Stromal Cells Covering Omental Fat-Associated Lymphoid Clusters Trigger Formation of Neutrophil Aggregates to Capture Peritoneal Contaminants.

The omentum is a visceral adipose tissue rich in fat-associated lymphoid clusters (FALCs) that collects peritoneal contaminants and provides a first layer of immunological defense within the abdomen. Here, we investigated the mechanisms that mediate the capture of peritoneal contaminants during peritonitis. Single-cell RNA sequencing and spatial analysis of omental stromal cells revealed that the surface of FALCs were covered by CXCL1+ mesothelial cells, which we termed FALC cover cells. Blockade of CXCL1 inhibited the recruitment and aggregation of neutrophils at FALCs during zymosan-induced peritonitis. Inhibition of protein arginine deiminase 4, an enzyme important for the release of neutrophil extracellular traps, abolished neutrophil aggregation and the capture of peritoneal contaminants by omental FALCs. Analysis of omental samples from patients with acute appendicitis confirmed neutrophil recruitment and bacterial capture at FALCs. Thus, specialized omental mesothelial cells coordinate the recruitment and aggregation of neutrophils to capture peritoneal contaminants.

Expression of Calretinin in the Cecal Muscularis Interna: Observation and Hypothetical Relevance to Appendicitis.

BACKGROUND: The unexpected observation of calretinin immunoreactivity in smooth muscle cells in the muscularis propria of the cecum led to a more detailed examination of calretinin expression and its possible relationship to propulsive contractile activity around the vermiform appendix. METHODS: Immunohistochemistry and RNA in situ hybridization were performed to analyze calretinin expression in intestinal samples from 33 patients at ages ranging from mid-gestation fetuses to adults, as well as in some potentially relevant animal models. Dual immunolabeling was done to compare calretinin localization with markers of smooth muscle and interstitial cells of Cajal. RESULTS: Calretinin expression was observed consistently in the innermost smooth muscle layers of the muscularis interna in the human cecum, appendiceal base, and proximal ascending colon, but not elsewhere in the intestinal tract. Calretinin-positive smooth muscle cells did not co-express markers located in adjacent interstitial cells of Cajal. Muscular calretinin immunoreactivity was not detected in the ceca of mice or macaques, species which lack appendices, nor in the rabbit cecum or appendix. CONCLUSIONS: Localized expression of calretinin in cecal smooth muscle cells may reduce the likelihood of retrograde, calcium-mediated propulsive contractions from the proximal colon and suppress pro-inflammatory fecal stasis in the appendix.

Investigation of VGLL3 and sub-target genes in the aetiology of paediatric acute appendicitis: a prospective case-control study.

PURPOSE: Vestigial like family member 3 (VGLL3) and its sub-target genes show considerable transcriptomic overlap in terms of several autoimmune and inflammatory diseases. Herein, we investigated the role of VGLL3 rs13074432 polymorphism and its sub-target genes in the aetiology of acute appendicitis (AA). METHODS: In this prospective case-control study, we included 250 patients (age, 0-18 years) who underwent appendectomy with the diagnosis of AA (patient group; blood and appendix tissue samples) and 200 healthy children (control group; only blood samples) without appendectomy. ELISA method was used for protein-level detection of VGLL3 and sub-target genes expression change in obtained tissue samples, and real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used for mRNA level detection. Genotyping analyses were performed on DNA samples isolated from blood using TaqMan SNP genotyping test. RESULTS: The frequency of TT variant genotype (p < 0.001) and T allele (p = 0.002) showed a significant decrease in the patient group compared with the control group. No significant correlation was observed between the expression of VGLL3 in the appendiceal tissue and patient clinical and demographic data (p > 0.050). CONCLUSION: This study revealed that the VGLL3 gene and its sub-target genes are associated with AA aetiology.

[Detection of SARS-CoV-2 RNA in the mucosa of the appendices of children with COVID-19]. / Vyyavlenie RNK SARS-CoV-2 v slizistoi obolochke cherveobraznogo otrostka u detei s COVID-19.

Background. The novel coronavirus infection (COVID-19) often manifests in children as diarrhea, vomiting, abdominal pain, and some children develop acute appendicitis. To elucidate the role of SARS-CoV-2 in the development of acute appendicitis, a more detailed study of the presence of its genetic material in the tissue of the appendix. OBJECTIVE: Determination of SARS-CoV-2 RNA in appendices of children with COVID-19 by fluorescence in situ hybridization (FISH). MATERIAL AND METHODS: A retrospective analysis of case histories and morphological analysis using FISH of appendices of pediatric patients with established clinical diagnosis of acute appendicitis and confirmed infection with SARS-CoV-2 was performed. The material was divided into 3 groups: 1st -appendices obtained during appendectomy in children with established clinical diagnosis of «coronavirus infection¼ (COVID-19, PCR+) (n=42; mean age 10.8 years); 2nd - appendices of children (n=55; mean age 9.7 years) with acute appendicitis obtained before the onset of the COVID-19 pandemic; 3rd (control) group (n=38; mean age 10.3 years) - autopsy material of the appendices (intact). RESULTS: In all samples of the appendices of the 1st group, a positive SARS-CoV-2 viral RNA signal was noted in the cytoplasm of most epithelial cells and single immunocompetent cells. The signal intensity remained the same in all slides, regardless of age. In all samples obtained from patients without COVID-19 (groups 2 and 3), confocal microscopy did not reveal a signal, which indicates successful adaptation of the FISH method in this study and excludes the false positive results. CONCLUSION: In the epithelium of the appendices of children of different age with COVID-19, the FISH method revealed SARS-CoV-2 RNA, which does not exclude the association between viral invasion and the development of acute appendicitis.

Differential expression of Chitinase 3-Like 1 protein in appendicitis and appendix carcinomas.

BACKGROUND: Chitinase 3-Like 1 (CHI3L1) has been used as an inflammatory biomarker for a variety of diseases, but its expression in acute appendicitis and appendix carcinomas remains unclear. METHODS: Sixty cases of patients were studied, including 46 acute appendicitis and 14 appendix carcinomas. We divided the acute appendicitis group into acute uncomplicated appendicitis (AUA), suppurative appendicitis (SA), and gangrenous appendicitis (GA). The appendix carcinoma group was divided into appendiceal neuroendocrine neoplasms (ANENs) and appendiceal mucinous neoplasms (AMN). Controls were 32 healthy donors. Blood neutrophil to lymphocyte ratio (NLR), CHI3L1, C-reactive protein (CRP), interleukin-6 (IL-6), and serum amyloid A (SAA) were measured in the patients. Meanwhile, immunohistochemistry and immunofluorescence were used to identify the expression level and location of CHI3L1 in different cell types in appendix tissues. RESULTS: Compared with the controls, CHI3L1 serum levels were up-regulated in SA, GA, and AMN groups, while no significant difference was observed in the AUA and ANEN groups. Immunofluorescence revealed that CHI3L1 expression was high in macrophages and adenocarcinoma cells of appendix tissues but not in the neuroendocrine carcinoma tissues. Moreover, levels of NLR and CRP in the SA and GA groups were considerably higher than in the control group. IL-6 and SAA in SA, GA, ANENs, and AMN groups were also increased compared with the control group. In addition, CHI3L1 displayed good performance in predicting appendicitis, with an AUC of 0.862. CONCLUSION: CHI3L1 was highly expressed in acute appendicitis and appendiceal mucinous neoplasms, which can be used as a novel biomarker predicting appendicitis.

The application of artificial intelligence methods to gene expression data for differentiation of uncomplicated and complicated appendicitis in children and adolescents - a proof of concept study.

BACKGROUND: Genome wide gene expression analysis has revealed hints for independent immunological pathways underlying the pathophysiologies of phlegmonous (PA) and gangrenous appendicitis (GA). Methods of artificial intelligence (AI) have successfully been applied to routine laboratory and sonographic parameters for differentiation of the inflammatory manifestations. In this study we aimed to apply AI methods to gene expression data to provide evidence for feasibility. METHODS: Modern algorithms from AI were applied to 56.666 gene expression data sets from 13 patients with PA and 16 with GA aged 7-17 years by using resampling methods (bootstrap). Performance with respect to sensitivities and specificities where investigated with receiver operating characteristic (ROC) analysis. RESULTS: Within the experimental setting a best performing discriminatory biomarker signature consisting of a set of 4 genes could be defined: ERGIC and golgi 3, regulator of G-protein signaling 2, Rho GTPase activating protein 33, and Golgi Reassembly Stacking Protein 2. ROC analysis showed a mean area under the curve of 84%. CONCLUSIONS: Gene expression based application of AI methods is feasible and represents a promising approach for future discriminatory diagnostics in children with acute appendicitis.

Genetic polymorphism patterns suggest a genetic driven inflammatory response as pathogenesis in appendicitis.

PURPOSE: The pathogenesis of appendicitis is not well understood. Environmental factors are regarded most important, but epidemiologic findings suggest a role of inflammatory and genetic mechanisms. This study determines the association of single nucleotide polymorphisms (SNPs) of inflammatory genes with appendicitis. METHODS: As part of a larger prospective study on the diagnostic value of inflammatory variables in appendicitis, the genotype frequency of 28 polymorphisms in 26 inflammatory response genes from the appendicitis and control patients was analyzed in blood samples from 343 patients, 100 with appendicitis, and 243 with non-specific abdominal pain, using TaqMan SNP genotyping assays. RESULTS: Associations with appendicitis were found for SNPs IL-13 rs1800925 with odds ratio (OR) 6.02 (95% CI 1.52-23.78) for T/T versus C/C + T/T, for IL-17 rs2275913 with OR 2.38 (CI 1.24-4.57) for A/A vs G/G + GA, for CCL22 rs223888 with OR 0.12 (0.02-0.90), and for A/A vs G/G + GA. Signs of effect modification of age for the association with appendicitis were found for IL-13 rs1800925 and CTLA4 rs3087243. Stratified analysis showed difference in association with severity of disease for IL-17 rs2275913 and CD44 rs187115. CONCLUSIONS: The association of gene variants on risk of appendicitis and its severity suggest an etiologic role of genetically regulated inflammatory response. This may have implications for understanding the prognosis of untreated appendicitis as a possible self-limiting disorder and for understanding the inverse association of appendicitis with ulcerative colitis.

Low mitochondrial DNA copy number of resected cecum appendix correlates with high severity of acute appendicitis.

BACKGROUND/PURPOSE: The roles of mitochondrial DNA alterations in acute appendicitis (AA) remain unclear. We evaluated the alterations of mtDNA copy number and mtDNA integrity [proportion of mtDNA templates without 8-hydroxyl-2'-deoxyguanosine (8-OHdG)] of the resected cecum appendixes in clinically suspected acute appendicitis (CSAA). METHODS: A total of 228 CSAA patients, including 50 harbored negative AA (NAA), 155 true AA (TAA) without rupture and 23 TAA with rupture, who underwent appendectomies were enrolled. Tissues of resected cecum appendixes from the paraffin-embedded pathological blocks were subjected to DNA extraction, and their mtDNA copy number and mtDNA integrity were determined by quantitative real-time polymerase chain reaction (Q-PCR). RESULTS: During the progression of disease severity from NAA to TAA without rupture and further TAA with rupture, increases of white blood cell (WBC) counts (p = 0.001), positive bacterial culture rates in turbid ascites (p = 0.016) and area (p < 0.001)/or volume (p < 0.001) indices of resected cecum appendixes were noted among CSAA patients. On the contrary, decrease of mtDNA copy number (p = 0.003) was observed during disease progression of CSAA patients, especially in female patients (p = 0.007). Furthermore, lower mtDNA copy numbers were correlated with higher WBC counts (p = 0.001) and larger area (p = 0.003) or volume (p < 0.001) indices of the resected cecum appendixes. However, such an alteration was not observed in mtDNA integrity of resected cecum appendixes. CONCLUSION: We conclude that a low mtDNA copy number of the resected cecum appendix may reflect high severity of acute appendicitis.

Familial Risk of Appendicitis: A Nationwide Population Study.

OBJECTIVE: To investigate the familial risk of appendicitis in the general population. STUDY DESIGN: A nationwide, cross-sectional study consisting of 24 349 599 Taiwan National Health Insurance beneficiaries in 2015 was conducted. Among them, 788 042 individuals had at least 1 first-degree relative with appendicitis. The familial relative risks (RRs) of appendicitis and familial transmission were estimated. RESULTS: The overall RR (95% CI) of appendicitis in individuals with any affected first-degree relatives was 1.67 (1.64-1.71) compared with the general population. The RRs for individuals with an affected twin, sibling, offspring, and parent were 3.40 (2.66-4.35), 1.98 (1.92-2.04), 1.55 (1.51-1.59), and 1.54 (1.50-1.58), respectively. The RRs for individuals with 1, 2, 3 or more affected first-degree relatives were 1.65 (1.62-1.68), 2.63 (2.37-2.91), and 6.70 (4.22-10.63), respectively. Furthermore, there was an age-dependent trend of the RRs, with the greatest RR in the youngest group. The estimated familial transmission (genetic plus shared environmental contribution to the total phenotypic variance of appendicitis) was 23.2%. CONCLUSION: Individuals with a family history of appendicitis have an increased risk of appendicitis. This risk is age-dependent and related to the genetic distance and numbers of affected relatives.

Reduced risk of UC in families affected by appendicitis: a Danish national cohort study.

OBJECTIVE: The possible aetiological link between appendicitis and UC remains unclear. In order to investigate the hereditary component of the association, we studied the risk of UC in family members of individuals with appendicitis. DESIGN: A cohort of 7.1 million individuals was established by linkage of national registers in Denmark with data on kinship and diagnoses of appendicitis and UC. Poisson regression models were used to calculate first hospital contact rate ratios (RR) for UC with 95% CIs between individuals with or without relatives with a history of appendicitis. RESULTS: During 174 million person-years of follow-up between 1977 and 2011, a total of 190 004 cohort members developed appendicitis and 45 202 developed UC. Individuals having a first-degree relative with appendicitis before age 20 years had significantly reduced risk of UC (RR 0.90; 95% CI 0.86 to 0.95); this association was stronger in individuals with a family predisposition to UC (RR 0.66; 95% CI 0.51 to 0.83). CONCLUSIONS: Individuals with a first-degree relative diagnosed with appendicitis before age 20 years are at reduced risk of UC, particularly when there is a family predisposition to UC. Our findings question a previously hypothesised direct protective influence of appendicitis on inflammation of the large bowel. Rather, genetic or environmental factors linked to an increased risk of appendicitis while being protective against UC may explain the repeatedly reported reduced relative risk of UC in individuals with a history of appendicitis.

Mining and exploration of appendicitis nursing targets: An observational study.

Appendicitis is an inflammation caused by obstruction of the appendiceal lumen or termination of blood supply leading to appendiceal necrosis followed by secondary bacterial infection. The relationship between TYROBP gene and the nursing of appendicitis remains unclear. The appendicitis dataset GSE9579 profile was downloaded from the gene expression omnibus database generated from GPL571. Differentially expressed genes were screened, followed by weighted gene co-expression network analysis, functional enrichment analysis, gene set enrichment analysis, construction and analysis of protein-protein interaction network, Comparative Toxicogenomics Database analysis, and immune infiltration analysis. Heatmaps of gene expression levels were plotted. A total of 1570 differentially expressed genes were identified. According to gene ontology analysis, they were mainly enriched in organic acid metabolic process, condensed chromosome kinetochore, oxidoreductase activity. In Kyoto Encyclopedia of Gene and Genome analysis, they mainly concentrated in metabolic pathways, P53 signaling pathway, PPAR signaling pathway. The soft threshold power in weighted gene co-expression network analysis was set to 12. Through the construction and analysis of protein-protein interaction network, 5 core genes (FCGR2A, IL1B, ITGAM, TLR2, TYROBP) were obtained. Heatmap of core gene expression levels revealed high expression of TYROBP in appendicitis samples. Comparative Toxicogenomics Database analysis found that core genes (FCGR2A, IL1B, ITGAM, TLR2, TYROBP) were closely related to abdominal pain, gastrointestinal dysfunction, fever, and inflammation occurrence. TYROBP gene is highly expressed in appendicitis, and higher expression of TYROBP gene indicates worse prognosis. TYROBP may serve as a molecular target for appendicitis and its nursing.

Gene Expression Profiling in Pediatric Appendicitis.

Importance: Appendicitis is the most common indication for urgent surgery in the pediatric population, presenting across a range of severity and with variable complications. Differentiating simple appendicitis (SA) and perforated appendicitis (PA) on presentation may help direct further diagnostic workup and appropriate therapy selection, including antibiotic choice and timing of surgery. Objective: To provide a mechanistic understanding of the differences in disease severity of appendicitis with the objective of developing improved diagnostics and treatments, specifically for the pediatric population. Design, Setting, and Participants: The Gene Expression Profiling of Pediatric Appendicitis (GEPPA) study was a single-center prospective exploratory diagnostic study with transcriptomic profiling of peripheral blood collected from a cohort of children aged 5 to 17 years with abdominal pain and suspected appendicitis between November 2016 and April 2017 at the Alberta Children's Hospital in Calgary, Alberta, Canada, with data analysis reported in August 2023. There was no patient follow-up in this study. Exposure: SA, PA, or nonappendicitis abdominal pain. Main Outcomes and Measures: Blood transcriptomics was used to develop a hypothesis of underlying mechanistic differences between SA and PA to build mechanistic hypotheses and blood-based diagnostics. Results: Seventy-one children (mean [SD] age, 11.8 [3.0] years; 48 [67.6%] male) presenting to the emergency department with abdominal pain and suspected appendicitis were investigated using whole-blood transcriptomics. A central role for immune system pathways was revealed in PA, including a dampening of major innate interferon responses. Gene expression changes in patients with PA were consistent with downregulation of immune response and inflammation pathways and shared similarities with gene expression signatures derived from patients with sepsis, including the most severe sepsis endotypes. Despite the challenges in identifying early biomarkers of severe appendicitis, a 4-gene signature that was predictive of PA compared to SA, with an accuracy of 85.7% (95% CI, 72.8-94.1) was identified. Conclusions: This study found that PA was complicated by a dysregulated immune response. This finding should inform improved diagnostics of severity, early management strategies, and prevention of further postsurgical complications.

Causal effects of gut microbiota on appendicitis: a two-sample Mendelian randomization study.

Background: Previous research has posited a potential correlation between the gut microbiota and the onset of appendicitis; however, the precise causal connection between appendicitis and the gut microbiota remains an unresolved and contentious issue. Methods: In this investigation, we performed a Mendelian randomization (MR) analysis employing publicly accessible summary data extracted from genome-wide association studies (GWAS) to elucidate the potential causal nexus between the gut microbiota and the development of appendicitis. We initially identified instrumental variables (IVs) through a comprehensive array of screening methodologies, subsequently executing MR analyses using the Inverse Variance Weighted (IVW) technique as our primary approach, supplemented by several alternative methods such as MR Egger, weighted median, simple mode, and weighted mode. Additionally, we implemented a series of sensitivity analysis procedures, encompassing Cochran's Q test, MR-Egger intercept test, Mendelian Randomized Polymorphism Residual and Outlier (MR-PRESSO) test, and a leave-one-out test, to affirm the robustness and validity of our findings. Results: Our investigation indicates that an elevated prevalence of Deltaproteobacteria, Christensenellaceae, Desulfovibrionaceae, Eubacterium ruminantium group, Lachnospiraceae NK4A136 group, Methanobrevibacter, Desulfovibrionales, and Euryarchaeota is inversely associated with the risk of appendicitis. Conversely, we observed a positive correlation between an increased abundance of Family XIII, Howardella, and Veillonella and the susceptibility to appendicitis. Sensitivity analyses have corroborated the robustness of these findings, and Mendelian randomization analyses provided no indications of reverse causality. Conclusion: Our Mendelian randomization (MR) analysis has unveiled potential advantageous or detrimental causal associations between the gut microbiota and the occurrence of appendicitis. This study offers novel theoretical and empirical insights into the understanding of appendicitis pathogenesis, along with its implications for preventive and therapeutic strategies.

Detection of Enterobius vermicularis in archived formalin-fixed paraffin-embedded (FFPE) appendectomy blocks: It's potential to compare genetic variations based on mitochondrial DNA (cox1) gene.

Acute appendicitis represents one of the most common causes of emergency abdominal surgery worldwide. Meanwhile, Enterobius vermicularis has been suggested as one of the probable causes of appendicitis. In this study, the morphological characteristics of the remnant pinworms and pathologic changes were explored in old-archived FFPE tissues of appendectomies. Moreover, we provide the first molecular identification, genetic, and haplotype variation of this nematode from the old-archived FFPE tissue section of appendectomy using the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene. Seventeen FFPE appendectomies with E. vermicularis infection, stored over 12-22 years, were collected from two different geographical areas of Iran. In the histopathological examination, tissue changes were observed in thirteen cases (76.4%) and inflammation in four blocks (23.5%). After DNA extraction, the cox1 gene was amplified in twelve (70.6%) cases using the nested polymerase chain reaction (PCR). Phylogenetic analysis and a median-joining network of 78 available cox1 sequences of E. vermicularis revealed 59 haplotypes. We identified five haplotypes that fell into type B. All Haplotypes are novel except for two haplotypes, Hap32 and Hap37, identical to E. vermicularis sequences from Iran, Greece, and Germany. The ranges of diversity distance and haplotype diversity within the isolates were 0-1.9% and HD:0.643-0.667, subsequently. Overall, the absence of inflammation or even tissue changes in some sections can suggest the possible non-inflammatory role of E. vermicularis in appendicitis. Although FFPE material suffers from PCR inhibition, we could successfully use nested PCR to characterize E. vermicularis in old-archived appendectomy blocks and suggest this method as a complementary diagnosis technique in pathology. While the predominant type was B in the Middle East and Europe, further studies on a larger sample size from different geographical regions could probably confirm the results obtained in the present study.

Molecular and phylogenetic analysis of E. vermicularis in appendectomy specimens from Iran.

Human infection with Enterobius vermicularis occurs worldwide, particularly in children. The role of E. vermicularis in appendicitis is neglected. This study was designed to investigate genotypes of E. vermicularis detected from appendectomy specimens in the human population from Iran and clarify the intra-species variation of the parasite. Seventy appendectomies for acute clinical appendicitis isolates from Azerbaijan and North Khorasan of Iran were used in the present study. The genetic information of Tehran and Hamedan regions was also obtained from GenBank for comparison and analysis. The nucleotide sequence of cytochrome c oxidase subunit 1 (cox1) gene was analyzed to perform genetic differentiation, haplotype network analysis, and population structure. Phylogenetic analysis of all the isolates were included in type B haplogroup. The number of haplotypes in all geographical locations of Iran is not much. Network analysis of sequences for regions such as Thailand, Iran, Denmark, and Poland show three classified subtypes B1, B2, and B3 in the B haplogroup. It seems that the haplotypes of E. vermicularis detected from appendectomy are B type, and divided into three subtypes. Further research using another genetic marker is required to elucidate the genetic variation of the parasites in detail.

Increased levels of deleted in malignant brain tumours 1 (DMBT1) in active bacteria-related appendicitis.

AIMS: Deleted in malignant brain tumours 1 (DMBT1; gp340) is a secreted glycoprotein which is found in the surface lining epithelia of human small and large intestine. DMBT1 is suggested to play a role in enterocyte differentiation and surface protection from intestinal bacteria. The aim of this study was to elucidate DMBT1 expression in bacteria-related active intestinal inflammation such as appendicitis. METHODS AND RESULTS: mRNA and protein levels of DMBT1 were analysed in surgical resections of 50 appendices (active inflammation: n = 25). In non-actively inflamed appendices, inter-individual differences in basal DMBT1 levels of enterocytes and some non-epithelial cells were found. In active appendicitis, enterocytic DMBT1 mRNA expression was increased approximately fivefold, which was paralleled by a corresponding increase of cytoplasmic and secreted DMBT1 protein levels. Increased DMBT1 expression was predominant in enterocytes adjacent to erosive lesions or ulcers. CONCLUSIONS: Our data demonstrate that bacteria-related active inflammation results in a sharp increase of DMBT1 levels in enterocytes. These findings substantiate the view that DMBT1 is of functional relevance for host defence and modulation of the course of intestinal bacteria-related inflammatory responses.

Distribution of RET proto-oncogene variants in children with appendicitis.

BACKGROUND: In addition to patient-related systemic factors directing the immune response, the pathomechanisms of appendicitis (AP) might also include insufficient drainage leading to inflammation caused by decreased peristalsis. Genetic predisposition accounts for 30%-50% of AP. M. Hirschsprung (HSCR), also characterized by disturbed peristalsis, is associated with variants in the RET proto-oncogene. We thus hypothesized that RET variants contribute to the etiology of AP. METHODS: DNA from paraffin-embedded appendices and clinical data of 264 children were analyzed for the RET c.135A>G variant (rs1800858, NC_000010.11:g.43100520A>G). In 46 patients with gangrenous or perforated AP (GAP), peripheral blood DNA was used for RET sequencing. RESULTS: Germline mutations were found in 13% of GAP, whereas no RET mutations were found in controls besides the benign variant p.Tyr791Phe (NC_000010.11:g.43118460A>T). In GAP, the polymorphic G-allele in rs2435352 (NC_000010.11:g.43105241A>G) in intron 4 was underrepresented (p = 0.0317). CONCLUSION: Our results suggest an impact of the RET proto-oncogene in the etiology of AP. Mutations were similar to patients with HSCR but no clinical features of HSCR were observed. The pathological phenotypes in both populations might thus represent a multigenic etiology including RET germline mutations with phenotypic heterogeneity and incomplete penetrance.

Impact of Human Genetic Variation on C-Reactive Protein Concentrations and Acute Appendicitis.

Background: Acute appendicitis is one of the most common abdominal emergencies worldwide. Both environmental and genetic factors contribute to the disease. C-reactive protein (CRP) is an important biomarker in the diagnosis of acute appendicitis. CRP concentrations are significantly affected by genetic variation. However, whether such genetic variation is causally related to appendicitis risk remains unclear. In this study, the causal relationship between single-nucleotide polymorphisms (SNPs) associated with circulating CRP concentrations and the risk and severity of acute appendicitis was investigated. Methods: CRP concentrations in serum of appendicitis patients (n = 325) were measured. Appendicitis was categorized as complicated/uncomplicated and gangrenous/non-gangrenous. Imputed SNP data (n = 287) were generated. A genome-wide association study (GWAS) on CRP concentrations and appendicitis severity was performed. Intersection and colocalization of the GWAS results were performed with appendicitis and CRP-associated loci from the Pan-UKBB cohort. A functional-genomics approach to prioritize genes was employed. Results: Thirteen percent of significant CRP quantitative trait loci (QTLs) that were previously identified in a large cohort of healthy individuals were replicated in our small patient cohort. Significant enrichment of CRP-QTLs in association with appendicitis was observed. Among these shared loci, the two top loci at chromosomes 1q41 and 8p23.1 were characterized. The top SNP at chromosome 1q41 is located within the promoter of H2.0 Like Homeobox (HLX) gene, which is involved in blood cell differentiation, and liver and gut organogeneses. The expression of HLX is increased in the appendix of appendicitis patients compared to controls. The locus at 8p23.1 contains multiple genes, including cathepsin B (CTSB), which is overexpressed in appendix tissue from appendicitis patients. The risk allele of the top SNP in this locus also increases CTSB expression in the sigmoid colon of healthy individuals. CTSB is involved in collagen degradation, MHC class II antigen presentation, and neutrophil degranulation. Conclusions: The results of this study prioritize HLX and CTSB as potential causal genes for appendicitis and suggest a shared genetic mechanism between appendicitis and CRP concentrations.

Evaluation of the Rho-kinase gene expression and polymorphisms in adult patients with acute appendicitis: a differential impact of gender.

OBJECTIVE: Acute appendicitis represents one of the most common causes of acute intra-abdominal emergencies worldwide. In this case-control study, we aimed to investigate associations of Rho-kinase gene expression and polymorphisms with acute appendicitis in a Turkish population. We also aimed to study the effects of gender on these parameters. METHODS: A total of 93 unrelated patients with acute appendicitis and 93 healthy controls in the Department of Emergency Medicine, Erciyes University, between June 2019 and June 2021 were included in this study. Genomic DNA was isolated from peripheral leukocytes, and the LightCycler 480 II real-time polymerase chain reaction was utilized to detect Rho-kinase1 gene rs35996865 and Rho-kinase2 gene rs2230774 (Thr431Asn) polymorphisms. Quantitative real-time polymerase chain reaction was applied to determine Rho-kinase1 and Rho-kinase2 gene expressions. RESULTS: There was a marked increase in Rho-kinase1, but not in Rho-kinase2, mRNA expression, and this increase was evident only in male patients (p=0.0008). No significant differences were found in allele and genotype frequencies for Rho-kinase1 gene rs35996865 and Rho-kinase2 gene rs2230774 polymorphisms between the patients with acute appendicitis and the control group. CONCLUSIONS: Our data imply that Rho-kinase1 (rs35996865) and Rho-kinase2 (rs2230774) gene variants are not risk factors for the development of acute appendicitis in the Turkish population. However, increased mRNA expression of the Rho-kinase1 gene in males indicated that Rho-kinase1 is involved in the pathogenesis of acute appendicitis in a gender-specific way.

Association Between NEDD4L Variation and the Genetic Risk of Acute Appendicitis: A Multi-institutional Genome-Wide Association Study.

Importance: The familial aspect of acute appendicitis (AA) has been proposed, but its hereditary basis remains undetermined. Objective: To identify genomic variants associated with AA. Design, Setting, and Participants: This genome-wide association study, conducted from June 21, 2019, to February 4, 2020, used a multi-institutional biobank to retrospectively identify patients with AA across 8 single-nucleotide variation (SNV) genotyping batches. The study also examined differential gene expression in appendiceal tissue samples between patients with AA and controls using the GSE9579 data set in the National Institutes of Health's Gene Expression Omnibus repository. Statistical analysis was conducted from October 1, 2019, to February 4, 2020. Main Outcomes and Measures: Single-nucleotide variations with a minor allele frequency of 5% or higher were tested for association with AA using a linear mixed model. The significance threshold was set at P = 5 × 10-8. Results: A total of 29 706 patients (15 088 women [50.8%]; mean [SD] age at enrollment, 60.1 [17.0] years) were included, 1743 of whom had a history of AA. The genomic inflation factor for the cohort was 1.003. A previously unknown SNV at chromosome 18q was found to be associated with AA (rs9953918: odds ratio, 0.99; 95% CI, 0.98-1.00; P = 4.48 × 10-8). This SNV is located in an intron of the NEDD4L gene. The heritability of appendicitis was estimated at 30.1%. Gene expression data from appendiceal tissue donors identified NEDD4L to be among the most differentially expressed genes (14 of 22 216 genes; ß [SE] = -2.71 [0.44]; log fold change = -1.69; adjusted P = .04). Conclusions and Relevance: This study identified SNVs within the NEDD4L gene as being associated with AA. Nedd4l is involved in the ubiquitination of intestinal ion channels and decreased Nedd4l activity may be implicated in the pathogenesis of AA. These findings can improve the understanding of the genetic predisposition to and pathogenesis of AA.