Transcriptome-wide isoform-level dysregulation in ASD, schizophrenia, and bipolar disorder
題目：自閉癥、精神分裂癥和躁郁癥中的轉錄組范圍上的==異構體水平==的失調
主要作者及第一單位：
Michael J. Gandal1,2,3,4,, Pan Zhang5,, Evi Hadjimichael6,7,8,9,, […] Chunyu Liu10,17,27, Lilia M.Iakoucheva5,, Dalila Pinto6,7,8,9,*, Daniel H. Geschwind1,2,3,4,
- 1Department of Psychiatry, Semel Institute, David Geffen School of Medicine, University of California, Los Angeles, 695 Charles E. Young Drive South, Los Angeles, CA 90095, USA.
發(fā)表期刊及時間：
Science 14 Dec 2018: Vol. 362, Issue 6420, eaat8127 DOI: 10.1126/science.aat8127
摘要：
Most genetic risk for psychiatric disease lies in regulatory regions, implicating pathogenic dysregulation of gene expression and splicing. However, comprehensive assessments of transcriptomic organization in diseased brains are limited. In this work, we integrated genotypes and RNA sequencing in brain samples from 1695 individuals with autism spectrum disorder (ASD), schizophrenia, and bipolar disorder, as well as controls. More than 25% of the transcriptome exhibits ==differential splicing==（差異剪接） or expression, with isoform-level changes capturing the largest disease effects and genetic enrichments. ==Coexpression networks== (共表達網(wǎng)絡) isolate disease-specific neuronal alterations, as well as microglial, astrocyte, and interferon-response modules defining previously unidentified neural-immune mechanisms. We integrated genetic and genomic data to perform a transcriptome-wide association study, prioritizing disease loci likely mediated by ==cis effects== （順式作用） on brain expression. This transcriptome-wide characterization of the molecular pathology across three major psychiatric disorders provides a comprehensive resource for ==mechanistic insight== (洞察機制) and therapeutic development.
大多數(shù)精神疾病的基因風險存在于調控區(qū)域诵棵，涉及基因表達和剪接的致病性失調舶胀。然而，患病者大腦轉錄組的全面評估是有限的网梢。在這項工作中震缭，我們整合了 1695 例自閉癥、精神分裂和躁狂抑郁癥患者和對照組的大腦樣本的基因型和 RNA 測序战虏。超過 25%的轉錄組表現(xiàn)出剪接或表達上的差異拣宰，亞型水平的變化捕獲了最大的疾病影響和遺傳富集。共表達網(wǎng)絡分離出疾病特異性的神經(jīng)元改變烦感，以及定義先前不確定的神經(jīng)元免疫機制的小膠質細胞巡社、星形膠質細胞和干擾素反應模塊。我們整合了遺傳和基因組數(shù)據(jù)手趣，進行了全轉錄組關聯(lián)研究重贺，優(yōu)先考慮在大腦表達中可能由順式作用調節(jié)的疾病位點。這個在三種主要的神經(jīng)疾病的轉錄組范圍分子病理學的特征回懦，為洞察機制和治療發(fā)展提供了全面的資源气笙。
圖表選析：
The PsychENCODE cross-disorder transcriptomic resource.
Human brain RNA-seq was integrated with ==genotypes== (基因型) across individuals with ASD, SCZ, BD, and controls, identifying pervasive dysregulation, including protein-coding, noncoding, splicing, and isoform-level changes. Systems-level and integrative genomic analyses prioritize previously unknown neurogenetic mechanisms and provide insight into the molecular neuropathology of these disorders.
PsychENCODE ：跨疾病轉錄組資源
被測試者中51個患自閉癥，559個患精神分裂癥怯晕，222個患雙向情感障礙和936個正常人潜圃，他們的基因型以及大腦的RNA序列被整合分析并用以識別普遍性的失調（左側圖表12），包括蛋白編碼舟茶、非編碼谭期、剪接和異構體水平的改變（右側圖表1234）。系統(tǒng)水平上和整合基因組分析優(yōu)先考慮以前未知的神經(jīng)遺傳機制吧凉，并提供深入了解這些疾病的分子神經(jīng)病理學隧出。
Fig. 1 Gene and isoform expression dysregulation in brain samples from individuals with psychiatric disorders.
(A) ==DE==（DE: Differential Expression， 差異表達） effect size ==(|log2FC|)== （FC：Fold Change阀捅， 倍數(shù)變化）histograms are shown for protein-coding, ==lncRNA== （long non-coding RNA胀瞪， 長非編碼RNA）, and ==pseudogene==（假基因） biotypes up- or down-regulated ==(FDR < 0.05)== (FDR: false discovery rate ，錯誤發(fā)現(xiàn)率) in disease. Isoform-level changes ==(DTE; blue)== (DTE: differential transcript expression) show larger effect sizes than at the gene level ==(DGE; red)==（differential gene expression）, particularly for protein-coding biotypes in ASD and SCZ. (B) A literature-based comparison shows that the number of DE genes detected is dependent on study sample size for each disorder. (C) ==Venn diagrams==（韋恩圖） depict overlap among up- or down-regulated genes and isoforms across disorders. (D) ==Gene ontology enrichments== （基因本體富集分析） are shown for differentially expressed genes or isoforms. The top five pathways are shown for each disorder. (E) Heatmap depicting cell type specificity of enrichment signals. Differentially expressed features show substantial enrichment for known ==CNS== （CNS: central nervous system ，中樞神經(jīng)系統(tǒng)） cell type markers, defined at the gene level from single-cell RNA-seq. (F) Annotation of 944 ==ncRNAs==（ncRNA： non-coding RNA非編碼RNA） DE in at least one disorder. From left to right: Sequence-based characterization of ncRNAs for measures of human selective constraint; brain developmental expression trajectories are similar across each disorder (colored lines represent mean trajectory across disorders); tissue specificity; and CNS cell type expression patterns.
圖1. 精神病患者腦組織基因及異構體表達異常的研究
（A）DE 效應大小(|log2FC|)直方圖顯示蛋白質編碼凄诞、lncRNA 和假基因生物型在疾病中上調或下調
(FDR<0.05)圆雁。異構體水平變化（DTE；藍色）比基因水平（DGE帆谍；紅色）顯示出更大的效應大小伪朽，特
別是對于 ASD 和 SCZ 中的蛋白質編碼生物型。（B）基于文獻的比較表明汛蝙，DE 基因的檢測數(shù)量取決于每
個疾病的研究樣本大小烈涮。（C）Venn 圖描繪了跨疾病的上調或下調基因及異構體之間的重疊部分。（D）對差異表達的基因或異構體進行了基因本體的富集分析窖剑。顯示了每種疾病的前五條通路都跃脊。（E）熱力圖描述富集信號的細胞類型特異性。差異表達的特征顯示已知 CNS 細胞類型標記物大量富集苛吱，這些標記物是從單細胞 RNA-seq的基因水平進行定義的酪术。（F）注釋了944個ncRNA在至少一種疾病中差異表達。從左到右：基于序列的非編碼RNA在人類選擇性限制措施中的特征翠储；大腦發(fā)育表達軌跡在每個疾病中是相似的（有色線代表障礙的平均軌跡）绘雁；組織特異性；和 CNS 細胞類型表達模式援所。
Fig. 3 Overlap and genetic enrichment among dysregulated transcriptomic features.
(A) Scatterplots demonstrate overlap among dysregulated transcriptomic features, summarized by their first principal component across subjects (R2 values; P < 0.05). ==PRS== (polygenic risk scores , 多基因風險評分) show greatest association with differential transcript signal in SCZ. (B) ==SNP== （SNP：Single* Nucleotide Polymorphism 庐舟，單核苷酸多態(tài)性） heritability in SCZ is enriched among multiple differentially expressed transcriptomic features, with down-regulated isoforms showing the most substantial association via stratified LD-score regression. (C**) Several individual genes and isoforms exhibit genome-wide significant associations with disease PRS. Plots are split by direction of association with increasing PRS. In ASD, most associations localize to the 17q21.31 locus, harboring a common inversion polymorphism.
圖 3 異常的轉錄組特征之間的重疊和基因富集
（A）散點圖顯示失調的轉錄組學特征之間的重疊，通過它們在受試者中的第一主成分總結（R2值; P <0.05）住拭。PRS（疾病的多基因風險評分）顯示 其在SCZ 中與差異轉錄物信號有最大關聯(lián)挪略。（B）SCZ 中的 SNP 遺傳多樣性在多種差異表達的轉錄組特征中富集，其中下調的異構體通過分層 LD 分數(shù)回歸顯示最顯著的關聯(lián)滔岳。（C）幾種單個基因和異構體在疾病 PRS 中表現(xiàn)出全基因組顯著相關性杠娱。根據(jù)與 PRS 相關聯(lián)的方向對圖進行分割。在 ASD 中谱煤，大多數(shù)關聯(lián)定位于 17q21.31 基因座摊求，具有共同的染色體倒置多態(tài)性
Fig. 4 Transcriptome-wide association.
Results from a TWAS prioritize genes whose cis-regulated expression in brain is associated with disease. Plots show conditionally-independent TWAS prioritized genes, with lighter shades depicting marginal associations. The sign of TWAS z-scores indicates predicted direction of effect. Genes significantly up- or down-regulated in diseased brain are shown with arrows, indicating directionality. (A) In SCZ, 193 genes (164 outside of MHC) are prioritized at Bonferroni-corrected P < 0.05, including 107 genes with conditionally independent signals. Of these, 23 are also differentially expressed in SCZ brains with 11 in the same direction as predicted. (B) Seventeen genes are prioritized in BD, of which 15 are conditionally independent. (C) In ASD, a TWAS prioritizes 12 genes, of which 5 are conditionally independent.
圖4 轉錄組范圍的關聯(lián)(分析結果)
TWAS的結果優(yōu)先考慮那些與疾病相關的在腦中順式調節(jié)表達的基因。上圖顯示了條件獨立的TWAS優(yōu)先考慮的基因刘离，用較淺的陰影標示邊緣關聯(lián)室叉。TWAS的z值表示效果的預測方向。用箭頭表示在患者腦中顯著上調或下調的基因硫惕，標示方向茧痕。（A）在精神分裂癥中，在Bonferroni校正P<0.05的結果中恼除，193個基因（在MHC外有164個）被優(yōu)先選出來踪旷，包括107個具有條件獨立信號的基因。其中，在SCZ腦中差異表達的23個基因中的11個與預測的方向相同埃脏。（B）在BD中有17個基因被放在前面搪锣，其中15個是條件獨立的秋忙。（C）在ASD中彩掐，TWAS優(yōu)先考慮了12個基因，其中5個是條件獨立的灰追。
Fig. 5 Gene and isoform coexpression networks capture shared and disease-specific cellular processes and interactions.
(A) Coexpression networks demonstrate pervasive dysregulation across psychiatric disorders. Hierarchical clustering shows that separate gene- and isoform-based networks are highly overlapping, with greater specificity conferred at the isoform level. Disease associations are shown for each module (==linear regression== （線性回歸） β value, FDR < 0.05, –P* < 0.05). Module enrichments (FDR < 0.05) are shown for major CNS cell types. Enrichments are shown for GWAS results from SCZ , using stratified LD score regression (FDR < 0.05, –P < 0.05). (B) Coexpression modules capture specific cellular identities and biological pathways. Colored circles represent module DE effect size in disease, with red outlines representing GWAS enrichment in that disorder. Modules are organized and labeled based on CNS cell type and top gene ontology enrichments. (C) Examples of specific modules dysregulated across disorders, with the top 25 hub genes shown. Edges represent coexpression (Pearson correlation > 0.5) and known protein-protein interactions. Nodes are colored to represent disorders in which that gene is differentially expressed (*FDR < 0.05).
圖 5 基因和異構體共表達網(wǎng)絡捕獲共享和疾病特有的細胞過程和相互作用堵幽。（A）共表達網(wǎng)絡顯示了精神障礙的普遍失調。分層聚類展示了分離的以基因和以異構體為基礎的網(wǎng)絡高度重疊弹澎，并且賦予異構體水平上更大的特異性朴下。每個模塊都顯示出疾病的相關性（線性回歸β值，F(xiàn)DR<0.05,-P<0.05）苦蒿。展示了主要的中樞神經(jīng)細胞類型的模塊富集分析（FDR<0.05）殴胧。用分層的 LD 評分回歸分析（FDR<0.05,-P<0.05）從精神分裂癥（59）、雙向情感障礙（97）和自閉癥（38）患者所得的全轉錄組關聯(lián)研究結果顯示了富集佩迟。（B）共表達模塊捕獲特定的細胞特征和生物學路徑团滥。有色的圓形代表了疾病中模塊 DE 影響大小，紅色輪廓代表了相關疾病的全轉錄組關聯(lián)研究的富集程度报强【逆ⅲ基于中樞神經(jīng)細胞類型對模塊進行組織和標記，基因本體的多樣性達到頂峰秉溉。（C）疾病中特定模塊失調的例子力惯，最核心的 25 個基因也展示了出來。邊緣代表了共表達（Pearson 相關性>0.05）和已知的蛋白-蛋白相互作用召嘶。有色的節(jié)點代表了在疾病中有差異表達的基因（FDR<0.05）
Fig. 7 Distinct neural-immune trajectories in disease.
(A) Coexpression networks refine the neural-immune/inflammatory processes up-regulated in ASD, SCZ, and BD. Previous work has identified specific contributions to this signal from astrocyte and microglial populations (13, 19). Here, we identify additional contributions from distinct IFN-response and NFkB signaling modules. (B) Eigengene-disease associations are shown for each of four identified neural-immune module pairs. The astrocyte and IFN-response modules are up-regulated in ASD and SCZ. NFkB signaling is elevated across all three disorders. The microglial module is up-regulated in ASD and down-regulated in SCZ and BD. (C) Top hub genes for each module are shown, along with edges supported by coexpression (light gray; Pearson correlation > 0.5) and known protein-protein interactions (dark lines). Nodes follow the same coloring scheme as in Fig. 5C. Hubs in the astrocyte module (geneM3/isoM1) include several canonical, specific astrocyte markers, including SOX9, GJA1, SPON1, and NOTCH2. Microglial module hub genes include canonical, specific microglial markers, including AIF1, CSF1R, TYROBP, and TMEM119. The NFkB module includes many known downstream transcription factor targets (JAK3, STAT3, JUNB, and FOS) and upstream activators (IL1R1, nine TNF receptor superfamily members) of this pathway. (D) The top four GO enrichments are shown for each module. (E) Module enrichment for known cell type–specific marker genes, collated from sequencing studies of neural-immune cell types (98–102). (F) Module eigengene expression across age demonstrates distinct and dynamic neural-immune trajectories for each disorder.
圖7 疾病中獨特的神經(jīng)-免疫軌跡
(A) 共表達網(wǎng)絡完善了在ASD父晶、SCZ和BD中上調的神經(jīng)-免疫/炎癥過程。之前的工作已經(jīng)從星狀膠質細胞和小神經(jīng)膠質細胞群體中證實了對這個信號的特殊貢獻（13弄跌，19）诱建。這次我們驗證了來自明顯的干擾素應答和核轉錄因子信號模塊的更多的貢獻。（B）四個被鑒別的神經(jīng)免疫模塊對均分別顯示了固有基因和疾病的聯(lián)系碟绑。星形膠質細胞和干擾素應答模塊在ASD和SCZ中上調俺猿。核轉錄因子信號在三種精神障礙中均上調。小神經(jīng)膠質細胞模塊在ASD中上調格仲，在另外兩種病中下調押袍。（C）每個模塊中最高的中心基因被展示出來，還有共表達支持的邊界（淺灰色凯肋，皮爾森相關系數(shù) > 0.5）和已知的蛋白-蛋白互作（黑色線）谊惭。節(jié)點遵循圖5C中的配色組合。星形膠質細胞模塊中的中樞（geneM3 / isoM1）包括幾種經(jīng)典的特異性星形膠質細胞標記物，包括SOX9圈盔，GJA1豹芯，SPON1和NOTCH2。小神經(jīng)膠質細胞模塊中的樞紐基因包括幾種經(jīng)典的特異性小神經(jīng)膠質細胞標志物驱敲，包括AIF1, CSF1R, TYROBP和TMEM119铁蹈。NFkB模型包括許多在這個通路中已知的下游轉錄因子靶點（JAK3, STAT3, JUNB和FOS）和上游激活物（IL1R1，九個TNF受體超家族成員）众眨。（D）每個模塊都會顯示前四個GO富集通路握牧。（E）已知細胞類型特異標志物基因的模塊富集，從對神經(jīng)免疫細胞類型（98-102）測序研究中搜集而來娩梨。（F）跨年齡的模塊固有基因表達闡述了獨特的沿腰、動態(tài)的每種疾病的神經(jīng)免疫軌跡
Fig. 8 LncRNA annotation, ANK2 isoform switching, and microexon enrichment.
(A) FISH images demonstrate interneuron expression for two poorly annotated lincRNAs—LINC00643 and LINC01166—in area 9 of adult human prefrontal cortex. Sections were labeled with GAD1 probe (green) to indicate GABAergic neurons and lncRNA (magenta) probes for LINC00643 (left) or for LINC01166 (right). All sections were counterstained with DAPI (blue) to reveal cell nuclei. Lipofuscin autofluorescence is visible in both the green and red channels and appears orange. Scale bar, 10 μm. FISH was repeated at least twice on independent samples (table S9) (21), with similar results (see also fig. S16). (B) ANK2 isoforms ANK2-006 and ANK2-013 show significant DTU in SCZ and ASD, respectively (FDR < 0.05). (C) Exon structure of ANK2 highlighting (dashed lines) the ANK2-006 and ANK2-013 isoforms. (Inset) These isoforms have different protein domains and carry different microexons. ANK2-006 is affected by multiple ASD DNMs, while ANK2-013 could be entirely eliminated by a de novo CNV deletion in ASD. (D) Disease-specific coexpressed PPI network. Both ANK2-006 and ANK2-013 interact with NRCAM. The ASD-associated isoform ANK2-013has two additional interacting partners, SCN4B and TAF9. (E) As a class, switch isoforms are significantly enriched for microexon(s). In contrast, exons of average length are not enriched among switch isoforms. The y axis displays odds ratio on a log2 scale. P values are calculated using logistic regression and corrected for multiple comparisons. (F*) Enrichment of 64 genes with switch isoforms for: ASD risk loci (81); CHD8 targets (103); FMRP targets (33); mutationally constraint genes (104); syndromic and highly ranked (1 and 2) genes from SFARI Gene database; vulnerable ASD genes (105); genes with probability of loss-of-function intolerance (pLI) > 0.99 as reported by the Exome Aggregation Consortium (106); genes with likely-gene-disruption (LGD) or LGD plus missense de novo mutations (DNMs) found in patients with neurodevelopmental disorders (21).
圖8LncRNA注釋，ANK2同種型轉換和微外顯子富集狈定。
A）FISH圖像顯示成人人類前額皮質區(qū)域9中兩個注釋不足的lincRNA-LINC00643和LINC01166-的中間神經(jīng)元表達颂龙。用GAD1探針（綠色）標記切片以指示用于LINC00643（左）或用于LINC01166（右）的gama氨基丁酸能神經(jīng)元和lncRNA（品紅色）探針。所有切片用DAPI（藍色）復染以顯示細胞核纽什。 Lipofuscin自發(fā)熒光在綠色和紅色通道中均可見措嵌，并呈橙色。比例尺稿湿，10μm铅匹。在獨立樣品上重復FISH至少兩次（表S9）（21），具有類似的結果（也參見圖S16）饺藤。 （B）ANK2同種型ANK2-006和ANK2-013分別在SCZ和ASD中顯示顯著的DTU（* FDR <0.05）包斑。 （C）ANK2的外顯子結構突出顯示（虛線）ANK2-006和ANK2-013同種型。 （插圖）這些同種型具有不同的蛋白質結構域并攜帶不同的微外顯子涕俗。 ANK2-006受多個ASD 新生突變的影響罗丰，而ANK2-013可以通過ASD中的從頭拷貝數(shù)變異缺失完全消除。 （D）疾病特異性共表達的PPI網(wǎng)絡再姑。 ANK2-006和ANK2-013都與NRCAM相互作用萌抵。 ASD相關同種型ANK2-013具有兩個額外的相互作用配偶體SCN4B和TAF9。 （E）作為一個類元镀，開關同種型顯著富集微外顯子绍填。相反，平均長度的外顯子不會在開關同種型中富集栖疑。 y軸以log2標度顯示優(yōu)勢比讨永。使用邏輯回歸計算P值并校正多重比較。 （F）用開關同種型富集64個基因：ASD風險基因座（81）; CHD8目標（103）; FMRP目標（33）;突變約束基因（104）;來自SFARI基因數(shù)據(jù)庫的綜合征和高度排名（1和2）基因;脆弱的ASD基因（105）; Exome Aggregation Consortium（106）報告的功能喪失不耐受概率（pLI）> 0.99的基因;神經(jīng)發(fā)育障礙患者中發(fā)現(xiàn)LGD或LGD加新生突變的基因（21）遇革。
翻譯小組：
陳凱星卿闹、鄧俊瑋揭糕、王俊豪、黃敬潼锻霎、黃子亮著角、葉名琛、李碧琪旋恼、渠夢葳吏口、鄭凌伶
