Abnormal function of EPHA2/p.R957P mutant in congenital cataract.

AIM: To identify genetic defects in a Chinese family with congenital posterior polar cataracts and assess the pathogenicity. METHODS: A four-generation Chinese family affected with autosomal dominant congenital cataract was recruited. Nineteen individuals took part in this study including 5 affected and 14 unaffected individuals. Sanger sequencing targeted hot-spot regions of 27 congenital cataract-causing genes for variant discovery. The pathogenicity of the variant was evaluated by the guidelines of American College of Medical Genetics and InterVar software. Confocal microscopy was applied to detect the subcellular localization of fluorescence-labeled ephrin type-A receptor 2 (EPHA2). Co-immunoprecipitation assay was implemented to estimate the interaction between EphA2 and other lens membrane proteins. The mRNA and protein expression were analyzed by reverse transcription-polymerase chain reaction (qRT-PCR) and Western blotting assay, respectively. The cell migration was analyzed by wound healing assay. Zebrafish model was generated by ectopic expression of human EPHA2/p.R957P mutant to demonstrate whether the mutant could cause lens opacity in vivo. RESULTS: A novel missense and pathogenic variant c.2870G>C was identified in the sterile alpha motif (SAM) domain of EPHA2. Functional studies demonstrated the variant's impact: reduced EPHA2 protein expression, altered subcellular localization, and disrupted interactions with other lens membrane proteins. This mutant notably enhanced human lens epithelial cell migration, and induced a central cloudy region and roughness in zebrafish lenses with ectopic expression of human EPHA2/p.R957P mutant under differential interference contrast (DIC) optics. CONCLUSION: Novel pathogenic c.2870G>C variant of EPHA2 in a Chinese congenital cataract family contributes to disease pathogenesis.

A recognition survey of granular corneal dystrophy type 2 genetic detection in China.

AIM: To evaluate the feasibility of promoting genetic detection for granular corneal dystrophy type 2 (GCD2) by a questionnaire conducted among citizens in five cities in China. METHODS: The data were collected by questionnaire, and analyzed by Chi-square test and one-tailed t test in IBM SPSS statistics. RESULTS: Based on the survey data on the awareness of GCD2 genetic detection in this study and the positive predictive analysis report of the citizens in five cities in China, the vast majority (84.2%) of respondents had never heard of it and did not know that GCD2 patients have been prohibited from performing excimer surgery that can deteriorate GCD2 patients' condition even leading to blindness. Though 3.4% of patients understood GCD2 very much, they have no idea that GCD2 could not be 100% accuracy diagnosed by the conventional inspection methods. CONCLUSION: It is feasible and necessary to use GCD2 genetic detection as an excimer preoperative examination project. In order to promote the development of detection project, a few improvements should be carried out in terms of the promoting efforts, costs, and research progress.

A recurrent G367R mutation in MYOC associated with juvenile open angle glaucoma in a large Chinese family.

AIM: To identify the mutations of MYOC, OPTN, CYP1B1 and WDR36 in a large Chinese family affected by juvenile open angle glaucoma (JOAG). METHODS: Of 114 members of one family were recruited in this study. Blood samples from twelve members of this pedigree were collected for further research. As a control, 100 unrelated subjects were recruited from the same hospital. The exon and flanking intron sequences of candidate genes were amplified using the polymerase chain reaction and direct DNA sequencing. RESULTS: The proband (III:10) was a seventy-three years old woman with binocular JOAG at the age of 31. A recurrent heterozygous mutation (c.1099G>A) of MYOC was identified in the three JOAG patients and another suspect. This transition was located in the first base pair of codon 367 (GGA>AGA) in exon 3 of MYOC and was predicted to be a missense substitution of glycine to arginine (p.G367R) in myocilin. Mutations in OPTN, CYP1B1 or WDR36 were not detected in this study. The G367R mutation was not present in unaffected family members or in 100 ethnically matched controls. Other variants of the coding regions of candidate genes were not detected in all participants. To date, this family was the largest to have been identified as carrying a certain MYOC mutation in China, further evidence of a founder effect for the G367R MYOC mutant was provided by our data. CONCLUSION: A MYOC c.1099G>A mutation in an autosomal dominant JOAG family is identified and the characteristic phenotypes among the patients are summarized. Genetic testing could be utilized in high-risk populations and be helpful not only for genetic counseling, but also for early diagnosis and treatment of affected patients or carriers of inherited JOAG.

Overexpression of AQP5 Was Detected in Axillary Sweat Glands of Primary Focal Hyperhidrosis Patients.

BACKGROUND: The expression of aquaporin 5 (AQP5) in human axillary sweat glands has never been studied so far. OBJECTIVE: To detect the expression of AQP5 in axillary sweat glands of patients with primary focal hyperhidrosis (PFH) relative to control subjects. METHODS: The morphological characteristics and the number of sweat coils in axillary sweat glands were compared between two groups by using transmission electron microscopy. The expression of AQP5 was detected by immunohistochemistry, Western blot analysis, and real-time transcription polymerase chain reaction. RESULTS: There were no significant differences between the two groups in terms of morphological characteristics and the number of sweat coils in axillary sweat glands. The expressions of AQP5 protein and AQP5 mRNA were significantly higher in the patient group than in the control group. CONCLUSION: AQP5 is involved in the secretion of human axillary sweat glands. The overexpression of AQP5 in sweat glands is probably one pathogenetic mechanism underlying PFH.

Efficient removal of uranium from aqueous solution by zero-valent iron nanoparticle and its graphene composite.

Zero-valent iron nanoparticle (ZVI-np) and its graphene composites were prepared and applied in the removal of uranium under anoxic conditions. It was found that solutions containing 24 ppm U(VI) could be completely cleaned up by ZVI-nps, regardless of the presence of NaHCO3, humic acid, mimic groundwater constituents or the change of solution pH from 5 to 9, manifesting the promising potential of this reactive material in permeable reactive barrier (PRB) to remediate uranium-contaminated groundwater. In the measurement of maximum sorption capacity, removal efficiency of uranium kept at 100% until C0(U) = 643 ppm, and the saturation sorption of 8173 mg U/g ZVI-nps was achieved at C0(U) = 714 ppm. In addition, reaction mechanisms were clarified based on the results of SEM, XRD, XANES, and chemical leaching in (NH4)2CO3 solution. Partially reductive precipitation of U(VI) as U3O7 was prevalent when sufficient iron was available; nevertheless, hydrolysis precipitation of U(VI) on surface would be predominant as iron got insufficient, characterized by releases of Fe(2+) ions. The dissolution of Fe(0) cores was assigned to be the driving force of continuous formation of U(VI) (hydr)oxide. The incorporation of graphene supporting matrix was found to facilitate faster removal rate and higher U(VI) reduction ratio, thus benefitting the long-term immobilization of uranium in geochemical environment.

Formation of N-heterocyclic diphosphine ligands from Ag(I)-assisted condensation reactions between bdppeda and formaldehyde and their binuclear silver(I) complexes.

The reaction of [Ag(MeCN)(4)]ClO(4) with N,N,N',N'-tetra(diphenylphosphanylmethyl)ethylenediamine (dppeda) in CH(2)Cl(2)/MeOH afforded an unexpected cationic binuclear complex [Ag(2)(L(1))(2)(η,η-µ-ClO(4))(2)](ClO(4))(2) (L(1) = N,N'-bis(diphenylphosphanylmethyl)-3H-4,5-dihydroimidazole-1-ium) (1). Compound 1 was also prepared in high yield from reactions of [Ag(MeCN)(4)]ClO(4) with N,N'-bis(diphenylphosphanylmethyl)ethylenediamine (bdppeda) in the presence of formaldehyde (HCHO) or formic acid (HCOOH). Analogous reactions of AgCl with bdppeda and HCHO resulted in the formation a neutral binuclear complex [Ag(2)(L(2))(2)(µ-Cl)(2)] (L(2) = N,N-bis(diphenylphosphanylmethyl)-tetrahydroimidazole) (2). Treatment of 1 with concentrated HCl gave rise to a partially anion-exchanged product [Ag(2)(L(1))(2)(µ-Cl)(2)](ClO(4))(2) (3). Compounds 1 and 3 have a similar cationic binuclear structure, in which a [Ag(2)(η,η-µ-ClO(4))(2)] or [Ag(2)(µ-Cl)(2)] ring is sandwiched by two in situ-formed cationic L(1) ligands. The L(1) ligand may be generated by the Ag(I)-assisted condensation reaction between bdppeda and HCHO or HCOOH. Compound 2 holds a neutral binuclear structure, in which a [Ag(2)(µ-Cl)(2)] ring is connected by two in situ-formed L(2) ligands from its top and bottom sites. The neutral ligand L(2) may be produced from another Ag(I)-assisted condensation reaction between bdppeda and HCHO. The in situ formation of the L(1) and L(2) ligands provides a new route to the N-heterocyclic diphosphine ligands, and an interesting insight into the coordination chemistry of their metal complexes.

A novel mutation impairing the tertiary structure and stability of γC-crystallin (CRYGC) leads to cataract formation in humans and zebrafish lens.

Congenital cataract is one of the leading causes of human blindness. In this study, we identified a novel, heterozygous c.385G

[A novel PAX6 mutation (c.1286delC) in the patients with hereditary congenital aniridia.].

To study the molecular genetic mechanism of hereditary congenital aniridia, the entire coding exons (exon 4-13) of PAX6 gene and the flanking exon-intron junctions were amplified through PCR from the genomic DNA of all the two patients in a Chinese family with aniridia. PCR products were purified from agarose gel and sequenced. In both patients, a novel deletion mutation (c. 1286delC) in exon 11 was identified. Compared with the normal product of PAX6 gene, this mutation caused frame shifting, and generated a novel 55 amino acid peptide from codon 309. This deletion also resulted in a premature termination codon (PTC) and preterminated peptide synthesis. Meanwhile, this mutation was absent in all the unaffected family members and 50 normal control individuals through PCR-RFLP.

[Rapid genetic screening of Leber's hereditary optic neuropathy with mtDNA G11778A mutation by AS-PCR with whole blood].

OBJECTIVE: Rapid Genetic Screening of Leber's hereditary optic neuropathy (LHON) with mtDNA G11778A mutation by allele-specific polymerase chain reaction (AS-PCR) with whole blood. METHODS: Whole blood with anticoagulant was used as a template of AS-PCR for the analysis of LHON with mtDNA G11778A point mutation. The amplified DNA fragment was directly observed by electrophoretogram with ethidium bromide stained. RESULTS: The accuracy was 100% by using this method in 24 blood samples tested, and the specific of PCR of which used whole blood as template was better than one of the purified mtDNA. The reliability of the method for screening of LHON with mtDNA G11778A mutation was checked by double-blind test in 22 blood samples. CONCLUSION: This method does not need purified DNA from blood and only required one step of PCR. Thus, it is very simple, rapid and accurate for the clinical genetic screening of LHON with mtDNA G11778A point mutation.

[Expression of recombinant human BMP-6 in Escherichia coli and its purification and bioassay in vitro].

To purify the recombinant human BMP-6 protein and to establish its in vitro bioassay method. The cDNA encoding the mature peptide of hBMP-6 protein was amplified by reverse transcription-polymerase chain reaction (RT-PCR), using human placental mRNA as template, and subcloned into the high-expression vector pET-15b under the control of T7 lac promoter. The resulting construct, pET-BMP6, was then transformed into an Escherichia coli strain BL21 (DE3) for the production of recombinant hBMP-6 protein (rhBMP-6). After 4 hours of induction by isopropyl-beta-D-thiogalactoside (IPTG), rhBMP-6 (approximately 15kD) was expressed and formed inclusion bodies, contributing up to 10% of the total bacterial protein. The inclusion bodies were isolated and redissolved in 8mol/L urea, and the denatured rhBMP-6 was purified to 95% purity by CM-Cellulose 32 ion exchange chromatography (IEC). The osteoinductivity of rhBMP-6 was measured by the expression of some of the osteoblast differentiation marker genes in rhBMP-6-treated C3H10T1/2 cells as reflected by determinations of alkaline phosphatase (ALPase) activity and semi-quantitative RT-PCR. At the end of the purification process, about 80% of rhBMP-6 formed disulphide-linked homodimers after refolding during renaturation. The apparent size of the protein was 30kD on non-reducing SDS-PAGE, similar to that of the native form of hBMP-6. The enzyme assays showed that the ALPase activity was increased in a dose-dependent manner with the treatment of rhBMP-6. After the addition of 300ng/mL of rhBMP-6, the ALPase activity of C3H10T1/2 cells increased significantly. The activity of rhBMP-6 used was comparable to about 70% of that of the standard hBMP-6 derived from eukaryotic cells. RNA extraction data also showed rhBMP-6 stimulated expression of osteoblast marker genes, including type I collagen, osterix, and osteocalcin in a time-dependent manner. After 5 days of treatment, their level of expression was increased to 3 times that of controls. Bone morphogenetic protein (BMP)-6, a member of the 60A subgroup of the bone morphogenetic protein (BMPs) family, plays a pivotal role in bone formation. Previous evidence showed that BMP-6 is selectively up-regulated by estrogen, suggesting its potential role in the treatment of osteoporotic fractures, especially for menopausal osteoporosis. Our present study demonstrates that the recombinant hBMP-6 produced in Escherichia coli is able to induce pre-osteoblastic cells to differentiate into osteoblasts in vitro, and analysis of mRNA expression of type I collagen, osterix, and osteocalcin can be also a method for measuring the osteoinductivity of BMP. This provides the basis for further studies on ectopic bone formation in the body and for the development of auxiliary drugs for the treatment of osteoporotic fractures.