Screening the pathogenic causes of congenital cataract via whole exome sequencing technology in three families: Molecular genetics of congenital cataract.

Congenital cataract is the commonest cause of visual impairment and blindness in children worldwide. Among congenital cataract cases, ~25% are caused by genetic defects, while several genetic mutations have been identified in hereditary cataract. In the present study, a patient with cataract underwent clinical ophthalmic examination and pedigree analysis. Whole exome sequencing and Sanger sequencing were performed to identify and verify gene mutations. The frequency, conservation, pathogenicity and hydrophobicity of the mutated amino acids were analyzed by bioinformatics analysis. The clinical examination and investigation verified that the probands of family A and C suffered from nuclear cataracts. In addition, the proband of family B was diagnosed with white punctate opacity. The pattern of inheritance was autosomal dominant. The sequencing analysis results revealed a mutation c.592-c593insG (p.W198Wfs*22) in exon 6 of CRYBA1/A3, a known mutation c.463C > T (p.Q155X) in exon 6 of CRYBB2 and a third mutation c.865­c.866insC (p.T289Tfs*91) in exon 2 of GJA8. Each variant was co­segregated with disease in family And the mutation frequency in the database was <0.01. It has been reported that the mutation sites are highly conserved among different species, thus greatly affecting the sequence and structure of a protein, while exhibiting high pathogenicity in theory. The two crystallin gene mutations could notably enhance the local hydrophobicity of the protein, eventually resulting in its reduced solubility and destruction of lens transparency. The current study identified pathogenic genes in three families with congenital cataract and analyzed the association between mutation sites and different cataract phenotypes. Overall, the results could expand the genotype spectrum of congenital cataract and provide evidence for its clinical diagnosis.

Efficacy and Safety of Tocilizumab Treatment COVID-19 Patients: A Case-Control Study and Meta-Analysis.

INTRODUCTION: As the pandemic progresses, the pathophysiology of COVID-19 is becoming more apparent, and the potential for tocilizumab is increasing. However, the clinical efficacy and safety of tocilizumab in the treatment of COVID-19 patients remain unclear. METHODS: To assess the efficacy and safety of tocilizumab treatment in COVID-19 patients, we performed a retrospective case-control study. The study was conducted, including 95 patients treated with tocilizumab plus standard treatment and matched controls with 95 patients treated with standard treatment therapy by propensity score from February to April 2020. We searched some databases using the search terms for studies published from January 1, 2020, to June 1, 2021. RESULTS: Our case-control study found a lower mortality rate in the tocilizumab treatment group than in the standard treatment group (9.47% versus 16.84%, P = 0.134), but the results were not statistically significant. We also found that the mortality rate in tocilizumab treatment groups was significantly lower than in the standard treatment group in the stratified ICU analysis (OR 0.52, 95% CI 0.44-0.61, P = 0.048 and OR 0.31, 95% CI 0.10-0.99, P = 0.044). We selected 49 studies (including 6568 cases and 11,660 controls) that met the inclusion criteria in the meta-analysis. In the overall analysis, we performed a meta-analysis that showed significantly decreased mortality after patients received tocilizumab (OR 0.81, 95% CI 0.69-0.95, P = 0.008). We also revealed significant associations within some subgroups. The sequential trial analysis showed a true-positive result. No significant associations were observed between tocilizumab and elevated secondary infection risk, discharge, adverse events, and mechanical ventilation in the overall analysis. CONCLUSION: Tocilizumab significantly decreased mortality in COVID-19 patients with no increased discharge, secondary infection risk, adverse events, and mechanical ventilation in a meta-analysis. Our data suggest that clinicians should pay attention to tocilizumab therapy as an effective and safe treatment for COVID-19 patients.

The paradoxical problem with COVID-19 ocular infection: Moderate clinical manifestation and potential infection risk.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which induced mainly the respiratory damage also caused ocular surface symptoms. However, the detailed description of ocular manifestations, severity fluctuations in confirmed COVID-19 adult patients still lacked. We analyzed onset clinical symptoms and duration, ocular symptoms, needs for medication, outcomes in 28 conjunctivitis patients who were extracted from 3198 COVID-19 patients hospitalized in Huoshenshan Hospital and Taikangtongji Hospital, Wuhan, China. The expression levels of ACE2, TMPRSS2, ANPEP, DPP4, NRP1 on fetal and adult ocular surface and mouse lacrimal glands were assessed by single cell seq analysis. Our results indicated that conjunctivitis was a rare and self-limited complication in adults with COVID-19 while the existence of coronavirus receptors on human ocular surface and mouse lacrimal glands indicated the risk of SARS-CoV-2 infection. Our research firstly examined SARS-CoV-2 receptors, including the new discovered one, NRP1, on the fetal ocular surface and in the mouse lacrimal glands.

[Correlation between microRNA-34b/c single nucleotide polymorphism rs4938723 and male infertility].

OBJECTIVE: To investigate the relationship between microRNA-34b/c single nucleotide polymorphism (SNP) rs4938723 and the risk of male infertility. METHODS: This case-control study included 553 males aged 19－40 (29.42 ± 5.09) years with idiopathic infertility, 153 with azoospermia and 400 with oligoasthenospermia, and another 332 normal fertile men aged 19－40 (28.5 4 ± 4.63) years as controls. We collected the clinical data and peripheral blood samples from the subjects, genotyped microRNA-34b/c rs4938723 by Sequenom MassARRAY, and analyzed the relationship between the genotypes of microRNA-34b/c rs4938723 and the risk of male infertility using the logistic regression model. RESULTS: Statistically significant differences were observed between the infertility patients and normal controls in sperm concentration (ï¼»18.71 ± 15.19ï¼½ vs ï¼»79.91 ± 43.96ï¼½ × 106/ml, P < 0.01), the percentage of progressively motile sperm (ï¼»13.27 ± 24.52ï¼½% vs ï¼»42.82 ± 8.86ï¼½%, P < 0.01) and the level of follicle stimulating hormone (ï¼»16.09 ± 17.37ï¼½ vs ï¼»12.20 ± 4.73ï¼½ IU/L, P < 0.01). Compared with the TT genotype, the TC and CC genotypes showed no correlation with male infertility, nor did the genetic locus in the subgroup analysis. CONCLUSIONS: No correlation was found between microRNA-34b/c SNP rs4938723 and male infertility, which, however, needs to be further verified by larger-sized samples.

[Detection of DPY19L2 gene mutation in 2 cases of globozoospermia].

OBJECTIVE: To investigate the mutation of the DPY19L2 gene in patients with globozoospermia. METHODS: We collected the clinical data and peripheral blood from 2 patients with globozoospermia and screened for mutation of the DPY19L2 gene by PCR amplification and DNA sequencing technology. RESULTS: The sperm from the 2 globozoospermia patients were round morphologically under the light microscope, with deeply stained nuclei but no acrosome. Electron microscopy showed the sperm with a large round head but no acrosomal structure, the nuclei enveloped by a single layer of membrane and the cytoplasm dispersed. PCR amplification revealed homozygous deletion of Exon 5, Exon6 and Exon15 in the DPY19L2 gene in both the patients. CONCLUSIONS: This study proved that the homozygous mutation of DPY19L2 could lead to globozoospermia, which has an important significance for researches on the molecular mechanisms and gene diagnosis of the disease as well as for clinicians in genetic counseling and treatment.

Association of single-nucleotide polymorphisms in antioxidant genes and their gene-gene interactions with risk of male infertility in a Chinese population.

The antioxidant defense system protects DNA from the damaging effects of oxidative stress and is hypothesized to be associated with an increased risk of male infertility. Polymorphisms in antioxidant genes and the gene-gene interactions associated with the antioxidant system may increase the potential risk of male infertility. In the present case-controlled study, the individual link between seven gene polymorphisms (NQO1 rs1800566, SOD2 rs4880, GSTM3 rs1571858, rs3814309, rs7483, GSTM5 rs11807 and GSTP1 rs1695) and the risk of male infertility was investigated. A total of 248 idiopathic infertility patients and 310 fertile controls were selected, and genotyping was performed using the Mass ARRAY platform. There were no significant associations between the seven polymorphisms and risk of male infertility. However, the analysis of gene-gene interactions showed a decreased risk of male infertility in GSTM3 rs3814309/NQO1 rs1800566 [CC x CT/TT; odds ratio (OR)=0.56, 95% confidence interval (CI)=0.34-0.92; P=0.022), and a significant association between a gene-gene interaction in GSTM3 rs1571858/NQO1 rs1800566 and azoospermia (AG/GG x CC; OR=3.84, 95% CI=1.25-11.81; P=0.019).

Potential False-Positive and False-Negative Results for COVID-19 IgG/IgM Antibody Testing After Heat-Inactivation.

Objectives: With the worldwide spread of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), various antibody detection kits have been developed to test for SARS-CoV-2- specific IgG, IgM, and total antibody. However, the use of different testing methods under various heat-inactivation conditions might affect the COVID-19 detection results. Methods: Seven different antibody detection kits produced by four manufacturers for detection of SARS-CoV-2 IgG, IgM, and total antibody were tested at Wuhan Huoshenshan Hospital, China. Most of the kits used the indirect immunity, capture, and double-antigen sandwich methods. The effects of various heat-inactivation conditions on SARS-CoV-2-specific IgG, IgM, and total antibody detection were analyzed for the different test methods. Results: Using the indirect immunity method, values for SARS-CoV-2 IgG antibody significantly increased and those for IgM antibody decreased with increasing temperature of heat-inactivation using indirect immunity method. However, values for SARS-CoV-2 IgM and total antibody showed no change when the capture and double-antigen sandwich methods were used. The changes in IgG and IgM antibody values with the indirect immunity method indicated that heat-inactivation could affect COVID-19 detection results obtained using this method. In particular, 18 (22.2%) SARS-CoV-2 IgM positive samples were detected as negative with heat-inactivation at 65°C for 30 min, and one (25%) IgG negative sample was detected as positive after heat-inactivation at 56°C for 60 min and 60°C for 30 min. Conclusions: Heat-inactivation could increase SARS-CoV-2 IgG antibody values, and decrease IgM antibody values, causing potential false-positive or false-negative results for COVID-19 antibody detection using the indirect immunity method. Thus, before conducting antibody testing, the testing platforms should be evaluated in accordance with the relevant requirements to ensure accurate COVID-19 detection results.

Heavy Metal Exposure Leads to Rapid Changes in Cellular Biophysical Properties.

Biophysical properties of cells, such as cell mechanics, cell shape, and cell migration, are emerging hallmarks for characterizing various cell functions. Conversely, disruptions to these biophysical properties may be used as reliable indicators of disruptions to cell homeostasis, such as in the case of chemical-induced toxicity. In this study, we demonstrate that treatment of lead(II) nitrate and cadmium nitrate leads to dosage-dependent changes in a collection of biophysical properties, including cellular traction forces, focal adhesions, mechanical stiffness, cell shape, migration speed, permeability, and wound-healing efficacy in mammalian cells. As those changes appear within a few hours after the treatment with a trace amount of lead/cadmium, our results highlight the promise of using biophysical properties to screen environmental chemicals to identify potential toxicants and establish dose response curves. Our systematic and quantitative characterization of the rapid changes in cytoskeletal structure and cell functions upon heavy metal treatment may inspire new research on the mechanisms of toxicity.

Biomechanical Microenvironment Regulates Fusogenicity of Breast Cancer Cells.

Fusion of cancer cells is thought to contribute to tumor development and drug resistance. The low frequency of cell fusion events and the instability of fused cells have hindered our ability to understand the molecular mechanisms that govern cell fusion. We have demonstrated that several breast cancer cell lines can fuse into multinucleated giant cells in vitro, and the initiation and longevity of fused cells can be regulated solely by biophysical factors. Dynamically tuning the adhesive area of the patterned substrates, reducing cytoskeletal tensions pharmacologically, altering matrix stiffness, and modulating pattern curvature all supported the spontaneous fusion and stability of these multinucleated giant cells. These observations highlight that the biomechanical microenvironment of cancer cells, including the matrix rigidity and interfacial curvature, can directly modulate their fusogenicity, an unexplored mechanism through which biophysical cues regulate tumor progression.

[Correlation of single nucleotide polymorphisms rs995030 and rs4474514 of the KITLG gene with male infertility].

OBJECTIVE: To investigate the correlation of the single nucleotide polymorphisms rs995030 and rs4474514 of the tyrosine kinase receptor-specific ligand (KITLG) gene with the risk of male infertility. METHODS: This study included 360 patients with idiopathic male infertility and 338 healthy fathers as controls, all from the surrounding areas of Nanjing. According to the 5th edition of the WHO Laboratory Manual for the Examination and Processing of Human Semen, we divided the infertility patients into an azoospermia (n = 143), a severe oligozoospermia (n = 159), and an oligozoospermia group (n = 58). We obtained the basic clinical data on all the subjects, collected genomic DNA from the peripheral blood of the patients, determined the genotypes of the KITLG gene rs995030 and rs4474514 by sequence mass-array, and analyzed the correlation between the two-point gene polymorphism and male infertility by logistic regression analysis. RESULTS: Statistically significant differences were observed between the infertility patients and normal fertile controls in sperm concentration (ï¼»13.23 ± 24.52ï¼½ vs ï¼»78.74 ± 61.25ï¼½ ×106/ml, P < 0.01), the percentage of progressively mobile sperm (ï¼»18.71 ± 15.19ï¼½% vs ï¼»39.36 ± 9.75ï¼½%, P < 0.01), and the level of FSH (ï¼»16.09 ± 17.31ï¼½ vs ï¼»4.56 ± 2.41ï¼½ IU/L, P < 0.01), but not between the genotypes and male infertility, and no correlation was found in subgroup analysis. CONCLUSIONS: The single nucleotide polymorphisms rs995030 and rs4474514 of the KITLG gene were not significantly correlated with male infertility, which is to be further verified by more studies with samples of larger size and expanded selection range.

[Correlation of the single nucleotide polymorphism rs662 of PON1 with the risk of male infertility].

Objective: To investigate the correlation between the single nucleotide polymorphism (SNP) rs662 of the paraoxonase 1 gene (PON1) and the risk of male infertility. METHODS: This case-control study included 403 male idiopathic infertility patients aged 29.00 ± 4.48 years in the case group and 329 normal fertile men aged 28.28 ± 4.08 years as healthy controls. We obtained DNA from the peripheral venous blood of the subjects, genotyped the SNP rs662 of PON1 by Sequenom MassArray, and analyzed the association between different genotypes of PON1 rs662 and male infertility using the logistic regression model. RESULTS: Compared with the normal controls, the infertility patients showed a significantly increased level of follicle-stimulating hormone (FSH) (ï¼»16.30 ± 17.76ï¼½ vs ï¼»4.72 ± 2.51ï¼½ U/L, P < 0.01) but a decreased percentage of progressively motile sperm (PMS) (ï¼»7.40 ± 14.17ï¼½ % vs ï¼»41.93 ± 9.06ï¼½ %, P < 0.01) and sperm concentration (ï¼»2.74 ± 3.64ï¼½ vs ï¼»75.83 ± 63.66ï¼½ ×106/ml, P < 0.01). Statistically significant differences were not found in the other parameters between the two groups of subjects, nor in the correlation of male infertility with the heterozygous genotype GA versus the wild homozygous genotype GG (OR = 0.98, 95% CI: 0.63－1.53, P = 0.923) or the homozygous genotype AA versus the wild homozygous genotype GG (OR = 0.87, 95% CI: 0.56－1.34, P = 0.525). CONCLUSIONS: The SNP rs662 of PON1 was not correlated with male infertility, which, however, needs to be confirmed by further studies with larger samples from a larger area.

[Correlation of the single nucleotide polymorphisms rs34349826 and rs6521 of the LHB gene with male infertility in Chinese men].

Objective: To study the relationship of the single nucleotide polymorphisms (SNP) rs34349826 (c.104 A>G) and rs6521 (c.114 C>G) of the luteinizing hormone beta-subunit (LHB) gene with male infertility in Chinese men. METHODS: This case-control study included 405 males with primary infertility (the infertility group) and 424 normal fertile men (the control group), the former again divided into subgroups of oligospermia, severe oligozoospermia and azoospermia according to the sperm concentration. Clinical data were collected from all the subjects and genomic DNA obtained from their peripheral blood for genotyping rs34349826 and rs6521 of the LHB gene by Sequence MassArray. We analyzed the correlation of male infertility with the SNPs of the two loci using the logistic regression model as well as its association with their haplotype combination with the SHEsis online software. RESULTS: There were statistically significant differences between the control and infertility groups in the semen volume (ï¼»3.51 ± 1.36ï¼½ vs ï¼»3.74 ± 1.71ï¼½ ml, P <0.05), sperm concentration (ï¼»79.21 ± 61.60ï¼½ vs ï¼»27.37 ± 30.80ï¼½ ×106/ml, P <0.01), percentage of progressively motile sperm (ï¼»39.40 ± 9.64ï¼½ % vs ï¼»11.90 ± 14.72ï¼½ %, P <0.01), and levels of serum luteinizing hormone (LH) (ï¼»3.29 ± 1.39ï¼½ vs ï¼»6.25 ± 4.83ï¼½ IU/L, P <0.01) and follicle-stimulating hormone (FSH) (ï¼»4.56 ± 2.31ï¼½ vs ï¼»15.64 ± 17.03ï¼½ IU/L, P <0.01). Logistic regression analysis revealed no correlation between male infertility and the genotypes of the rs34349826 and rs6521 loci of the LHB gene, and similar results were found in the subgroups of the infertile males. SHEsis analysis on the haplotypes of the rs34349826 and rs6521 loci showed the GG genotype combination to be a protective factor against male infertility. CONCLUSIONS: The rs34349826 and rs6521 loci of the LHB gene were not related to male infertility, which can be further confirmed by larger-sample studies. The GG genotype combination is a protective factor against male infertility.

A novel compound heterozygous mutation of the SMARCAL1 gene leading to mild Schimke immune-osseous dysplasia: a case report.

BACKGROUND: Schimke immune-osseous dysplasia (SIOD, OMIM 242900) is characterized by spondyloepiphyseal dysplasia, T-cell deficiency, renal dysfunction and special facial features. SMARCAL1 gene mutations are determined in approximately 50% of patients diagnosed with SIOD. CASE PRESENTATION: The case presented here is that of a 6-year-old boy who was born at 33 weeks to healthy, non-consanguineous Chinese parents. He presented with short stature (95 cm; <3rd percentile) and proteinuria. Initially suspected of having IgM nephropathy, the patient was finally diagnosed with mild Schimke immune-osseous dysplasia. One novel mutation (p.R817H) and one well-known mutation (p.R645C) was identified in the SMARCAL1 gene. CONCLUSION: This report describes a clinical and genetic diagnostic model of mild SIOD. It also highlights the importance of molecular testing or clinical diagnosis and the guidance it provides in disease prognosis.

Polymorphisms of protamine genes contribute to male infertility susceptibility in the Chinese Han population.

Protamine (PRM) plays important roles in the packaging of DNA within the sperm nucleus. To investigate the role of PRM1/2 and transition protein 1 (TNP1) polymorphisms in male infertility, 636 infertile men and 442 healthy individuals were recruited into this case-controlled study of the Chinese Han population, using MassARRAY technology to analyze genotypes. Our analysis showed that there were no significant differences between controls and infertile cases among the five single nucleotide polymorphisms identified in PRM1, PRM2 and TNP1 [rs737008 (G/A), rs2301365 (C/A), rs2070923 (C/A), rs1646022 (C/G) and rs62180545 (A/G)]. However, we found that the PRM1 and PRM2 haplotypes GCTGC, TCGCA and TCGCC exhibited significant protective effects against male infertility compared to fertile men, while TCGGA, GCTCC and TCGGC represented significant risk factors for spermatogenesis. Our data showed that rs737008 and rs2301365 in PRM1, and rs1646022 in PRM2, were significantly associated with male infertility and that gene-gene interaction played a role in male infertility. A linkage disequilibrium plot for the five SNPs showed that rs737008 was strongly linked with both rs2301365 and rs2070923. These findings are likely to help improve our understanding of the etiology of male infertility. Further studies should include a larger number of genes and SNPs, particularly growing critical genes; such studies will help us to unravel the effect of individual genetic factors upon male infertility.

Application of next­generation sequencing for molecular diagnosis in a large family with osteogenesis imperfecta type I.

Increased bone fragility and low bone mass are common features of osteogenesis imperfecta (OI), which is associated with connective tissue. Its type is distinguished by clinical phenotypes and molecular genetics. Although fifteen types (I­XV) of OI have been identified at present, the majority of patients are diagnosed as OI type I­IV. Type I collagen is responsible for OI type I­IV, consists of α1 (I) and α2 (I) chains and is encoded by COL1A1 and COL1A2. To identify the pathogenic gene of a large Chinese family with OI type I and explain genetic heterogeneity of the patients, next­generation sequencing (NGS) was conducted in a female with OI type I and her affected niece and daughter to search for the mutation. Subsequently, it was confirmed in other family members by Sanger sequencing. Analysis of COL1A1 gene identified a splicing mutation (c.471+1G>A, also termed IVS5+1G>A) that converted the 5' end of intron 5 from GT to AT. The current study aimed to investigate why there are different phenotypes with the same mutation observed within the same OI pedigree, and the results suggested that there may be environmental factors involved. The present study provided genetic counseling and prenatal diagnosis for the family members, however additionally provided insight into the phenotype­genotype association in OI.

The susceptibility of FSHB -211G > T and FSHR G-29A, 919A > G, 2039A > G polymorphisms to men infertility: an association study and meta-analysis.

BACKGROUND: Male infertility is a complex disorder caused by genetic, developmental, endocrine, or environmental factors as well as unknown etiology. Polymorphisms in the follicle stimulating hormone beta subunit (FSHB) (rs10835638, c.-211G > T) and follicle stimulating hormone receptor (FSHR) (rs1394205, c.-29G > A; rs6165, c.919A > G; rs6166, c.2039 A > G) genes might disturb normal spermatogenesis and affect male reproductive ability. METHODS: To further ascertain the aforementioned effects, we conducted a case-control study of 255 infertile men and 340 fertile controls from South China using the Mass ARRAY method, which was analyzed by the t-tests and logistic regression analysis using SPSS for Windows 14.0. In addition, a meta-analysis was performed by combining our results with previous reports using STATA 12.0. RESULTS: In the FSHB or FSHR gene single nucleotide polymorphism (SNP) evaluation, no statistically-significant difference was found in the frequency of allelic variants or in genotype distribution between cases and controls. However, a significant association for the comparison of GAA (P: 0.022, OR: 0.63, 95%CI: 0.43-0.94) was seen between the oligozoospermia and controls in haplotype analysis of rs1394205/rs6165/rs6166. In the meta-analysis, rs6165G allele and rs6166 GG genotype were associated with increased risk of the male infertility. CONCLUSIONS: This study suggested that FSHR GAA haplotype would exert protective effects against male sterility, which indicated that the combination of three SNP genotypes of FSHR was predicted to have a much stronger impact than either one alone. Then in the meta-analysis, a significant association was seen between FSHR rs6165, rs6166 polymorphisms and male infertility. In terms of male infertility with multifactorial etiology, further studies with larger sample sizes and different ethnic backgrounds or other risk factors are warranted to clarify the potential role of FSHB and FSHR polymorphisms in the pathogenesis of male infertility.

[Single nucleotide polymorphism of the TP53 gene is not correlated with male infertility].

OBJECTIVE: To investigate the correlation of the single nucleotide polymorphism (SNP) rs1042522 of the tumor protein p53 (TP53) gene with the risk of male infertility. METHODS: This casecontrol study included 380 male patients with idiopathic infertility and 398 normal fertile men as controls from the Nanjing area. We genotyped the SNP rs1042522 of the TP53 gene by Sequence Mass Array and analyzed the correlation of the SNP with male infertility using the logistic regression model. RESULTS: Compared with the normal controls, the patients with idiopathic infertility showed significantly decreased sperm concentration (ï¼»77.34±49.24ï¼½ vs ï¼»13.13±24.96ï¼½ ×106/ml), percentage of progressively motile sperm (ï¼»42.55±9.57ï¼½ vs ï¼»10.38±5.57ï¼½%), serum testosterone level (ï¼»14.07±5.36ï¼½ vs ï¼»11.89±4.50ï¼½ nmol/L), and folliclestimulating hormone level (ï¼»16.80±18.20ï¼½ vs ï¼»4.55±7.17ï¼½ U/L) (P < 0.05) but no statistically significant differences in other parameters. No correlation was observed between the SNP frequencies and male infertility and similar results were found in the subgroups of the cases. CONCLUSIONS: SNP rs1042522 of the TP53 gene is not significantly correlated with the risk of male infertility.