splicing in human basal ganglia are enriched for disease-relevant information

International Parkinson’s Disease Genomics Consortium (IPDGC), UK Brain Expression Consortium (UKBEC).Genome-wide association studies have generated an increasing number of common genetic variants associated with neurological and psychiatric disease risk. An improved understanding of the genetic control of gene expression in human brain is vital considering this is the likely modus operandum for many causal variants. However, human brain sampling complexities limit the explanatory power of brain-related expression quantitative trait loci (eQTL) and allele-specific expression (ASE) signals. We address this, using paired genomic and transcriptomic data from putamen and substantia nigra from 117 human brains, interrogating regulation at different RNA processing stages and uncovering novel transcripts. We identify disease-relevant regulatory loci, find that splicing eQTLs are enriched for regulatory information of neuron-specific genes, that ASEs provide cell-specific regulatory information with evidence for cellular specificity, and that incomplete annotation of the brain transcriptome limits interpretation of risk loci for neuropsychiatric disease. This resource of regulatory data is accessible through our web server, http://braineacv2.inf.um.es/.Mina Ryten, David Zhang, and Karishma D’Sa were supported by the UK Medical Research Council (MRC) through the award of Tenure-track Clinician Scientist Fellowship to Mina Ryten (MR/N008324/1). Sebastian Guelfi was supported by Alzheimer’s Research UK through the award of a PhD Fellowship (ARUK-PhD2014-16). Regina Reynolds was supported through the award of a Leonard Wolfson Doctoral Training Fellowship in Neurodegeneration

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

Human-lineage-specific genomic elements are associated with 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 to be more abundant in Alzheimer?s disease with more severe tau and amyloid pathological burden. Thus, we demonstrate potential association of human-lineage-specific sequences in brain development and neurological disease.FUNDING: Acknowledgements The authors are grateful to the participants in the Religious Order Study, the Memory and Aging Project. Z.C. and R.H.R. were supported by grants from the Leonard Wolfson Foundation. M.R. was supported by the United Kingdom Medical Research Council (MRC) through the award of a Tenure Track Clinician Scientist Fellowship (MR/ N008324/1). J.H. was supported by the UK Dementia Research Institute which receives its funding from DRI Limited, funded by the UK Medical Research Council, Alzheimer’s Society and Alzheimer’s Research UK. J.H. has also been funded by the Medical Research Council (award MR/N026004/1), Wellcome Trust (award 202903/Z/16/Z), Dolby Family Fund and National Institute for Health Research University College London Hospitals Biomedical Research Centre. J.B. is supported through the Science and Technology Agency, Séneca Foundation, CARM, Spain (research project 00007/COVI/20)

Identification and biochemical analysis of a novel APOB mutation that causes autosomal dominant hypercholesterolemia

Patients with autosomal dominant hypercholesterolemia (ADH) have a high risk of developing cardiovascular disease that can be effectively treated using statin drugs. Molecular diagnosis and family cascade screening is recommended for early identification of individuals at risk, but up to 40% of families have no mutation detected in known genes. This study combined linkage analysis and exome sequencing to identify a novel variant in exon 3 of APOB (Arg50Trp). Mass spectrometry established that low-density lipoprotein (LDL) containing Arg50Trp APOB accumulates in the circulation of affected individuals, suggesting defective hepatic uptake. Previously reported mutations in APOB causing ADH have been located in exon 26. This is the first report of a mutation outside this region causing this phenotype, therefore, more extensive screening of this large and highly polymorphic gene may be necessary in ADH families. This is now feasible due to the high capacity of recently available sequencing platforms

Specialist multidisciplinary input maximises rare disease diagnoses from whole genome sequencing

Diagnostic whole genome sequencing (WGS) is increasingly used in rare diseases. However, standard, semi-automated WGS analysis may overlook diagnoses in complex disorders. Here, we show that specialist multidisciplinary analysis of WGS, following an initial 'no primary findings' (NPF) report, improves diagnostic rates and alters management. We undertook WGS in 102 adults with diagnostically challenging primary mitochondrial disease phenotypes. NPF cases were reviewed by a genomic medicine team, thus enabling bespoke informatic approaches, co-ordinated phenotypic validation, and functional work. We enhanced the diagnostic rate from 16.7% to 31.4%, with management implications for all new diagnoses, and detected strong candidate disease-causing variants in a further 3.9% of patients. This approach presents a standardised model of care that supports mainstream clinicians and enhances diagnostic equity for complex disorders, thereby facilitating access to the potential benefits of genomic healthcare. This research was made possible through access to the data and findings generated by the 100,000 Genomes Project: http://www.genomicsengland.co.uk