Introduction

There is an urgent and unmet need for novel therapeutic approaches of acetaminophen-induced liver injury (AILI) in current clinical practice. We recently has revealed the beneficial role of hepatocyte-specific Mas activation dependent fatty acid oxidation in mouse AILI^[1], which is the first study of Mas signaling in AILI and also highlighted by the editorial of Journal of Hepatology^[2]. However, considering the broad expression pattern of Mas, it is important to investigate whether targeting other cell types could also have protective effects against AILI, which has important implications for the efficacy and safety of future drug development.

Aims & Methods

Multiple cell-specific Mas1-knockout mice (LysM^creMas1^f/f, Alb^creMas1^f/f, Cdh5^creMas1^f/f, Lrat^creMas1^f/f and Cd4^creMas1^f/f mice) were used for in vivo analyses. Single-cell RNA sequencing (scRNA-seq), spatial transcriptomics (ST) and intravital imaging were used to reveal cellular interactions in complex pathophysiological microenvironments. Cdh5^crePkm^f/+mice, metabolic flux analysis, pharmacological compounds, genetic tools, cell co-culture and human liver samples were used to validate the discoveries of bioinformatics analysis.

Results

By generating multiple cell-specific Mas1-knockout mice and using multi-omic analysis, we have further identified the myeloid-Mas signaling as the critical driver of immunometabolic landscape in the progression of AILI. Notably, the AILI-imprinted immunometabolic landscape, consisting of CD31⁺MYC⁺CD63⁺ endothelial cells (ECs), F4/80⁺MMP12⁺ macrophages (Mψ) and monocytes, which is firstly described and visualized by the hour-long intravital imaging in living mice. By incorporating the paradigm with a computational imaging framework, a novel intravital microscopy that fulfills the unmet requirement for hour-long, high-speed three-dimension (3D) subcellular observations in vivo^[3], we demonstrated that evaluating the behaviors of immune cells in situ is an effective way to reveal complicated immune processes and understand disease pathogenesis. The deficiency of myeloid-Mas could cause a significant up-regulation of TNF and NF-κB signaling in monocytes, and the subsequent release of TNF-α to induce the pro-inflammatory phenotype of ECs possibly through metabolic reprogramming. Human blood monocytes (CD14⁺ cells) were isolated to verify the clinical relevance. Moreover, MYC⁺ ECs could produce abundant glycolytic metabolites to enhance glycolysis in adjacent Mψ and regulate their function. Glycolysis was demonstrated to be the key metabolic pathway for intercellular interactions. Importantly, the myeloid-Mas driven landscape established in mouse AILI, was perfectly mapped and enriched in human AILI, whose landscape also consists of three cell counterparts including MYC⁺ ECs, MMP12⁺ Mψ and CD14⁺ monocytes. Then, the k-means algorithm was applied to conduct an unsupervised clustering, suggesting the probability of 83% to identify advanced AILI by the microenvironmental characteristic.

Conclusion

Our findings offer a new insight into Mas-targeted therapeutic strategies against AILI, which should be designed based on the specific cellular and metabolic pathway. Our data show a great potential for the clinical transformation of “disease-imprinted microenvironment” into diagnostic and severity stratified models through artificial intelligence in the future. By combining scRNA-seq, ST and intravital imaging, our work provides a comprehensive paradigm for studying mammalian cellular interactions in complex pathophysiological microenvironments.

References

[1] Chen S, Lu Z, Jia H, et al. Hepatocyte-specific Mas activation enhances lipophagy and fatty acid oxidation to protect against acetaminophen-induced hepatotoxicity in mice. Journal of Hepatology, 2023, 78(3): 543-557.
[2] Nogueiras R, Aspichueta P. Expanding the roles of the renin–angiotensin system: drug-induced liver injury. Journal of Hepatology, 2023, 78(3): 457-459.
[3] Wu J, Lu Z, Jiang D, et al. Iterative tomography with digital adaptive optics permits hour-long intravital observation of 3D subcellular dynamics at millisecond scale. Cell, 2021, 184(12): 3318-3332. e17.

Introduction

Rare early onset cases of diabetes stem from single gene mutations, whereas frequent gene variants can also contribute to multifactorial diabetes, indicating shared disease mechanisms. Our recent study has identified ONECUT1 as a gene implicated in both monogenic and multifactorial diabetes, establishing it as crucial target for investigating fundamental signaling pathways in diabetes pathology [1,2]. However, numerous diabetic characteristics remain unexplained by typical genetic alterations, highlighting the underestimated significance of the non-coding genome. We have identified a neonatal diabetes patient carrying a heterozygous ONECUT1 truncating variant. Remarkably, the patient exhibited a phenotype akin to individuals with homozygous ONECUT1 truncating mutations. Upon further examination, we detected an additional heterozygous deletion in the non-coding genome, leading us to hypothesize that it could impact the regulation of the remaining ONECUT1 allele. Consequently, the dysregulation of the ONECUT1-dependent transcriptional machinery crucial for proper beta-cell development occurs, ultimately leading to the severe diabetic phenotype observed in the patient.

Aims & Methods

Following deletion of specific genomic regions in human embryonic stem cells, we initiated their differentiation into the pancreatic endocrine lineage and meticulously characterized the developmental stages.

Results

Importantly, we successfully replicated the patient’s diabetic phenotype, elucidating deficiencies in both pancreatic development and beta-cell functionality. The differentiation of pancreatic progenitors was notably hindered by the loss of the regulatory region associated with nearly complete ablation of ONECUT1. Moreover, the stem cell-derived islets exhibited a reduced number of beta-cells as well as impaired insulin secretion, even in heterozygous knockouts.

Conclusion

Altogether, our ongoing comprehensive mechanistic characterization provides insights into the precise role of the identified regulatory element in pancreatic beta-cell development and diabetes pathophysiology, emphasizing the significance of the non-coding genome. By unveiling unknown molecular pathomechanisms linked to alterations in regulatory sequences, we contribute to a deeper understanding of the pancreatic transcriptional network and establish the foundation for personalized diabetes treatment.

References

[1] Philippi, A., Heller, S., Costa, I.G. et al. Mutations and variants of ONECUT1 in diabetes. Nat Med 27, 1928–1940 (2021). https://doi.org/10.1038/s41591-021-01502-7
[2] Heller, S., Li, Z., Lin, Q. et al. Transcriptional changes and the role of ONECUT1 in hPSC pancreatic differentiation. Commun Biol 4, 1298 (2021). https://doi.org/10.1038/s42003-021-02818-3

Introduction

Gastroparesis is a severe medical condition characterized by delayed gastric emptying and symptoms of nausea, vomiting, bloating, fullness after meals, and abdominal pain. Thus far, little is known about the underlying genetic risk factors for nausea in gastroparesis patients. We explored the association of genetic variants with nausea severity in a set of WGS data from a clinical gastroparesis study.

Aims & Methods

Blood samples were obtained from adults with a diagnosis of diabetic or idiopathic gastroparesis, having evidence of delayed gastric emptying, and moderate to severe nausea, daily average nausea score of >2.5, at least one episode of vomiting, PAGI-SYM nausea score of ≥2 at screening, and controlled blood glucose levels. WGS was conducted with 30x read depth using whole blood samples. Nausea score was assessed from a daily symptom dairy with a range from 0 mild to 5 severe. GWAS using linear model was conducted on variants with MAF ≥0.01 with adjustment of the subject’s age, sex, BMI, etiology, and the first 3 PCs.

Results

Variant clusters from multiple genes are significantly associated with baseline nausea score at p=1e-5 level. Among the top scoring variants are loci within ATF3, PP2D1, CNTN4, BMP2, ABL1, CTNNA3 and KIF6. Interestingly, upon comparing our results with a published transcriptomic analysis that included both diabetic and idiopathic gastroparesis subjects, we found that ATF3 and BMP2 were overlapping with genes differentially expressed in both diabetic and idiopathic gastroparesis when compared with controls. ABL1 was however differentially expressed in diabetic cases whereas CNTN4 was differentially expressed in idiopathic controls.
We expanded the analysis with single gene tests which shows that ATF3 is significant especially for MAF>0.05 variants (SKAT p=0.02). The top variant in ATF3 is rs79151334 (beta= 0.68, p=5.76e-6), whose minor allele associates with higher nausea score.

Conclusion

Additionally, we report new potential associations CTNNA3, PP2D1, and KIF6 as well as a cluster of variants between CDH2 and MIR302F, that associated with baseline nausea score. Among those, both CTNNA3 and CDH2 encode cadherin related proteins, suggesting this type of junction could play a role in the underlying mechanism.

Disclosure

I am an employee at Vanda Pharmaceuticals Inc.

Introduction

Individuals with a germline CDKN2A pathogenic variant (PV) have an increased lifetime risk of up to 70% for melanoma and up to 20% for pancreatic cancer, and are recommended to undergo skin and pancreatic cancer surveillance. Little is known about the psychosocial issues associated with this hereditary cancer syndrome.

Aims & Methods

This cross-sectional study included confirmed CDKN2A PV carriers and those with a 50% risk of the PV (at-risk carriers) under cancer surveillance. Individuals ≥18 years were identified from the skin and pancreatic cancer surveillance programs at the Leiden University Medical Center and invited for a one-time questionnaire with study-specific questions ​concerning genetic testing, family planning, and preimplantation genetic testing (PGT).

Results

A total of 537 individuals were screened for eligibility, of whom 208 of 366 (57%) confirmed carriers (56% female, median age 54 years [IQR 46-63]) and 39 of 171 (23%) at-risk carriers (59% female, median age of 26 years [IQR 22-32]) participated in the study. Primary motivations for genetic testing were to gain control over their personal and children's cancer risk, as well as increasing cancer surveillance practices. In contrast, concerns about obtaining a mortgage and life insurance were frequently cited as reasons for postponing genetic testing. Family planning decisions remained largely unaffected in both confirmed and at-risk carriers; however, the majority of confirmed carriers were still unaware of their familial or personal cancer risk when starting a family. More than 60% of the participants were unfamiliar with PGT and only a minority (19% of confirmed carriers and 10% of at-risk carriers) would be open to considering PGT as a reproductive option (Table 1).

Table 1. Attitudes toward PGT

Conclusion

This study found different attitudes toward genetic testing, family planning, and PGT among individuals affected by the CDKN2A PV. Understanding these different attitudes can help clinicians to address the complexities surrounding these issues, especially for younger individuals facing difficult decisions about the timing of genetic testing, family planning, and the potential use of reproductive assisted options.

Introduction

Non-alcoholic fatty liver disease (NAFLD) is a widespread clinical condition that affects about 1/4th adults globally. It can lead to liver fibrosis, cirrhosis, and hepatocellular carcinoma (HCC). More than one-third of Egyptians are affected by NAFLD. Unfortunately, many patients diagnosed with NAFLD after cirrhosis has already occurred.
Single Nucleotide Polymorphisms (SNPs) associated with NAFLD have been proposed to identify the causal variants of the disease and also as potential pharmacological targets. Heritability of NAFLD estimates range from 20% to 70% based on ethnicity, and environmental factors. The first gene variant found to be a consistent genetic risk factor for NAFLD was Patatin-like phospholipase domain containing 3 (PNPLA3) rs738409. In addition, other studies have shown that, Transmembrane 6 superfamily 2 (TM6SF2) rs58542926 and Hydroxysteroid 17-beta dehydrogenase 13 (HSD17B13) rs9992651 also affect risk of NAFLD developmen. Some studies also show a role for Glucokinase regulatory protein (GCKR) rs1260326.

Aims & Methods

We aimed to investigate genetic risk factors for NAFLD occurrence in Egyptian patients.
Cases of clinically diagnosed NAFLD (n= 205) and controls (n= 187) were recruited. A subgroup of individuals with NAFLD that had progressed to liver fibrosis (n= 131) was compared with the control individuals. TaqMan-based genotyping assays were employed to explore the association of selected SNPs (PNPLA3 rs738409, TM6SF2 rs58542926, HSD17B13 rs9992651 and GCKR rs1260326) with NAFLD in Egyptian patients.

Results

NAFLD cases versus controls genotype data: rs738409 -genotype showed statistical significance. Univariate analysis for TM6SF2 rs58542926, HSD17B13 rs9992651, and GCKR rs1260326 did not show statistical significance.
After adjusting for the other risk factors -age, gender, BMI, and waist circumference, the association of PNPLA3 rs738409 -genotype, and allele models- with the variant G allele more common in NAFLD remained statistically significant with an increased odds ratio. PNPLA3 rs738409 GG carriers have 5.296 times NAFLD risk than CC carriers. For GCKR rs1260326 genotype model, after correction for the other risk factors, the variant T allele acquired statistical significance.
Fibrotic-NAFLD cases versus controls genotype data: Univariate analysis for PNPLA3 rs738409 showed statistical significance. The univariate analysis for TM6SF2 rs58542926, HSD17B13 rs9992651, and GCKR rs1260326 did not show statistical significance.
After adjusting for the other risk factors -age, gender, BMI, and waist circumference, the association of PNPLA3 rs738409 -genotype, and allele models remained statistically significance with an increased odds ratio. Of note, PNPLA3 rs738409 GG carriers have 9.267 times higher risk of Fibrotic-NAFLD than CC carriers.The association of HSD17B13 rs9992651 -genotype model-with the variant A allele protective against fibrosis acquired statistical significance and decreased odds ratio. Notably, HSD17B13 rs9992651 GA carriers have a fibrosis risk 0.487 times less than GG carriers. The association of GCKR rs1260326 -genotype model, after correction for the other risk factors acquired statistical significance and an increased odds ratio.

Conclusion

We discovered that in our study population, PNPLA3 rs738409 and GCKR rs1260326 variants increase the risk of NAFLD generally as well as its progression to fibrosis. On the other hand, the HSD17B13 rs9992651 variant may protect against fibrosis development in NAFLD.
Nehal Elmansoury shares the first authorship with Ahmed Kamal.

Introduction

Inflammatory bowel disease (IBD), consisting of Crohn’s disease (CD) and Ulcerative colitis (UC), is a group of chronic, relapsing disorders in the gastrointestinal tract. The disease mechanisms are largely unknown. We hypothesized that the interaction of genetic variants and tobacco smoking can influence the disease risk of IBD and impact the disease progression in CD and UC differently.

Aims & Methods

3530 IBD patients were included in the present study via Parelsnoer IBD Biobank (PSI), a Dutch prospective, nationwide biobank of IBD patients. Phenotypic data on smoking status at the time of diagnosis was obtained through the PSI questionnaire. Genome-wide genotyping was conducted on all PSI participants using the Global Screening Array and divided into two independent datasets, the discovery (N=990) and replication cohort (N=2540). Case-only study design was applied to estimate the interaction effects for IBD, CD, and UC separately with subsequent meta-analysis between discovery and replication cohorts.

Results

572 lead single nucleotide polymorphisms (SNPs) were demonstrated with interaction effect of smoking (meta-analysis P < 5e-5, heterogeneity Cochrane Q test P > 0.05). 30 interacting SNPs showed distinct opposite effects between CD and UC (the ratio of interaction effect sizes between CD and UC > 5 or < 0.1). Our analysis also confirmed 9 previously reported interacting SNPs in the regions of NOTCH4, TSBP1-AS1, HLA-DRA, HLA-DRA-HLA-DRB5, HLA-DRB9, DNMT3A, MUC22, and CCR3.

Conclusion

Our present study identified a large number of new interaction loci for IBD on a genome wide scale and shed new light on the disease mechanisms.

Disclosure

No conflict of interest