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The exchange of genes between cells is known to play an important physiological and pathological role in many organisms. We show that circulating tumor DNA (ctDNA) facilitates cell-specific gene transfer between human cancer cells and explain part of the mechanisms behind this phenomenon. As ctDNA migrates into the nucleus, genetic information is transferred. Cell targeting and ctDNA integration require ERVL, SINE or LINE DNA sequences. Chemically manufactured AluSp and MER11C sequences replicated multiple myeloma (MM) ctDNA cell targeting and integration. Additionally, we found that ctDNA may alter the treatment response of MM and pancreatic cancer models. This study shows that retrotransposon DNA sequences promote cancer gene transfer. However, because cell-free DNA has been detected in physiological and other pathological conditions, our findings have a broader impact than just cancer. Furthermore, the discovery that transposon DNA sequences mediate tissue-specific targeting will open up a new avenue for the delivery of genes and therapies.
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ADN Tumoral Circulante , Elementos Transponibles de ADN , Humanos , ADN Tumoral Circulante/genética , ADN Tumoral Circulante/sangre , Elementos Transponibles de ADN/genética , Línea Celular Tumoral , Mieloma Múltiple/genética , Mieloma Múltiple/terapia , Animales , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/terapia , Ratones , Especificidad de Órganos/genética , Retroelementos/genética , Técnicas de Transferencia de GenRESUMEN
BACKGROUND: Short tandem repeats (STRs) are widely distributed across the human genome and are associated with numerous neurological disorders. However, the extent that STRs contribute to disease is likely under-estimated because of the challenges calling these variants in short read next generation sequencing data. Several computational tools have been developed for STR variant calling, but none fully address all of the complexities associated with this variant class. RESULTS: Here we introduce LUSTR which is designed to address some of the challenges associated with STR variant calling by enabling more flexibility in defining STR loci, allowing for customizable modules to tailor analyses, and expanding the capability to call somatic and multiallelic STR variants. LUSTR is a user-friendly and easily customizable tool for targeted or unbiased genome-wide STR variant screening that can use either predefined or novel genome builds. Using both simulated and real data sets, we demonstrated that LUSTR accurately infers germline and somatic STR expansions in individuals with and without diseases. CONCLUSIONS: LUSTR offers a powerful and user-friendly approach that allows for the identification of STR variants and can facilitate more comprehensive studies evaluating the role of pathogenic STR variants across human diseases.
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Genoma Humano , Repeticiones de Microsatélite , Humanos , Repeticiones de Microsatélite/genética , Células Germinativas , Secuenciación de Nucleótidos de Alto RendimientoRESUMEN
The risk of developing Alzheimer's disease (AD) significantly increases in individuals carrying the APOEε4 allele. Elderly cognitively healthy individuals with APOEε4 also exist, suggesting the presence of cellular mechanisms that counteract the pathological effects of APOEε4; however, these mechanisms are unknown. We hypothesized that APOEε4 carriers without dementia might carry genetic variations that could protect them from developing APOEε4-mediated AD pathology. To test this, we leveraged whole-genome sequencing (WGS) data in the National Institute on Aging Alzheimer's Disease Family Based Study (NIA-AD FBS), Washington Heights/Inwood Columbia Aging Project (WHICAP), and Estudio Familiar de Influencia Genetica en Alzheimer (EFIGA) cohorts and identified potentially protective variants segregating exclusively among unaffected APOEε4 carriers. In homozygous unaffected carriers above 70 years old, we identified 510 rare coding variants. Pathway analysis of the genes harboring these variants showed significant enrichment in extracellular matrix (ECM)-related processes, suggesting protective effects of functional modifications in ECM proteins. We prioritized two genes that were highly represented in the ECM-related gene ontology terms, (FN1) and collagen type VI alpha 2 chain (COL6A2) and are known to be expressed at the blood-brain barrier (BBB), for postmortem validation and in vivo functional studies. An independent analysis in a large cohort of 7185 APOEε4 homozygous carriers found that rs140926439 variant in FN1 was protective of AD (OR = 0.29; 95% CI [0.11, 0.78], P = 0.014) and delayed age at onset of disease by 3.37 years (95% CI [0.42, 6.32], P = 0.025). The FN1 and COL6A2 protein levels were increased at the BBB in APOEε4 carriers with AD. Brain expression of cognitively unaffected homozygous APOEε4 carriers had significantly lower FN1 deposition and less reactive gliosis compared to homozygous APOEε4 carriers with AD, suggesting that FN1 might be a downstream driver of APOEε4-mediated AD-related pathology and cognitive decline. To validate our findings, we used zebrafish models with loss-of-function (LOF) mutations in fn1b-the ortholog for human FN1. We found that fibronectin LOF reduced gliosis, enhanced gliovascular remodeling, and potentiated the microglial response, suggesting that pathological accumulation of FN1 could impair toxic protein clearance, which is ameliorated with FN1 LOF. Our study suggests that vascular deposition of FN1 is related to the pathogenicity of APOEε4, and LOF variants in FN1 may reduce APOEε4-related AD risk, providing novel clues to potential therapeutic interventions targeting the ECM to mitigate AD risk.
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Enfermedad de Alzheimer , Fibronectinas , Anciano , Animales , Humanos , Enfermedad de Alzheimer/genética , Fibronectinas/genética , Variación Genética/genética , Gliosis , Pez CebraRESUMEN
Genetic variants and epigenetic features both contribute to the risk of Alzheimer's disease (AD). We studied the AD association of CpG-related single nucleotide polymorphisms (CGS), which act as a hub of both the genetic and epigenetic effects, in Caribbean Hispanics (CH) and generalized the findings to Non-Hispanic Whites (NHW). First, we conducted a genome-wide, sliding-window-based association with AD, in 7,155 CH and 1,283 NHW participants. Next, using data from the dorsolateral prefrontal cortex in 179 CH brains, we tested the cis- and trans-effects of AD-associated CGS on brain DNA methylation to mRNA expression. For the genes with significant cis- and trans-effects, we investigated their enriched pathways. We identified six genetic loci in CH with CGS dosage associated with AD at genome-wide significance levels: ADAM20 (Score = 55.19, P = 4.06 × 10-8), the intergenic region between VRTN and SYNDIG1L (Score = - 37.67, P = 2.25 × 10-9), SPG7 (16q24.3) (Score = 40.51, P = 2.23 × 10-8), PVRL2 (Score = 125.86, P = 1.64 × 10-9), TOMM40 (Score = - 18.58, P = 4.61 × 10-8), and APOE (Score = 75.12, P = 7.26 × 10-26). CGSes in PVRL2 and APOE were also significant in NHW. Except for ADAM20, CGSes in the other five loci were associated with CH brain methylation levels (mQTLs) and CGSes in SPG7, PVRL2, and APOE were also mQTLs in NHW. Except for SYNDIG1L (P = 0.08), brain methylation levels in the other five loci affected downstream mRNA expression in CH (P < 0.05), and methylation at VRTN and TOMM40 were also associated with mRNA expression in NHW. Gene expression in these six loci were also regulated by CpG sites in genes that were enriched in the neuron projection and glutamatergic synapse pathways (FDR < 0.05). DNA methylation at all six loci and mRNA expression of SYNDIG1 and TOMM40 were significantly associated with Braak Stage in CH. In summary, we identified six CpG-related genetic loci associated with AD in CH, harboring both genetic and epigenetic risks. However, their downstream effects on mRNA expression maybe ethnic specific and different from NHW.
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Enfermedad de Alzheimer , Encéfalo , Epigénesis Genética , Predisposición Genética a la Enfermedad , Estudio de Asociación del Genoma Completo , Hispánicos o Latinos , Polimorfismo de Nucleótido Simple , Población Blanca , Humanos , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Enfermedad de Alzheimer/etnología , Femenino , Masculino , Anciano , Población Blanca/genética , Encéfalo/patología , Hispánicos o Latinos/genética , Anciano de 80 o más Años , Metilación de ADN , Autopsia , Región del Caribe/etnologíaRESUMEN
BACKGROUND: Alzheimer's disease (AD) biomarkers can help differentiate cognitively unimpaired (CU) individuals from mild cognitive impairment (MCI) and dementia. The role of AD biomarkers in predicting cognitive impairment and AD needs examination. METHODS: In 628 CU individuals from a multi-ethnic cohort, amyloid beta (Aß)42, Aß40, phosphorylated tau-181 (p-tau181), glial fibrillary acidic protein (GFAP), and neurofilament light chain (NfL) were measured in plasma. RESULTS: Higher baseline levels of p-tau181/Aß42 ratio were associated with an increased risk of incident dementia. A biomarker pattern (with elevated Aß42/Aß40 but low p-tau181/Aß42) was associated with decreased dementia risk. Compared to CU, participants who developed MCI or dementia had a rapid decrease in this protective biomarker pattern reflecting AD-specific pathological change. DISCUSSION: Elevated levels of AD biomarker p-tau181/Aß42, by itself or combined with a low Aß42/Aß40 level, predicts clinically diagnosed AD. Individuals with a rapid change in these biomarkers may need close monitoring for the potential downward trajectory of cognition. HIGHLIGHTS: We discuss a multi-ethnic, urban community study of elderly individuals. The study consisted of a longitudinal assessment over 6 years with repeated clinical assessments. The study used blood-based biomarkers as predictors of mild cognitive impairment and Alzheimer's disease.
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Enfermedad de Alzheimer , Disfunción Cognitiva , Humanos , Anciano , Péptidos beta-Amiloides , Washingtón , Proteínas tau , Disfunción Cognitiva/diagnóstico , Envejecimiento , BiomarcadoresRESUMEN
INTRODUCTION: Late-onset Alzheimer's disease (LOAD) has a strong genetic component. Participants in Long-Life Family Study (LLFS) exhibit delayed onset of dementia, offering a unique opportunity to investigate LOAD genetics. METHODS: We conducted a whole genome sequence analysis of 3475 LLFS members. Genetic associations were examined in six independent studies (N = 14,260) with a wide range of LOAD risk. Association analysis in a sub-sample of the LLFS cohort (N = 1739) evaluated the association of LOAD variants with beta amyloid (Aß) levels. RESULTS: We identified several single nucleotide polymorphisms (SNPs) in tight linkage disequilibrium within the MTUS2 gene associated with LOAD (rs73154407, p = 7.6 × 10-9). Association of MTUS2 variants with LOAD was observed in the five independent studies and was significantly stronger within high levels of Aß42/40 ratio compared to lower amyloid. DISCUSSION: MTUS2 encodes a microtubule associated protein implicated in the development and function of the nervous system, making it a plausible candidate to investigate LOAD biology. HIGHLIGHTS: Long-Life Family Study (LLFS) families may harbor late onset Alzheimer's dementia (LOAD) variants. LLFS whole genome sequence analysis identified MTUS2 gene variants associated with LOAD. The observed LLFS variants generalized to cohorts with wide range of LOAD risk. The association of MTUS2 with LOAD was stronger within high levels of beta amyloid. Our results provide evidence for MTUS2 gene as a novel LOAD candidate locus.
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Enfermedad de Alzheimer , Humanos , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Predisposición Genética a la Enfermedad , Estudio de Asociación del Genoma Completo/métodos , Proteínas Asociadas a Microtúbulos , Polimorfismo de Nucleótido Simple/genética , Análisis de SecuenciaRESUMEN
INTRODUCTION: Despite a two-fold risk, individuals of African ancestry have been underrepresented in Alzheimer's disease (AD) genomics efforts. METHODS: Genome-wide association studies (GWAS) of 2,903 AD cases and 6,265 controls of African ancestry. Within-dataset results were meta-analyzed, followed by functional genomics analyses. RESULTS: A novel AD-risk locus was identified in MPDZ on chromosome (chr) 9p23 (rs141610415, MAF = 0.002, P = 3.68×10-9). Two additional novel common and nine rare loci were identified with suggestive associations (P < 9×10-7). Comparison of association and linkage disequilibrium (LD) patterns between datasets with higher and lower degrees of African ancestry showed differential association patterns at chr12q23.2 (ASCL1), suggesting that this association is modulated by regional origin of local African ancestry. DISCUSSION: These analyses identified novel AD-associated loci in individuals of African ancestry and suggest that degree of African ancestry modulates some associations. Increased sample sets covering as much African genetic diversity as possible will be critical to identify additional loci and deconvolute local genetic ancestry effects. HIGHLIGHTS: Genetic ancestry significantly impacts risk of Alzheimer's Disease (AD). Although individuals of African ancestry are twice as likely to develop AD, they are vastly underrepresented in AD genomics studies. The Alzheimer's Disease Genetics Consortium has previously identified 16 common and rare genetic loci associated with AD in African American individuals. The current analyses significantly expand this effort by increasing the sample size and extending ancestral diversity by including populations from continental Africa. Single variant meta-analysis identified a novel genome-wide significant AD-risk locus in individuals of African ancestry at the MPDZ gene, and 11 additional novel loci with suggestive genome-wide significance at P < 9×10-7. Comparison of African American datasets with samples of higher degree of African ancestry demonstrated differing patterns of association and linkage disequilibrium at one of these loci, suggesting that degree and/or geographic origin of African ancestry modulates the effect at this locus. These findings illustrate the importance of increasing number and ancestral diversity of African ancestry samples in AD genomics studies to fully disentangle the genetic architecture underlying AD, and yield more effective ancestry-informed genetic screening tools and therapeutic interventions.
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INTRODUCTION: Clinical research in Alzheimer's disease (AD) lacks cohort diversity despite being a global health crisis. The Asian Cohort for Alzheimer's Disease (ACAD) was formed to address underrepresentation of Asians in research, and limited understanding of how genetics and non-genetic/lifestyle factors impact this multi-ethnic population. METHODS: The ACAD started fully recruiting in October 2021 with one central coordination site, eight recruitment sites, and two analysis sites. We developed a comprehensive study protocol for outreach and recruitment, an extensive data collection packet, and a centralized data management system, in English, Chinese, Korean, and Vietnamese. RESULTS: ACAD has recruited 606 participants with an additional 900 expressing interest in enrollment since program inception. DISCUSSION: ACAD's traction indicates the feasibility of recruiting Asians for clinical research to enhance understanding of AD risk factors. ACAD will recruit > 5000 participants to identify genetic and non-genetic/lifestyle AD risk factors, establish blood biomarker levels for AD diagnosis, and facilitate clinical trial readiness. HIGHLIGHTS: The Asian Cohort for Alzheimer's Disease (ACAD) promotes awareness of under-investment in clinical research for Asians. We are recruiting Asian Americans and Canadians for novel insights into Alzheimer's disease. We describe culturally appropriate recruitment strategies and data collection protocol. ACAD addresses challenges of recruitment from heterogeneous Asian subcommunities. We aim to implement a successful recruitment program that enrolls across three Asian subcommunities.
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Enfermedad de Alzheimer , Pueblos de América del Norte , Humanos , Enfermedad de Alzheimer/genética , Proyectos Piloto , Asiático/genética , Canadá , Factores de RiesgoRESUMEN
Background: High Lp(a) levels contribute to atherosclerotic cardiovascular disease and are tightly regulated by the LPA gene . Lp(a) levels have an inverse correlation with LPA Kringle IV Type-2 (KIV-2) copy number (CN). Black (B) and Hispanic (H) individuals exhibit higher levels of Lp(a), and rates of CVD compared to non-Hispanic Whites (NHW). Therefore, we investigated genetic variations in the LPA KIV-2 region across three ancestries and their associations with metabolic risk factors. Methods: Using published pipelines, we analyzed a multi-ethnic whole exome dataset comprising 3,817 participants from the Washington Heights and Inwood Columbia Aging Project (WHICAP): 886 [NHW (23%), 1,811 Caribbean (C) H (47%), and 1,120 B individuals (29%). Rare and common variants (alternative allele carrier frequency, CF < 0.01 or > 0.99 and 0.01 < CF < 0.99, respectively) were identified and KIV-2 CN estimated. The associations of variants and CN with history of heart disease, hypertension (HTN), stroke, lipid levels and clinical diagnosis of Alzheimer's disease (AD) was assessed. A small pilot provided in-silico validation of study findings. Results: We report 1421 variants in the LPA KIV-2 repeat region, comprising 267 exonic and 1154 intronic variants. 61.4% of the exonic variants have not been previously described. Three novel exonic variants significantly increase the risk of HTN across all ethnic groups: 4785-C/A (frequency = 78%, odds ratio [OR] = 1.45, p = 0.032), 727-T/C (frequency = 96%, OR = 2.11, p = 0.032), and 723-A/G (frequency = 96%, OR = 1.97, p = 0.038). Additionally, six intronic variants showed associations with HTN: 166-G/A, 387-G/C, 402-G/A, 4527-A/T, 4541-G/A, and 4653-A/T. One intronic variant, 412-C/T, was associated with decreased blood glucose levels (frequency = 72%, ß = -14.52, p = 0.02).Three of the associations were not affected after adjusting for LPA KIV-2 CN: 412-C/T (ß = -14.2, p = 0.03), 166-G/A (OR = 1.41, p = 0.05), and 387-G/C (OR = 1.40, p = 0.05). KIV CN itself was significantly associated with 314 variants and was negatively correlated with plasma total cholesterol levels. Conclusions: In three ancestry groups, we identify novel rare and common LPA KIV-2 region variants. We report new associations of variants with HTN and Glucose levels. These results underscore the genetic complexity of the LPA KIV-2 region in influencing cardiovascular and metabolic health, suggesting potential genetic regulation of pathways that can be studied for research and therapeutic interventions.
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Introduction: T cells, known for their ability to respond to an enormous variety of pathogens and other insults, are increasingly recognized as important mediators of pathology in neurodegeneration and other diseases. T cell gene expression phenotypes can be regulated by disease-associated genetic variants. Many complex diseases are better represented by polygenic risk than by individual variants. Methods: We first compute a polygenic risk score (PRS) for Alzheimer's disease (AD) using genomic sequencing data from a cohort of Alzheimer's disease (AD) patients and age-matched controls, and validate the AD PRS against clinical metrics in our cohort. We then calculate the PRS for several autoimmune disease, neurological disorder, and immune function traits, and correlate these PRSs with T cell gene expression data from our cohort. We compare PRS-associated genes across traits and four T cell subtypes. Results: Several genes and biological pathways associated with the PRS for these traits relate to key T cell functions. The PRS-associated gene signature generally correlates positively for traits within a particular category (autoimmune disease, neurological disease, immune function) with the exception of stroke. The trait-associated gene expression signature for autoimmune disease traits was polarized towards CD4+ T cell subtypes. Discussion: Our findings show that polygenic risk for complex disease and immune function traits can have varying effects on T cell gene expression trends. Several PRS-associated genes are potential candidates for therapeutic modulation in T cells, and could be tested in in vitro applications using cells from patients bearing high or low polygenic risk for AD or other conditions.
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Enfermedad de Alzheimer , Enfermedades Autoinmunes , Humanos , Enfermedad de Alzheimer/genética , Fenotipo , Riesgo , Transducción de Señal/genéticaRESUMEN
Background: We profiled circulating plasma metabolites to identify systemic biochemical changes in clinical and biomarker-assisted diagnosis of Alzheimer's disease (AD). Methods: We used an untargeted approach with liquid chromatography coupled to high-resolution mass spectrometry to measure small molecule plasma metabolites from 150 clinically diagnosed AD patients and 567 age-matched healthy elderly of Caribbean Hispanic ancestry. Plasma biomarkers of AD were measured including P-tau181, Aß40, Aß42, total-tau, neurofilament light chain (NfL) and glial fibrillary acidic protein (GFAP). Association of individual and co-abundant modules of metabolites were tested with clinical diagnosis of AD, as well as biologically-defined AD pathological process based on P-tau181 and other biomarker levels. Results: Over 6000 metabolomic features were measured with high accuracy. First principal component (PC) of lysophosphatidylcholines (lysoPC) that bind to or interact with docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA) and arachidonic acid (AHA) was associated with decreased risk of AD (OR = 0.91 [0.89-0.96], p = 2e-04). Association was restricted to individuals without an APOE ε4 allele (OR = 0.89 [0.84-0.94], p = 8.7e-05). Among individuals carrying at least one APOE ε4 allele, PC4 of lysoPCs moderately increased risk of AD (OR = 1.37 [1.16-1.6], p = 1e-04). Essential amino acids including tyrosine metabolism pathways were enriched among metabolites associated with P-tau181 levels and heparan and keratan sulfate degradation pathways were associated with Aß42/Aß40 ratio. Conclusions: Unbiased metabolic profiling can identify critical metabolites and pathways associated with ß-amyloid and phosphotau pathology. We also observed an APOE-ε4 dependent association of lysoPCs with AD and biologically based diagnostic criteria may aid in the identification of unique pathogenic mechanisms.
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This article presents a practical guide to mass spectrometry-based data-independent acquisition and label-free quantification for proteomics analysis applied to cerebrospinal fluid, offering a robust and scalable approach to probing the proteomic composition of the central nervous system. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Cerebrospinal fluid sample collection and preparation for mass spectrometry analysis Basic Protocol 2: Mass spectrometry sample analysis with data-independent acquisition Support Protocol: Data-dependent mass spectrometry and spectral library construction Basic Protocol 3: Analysis of mass spectrometry data.
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Proteoma , Proteómica , Humanos , Proteómica/métodos , Proteoma/análisis , Espectrometría de Masas/métodos , Proteínas del Líquido Cefalorraquídeo/químicaRESUMEN
Glycated hemoglobin (HbA1c) indicates average glucose levels over three months and is associated with insulin resistance and type 2 diabetes (T2D). Longitudinal changes in HbA1c (ΔHbA1c) are also associated with aging processes, cognitive performance, and mortality. We analyzed ΔHbA1c in 1,886 non-diabetic Europeans from the Long Life Family Study to uncover gene variants influencing ΔHbA1c. Using growth curve modeling adjusted for multiple covariates, we derived ΔHbA1c and conducted linkage-guided sequence analysis. Our genome-wide linkage scan identified a significant locus on 17p12. In-depth analysis of this locus revealed a variant rs56340929 (explaining 27% of the linkage peak) in the ARHGAP44 gene that was significantly associated with ΔHbA1c. RNA transcription of ARHGAP44 was associated with ΔHbA1c. The Framingham Offspring Study data further supported these findings on the gene level. Together, we found a novel gene ARHGAP44 for ΔHbA1c in family members without T2D. Follow-up studies using longitudinal omics data in large independent cohorts are warranted.
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We developed an imputation panel for Alzheimer's disease (AD) and related dementias (ADRD) using whole-genome sequencing (WGS) data from the Alzheimer's Disease Sequencing Project (ADSP). Recognizing the significant associations between structural variants (SVs) and AD, and their underrepresentation in existing public reference panels, our panel uniquely integrates single nucleotide variants (SNVs), short insertions and deletions (indels), and SVs. This panel enhances the imputation of disease susceptibility, including rare AD-associated SNVs, indels, and SVs, onto genotype array data, offering a cost-effective alternative to whole-genome sequencing while significantly augmenting statistical power. Notably, we discovered 10 rare indels nominal significant related to AD that are absent in the TOPMed-r2 panel and identified three suggestive significant (p-value < 1E-05) AD-associated SVs in the genes EXOC3L2 and DMPK, were identified. These findings provide new insights into AD genetics and underscore the critical role of imputation panels in advancing our understanding of complex diseases like ADRD.
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The risk of developing Alzheimer's disease (AD) significantly increases in individuals carrying the APOEε4 allele. Elderly cognitively healthy individuals with APOEε4 also exist, suggesting the presence of cellular mechanisms that counteract the pathological effects of APOEε4 ; however, these mechanisms are unknown. We hypothesized that APOEε4 carriers without dementia might carry genetic variations that could protect them from developing APOEε4- mediated AD pathology. To test this, we leveraged whole genome sequencing (WGS) data in National Institute on Aging Alzheimer's Disease Family Based Study (NIA-AD FBS), Washington Heights/Inwood Columbia Aging Project (WHICAP), and Estudio Familiar de Influencia Genetica en Alzheimer (EFIGA) cohorts and identified potentially protective variants segregating exclusively among unaffected APOEε4 carriers. In homozygous unaffected carriers above 70 years old, we identified 510 rare coding variants. Pathway analysis of the genes harboring these variants showed significant enrichment in extracellular matrix (ECM)-related processes, suggesting protective effects of functional modifications in ECM proteins. We prioritized two genes that were highly represented in the ECM-related gene ontology terms, (FN1) and collagen type VI alpha 2 chain ( COL6A2 ) and are known to be expressed at the blood-brain barrier (BBB), for postmortem validation and in vivo functional studies. The FN1 and COL6A2 protein levels were increased at the BBB in APOEε4 carriers with AD. Brain expression of cognitively unaffected homozygous APOEε4 carriers had significantly lower FN1 deposition and less reactive gliosis compared to homozygous APOEε4 carriers with AD, suggesting that FN1 might be a downstream driver of APOEε4 -mediated AD-related pathology and cognitive decline. To validate our findings, we used zebrafish models with loss-of-function (LOF) mutations in fn1b - the ortholog for human FN1 . We found that fibronectin LOF reduced gliosis, enhanced gliovascular remodeling and potentiated the microglial response, suggesting that pathological accumulation of FN1 could impair toxic protein clearance, which is ameliorated with FN1 LOF. Our study suggests vascular deposition of FN1 is related to the pathogenicity of APOEε4 , LOF variants in FN1 may reduce APOEε4 -related AD risk, providing novel clues to potential therapeutic interventions targeting the ECM to mitigate AD risk.
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Detecting structural variants (SVs) in whole-genome sequencing poses significant challenges. We present a protocol for variant calling, merging, genotyping, sensitivity analysis, and laboratory validation for generating a high-quality SV call set in whole-genome sequencing from the Alzheimer's Disease Sequencing Project comprising 578 individuals from 111 families. Employing two complementary pipelines, Scalpel and Parliament, for SV/indel calling, we assessed sensitivity through sample replicates (N = 9) with in silico variant spike-ins. We developed a novel metric, D-score, to evaluate caller specificity for deletions. The accuracy of deletions was evaluated by Sanger sequencing. We generated a high-quality call set of 152,301 deletions of diverse sizes. Sanger sequencing validated 114 of 146 detected deletions (78.1%). Scalpel excelled in accuracy for deletions ≤100 bp, whereas Parliament was optimal for deletions >900 bp. Overall, 83.0% and 72.5% of calls by Scalpel and Parliament were validated, respectively, including all 11 deletions called by both Parliament and Scalpel between 101 and 900 bp. Our flexible protocol successfully generated a high-quality deletion call set and a truth set of Sanger sequencing-validated deletions with precise breakpoints spanning 1-17,000 bp.
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Enfermedad de Alzheimer , Humanos , Enfermedad de Alzheimer/genética , Secuenciación Completa del Genoma/métodosRESUMEN
The relationship between genetic variation and gene expression in brain cell types and subtypes remains understudied. Here, we generated single-nucleus RNA sequencing data from the neocortex of 424 individuals of advanced age; we assessed the effect of genetic variants on RNA expression in cis (cis-expression quantitative trait loci) for seven cell types and 64 cell subtypes using 1.5 million transcriptomes. This effort identified 10,004 eGenes at the cell type level and 8,099 eGenes at the cell subtype level. Many eGenes are only detected within cell subtypes. A new variant influences APOE expression only in microglia and is associated with greater cerebral amyloid angiopathy but not Alzheimer's disease pathology, after adjusting for APOEε4, providing mechanistic insights into both pathologies. Furthermore, only a TMEM106B variant affects the proportion of cell subtypes. Integration of these results with genome-wide association studies highlighted the targeted cell type and probable causal gene within Alzheimer's disease, schizophrenia, educational attainment and Parkinson's disease loci.
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Enfermedad de Alzheimer , Humanos , Enfermedad de Alzheimer/metabolismo , Estudio de Asociación del Genoma Completo/métodos , Encéfalo/metabolismo , Sitios de Carácter Cuantitativo/genética , Variación Genética/genética , Proteínas de la Membrana/genética , Proteínas del Tejido Nervioso/genéticaRESUMEN
Alzheimer's disease (AD) remains a complex challenge characterized by cognitive decline and memory loss. Genetic variations have emerged as crucial players in the etiology of AD, enabling hope for a better understanding of the disease mechanisms; yet the specific mechanism of action for those genetic variants remain uncertain. Animal models with reminiscent disease pathology could uncover previously uncharacterized roles of these genes. Using CRISPR/Cas9 gene editing, we generated a knockout model for abca7, orthologous to human ABCA7 - an established AD-risk gene. The abca7 +/- zebrafish showed reduced astroglial proliferation, synaptic density, and microglial abundance in response to amyloid beta 42 (Aß42). Single-cell transcriptomics revealed abca7 -dependent neuronal and glial cellular crosstalk through neuropeptide Y (NPY) signaling. The abca7 knockout reduced the expression of npy, bdnf and ngfra , which are required for synaptic integrity and astroglial proliferation. With clinical data in humans, we showed reduced NPY in AD correlates with elevated Braak stage, predicted regulatory interaction between NPY and BDNF , identified genetic variants in NPY associated with AD, found segregation of variants in ABCA7, BDNF and NGFR in AD families, and discovered epigenetic changes in the promoter regions of NPY, NGFR and BDNF in humans with specific single nucleotide polymorphisms in ABCA7 . These results suggest that ABCA7-dependent NPY signaling is required for synaptic integrity, the impairment of which generates a risk factor for AD through compromised brain resilience.
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Background: Both genetic variants and epigenetic features contribute to the risk of Alzheimer's disease (AD). We studied the AD association of CpG-related single nucleotide polymorphisms (CGS), which act as the hub of both the genetic and epigenetic effects, in Hispanics decedents and generalized the findings to Non-Hispanic Whites (NHW) decedents. Methods: First, we derived the dosage of the CpG site-creating allele of multiple CGSes in each 1 KB window across the genome and we conducted a sliding window association test with clinical diagnosis of AD in 7,155 Hispanics (3,194 cases and 3,961 controls) using generalized linear mixed models with the adjustment of age, sex, population structure, genomic relationship matrix, and genotyping batches. Next, using methylation and bulk RNA-sequencing data from the dorsolateral pre-frontal cortex in 150 Hispanics brains, we tested the cis- and trans-effects of AD associated CGS on brain DNA methylation to mRNA expression. For the genes with significant cis- and trans-effects, we checked their enriched pathways. Results: We identified six genetic loci in Hispanics with CGS dosage associated with AD at genome-wide significance levels: ADAM20 (Score=55.2, P= 4.06×10 -8 ), between VRTN (Score=-19.6, P= 1.47×10 -8 ) and SYNDIG1L (Score=-37.7, P= 2.25×10 -9 ), SPG7 (16q24.3) (Score=40.5, P= 2.23×10 -8 ), PVRL2 (Score=125.86, P= 1.64×10 -9 ), TOMM40 (Score=-18.58, P= 4.61×10 -8 ), and APOE (Score=75.12, P= 7.26×10 -26 ). CGSes in PVRL2 and APOE were also genome-wide significant in NHW. Except for ADAM20 , CGSes in all the other five loci were associated with Hispanic brain methylation levels (mQTLs) and CGSes in SPG7, PVRL2, and APOE were also mQTLs in NHW. Except for SYNDIG1L ( P =0.08), brain methylation levels in all the other five loci affected downstream RNA expression in the Hispanics ( P <0.05), and methylation at VRTN and TOMM40 were also associated with RNA expression in NHW. Gene expression in these six loci were also regulated by CpG sites in genes that were enriched in the neuron projection and synapse (FDR<0.05). Conclusions: We identified six CpG associated genetic loci associated with AD in Hispanics, harboring both genetic and epigenetic risks. However, their downstream effects on mRNA expression maybe ethnic specific and different from NHW.
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BACKGROUND: Few rare variants have been identified in genetic loci from genome wide association studies of Alzheimer's disease (AD), limiting understanding of mechanisms and risk assessment, and genetic counseling. METHODS: Using genome sequencing data from 197 families in The NIA Alzheimer's Disease Family Based Study, and 214 Caribbean Hispanic families, we searched for rare coding variants within known GWAS loci from the largest published study. RESULTS: Eighty-six rare missense or loss of function (LoF) variants completely segregated in 17.5% of families, but in 91 (22.1%) of families APOE-e4 was the only variant segregating. However, in 60.3% of families neither APOE-e4 nor missense or LoF variants were found within the GWAS loci. DISCUSSION: Although APOE-ε4 and several rare variants were found to segregate in both family datasets, many families had no variant accounting for their disease. This suggests that familial AD may be the result of unidentified rare variants.