Positive selection of somatically mutated clones identifies adaptive pathways in metabolic liver disease

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Somatic mutations are common in most individuals, and mutation burden increases with age and chronic tissue damage.

“Somatic mutations are common in most individuals, and there is accumulating evidence that mutation burden increases with age and chronic tissue damage1-4.”

• cites [1] Martincorena, 2015, Somatic mutation in cancer and normal cells, Science sourceNot checked — no full text of the source
• cites [2] Brunner, 2019, Somatic mutations and clonal dynamics in healthy and cirrhotic human liver, Nature sourcePartly supported

  The Abstract reports somatic mutations in normal liver, a higher mutation burden in cirrhotic than normal liver, and a signature that accumulates with age. The study examined liver samples from 14 people, so it does not establish that somatic mutations are common in most individuals generally; its estimated age-related increase in overall mutation burden also had wide confidence intervals and was not statistically significant. (LLM, conf=0.85)

  Experimental — accuracy not yet measured
• cites [3] Zhu, 2019, Somatic mutations increase hepatic clonal fitness and regeneration in chronic liver disease, Cell sourcePartly supported

  The Introduction (page unavailable) says aging is associated with extensive somatic mutagenesis across multiple tissues. The Results and Discussion (page unavailable) report that mutation counts increase with fibrosis and damage in diseased liver. The copy does not establish that somatic mutations are common in most individuals or that chronic damage increases mutation burden across tissues generally; in this liver cohort, mutation count was not associated with age. (LLM, conf=0.84)

  Experimental — accuracy not yet measured
• cites [4] Lawson, 2020, Extensive heterogeneity in somatic mutation and selection in the human bladder, Science sourceNot checked — no full text of the source

May combine two claims.

The distribution of sgRNAs between plasmids/AAVs and chow/WD livers were very similar, suggesting most sgRNAs did not change in frequency and indicating a lack of genetic drift.

“We compared sgRNA distributions from different screening steps and found the distribution between plasmids/AAVs (the starting points) and chow/WD livers (the endpoints) were very similar (Figure S2E and Table S3), suggesting that most of the sgRNAs in the screen did not change in frequency, which is also true for other CRISPR screens14, and indicated a lack of genetic drift.”

• cites [14] Li, 2014, MAGeCK enables robust identification of essential genes from genome-scale CRISPR/Cas9 knockout screens, Genome Biol. sourcePartly supported

  In Materials and methods (The MAGeCK algorithm; page unavailable), the paper assumes that most sgRNAs have no effect on selection, but cautions that this may not hold in some scenarios. It does not establish that sgRNA frequencies remained stable in other CRISPR screens; the plasmid/AAV and liver comparison is the citing authors’ own finding, not a result attributed to this source. (LLM, conf=0.82)

  Experimental — accuracy not yet measured

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Dgat2 and Gpam are critical enzymes in triglyceride synthesis, and Dgat2 is currently being inhibited in NASH clinical trials.

“Dgat2 and Gpam are critical enzymes in triglyceride synthesis, and Dgat2 is currently being inhibited in NASH clinical trials15,16.”

• cites [15] Calle, 2021, ACC inhibitor alone or co-administered with a DGAT2 inhibitor in patients with non-alcoholic fatty liver disease: two parallel, placebo-controlled, randomized phase 2a trials, Nat. Med. sourceNot checked — no full text of the source
• cites [16] Yenilmez, 2022, An RNAi therapeutic targeting hepatic DGAT2 in a genetically obese mouse model of nonalcoholic steatohepatitis, Mol. Ther. sourcePartly supported

  The Introduction identifies DGAT2 as catalyzing the final step of triglyceride synthesis and reports human trials targeting it; the Discussion also mentions a phase 2 trial of a DGAT2 inhibitor (page numbers unavailable). Our copy does not establish Gpam’s role in triglyceride synthesis, so it supports only the DGAT2 portion of the claim. (LLM, conf=0.86)

  Experimental — accuracy not yet measured

May combine two claims.

Several genes were previously connected with fatty liver disease such as Nr3c1, Nr3c2, Zbtb16, Dnmt1, and Keap1, but most of the top genes had no connections with liver disease or NASH.

“Several genes were previously connected with fatty liver disease such as the glucocorticoid receptor Nr3c128,29, the mineralocorticoid receptor Nr3c230,31, the transcription factor Zbtb1632, the DNA methyltransferase Dnmt133, and the E3 ubiquitin ligase adaptor Keap134,35, but most of the top genes had no connections with liver disease or NASH.”

• cites [28] Wang, 2020, Hepatic miR-192-3p reactivation alleviates steatosis by targeting glucocorticoid receptor, JHEP Rep. sourceSupported
• cites [29] Moreira, 2014, Influence of the A3669G glucocorticoid receptor gene polymorphism on the metabolic profile of pediatric patients with congenital adrenal hyperplasia, Int. J. Endocrinol. sourceCould not verify

  The Results (§3.2) link an NR3C1 polymorphism to higher LDL cholesterol in pediatric patients with congenital adrenal hyperplasia. The cited paper does not address whether NR3C1 is connected with fatty liver disease or NASH; searches for fatty liver, hepatic steatosis, liver, and NASH found no passage establishing that connection. The other genes and the statement about most top genes are not attributed to this citation. (LLM, conf=0.77)

  Experimental — accuracy not yet measured
• cites [30] Pizarro, 2015, Beneficial effects of mineralocorticoid receptor blockade in experimental non-alcoholic steatohepatitis, Liver Int. sourceSupported
• cites [31] Wada, 2013, Eplerenone ameliorates the phenotypes of metabolic syndrome with NASH in liver-specific SREBP-1c Tg mice fed high-fat and high-fructose diet, Am. J. Physiol. Endocrinol. Metab. sourceNot checked — no full text of the source
• cites [32] Liška, 2017, Downregulation of plzf gene ameliorates metabolic and cardiac traits in the spontaneously hypertensive rat, Hypertension sourceNot checked — no full text of the source
• cites [33] Kutay, 2012, Reduced susceptibility of DNA methyltransferase 1 hypomorphic (Dnmt1N/+) mice to hepatic steatosis upon feeding liquid alcohol diet, PLoS One sourceSupported
• cites [34] Mohs, 2021, Hepatocyte-specific NRF2 activation controls fibrogenesis and carcinogenesis in steatohepatitis, J. Hepatol. sourceSupported
• cites [35] Ramadori, 2016, Hepatocyte-specific Keap1 deletion reduces liver steatosis but not inflammation during non-alcoholic steatohepatitis development, Free Radic. Biol. Med. sourceNot checked — no full text of the source

Probably not a substantial claim: may not pass S3 (load-bearing).

Mboat7 encodes a phospholipid synthetic enzyme identified through GWAS.

“Membrane-bound O-acyltransferase 7 (Mboat7) encodes a phospholipid synthetic enzyme identified through GWAS7,8.”

• cites [7] Buch, 2015, A genome-wide association study confirms PNPLA3 and identifies TM6SF2 and MBOAT7 as risk loci for alcohol-related cirrhosis, Nat. Genet. sourceNot checked — no full text of the source
• cites [8] Xia, 2021, Hepatic deletion of Mboat7 (LPIAT1) causes activation of SREBP-1c and fatty liver, J. Lipid Res. sourceSupported

May combine two claims.

Irs1 is an insulin receptor substrate and when lost, leads to suppressed lipogenesis due to reduced insulin signaling.

“Irs1 is an insulin receptor substrate and when lost, leads to suppressed lipogenesis due to reduced insulin signaling20-22.”

• cites [20] Sun, 1991, Structure of the insulin receptor substrate IRS-1 defines a unique signal transduction protein, Nature sourceNot checked — no full text of the source
• cites [21] Kubota, 2008, Dynamic functional relay between insulin receptor substrate 1 and 2 in hepatic insulin signaling during fasting and feeding, Cell Metab. sourcePartly supported

  The Results (page unavailable) show that liver-specific Irs1 loss reduced insulin-signaling markers and SREBP1c expression after refeeding. This supports the proposed link, but the paper does not establish suppressed lipogenesis itself, and the reported effect is specific to the liver under refeeding conditions rather than Irs1 loss generally. (LLM, conf=0.84)

  Experimental — accuracy not yet measured
• cites [22] Kubota, 2016, Differential hepatic distribution of insulin receptor substrates causes selective insulin resistance in diabetes and obesity, Nat. Commun. sourceSupported

May combine two claims.

Acvr2a and Gpam are two of the most recurrently mutated genes in cirrhotic tissues from alcoholic liver disease and NASH patients.

“Interestingly, Acvr2a and Gpam are two of the most recurrently mutated genes in cirrhotic tissues from alcoholic liver disease and NASH patients6.”

• cites [6] Ng, 2021, Convergent somatic mutations in metabolism genes in chronic liver disease, Nature sourceCould not verify

  No passage from our copy addresses whether ACVR2A and GPAM are among the most recurrently mutated genes in cirrhotic tissue from patients with alcoholic liver disease or NASH. Searches for both gene names, cirrhosis, and related descriptions returned only bibliographic material or no matching passage. (LLM, conf=0.68)

  Experimental — accuracy not yet measured

May combine two claims.

MOSAICS is distinct from previous Fah KO based regeneration screening systems because it is designed to assess a much higher density of mutant clones during homeostasis, and does not require rapid proliferation based selection of FAH expressing clones.

“MOSAICS is distinct from previous Fah KO based regeneration screening systems9,10 because it is designed to assess a much higher density of mutant clones during homeostasis, and does not require rapid proliferation based selection of FAH expressing clones.”

• cites [9] Wuestefeld, 2013, A direct in vivo RNAi screen identifies MKK4 as a key regulator of liver regeneration, Cell sourceNot checked — no full text of the source
• cites [10] Jia, 2022, In vivo CRISPR screening identifies BAZ2 chromatin remodelers as druggable regulators of mammalian liver regeneration, Cell Stem Cell sourceSupported

May combine two claims.

Bcl6 is a transcriptional repressor that is enriched in the fed state and cooperates with PPARα to suppress the induction of fasting transcription, and Bcl6 loss has been shown to increase insulin sensitivity and repress NASH.

“Bcl6 is a transcriptional repressor that is enriched in the fed state and cooperates with PPARα to suppress the induction of fasting transcription, and Bcl6 loss has been shown to increase insulin sensitivity and repress NASH36-38.”

• cites [36] Senagolage, 2018, Loss of transcriptional repression by BCL6 confers insulin sensitivity in the setting of obesity, Cell Rep. sourcePartly supported

  The Introduction identifies BCL6 as a transcriptional repressor, and the Results section on adipocyte BCL6 deficiency reports improved liver insulin sensitivity and reduced hepatic steatosis and inflammatory markers in Bcl6AKO mice. The copy does not establish that BCL6 is enriched in the fed state or cooperates with PPARα to suppress fasting transcription. It also does not report that BCL6 loss represses NASH specifically; reduced steatosis and inflammatory markers do not establish that broader claim. (LLM, conf=0.88)

  Experimental — accuracy not yet measured
• cites [37] Chikada, 2020, Liver-specific knockout of B cell lymphoma 6 suppresses progression of non-alcoholic steatohepatitis in mice, Sci. Rep. sourcePartly supported

  The Introduction identifies Bcl6 as a transcriptional repressor, and the Abstract reports that liver-specific Bcl6 knockout attenuated NASH-like changes in mice fed a CDAHFD diet. The Introduction says only that prior work found Bcl6 regulates insulin sensitivity; it does not state the direction of the effect. Our copy does not establish enrichment in the fed state or cooperation with PPARα to suppress fasting transcription. Page numbers were not available in the indexed passages. (LLM, conf=0.88)

  Experimental — accuracy not yet measured
• cites [38] Sommars, 2019, Dynamic repression by BCL6 controls the genome-wide liver response to fasting and steatosis, eLife sourcePartly supported

  The Abstract describes BCL6 as a fed-state repressor that converges with PPARα to suppress fasting transcription; the Discussion clarifies that they bind independently and BCL6 opposes PPARα rather than cooperating with it. The Results ('Bcl6 ablation enhances hepatic lipid catabolism and reduces steatosis'; page not provided) report only a trend toward enhanced insulin responsiveness and protection from steatosis in mice. They do not establish increased insulin sensitivity or repression of NASH. (LLM, conf=0.89)

  Experimental — accuracy not yet measured

Classified as probably not a substantial claim.

Transcription factor gene expression levels do not always reflect their functional importance.

“Since transcription factor gene expression levels do not always reflect their functional importance, we chose those with the highest or lowest activities based on the induction or suppression of their downstream targets (See methods for details)26,27.”

• cites [26] Subramanian, 2005, Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles, Proc. Natl. Acad. Sci. USA sourcePartly supported

  The Abstract says single-gene expression analysis can miss important pathway effects, and the Results identify gene sets associated with p53 function, including downstream targets. This supports looking beyond individual genes, but the paper does not directly compare transcription factors’ own expression levels with their functional importance. (LLM, conf=0.78)

  Experimental — accuracy not yet measured
• cites [27] Liberzon, 2011, Molecular signatures database (MSigDB) 3.0, Bioinformatics sourceCould not verify

  The Introduction discusses gene-expression signatures, and Results section 2 describes gene sets of transcription-factor targets, but neither says whether a transcription factor’s expression reflects its functional importance. Searches for the quoted claim and for activity inferred from downstream targets found no passage addressing that comparison. (LLM, conf=0.72)

  Experimental — accuracy not yet measured

Probably not a substantial claim: may not pass S3 (load-bearing).

Gender differences in the progression of NASH have also been observed in Bcl6 deficient mice.

“Gender differences in the progression of NASH have also been observed in Bcl6 deficient mice40.”

• cites [40] Nikkanen, 2022, An evolutionary trade-off between host immunity and metabolism drives fatty liver in male mice, Science sourcePartly supported

  The passage on hepatic Bcl6 deletion (section labeled Abstract; page unavailable) reports that deleting Bcl6 reduced fatty-liver features in male mice and reduced hepatic triglycerides more subtly in females. That supports a sex difference in fatty-liver outcomes, but the paper does not establish a sex difference in NASH progression in Bcl6-deficient mice. (LLM, conf=0.88)

  Experimental — accuracy not yet measured

Probably not a substantial claim: may not pass S3 (load-bearing).

Some of the most important human metabolic disease genes have been identified using germline genetic methods such as GWAS and whole exome sequencing.

“Some of the most important human metabolic disease genes have been identified using germline genetic methods such as GWAS and whole exome sequencing42-44.”

• cites [42] Romeo, 2008, Genetic variation in PNPLA3 confers susceptibility to nonalcoholic fatty liver disease, Nat. Genet. sourceSupported
• cites [43] Abul-Husn, 2018, A protein-truncating HSD17B13 variant and protection from chronic liver disease, N. Engl. J. Med. sourceSupported
• cites [44] Cohen, 2006, Sequence variations in PCSK9, low LDL, and protection against coronary heart disease, N. Engl. J. Med. sourcePartly supported

  The Methods and Results describe a study of inherited PCSK9 variants associated with lower LDL cholesterol and reduced coronary heart disease risk. The Genotyping section specifies targeted TaqMan assays for known variants; the paper does not say that PCSK9 was identified by GWAS or whole-exome sequencing. (LLM, conf=0.86)

  Experimental — accuracy not yet measured

May combine two claims.

AZ505 is a selective SMYD2 inhibitor with an IC50 of 0.12 μM and greater than 600-fold selectivity.

“We used AZ505, a selective SMYD2 inhibitor with an IC50 of 0.12 μM41 (>600 fold selectivity).”

• cites [41] Ferguson, 2011, Structural basis of substrate methylation and inhibition of SMYD2, Structure sourcePartly supported

  The HTS Assay and Hit Validation section reports AZ505’s IC50 as 0.12 (rendered as “mM” in our copy), and the Cofactor Binding Site section reports no potency against SMYD3. The paper describes a selectivity profile, but the passages do not establish the claimed greater-than-600-fold figure. (LLM, conf=0.85)

  Experimental — accuracy not yet measured

Probably not a substantial claim: may not pass S3 (load-bearing).

A similar linear regression-based model was previously proposed to predict transcription factor regulatory activities and motifs from yeast gene expression data.

“A similar linear regression-based model was previously proposed to predict transcription factor regulatory activities and motifs from yeast gene expression data56.”

• cites [56] Conlon, 2003, Integrating regulatory motif discovery and genome-wide expression analysis, Proc. Natl. Acad. Sci. USA sourceSupported

May combine two claims.

The somatic mutations from human liver tissues were the most positively selected in mouse models of fatty liver, but were not selected for in the absence of disease.

“The somatic mutations from human liver tissues were also the most positively selected in mouse models of fatty liver, but were not selected for in the absence of disease.”

May combine two claims.

H&E showed macroscopic lipid droplets in pericentral hepatocytes of WD fed Mboat7 KO livers, while only microscopic lipid droplets were observed in WD fed WT livers.

“H&E showed macroscopic lipid droplets in pericentral hepatocytes of WD fed Mboat7 KO livers, while only microscopic lipid droplets were observed in WD fed WT livers (Figure 1B).”

May combine two claims.

After 6 months, control sgRNAs in WD fed livers decreased in abundance compared to chow fed livers, whereas each of the guides against Acvr2a, Irs1, Srebf1, and Dgat2 were positively selected.

“After 6 months, the control sgRNAs in the WD fed livers decreased in abundance compared to those in chow fed livers, whereas each of the guides against Acvr2a, Irs1, Srebf1, and Dgat2 were positively selected (Figure 3F and Table S2).”

May combine two claims.

In the context of WD, Mboat7 deficiency led to increased transaminitis, liver lipids, and lipidemia.

“In the context of WD, Mboat7 deficiency also led to increased transaminitis (Figure 1C,D), liver lipids (Figure 1E,F), and lipidemia (Figure S1E,F).”

May combine two claims.

Whole-liver Gpam KO mice showed a trend toward decreased liver/body weight ratios, decreased steatosis, and a trend toward improved transaminitis after 3 months of WD.

“KO mice showed a trend toward decreased liver/body weight ratios (Figure 4E), decreased steatosis (Figure 4F), and a trend toward improved transaminitis (Figure S3F,G).”

May combine two claims.

After 4 months of tracing with chow diets, Tomato+ clones increased in both Mboat7+/+ and Mboat7f/f mice similarly, indicating a neutral effect of Mboat7 loss under non-NASH conditions.

“After 4 months of tracing with chow diets, Tomato+ clones increased in both Mboat7+/+ and Mboat7f/f mice similarly (Figure 1I,J middle column), indicating a neutral effect of Mboat7 loss in the liver under non-NASH conditions.”

May combine two claims.

KO livers had reduced macrovesicular steatosis, microvesicular steatosis, and hepatocyte hypertrophy.

“This showed that KO livers had reduced macrovesicular steatosis, microvesicular steatosis, and hepatocyte hypertrophy (Figure 4G).”

May combine two claims.

The somatic mutations did not cause rapid cancer development, as no gross liver tumors were detected.

“we did not detect gross liver tumors, suggesting that the somatic mutations did not cause rapid cancer development.”

May combine two claims.

After 4 months of tracing with WD, there was a modest decrease in Tomato+ cells in Mboat7+/+ mice, but a more substantial decrease in Mboat7f/f mice.

“After 4 months of tracing with WD, we observed a modest decrease in the percentage of Tomato+ cells in Mboat7+/+ mice, but a more substantial decrease in Mboat7f/f mice (Figure 1I,J right column).”

The quote only partly states this claim.

After 6 months of WD, there was an increase in Gpam KO clones compared to WT.

“we observed an increase in Gpam KO clones in the setting of WD (Figure 4C,D, right column).”

May combine two claims.

Increasing amounts of MOSAICS AAV could delete Pten or activate Tomato in an increasing number of hepatocytes.

“Increasing amounts of AAV could delete Pten or activate Tomato in an increasing number of hepatocytes (Figure 2C-E), showing that this system could be titrated to generate either mosaicism or liver-wide gene deletion.”

May combine two claims.

In WT livers, some apoptotic hepatocytes were filled with macroscopic lipid droplets, whereas in KO livers, apoptotic hepatocytes did not have these characteristics.

“In WT livers, some apoptotic hepatocytes were filled with macroscopic lipid droplets, whereas in KO livers, the apoptotic hepatocytes did not have these characteristics (Figure S3L).”

May combine two claims.

Liver triglyceride but not cholesterol levels were decreased in the WD fed Gpam KO group.

“Liver triglyceride but not cholesterol levels were decreased in the WD fed KO group (Figure 4H,I), consistent with Gpam’s role as a rate limiting enzyme in triglyceride synthesis.”

• AssumptionRests on an assumption: GPAM catalyzes the acylation of glycerol-3-phosphate with acyl-coenzyme A to generate CoA and lysophosphatidic acid, the rate-limiting step in triacylglycerol synthesis.

May combine two claims.

Plasma cholesterol but not triglyceride was decreased in the Gpam KO group.

“Interestingly, plasma cholesterol but not triglyceride was decreased in the KO group (Figure S3H,I).”

May combine two claims.

Proliferating hepatocytes in WT livers were more likely to be found near portal triads, whereas in KO livers, proliferating hepatocytes were more evenly distributed between portal and central regions, suggesting that steatotic hepatocytes were less likely to divide.

“However, proliferating hepatocytes in WT livers were more likely to be found near portal triads, where steatosis was less pronounced, whereas in KO livers, proliferating hepatocytes were more evenly distributed between portal and central regions (Figure 4K,L), suggesting that steatotic hepatocytes were less likely to divide.”

May combine two claims.

No improvements for ALT and AST were seen in female Tbx3 KO mice, but modest improvements were seen in Smyd2 KO mice.

“We did not see improvements for ALT and AST in Tbx3 KO mice but did see modest improvements in Smyd2 KO mice (Figure S8G).”

May combine two claims.

Protection against fatty liver disease in these KO mice was evident from improved plasma lipids, decreased liver lipid content, and reduced steatosis.

“Furthermore, the protection against fatty liver disease was evident from improved plasma lipids, decreased liver lipid content (Figure 5G-I and Figure S6D-F), and reduced steatosis (Figure 5J and Figure S7A,B).”

May combine two claims. The quote only partly states this claim.

A trend towards decreased liver/body weight ratios was observed in Tbx3 KO livers and a significant decrease in Smyd2 KO livers in females.

“We observed a trend towards decreased liver/body weight ratios in Tbx3 KO livers and a significant decrease in Smyd2 KO livers (Figure S8F).”

May combine two claims.

RNA-seq of control and KO livers showed massive expression changes in Irs1 KO livers and fewer changes in Tbx3 and Bcl6 KO livers.

“We observed massive expression changes in Irs1 KO livers and fewer changes in Tbx3 and Bcl6 KO livers (Figure 6A)”

May combine two claims.

Significant decreases of ALT and AST were observed in male Tbx3 KO mice, suggesting reduced liver damage.

“Significant decreases of ALT and AST were also observed (Figure S8B), suggesting reduced liver damage.”

May combine two claims.

In male Smyd2 KO mice, liver/body weight ratios decreased, but liver and body weights were unchanged, and ALT/AST levels were unchanged.

“In male Smyd2 KO mice, liver/body weight ratios decreased, but liver and body weights were unchanged (Figure S8A), and ALT/AST levels were unchanged (Figure S8B).”

May combine two claims.

Reduced plasma cholesterol was observed in Smyd2 KO mice and increased plasma triglyceride in Tbx3 KO mice, while other KO models showed modest or no changes.

“We also observed reduced plasma cholesterol in Smyd2 KO mice and increased plasma triglyceride in Tbx3 KO mice, while the other KO models showed modest or no changes in these parameters (Figure S6E,F).”

May combine two claims.

Srebf1 KO livers showed fewer gene expression changes compared with Irs1, Tbx3, and Bcl6 KO livers, and Smyd2 KO livers showed the smallest number of expression changes.

“Srebf1 KO livers showed fewer gene expression changes compared with Irs1, Tbx3, and Bcl6 KO livers; Smyd2 KO livers showed the smallest number of expression changes (Figure 6A).”

May combine two claims.

In male Tbx3 KO mice, liver weight and liver/body weight ratios decreased, while body weights were unchanged.

“In male Tbx3 KO mice, liver weight and liver/body weight ratios decreased, while body weights were unchanged (Figure S8A).”

May combine two claims.

There were no improvements for fibrosis in female Tbx3 KO livers but a trend of decreased fibrosis in Smyd2 KO livers.

“There were no improvements for fibrosis in Tbx3 KO livers but a trend of decreased fibrosis in Smyd2 KO livers (Figure S8I,J).”

May combine two claims.

While Tbx3 and Smyd2 KO mice had reduced liver cholesterol, Bcl6 KO mice had increased liver cholesterol.

“While Tbx3 and Smyd2 KO mice had reduced liver cholesterol, Bcl6 KO mice had increased liver cholesterol (Figure 5I).”

May combine two claims.

In plasma, both Tbx3 and Smyd2 KOs showed moderate increases in triglyceride but cholesterol levels remained similar to controls.

“In the plasma, both Tbx3 and Smyd2 KOs showed moderate increases in triglyceride but cholesterol levels remained similar to controls (Figure S8C).”

May combine two claims.

Among all 20 KO mice associated with the transcription and epigenetic factor screens, more than half showed significant reductions in liver mass and none showed significant increases.

“Among all 20 KO mice associated with the transcription and epigenetic factor screens (Table S1), more than half of the KO models showed significant reductions in liver mass and none showed significant increases (Figure S6C).”

May combine two claims.

These three KO models showed the most significant reductions in liver injury as measured by ALT, a trend toward reduced AST, and the most significant reductions in liver triglyceride levels.

“These three KO models showed the most significant reductions in liver injury as measured by ALT (Figure 5G), a trend toward reduced AST (Figure S6D), and the most significant reductions in liver triglyceride levels (Figure 5H).”

May combine two claims.

Plasma triglyceride levels were increased in female Tbx3 KO but not Smyd2 KO mice, while cholesterol in both KO cohorts was unaffected.

“Plasma triglyceride levels were increased in Tbx3 KO but not in Smyd2 KO mice, while cholesterol in both KO cohorts were unaffected (Figure S8H).”

May combine two claims.

Deletion of Tbx3 and Bcl6, as well as Smyd2, showed significant reductions in body weights and the most substantial reductions in liver/body weight ratios compared to controls after 3 months of WD.

“Among all KO models, deletion of the transcription factors Tbx3 and Bcl6, as well as the epigenetic factor Smyd2, showed significant reductions in body weights (Figure 5E) and the most substantial reductions in liver/body weight ratios compared to controls after 3 months of WD (dark dots in Figure 5F).”

May combine two claims.

In WD-fed WT mice, two months of AZ505 treatment significantly reduced body weight, liver weight, and liver/body weight ratios compared with vehicle treatment.

“we observed significant reductions in body weight, liver weight, and liver/body weight ratios in AZ505 vs. vehicle treated mice (Figure 7B-D)”

May combine two claims.

AZ505-treated WD mice had smaller, less pale livers and decreased steatosis on histology compared with vehicle-treated livers.

“Smaller, less pale livers were observed in AZ505 treated mice, and liver histology showed decreased steatosis in contrast to a wide range of macro and microscopic lipid droplet deposition in vehicle treated livers (Figure 7E).”

May combine two claims.

In chow-fed mice, AZ505 treatment caused no changes in liver histology, AST/ALT, or plasma lipids.

“No changes were observed in liver histology (Figure 7F), AST/ALT (Figure 7G,H), or plasma lipids (Figure 7I,J).”

May combine two claims.

These mutations mitigated lipotoxic phenotypes, thereby increasing the survival of hepatocyte clones.

“Mechanistically, these mutations mitigated lipotoxic phenotypes, thereby increasing the survival of hepatocyte clones.”

Probably not a substantial claim: may not pass S3 (load-bearing).

SgRNA distributions were largely maintained before and after screening.

“SgRNA distributions were largely maintained before and after screening (Figure S5G,H and Table S3).”

May combine two claims.

AAV8-TBG-Cre efficiently deleted the floxed exon and depleted intact Mboat7 mRNA.

“We observed efficient deletion of the floxed exon and depletion of the intact Mboat7 mRNA (Figure S1A,B) after AAV8-TBG-Cre.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Srebf1 is a major transcriptional regulator of lipogenesis.

“Srebf1 is a major transcriptional regulator of lipogenesis17-19.”

• cites [17] Wang, 1993, Nuclear protein that binds sterol regulatory element of low density lipoprotein receptor promoter. II. Purification and characterization, J. Biol. Chem. sourceNot checked — no full text of the source
• cites [18] Briggs, 1993, Nuclear protein that binds sterol regulatory element of low density lipoprotein receptor promoter. I. Identification of the protein and delineation of its target nucleotide sequence, J. Biol. Chem. sourceNot checked — no full text of the source
• cites [19] Brown, 1997, The SREBP pathway: regulation of cholesterol metabolism by proteolysis of a membrane-bound transcription factor, Cell sourceNot checked — no full text of the source

Classified as probably not a substantial claim. May combine two claims.

Seven to eight independent mice were used for each group, and results from individual mice were consistent.

“Seven to eight independent mice were used for each group, and the results from individual mice were consistent (Table S2).”

May combine two claims.

CRISPR liver-wide conditional deletion was an effective and rapid method of organ level validation.

“This showed that CRISPR liver-wide conditional deletion was an effective and rapid method of organ level validation.”

May combine two claims.

The phenotypes of Tbx3, Bcl6, and Smyd2 KO mice were comparable to Srebf1 and Dgat2 KO mice, suggesting potent regulation of lipid metabolism by these three genes.

“The phenotypes of Tbx3, Bcl6, and Smyd2 KO mice were comparable to Srebf1 and Dgat2 KO mice, suggesting potent regulation of lipid metabolism by these three genes.”

May combine two claims.

The study was underpowered to detect statistical differences in fibrosis in KOs vs. controls, but trends toward decreased fibrosis were observed in both KO models.

“Although the study was underpowered to detect statistical differences in fibrosis in KOs vs. controls, we observed trends toward decreased fibrosis in both KO models (Figure S8D,E).”

May combine two claims.

Both KO models protect the liver from steatosis, but male Tbx3 KO had more pronounced effects under long-term WD.

“These data showed that both KO models protect the liver from steatosis, but male Tbx3 KO had more pronounced effects under long-term WD (Figure S8D).”

Probably not a substantial claim: may not pass S3 (load-bearing).

The increase in cholesterol synthesis genes in Srebf1 KO livers was potentially due to a compensatory effect on cholesterol production.

“this increase was potentially due to a compensatory effect on cholesterol production”

• AssumptionRests on an assumption: Srebpl is a master transcription factor for cholesterol synthesis.

May combine two claims.

Multiple lines of evidence suggest that these mutations do not promote cancer since many of these genes were not identified in large cancer sequencing efforts, and in vivo experiments did not reveal increased tumorigenesis.

“multiple lines of evidence suggest that these mutations do not promote cancer since many of these genes were not identified in large cancer sequencing efforts, and our in vivo experiments did not reveal increased tumorigenesis.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Image analysis for quantification was blinded.

“Image analysis for quantification was blinded.”

Probably not a substantial claim: may not pass S4 (based).

Because adaptive mutations are rare and clonal expansion takes decades, it is likely that beneficial clones cannot expand enough to prevent or reverse NASH development within the lifetime of the patient.

“Because adaptive mutations are rare and clonal expansion takes decades, it is likely that beneficial clones cannot expand enough to prevent or reverse NASH development within the lifetime of the patient.”

• AssumptionRests on an assumption: Adaptive mutations are rare and clonal expansion takes decades.

Probably not a substantial claim: may not pass S3 (load-bearing).

All experiments were done in an age and sex controlled fashion unless otherwise noted.

“All experiments were done in an age and sex controlled fashion unless otherwise noted.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Some of the genes identified in this study, when deleted or inhibited, may not cause improvements in all metabolic parameters associated with fatty liver disease.

“Some of the genes identified in this study, when deleted or inhibited, may not cause improvements in all metabolic parameters associated with fatty liver disease.”

Probably not a substantial claim: may not pass S3 (load-bearing).

All mice used in this study were male except for those in Figure S8F-J.

“All mice used in this study were male except for those in Figure S8F-J.”

Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

Some of the hits may not have an impact on late stage NASH features.

“some of the hits may not have an impact on late stage NASH features.”

Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

In humans, these fitness improvements are enough to drive measurable levels of positive selection over decades.

“In humans, these fitness improvements are enough to drive measurable levels of positive selection over decades.”

May combine two claims.

Somatic mosaicism will likely reveal adaptive mechanisms and potential therapeutic targets.

“Nevertheless, somatic mosaicism will likely reveal adaptive mechanisms and potential therapeutic targets.”

Probably not a substantial claim: may not pass S3 (load-bearing).

The data in most panels reflect multiple experiments performed on different days using mice derived from different litters.

“The data in most panels reflect multiple experiments performed on different days using mice derived from different litters.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Each sgRNA maintained a greater than 1000-fold representation during MOSAICS library construction.

“Each sgRNA maintained a >1000-fold representation during construction.”

Probably not a substantial claim: may not pass S3 (load-bearing).

The ultimate clinical significance of the genes identified is unknown.

“the ultimate clinical significance of the genes identified is unknown.”

Probably not a substantial claim: may not pass S4 (based).

Germline mutations are limited in the amount of genetic space that can be explored because lethal developmental phenotypes select against mutations that could have a benefit in specific tissues later in life.

“germline mutations are limited in the amount of genetic space that can be explored because lethal developmental phenotypes select against mutations that could have a benefit in specific tissues later in life.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

The PCR cycle number for amplicon library preparation was pre-optimized using the same PCR reactions with a smaller volume, and cycle numbers giving a weak but sharp band on the DNA gel were used.

“The PCR cycle number was pre-optimized using the same PCR reactions with a smaller volume.”

• Also stated in: § METHOD DETAILS · plain

  “The cycle numbers that gave a weak but sharp band on the DNA gel were used.”

May combine two claims.

The MOSAICS technology allows for in vivo screening with much greater clone density, and in a low-proliferation setting, in contrast to the Fah KO screening model.

“The advance associated with this technology is that it allows for in vivo screening with a much greater clone density, and in a low-proliferation setting, in contrast to the Fah KO screening model.”

• AssumptionRests on an assumption: Metabolic disease has not identified a facile method of gene discovery using phenotypic screens, in vitro or in vivo.
• AssumptionRests on an assumption: It is difficult to phenotype many genetic strains of mice or humans, but it is much easier to phenotype thousands of genetically distinct and competing cells.