整合小鼠和人类遗传数据可鉴定胆固醇代谢中的致病基因(Cell Metabolism)

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Genome-Wide Mouse Liver Co-expression Network Analysis

• These data are collected from more than 900 unique mice and are composed of 12 distinct datasets based on sex, dietary intervention, and molecular trait
• In 11 of 12 datasets, we were able to identify a module of genes significantly enriched for the Gene Ontology (GO) term, cholesterol biosynthetic process
• With the 2,435unique genes, we identified genes that were replicated acrosscholesterol modules in more than three datasets at the transcript level, more than two datasets at the protein level, or present in both transcript and protein datasets. From this analysis, we were able to identify 112 replicated genes.
• We also intersected the 112 replicated genes with publicly available sterol regulatory element-binding proteins 1 and 2 (SREBP1 and SREBP2) genome-wide chromatin immunoprecipitation sequencing (ChIP-seq) data and found 43 genes with significant ChIP-seq peaks.
• Collectively,through mouse liver co-expression network analysis, we identi-fied a conserved cholesterol module, and our replication analysis identified 112 genes that capture known cholesterol and lipid metabolism genes, providing proof of concept.

Cross-Reference with Human Lipid GWAS Data

• Next, we cross-referenced the 112 replicated genes from the mouse co-expression networks with the publicly available Global Lipid Genetics Consortium (GLGC) human GWAS data for plasma TC, LDL cholesterol, HDL cholesterol, and TG levels (Willer et al., 2013). Projecting the 112 replicated genes from the mouse onto the human genome, we identified single-nucleotide polymorphisms (SNPs) within 200 kilobases (kb) of the gene start and stop position that showed association.
• , we were able to identify 48 genes that met our criteria (Nelson et al., 2017; Wang et al., 2016). Among the 48 genes, 18 genes had SNPs at a genome-wide significant level and 30 genes had SNPs at a sub-threshold significant level.
• In total, we identified 48 genes showing replication in mouse liver networks and association with plasma lipid traits in humans
• Among these 48 replicated and cross-referenced genes and the six X chromosome genes, 25 have no previously identified role in cholesterol and lipid metabolism. Out of these 25 genes, 21 genes show genome-wide significant or subthreshold association with lipid traits, and four genes are on the X chromosome. Finally, 25 total genes were prioritized for further human GWAS replication analysis and functional testing

Human GWAS Replication for Prioritized Genes

• With the 25 prioritized genes, we looked for replication of association in two additional human lipid GWAS datasets, Million Veteran Program (MVP) and the UK Biobank (UKBB)
• Seven genes(SESN1, BCAP29, LRPAP1, TLCD1, PRSS8, PTPRJ, and RRAS) of the 25 genes were associated with at least one human plasma lipid trait in all GWAS datasets we cross-referenced
• Of the 25 prioritized genes, 13 genes show signifi- cant cis eQTL in human livers; however, none showed significant co-localization with the GWAS signal

Functional Testing of Prioritized Genes

• We performed six functional tests on the 25 prioritized genes to determine their involvement in cholesterol metabolism
• Based on functional studies, we identified three genes (Aldoc, Sesn1, and Sigmar1) that showed significant response across all six functional tests performed (Figure 2D), indicating their functional involvement in cholesterol metabolism in mice and cells.

Identification and Prioritization of Sesn1

• Combining human lipid GWAS replication and functional testing results, the only common gene is SESN1 (Sestrin1) (Figure 2D). SESN1 belongs to the Sestrin protein family (SESN1, SESN2, and SESN3) and has never been implicated or reported to be involved in cholesterol metabolism. In human lipid GWAS data, SNPs within or nearby SESN1 are associated with at least one plasma lipid trait across all GWAS datasets we analyzed。
• Taken together, the association of SESN1 with plasma lipid traits is highly replicated across multiple independent human GWAS datasets.
• 全外显子组测序（WholeExomeSequencing，WES），是指利用序列捕获技术将全基因组外显子区域DNA捕获富集后进行高通量测序，能够直接发现与蛋白质功能变异相关的遗传变异，虽然外显子只占基因组的1%，但人类基因组的蛋白编码区大约包含85%的致病突变，测序深度更高，数据更有效，变异检测更准确！
• In summary, using the GLGC and UKBB exome datasets, we did not identify any low-frequency or rare exome SNPs within SESN1 associated with plasma TC.We also analyzed three publicly available liver eQTL datasets but did not identify a significant cis eQTL for SESN1 expression

Sesn1 Is Transcriptional Regulated by Cholesterol

• Based on our systematic stepwise approach, we pinpoint SESN1 as a candidate causal gene and then initiated biochemical studies focused on the role of SESN1 in cholesterol metabolism.
• . In mice, liver expression levels of Sesn1, but not the other Sestrin family members (Sesn2 and Sesn3), are repressed after 7 days of cholesterol feeding(胆固醇含量过高抑制了SESN1的表达)
• Transcriptional regulation of Sesn1 by cholesterol is characteristic of a SREBP2 target gene (Horton et al., 1998; Seo et al., 2011). Consistent with SREBP2 regulation, overexpression of a truncated constitutively active SREBP2 upregulated Sesn1 expression, whereas silencing or inhibition of SREBP2 blocked the response of Sesn1 to cellular cholesterol depletion
• Confirming our finding that Sesn1 transcription is regulated by SREBP2 in cells, Sesn1 has been found to contain a SREBP2 ChIP-seq peak (Table S4), supporting that Sesn1 is a SREBP2 target gene.

Sesn1 Is Required to Maintain Plasma Cholesterol Levels

• （基因敲除验证）When fed a cholesterol diet, both Sesn1
  /
  and Sesn1+/
  micehave higher levels of plasma TC than Sesn1+/+ mice at 4, 8,and 12 weeks。（基因表达与血浆脂质含量负相关，Our data show that Sesn1 is required to control adaptation during cholesterol feeding and after refeeding）

Sesn1 Functions Independent of Cholesterol Uptake or SREBP2 Signaling

• demonstrating that Sesn1 regulates plasma cholesterol levels independent of the LDLR. We next investigated SREBP2 signaling, which is a major transcriptional regulator of cholesterol metabolism
• silencing Sesn2 or Sesn3 genes did not alter cellular cholesterol levels (Figures S5D and S5E), demonstrating that Sesn1 is the only Sestrin protein member that affects cholestero
• Taken together, Sesn1 affects plasma and cellular cholesterol independent of DLR, SREBP2, and LXR.

Liver-Specific Function of Sesn1

• Furthermore, deficiency of Sesn1 in liver esulted in higher plasma cholesterol levels after refeeding (Figure 6B). Taken together, liver-specific deletion of Sesn1 impairs lasma cholesterol adaptation to cholesterol feeding and afterrefeeding.

Sesn1 Regulates Cholesterol Biosynthesis

• we treated mice ith lovastatin to inhibit Hmgcr activity and block cholesterolbiosynthesis. In this experiment, we observed a significant reduction in plasma cholesterol levels in cholesterol-fed Sesn1+/
  and Sesn1
  /
  mice but not in control mice (Sesn1+/+)
• Overall, the data demonstrate that the effect of Sesn1 on plasma cholesterol is through regulation of cholesterol biosynthesis in a liver-specific manner
• Altogether, our studies indicate Sesn1 functions to repress cholesterol biosynthesis likely through activating AMP kinase, which can phosphorylate Hmgcr and inhibit its activity

DISCUSSION

Here, we present an approach that integrates mouse liver coexpression network analysis with human plasma lipid GWAS data to identify causal genes that drive associations with plasma levels of TC, LDL cholesterol, HDL cholesterol, and TG