Discrete class I molecules on brain endothelium differentially regulate neuropathology in experimental cerebral malaria.

Cerebral malaria is the deadliest complication that can arise from Plasmodium infection. CD8 T-cell engagement of brain vasculature is a putative mechanism of neuropathology in cerebral malaria. To define contributions of brain endothelial cell major histocompatibility complex (MHC) class I antigen-presentation to CD8 T cells in establishing cerebral malaria pathology, we developed novel H-2Kb LoxP and H-2Db LoxP mice crossed with Cdh5-Cre mice to achieve targeted deletion of discrete class I molecules, specifically from brain endothelium. This strategy allowed us to avoid off-target effects on iron homeostasis and class I-like molecules, which are known to perturb Plasmodium infection. This is the first endothelial-specific ablation of individual class-I molecules enabling us to interrogate these molecular interactions. In these studies, we interrogated human and mouse transcriptomics data to compare antigen presentation capacity during cerebral malaria. Using the Plasmodium berghei ANKA model of experimental cerebral malaria (ECM), we observed that H-2Kb and H-2Db class I molecules regulate distinct patterns of disease onset, CD8 T-cell infiltration, targeted cell death and regional blood-brain barrier disruption. Strikingly, ablation of either molecule from brain endothelial cells resulted in reduced CD8 T-cell activation, attenuated T-cell interaction with brain vasculature, lessened targeted cell death, preserved blood-brain barrier integrity and prevention of ECM and the death of the animal. We were able to show that these events were brain-specific through the use of parabiosis and created the novel technique of dual small animal MRI to simultaneously scan conjoined parabionts during infection. These data demonstrate that interactions of CD8 T cells with discrete MHC class I molecules on brain endothelium differentially regulate development of ECM neuropathology. Therefore, targeting MHC class I interactions therapeutically may hold potential for treatment of cases of severe malaria.

Towards defining morphologic parameters of normal parous and nulliparous breast tissues by artificial intelligence.

BACKGROUND: Breast terminal duct lobular units (TDLUs), the source of most breast cancer (BC) precursors, are shaped by age-related involution, a gradual process, and postpartum involution (PPI), a dramatic inflammatory process that restores baseline microanatomy after weaning. Dysregulated PPI is implicated in the pathogenesis of postpartum BCs. We propose that assessment of TDLUs in the postpartum period may have value in risk estimation, but characteristics of these tissues in relation to epidemiological factors are incompletely described. METHODS: Using validated Artificial Intelligence and morphometric methods, we analyzed digitized images of tissue sections of normal breast tissues stained with hematoxylin and eosin from donors ≤ 45 years from the Komen Tissue Bank (180 parous and 545 nulliparous). Metrics assessed by AI, included: TDLU count; adipose tissue fraction; mean acini count/TDLU; mean dilated acini; mean average acini area; mean "capillary" area; mean epithelial area; mean ratio of epithelial area versus intralobular stroma; mean mononuclear cell count (surrogate of immune cells); mean fat area proximate to TDLUs and TDLU area. We compared epidemiologic characteristics collected via questionnaire by parity status and race, using a Wilcoxon rank sum test or Fisher's exact test. Histologic features were compared between nulliparous and parous women (overall and by time between last birth and donation [recent birth: ≤ 5 years versus remote birth: > 5 years]) using multivariable regression models. RESULTS: Normal breast tissues of parous women contained significantly higher TDLU counts and acini counts, more frequent dilated acini, higher mononuclear cell counts in TDLUs and smaller acini area per TDLU than nulliparas (all multivariable analyses p < 0.001). Differences in TDLU counts and average acini size persisted for > 5 years postpartum, whereas increases in immune cells were most marked ≤ 5 years of a birth. Relationships were suggestively modified by several other factors, including demographic and reproductive characteristics, ethanol consumption and breastfeeding duration. CONCLUSIONS: Our study identified sustained expansion of TDLU numbers and reduced average acini area among parous versus nulliparous women and notable increases in immune responses within five years following childbirth. Further, we show that quantitative characteristics of normal breast samples vary with demographic features and BC risk factors.

Chromoanasynthesis is a common mechanism that leads to ERBB2 amplifications in a cohort of early stage HER2+ breast cancer samples.

BACKGROUND: HER2 positive (HER2+) breast cancers involve chromosomal structural alterations that act as oncogenic driver events. METHODS: We interrogated the genomic structure of 18 clinically-defined HER2+ breast tumors through integrated analysis of whole genome and transcriptome sequencing, coupled with clinical information. RESULTS: ERBB2 overexpression in 15 of these tumors was associated with ERBB2 amplification due to chromoanasynthesis with six of them containing single events and the other nine exhibiting multiple events. Two of the more complex cases had adverse clinical outcomes. Chromosomes 8 was commonly involved in the same chromoanasynthesis with 17. In ten cases where chromosome 8 was involved we observed NRG1 fusions (two cases), NRG1 amplification (one case), FGFR1 amplification and ADAM32 or ADAM5 fusions. ERBB3 over-expression was associated with NRG1 fusions and EGFR and ERBB3 expressions were anti-correlated. Of the remaining three cases, one had a small duplication fully encompassing ERBB2 and was accompanied with a pathogenic mutation. CONCLUSION: Chromoanasynthesis involving chromosome 17 can lead to ERBB2 amplifications in HER2+ breast cancer. However, additional large genomic alterations contribute to a high level of genomic complexity, generating the hypothesis that worse outcome could be associated with multiple chromoanasynthetic events.

The eSNV-detect: a computational system to identify expressed single nucleotide variants from transcriptome sequencing data.

Rapid development of next generation sequencing technology has enabled the identification of genomic alterations from short sequencing reads. There are a number of software pipelines available for calling single nucleotide variants from genomic DNA but, no comprehensive pipelines to identify, annotate and prioritize expressed SNVs (eSNVs) from non-directional paired-end RNA-Seq data. We have developed the eSNV-Detect, a novel computational system, which utilizes data from multiple aligners to call, even at low read depths, and rank variants from RNA-Seq. Multi-platform comparisons with the eSNV-Detect variant candidates were performed. The method was first applied to RNA-Seq from a lymphoblastoid cell-line, achieving 99.7% precision and 91.0% sensitivity in the expressed SNPs for the matching HumanOmni2.5 BeadChip data. Comparison of RNA-Seq eSNV candidates from 25 ER+ breast tumors from The Cancer Genome Atlas (TCGA) project with whole exome coding data showed 90.6-96.8% precision and 91.6-95.7% sensitivity. Contrasting single-cell mRNA-Seq variants with matching traditional multicellular RNA-Seq data for the MD-MB231 breast cancer cell-line delineated variant heterogeneity among the single-cells. Further, Sanger sequencing validation was performed for an ER+ breast tumor with paired normal adjacent tissue validating 29 out of 31 candidate eSNVs. The source code and user manuals of the eSNV-Detect pipeline for Sun Grid Engine and virtual machine are available at http://bioinformaticstools.mayo.edu/research/esnv-detect/.

VPS35 mutations in Parkinson disease.

The identification of genetic causes for Mendelian disorders has been based on the collection of multi-incident families, linkage analysis, and sequencing of genes in candidate intervals. This study describes the application of next-generation sequencing technologies to a Swiss kindred presenting with autosomal-dominant, late-onset Parkinson disease (PD). The family has tremor-predominant dopa-responsive parkinsonism with a mean onset of 50.6 ± 7.3 years. Exome analysis suggests that an aspartic-acid-to-asparagine mutation within vacuolar protein sorting 35 (VPS35 c.1858G>A; p.Asp620Asn) is the genetic determinant of disease. VPS35 is a central component of the retromer cargo-recognition complex, is critical for endosome-trans-golgi trafficking and membrane-protein recycling, and is evolutionarily highly conserved. VPS35 c.1858G>A was found in all affected members of the Swiss kindred and in three more families and one patient with sporadic PD, but it was not observed in 3,309 controls. Further sequencing of familial affected probands revealed only one other missense variant, VPS35 c.946C>T; (p.Pro316Ser), in a pedigree with one unaffected and two affected carriers, and thus the pathogenicity of this mutation remains uncertain. Retromer-mediated sorting and transport is best characterized for acid hydrolase receptors. However, the complex has many types of cargo and is involved in a diverse array of biologic pathways from developmental Wnt signaling to lysosome biogenesis. Our study implicates disruption of VPS35 and retromer-mediated trans-membrane protein sorting, rescue, and recycling in the neurodegenerative process leading to PD.

Translation initiator EIF4G1 mutations in familial Parkinson disease.

Genome-wide analysis of a multi-incident family with autosomal-dominant parkinsonism has implicated a locus on chromosomal region 3q26-q28. Linkage and disease segregation is explained by a missense mutation c.3614G>A (p.Arg1205His) in eukaryotic translation initiation factor 4-gamma (EIF4G1). Subsequent sequence and genotype analysis identified EIF4G1 c.1505C>T (p.Ala502Val), c.2056G>T (p.Gly686Cys), c.3490A>C (p.Ser1164Arg), c.3589C>T (p.Arg1197Trp) and c.3614G>A (p.Arg1205His) substitutions in affected subjects with familial parkinsonism and idiopathic Lewy body disease but not in control subjects. Despite different countries of origin, persons with EIF4G1 c.1505C>T (p.Ala502Val) or c.3614G>A (p.Arg1205His) mutations appear to share haplotypes consistent with ancestral founders. eIF4G1 p.Ala502Val and p.Arg1205His disrupt eIF4E or eIF3e binding, although the wild-type protein does not, and render mutant cells more vulnerable to reactive oxidative species. EIF4G1 mutations implicate mRNA translation initiation in familial parkinsonism and highlight a convergent pathway for monogenic, toxin and perhaps virally-induced Parkinson disease.

Senescent and disease-associated microglia are modifiable features of aged brain white matter.

Brain white matter tracts undergo structural and functional changes linked to late-life cognitive decline, but the cellular and molecular contributions to their selective vulnerability are not well defined. In naturally aged mice, we demonstrate that senescent and disease-associated microglia (DAM) phenotypes converge in hippocampus-adjacent white matter. Through gold-standard gene expression and immunolabeling combined with high-dimensional spatial mapping, we identified microglial cell fates in aged white matter characterized by aberrant morphology, microenvironment reorganization, and expression of senescence and DAM markers, including galectin 3 (GAL3/Lgals3), B-cell lymphoma 2 (Bcl2), and cyclin dependent kinase inhibitors, including Cdkn2a/p16ink4a. Pharmacogenetic or pharmacological targeting of p16ink4a or BCL2 reduced white matter GAL3+ DAM abundance and rejuvenated microglial fimbria organization. Our results demonstrate dynamic changes in microglial identity in aged white matter that can be reverted by senotherapeutic intervention to promote homeostatic maintenance in the aged brain.

Distinct spatial immune microlandscapes are independently associated with outcomes in triple-negative breast cancer.

The utility of spatial immunobiomarker quantitation in prognostication and therapeutic prediction is actively being investigated in triple-negative breast cancer (TNBC). Here, with high-plex quantitative digital spatial profiling, we map and quantitate intraepithelial and adjacent stromal tumor immune protein microenvironments in systemic treatment-naïve (female only) TNBC to assess the spatial context in immunobiomarker-based prediction of outcome. Immune protein profiles of CD45-rich and CD68-rich stromal microenvironments differ significantly. While they typically mirror adjacent, intraepithelial microenvironments, this is not uniformly true. In two TNBC cohorts, intraepithelial CD40 or HLA-DR enrichment associates with better outcomes, independently of stromal immune protein profiles or stromal TILs and other established prognostic variables. In contrast, intraepithelial or stromal microenvironment enrichment with IDO1 associates with improved survival irrespective of its spatial location. Antigen-presenting and T-cell activation states are inferred from eigenprotein scores. Such scores within the intraepithelial compartment interact with PD-L1 and IDO1 in ways that suggest prognostic and/or therapeutic potential. This characterization of the intrinsic spatial immunobiology of treatment-naïve TNBC highlights the importance of spatial microenvironments for biomarker quantitation to resolve intrinsic prognostic and predictive immune features and ultimately inform therapeutic strategies for clinically actionable immune biomarkers.

Evidence of cerebellar TDP-43 loss of function in FTLD-TDP.

Frontotemporal lobar degeneration with TDP-43 pathology (FTLD-TDP) is a neurodegenerative disease primarily affecting the frontal and/or temporal cortices. However, a growing body of evidence suggests that the cerebellum contributes to biochemical, cognitive, and behavioral changes in FTLD-TDP. To evaluate cerebellar TDP-43 expression and function in FTLD-TDP, we analyzed TDP-43 protein levels and the splicing of a TDP-43 target, STMN2, in the cerebellum of 95 FTLD-TDP cases and 25 non-neurological disease controls. Soluble TDP-43 was decreased in the cerebellum of FTLD-TDP cases but a concomitant increase in insoluble TDP-43 was not seen. Truncated STMN2 transcripts, an indicator of TDP-43 dysfunction, were elevated in the cerebellum of FTLD-TDP cases and inversely associated with TDP-43 levels. Additionally, lower cerebellar TDP-43 associated with a younger age at disease onset. We provide evidence of TDP-43 loss of function in the cerebellum in FTLD-TDP, supporting further investigation into this understudied brain region.

Rejuvenation of the aged brain immune cell landscape in mice through p16-positive senescent cell clearance.

Cellular senescence is a plausible mediator of inflammation-related tissue dysfunction. In the aged brain, senescent cell identities and the mechanisms by which they exert adverse influence are unclear. Here we used high-dimensional molecular profiling, coupled with mechanistic experiments, to study the properties of senescent cells in the aged mouse brain. We show that senescence and inflammatory expression profiles increase with age and are brain region- and sex-specific. p16-positive myeloid cells exhibiting senescent and disease-associated activation signatures, including upregulation of chemoattractant factors, accumulate in the aged mouse brain. Senescent brain myeloid cells promote peripheral immune cell chemotaxis in vitro. Activated resident and infiltrating immune cells increase in the aged brain and are partially restored to youthful levels through p16-positive senescent cell clearance in female p16-InkAttac mice, which is associated with preservation of cognitive function. Our study reveals dynamic remodeling of the brain immune cell landscape in aging and suggests senescent cell targeting as a strategy to counter inflammatory changes and cognitive decline.

Characteristics and Spatially Defined Immune (micro)landscapes of Early-stage PD-L1-positive Triple-negative Breast Cancer.

PURPOSE: Programmed death ligand 1 [PD-(L)1]-targeted therapies have shown modest survival benefit in triple-negative breast cancer (TNBC). PD-L1+ microenvironments in TNBC are not well characterized and may inform combinatorial immune therapies. Herein, we characterized clinicopathologic features, RNA-based immune signatures, and spatially defined protein-based tumor-immune microenvironments (TIME) in early-stage PD-L1+ and PD-L1- TNBC. EXPERIMENTAL DESIGN: From a large cohort of chemotherapy-naïve TNBC, clinicopathologic features, deconvoluted RNA immune signatures, and intraepithelial and stromal TIME (Nanostring GeoMX) were identified in subsets of PD-L1+ and PD-L1- TNBC, as defined by FDA-approved PD-L1 companion assays. RESULTS: 228 of 499 (46%) TNBC were PD-L1+ (SP142: ≥1% immune cells-positive). Using PD-L1 22C3, 46% had combined positive score (CPS) ≥ 1 and 16% had CPS ≥10. PD-L1+ TNBC were higher grade with higher tumor-infiltrating lymphocytes (TIL; P < 0.05). PD-L1 was not associated with improved survival following adjustment for TILs and other variables. RNA profiles of PD-L1+ TNBC had increased dendritic cell, macrophage, and T/B cell subset features; and decreased myeloid-derived suppressor cells. PD-L1+ stromal and intraepithelial TIMEs were highly enriched in IDO-1, HLA-DR, CD40, and CD163 compared with PD-L1-TIME, with spatially specific alterations in CTLA-4, Stimulator of Interferon Genes (STING), and fibronectin. Macrophage- and antigen presentation-related proteins correlated most strongly with PD-L1 protein. CONCLUSIONS: In this early-stage TNBC cohort, nearly 50% were PD-L1+ (SP142 companion assay) while 16% were PD-L1+ with the 22C3 companion assay. PD-L1+ TNBC had specific myeloid-derived and lymphoid features. Spatially defined PD-L1+ TIME were enriched in several clinically actionable immune proteins. These data may inform future studies on combinatorial immunotherapies for patients with PD-L1+ TNBC.See related commentary by Symmans, p. 5446.

LINGO1 and LINGO2 variants are associated with essential tremor and Parkinson disease.

Genetic variation in the leucine-rich repeat and Ig domain containing 1 gene (LINGO1) was recently associated with an increased risk of developing essential tremor (ET) and Parkinson disease (PD). Herein, we performed a comprehensive study of LINGO1 and its paralog LINGO2 in ET and PD by sequencing both genes in patients (ET, n=95; PD, n=96) and by examining haplotype-tagging single-nucleotide polymorphisms (tSNPs) in a multicenter North American series of patients (ET, n=1,247; PD, n= 633) and controls (n=642). The sequencing study identified six novel coding variants in LINGO1 (p.S4C, p.V107M, p.A277T, p.R423R, p.G537A, p.D610D) and three in LINGO2 (p.D135D, p.P217P, p.V565V), however segregation analysis did not support pathogenicity. The association study employed 16 tSNPs at the LINGO1 locus and 21 at the LINGO2 locus. One variant in LINGO1 (rs9652490) displayed evidence of an association with ET (odds ratio (OR) =0.63; P=0.026) and PD (OR=0.54; P=0.016). Additionally, four other tSNPs in LINGO1 and one in LINGO2 were associated with ET and one tSNP in LINGO2 associated with PD (P<0.05). Further analysis identified one tSNP in LINGO1 and two in LINGO2 which influenced age at onset of ET and two tSNPs in LINGO1 which altered age at onset of PD (P<0.05). Our results support a role for LINGO1 and LINGO2 in determining risk for and perhaps age at onset of ET and PD. Further studies are warranted to confirm these findings and to determine the pathogenic mechanisms involved.

LRRK2 variation and Parkinson's disease in African Americans.

The global impact of LRRK2 mutations is yet to be realized with a lack of studies in specific ethnic groups, including those of Asian and African descent. Herein, we investigated the frequency of common LRRK2 variants by complete exon sequencing in a series of publicly available African American Parkinson's disease patients. Our study identified three novel synonymous exonic variants and 13 known coding variations; however, there did not appear to be any frequent (>5%) pathogenic mutations. Given the ethnic-specific LRRK2 variation previously identified in PD further studies in under-represented populations are warranted.

Comprehensive sequencing of the LRRK2 gene in patients with familial Parkinson's disease from North Africa.

The LRRK2 gene is a key player in Parkinson's disease (PD), however prevalence and pathogenicity of LRRK2 variants remain to be investigated in ethnically diverse populations. Herein, we performed comprehensive sequencing of the LRRK2 gene in 92 Tunisian probands with familial PD. We then performed an association study using all identified variants in a series of 167 Lrrk2 p.G2019S-negative patients with sporadic PD and 365 Lrrk2 p.G2019S-negative healthy control subjects, all from the same Arab-Berber ethnicity. We identified one novel coding substitution (p.M2408I) and 24 known coding changes. Only the Lrrk2 p.G2019S mutation segregated with disease within families and was found in 39% of familial probands. None of the variants displayed significant association with risk for sporadic PD, however a trend was observed for Lrrk2 p.Y2189C. The present study underscores the importance of the LRRK2 gene in the Tunisian PD population.

Novel pathogenic LRRK2 p.Asn1437His substitution in familial Parkinson's disease.

Genealogical investigation of a large Norwegian family (F04) with autosomal dominant parkinsonism has identified 18 affected family members over four generations. Genetic studies have revealed a novel pathogenic LRRK2 mutation c.4309 A>C (p.Asn1437His) that co-segregates with disease manifestation (LOD = 3.15, θ = 0). Affected carriers have an early age at onset (48 ± 7.7 SD years) and are clinically asymmetric and levodopa responsive. The variant was absent in 623 Norwegian control subjects. Further screening of patients from the same population identified one additional affected carrier (1 of 692) with familial parkinsonism who shares the same haplotype. The mutation is located within the Roc domain of the protein and enhances GTP-binding and kinase activity, further implicating these activities as the mechanisms that underlie LRRK2-linked parkinsonism.

Elevated methylation levels, reduced expression levels, and frequent contractions in a clinical cohort of C9orf72 expansion carriers.

BACKGROUND: A repeat expansion in the C9orf72-SMCR8 complex subunit (C9orf72) is the most common genetic cause of two debilitating neurodegenerative diseases: amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Currently, much remains unknown about which variables may modify these diseases. We sought to investigate associations between C9orf72 promoter methylation, RNA expression levels, and repeat length, their potential effects on disease features, as well as changes over time and within families. METHODS: All samples were obtained through the ALS Center at Mayo Clinic Florida. Our primary cohort included 75 unrelated patients with an expanded C9orf72 repeat, 33 patients who did not possess this expansion, and 20 control subjects without neurodegenerative diseases. Additionally, 67 members from 17 independent C9orf72 families were selected of whom 33 harbored this expansion. Longitudinally collected samples were available for 35 C9orf72 expansion carriers. To increase our understanding of C9orf72-related diseases, we performed quantitative methylation-sensitive restriction enzyme-based assays, digital molecular barcoding, quantitative real-time PCR, and Southern blotting. RESULTS: In our primary cohort, higher methylation levels were observed in patients with a C9orf72 repeat expansion than in patients without this expansion (p = 1.7e-13) or in control subjects (p = 3.3e-07). Moreover, we discovered that an increase in methylation levels was associated with a decrease in total C9orf72 transcript levels (p = 5.5e-05). These findings aligned with our observation that C9orf72 expansion carriers had lower expression levels of total C9orf72 transcripts than patients lacking this expansion (p = 3.7e-07) or control subjects (p = 9.1e-05). We also detected an elevation of transcripts containing intron 1a (upstream of the repeat) in patients carrying a C9orf72 repeat expansion compared to (disease) controls (p ≤ 0.01), an indication of abortive transcripts and/or a switch in transcription start site usage. While methylation and expression levels were relatively stable over time, fluctuations were seen in repeat length. Interestingly, contractions occurred frequently in parent-offspring transmissions (> 50%), especially in paternal transmissions. Furthermore, smaller repeat lengths were detected in currently unaffected individuals than in affected individuals (p = 8.9e-04) and they were associated with an earlier age at collection (p = 0.008). CONCLUSIONS: In blood from C9orf72 expansion carriers, we found elevated methylation levels, reduced expression levels, and unstable expansions that tend to contract in successive generations, arguing against anticipation.

Association of Genetic Variants at TRPC6 With Chemotherapy-Related Heart Failure.

Background: Our previous GWAS identified genetic variants at six novel loci that were associated with a decline in left ventricular ejection fraction (LVEF), p < 1 × 10-5 in 1,191 early breast cancer patients from the N9831 clinical trial of chemotherapy plus trastuzumab. In this study we sought replication of these loci. Methods: We tested the top loci from the GWAS for association with chemotherapy-related heart failure (CRHF) using 26 CRHF cases from N9831 and 984 patients from the Mayo Clinic Biobank which included CRHF cases (N = 12) and control groups of patients treated with anthracycline +/- trastuzumab without HF (N = 282) and patients with HF that were never treated with anthracycline or trastuzumab (N = 690). We further examined associated loci in the context of gene expression and rare coding variants using a TWAS approach in heart left ventricle and Sanger sequencing, respectively. Doxorubicin-induced apoptosis and cardiomyopathy was modeled in human iPSC-derived cardiomyocytes and endothelial cells and a mouse model, respectively, that were pre-treated with GsMTx-4, an inhibitor of TRPC6. Results: TRPC6 5' flanking variant rs57242572-T was significantly more frequent in cases compared to controls, p = 0.031, and rs61918162-T showed a trend for association, p = 0.065. The rs61918162 T-allele was associated with higher TRPC6 expression in the heart left ventricle. We identified a single TRPC6 rare missense variant (rs767086724, N338S, prevalence 0.0025% in GnomAD) in one of 38 patients (2.6%) with CRHF. Pre-treatment of cardiomyocytes and endothelial cells with GsMTx4 significantly reduced doxorubicin-induced apoptosis. Similarly, mice treated with GsMTx4 had significantly improved doxorubicin-induced cardiac dysfunction. Conclusions: Genetic variants that are associated with increased TRPC6 expression in the heart and rare TRPC6 missense variants may be clinically useful as risk factors for CRHF. GsMTx-4 may be a cardioprotective agent in patients with TRPC6 risk variants. Replication of the genetic associations in larger well-characterized samples and functional studies are required.

Truncated stathmin-2 is a marker of TDP-43 pathology in frontotemporal dementia.

No treatment for frontotemporal dementia (FTD), the second most common type of early-onset dementia, is available, but therapeutics are being investigated to target the 2 main proteins associated with FTD pathological subtypes: TDP-43 (FTLD-TDP) and tau (FTLD-tau). Testing potential therapies in clinical trials is hampered by our inability to distinguish between patients with FTLD-TDP and FTLD-tau. Therefore, we evaluated truncated stathmin-2 (STMN2) as a proxy of TDP-43 pathology, given the reports that TDP-43 dysfunction causes truncated STMN2 accumulation. Truncated STMN2 accumulated in human induced pluripotent stem cell-derived neurons depleted of TDP-43, but not in those with pathogenic TARDBP mutations in the absence of TDP-43 aggregation or loss of nuclear protein. In RNA-Seq analyses of human brain samples from the NYGC ALS cohort, truncated STMN2 RNA was confined to tissues and disease subtypes marked by TDP-43 inclusions. Last, we validated that truncated STMN2 RNA was elevated in the frontal cortex of a cohort of patients with FTLD-TDP but not in controls or patients with progressive supranuclear palsy, a type of FTLD-tau. Further, in patients with FTLD-TDP, we observed significant associations of truncated STMN2 RNA with phosphorylated TDP-43 levels and an earlier age of disease onset. Overall, our data uncovered truncated STMN2 as a marker for TDP-43 dysfunction in FTD.

Genomic investigation of alpha-synuclein multiplication and parkinsonism.

OBJECTIVE: Copy number variation is a common polymorphic phenomenon within the human genome. Although the majority of these events are non-deleterious they can also be highly pathogenic. Herein we characterize five families with parkinsonism that have been identified to harbor multiplication of the chromosomal 4q21 locus containing the alpha-synuclein gene (SNCA). METHODS: A methodological approach using fluorescent in situ hybridization and Affymetrix (Santa Clara, CA) 250K SNP microarrays was used to characterize the multiplication in each family and to identify the genes encoded within the region. The telomeric and centromeric breakpoints of each family were further narrowed using semiquantitative polymerase chain reaction with microsatellite markers and then screened for transposable repeat elements. RESULTS: The severity of clinical presentation is correlated with SNCA dosage and does not appear to be overtly affected by the presence of other genes in the multiplicated region. With the exception of the Lister kindred, in each family the multiplication event appears de novo. The type and position of Alu/LINE repeats are also different at each breakpoint. Microsatellite analysis demonstrates two genomic mechanisms are responsible for chromosome 4q21 multiplications, including both SNCA duplication and recombination. INTERPRETATION: SNCA dosage is responsible for parkinsonism, autonomic dysfunction, and dementia observed within each family. We hypothesize dysregulated expression of wild-type alpha-synuclein results in parkinsonism and may explain the recent association of common SNCA variants in sporadic Parkinson's disease. SNCA genomic duplication results from intraallelic (segmental duplication) or interallelic recombination with unequal crossing over, whereas both mechanisms appear to be required for genomic SNCA triplication.

Expanding the clinical phenotype of SNCA duplication carriers.

SNCA duplication is a recognized cause of familial Parkinson's disease (PD). We aimed to explore the genetic and clinical variability in the disease manifestation. Molecular characterization was performed using real-time PCR, SNP arrays, and haplotype analysis. We further studied those patients who were found to harbor SNCA duplication with olfactory function tests, polysomnography, and PET. We identified four new families and one sporadic patient with SNCA duplication. Eleven symptomatic patients from these four families presented with parkinsonism, of which three subsequently developed dementia. The lifetime estimate of overall penetrance was 43.8%. FDG-PET study of symptomatic patients showed hypometabolism in the occipital lobe, whereas asymptomatic carriers of SNCA duplication demonstrated normal glucose metabolism. Symptomatic patients showed abnormal olfactory function and polysomnography and asymptomatic carriers showed normal results. The clinical features of SNCA duplication include parkinsonism with or without dementia. Asymptomatic carriers displayed normal test results with the eldest individual aged 79 years; thus, even a carrier of SNCA duplication may escape the development of PD. This difference in age-associated penetrance may be due to the genetic background or environmental exposures. Further studies of SNCA duplication carriers will help identify disease-modifiers and may open novel avenues for future treatment.