Background
Microtubule associated protein tau (MAPT) is involved in assembly and stabilization of microtubules. However, mutated or misfolded forms are commonly found to aggregate in Frontotemporal ...dementia (FTD), Progressive supranuclear palsy (PSP), Corticobasal degeneration (CBD) etc. singly or with another protein as in Alzheimer’s disease (AD). The second strongest signal in MAPT locus after H1 haplotype, as identified by multiple GWAS on AD, PD, PSP and CBD, is from MAPT intronic SNP rs242557. It is part of a FANTOM5 enhancer in eye and brain and evolutionarily conserved from humans to armadillo. While it has been hypothesized that rs242557 could exert an effect on MAPT splicing, the results are contradictory. Strong, Independent signals from MAPT neighboring genes KANSL1, NSF LRR37A and WNT3 have also been noted in some of these diseases. In this study we investigate the role of the enhancer region encompassing rs242557 in gene regulation within MAPT locus.
Method
Established CRISPR‐Cas9 editing protocols were used to delete rs242557 and its flanking sequences in human microglial clone 3 (HMC3) and human neuroblastoma (NBL‐S) cell lines. Heterozygous and homozygous clones with deleted rs242557 and its flanking sequences were individually selected for the study. Cells that went through the entire process of genome editing protocol without the presence of any guide RNA were used as wild type controls. cDNA, synthesized from extracted RNA were used to study expression of MAPT isoforms and its neighboring genes by qRT‐PCR, while western blots were performed from the corresponding proteins.
Result
HMC‐3 cells had significant reduction (p=0.0001) of Nsf, Kansl1, Kansl1‐AS1, LRR37A, LRR37A2 and LRR37A3 transcripts in both rs242557 homozygous and heterozygous deletion clones. Total MAPT, MAPT isoforms (3R, 4R, 0N, 1N and 2N) and CRHR1 were also low with some having ct values beyond 33. At the protein level reduced expression of NSF and KANSL1 were noted in the homozygotes and some heterozygotes. No significant alteration was observed in the NBL‐S clones. Experiments are currently in progress for the remaining neighboring proteins.
Conclusion
Our results indicate that the enhancer encompassing rs242557 alters expression of MAPT and several of its neighbors in HMC‐3 but not NBL‐S cells.
The accumulation of unfolded or misfolded proteins in the endoplasmic reticulum (ER) results in the condition called “ER stress,” which induces the unfolded protein response (UPR), a complex cellular ...process that includes changes in expression of many genes. Failure to restore homeostasis in the ER is associated with human diseases. To identify the underlying changes in gene expression in response to ER stress, we induced ER stress in human B cells and then measured gene expression at ten time points. We followed up those results by studying cells from 60 unrelated people. We rediscovered genes that were known to play a role in the ER-stress response and uncovered several thousand genes that are not known to be involved. Two of these are VLDLR and INHBE, which showed significant increase in expression after ER stress in B cells and in primary fibroblasts. To study the links between UPR and disease susceptibility, we identified ER-stress-responsive genes that are associated with human diseases and assessed individual differences in the ER-stress response. Many of the UPR genes are associated with Mendelian disorders, such as Wolfram syndrome, and complex diseases, including amyotrophic lateral sclerosis and diabetes. Data from two independent samples showed extensive individual variability in ER-stress response. Additional analyses with monozygotic twins revealed significant correlations within twin pairs in their responses to ER stress, thus showing evidence for heritable variation among individuals. These results have implications for basic understanding of ER function and its role in disease susceptibility.
Based on emerging evidence on the role for specific single-nucleotide variants (SNVs) in
EIF2AK3
encoding the integrated stress response kinase PERK, in neurodegeneration, we assessed the association ...of
EIF2AK3
SNVs with neurocognitive performance in people with HIV (PWH) using a candidate gene approach. This retrospective study included the CHARTER cohort participants, excluding those with severe neuropsychiatric comorbidities. Genome-wide data previously obtained for 1047 participants and targeted sequencing of 992 participants with available genomic DNA were utilized to interrogate the association of three noncoding and three coding
EIF2AK3
SNVs with the continuous global deficit score (GDS) and global neurocognitive impairment (NCI; GDS ≥ 0.5) using univariable and multivariable methods, with demographic, disease-associated, and treatment characteristics as covariates. The cohort characteristics were as follows: median age, 43.1 years; females, 22.8%; European ancestry, 41%; median CD4 + T cell counts, 175/µL (nadir) and 428/µL (current). At first assessment, 70.5% used ART and 68.3% of these had plasma HIV RNA levels ≤ 200 copies/mL. All three noncoding
EIF2AK3
SNVs were associated with GDS and NCI (all
p
< 0.05). Additionally, 30.9%, 30.9%, and 41.2% of participants had at least one risk allele for the coding SNVs rs1805165 (G), rs867529 (G), and rs13045 (A), respectively. Homozygosity for all three coding SNVs was associated with significantly worse GDS (
p
< 0.001) and more NCI (
p
< 0.001). By multivariable analysis, the rs13045 A risk allele, current ART use, and Beck Depression Inventory-II value > 13 were independently associated with GDS and NCI (
p
< 0.001) whereas the other two coding SNVs did not significantly correlate with GDS or NCI after including rs13045 in the model. The coding
EIF2AK3
SNVs were associated with worse performance in executive functioning, motor functioning, learning, and verbal fluency. Coding and non-coding SNVs of
EIF2AK3
were associated with global NC and domain-specific performance. The effects were small-to-medium in size but present in multivariable analyses, raising the possibility of specific SNVs in
EIF2AK3
as an important component of genetic vulnerability to neurocognitive complications in PWH.
Graphical Abstract
Microtubule Associated Protein Tau (MAPT) forms proteopathic aggregates in several diseases. The G273R tau mutation, located in the first repeat region, was found by exome sequencing in a patient who ...presented with dementia and parkinsonism. We herein return to pathological examination which demonstrated tau immunoreactivity in neurons and glia consistent of mixed progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD) features. To rationalize the pathological findings, we used molecular biophysics to characterize the mutation in more detail in vitro and in Drosophila. The G273R mutation increases the aggregation propensity of 4-repeat (4R) tau and alters the tau binding affinity towards microtubules (MTs) and F-actin. Tau aggregates in PSP and CBD are predominantly 4R tau. Our data suggest that the G273R mutation induces a shift in pool of 4R tau by lower F-actin affinity, alters the conformation of MT bound 4R tau, while increasing chaperoning of 3R tau by binding stronger to F-actin. The mutation augmented fibrillation of 4R tau initiation in vitro and in glial cells in Drosophila and showed preferential seeding of 4R tau in vitro suggestively causing a late onset 4R tauopathy reminiscent of PSP and CBD.
•G273R tau mutation was found in a patient with dementia and parkinsonism.•Tau pathology in neurons and glia resembled mixed PSP and CBD.•The G273R mutation increased the aggregation of 4R tau in vitro &Drosophila model.•The 4R tau G273R mutation selectively seeded 4R tau in vitro.
IMPORTANCE High-prevalence foci of amyotrophic lateral sclerosis (ALS) and parkinsonism-dementia complex (PDC) exist in Japanese on the Kii Peninsula of Japan and in the Chamorros of Guam. Clinical ...and neuropathologic similarities suggest that the disease in these 2 populations may be related. Recent findings showed that some of the Kii Peninsula ALS cases had pathogenic C9orf72 repeat expansions, a genotype that causes ALS in Western populations. OBJECTIVES To perform genotyping among Guam residents to determine if the C9orf72 expanded repeat allele contributes to ALS-PDC in this population and to evaluate LRRK2 for mutations in the same population. DESIGN AND SETTING Case-control series from neurodegenerative disease research programs on Guam that screened residents for ALS, PDC, and dementia. PARTICIPANTS Study participants included 24 with ALS and 22 with PDC and 43 older control subjects with normal cognition ascertained between 1956 and 2006. All but one participant were Chamorro, the indigenous people of Guam. A single individual of white race/ethnicity with ALS was ascertained on Guam during the study. MAIN OUTCOMES AND MEASURES Participants were screened for C9orf72 hexanucleotide repeat length. Participants with repeat numbers in great excess of 30 were considered to have pathogenic repeat expansions. LRRK2 was screened for point mutations by DNA sequencing. RESULTS We found a single individual with an expanded pathogenic hexanucleotide repeat. This individual of white race/ethnicity with ALS was living on Guam at the time of ascertainment but had been born in the United States. All Chamorro participants with ALS and PDC and control subjects had normal repeats, ranging from 2 to 17 copies. No pathogenic LRRK2 mutations were found. CONCLUSIONS AND RELEVANCE Unlike participants with ALS from the Kii Peninsula, C9orf72 expansions do not cause ALS-PDC in Chamorros. Likewise, LRRK2 mutations do not cause Guam ALS-PDC.
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.
Using a selective screening strategy to enrich for active L1 elements, we isolated 13 full-length elements from a human genomic library. We tested these and two previously-isolated L1s (L1.3 and ...L1.4) for reverse transcriptase (RT) activity and the ability to retrotranspose in HeLa cells. Of the 13 newly-isolated L1s, eight had RT activity and three were able to retrotranspose. L1.3 and L1.4 possessed RT activity and retrotransposed at remarkably high frequencies. These studies bring the number of characterized active human L1 elements to seven. Based on these and other data, we estimate that 30-60 active L1 elements reside in the average diploid genome.
Progressive supranuclear palsy (PSP) is a rare neurodegenerative disease characterized by the accumulation of aggregated tau proteins in astrocytes, neurons, and oligodendrocytes. Previous ...genome-wide association studies for PSP were based on genotype array, therefore, were inadequate for the analysis of rare variants as well as larger mutations, such as small insertions/deletions (indels) and structural variants (SVs).BACKGROUNDProgressive supranuclear palsy (PSP) is a rare neurodegenerative disease characterized by the accumulation of aggregated tau proteins in astrocytes, neurons, and oligodendrocytes. Previous genome-wide association studies for PSP were based on genotype array, therefore, were inadequate for the analysis of rare variants as well as larger mutations, such as small insertions/deletions (indels) and structural variants (SVs).In this study, we performed whole genome sequencing (WGS) and conducted association analysis for single nucleotide variants (SNVs), indels, and SVs, in a cohort of 1,718 cases and 2,944 controls of European ancestry. Of the 1,718 PSP individuals, 1,441 were autopsy-confirmed and 277 were clinically diagnosed.METHODIn this study, we performed whole genome sequencing (WGS) and conducted association analysis for single nucleotide variants (SNVs), indels, and SVs, in a cohort of 1,718 cases and 2,944 controls of European ancestry. Of the 1,718 PSP individuals, 1,441 were autopsy-confirmed and 277 were clinically diagnosed.Our analysis of common SNVs and indels confirmed known genetic loci at MAPT, MOBP, STX6, SLCO1A2, DUSP10, and SP1, and further uncovered novel signals in APOE, FCHO1/MAP1S, KIF13A, TRIM24, TNXB, and ELOVL1. Notably, in contrast to Alzheimer's disease (AD), we observed the APOE ε2 allele to be the risk allele in PSP. Analysis of rare SNVs and indels identified significant association in ZNF592 and further gene network analysis identified a module of neuronal genes dysregulated in PSP. Moreover, seven common SVs associated with PSP were observed in the H1/H2 haplotype region (17q21.31) and other loci, including IGH, PCMT1, CYP2A13, and SMCP. In the H1/H2 haplotype region, there is a burden of rare deletions and duplications (P = 6.73 × 10-3) in PSP.RESULTSOur analysis of common SNVs and indels confirmed known genetic loci at MAPT, MOBP, STX6, SLCO1A2, DUSP10, and SP1, and further uncovered novel signals in APOE, FCHO1/MAP1S, KIF13A, TRIM24, TNXB, and ELOVL1. Notably, in contrast to Alzheimer's disease (AD), we observed the APOE ε2 allele to be the risk allele in PSP. Analysis of rare SNVs and indels identified significant association in ZNF592 and further gene network analysis identified a module of neuronal genes dysregulated in PSP. Moreover, seven common SVs associated with PSP were observed in the H1/H2 haplotype region (17q21.31) and other loci, including IGH, PCMT1, CYP2A13, and SMCP. In the H1/H2 haplotype region, there is a burden of rare deletions and duplications (P = 6.73 × 10-3) in PSP.Through WGS, we significantly enhanced our understanding of the genetic basis of PSP, providing new targets for exploring disease mechanisms and therapeutic interventions.CONCLUSIONSThrough WGS, we significantly enhanced our understanding of the genetic basis of PSP, providing new targets for exploring disease mechanisms and therapeutic interventions.
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