Objective To investigate the effects of TLR7 on imiquimod induced apoptosis of THP-1 derived macrophages.Methods Three cell lines ( THP-1 derived macrophages, MDCK cell line and HUVEC cell line) with different capabilities of expressing TLR7 were selected.The survival rates of cells af-ter the treatment with different concentrations of imiquimod were detected by MTT assay.The levels of IL-6 in the supernatants of TLR7 inhibitor chloroquine or TLR7-siRNA treated cells were detected by enzyme-linked immunosorbent assay.The apoptosis of cells was detected by flow cytometry after inhibiting the ex-pression of TLR7.Results Imiquimod induced the apoptosis of THP-1 derived macrophages, MDCK cell lines and HUVEC cell lines.The levels of IL-6 were significantly decreased as the expression of TLR7 was inhibited by treating THP-1 derived macrophages with chloroquine or TLR7-siRNA.Treating THP-1 derived macrophages with chloroquine or TLR7-siRNA did not affect the cell apoptosis induced by imiquimod.Con-clusion Imiquimod could induce the apoptosis of THP-1 derived macrophages through TLR7 independent pathway.

Objective:To summarize the clinical manifestations of Autosomal dominant complex neurodevelopmental disorders due to variants of EEF1A2 gene and explore their pathogenic mechanisms. Methods:A child who had visited Luoyang Maternal and Child Health Care Hospital in July 2021 for global developmental delay was selected as the study subject. Clinical data of the child was reviewed. The child was subjected to whole exome sequencing, and relevant literature was reviewed. This study has been approved by the Medical Ethics Committee of Luoyang Maternal and Child Health Care Hospital (No. YCCZ-KS-KY-2021-03).Results:The patient, a 2-year-and-4-month-old girl, had presented with global developmental delay, gait instability, low limb muscle strength, and absence language development. Her parents were both healthy and denied relevant family history. Genetic testing revealed that she has harbored a de novo heterozygous c. 44A>G (p.H15R) missense variant of the EEF1A2 gene (NM_001958.5), which was unreported previously. Based on the guidelines from the American College of Medical Genetics and Genomics, the variant was rated as pathogenic. Conclusion:The c. 44A>G (p.H15R) variant of the EEF1A2 gene probably underlay the pathogenesis in this patient. Above finding has also enriched the mutational spectrum of the EEF1A2 gene.

Objective:To explore the characteristics of phenylalanine hydroxylase ( PAH) gene variants and prenatal diagnosis for 43 Chinese pedigrees affected with Phenylketonuria (PKU). Methods:Forty three PKU pedigrees diagnosed at the First Affiliated Hospital of Zhengzhou University between 2019 and 2021 were selected as the study subjects. Variants of the PAH gene of the probands were screened by high-throughput sequencing, and candidate variants were verified by Sanger sequencing. Negative cases were further analyzed by multiplex ligation-dependent probe amplification (MLPA) to detect large fragment deletions and duplications of the PAH gene. For 43 women undergoing subsequent pregnancy, Sanger sequencing, MLPA, combined with short tandem repeats (STR) sequence-based linkage analysis, were carried out for prenatal diagnosis. Results:Among the 86 alleles carried by the 43 probands, 78 nucleotide variants (90.70%) and 3 large deletions (3.49%) were found based on high-throughput sequencing and MLPA. The 81 mutant alleles had included 21 missense variants, 5 splice site variants, 4 nonsense variants, 2 microdeletions, 1 insertional variant and 2 large fragment deletions. Relatively common variants have included p. Arg243Gln (23.26%), p. Arg111Ter (8.14%), EX6-96A>G (6.98%), p. Val399Val (5.81%) and p. Arg413Pro (4.65%). Most of the variants were located in exons 7, 11, 3, 6 and 12. For the 43 families undergoing prenatal diagnosis, 9 fetuses (20.45%) were diagnosed with PKU, 20 (45.45%) were heterozygous carriers, and 15 (34.09%) did not carry the same pathogenic allele as the proband. All neonates were followed up till 6 months old, and the accuracy of prenatal diagnosis was 100%.Conclusion:The combination of high-throughput sequencing, Sanger sequencing, MLPA and linkage analysis can increase the diagnostic rate of PKU and attain accurate prenatal diagnosis.

Objective:To carry out genetic testing on a child diagnosed with Very-long-chain acyl-CoA dehydrogenase deficiency (VLADD) in order to provide a basis for genetic counseling and prenatal diagnosis for his family.Methods:Whole exome sequencing was performed for the proband. Candidate variant sites in the ACADVL gene were verified by Sanger sequencing, and their pathogenicity was predicted based on the guidelines from the American College of Medical Genetics and Genomics (ACMG). Prenatal diagnosis was performed on the fetus upon subsequent pregnancy. This study was approved by Medical Ethics Committe of the Luoyang Maternal and Child Health Care Hospital (Ethics No.LYFY-YCCZ-2021003). Results:The proband was found to harbor compound heterozygous variants of the ACADVL gene, namely c. 1532G>A and 1827+ 2_1827+ 12del, which were inherited from his mother and father, and classified as likely pathogenic and pathogenic, respectively. By combining the clinical manifestations of the proband and the results of blood tandem mass spectrometry and genetic testing, the child was ultimately diagnosed as cardiomyopathy type VLADD. Prenatal diagnosis showed that the fetus has carried the same compound heterozygous variants, and the couple had opted to terminate the pregnancy. Conclusion:The c. 1532G>A/1827+ 2_1827+ 12del compound heterozygous variants of the ACADVL gene probably underlay the pathogenesis of VLADD in this pedigree. The discovery of the 1827+ 2_1827+ 12del variant has enriched the mutational spectrum of the ACADVL gene.

Objective:To explore the genotype-phenotype relationship of Wilson's disease (WD) and further study the mutation spectrum in the ATP7B gene.Methods:The clinical data and genetic test results of 115 cases with WD diagnosed in the First Affiliated Hospital of Zhengzhou University from 2015 to 2022 were retrospectively analyzed. The rank sum test was used for quantitative data comparison, and χ2 test was used for count data comparison. Multivariate logistic regression was used to analyze the relationship between patients' genotype and phenotype. Results:The onset of liver manifestations (hepatic type) accounted for 60.9%, neurological symptoms (cerebral type) for 13.0%, and mixed hepato-cerebral symptoms for 26.1%. Presymptomatic individuals (hepatic types) accounted for 62.9%. Next-generation sequencing- diagnosed WD cases accounted for 87.8%. Combined multiplex ligation-dependent probe amplification assay-diagnosed WD cases accounted for 89.6%. A single case with a detected pathogenic locus accounted for 10.4%. The diagnostic rate of WD by genetic testing combined with clinical data was 100%. A total of 76 ATP7B mutations were detected, and the top three mutation frequencies were c.2333G>T (p.Arg778Leu) (30.7%), c.2975C>T (p.Pro992Leu) (7.3%), and c.2621C>T (p.Ala874Val) (6.4%). The mutations were mainly distributed in exons 8, 11-13, and 15-18, accounting for more than 90% of the total mutations. Eight new mutations were found, including c.3724G>A (p.Glu1242Lys), c.3703G>C (p.Gly1235Arg), c.3593T>C (p.Val1198Ala), c.2494A>C (p.Lys832Gln), c.1517T>A (p.Ile506Lys), c.484G>T (p.Glu162Ter), c.1870-49A>G, and the missing of exons 10-21. Liver histopathology showed cellular edema, degeneration, inflammation, and necrosis, as well as a 42.8% copper staining positive rate. Genotype-phenotype analysis showed that the p.Arg778Leu mutation had higher alanine aminotransferase (ALT) levels than those carrying other mutations ( P=0.024), while the homozygous mutation of p.Arg778Leu was associated with cerebral-type patients ( P=0.027). Conclusion:Genetic testing plays an important role in the diagnosis of WD. p.Arg778Leu is the first high-frequency mutation in the Chinese population, and patients carrying it have higher ALT levels. The p.Arg778Leu homozygous mutation is prone to causing cerebral-type WD. This study expands the ATP7B gene mutation spectrum.

Objective:To establish and validate a preoperative differentiateon model of intrahepatic cholangiocarcinoma (ICC) and combined hepatocellular carcinoma (CHC) based on the inflammatory markers and conventional clinical indicators.Methods:The clinical data of 116 patients with ICC or CHC admitted to Henan Provincial People's Hospital from January 2018 to March 2023 were retrospectively analyzed, including 74 males and 42 females, aged (58.5±9.4) years old. The data of 83 patients were used to establish the differentiation model as the training group, including 50 cases of ICC and 33 cases of CHC. The data of 33 patients were used to validate the model as the validation group, including 20 cases of ICC and 13 cases of CHC. The clinical data including the platelet-to-lymphocyte ratio (PLR), systemic immune inflammation index (SII), prognostic inflammatory index (PII), prognostic nutritional index (PNI), neutrophil-to-lymphocyte ratio (NLR) and lymphocyte-to-monocyte ratio (LMR) were collected and analyzed. The receiver operating characteristic (ROC) curve was used to analyze the best cut-off values of PLR, SII, PII, PNI, NLR and LMR. Univariate and multivariate logistic regression analysis were used to determine the differential factors between ICC and CHC. The R software was used to draw the nomogram, calculate the area under the curve (AUC) to evaluate the model accuracy, and draw the calibration chart and the decision curve to evaluate the predictive efficacy of the model.Results:Univariate logistic regression analysis showed that liver cirrhosis, history of hepatitis, alpha fetoprotein, carbohydrate antigen 199, gamma-glutamyltransferase (GGT), PLR, PNI and inflammation score (IS) could be used to differentiate ICC from CHC (all P<0.05). The indicators identified in univariate analysis were included in multivariate logistic regression analysis. The results showed that absence of liver cirrhosis, GGT>60 U/L, PNI>49.53, and IS<2 indicated the pathology of ICC (all P<0.05). Based on the above four factors, a nomogram model was established to differentiate the ICC and CHC. The AUC of ROC curve of the nomogram model in the training and validation groups were 0.851 (95% CI: 0.769-0.933) and 0.771 (95% CI: 0.594-0.949), respectively. The sensitivities were 0.760 and 0.750, and the specificities were 0.818 and 0.769, respectively. The calibration chart showed that the predicted curve fitted well to the reference line. The decision curve showed that the model has a clear positive net benefit. Conclusion:The nomogram model based on inflammatory markers showed a good differentiation performance of ICC and CHC, which could benefits the individualized treatment.