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(zhuǎn)錄組范圍上的==異構(gòu)體水平==的失調(diào)

主要作者及第一單位：

Michael J. Gandal^1,2,3,4,, Pan Zhang⁵,, Evi Hadjimichael^6,7,8,9,, […] Chunyu Liu^10,17,27, Lilia M.Iakoucheva⁵,, Dalila Pinto^6,7,8,9,*, Daniel H. Geschwind^1,2,3,4,

1. 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(shí)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(dá)網(wǎng)絡(luò)) 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(jī)制) and therapeutic development.

大多數(shù)精神疾病的基因風(fēng)險(xiǎn)存在于調(diào)控區(qū)域，涉及基因表達(dá)和剪接的致病性失調(diào)。然而，患病者大腦轉(zhuǎn)錄組的全面評(píng)估是有限的。在這項(xiàng)工作中，我們整合了 1695 例自閉癥、精神分裂和躁狂抑郁癥患者和對(duì)照組的大腦樣本的基因型和 RNA 測(cè)序。超過(guò) 25%的轉(zhuǎn)錄組表現(xiàn)出剪接或表達(dá)上的差異，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(dá)網(wǎng)絡(luò)分離出疾病特異性的神經(jīng)元改變，以及定義先前不確定的神經(jīng)元免疫機(jī)制的小膠質(zhì)細(xì)胞、星形膠質(zhì)細(xì)胞和干擾素反應(yīng)模塊。我們整合了遺傳和基因組數(shù)據(jù)，進(jìn)行了全轉(zhuǎn)錄組關(guān)聯(lián)研究，優(yōu)先考慮在大腦表達(dá)中可能由順式作用調(diào)節(jié)的疾病位點(diǎn)。這個(gè)在三種主要的神經(jīng)疾病的轉(zhuǎn)錄組范圍分子病理學(xué)的特征，為洞察機(jī)制和治療發(fā)展提供了全面的資源。

圖表選析：

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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(zhuǎn)錄組資源
被測(cè)試者中51個(gè)患自閉癥，559個(gè)患精神分裂癥，222個(gè)患雙向情感障礙和936個(gè)正常人，他們的基因型以及大腦的RNA序列被整合分析并用以識(shí)別普遍性的失調(diào)（左側(cè)圖表12），包括蛋白編碼、非編碼、剪接和異構(gòu)體水平的改變（右側(cè)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(jī)制，并提供深入了解這些疾病的分子神經(jīng)病理學(xué)。

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Fig. 1 Gene and isoform expression dysregulation in brain samples from individuals with psychiatric disorders.

(A) ==DE==（DE: Differential Expression， 差異表達(dá)） effect size ==(|log2FC|)== （FC：Fold Change， 倍數(shù)變化）histograms are shown for protein-coding, ==lncRNA== （long non-coding RNA， 長(zhǎng)非編碼RNA）, and ==pseudogene==（假基因） biotypes up- or down-regulated ==(FDR < 0.05)== (FDR: false discovery rate ，錯(cuò)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(gòu)體表達(dá)異常的研究
（A）DE 效應(yīng)大小(|log2FC|)直方圖顯示蛋白質(zhì)編碼、lncRNA 和假基因生物型在疾病中上調(diào)或下調(diào)
(FDR<0.05)。異構(gòu)體水平變化（DTE；藍(lán)色）比基因水平（DGE；紅色）顯示出更大的效應(yīng)大小，特
別是對(duì)于 ASD 和 SCZ 中的蛋白質(zhì)編碼生物型。（B）基于文獻(xiàn)的比較表明，DE 基因的檢測(cè)數(shù)量取決于每
個(gè)疾病的研究樣本大小。（C）Venn 圖描繪了跨疾病的上調(diào)或下調(diào)基因及異構(gòu)體之間的重疊部分。（D）對(duì)差異表達(dá)的基因或異構(gòu)體進(jìn)行了基因本體的富集分析。顯示了每種疾病的前五條通路都。（E）熱力圖描述富集信號(hào)的細(xì)胞類型特異性。差異表達(dá)的特征顯示已知 CNS 細(xì)胞類型標(biāo)記物大量富集，這些標(biāo)記物是從單細(xì)胞 RNA-seq的基因水平進(jìn)行定義的。（F）注釋了944個(gè)ncRNA在至少一種疾病中差異表達(dá)。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；大腦發(fā)育表達(dá)軌跡在每個(gè)疾病中是相似的（有色線代表障礙的平均軌跡）；組織特異性；和 CNS 細(xì)胞類型表達(dá)模式。

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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(fēng)險(xiǎn)評(píng)分) show greatest association with differential transcript signal in SCZ. (B) ==SNP== （SNP：Single* Nucleotide Polymorphism ，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(zhuǎn)錄組特征之間的重疊和基因富集
（A）散點(diǎn)圖顯示失調(diào)的轉(zhuǎn)錄組學(xué)特征之間的重疊，通過(guò)它們?cè)谑茉囌咧械牡谝恢鞒煞挚偨Y(jié)（R2值; P <0.05）。PRS（疾病的多基因風(fēng)險(xiǎn)評(píng)分）顯示 其在SCZ 中與差異轉(zhuǎn)錄物信號(hào)有最大關(guān)聯(lián)。（B）SCZ 中的 SNP 遺傳多樣性在多種差異表達(dá)的轉(zhuǎn)錄組特征中富集，其中下調(diào)的異構(gòu)體通過(guò)分層 LD 分?jǐn)?shù)回歸顯示最顯著的關(guān)聯(lián)。（C）幾種單個(gè)基因和異構(gòu)體在疾病 PRS 中表現(xiàn)出全基因組顯著相關(guān)性。根據(jù)與 PRS 相關(guān)聯(lián)的方向?qū)D進(jìn)行分割。在 ASD 中，大多數(shù)關(guān)聯(lián)定位于 17q21.31 基因座，具有共同的染色體倒置多態(tài)性

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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(zhuǎn)錄組范圍的關(guān)聯(lián)(分析結(jié)果)
TWAS的結(jié)果優(yōu)先考慮那些與疾病相關(guān)的在腦中順式調(diào)節(jié)表達(dá)的基因。上圖顯示了條件獨(dú)立的TWAS優(yōu)先考慮的基因，用較淺的陰影標(biāo)示邊緣關(guān)聯(lián)。TWAS的z值表示效果的預(yù)測(cè)方向。用箭頭表示在患者腦中顯著上調(diào)或下調(diào)的基因，標(biāo)示方向。（A）在精神分裂癥中，在Bonferroni校正P<0.05的結(jié)果中，193個(gè)基因（在MHC外有164個(gè)）被優(yōu)先選出來(lái)，包括107個(gè)具有條件獨(dú)立信號(hào)的基因。其中，在SCZ腦中差異表達(dá)的23個(gè)基因中的11個(gè)與預(yù)測(cè)的方向相同。（B）在BD中有17個(gè)基因被放在前面，其中15個(gè)是條件獨(dú)立的。（C）在ASD中，TWAS優(yōu)先考慮了12個(gè)基因，其中5個(gè)是條件獨(dú)立的。

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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(gòu)體共表達(dá)網(wǎng)絡(luò)捕獲共享和疾病特有的細(xì)胞過(guò)程和相互作用。（A）共表達(dá)網(wǎng)絡(luò)顯示了精神障礙的普遍失調(diào)。分層聚類展示了分離的以基因和以異構(gòu)體為基礎(chǔ)的網(wǎng)絡(luò)高度重疊，并且賦予異構(gòu)體水平上更大的特異性。每個(gè)模塊都顯示出疾病的相關(guān)性（線性回歸β值，F(xiàn)DR<0.05,-P<0.05）。展示了主要的中樞神經(jīng)細(xì)胞類型的模塊富集分析（FDR<0.05）。用分層的 LD 評(píng)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(zhuǎn)錄組關(guān)聯(lián)研究結(jié)果顯示了富集。（B）共表達(dá)模塊捕獲特定的細(xì)胞特征和生物學(xué)路徑。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(guān)疾病的全轉(zhuǎn)錄組關(guān)聯(lián)研究的富集程度。基于中樞神經(jīng)細(xì)胞類型對(duì)模塊進(jìn)行組織和標(biāo)記，基因本體的多樣性達(dá)到頂峰。（C）疾病中特定模塊失調(diào)的例子，最核心的 25 個(gè)基因也展示了出來(lái)。邊緣代表了共表達(dá)（Pearson 相關(guān)性>0.05）和已知的蛋白-蛋白相互作用。有色的節(jié)點(diǎn)代表了在疾病中有差異表達(dá)的基因（FDR<0.05）

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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(dú)特的神經(jīng)-免疫軌跡
(A) 共表達(dá)網(wǎng)絡(luò)完善了在ASD、SCZ和BD中上調(diào)的神經(jīng)-免疫/炎癥過(guò)程。之前的工作已經(jīng)從星狀膠質(zhì)細(xì)胞和小神經(jīng)膠質(zhì)細(xì)胞群體中證實(shí)了對(duì)這個(gè)信號(hào)的特殊貢獻(xiàn)（13，19）。這次我們驗(yàn)證了來(lái)自明顯的干擾素應(yīng)答和核轉(zhuǎn)錄因子信號(hào)模塊的更多的貢獻(xiàn)。（B）四個(gè)被鑒別的神經(jīng)免疫模塊對(duì)均分別顯示了固有基因和疾病的聯(lián)系。星形膠質(zhì)細(xì)胞和干擾素應(yīng)答模塊在ASD和SCZ中上調(diào)。核轉(zhuǎn)錄因子信號(hào)在三種精神障礙中均上調(diào)。小神經(jīng)膠質(zhì)細(xì)胞模塊在ASD中上調(diào)，在另外兩種病中下調(diào)。（C）每個(gè)模塊中最高的中心基因被展示出來(lái)，還有共表達(dá)支持的邊界（淺灰色，皮爾森相關(guān)系數(shù) > 0.5）和已知的蛋白-蛋白互作（黑色線）。節(jié)點(diǎn)遵循圖5C中的配色組合。星形膠質(zhì)細(xì)胞模塊中的中樞（geneM3 / isoM1）包括幾種經(jīng)典的特異性星形膠質(zhì)細(xì)胞標(biāo)記物，包括SOX9，GJA1，SPON1和NOTCH2。小神經(jīng)膠質(zhì)細(xì)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(zhì)細(xì)胞標(biāo)志物，包括AIF1, CSF1R, TYROBP和TMEM119。NFkB模型包括許多在這個(gè)通路中已知的下游轉(zhuǎn)錄因子靶點(diǎn)（JAK3, STAT3, JUNB和FOS）和上游激活物（IL1R1，九個(gè)TNF受體超家族成員）。（D）每個(gè)模塊都會(huì)顯示前四個(gè)GO富集通路。（E）已知細(xì)胞類型特異標(biāo)志物基因的模塊富集，從對(duì)神經(jīng)免疫細(xì)胞類型（98-102）測(cè)序研究中搜集而來(lái)。（F）跨年齡的模塊固有基因表達(dá)闡述了獨(dú)特的、動(dòng)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(zhuǎn)換和微外顯子富集。
A）FISH圖像顯示成人人類前額皮質(zhì)區(qū)域9中兩個(gè)注釋不足的lincRNA-LINC00643和LINC01166-的中間神經(jīng)元表達(dá)。用GAD1探針（綠色）標(biāo)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(lán)色）復(fù)染以顯示細(xì)胞核。 Lipofuscin自發(fā)熒光在綠色和紅色通道中均可見(jiàn)，并呈橙色。比例尺，10μm。在獨(dú)立樣品上重復(fù)FISH至少兩次（表S9）（21），具有類似的結(jié)果（也參見(jiàn)圖S16）。 （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）。 （C）ANK2的外顯子結(jié)構(gòu)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(zhì)結(jié)構(gòu)域并攜帶不同的微外顯子。 ANK2-006受多個(gè)ASD 新生突變的影響，而ANK2-013可以通過(guò)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(dá)的PPI網(wǎng)絡(luò)。 ANK2-006和ANK2-013都與NRCAM相互作用。 ASD相關(guān)同種型ANK2-013具有兩個(gè)額外的相互作用配偶體SCN4B和TAF9。 （E）作為一個(gè)類，開(kāi)關(guān)同種型顯著富集微外顯子。相反，平均長(zhǎng)度的外顯子不會(huì)在開(kāi)關(guān)同種型中富集。 y軸以log2標(biāo)度顯示優(yōu)勢(shì)比。使用邏輯回歸計(jì)算P值并校正多重比較。 （F）用開(kāi)關(guān)同種型富集64個(gè)基因：ASD風(fēng)險(xiǎn)基因座（81）; CHD8目標(biāo)（103）; FMRP目標(biāo)（33）;突變約束基因（104）;來(lái)自SFARI基因數(shù)據(jù)庫(kù)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(bào)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）。

翻譯小組：

陳凱星、鄧俊瑋、王俊豪、黃敬潼、黃子亮、葉名琛、李碧琪、渠夢(mèng)葳、鄭凌伶