[Genetic analysis of two families with abnormal findings upon prenatal diagnosis].

OBJECTIVE: To carry out genetic analysis on two families with carriers of small terminal translocations using karyotyping analysis and genomic copy number variation sequencing (CNV-seq). METHODS: Two couples undergoing prenatal diagnosis at the Tianjin Central Hospital of Obstetrics and Gynecology respectively on April 12, 2020 and December 17, 2021 were selected as the study subjects. With informed consent, amniotic fluid and peripheral blood samples were collected and subjected to conventional karyotyping and CNV-seq analysis for the detection of chromosomal microdeletion/duplications. RESULTS: Both couples had given births to children with chromosomal aberrations previously, and both fetuses were found to have abnormal karyotypes. CNV-seq showed that they had harbored microdeletion/duplications, and their mothers had both carried balanced translocations involving terminal fragments of chromosomes. CONCLUSION: For fetuses with small chromosomal segmental abnormalities, their parental origin should be traced, and the diagnosis should be confirmed with combined genetic techniques.

Mutation spectrum of COL1A1/COL1A2 screening by high-resolution melting analysis of Chinese patients with osteogenesis imperfecta.

High-resolution melting (HRM) analysis has been shown to be a time-saving method for the screening of genetic variants. To increase the precision of the diagnosis of osteogenesis imperfecta (OI), we used HRM to explore COL1A1/COL1A2 mutations in 87 Chinese OI patients and to perform population-based studies of the relationships between their genotypes and phenotypes. Peripheral blood samples were collected from the 87 non-consanguineous probands. The coding regions and exon boundaries of COL1A1/COL1A2 were detected by HRM and confirmed by Sanger sequencing. The functional effects of mutations were predicted through bioinformatic tools. Mutations were detected in 70.3% of familial cases and 40% of sporadic cases (p < 0.01). Compared with COL1A1 mutations, patients with COL1A2 mutations were more prone to severe phenotypes. Helical mutations (caused by substitution of the glycine within the Gly-X-Y triplet domain) were more likely to occur in patients with type III and IV (p < 0.05). Haploinsufficiency mutations (caused by frameshift, nonsense, and splice-site mutations) appeared more frequently in patients with type I (p < 0.05). Compared with the Sanger sequencing and whole exome sequencing (WES), HRM was found to reduce total costs by 78%- 80% in patients who had a positive HRM separate melting curve. Our findings suggest that HRM would greatly benefit small and understaffed hospitals and laboratories, and would facilitate the accurate diagnosis and early treatment of OI in remote and less developed regions.

Pretreatment with interleukin 35-engineered mesenchymal stem cells protected against lipopolysaccharide-induced acute lung injury via pulmonary inflammation suppression.

Acute lung injury (ALI)-triggered pulmonary injury has been associated with high mortality, despite advances in drug treatment and supportive therapy. Remarkable progress has been made in attenuating the inflammatory injury associated with ALI using mesenchymal stem cells (MSCs)-based cell and gene therapy. However, to date, the benefits of interleukin-35 (IL-35)-modified MSCs in ALI intervention have not been investigated. In the present study, adult male C57BL/6 mice randomly received intravenous infusion of adipose-derived mesenchymal stem cells (ADSCs) constitutively expressing IL-35 (IL-35-GFP-ADSCs) or GFP (GFP-ADSCs) via retrovirus-mediated transduction (8 × 105 cells per mice) or isotonic saline 7 days before ALI modeling to investigate the effect and related mechanism. ALI was performed by lipopolysaccharide (LPS) inhalation for 24 h. Normal mice served as the sham group. The results indicated that compared with GFP-ADSCs, IL-35-modified ADSCs significantly increased cellular and pulmonary IL-10 and IL-35 production. Pretreatment with IL-35-ADSCs markedly reduced body weight loss, pulmonary wet/dry weight ratio and pathological injury. The PO2 was rescued to normal levels in mice that received IL-35-ADSCs. IL-35-ADSCs infusion apparently inhibited IL-6 release, protein leakage and MPO activity but greatly elevated IL-35 level in the bronchoalveolar lavage fluid (BALF). Splenic regulatory T cells in IL-35-ADSCs-pretreated mice got effective increase. Moreover, IL-35-ADSCs pretreatment remarkably inhibited neutrophil and macrophage infiltration and greatly decreased IL-6, tumor necrosis factor α (TNF-α) and Toll-like receptor 4 (TLR4) expression. In conclusion, pretreatment with IL-35-engineered ADSCs provided effective protection against LPS-induced ALI through suppression of pulmonary inflammation and, thus, might be a promising strategy to improve outcomes after ALI. The enhanced paracrine and immunosuppressive capacity of IL-35-ADSCs might contribute to their beneficial effects. However, further studies are needed to illuminate the detailed mechanism.

Pharmacokinetic Properties of Arsenic Species after Intravenous and Intragastrical Administration of Arsenic Trioxide Solution in Cynomolgus Macaques Using HPLC-ICP-MS.

A rapid and sensitive method was established for arsenic (As) speciation based on high performance liquid chromatography coupled to inductively coupled plasma mass spectrometry (HPLC-ICP-MS). This method was validated for the quantification of four arsenic species, including arsenite (AsIII), arsenate (AsV), monomethylarsonic acid (MMAV) and dimethylarsinic acid (DMAV) in cynomolgus macaque plasma. Separation was achieved in just 3.7 min with an alkyl reverse phase column and highly aqueous mobile phase containing 20 mM citric acid and 5 mM sodium hexanesulfonate (pH = 4.3). The calibration curves were linear over the range of 5⁻500 ng·mL-1 (measured as As), with r > 0.99. The above method was validated for selectivity, precision, accuracy, matrix effect, recovery, carryover effect and stability, and applied in a comparative pharmacokinetic study of arsenic species in cynomolgus macaque samples following intravenous and intragastrical administration of arsenic trioxide solution (0.80 mg·kg-1; 0.61 mg·kg-1 of arsenic); in addition, the absolute oral bioavailability of the active ingredient AsIII of arsenic trioxide in cynomolgus macaque samples was derived as 60.9 ± 16.1%.

[Genetic mutation and clinical features of osteogenesis imperfecta type V].

OBJECTIVE: To explore genetic mutations and clinical features of osteogenesis imperfecta type V. METHODS: Clinical record of five patients (including one familial case) with osteogenesis imperfecta type V were retrospectively analyzed. Peripheral blood samples of the patients, one family member, as well as healthy controls were collected. Mutation of IFITM5 gene was identified by PCR amplification and Sanger sequencing. RESULTS: A heterozygous mutation (c.-14C>T) in the 5-UTR of the IFITM5 gene was identified in all of the patients and one mother. The clinical findings included frequent fractures and spine and/or extremities deformities, absence of dentinogenesis imperfecta, absence of hearing impairment, and blue sclera in 1 case. Radiographic findings revealed calcification of the interosseous membrane between the radius-ulna in all cases. Hyperplastic callus formation was found in 3 cases. Four had radial-head dislocation. CONCLUSION: A single heterozygous mutation c.-14C>T was found in the 5-UTR of the IFITM5 gene in 5 patients with osteogensis imperfecta type V. The patients showed specific radiological features including calcification of interosseous membrane, hyperplastic callus formation, and radial-head dislocation.

[Analysis of type IV osteogenesis imperfecta caused by two mutations occurred simultaneously in COL1A1 gene in a Chinese child].

OBJECTIVE: To detect potential mutations of COL1A1 and COL1A2 genes with polymerase chain reaction-high-resolution melting analysis(PCR-HRMA) in a proband diagnosed with osteogenesis imperfecta (OI). METHODS: Peripheral blood samples were collected from the proband and members of his family as well as healthy controls. The mutations were detected by PCR-HRMA and confirmed by direct sequencing. Potential effects of the mutations were predicted using softwares including PolyPhen, SIFT and Align GVGD. RESULTS: The PCR-HRMA has indicated mutations in exon 45 of the COL1A1 gene in the proband as well as his parents, which were presented as the difference in the melting curves between the patients and the control samples. Sequencing analysis confirmed that the proband has carried two heterozygous mutations (c.3235G>A, p.Gly1079Ser and c.3247G>A, p.Ala1083Thr) in exon 45 of the COL1A1 gene. Among them, c.3235G>A was predicted to have impeded alpha helix structure domain, which was inherited from the father who also had OI. c.3247G>A was inherited from mother who had a normal phenotype. All three softwares predicted that the c.3235G>A mutation can interfere with the function of the protein, while the c.3247G>A may have a benign effect by PolyPhen analysis. CONCLUSION: The study identified two mutations (c.3235G>A and c.3247G>A) occurred simultaneously in COL1A1 gene in a case. The case is the first reported in human collagen mutation database. As identified,mutation of c.3235G>A may be the major cause of the disease in the proband.

Improvement of Inflammation and Abnormal Vascularization by TSP1 Treatment Combined with ADSCs Transplantation in Mice with Induced Polycystic Ovary Syndrome.

Polycystic ovary syndrome (PCOS) is a common gynecological endocrine disease with a certain degree of chronic inflammation and abnormal ovarian angiogenesis in reproductive women. Mesenchymal stem cells (MSCs) have potent immunomodulatory properties to regulate ovarian function, while thrombospondin 1 (TSP1) improves the abnormal formation of ovarian vessels. The present study investigated the efficacy of the combined use of adipose-derived mesenchymal stem cells (ADSCs) and TSP1 in PCOS mice. The PCOS model is established using dehydroepiandrosterone (DHEA) by subcutaneous injection. Ovarian apoptosis is assessed using terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL). Real-time quantitative PCR (RT-PCR) and western blotting are used to detect the expression of inflammatory factors and the levels of angiogenesis-related factors in ovarian tissues. Inflammatory cells count and ovarian angiogenesis are evaluated by immunofluorescence staining. This research shows that TSP1 and ADSCs treatment can significantly reduce the inflammatory state of PCOS mice, relieve the degree of ovarian cell apoptosis, optimize the ovarian histological manifestations, and restore the levels of related hormones. The proportion of CD31-positive cells in PCOS mice returned to near-normal levels. The synergistic use of ADSCs and TSP1 therapy can exert a more impressive effect by inhibiting the ovarian inflammatory response and regulating the balance of angiogenesis than the single application in PCOS mice.

The causal relationship between anti-diabetic drugs and gastrointestinal disorders: a drug-targeted mendelian randomization study.

BACKGROUND: The incidence of diabetic gastrointestinal diseases is increasing year by year. This study aimed to investigate the causal relationship between antidiabetic medications and gastrointestinal disorders, with the goal of reducing the incidence of diabetes-related gastrointestinal diseases and exploring the potential repurposing of antidiabetic drugs. METHODS: We employed a two-sample Mendelian randomization (TSMR) design to investigate the causal association between antidiabetic medications and gastrointestinal disorders, including gastroesophageal reflux disease (GERD), gastric ulcer (GU), chronic gastritis, acute gastritis, Helicobacter pylori infection, gastric cancer (GC), functional dyspepsia (FD), irritable bowel syndrome (IBS), ulcerative colitis (UC), Crohn's disease (CD), diverticulosis, and colorectal cancer (CRC). To identify potential inhibitors of antidiabetic drug targets, we collected single-nucleotide polymorphisms (SNPs) associated with metformin, GLP-1 receptor agonists, SGLT2 inhibitors, DPP-4 inhibitors, insulin, and its analogs, thiazolidinediones, sulfonylureas, and alpha-glucosidase inhibitors from published genome-wide association study statistics. We then conducted a drug-target Mendelian randomization (MR) analysis using inverse variance weighting (IVW) as the primary analytical method to assess the impact of these inhibitors on gastrointestinal disorders. Additionally, diabetes was selected as a positive control. RESULTS: Sulfonylureas were found to significantly reduce the risk of CD (IVW: OR [95% CI] = 0.986 [0.978, 0.995], p = 1.99 × 10- 3), GERD (IVW: OR [95% CI] = 0.649 [0.452, 0.932], p = 1.90 × 10- 2), and chronic gastritis (IVW: OR [95% CI] = 0.991 [0.982, 0.999], p = 4.50 × 10- 2). However, they were associated with an increased risk of GU development (IVW: OR [95%CI] = 2 0.761 [1.259, 6.057], p = 1 0.12 × 10- 2). CONCLUSIONS: The results indicated that sulfonylureas had a positive effect on the prevention of CD, GERD, and chronic gastritis but a negative effect on the development of gastric ulcers. However, our research found no causal evidence for the impact of metformin, GLP-1 agonists, SGLT2 inhibitors, DPP 4 inhibitors, insulin and its analogs, thiazolidinediones, or alpha-glucosidase inhibitors on gastrointestinal diseases.

Large chromosomal deletions and impaired homologous recombination repairing in HEK293T cells exposed to polychlorinated biphenyl 153.

BACKGROUND: Polychlorinated biphenyls (PCBs) are persistent pollutants with carcinogenesis and mutagenesis effects which have been closely associated with PCBs-induced DNA damage. However, the detailed DNA damage events and corresponding pathway alterations under PCBs poisoning is still not well understood. METHODS: Whole-genome sequencing (WGS) and RNA sequencing (RNA-seq) were used to explore genome wide variations and related pathway changes in HEK293T cells that challenged by 15 µM PCB153 for 96 h in vitro. Double strand breaks (DSBs) were measured by 53BP1 foci detection, altered pathways were confirmed by quantitative real-time PCR (qPCR). RESULTS: The results indicated that abundant copy number variations (CNVs), including four duplications and 30 deletions, occurred in PCB153-exposed HEK293T cells. Multiple large fragment deletions (>1 Mb) involving up to 245 Mb regions on many chromosomes. Missense mutations were found in six tumor susceptibility genes, two of which are key members participating in homologous recombination (HR) repair response, BRCA1 and BRCA2. RNA-seq data showed that PCB153 poisoning apparently suppressedHR repairing genes. Besides, 15 µM PCB153 exposure significantly increased 53BP1 foci formation and effectively reduced BRCA1, RAD51B and RAD51C expression, indicating an elevated DSBs and impaired HR repairing. CONCLUSION: This study firstly reported multiple large chromosomal deletions and impaired HR repairing in PCB153-exposed HEK293T cells, which provided a new insight into the understanding of early response and the mechanism underlying PCB153 genotoxicity. The chromosomal instabilities might be related to the impaired HR repairing that induced by PCB153; however, further investigations, especially on actual toxic effects of human body, are needed to confirm such speculation.

Modification of COL1A1 in Autologous Adipose Tissue-Derived Progenitor Cells Rescues the Bone Phenotype in a Mouse Model of Osteogenesis Imperfecta.

Osteogenesis imperfecta (OI) is a congenital genetic disorder mainly manifested as bone fragility and recurrent fracture. Mutation of COL1A1/COL1A2 genes encoding the type I collagen are most responsible for the clinical patients. Allogenic mesenchymal stem cells (MSCs) provide the potential to treat OI through differentiation into osteoblasts. Autologous defective MSCs have not been utilized in OI treatment mainly because of their impaired osteogenesis, but the latent mechanism has not been well understood. Here, the relative signaling abnormality of adipose-derived mesenchymal stem cells (ADSCs) isolated from OI type I mice (Col1a1+/-365 mice) was explored. Autologous ADSCs transfected by retrovirus carrying human COL1A1 gene was first utilized in OI therapy. The results showed that decreased activity of Yes-associated protein (YAP) due to hyperactive upstream Hippo kinases greatly contributed to the weakened bone-forming capacity of defective ADSCs. Recovered collagen synthesis of autologous ADSCs by COL1A1 gene modification normalized Hippo/YAP signaling and effectively rescued YAP-mediated osteogenesis. And the COL1A1 gene engineered autologous ADSCs efficaciously improved the microstructure, enhanced the mechanical properties and promoted bone formation of Col1a1+/-365 mice after femoral bone marrow cavity delivery and might serve as an alternative source of stem cells in OI treatment. © 2021 American Society for Bone and Mineral Research (ASBMR).

The synergistic effect of NELL1 and adipose-derived stem cells on promoting bone formation in osteogenesis imperfecta treatment.

BACKGROUND: Osteogenesis imperfecta (OI) is a rare genetic disorder characterized by bone fragility and deformity. Mesenchymal stem cells (MSCs) infusion can improve bone performance mainly due to their differentiation into osteoblasts in OI therapy. The osteoinductive activity of NELL1 have benefited various bone defect and osteoporotic models by promoting bone formation. The present study investigated the efficacy of combined use of NELL1 and adipose-derived mesenchymal stem cells (ADSCs) in OI treatment. METHODS: Lentiviral vector carrying mouse Nell1 gene was constructed and lentivirus were used to infect ADSCs. The osteogenic capacity of MC3T3-E1 and ADSCs stimulated by recombinant mouse NELL1 protein (rmNELL1) and Nell1 gene genetically modified ADSCs (lenti-Nell1-ADSCs) were estimated by real-time quantitative PCR. Thirty adult male OI type I mice with single Col1a1 gene knockout were randomly divided into five groups and received intravenously injected PBS, rmNELL1 (1.25 mg/Kg), ADSCs (2 × 105 cells per mice), rmNELL1 (1.25 mg/Kg) combined with ADSCs (2 × 105 cells per mice), or lenti-Nell1-ADSCs (2 × 105 cells per mice) respectively. Six wildtype (WT) mice served as positive control. Bone formation was examined after 4 weeks using micro-CT, histological and immunohistochemical methods. RESULTS: Three osteoblast related genes of MC3T3-E1 and ADSCs were significantly up-regulated by rmNELL1 in vitro. Lenti-Nell1-ADSCs showed greatly enhanced osteogenic differentiation capacity. The infused lenti-Nell1-ADSCs could migrate to femur and differentiate into ALPL-positive cells. Systemic administration of rmNELL1 combined with ADSCs or lenti-Nell1-ADSCs markedly improved the femoral microstructure and promoted bone formation through increasing the ALPL and osteocalcin (OCN) expression, much better than mice that received single rmNELL1 or ADSCs. And Nell1 gene engineered ADSCs achieved slightly better outcomes than that of combinative use of rmNELL1 and ADSCs. CONCLUSIONS: NELL1 and ADSCs exhibited synergistic effect on stimulating bone formation of OI mice, which might provide an alternative strategy in OI treatment. Compared with dose escalation or multiple administration of rmNELL1, lentivirus-mediated long term expression of NELL1 might be more feasible and convenient. However, further studies are needed to confirm the safety and optimize the therapeutic regime.

Interleukin 35 induced Th2 and Tregs bias under normal conditions in mice.

OBJECTIVE: The benefits of IL-35 treatment have been verified in multiple animal models of diseases, while its influence on T cells immunity under normal condition still needs to be elucidated. The present study was designed to investigate the effects modulating IL-35 levels in vivo and in vitro on T cells, response and also the effects on T cells subsets in normal mice. METHODS: A plasmid pMSCV-IL-35-GFP carrying mouse linear IL-35 fragment with two subunits joint together was constructed and the heterodimer expression was confirmed. Normal mice were randomly divided into three groups and received an intravenous injection of PBS, pMSCV-GFP and pMSCV-IL-35-GFP respectively. After 72 h, spleen tissues and peripheral blood were harvested for following analysis. Meanwhile, splenic T cells were isolated and incubated with 10, 30, or 50 ng/mL recombinant IL-35 factor for 24 h with the addition of anti-CD3/CD28 in vitro. T-cell subsets were assessed by Fluorescence activated cell sorting (FACS) and related cytokines together with effector molecules were determined by real time PCR. RESULTS: Western blotting confirmed a 52 kDa band in the cell lysate of HEK 293T transducted with pMSCV-IL-35-GFP plasmid, indicating a successful expression of IL-35. Ebi3 and IL-12A, two subunits of IL-35, could be identified 72 h post DNA injection. IL-35 upregulation in vivo effectively inhibit CD4+ and CD8+ T cell proliferation and Th1 cytokine secretion. Effector molecules of CD8+ T cells were also remarkably suppressed. On the contrary, high level of IL-35 significantly induced CD4+ CD25+ Tregs and Th2 enhancement. The in vitro study provided similar results. CONCLUSION: The results indicated Th1 and CD8+ T cell inhibition and Th2 and Tregs bias in the presence of IL-35 under a normal state which partly contributed to its therapeutic potential.

The correlation between the expression of genes involved in drug metabolism and the blood level of tacrolimus in liver transplant receipts.

Immunosuppressive medications, such as tacrolimus and mycophenolate mofetil, are commonly used for reducing the risk of organ rejection in receipts of allogeneic organ transplant. The optimal dosages of these drugs are required for preventing rejection and avoiding toxicity to receipts. This study aimed to identify the correlation between the expression profiling of genes involved in drug metabolism and the blood level of tacrolimus in liver transplant receipts. Sixty-four liver transplant receipts were enrolled in this retrospective study. Receipts were divided into low (2-5.9 ng/ml) and high (6-15 ng/ml) tacrolimus groups. Clinical assessment showed that the blood level of tacrolimus was inversely correlated with the liver function evaluated by blood levels of total bilirubin and creatinine. Compared to the high tacrolimus group, expression levels of six cytochrome P450 enzymes, CYP1A1, CYP2B6, CYP3A5, CYP4A11, CYP19A1, and CYP17A1 were significantly higher in the low tacrolimus group. The expression levels of these genes were negatively correlated with the tacrolimus blood level. Enzyme assays showed that CYP3A5 and CYP17A1 exerted direct metabolic effects on tacrolimus and mycophenolate mofetil, respectively. These results support clinical application of this expression profiling of genes in drug metabolism for selection of immunosuppressive medications and optimal dosages for organ transplant receipts.