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. 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. 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. 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.

INTRODUCTION:Genome-wide association studies (GWAS) studies in Alzheimer's disease (AD) demonstrate ancestry-specific loci. Previous studies in the regulatory architecture have only been conducted in Europeans (EUs), thus studies in additional ancestries are needed. Given the prevalence of AD genes expressed in microglia, we initiated our studies in induced pluripotent stem cell (iPSC) -derived microglia. METHODS:We created iPSC-derived microglia from 13 individuals of either high Amerindian (AI), African (AF), or EU global ancestry, including both AD and controls. RNA-seq, ATAC-seq, and pathway analyses were compared between ancestries in both AD and non-AD genes. RESULTS:Twelve AD genes were differentially expressed genes (DEGs) and/or accessible between ancestries, including ABI3, CTSB, and MS4A6A. A total of 5% of all genes had differential ancestral expression, but differences in accessibility were less than 1%. The DEGs were enriched in known AD pathways. DISCUSSION:This resource will be valuable in evaluating AD in admixed populations and other neurological disorders and understanding the AD risk differences between populations. HIGHLIGHTS:First comparison of the genomics of AI, AF, and EU microglia. Report differences in expression and accessibility of AD genes between ancestries. Ancestral expression differences are greater than differences in accessibility. Good transcriptome correlation was seen between brain and iPSC-derived microglia. Differentially expressed AD genes were in known AD pathways.

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. 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. 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. 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.

In the US, African Americans (admixed with African and European) followed by Hispanics (admixed with Amerindian, African, and European) are the most affected groups compared to non-Hispanic Whites (NHW). While genetic diversity and admixture play crucial roles in disease risk, the ancestry-specific mechanisms remain poorly understood with most AD-related studies focusing on NHW. Despite the recent field efforts to include genetically admixed populations, there continues to be a lack of functional studies in AD across the different cell types in these populations. Given the importance of Microglia in AD, we here characterize the genetic regulatory architecture (GRA) on iPSC-derived Microglia (MGL) in African and Amerindian genomes. iPSC lines derived from controls and AD patients with >90% genomic content from different ancestries (Amerindian, African, and European) were differentiated into MGL. We performed bulk RNA-seq and ATAC-seq, followed by differential expression and accessibility analyses to study the GRA of these admixed populations and its contributions to AD. We identified 1,103 differentially expressed genes (DEGs) and 267 differentially accessible genes (DAGs) across ancestries. We observed the most differences on both chromatin accessibility and gene expression levels between AI and AF. On the chromatin level and in the context of AD, we observed 2 DAGs ( PRDM7 and SCIMP ) between AI and AF, and 1 DAG between AI and EU ( PRDM7 ). In addition, we identified 10 AD-risk modifying genes that are differentially expressed between AI and AF ancestries ( ABI3, CTSB, JAZF1, MS4A6A, PILRA, PLEKHA1, RASGEF1C, SORL1, TREM2 , and TREML2 ) and 3 DEGs between AI and EU ( JAZF1, MS4A6A , and SORL1 ). We identified several DEGs to be involved in lipid metabolism, cholesterol biosynthesis and metabolism, lysosomal activity, and immune response - all highly relevant processes in AD pathology. We provide new insights into ancestry-specific genetic risk factors in AD pathophysiology. Here, we report novel transcriptomic and chromatin accessibility data in microglia of AI and AF ancestries that potentially contribute to a differential genetic risk in AD in the different ancestries. Interestingly, those ancestries with greatest migratory differences revealed the largest DEG.

APOEε4 is the strongest genetic risk factor for Alzheimer's disease (AD) with approximately 50% of AD patients carrying at least one APOEε4 allele. Our group identified a protective interaction between APOEε4 with the African-specific A allele of rs10423769, which reduces the AD risk effect of APOEε4 homozygotes by approximately 75%. The protective variant lies 2Mb from APOE in a region of segmental duplications (SD) of chromosome 19 containing a cluster of pregnancy specific beta-1 glycoprotein genes (PSGs) and a long non-coding RNA. Using both short and long read sequencing, we demonstrate that rs10423769_A allele lies within a unique single haplotype inside this region of segmental duplication. We identified the protective haplotype in all African ancestry populations studied, including both West and East Africans, suggesting the variant has an old origin. Long-read sequencing identified both structural and DNA methylation differences between the protective rs10423769_A allele and non-protective haplotypes. An expanded variable number tandem repeat (VNTR) containing multiple MEF2 family transcription factor binding motifs was found associated with the protective haplotype (p-value = 2.9e-10). These findings provide novel insights into the mechanisms of this African-origin protective variant for AD in APOEε4 carriers and supports the importance of including all ancestries in AD research.

Delayed fatherhood results in a higher risk of inheriting a new germline mutation that might result in a congenital disorder in the offspring. In particular, some FGFR3 mutations increase in frequency with age, but there are still a large number of uncharacterized FGFR3 mutations that could be expanding in the male germline with potentially early- or late-onset effects in the offspring. Here, we used digital polymerase chain reaction to assess the frequency and spatial distribution of 10 different FGFR3 missense substitutions in the sexually mature male germline. Our functional assessment of the receptor signaling of the variants with biophysical methods showed that 9 of these variants resulted in a higher activation of the receptor´s downstream signaling, resulting in 2 different expansion behaviors. Variants that form larger subclonal expansions in a dissected postmortem testis also showed a positive correlation of the substitution frequency with the sperm donor's age, and a high and ligand-independent FGFR3 activation. In contrast, variants that measured high FGFR3 signaling and elevated substitution frequencies independent of the donor's age did not result in measurable subclonal expansions in the testis. This suggests that promiscuous signal activation might also result in an accumulation of mutations before the sexual maturation of the male gonad with clones staying relatively constant in size throughout time. Collectively, these results provide novel insights into our understanding of the mutagenesis of driver mutations and their resulting mosaicism in the male germline with important consequences for the transmission and recurrence of associated disorders.

Advanced paternal age increases the risk of transmitting de novo germline mutations, particularly missense mutations activating the receptor tyrosine kinase (RTK) signalling pathway, as exemplified by the FGFR3 mutation, which is linked to achondroplasia (ACH). This risk is attributed to the expansion of spermatogonial stem cells carrying the mutation, forming sub-clonal clusters in the ageing testis, thereby increasing the frequency of mutant sperm and the number of affected offspring from older fathers. While prior studies proposed a correlation between sub-clonal cluster expansion in the testis and elevated mutant sperm production in older donors, limited data exist on the universality of this phenomenon. Our study addresses this gap by examining the testis-expansion patterns, as well as the increases in mutations in sperm for two FGFR3 variants—c.1138G>A (p.G380R) and c.1948A>G (p.K650E)—which are associated with ACH or thanatophoric dysplasia (TDII), respectively. Unlike the ACH mutation, which showed sub-clonal expansion events in an aged testis and a significant increase in mutant sperm with the donor’s age, as also reported in other studies, the TDII mutation showed focal mutation pockets in the testis but exhibited reduced transmission into sperm and no significant age-related increase. The mechanism behind this divergence remains unclear, suggesting potential pleiotropic effects of aberrant RTK signalling in the male germline, possibly hindering differentiation requiring meiosis. This study provides further insights into the transmission risks of micro-mosaics associated with advanced paternal age in the male germline.

Background: This study aims to elucidate ancestry-specific changes to the genomic regulatory architecture in induced pluripotent stem cell (iPSC)-derived oligodendroglia, focusing on their implications for Alzheimer's disease (AD). This work addresses the lack of diversity in previous iPSC studies by including ancestries that contribute to African American (European/African) and Hispanic/Latino populations (Amerindian/African/European). Methods: We generated 12 iPSC lines—four African, four Amerindian, and four European— from both AD patients and non-cognitively impaired individuals, with varying APOE genotypes (APOE3/3 and APOE4/4). These lines were differentiated into neural spheroids containing oligodendrocyte lineage cells. Single-nuclei RNA sequencing and ATAC sequencing were employed to analyze transcriptional and chromatin accessibility profiles, respectively. Differential gene expression, chromatin accessibility, and Hi-C analyses were conducted, followed by pathway analysis to interpret the results. Results: We identified ancestry-specific differences in gene expression and chromatin accessibility. Notably, numerous AD GWAS-associated genes were differentially expressed across ancestries. The largest number of differentially expressed genes (DEGs) were found in European vs. Amerindian and African vs. Amerindian iPSC-derived oligodendrocyte progenitor cells (OPCs). Pathway analysis of APOE4/4 carriers vs APOE3/3 carriers exhibited upregulation of a large number of disease and metabolic pathways in APOE4/4 individuals of all ancestries. Of particular interest was that APOE4/4 carriers had significantly upregulated cholesterol biosynthesis genes relative to APOE3/3 individuals across all ancestries, strongest in iOPCs. Comparison of iOPC and iOL transcriptome data with corresponding human frontal cortex data demonstrated a high correlation (R2 > 0.85). Conclusions: This research emphasizes the importance of including diverse ancestries in AD research to uncover critical gene expression differences between populations and ancestries that may influence disease susceptibility and therapeutic interventions. The upregulation of cholesterol biosynthesis genes in APOE4/4 carriers of all three ancestries supports the concept that APOE4 may produce disease effects early in life, which could have therapeutic implications as we move forward towards specific therapy for APOE4 carriers. These findings and the high correlation between brain and iPSC-derived OPC and OL transcriptomes support the relevance of this approach as a model for disease study.

Alzheimer's disease (AD) risk differs between population groups, with African Americans and Hispanics being the most affected groups compared to non-Hispanic Whites. Genetic factors contribute significant risk to AD, but the genetic regulatory architectures (GRA) have primarily been studied in Europeans. Many AD genes are expressed in microglia; thus, we explored the impact of genetic ancestry (Amerindian (AI), African (AF), and European (EU)) on the GRA in iPSC-derived microglia from 13 individuals (~4 each with high global ancestry, AD and controls) through ATAC-seq and RNA-seq analyses. We identified several differentially accessible and expressed genes (2 and 10 AD-related, respectively) between ancestry groups. We also found a high correlation between the transcriptomes of iPSC-derived and brain microglia, supporting their use in human studies. This study provides valuable insights into genetically diverse microglia beyond the analysis of AD. ### Competing Interest Statement The authors have declared no competing interest.

The APOEε4 confers the highest genetic risk factor for Alzheimer’s disease (AD). Specifically, Non-Hispanic Whites (NHW) are more susceptible to developing AD than African American (AA) individuals (OR∼15 vs 8, respectively). Local ancestry (LA) surrounding the APOE region has been implicated in this risk difference, with APOEε4 carriers of European LA (ELA) having increased chromatin accessibility and higher APOEε4 expression than individuals of African LA (ALA). In contrast, carriers of APOEε3, the most common allele, are not predisposed to AD. We here sought to investigate whether this differential accessibility and gene expression are solely due to APOE LA or whether the APOE genotype also contributes to the different disease risk. We screened 94 brains from individuals self-identified as either AA or NHW of which we identified a total of 22 AD autopsy samples (18 with ELA and 4 with ALA) by GSA, all homozygous for LA and APOEε3. We performed single nuclei ATAC-seq and single nuclei RNA sequencing (snRNA-seq) using frozen frontal cortex using the 10x Genomics. To date, we have performed snRNA-seq in a total of 16,099 nuclei for four brains (2 ELA and 2 ALA). We identified 27 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 8 clusters (primarily neurons) which had a greater than 2-fold difference in ELA. Preliminary results show that APOEε3 carriers with ELA have a significantly higher APOE expression in astrocytes (cluster 11) than those of ALA similar to previous reports in APOEε4 carriers (Griswold, A. et al, (2021)). Interestingly, we also observed an increase in APOE expression in ALA for one oligodendrocyte cluster and four neuronal clusters (inhibitory and excitatory neurons) independent of those mentioned above. We report novel insights on the effect of APOE LA and genotype and its contributions to Alzheimer’s disease risk. Specifically, as APOEε3 allele expression follows a similar pattern of overexpression in ELA vs ALA as observed for APOEε4 carriers, our data support that LA and not allele status contribute to the differential APOE expression observed among different ancestral populations.

Amyloid β (Aβ) accumulation is a hallmark of Alzheimer's disease. In adult Drosophila brains, human Aβ overexpression harms climbing and lifespan. It's uncertain whether Aβ is intrinsically toxic or activates downstream neurodegeneration pathways. Our study uncovers a novel protective role against Aβ toxicity: intra-endoplasmic reticulum (ER) protein accumulation with a focus on laminin and collagen subunits. Despite high Aβ, laminin B1 (LanB1) overexpression robustly counters toxicity, suggesting a potential Aβ resistance mechanism. Other laminin subunits and collagen IV also alleviate Aβ toxicity; combining them with LanB1 augments the effect. Imaging reveals ER retention of LanB1 without altering Aβ secretion. LanB1's rescue function operates independently of the IRE1α/XBP1 ER stress response. ER-targeted GFP overexpression also mitigates Aβ toxicity, highlighting broader ER protein retention advantages. Proof-of-principle tests in murine hippocampal slices using mouse Lamb1 demonstrate ER retention in transduced cells, indicating a conserved mechanism. Though ER protein retention generally harms, it could paradoxically counter neuronal Aβ toxicity, offering a new therapeutic avenue for Alzheimer's disease.

Increasing ethnic/ancestral diversity in genetic studies is critical for defining the genetic architecture of Alzheimer disease (AD). Amerindian (AI) populations are substantially underrepresented in AD genetic studies. The Peruvian population, with up to ∼80% of AI ancestry, provides a unique opportunity to assess the role of AI ancestry in AD. We aimed to conduct a genome-wide association study and comprehensive analyzes to identify novel ancestry-specific AD susceptibility loci and characterize the known AD genetic risk loci in the Peruvian population. 528 individuals were included in these analyses (175 AD, 353 cognitively unimpaired). Array genotype was imputed to the NHLBI TOPMedv5 haplotype reference panel. We used a generalized linear mixed model adjusting for sex, age, and population substructure (model-1), and adjusting for APOE-ε4 allele dosage (model-2). Both models included a genetic relationship matrix as a random effect to account for cryptic relatedness. To determine if the associations were ancestry-specific, we further explored interactions between top genetic variants and local ancestry. Single nuclei ATACseq and RNAseq, and Hi-C analysis was performed to identify potential interactions with the associated locus. We identified a novel genome-wide significant signal (rs2625222; P =3.5×10-8) within the Neurofascin gene ( NFASC) on chromosome 1. Local ancestry approach estimated both European and African ancestral backgrounds at the NFASC locus. Functional studies found that rs2625222 lies within an open ATAC peak and is expressed in oligodendrocytes and neurons. Hi-C studies revealed strong loops between rs2625222 region and the promoter of NFASC in neurons and oligodendrocytes. We also replicated three known AD loci: APOE , TREML2 , and CLU . This study identified a novel locus associated with risk for AD in the Peruvian population. Functional studies strongly support that NFASC is the targeted gene of this risk association. These findings emphasize the importance of including diverse populations in genetic studies of AD. Author summary Alzheimer Disease (AD), the most common type of dementia in older adults, has a complex etiology with a strong genetic predisposition. Despite Genome-Wide Association Studies (GWAS) identifying over 75 loci associated with AD, these have predominantly focused on the non-Hispanic White population. Genetic studies of diverse populations, such as the Peruvian population with its significant Amerindian ancestral background, are crucial for identifying ancestry-specific genetic risk and protective loci associated with AD. Our objective in this study was to conduct a GWAS and comprehensive analyses of 528 Peruvian individuals to identify new ancestry-specific AD risk loci. In this study, we identified a genome-wide significant novel signal within the NFASC gene, which has a crucial role in central and peripheral nervous systems, on chromosome 1. This locus showed both European and African ancestral backgrounds. Our functional analyses strongly support that the NFASC gene itself is the primary gene tagged by the risk-associated signal. These findings emphasize the inclusion of admixed populations in genetic studies provides an important opportunity to assess the role of different ancestries in AD. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This investigation was supported by grants R01AG070864 (MAPV, GWB, FR), RF1AG082009 (GT), U19AG074865 (MAPV, GT, JMV), R56AG069118 (GT), U01AG058654 (ERM, MAPV), RF1AG054074 (MAPV, GWB), U01AG057659 (MAPV), RF1AG059018 (JMV), and U01AG072579 (JMV, DMD, AJG) from the National Institutes on Aging of NIH, A2018556F (FR) grant from the BrightFocus Foundation, and 21A18 (KC) grant from the Florida Department of Health Ed and Ethel Moore Alzheimer's Disease Research Program. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Informed consent was obtained from all participants, and the study protocols were approved by the Ethical Research Committee of the Instituto Nacional de Ciencias Neurologicas (INCN), and Instituto Peruano de Neurociencias (IPN) Lima, the University of Miami's Institutional Review Board, and the Columbia University Review Board. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes Data are available through the National Institute on Aging Genetics of Alzheimers Disease Data Storage Site (NIAGADS) Data Sharing Service (DSS).

The effect of variants associated with Alzheimer’s disease (AD) can be influenced by ancestry, which determines the genomic regulatory architecture (GRA). A global understanding of GRA in the context of AD is imperative to interpret the variability associated with AD risk genes across populations. Most studies up to date have focused on studying GRA in European ancestry, in this study we aimed at determining the GRA in African, Amerindian, and European ancestries. Since GRA is cell specific, we developed a human induced pluripotent cells (hiPSC) based model for oligodendrocytes (OLs), a cell type which has limited studies focused on AD. Cells from AD patients or non-cognitively impaired controls with >90% of either Amerindian, African or European global ancestry were differentiated using a modified multi-stage protocol that promotes the development and enrichment of oligodendrocytes in neural spheroids. After terminal differentiation, cells were collected and lysed to isolate nuclei for Multiomic profiling of chromatin accessibility and transcriptome using Single Cell ATAC and Single Cell RNA-seq. Additionally, we examined chromatin interactions using Hi-C analyses. We identified oligodendrocyte lineage cells at different stages of development ranging from dividing cells with transcriptional profiles consistent with those of oligodendrocyte precursor cells (OPC) to mature myelinating oligodendrocytes. We compared the oligodendrocytes clusters across ancestries, cases versus controls and APOE genotypes to characterize the genomic landmarks and signatures associated with AD related GWAS loci. Astrocytes and neurons were also derived within our 3D spheroids, allowing us to study ancestry-related cell type specific changes in GRA. Our results provide ancestry-specific insights into oligodendrocyte chromatin structure and gene regulation in the context of AD. These results offer an integrated view of the GRA of a previously overlooked cell lineage that constitute a large population in the central nervous system and is compromised during AD in terms of abundance and function. This will expand the available functional resources for gene identification studies in African American and Hispanic/Latino studies.

Genomic regulatory architecture (GRA) has been primarily studied in European ancestry. As part of the functional Consortium of the Alzheimer Disease Sequencing Project, we are determining the GRA in African and Amerindian ancestries. Oligodendrocytes (OLs) are the largest glial population in the adult central nervous system. While their main role is to support neuronal metabolism and connectivity, few studies have examined their importance in Alzheimer’s disease (AD), despite reports of low numbers of oligodendrocytes and reduced myelin in the early stages of AD and a demonstrated role of myelinating oligodendrocytes in learning and memory. Several studies have reported the derivation of OLs from pluripotent cells, such as induced pluripotent stem cells (iPSC) to be challenging. Here, we optimized a protocol to derive oligodendrocytes from iPSC for studies in AD patients with different ancestries and how ancestry-specific genomic differences drive the onset and pathogenesis of AD. iPSC lines derived from AD patients were cultured and differentiated into oligodendrocytes using different induction media, as well as different seeding densities. The cells in each treatment were compared at multiple time points using immunocytochemistry (ICC) and qRT-PCR for oligodendroglia lineage markers with the goal of identifying the culture conditions that increase the yield of O4 + cells and myelinating oligodendrocytes. Our results showed that increasing the initial seeding density positively correlates with the number of Olig2 + cells that subsequently transitioned into mature O4+ cells capable of producing myelin. Additionally, we showed that the addition of N2 supplement to the induction media was necessary to maintain the cell viability during the initial stage of differentiation. We have optimized a protocol to derive OLs from human iPSC lines. We determined that both the seeding density and the media supplements used during the initial stage of differentiation directly influence viability and, consequently the amount Olig2 + cells that could be obtained to be terminally differentiated into O4 + and myelinating cells. Our optimized protocol will be used to evaluate the GRA and functionality of potential GWAS driving loci in cultured oligodendrocytes from individuals of African and Amerindian ancestries.

De novo mutations (DNMs) are important players in heritable diseases and evolution. Of particular interest are highly recurrent DNMs associated with congenital disorders that have been described as selfish mutations expanding in the male germline, thus becoming more frequent with age. Here, we have adapted duplex sequencing (DS), an ultradeep sequencing method that renders sequence information on both DNA strands; thus, one mutation can be reliably called in millions of sequenced bases. With DS, we examined ∼4.5 kb of the FGFR3 coding region in sperm DNA from older and younger donors. We identified sites with variant allele frequencies (VAFs) of 10−4 to 10−5, with an overall mutation frequency of the region of ∼6 × 10−7. Some of the substitutions are recurrent and are found at a higher VAF in older donors than in younger ones or are found exclusively in older donors. Also, older donors harbor more mutations associated with congenital disorders. Other mutations are present in both age groups, suggesting that these might result from a different mechanism (e.g., postzygotic mosaicism). We also observe that independent of age, the frequency and deleteriousness of the mutational spectra are more similar to COSMIC than to gnomAD variants. Our approach is an important strategy to identify mutations that could be associated with a gain of function of the receptor tyrosine kinase activity, with unexplored consequences in a society with delayed fatherhood.

Delayed fatherhood results in a higher risk to inherit a new germline mutation that might result in a congenital disorder in the offspring. In particular, some FGFR3 mutations increase in frequency with age, but there are still a large number of uncharacterized FGFR3 mutations that could be expanding in the male germline with potentially early or late-onset effects in the offspring. Here, we investigated the mutation frequency in the DNA of human testis and sperm and the activation state of the expressed mutant protein of eight different FGFR3 variants categorized by ClinVar as deleterious, benign, or not reported. Overall, the ligand-independent activation of the mutant protein resulted in a increased number of mutant sperm; although, strong activating mutations did not necessarily result in the highest frequencies. Moreover, only two mutants c.952G>A and c.1620C>A showed an increase with the donor’s age; the latter also forming larger clonal expansions in the testis. We also showed that the prediction of deleteriousness of a mutation is not always accurate, and similar in silico scores can reflect either a gain-of-function or loss-of-function. Our approach led to the discovery of two novel variants c.1261G>A and c.952G>A to have promiscuous FGFR3 activation and increased mutation frequencies in the male germline. The large fraction of donors with mutations suggests a high de novo rate potentially explained by a selective advantage before the maturation of the male germline. This sequence-function study provides important data for the evaluation and interpretation of variants with relevant clinical implications.

Accumulation of Aβ in the brain is one of the hallmarks of Alzheimer’s disease (AD). In the adult Drosophila brain, human Aβ over-expression is toxic and leads to deterioration of climbing ability and shortened lifespan. However, it remains unknown if Aβ is inherently toxic or if it triggers toxic downstream pathways that lead to neurodegeneration. Here, we describe a novel, and previously unidentified, protective role of intracellular laminin chain accumulation. Despite high Aβ levels, over-expression of the extracellular matrix protein subunit Laminin B1 (LanB1) resulted in a robust rescue of toxicity, highlighting a potential protective mechanism of resistance to Aβ. Over-expression of other Laminin subunits and a Collagen IV subunit also significantly rescued Aβ toxicity, while combining LanB1 with these subunits led to an even larger rescue. Imaging revealed that LanB1 was retained in the ER but had no effect on the secretion of Aβ into the extracellular milieu. LanB1 rescued toxicity independently of the IRE1α/XBP1-mediated branch of the ER stress response. Interestingly, over-expression of ER-targeted GFP also rescued Aβ toxicity, indicating a potentially broader benefit of ER protein retention. Finally, in proof-of-principle lentiviral transduction experiments using murine organotypic hippocampal slice cultures, over-expression of mouse Lamb1 resulted in ER-retention in transduced cells, highlighting a conserved mechanism. Typically, retention of proteins in the ER is detrimental to cellular health, but in the context of neuronal Aβ toxicity it may prove to be beneficial and a new therapeutic avenue for AD.