[Development and application of a method for molecular diagnosis of 21-hydroxylase deficiency].

OBJECTIVE: To develop a method for elucidating genetic basis of 21-hydroxylase deficiency. METHODS: Sanger sequencing of entire 21-hydroxylase coding gene CYP21A2 was carried out to detect point mutations, and multiplex ligation-dependent probe amplification (MLPA) and locus-specific PCR/enzyme restriction method were used to detect large deletions and conversion mutations. RESULTS: Nine children were analyzed. Point mutations of the CYP21A2 gene have been identified as: IVS2 13A/C>G (9 alleles), p.Arg356Trp (1 allele), Cluster E6 (1 allele), p.Gln318X (1 allele), and Prom conv (1 allele). While the former 4 mutations are pathogenic, the role of Prom conv mutation in the pathogenesis was uncertain. Three cases had entire CYP21A2 gene deletions (3 alleles), three had CYP21A1P/CYP21A2 chimeric mutations (3 alleles). The genotypes of all patients were determined. And all of the mutations were inherited from parents. CONCLUSION: A rational method for detecting point mutations and large deletions/conversions of CYP21A2 gene has been established.

[Molecular diagnosis of OTC gene mutation in a Chinese family with ornithine transcarbamylase deficiency].

OBJECTIVE: To detect potential mutations of OTC gene in a male infant affected with ornithine transcarbamylase deficiency. METHODS: Genomic DNA were isolated from peripheral blood samples of family members and 100 healthy individuals. Potential mutations of the 10 exons of OTC gene were screened with PCR and Sanger sequencing. RESULTS: A homozygous missense mutation c.917G>C in exon 9, which results in p.R306T, was identified in the infant. Sequencing of the mother and two female members of the family indicated a heterozygous status for the same mutation. The same mutation was not found in other members of the family and 100 healthy controls. CONCLUSION: A missense mutation c.917G>C in the OTC gene is responsible for the pathogenesis of the disease. Identification of the mutation can facilitate prenatal diagnosis and genetic counseling for the family.

[Prenatal diagnosis of 22q11.2 microdeletion by multiplex ligation-dependent probe amplification].

OBJECTIVE: To explore the clinical value of multiplex ligation-dependent probe amplification (MLPA) technique performed in prenatal diagnosis of chromosome 22q11.2 microdeletion. METHODS: MLPA was performed to detect chromosome 22q11.2 mircodeletion in 62 fetuses with congenital heart defects by fetal echocardiography and a normal karyotype by standard G-banding analysis.For a 22q11.2 mircodeletion fetus, his parents were detected to know if it is inherited or de novo. The microdeletion was confirmed by array-based comparative genomic hybridization (arrayCGH). RESULTS: MLPA revealed five 22q11.2 mircodeletions in the 62 fetuses, and the positive detection rate was 8% (5/62). Among these, 4 cases carried the 3M typically deletion which all are de novo, and 1 case carried the 1.5M non-typically deletion which was inherited from his father.arrayCGH confirmed the 22q11.2 microdeletions and delineated the precise location and size of microdeletions. CONCLUSION: MLPA has clinical value in prenatal diagnosis of 22q11.2 mircodeletion, which could provide important genetic information for genetic consulting, pregnancy management and intervention after birth.

[Array-based comparative genomic hybridization detection of copy number variations in a fetus with hypoplastic left-heart syndrome].

OBJECTIVE: To detect the copy number variations (CNVs) of a fetus with hypoplastic left-heart syndrome, and to assess the value of array-based comparative genomic hybridization (array-CGH) for molecular cytogenetic diagnosis. METHODS: The whole genome of a fetus with normal karyotype by G-banding was scanned and analyzed by array-CGH, and the CNVs was confirmed by multiplex ligation-dependent probe amplification (MLPA). RESULTS: Two submicroscopic CNVs [del(11)(q24.1-ter)(121951443-134449216, -12.50 Mb),dup(15)(q26.3)(96889082-100215359, -3.33 Mb)] were identified and mapped by array-CGH. MLPA test confirmed both CNVs. CONCLUSION: Del (11) (q24.1-ter) may contribute to hypoplastic left-heart syndrome of the fetus. For its high-resolution and high-accuracy, array-CGH has provided a powerful tool for detection of genomic imbalance.

[Prenatal diagnosis of two fetuses with de novo small supernumerary markers by single nucleotide polymorphism array based comparative genomic hybridization].

OBJECTIVE: To explore the origins of small de novo mosaic supernumerary marker chromosomes (mars) in two fetuses, and to assess the feasibility of single nucleotide polymorphism array-based comparative genomic hybridization (SNP-array CGH) for prenatal molecular cytogenetic diagnosis. METHODS: Two fetuses with de novo were identified. SNP-array marker chromosomes was applied to define the location and range of marker chromosomes. The karyotype of fetus I was determined to be 47,XX,+ mar[23]/46,XX[16], and that of fetus II was 47,XX,+ mar. Multiplex ligation-dependent probe amplification (MLPA) was applied to verify the genomic imbalance found in fetus II. The karyotypes of parents were normal in both families. RESULTS: SNP-array CGH has indicated a 8.3 Mb duplication at 9p21.1-p21.3 in fetus I, and a 10.8 Mb duplication at 15q11.2-q13.3 in fetus II. MLPA has also confirmed a 15q terminal duplication in fetus II. CONCLUSION: Above cases have illustrated that SNP-array CGH is a rapid, powerful and sensitive technique which may be used for identify the origins of marker chromosomes in prenatal diagnosis.

[Detection of cryptic copy number variations in a fetus with congenital heart disease by array-based comparative genomic hybridization].

OBJECTIVE: To detect the copy number variation (CNV) of a fetus with interrupted aortic arch and ventricular septal defect, in order to explore the underlying genetic causes of the congenital malformation, and investigate the feasibility of array-based comparative genomic hybridization (array-CGH) in molecular cytogenetic diagnosis. METHODS: The whole genome of the fetus with de novo apparently balanced translocations [46,XX,t(7;9)(q12;q21)] diagnosed by G-banding was scanned and analyzed by array-CGH, and the copy number variation was confirmed by fluorescence in situ hybridization (FISH). RESULTS: A pathologic submicroscopic CNV [del(22) (q11.2) (17 370 128-19 790 009, -2.42 Mb)] was identified and mapped by array-CGH. FISH test confirmed the microdeletion detected by array-CGH. CONCLUSION: The cryptic 22q11.2 deletion might be the reason leading to the congenital malformation of the fetus. This study provides evidence that apparently balanced translocations classified by conventional cytogenetic techniques may host additional submicroscopic CNVs which are not located at the breakpoints. Due to the high-resolution, high-throughput and high-accuracy, array-CGH is considered to be a powerful tool for submicroscopic CNVs detection.

[Application of multiplex ligation-dependent probe amplification for rapid detection of aneuploidies in prenatal diagnosis].

OBJECTIVE: To determine the applicability of multiplex ligation-dependent probe amplification (MLPA) for rapid detection of aneuploidies in prenatal diagnosis. METHODS: A total of 561 prenatal samples were analyzed in parallel by MLPA and traditional karyotyping. Another 20 clinical samples with known common chromosome abnormalities were also determined by MLPA to evaluate the accuracy and reliability of MLPA. The results obtained from MLPA were compared with that from traditional karyotyping. RESULTS: The results were available within 48 h. A total of 38 aneuploidies were identified by MLPA, including 20 cases of trisomy 21, 10 cases of trisomy 18, 1 case of trisomy 13, 4 cases of Turner syndrome, 1 case of Klinefelter syndrome, 1 case of 47, XYY trisomy and 1 case of 48,XYY, +18. MLPA was able to detect all the expected aneuploidies with 100% accuracy. The results obtained from MLPA agreed with traditional karyotyping. Among 561 prenatal samples, the results of 550 samples were concordant with those of karyotyping, and the coincidence rate of MLPA was 98.04%. CONCLUSION: MLPA is a rapid, simple and reliable method for detection of the most common chromosome aneuploidies in prenatal diagnosis. MLPA is a valuable tool in prenatal clinical practice.

[ASS1 mutation leading to citrullinemia I in a Chinese Han family].

OBJECTIVE: To investigate potential mutation of the ASS1 gene in a male infant with acute citrullinemia type I. METHODS: Genomic DNA was prepared from peripheral blood samples of the family members. Mutation analysis of the 14 ASS1 exons was carried out by PCR and direct DNA sequencing. RESULTS: A homozygous missense mutation of c.970G>A located in exon 13, which results in p.G324S, was identified in the child. Sequencing of the parents showed a heterozygous status for the same mutation. CONCLUSION: A missense mutation of c.970G>A in the ASS1 gene is responsible for the pathogenesis of the disease in the infant.

Combination of human plasminogen kringle 5 with ionizing radiation significantly enhances the efficacy of antitumor effect.

Antiangiogenic therapy could destroy tumor vasculature and inhibit tumor growth. It might inhibit tumor growth significantly when used as a single treatment modality and its therapeutic benefit may even be greater when used in combination with established treatment modalities such as radiation therapy (RT). In the present report, we investigated the effect of recombinant human plasminogen kringle 5 domain (rhK5) in combination with ionizing radiation on angiogenesis, tumor growth and survival in a murine Lewis lung carcinoma (LLC) tumor model. Combined treatment using rhK5 and radiotherapy displayed obvious suppressive effect on LLC tumor growth as compared with single treatment with either modality (p < 0.05), and resulted in a more additive effect on tumor growth delay in this model. In addition, combined treatment significantly enhanced the survival of mice and no toxic effect, such as weight loss, was observed. The significant antitumor effect of rhK5 plus radiation was associated with a direct suppression effect on early neoangiogenesis and tumor cell apoptosis. Furthermore, the expression of VEGF and HIF-1alpha in tumor tissue correlated well with decreased vessel density. The results suggest that rhK5 significantly enhances the antitumor activity of RT and could be a potent adjuvant therapeutic approach to improve the efficacy of radiotherapy for lung cancer.

Enhanced therapeutic effect by combination of tumor-targeting Salmonella and endostatin in murine melanoma model.

The growth of tumor is angiogenesis-dependent and it often contains hypoxia and necrotic areas. Salmonella VNP20009 could target and replicate in hypoxia and necrotic areas within tumor and induce antitumor effect. Angiogenesis inhibitor endostatin could reduce tumor angiogenesis and inhibit its growth. However, in the phase I trials of VNP20009 and endostatin at the maximum-tolerated dose, no antitumor effects for bacteria therapy and minor therapeutic effects for endostatin treatment were seen. The ineffectiveness of these agents in clinical trials suggests that the combination of these agents with synergic modalities might be necessary. Here we described antitumor effects mediated by the combination of VNP20009 with recombinant human endostatin in B16F10 murine melanoma model with the aim to exploit tumor-targeting of bacteria and anti-angiogenesis strategy to enhance therapeutic efficacy. Combination therapy of these agents significantly enhanced antitumor effects by inducing greater tumor growth inhibition, more severe tumor tissue necrosis as well as less blood vessel density than those induced by either of treatments. The findings suggest that the combination of tumor-targeting bacteria with angiogenesis inhibitor might be of value for the treatment of solid tumors.

1q25.2-q31.3 Deletion in a female with mental retardation, clinodactyly, minor facial anomalies but no growth retardation.

The reports of 1q25-32 deletion cases are rare. We reported here an 11-year-old Chinese Han female with an interstitial 1q25 deletion displaying mental retardation, clinodactyly of the 5th finger and minor facial anomalies. Notably, the patient did not present growth retardation which is quite common in patients with 1q25-32 deletion encompassing LHX4. The heterozygous deletion in this patient was characterized as 46,XX,del(1)(q25.2-q31.3) with a length of 20.5 Mb according to SNP-array test results. STRP (Short Tandem Repeat Polymorphism) analysis of the family trio indicated the genomic abnormality was de novo with paternal origin. After a genotype-phenotype analysis, we proposed here the loss of a 3.1 Mb critical region including 24 genes within 1q25.2 (chr1:174.5-177.6 Mb, build 36) may account for the mental retardation in patients with 1q25-32 deletion.

An improved strategy for high-level production of TEV protease in Escherichia coli and its purification and characterization.

Because of its stringent sequence specificity, tobacco etch virus (TEV) protease emerges as a useful reagent with wide application in the cleavage of recombinant fusion proteins. However, the solubility of TEV protease expressed in Escherichia coli is extremely low. In the present study, we introduced a more efficient system to improve and facilitate the soluble production of TEV protease in E. coli. Optimal expression of soluble His6-TEV was achieved by examining the contribution of chaperone co-expression and lower temperature fermentation. When further purified by Ni(2+) affinity chromatography, 65mg of His6-TEV was isolated with purity over 95% from 1L of culture. The enzyme activity of His6-TEV was generally characterized by using GST-EGFP and His6-L-TNF fusion protein as substrates, which contained a TEV cleavage site between two moieties.

Cross-linked polyethylenimine as potential DNA vector for gene delivery with high efficiency and low cytotoxicity.

Polyethylenimine (PEI) has been known as an efficient gene carrier with the highest cationic charge potential. High transfection efficiency of PEI, along with its cytotoxicity, strongly depends on its molecular weight. To enhance its gene delivery efficiency and minimize cytotoxicity, we have synthesized small cross-linked PEI with biodegradable linkages and evaluated their transfection efficiencies in vitro. In this study, branched PEI with a molecular weight of 800 Da was cross-linked by small diacrylate [1,4-butanediol diacrylate or ethyleneglycol dimethacrylate (EGDMA)] for 2-6 h. The efficiencies of the cross-linked PEI in in vitro transfection of plasmid DNA containing enhanced green fluorescent protein (EGFP) reporter gene were assessed in melanoma B16F10 cell line and other cell lines. Flow cytometry was used to quantify the cellular entry efficiency of plasmid and the transgene expression level. The cytotoxicities of the cross-linked PEI in these cells were evaluated by MTT assay. EGDMA-PEI 800-4h, a typical cross-linked PEI reported here, mediated a more efficient expression of reporter gene than the commercially available 25-kDa branched PEI control, and resulted in a 9-fold increase in gene delivery in B16F10 cells and a 16-fold increase in 293T cells, while no cytotoxicity was found at the optimized condition for gene delivery. Furthermore, the transfection activity of polyplexes was preserved in the presence of serum proteins.

Qualitative and quantitative analysis of active flavonoids in Flos Lonicerae by capillary zone electrophoresis coupled with solid-phase extraction.

Flavonoids are an important bioactive group in the commonly used herbal medicine Flos Lonicerae. A new method of capillary zone electrophoresis (CZE) coupled with solid-phase extraction (SPE) was developed for simultaneous assay of flavonoid aglycones and glycosides in Flos Lonicerae. Optimum CZE separation was achieved with a background electrolyte (BGE) solution consisting of 80 mM boric acid and 20 mM phosphate acid, adjusted to pH 8.1, with 15% acetonitrile (v/v) added, and applying a separation voltage of 28 kV. The SPE method was used for pretreating the complex matrix of botanical materials and good reproducibility was obtained when avicularin was used as internal standard. Linearity of the method was excellent with correlation coefficients (r2) in the range of 0.9995-0.9999 and detection limits were lower than 0.6 microg/mL for the four flavonoids. The obtained recoveries varied between 93 to 104% while the relative standard deviations (RSDs) were below 4.4% (n=3). The developed CZE method was successfully used for the separation of eight flavonoids and the quantification of the four flavonoids in five species of Flos Lonicerae.