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PurposeDetermination of genotypic/phenotypic features of GATAD2B-associated neurodevelopmental disorder (GAND).MethodsFifty GAND subjects were evaluated to determine consistent genotypic/phenotypic features. Immunoprecipitation assays utilizing in vitro transcription-translation products were used to evaluate GATAD2B missense variants' ability to interact with binding partners within the nucleosome remodeling and deacetylase (NuRD) complex.ResultsSubjects had clinical findings that included macrocephaly, hypotonia, intellectual disability, neonatal feeding issues, polyhydramnios, apraxia of speech, epilepsy, and bicuspid aortic valves. Forty-one novelGATAD2B variants were identified with multiple variant types (nonsense, truncating frameshift, splice-site variants, deletions, and missense). Seven subjects were identified with missense variants that localized within two conserved region domains (CR1 or CR2) of the GATAD2B protein. Immunoprecipitation assays revealed several of these missense variants disrupted GATAD2B interactions with its NuRD complex binding partners.ConclusionsA consistent GAND phenotype was caused by a range of genetic variants in GATAD2B that include loss-of-function and missense subtypes. Missense variants were present in conserved region domains that disrupted assembly of NuRD complex proteins. GAND's clinical phenotype had substantial clinical overlap with other disorders associated with the NuRD complex that involve CHD3 and CHD4, with clinical features of hypotonia, intellectual disability, cardiac defects, childhood apraxia of speech, and macrocephaly.

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BackgroundNeurofibromatosis type 2 (NF2) is a genetic tumor-predisposition disorder caused by NF2/merlin tumor suppressor gene inactivation. The hallmark of NF2 is formation of bilateral vestibular schwannomas (VS). Because merlin modulates activity of the Ras/Raf/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway, we investigated repurposing drugs targeting MEK1 and/or MEK2 as a treatment for NF2-associated schwannomas.MethodsMouse and human merlin-deficient Schwann cell lines (MD-MSC/HSC) were screened against 6 MEK1/2 inhibitors. Efficacious drugs were tested in orthotopic allograft and NF2 transgenic mouse models. Pathway and proteome analyses were conducted. Drug efficacy was examined in primary human VS cells with NF2 mutations and correlated with DNA methylation patterns.ResultsTrametinib, PD0325901, and cobimetinib were most effective in reducing MD-MSC/HSC viability. Each decreased phosphorylated pERK1/2 and cyclin D1, increased p27, and induced caspase-3 cleavage in MD-MSCs. Proteomic analysis confirmed cell cycle arrest and activation of pro-apoptotic pathways in trametinib-treated MD-MSCs. The 3 inhibitors slowed allograft growth; however, decreased pERK1/2, cyclin D1, and Ki-67 levels were observed only in PD0325901 and cobimetinib-treated grafts. Tumor burden and average tumor size were reduced in trametinib-treated NF2 transgenic mice; however, tumors did not exhibit reduced pERK1/2 levels. Trametinib and PD0325901 modestly reduced viability of several primary human VS cell cultures with NF2 mutations. DNA methylation analysis of PD0325901-resistant versus -susceptible VS identified genes that could contribute to drug resistance.ConclusionMEK inhibitors exhibited differences in antitumor efficacy resistance in schwannoma models with possible emergence of trametinib resistance. The results support further investigation of MEK inhibitors in combination with other targeted drugs for NF2 schwannomas.

Biallelic SORD mutations cause one of the most frequent forms of recessive hereditary neuropathy, estimated to affect ∼10 000 patients in North America and Europe alone. Pathogenic SORD loss-of-function changes in the encoded enzyme sorbitol dehydrogenase result in abnormally high sorbitol levels in cells and serum. How sorbitol accumulation leads to peripheral neuropathy remains to be elucidated. A reproducible animal model for SORD neuropathy is essential to illuminate the pathogenesis of SORD deficiency and for preclinical studies of potential therapies. Therefore, we have generated a Sord knockout (KO), Sord −/−, Sprague Dawley rat, to model the human disease and to investigate the pathophysiology underlying SORD deficiency. We have characterized the phenotype in these rats with a battery of behavioural tests as well as biochemical, physiological and comprehensive histological examinations. Sord −/− rats had remarkably increased levels of sorbitol in serum, CSF and peripheral nerve. Moreover, serum from Sord −/− rats contained significantly increased levels of neurofilament light chain, an established biomarker for axonal degeneration. Motor performance significantly declined in Sord −/− animals starting at ∼7 months of age. Gait analysis evaluated with video motion-tracking confirmed abnormal gait patterns in the hindlimbs. Motor nerve conduction velocities of the tibial nerves were slowed. Light and electron microscopy of the peripheral nervous system revealed degenerating myelinated axons, de- and remyelinated axons, and a likely pathognomonic finding—enlarged 'ballooned' myelin sheaths. These findings mainly affected myelinated motor axons; myelinated sensory axons were largely spared. In summary, Sord −/− rats develop a motor-predominant neuropathy that closely resembles the human phenotype. Our studies revealed novel significant aspects of SORD deficiency, and this model will lead to an improved understanding of the pathophysiology and the therapeutic options for SORD neuropathy. [ABSTRACT FROM AUTHOR]

We set out to determine whether expansions in the C9ORF72 repeat found in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) families are associated with Parkinson disease (PD). We determined the repeat size in a total of 889 clinically ascertained patients (including PD and essential tremor plus Parkinsonism (ETP)) and 1144 controls using a repeat-primed PCR assay. We found that large C9ORF72 repeat expansions (>30 repeats) were not contributing to PD risk. However, PD and ETP cases had a significant increase in intermediate (>20 to 30+) repeat copies compared to controls. Overall, 14 cases (13 PD, 1 ETP) and three controls had >20 repeat copies (Fisher's exact test p = 0.002). Further, seven cases and no controls had >23 repeat copies (p = 0.003). Our results suggest that intermediate copy numbers of the C9ORF72 repeat contribute to risk for PD and ETP. This also suggests that PD, ALS and FTD share some pathophysiological mechanisms of disease. Further studies are needed to elucidate the contribution of the C9ORF72 repeat in the overall PD population and to determine whether other common genetic risk factors exist between these neurodegenerative disorders.

Background: We previously identified a 44‐base pair deletion in (ATP‐binding cassette sub‐family A member 7) (ABCA7) that is significantly associated with Alzheimer's disease (AD) in African Americans (AA), producing a frameshift mutation resulting in a truncated protein (p.Arg578Alafs). ABCA7 is a lipid transporter across cellular membranes. While we have shown the mutant mRNA is present in neurons, whether it is translated into a stable protein is not known due to the lack of antibodies capable of recognizing the N‐terminus of endogenous ABCA7. The abrupt ABCA7 translation due to the deletion may alter the lipid metabolism, which can be investigated using isogenic iPSC and differentiated models. Methods: We analyzed single‐cell RNAseq (scRNAseq) data from spheroid cultures of 12 individuals. We generated a recombinant version of the ABCA7 gene with and without the deletion and bearing an N‐terminal flag tag which were transfected into HEK cells. We utilized CRISPR‐based genome editing using induced pluripotent stem cell (iPSC) lines from three independent AA‐cognitively unimpaired individuals (WT) to introduce the AA‐specific ABCA7 homozygous or heterozygous deletions. These were differentiated using standard protocols in the HIHG iPSC core into monocultures of induced neuron‐like cells. C‐terminal antibodies were used to assess the presence of native ABCA7 in HEK cells. Results: Examination of HEK cells revealed no detectable native ABCA7. The truncated ABCA7‐tagged protein appeared stable and localized in the plasma membrane as seen for the wild‐type protein (Figure 1). ScRNAseq confirmed that expression of ABCA7 is highest in neurons, identifying them as the iPSC differentiated cell of choice. We successfully generated both homozygous and heterozygous deletion isogenic lines in the three WT iPSC lines. Differentiated neurons from these isogenic lines have a normal phenotype, allowing for functional assays and lipidomic studies. Conclusion: The truncated protein p.Arg578Alafs appears to be expressed and stable in HEK cells, and surprisingly located in the plasma membrane despite the absence of most transmembrane domains. Our three isogenic iPSC pairs will be a great resource for studying the pathogenic effects of the ABCA7 truncation in differentiated cells. [ABSTRACT FROM AUTHOR]

Background: Genome‐wide association studies (GWAS) in Alzheimer's disease (AD) are consistently discovering genetic variants linked to the risk of developing this neurodegenerative condition. However, the effect size of the shared associated loci varies across populations as well as each population can have unique associations. This phenomenon could be explained by ancestry‐dependent changes in the genomic regulatory architecture (GRA) influencing the expression of these genes, similar to the effect of different local ancestry on the risk of AD in APOE4 carriers. Thus, understanding of GRA in the context of AD is imperative but currently most GRA data available is predominantly European, limiting our ability to comprehensively interpret the variability associated with AD risk genes across populations. For this study we focused on oligodendroglia, a cell lineage that has been historically overlooked but that is emerging as key players in AD due to their involvement in various pathological processes, including neuroinflammation, oxidative stress, and synaptic dysfunction. Here, we report ancestry‐dependent differences in the GRA of iPSC derived oligodendroglia with African, Amerindian, or European global ancestry. Method: We obtained PBMCs from individuals with Alzheimer's disease (AD) or without cognitive impairment, each with over 85% global ancestry of a specific ancestral background. These cells were then transformed into induced pluripotent stem cells (iPSC) and subsequently differentiated into oligodendroglia‐containing 3D neural cultures. On the 76th day of differentiation, we harvested and lysed the cells to isolate nuclei for Multiomic profiling including Single Cell ATAC and Single Cell RNA‐seq, we analyzed the chromatin accessibility and transcriptomes to identify ancestry‐dependent changes genome‐wide and in AD GWAS hits. Result: We found several AD GWAS hits differentially expressed between ancestries in OPCs and in the more mature oligodendrocyte population (including APP and CLU) and some differentially accessible peaks associated to some of these genes (predominantly PRDM7). Nevertheless, OPCs showed more ancestry‐specific regulation than the more mature oligodendrocytes. Conclusion: Our findings offer ancestry‐specific understanding of oligodendroglia chromatin changes and gene regulation in the context of AD. These results present a comprehensive perspective on the genetic regulatory architecture of oligodendroglia and constitute a resource for gene identification studies in the African American and Hispanic populations. [ABSTRACT FROM AUTHOR]

Background: Annotation of target genes of non‐coding GWAS loci remains a challenge since 1) regulatory elements identified by GWAS can be metabases away from its actual target, 2) one regulatory element can target multiple genes, and 3) multiple regulatory elements can target one gene. AD GWAS in populations with different ancestries have identified different loci, suggesting ancestry‐specific genetic risks. To understand the connection between associated loci (potential regulatory elements) and their target genes, we conducted Hi‐C analysis in frontal cortex of African American (AA) and Non‐Hispanic Whites (NHW) AD patients to map chromatin loops, which often represent enhancer‐promoter (EP) interactions. In our initial analyses, we applied HiC to uncover the regulatory architecture of rs3851179 linked to the embryonic ectoderm development gene (EED) in the ADSP gene verification list. Method: Hi‐C libraries were derived from four AA and four NHW donors matched for age, sex, and APOE genotype. The AA samples had 68∼88% African (AF) genome while NHW samples had >95% European (EU) genome. DeepLoop was used to generate robust maps of chromatin loops. Hi‐C data from each population were pooled to generate reference chromatin loop maps for the AF and EU genomes. Result: rs3851179 (OR = 0.9, P = 3.0 × 10−48) resides between EED and PICALM, about 80 Kb away from both with each gene transcribed in opposite directions. Although PICALM is designated as a causal gene, EED is designated a susceptibility gene by ADSP based on the transcription direction and distance from rs3851179. Hi‐C data from both AF and EU genome in frontal cortex revealed that rs3851179 co‐localizes with a loop anchor, connecting H3K27ac peaks (enhancer) overlapping rs3851179 to the proximal promoter of PICALM, suggesting that PICALM but not EED is the target gene, corroborated by the GTEX e‐QTL data. Conclusion: This initial application of Hi‐C data demonstrates the utility of chromatin regulatory maps in nominating target genes of GWAS hits in non‐coding loci. The generation and eventual application of Hi‐C maps in African and Amerindian genomes (which are part of the AA or American Hispanic genomic admixture) will be key dissecting ancestry‐specific genetic loci and, thereby, broadening diversity in AD research. [ABSTRACT FROM AUTHOR]

Background: The Apoliproprotein E (APOE) e4 allele is the most significant genetic risk factor for late‐onset Alzheimer disease (AD). However, the risk associated with the APOE e4 allele differs across populations with individuals of African ancestry having a reduced risk than individuals of European (EU) ancestry. Further, single‐nuclei RNAseq analysis in autopsy samples from AD APOEε4 homozygotes with EU Local Ancestry (LA) had a significantly increased APOEε4 expression compared to those with African LA, particularly in astrocytes. To test the functional consequences of elevated APOE4 expression on astrocytes, we examined the effect of APOE4 overexpression in inducible pluripotent stem cell (iPSC) derived astrocytes (iAstrocytes). Method: iPSC were derived from European and African LA AD individuals homozygous for APOEε4/ε4. These APOEε4/ε4 iPSC were differentiated into astrocytes (iAstrocytes) which were transduced with a lentiviral construct overexpressing APOEε4 or a control vector. RNAseq analysis was performed to identify differentially expressed genes followed by pathway analyses. Result: iPSC lines were derived, validated for pluripotency, and differentiated into iAstrocytes. Preliminary analysis of transcriptional effects ofApoEe4 overexpression in iAstrocytes showed alterations in gene expression patterns, including the down regulation of the inflammatory markers THBS1 and CLDN11 which had both been shown to be downregulated in AD, the mitochondrial‐related gene NDUFA4L2 shown to be dysregulated in AD, as well as SFRP2 (Wnt modulator involved in TREM2 processing). Upregulated genes included RGMA (accumulates in amyloid plaques), as well as TNC and CECR2. Pathway analysis showed an enrichment for extracellular matrix (ECM)‐receptor interaction, PI3K‐Akt signaling, and TGF‐beta signaling pathways. Interestingly, the transcriptional response to APOEε4 overexpression has an ancestry‐specific component in these iAstrocytes; with some genes dysregulated exclusively in African ancestry iAstrocytes such as ZBTB4 and PRND, and others only in European iAstrocytes, including MYBPHL and VWF. Conclusion: The overexpression of APOEε4 in iAstrocytes resulted in alteration in gene regulatory patterns, including the ECM and key signaling pathways across ancestries (EU and AF). In addition, gene expression differences were identified between ancestries. Further analyses of these ancestry‐specific transcriptional differences may help to elucidate the mechanisms that drive the differential AD risk seen in APOEε4 individuals across ancestries. [ABSTRACT FROM AUTHOR]

Background: Non‐Hispanic White APOE4 carriers have a higher risk of developing AD compared to African American APOE4 carriers. The local ancestry (LA) surrounding the APOE region was previously shown to be the primary factor in this risk difference. APOE4 carriers of European LA (ELA) have been found to have higher APOE4 expression and chromatin accessibility compared to African LA (ALA). We sought to investigate whether the LA around APOE3 has the same effect between ancestries. Differences between alleles could provide insight into ancestry‐specific regulatory areas controlling the APOE4 expression. Method: We identified AD autopsy samples by GSA, all homozygous for APOE3 and LA from 4 ADRC brain banks: Miami, Emory, Duke, and Rush. We performed single nuclei RNA sequencing (snRNA‐seq) on eight frozen frontal cortex (B9 area) samples using 10x Genomics. Result: We performed snRNA‐seq in a total of 51,462 nuclei from eight brains (4 ELA and 4 ALA). We identified 35 distinct cell clusters at a resolution of 0.6. The proportion of cells per cluster between ELA and ALA samples was similar for all clusters, except for cluster 32 (Excitatory Neurons) which had a greater than 2‐fold difference in ALA. Our data shows that APOE3 carriers with ELA have a significantly higher APOE expression in Excitatory Neurons (cluster 7) than those of ALA and contrary to our previous observations in APOE4 carriers (Griswold, A. et al, (2021)), APOE3 carriers of ALA express higher APOE in astrocytes (cluster 2) and Microglia (cluster 6). However, overall, comparison of APOE3 vs APOE4 carriers in this study demonstrated that APOE3 alleles have significantly lower gene expression than APOE4 carriers of the same LA. Conclusion: Our preliminary data suggest that the LA surrounding APOE3 is associated with different effects on APOE expression compared to APOE4 allele. Further, within the same LA, we observed that, overall, the different cell types express less APOE in APOE3 carriers compared to APOE4 carriers. Altogether, this may provide additional insight into the regulatory mechanisms affecting APOE4 expression. [ABSTRACT FROM AUTHOR]

Background: The effect size of APOE4 varies across genetic ancestries with African (AFR) local ancestry conferring a lower risk when compared to other ancestries. Recently, we identified a strong effect of the A allele of rs10423769 (with a minor allele frequency of 0.12 in AFR and 0.003 in Europeans), which lowers the risk of AD by 71% in APOE4 carriers. rs10423769 is 2 MB from APOEe4 in a region of segmental duplications (SD) containing a cluster of pregnancy specific beta‐1 glycoprotein genes (PSGs) and a lncRNA. To gain insights into the mechanism of protection, we characterized the haplotype harboring the variant and investigated differences in methylation and structural variation (SV) between haplotypes. Method: We used the Alzheimer's Disease Sequencing Project (ADSP) short read sequencing database to fine map and extend the protective haplotype using Haploview 4.2. In addition, long read sequencing (LRS) with the Oxford Nanopore PromethION was performed on 16 samples of individuals carrying one or two copies of rs10423769_A. LRS was used for SV calling with Sniffles2 and differential methylation analysis using Nanomethphase. Result: The minimum shared haplotype harboring rs10423769_A allele was ∼21kbp (chr19:43099521‐ 43120243). Though the region is rich in SDs, high quality LRS reads mapped uniquely to the region around the A allele confirming that the haplotype is unique and not duplicated elsewhere in the genome of the sequenced individuals. Moreover, the rs10423769_A haplotype harbors 23 differential CpG sites (fdr<0.05) compared to rs10423769_G, although no differential methylated region was identified. LRS revealed a 140bp insertion of repetitive MEF2 binding motifs. Initial examination demonstrated that the insertion was present in 80% of the A allele carriers. Conclusion: We have identified an AF‐specific haplotype protective against AD risk conferred by APOE ε4. Considering the distance from APOE and the complexity of the highly SD region where the variant is located, we have identified the minimum shared haplotype from AFR origin and are investigating the potential effects of SVs and differential CpG sites. Ongoing analyses include 3D chromatin structure and LRS at higher coverage to assemble and resolve the complex genomic context of the allele. [ABSTRACT FROM AUTHOR]

Background: Previous studies have reported that non‐Hispanic white (NHW) females carrying the APOE ε4 allele differ in risk of developing Alzheimer's disease (AD) when compared to men. Recent studies suggest the association between APOE ε4 and AD risk may be modified by age and its local ancestry in admixed populations. However, there is still scant evidence on how sex could interact with these factors. We aimed to investigate the association of sex, ancestry, APOE, and local ancestry with AD in multiple cohorts. Methods: A pooled case‐control study was undertaken from 50 independent datasets (n = 30,539;age≥55years) in the Alzheimer's Disease Genetics Consortium. Logistic regression models were used to study the associations between disease status and independent variables including age, sex, reported race (as a proxy of ancestry), and APOE genotype. Additionally, mixed models were implemented for the local ancestry APOE haplotype analyses among different ethnicities. A p‐value less than 0.05 was considered statistically significant. Results: Regardless of ancestry, men and women with APOE ε3/ε4 or ε4/ε4 genotypes showed no significant difference in the risk of developing AD. When comparing subjects with APOE ε3/ε4 or ε4/ε4 (vs. ε3/ε3), East‐Asian (EA) males (OR,5.56; 95% CI:4.26‐7.60) and females(OR,5.46:4.34‐6.86) had the strongest ORs; whilst the weakest risk was for South‐Asian (SA) men(OR,1.14:0.99‐2.57) among all races/ethnicities. Age‐stratified analyses showed that NHW females(OR,5.36:4.74‐6.07 vs. males: OR,4.72:4.10‐5.60) had a higher risk of developing AD between ages 66‐75; whereas African American (AA), EA and SA exhibited this pattern at 55‐64. In Hispanics, the relationship is reversed, with males aged 55‐65 years carrying APOE ε3/ε4 or ε4/ε4(OR,4.53:1.67‐12.30: vs. females: OR,3.03:1.59‐5.78) having a stronger association with AD risk than their counterpart. No differences were found when stratified by sex and age for APOE ε2 and AD. Local ancestry (European ‐EU‐; African ‐AF‐; Amerindian ‐AI‐; and EA) analyses are being conducted by ancestry and sex. Preliminary analyses suggest differences by sex and local ancestry for APOE ε4. Conclusions: Our findings suggest differences by sex and reported ancestry in the risk for developing AD in carriers of APOE ε4. Global, local ancestry and sex should be considered in predicting risk due to APOE alleles. [ABSTRACT FROM AUTHOR]