ObjectiveTo identify 3 the disease-causing genes and mutations of Leber congenital amaurosis (LCA), and to study the correlation of phenotype and genotype. MethodsA retrospective study. Four LCA patients and seven family members who were diagnosed by eye examination in Ning Xia Eye Hospital of People's Hospital of Ningxia Hui Autonomous Region from January to December 2021 were included in the study. Four patients were from 3 unrelated families. Detailed collection of medical history and family history were received. Related ophthalmologic examination were collected and genomic DNA was extracted from peripheral blood. Whole-exome sequencing method was used for genetic diagnosis. The identified variant was confirmed with Sanger sequencing. Potential pathogenic mutation was analyzed using software and conserved domain analysis and performed co-separated analysis between the family member and the proband. ResultsOf the 4 patients, 1 patient was males and 3 patients were females; the age was from 4 to 18 years. Nystagmus were seen in 3 cases, finger pressing eyes and night blindness was seen in 1 cases; electroretinogram showed 4 cases of extinction or near extinction. The foveal reflection was visible in all eyes, and there was no obvious abnormality in the peripheral retina. One eye had strong reflection signal with raised ellipsoid in macular area; two eyes had weak reflection signal faintly visible between retinal layers; 1 eye had increased blood vessel branches, peripheral retinal non-perfusion area with capillary leakage; annular strong autofluorescence in macular area 4 eyes. No obvious abnormality was found in the phenotypes of family members. Genetic testing showed that the proband of pedigree 1 (Ⅱ-1) was found a homozygous missense mutation in c.640A>T (p.C214S) (M1) of PRPH2 gene. The proband of pedigree 2 (Ⅱ-2) was found compound heterozygous mutation in c.1256G>A(p.R419Q) (M2) and c.1A>C (p.M1L) (M3) of TULP1 gene. The proband 3 (Ⅱ-1) and her sister (Ⅱ-2) were both found compound heterozygous mutation in c.1943T>C (p.L648P) (M4) and c.380C>T (p.P127L) (M5) of GUCY2D gene. The parents and sister (Ⅱ-1) of the proband in family 2 and the parents of the proband in family 3 were all carriers of the corresponding heterozygous variant. M1, M3, M4, M5 were novel mutations and unreported. The genotype and disease phenotype were co-segregated within the family. According to the analysis of pedigree and genetic testing results, all 3 families were autosomal recessive inheritance. The amino acid conservation analysis found that M1, M2, M3, M4, and M5 were highly conserved among species. The results of bioinformatics analysis were all pathogenic variants. ConclusionsPRPH2 gene M1, TULP1 gene M3, and GUCY2D gene M4, M5 were novel mutations and not been reported in the literature and database. This research expanded the gene mutation spectrum of LCA. The patients with LCA have available characterristics, including onset age, varying ocular fundus and severe visual impairment.
ObjectiveTo investigate the causal relationship between 731 kinds of immune cell phenotypes and positive-human epidermal growth factor receptor (HER+), negative-human epidermal growth factor receptor (HER–), negative-human epidermal growth factor receptor 2 (HER2–) breast cancer. MethodsGenome-wide association data using immune cell phenotype and breast cancer were used with inverse variance weighting as the primary analytical method; horizontal pleiotropy was tested using MR-PRESSO and outliers were corrected; in addition, the reliability of the obtained data was verified using Cochran’s Q test, Mendelian randomization (MR)- Egger regression and leave-one-out method were used to verify the reliability of the obtained data. ResultsFor HER+ breast cancer, CD3 on CD39+CD4+T cell [ OR=0.940, 95%CI (0.913, 0.968), P=0.019] was protective factor. For HER– breast cancer, no immune cell phenotype was found to be correlated with it. For HER2– breast cancer, CD3 on CD39+CD4+ T cell [OR=0.951, 95%CI (0.930, 0.973), P=0.014], CD3 on secreting CD4 regulatory T cell [OR=0.949, 95%CI (0.925, 0.974), P=0.023] were protective factors. ConclusionThere is a causal association between certain immune cell phenotypes and breast cancer, which may be predictive markers for early diagnosis of breast cancer and development of new immunotherapies.
ObjectiveTo explore the composition of intestinal microbiota between patients with fixed airflow obstruction asthma, reversible airflow obstruction asthma, and healthy control, and analyze the correlation between key differential bacterial distribution and clinical characteristics. MethodsFifteen patients with fixed airflow obstruction asthma (FAO) and 13 patients with reversible airflow obstruction asthma (RAO) were included, along with 11 matched healthy control subjects. Clinical data were collected, and lung function tests and induced sputum examination were performed. Blood and stool samples were tested to compare the gut microbiota status among the groups, and analyze the relationship between gut microbiota abundance and patients' blood routine, IgE levels, lung function, and induced sputum. Results The dominant bacterial compositions were similar in the three groups, but there were differences in the abundance of some species. Compared to the RAO group, the FAO group showed a significant increase in the genera of Bacteroides and Escherichia coli, while Pseudomonas was significantly decreased. The phylum Firmicutes was negatively correlated with the course of asthma, while the phylum Bacteroidetes and genus Bacteroides were positively correlated with the asthma course. Bacteroidetes was negatively correlated with Pre-BD FEV1/FVC, Pseudomonas was positively correlated with Pre-BD FEV1, Escherichia coli was negatively correlated with Post-BD FEV1/FVC, and Bacteroides was negatively correlated with Post-BD MMEF. The class Actinobacteria and the order Actinomycetales were negatively correlated with peripheral blood EOS%, while the order Enterobacteriales and the family Enterobacteriaceae were positively correlated with peripheral blood IgE levels. Furthermore, Actinobacteria and Actinomycetales were negatively correlated with induced sputum EOS%. Conclusions There are differences in the gut microbiota among patients with fixed airflow obstruction asthma, reversible airflow obstruction asthma, and healthy individuals. Bacteroides and Escherichia coli are enriched in the fixed airflow obstruction asthma group, while the Firmicutes are increased in the reversible airflow obstruction asthma group. These three microbiota may act together on Th2 cell-mediated inflammatory responses, influencing the process of airway remodeling, and thereby interfering with the occurrence of fixed airflow obstruction in asthma.
ObjectiveTo investigate the influences of lactic acid (LA), the final degradation product of polylactic acid (PLA) on the prol iferation and osteoblastic phenotype of osteoblast-l ike cells so as to provide theoretical basis for bone tissue engineering. MethodsRos17/2.8 osteoblast-l ike cells were harvested and divided into 3 groups. In groups A and B, the cells were cultured with the medium containing 4, 8, 16, 22, and 27 mmol/L L-LA and D, L-LA, respectively. In group C, the cells were cultured with normal medium (pH7.4). The cell prol iferation was determined with MTT method after 1, 3, and 5 days. The relative growth ratio (RGR) was calculated, and the cytotoxicity was evaluated according to national standard of China. In addition, the alkal ine phosphatase (ALP) activity of cells cultured with medium containing 4 mmol/L L-LA (group A), 4 mmol/ L D, L-LA (group B), and normal medium (group C) after 1 and 5 days were detected with ALP kits, and the relative ALP ratio (RAR) was calculated; after 21 days, the calcium nodules were tested with von Kossa staining method, and were quantitatively analyzed. ResultsWhen LA concentration was 4 mmol/L, the mean RGR of both groups A and B were all above 80%, and the cytotoxic grades were grade 0 or 1, which meant non-cytotoxicity. When LA concentration was 8 mmol/L and 16 mmol/ L, groups A and B showed cytotoxicity after 5 days and 3 days, respectively. When LA concentration was above 22 mmol/L, cell prol iferations of groups A and B were inhibited evidently after 1-day culture. At each LA concentration, RGR of group A was significantly higher than that of group B at the same culture time (P<0.05) except those at 4 mmol/L after 1-day and 3-day culture. After 1 day, the RAR of group A was significantly higher than that of group B on 1 day (144.1%±3.2% vs. 115.2%±9.8%, P<0.05) and on 5 days (129.6%±9.8% vs. 78.2%±6.9%, P<0.05). The results of von Kossa staining showed that the black gobbets in group A were obviously more than those of groups B and C. The staining area of group A (91.2%±8.2%) was significantly higher than that of groups B (50.3%±7.9%) and C (54.2%±8.6%) (P<0.05). ConclusionThe concentration and composition of LA have significant effects on the cell proliferation and osteoblastic phenotype of osteoblast-l ike cells.
ObjectiveTo study the clinicopathological features of mediastinum nodular sclerosis Hodgkin lymphoma (NSHL) in order to improve the recognition of it. MethodsThe clinical data of 3 cases of mediastinum NSHL between 2003 and 2012 were collected. Then we analyzed the carcinoma pathologic samples by pathomorphology, immunophenotypic phenotype, related gene rearrangement and situ hybridization with EBER. ResultsThe pathomorphologic results showed that broad fibrotic bands subdivided the lymphoid parenchyma into large nodules, the tumoral cells had distinct boundary with empty cytoplasm and small-to-medium-sized nucleoli, and the nodules contained inflammatory cell components. The immunophenotypic phenotype of the tumoral cells were CD15, CD30, PAX-5 and CD20 partly, but anaplastic lymphoma kinase, CD45, cytokeratin, CD79α and S-100 were not expressed. T cell receptor γ and IgH gene were no rearranged, and EBER in situ hybridization was not detected. ConclusionVarious lymphomas occur in the mediastinum and mediastinum NSHL is just one of them. Mastering its distinctive pathomorphology and immunophenotypic phenotype is highly significant for diagnosis, differential diagnosis and treatment of the disease.
ObjectiveTo analyze the drug-resistant phenotype and genotype characteristics of carbapenem-resistant Enterobacteriaceae (CRE) in a traditional Chinese medicine hospital from 2016 to 2018, to provide guidance for clinical rational drug use and effective anti-infection treatment.MethodsA total of 2 901 Enterobacteriaceae bacteria strains isolated from January 2016 to December 2018 were selected, and CRE strains were screened by microdilution test and Kirby-Bauer methods. CRE strains with successful seed preservation and detailed clinical data were selected for carbapenemase phenotype confirmation test, drug-resistant gene amplification, and sequencing comparison.ResultsThe 101 CRE strains collected between 2016 and 2018 were mainly Klebsiella pneumonia (73.27%, 74/101) and Escherichia coli (14.85%, 15/101), and the specimens were mainly from sputum (63.37%, 64/101) and catheter urine (11.88%, 12/101). The phenotypic test results of carbapenemase showed that 94 strains were positive in modified Hodge test, with a positive rate of 93.07%, 96 strains were positive in Carba NP test, with a positive rate of 95.05%, and 98 strains were positive in modified carbapenem inactivation method test, with a positive rate of 97.03%. Drug-resistant genes were detected in 92 (91.01%) of the 101 CRE strains, sequencing results showed that 66 (65.35%) carried blaKPC-2 gene, 4 (3.96%) carried blaKPC-19 gene, 9 (8.91%) carried blaNDM-1 gene, and 13 (12.87%) carried blaNDM-5 gene. No CRE strains carrying two resistance genes were detected. Among them, Klebsiella pneumoniae strains mainly carried blaKPC-2 gene (82.43%, 61/74), and Escherichia coli strains mainly carried blaNDM-5 gene (86.67%, 13/15), which were consistent with the main epidemic genotype in China.ConclusionsIn recent three years, the CRE strains in this hospital mainly included Klebsiella pneumoniae with blaKPC-2 gene and Escherichia coli with blaNDM-5 gene. According to the results of this test, we can reasonably select antimicrobial agents in combination with the drug sensitivity report from the microbial laboratory, so as to delay the growth of drug-resistant strains and prevent hospital transmission of multidrug-resistant bacteria.
ObjectiveTo study the relation between the clinical phenotype and neurological developmental quotient in children with epilepsy and GPR98 gene mutation. MethodsGenomic DNA was extracted from peripheral blood lymphocytes of the probands and other available members in the epilepsy families.Clinical datas and screened for mutations by next-generation sequencing conbined target sequencing technology and PCR and direct DNA sequencing were collected.Then, the relations between the clinical phenotype and developmental quotient in children with epilepsy and GPR98 gene mutation was analyzed. ResultsSeven novel GPR98 gene mutations were found in seven probands in 65 families, including six heterozygote missense mutations (c.6083C <、c.1969A < C、c.17531C < T、c.9069G < C、c.6661G < A and c.18496A < C) and one nonsense mutation (c.14224G < T). One of their parents carried the same GPR98 gene mutation as the proband's. The initial symptom of six cases was afebrile seizures and one showed febrile seizure, in which the main type seizure was generalized seizure.Moreover, was were significant difference between children with epilepsy and GPR98 gene mutations and healthy children in developmental quotient test(P < 0.01). ConclusionsThe main type of seizures in children with epilepsy and GPR98 gene mutations is generalized seizure. Furthermore, GPR98 gene mutations may be associated with psychomotor retardation.
ObjectiveTo observe and analyze the clinical phenotype and genetic characteristics of COL2A1 and COL11A1 de novo mutation (DNM) related Stickler syndrome type Ⅰ and Ⅱ patients. MethodsA family-based cohort study. From December 2023 to November 2024, 4 patients (all probands) with Stickler syndrome diagnosed by clinical and genetic testing in Department of Ophthalmology of People's Hospital of Ningxia Hui Autonomous Region and their parents (8 cases) were included in the study. The patients came from 4 unrelated families. A detailed medical history was taken, and the patients underwent best-corrected visual acuity (BCVA), refraction, and fundus color photography examinations. Systemic examinations included the oral and facial regions, skeletal, joints, and hearing. Peripheral venous blood samples were collected from the patients and their parents, and genomic DNA was extracted. Whole-exome sequencing was used to screen for pathogenic genes and their loci, which were then validated by Sanger sequencing and combined with segregation analysis in the families to identify candidate gene mutation sites. The candidate variants were assessed for pathogenicity according to the American College of Medical Genetics and Genomics (ACMG) criteria and guidelines for the classification of genetic variants. Additionally, cross-species conservation analysis was performed to determine the evolutionary conservation of wild-type amino acids, and protein three-dimensional modeling techniques were used to characterize the spatial conformational changes of the variant proteins and the alterations in their local hydrogen bond networks. ResultsAmong the 4 patients, there were 2 males and 2 females; their ages ranged from 3 to 12 years. There were 2 cases of Stickler syndrome type Ⅰ (proband of families 1 and 2) and 2 cases of type Ⅱ (proband of families 3 and 4). The diopters ranged from ?8.00 to?18.00 D. BCVA ranged from no light perception to 0.6-. There were 2 cases each of vitreous membrane-like and “bead-like” opacity. Three cases showed peripapillary atrophy arcs and leopard pattern changes in the retina; one case had bilateral retinal detachment with a large macular hole in the left eye, which had previously been treated with vitrectomy surgery. One case had bilateral sensorineural hearing loss. There were 3 cases of simple micrognathia; one case had a flat nasal bridge, short nose, midface depression, and micrognathia. Two cases had excessive elbow joint extension. The phenotypes of the parents of the 4 patients were normal. Genetic testing results revealed that the probands of families 1 and 2 carried COL2A1 gene c.85+1G>C (M1) splice site variant and c.3950_3951insA (p.M1317Ifs*48) (M2) frameshift variant, respectively; the probands of families 3 and 4 carried COL11A1 gene (NM_001854.4) c.2549 G>T (p.G850V) (M3) missense variant and c.3816+6T>C (M4) splice site variant, respectively. The parents did not carry the related gene variants. Among them, M2, M3, and M4 are newly reported DNM. According to the ACMG guidelines, they were all considered likely pathogenic. The cross-species conservation analysis results showed that the wild-type amino acid of the COL11A1 gene M3 missense variant was highly conserved across multiple different species. Protein local structure modeling analysis revealed that the COL2A1 gene M2 frameshift variant and the COL11A1 gene M3 missense variant significantly altered the tertiary structure conformation of the protein, leading to abnormal spatial arrangement and hydrogen bond network in the key functional domains ConclusionThe COL2A1 gene M1 splice site variant, M2 frameshift variant, and the COL11A1 gene M3 missense variant, M4 splice site variant are respectively the potential pathogenic genes for families 1, 2, and families 3, 4; leading to the onset of Stickler syndrome type Ⅰ in families 1 and 2, and type Ⅱ in families 3 and 4.
To aggressively proliferate and metastasize, cancer cells are in extreme need of energy supply and nutrients. Therefore, a promising cancer therapy strategy is developed to target its hallmark feature of metabolism. Recent findings revealed the regulatory role of caveolin-1 (Cav-1), a structural protein of caveolae, in cancer metabolism. And low Cav-1 expression in tumor stroma was proved to be a central player of cancer malignant phenotype. Here, we summarized the progressions of studies on Cav-1, mitochondria and cancer metabolism to indicate that the altered metabolism induced by Cav-1 and mitochondria association is a major cause of cancer malignant phenotype.
Objective To investigate the effects of in-vitro monolayer culture and three-dimensional (3-D) alginate microsphere culture on the differentiation of normal human nucleus pulposus cells (NPCs), and to discuss the regulatory mechanism of restoring the phenotype of dedifferentiated NPCs by culturing resveratrol (RES) in 3-D alginate microsphere. Methods Normal human nucleus pulposus tissues were harvested for culture and identification of NPCs from 6 patients with burst lumbar vertebra fracture. NPCs at passages 1, 3, 5, and 7 in the in-vitro monolayer culture were harvested to observe the morphology, cell aging, and proteoglycan expression. The cell proliferation rates of NPCs at passage 1 in-vitro in monolayer culture and in 3-D alginate microsphere culture were detected. NPCs at passage 7 were randomly divided into 3-D alginate microsphere control group (group A), RES group (group B), silent mating type information regulation 2 homolog 1 (SIRT1)- small interfering RNA (siRNA) + RES group (group C), and negative control-siRNA + RES group (group D); and NPCs in the in-vitro monolayer culture was monolayer control group (group E). After corresponding treatment, Western blot was used for determining the protein expressions of SIRT1, Aggrecan, and collagen type II; real-time fluorescence quantitative PCR was used for detecting SIRT1 mRNA expression. Results The cultured cells were identified to be NPCs. Morphological observation, senescence-associated β-galactosidase (SA-β-gal) staining, and toluidine blue staining showed that dedifferentiation of normal NPCs tended to occur under continuous in-vitro monolayer culture, which was more obvious with increase of passage number. NPCs in 3-D alginate microsphere culture showed significantly lower proliferation rate than NPCs in the in-vitro monolayer culture (P lt; 0.05), but it could significantly improve the protein expressions of collagen type II and Aggrecan in dedifferentiated NPCs, showing significantly difference between groups E and A (P lt; 0.05). The protein expressions of SIRT1, collagen type II, and Aggrecan in group B were significantly improved when compared with that in group A (P lt; 0.05). Real-time fluorescence quantitative PCR and Western blot showed that the expressions of SIRT1 mRNA and proteins in group C were significantly inhibited after transfected with SIRT1-siRNA when compared with those in groups B and D (P lt; 0.05), and the protein expressions of collagen type II and Aggrecan in group C were significantly lower than those in groups B and D (P lt; 0.05). Conclusion Continuous in-vitro monolayer culture could efficiently cultivate numerous seeding NPCs, but it is liable to dedifferentiate. In 3-D alginate microsphere culture, RES could restore the phenotype of dedifferentiated NPCs and synthesize more extracellular matrix, which is related to the regulation of SIRT1.