Mutations in NSUN2 Cause Autosomal- Recessive Intellectual Disability

With a prevalence between 1 and 3%, hereditary forms of intellectual disability (ID) are among the most important problems in health care. Particularly, autosomal-recessive forms of the disorder have a very heterogeneous molecular basis, and genes with an increased number of disease-causing mutations are not common. Here, we report on three different mutations (two nonsense mutations, c.679C>T [p.Gln227∗] and c.1114C>T [p.Gln372∗], as well as one splicing mutation, g.6622224A>C [p.Ile179Argfs∗192]) that cause a loss of the tRNA-methyltransferase-encoding NSUN2 main transcript in homozygotes. We identified the mutations by sequencing exons and exon-intron boundaries within the genomic region where the linkage intervals of three independent consanguineous families of Iranian and Kurdish origin overlapped with the previously described MRT5 locus. In order to gain further evidence concerning the effect of a loss of NSUN2 on memory and learning, we constructed a Drosophila model by deleting the NSUN2 ortholog, CG6133, and investigated the mutants by using molecular and behavioral approaches. When the Drosophila melanogaster NSUN2 ortholog was deleted, severe short-term-memory (STM) deficits were observed; STM could be rescued by re-expression of the wild-type protein in the nervous system. The humans homozygous for NSUN2 mutations showed an overlapping phenotype consisting of moderate to severe ID and facial dysmorphism (which includes a long face, characteristic eyebrows, a long nose, and a small chin), suggesting that mutations in this gene might even induce a syndromic form of ID. Moreover, our observations from the Drosophila model point toward an evolutionarily conserved role of RNA methylation in normal cognitive development

BOD1 Is Required for Cognitive Function in Humans and <i>Drosophila</i>

Here we report a stop-mutation in the BOD1 (Biorientation Defective 1) gene, which co-segregates with intellectual disability in a large consanguineous family, where individuals that are homozygous for the mutation have no detectable BOD1 mRNA or protein. The BOD1 protein is required for proper chromosome segregation, regulating phosphorylation of PLK1 substrates by modulating Protein Phosphatase 2A (PP2A) activity during mitosis. We report that fibroblast cell lines derived from homozygous BOD1 mutation carriers show aberrant localisation of the cell cycle kinase PLK1 and its phosphatase PP2A at mitotic kinetochores. However, in contrast to the mitotic arrest observed in BOD1-siRNA treated HeLa cells, patient-derived cells progressed through mitosis with no apparent segregation defects but at an accelerated rate compared to controls. The relatively normal cell cycle progression observed in cultured cells is in line with the absence of gross structural brain abnormalities in the affected individuals. Moreover, we found that in normal adult brain tissues BOD1 expression is maintained at considerable levels, in contrast to PLK1 expression, and provide evidence for synaptic localization of Bod1 in murine neurons. These observations suggest that BOD1 plays a cell cycle-independent role in the nervous system. To address this possibility, we established two Drosophila models, where neuron-specific knockdown of BOD1 caused pronounced learning deficits and significant abnormalities in synapse morphology. Together our results reveal novel postmitotic functions of BOD1 as well as pathogenic mechanisms that strongly support a causative role of BOD1 deficiency in the aetiology of intellectual disability. Moreover, by demonstrating its requirement for cognitive function in humans and Drosophila we provide evidence for a conserved role of BOD1 in the development and maintenance of cognitive features

Antibodies used in western blotting.

Neurodegenerative diseases encompass a group of debilitating conditions resulting from progressive nerve cell death. Of these, Alzheimer’s disease (AD) occurs most frequently, but is currently incurable and has limited treatment success. Late onset AD, the most common form, is highly heritable but is caused by a combination of non-genetic risk factors and many low-effect genetic variants whose disease-causing mechanisms remain unclear. By mining the FinnGen study database of phenome-wide association studies, we identified a rare variant, rs148726219, enriched in the Finnish population that is associated with AD risk and dementia, and appears to have arisen on a common haplotype with older AD-associated variants such as rs429358. The rs148726219 variant lies in an overlapping intron of the FosB proto-oncogene (FOSB) and ERCC excision repair 1 (ERCC1) genes. To understand the impact of this SNP on disease phenotypes, we performed CRISPR/Cas9 editing in a human induced pluripotent stem cell (hiPSC) line to generate isogenic clones harboring heterozygous and homozygous alleles of rs148726219. hiPSC clones differentiated into induced excitatory neurons (iNs) did not exhibit detectable molecular or morphological variation in differentiation potential compared to isogenic controls. However, global transcriptome analysis showed differential regulation of nearby genes and upregulation of several biological pathways related to neuronal function, particularly synaptogenesis and calcium signaling, specifically in mature iNs harboring rs148726219 homozygous and heterozygous alleles. Functional differences in iN circuit maturation as measured by calcium imaging were observed across genotypes. Edited mature iNs also displayed downregulation of unfolded protein response and cell death pathways. This study implicates a phenotypic impact of rs148726219 in the context of mature neurons, consistent with its identification in late onset AD, and underscores a hiPSC-based experimental model to functionalize GWAS-identified variants.</div

Secreted levels of amyloid beta from rs148726219-edited lines.

Expression of Aβ protein isoforms Aβ40 (A), Aβ38 (B), and Aβ42 (C) measured by MSD ELISA from media conditioned by BIONi010-C-13 parental, WT-2A1, HET-2D2, HET-2G6, HOM-2B11, and HOM-2H6 lines on day 23 of iN differentiation. The ratio of Aβ42/Aβ40 is also shown (D). Cells were untreated (blue bars), treated with vehicle control (DMSO; grey bars), or treated with DAPT to inhibit Aβ production (white bars) for 24 hrs prior to media collection. Values were normalized against a standard curve and expressed relative to total cell number, quantified as relative fluorescence units from Cell Titer Glo assays. n = 3 independent hiPSC-iNeuron differentiations (or n = 2 for HOM-2H6), with each represented by a black dot. No apparent differences in Aβ levels are observed between lines with heterozygous/homozygous alleles for rs148726219 and wild type lines. (PDF)</p

Principle component analysis plots of RNA-seq data.

PCA plots are colored by time point (A), colored by rs148726219-edited clone (B), and colored by BIONi010-C-13 parental line versus WT-2A1 clone (C). Samples cluster by time point, not by line. No apparent differences are detectable between the parental line and WT clone. (PDF)</p

Differentially expressed genes within 1 Mb of <i>FOSB</i>.

Differentially expressed genes within 1 Mb of FOSB.</p

Expression of genes within 1 Mb of rs148726219.

Expression values are expressed as fragments per kilobase per million mapped reads (FPKM) for WT-2A1 (green), rs148726219-heterozygous (HET-2D2, HET-2G6; orange), and homozygous (HOM-2B11, HOM-2H6; purple) clones at day 0, 2, 6, 13, and 23 of hiPSC-iNeuron differentiation. Four technical replicates for each clone are shown with each represented by a black dot. A. ERCC1. B. BCL3. C. PPP1R37. D. RTN2. E. DMPK. F. APOE. (PDF)</p

Calcium signaling imaging of HET-2D2 iNeurons.

Neurodegenerative diseases encompass a group of debilitating conditions resulting from progressive nerve cell death. Of these, Alzheimer’s disease (AD) occurs most frequently, but is currently incurable and has limited treatment success. Late onset AD, the most common form, is highly heritable but is caused by a combination of non-genetic risk factors and many low-effect genetic variants whose disease-causing mechanisms remain unclear. By mining the FinnGen study database of phenome-wide association studies, we identified a rare variant, rs148726219, enriched in the Finnish population that is associated with AD risk and dementia, and appears to have arisen on a common haplotype with older AD-associated variants such as rs429358. The rs148726219 variant lies in an overlapping intron of the FosB proto-oncogene (FOSB) and ERCC excision repair 1 (ERCC1) genes. To understand the impact of this SNP on disease phenotypes, we performed CRISPR/Cas9 editing in a human induced pluripotent stem cell (hiPSC) line to generate isogenic clones harboring heterozygous and homozygous alleles of rs148726219. hiPSC clones differentiated into induced excitatory neurons (iNs) did not exhibit detectable molecular or morphological variation in differentiation potential compared to isogenic controls. However, global transcriptome analysis showed differential regulation of nearby genes and upregulation of several biological pathways related to neuronal function, particularly synaptogenesis and calcium signaling, specifically in mature iNs harboring rs148726219 homozygous and heterozygous alleles. Functional differences in iN circuit maturation as measured by calcium imaging were observed across genotypes. Edited mature iNs also displayed downregulation of unfolded protein response and cell death pathways. This study implicates a phenotypic impact of rs148726219 in the context of mature neurons, consistent with its identification in late onset AD, and underscores a hiPSC-based experimental model to functionalize GWAS-identified variants.</div

<i>CAT</i> and <i>PCDHB5</i> are differentially expressed in rs148726219-edited cells throughout hiPSC-iN differentiation.

A.CAT expression measured by RNA-seq shown as fragments per kilobase per million mapped reads (FPKM) for WT-2A1 (green), rs148726219-heterozygous (orange), and homozygous (purple) clones at day 0, 2, 6, 13, and 23 of hiPSC-iNeuron differentiation. Four technical replicates for each clone are shown with each represented by a black dot. B. PCDHB5 expression measured by RNA-seq, as described in (A). C. Representative western blot of day 23 iNeuron lysates in BIONi010-C-13 parental, WT-2A1, HET-2D2, HET-2G6, HOM-2B11, and HOM-2H6 lines probed with an antibody against catalase (top). Protein was quantified relative to GAPDH loading control and is expressed as fold change relative to the average of WT-parental and WT-2A1 lines (bottom; additional WBs used for quantification are shown in S19 Fig). n = 3. ** p < 0.01, **** p < 0.0001 by one-way ANOVA with multiple comparisons.</p