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1. The phenotypic spectrum of pathogenic ATP1A1 variants expands: the novel p.P600R substitution causes demyelinating Charcot–Marie–Tooth disease

   https://doi.org/10.1007/s00415-023-11581-w  

   Cinarli Yuksel, Feride; Nicolaou, Paschalis; Spontarelli, Kerri; Dohrn, Maike F.; Rebelo, Adriana P.; Koutsou, Pantelitsa; Georghiou, Anthi; Artigas, Pablo; Züchner, Stephan L.; Kleopa, Kleopas A.; et al (February 2023, Journal of Neurology)

   Abstract BackgroundCharcot–Marie–Tooth disease (CMT) is a genetically and clinically heterogeneous group of inherited neuropathies. Monoallelic pathogenic variants inATP1A1were associated with axonal and intermediate CMT.ATP1A1encodes for the catalytic α1 subunit of the Na⁺/ K⁺ATPase. Besides neuropathy, other associated phenotypes are spastic paraplegia, intellectual disability, and renal hypomagnesemia. We hereby report the first demyelinating CMT case due to a novelATP1A1variant. MethodsWhole-exome sequencing on the patient’s genomic DNA and Sanger sequencing to validate and confirm the segregation of the identified p.P600RATP1A1variation were performed. To evaluate functional effects, blood-derived mRNA and protein levels ofATP1A1and the auxiliary β1 subunit encoded byATP1B1were investigated. The ouabain-survival assay was performed in transfected HEK cells to assess cell viability, and two-electrode voltage clamp studies were performed in Xenopus oocytes. ResultsThe variant was absent in the local and global control datasets, falls within a highly conserved protein position, and is in a missense-constrained region. The expression levels of ATP1A1 and ATP1B1 were significantly reduced in the patient compared to healthy controls. Electrophysiology indicated thatATP1A1^p.P600Rinjected Xenopus oocytes have reduced Na⁺/ K⁺ATPase function. Moreover, HEK cells transfected with a construct encodingATP1A1^p.P600Rharbouring variants that confers ouabain insensitivity displayed a significant decrease in cell viability after ouabain treatment compared to the wild type, further supporting the pathogenicity of this variant. ConclusionOur results further confirm the causative role ofATP1A1in peripheral neuropathy and broaden the mutational and phenotypic spectrum ofATP1A1-associated CMT. 

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2. Genomic Landscape of Chromosome X Factor VIII: From Hemophilia A in Males to Risk Variants in Females

   https://doi.org/10.3390/genes15121522  

   Morris, Olivia; Morris, Michele; Jobe, Shawn; Bhargava, Disha; Krueger, Jena M; Arora, Sanjana; Prokop, Jeremy W; Stenger, Cynthia (December 2024, Genes)

   Background: Variants within factor VIII (F8) are associated with sex-linked hemophilia A and thrombosis, with gene therapy approaches being available for pathogenic variants. Many variants within F8 remain variants of uncertain significance (VUS) or are under-explored as to their connections to phenotypic outcomes. Methods: We assessed data on F8 expression while screening the UniProt, ClinVar, Geno2MP, and gnomAD databases for F8 missense variants; these collectively represent the sequencing of more than a million individuals. Results: For the two F8 isoforms coding for different protein lengths (2351 and 216 amino acids), we observed noncoding variants influencing expression which are also associated with thrombosis risk, with uncertainty as to differences in females and males. Variant analysis identified a severe stratification of potential annotation issues for missense variants in subjects of non-European ancestry, suggesting a need for further defining the genetics of diverse populations. Additionally, few heterozygous female carriers of known pathogenic variants have sufficiently confident phenotyping data, leaving researchers unable to determine subtle, less defined phenotypes. Using structure movement correlations to known pathogenic variants for the VUS, we determined seven clusters of likely pathogenic variants based on screening work. Conclusions: This work highlights the need to define missense variants, especially those for VUS and from subjects of non-European ancestry, as well as the roles of these variants in women’s physiology. 

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3. Allele-specific NKX2-5 binding underlies multiple genetic associations with human electrocardiographic traits

   https://doi.org/10.1038/s41588-019-0499-3  

   Benaglio, Paola; D’Antonio-Chronowska, Agnieszka; Ma, Wubin; Yang, Feng; Young Greenwald, William W.; Donovan, Margaret K.; DeBoever, Christopher; Li, He; Drees, Frauke; Singhal, Sanghamitra; et al (September 2019, Nature Genetics)

   The cardiac transcription factor (TF) gene NKX2-5 has been associated with electrocardiographic (EKG) traits through genome-wide association studies (GWASs), but the extent to which differential binding of NKX2-5 at common regulatory variants contributes to these traits has not yet been studied. We analyzed transcriptomic and epigenomic data from induced pluripotent stem cell-derived cardiomyocytes from seven related individuals, and identified ~2,000 single-nucleotide variants associated with allele-specific effects (ASE-SNVs) on NKX2-5 binding. NKX2-5 ASE-SNVs were enriched for altered TF motifs, for heart-specific expression quantitative trait loci and for EKG GWAS signals. Using fine-mapping combined with epigenomic data from induced pluripotent stem cell–derived cardiomyocytes, we prioritized candidate causal variants for EKG traits, many of which were NKX2-5 ASE-SNVs. Experimentally characterizing two NKX2-5 ASE-SNVs (rs3807989 and rs590041) showed that they modulate the expression of target genes via differential protein binding in cardiac cells, indicating that they are functional variants underlying EKG GWAS signals. Our results show that differential NKX2-5 binding at numerous regulatory variants across the genome contributes to EKG phenotypes. 

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4. Discovery of target genes and pathways at GWAS loci by pooled single-cell CRISPR screens

   https://doi.org/10.1126/science.adh7699  

   Morris, John A.; Caragine, Christina; Daniloski, Zharko; Domingo, Júlia; Barry, Timothy; Lu, Lu; Davis, Kyrie; Ziosi, Marcello; Glinos, Dafni A.; Hao, Stephanie; et al (May 2023, Science)

   INTRODUCTION Genome-wide association studies (GWASs) have identified thousands of human genetic variants associated with diverse diseases and traits, and most of these variants map to noncoding loci with unknown target genes and function. Current approaches to understand which GWAS loci harbor causal variants and to map these noncoding regulators to target genes suffer from low throughput. With newer multiancestry GWASs from individuals of diverse ancestries, there is a pressing and growing need to scale experimental assays to connect GWAS variants with molecular mechanisms. Here, we combined biobank-scale GWASs, massively parallel CRISPR screens, and single-cell sequencing to discover target genes of noncoding variants for blood trait loci with systematic targeting and inhibition of noncoding GWAS loci with single-cell sequencing (STING-seq). RATIONALE Blood traits are highly polygenic, and GWASs have identified thousands of noncoding loci that map to candidate cis -regulatory elements (CREs). By combining CRE-silencing CRISPR perturbations and single-cell readouts, we targeted hundreds of GWAS loci in a single assay, revealing target genes in cis and in trans . For select CREs that regulate target genes, we performed direct variant insertion. Although silencing the CRE can identify the target gene, direct variant insertion can identify magnitude and direction of effect on gene expression for the GWAS variant. In select cases in which the target gene was a transcription factor or microRNA, we also investigated the gene-regulatory networks altered upon CRE perturbation and how these networks differ across blood cell types. RESULTS We inhibited candidate CREs from fine-mapped blood trait GWAS variants (from ~750,000 individual of diverse ancestries) in human erythroid progenitors. In total, we targeted 543 variants (254 loci) mapping to candidate CREs, generating multimodal single-cell data including transcriptome, direct CRISPR gRNA capture, and cell surface proteins. We identified target genes in cis (within 500 kb) for 134 CREs. In most cases, we found that the target gene was the closest gene and that specific enhancer-associated biochemical hallmarks (H3K27ac and accessible chromatin) are essential for CRE function. Using multiple perturbations at the same locus, we were able to distinguished between causal variants from noncausal variants in linkage disequilibrium. For a subset of validated CREs, we also inserted specific GWAS variants using base-editing STING-seq (beeSTING-seq) and quantified the effect size and direction of GWAS variants on gene expression. Given our transcriptome-wide data, we examined dosage effects in cis and trans in cases in which the cis target is a transcription factor or microRNA. We found that trans target genes are also enriched for GWAS loci, and identified gene clusters within trans gene networks with distinct biological functions and expression patterns in primary human blood cells. CONCLUSION In this work, we investigated noncoding GWAS variants at scale, identifying target genes in single cells. These methods can help to address the variant-to-function challenges that are a barrier for translation of GWAS findings (e.g., drug targets for diseases with a genetic basis) and greatly expand our ability to understand mechanisms underlying GWAS loci. Identifying causal variants and their target genes with STING-seq. Uncovering causal variants and their target genes or function are a major challenge for GWASs. STING-seq combines perturbation of noncoding loci with multimodal single-cell sequencing to profile hundreds of GWAS loci in parallel. This approach can identify target genes in cis and trans , measure dosage effects, and decipher gene-regulatory networks. 

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5. Developmental onset of enduring long‐term potentiation in mouse hippocampus

   https://doi.org/10.1002/hipo.23257  

   Ostrovskaya, Olga I.; Cao, Guan; Eroglu, Cagla; Harris, Kristen M. (September 2020, Hippocampus)

   Abstract Analysis of long‐term potentiation (LTP) provides a powerful window into cellular mechanisms of learning and memory. Prior work shows late LTP (L‐LTP), lasting >3 hr, occurs abruptly at postnatal day 12 (P12) in thestratum radiatumof rat hippocampal area CA1. The goal here was to determine the developmental profile of synaptic plasticity leading to L‐LTP in the mouse hippocampus. Two mouse strains and two mutations known to affect synaptic plasticity were chosen: C57BL/6J andFmr1^−/yon the C57BL/6J background, and 129SVE andHevin^−/−(Sparcl1^−/−) on the 129SVE background. Like rats, hippocampal slices from all of the mice showed test pulse‐induced depression early during development that was gradually resolved with maturation by 5 weeks. All the mouse strains showed a gradual progression between P10‐P35 in the expression of short‐term potentiation (STP), lasting ≤1 hr. In the 129SVE mice, L‐LTP onset (>25% of slices) occurred by 3 weeks, reliable L‐LTP (>50% slices) was achieved by 4 weeks, andHevin^−/−advanced this profile by 1 week. In the C57BL/6J mice, L‐LTP onset occurred significantly later, over 3–4 weeks, and reliability was not achieved until 5 weeks. Although some of theFmr1^−/ymice showed L‐LTP before 3 weeks, reliable L‐LTP also was not achieved until 5 weeks. L‐LTP onset was not advanced in any of the mouse genotypes by multiple bouts of theta‐burst stimulation at 90 or 180 min intervals. These findings show important species differences in the onset of STP and L‐LTP, which occur at the same age in rats but are sequentially acquired in mice. 

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