Molecular study and genotype-phenotype in Chinese female patients with 46, XY disorders of sex development.

OBJECTIVE: The rare condition 46, XY disorders of sex development (DSDs) is characterized by the female phenotype and male karyotype. We aimed to describe the genetic basis of 46, XY DSDs in nine patients and the genotype-phenotype relationships of the genes involved. METHODS: Targeted next-generation sequencing (NGS) was used to analyze the underlying hereditary etiology in nine female patients with 46, XY DSDs. In silico analyses were used to predict the effects of novel variants on the protein function of the identified genes. RESULTS: Primary amenorrhea with the absence of puberty, inguinal hernia, and clitoridauxe were common complaints. All enrolled patients had a differential etiology by genetic testing, and five novel genetic variants involved in four genes (SRY, AR, NR5A1, and LHCGR) were identified. A novel nonsense variant of SRY c.51C > G was found in XY patients without testicles. Two novel heterozygous variants, i.e. c.265A > T (Ile89Leu) and c.422T > C (Val141Ala), of the LHCGR gene were found in male pseudo-hermaphroditism. CONCLUSIONS: We expanded the genetic mutation spectrum and described in detail the genotype-phenotype relationships of 46, XY DSDs. DNA sequencing for SRY should be a priority in female patients with 46, XY DSDs. NGS is useful for clarifying genetic pathogenesis and could provide a basis for clinical diagnosis and treatments of patients with 46, XY DSDs.

Genetic investigation of 211 Chinese families expands the mutational and phenotypical spectra of hereditary retinopathy genes through targeted sequencing technology.

BACKGROUND: Hereditary retinopathy is a significant cause of blindness worldwide. Despite the discovery of many mutations in various retinopathies, a large number of patients remain genetically undiagnosed. Targeted next-generation sequencing of the human genome is a suitable approach for the molecular diagnosis of retinopathy. METHODS: We describe a cohort of 211 families from central China with various forms of retinopathy; 95 patients were investigated using multigene panel sequencing, and the other 116 with suspected Leber hereditary optic neuropathy (LHON) were tested by Sanger sequencing. The detected variation of targeted sequencing was verified by PCR-based Sanger sequencing. We performed a comprehensive analysis of the cases using sequencing data and ophthalmologic examination information. RESULTS: Potential causal mutations were identified in the majority of families with retinopathy (57.9% of 95 families) and suspected LHON (21.6% of 116 families). There were 68 variants of a certain significance distributed in 31 known disease-causing genes in the 95 families; 37 of the variants are novel and have not been reported to be related to hereditary retinopathy. The NGS panel solution provided a 45.3% potential diagnostic rate for retinopathy families, with candidate gene mutations of undefined pathogenicity revealed in another 12.6%of the families. CONCLUSION: Our study uncovered novel mutations and phenotypic aspects of retinopathy and demonstrated the genetic and clinical heterogeneity of related conditions. The findings show the detection rate of pathogenic variants in patients with hereditary retinopathy in central China as well as the diversity and gene distribution of these variants. The significance of molecular genetic testing for patients with hereditary retinopathy is also highlighted.

[Analysis of phenotype and CYP4V2 gene variants in two pedigrees affected with Bietti crystalline corneoretinal dystrophy].

OBJECTIVE: The CYP4V2 gene of two pedigrees affected with Bietti crystalline corneoretinal dystrophy was analyzed to indentify the cause of the disease and provide a basis for clinical diagnosis. METHODS: The probands were subjected to next generation sequencing (NGS). Suspected variants were verified by Sanger sequencing. Pathogenicity of the variants were searched through relevant databases and PubMed by following the ACMG guidelines. RESULTS: A homozygous variant in the CYP4V2 gene c. (802-8) _810delTCATACAGGTCATCGCTinsGC was detected in proband from pedigree 1, parents did not detect; CYP4V2 genes c. (802-8)_810delTCATACAGGTCATCGCTinsGC and c. 958 C>T (p.Arg320X) compound heterozygous variants existed in the proband of pedigree 2,both parents were variant carriers. The results of Sanger sequencing showed that the variant of CYP4V2 gene in the two families was consistent with the NGS sequencing. The c. (802-8)_810delTCATACAGGTCATCGCTinsGC of CYP4V2 gene was splicing variant, and both splicing variant and nonsense variant could produce truncated nonfunctional protein products. Based on standards and guidelines by American College of Medical Genetics and Genomics, the CYP4V2 genes c. (802-8)_810del TCATACAGGTCATCGCTinsGC and c. 958 C>T (p.Arg320X) were predicted to be pathogenic variants (PVS1+PS1+PM2+PM3). CONCLUSION: The homozygous variant c. (802-8) _810delTCATACAGGTCATCGCTinsGC and the complex heterozygous variants c. (802-8) _810delTCATACAGGTCATCGCTinsGC and c.958C>T (p.Arg320X) in CYP4V2 gene are the cause of the disease in the probands of two pedigrees , respectively.

[Clinical characteristics and variant analysis of five pedigrees with hereditary spastic paraplegia].

OBJECTIVE: To explore the clinical and genetic characteristics of five pedigrees affected with hereditary spastic paraplegia(HSP). METHODS: Clinical data of the five pedigrees was collected, and high-throughput sequencing was carried out to detect potential variants. Sanger sequencing were used to verify the results. RESULTS: The probands of pedigree 1 and 2 were found to harbor heterozygous SPAST gene variants, namely c.1196C>T and c.1523T>A. The proband of pedigree 3 harbored compound heterozygous variants of FA2H gene (c.61G>C and c.688G>A). Proband from pedigree 4 harbored compound heterozygous variants of SPG11 gene (c.6812+4_6812+7delAGTA and c.915delT). The proband of pedigree 5 harbored compound heterozygous variants of SPG7 gene (c.1703_1704delAG and c.1937-1G>C). Based on the American College of Medical Genetics and Genomics(ACMG) guidelines, all variants were predicted to be likely pathogenic. Among these, SPAST gene c.1523T>A, FA2H gene c.61.G>C, SPG11 gene splicing region c.6812+4_6812+7delAGTA, c.915delT, SPG7 gene c.1703_1704delAG and splicing region c.1937-1G>C variants were unreported previously. CONCLUSION: The probands of pedigrees 1 and 2 were diagnosed with autosomal dominant hereditary spastic paraplegia type 4, for which pedigree 2 showed incompletely penetrance. Pedigrees 3, 4, and 5 were diagnosed with autosomal recessive hereditary spastic paraplegia type 35, 11 and 7, respectively. Above result provided a reference for clinical diagnosis and genetic counseling for the affected pedigrees.

Additive effects of EGF and IL-1ß regulate tumor cell migration and invasion in gastric adenocarcinoma via activation of ERK1/2.

Growth and inflammatory factors are associated with poor prognosis in gastric adenocarcinoma (GA); however, the additive effects of growth and inflammatory factors in GA remain unclear. In this study, we investigated the ability of epidermal growth factor (EGF) and interleukin (IL-1ß) to activate extracellular signal-regulated kinase (ERK)1/2 in GA cells, and correlated the relationships between their roles with the metastatic potential both in GA cells and GA tissues. The effects of EGF, IL-1ß and EGF plus IL-1ß in AGS and MKN-45 GA cells were examined using western blotting, Transwell migration and invasion assays, immunocytochemical staining and an activator protein (AP)-1 luciferase reporter gene assay, and was further characterized in GA tissues by immunohistochemistry. The results exhibited that EGF and IL-1ß additively activated ERK1/2, increased migration and invasion than either EGF or IL-1ß alone in AGS and MKN-45 cells. The mechanisms were involved in upregulating MMP-9 expression through increasing AP-1 transcriptional activity via ERK1/2 pathway; these effects were dose-dependently inhibited by silencing ERK1/2 or using U0126. In vivo data also confirmed that the overexpression of p-ERK1/2 in GA tissues correlated well with the EGF, IL-1ß, EGF plus IL-1ß, and was associated with metastasis, which was well correlation with the expression of MMP-9 and c-fos (AP-1). The results demonstrate that growth and inflammatory factors play an important role in metastasis of GA by additively activating ERK-1/2 and AP-1, and upregulating MMP-9. As both cytokines contribute to the migration and invasion of GA cells, EGF/IL-1ß/ERK1/2 pathways may be key pathways closely associated with GA progression.

IL-1ß-induced activation of p38 promotes metastasis in gastric adenocarcinoma via upregulation of AP-1/c-fos, MMP2 and MMP9.

BACKGROUND: Interleukin-1ß (IL-1ß) has been implicated in the progression of gastric adenocarcinoma (GA); however, the molecular mechanisms of action of IL-1ß in GA are poorly characterized. P38 and JNK are the major MAPK family members that regulate IL-1ß signaling pathways. Here, we investigated the role of both p38 and JNK in IL-1ß-induced GA cell migration, invasion and metastatic potential. METHODS: The effects of IL-1ß-induced p38 and JNK activation in GA cells were determined using in vitro Transwell migration and invasion assays of MKN-45 and AGS cells, or an in vivo metastasis assay in nude mice. The IL-1ß-induced p38 signaling pathway was further characterized in GA cells. Activation of the IL-1ß/p38 signaling pathway was also assessed in human primary GA tissues by immunohistochemistry. RESULTS: IL-1ß-induced activation of p38 increased GA cell migration and invasion in vitro and promoted the metastatic potential of GA cells in vivo; these effects were attenuated by p38 siRNA or the p38 inhibitor SB202190. MMP2 or MMP9 siRNAs and the MMP2/9 inhibitor BiPS also inhibited IL-1ß-induced GA cell migration and invasion in vitro. IL-1ß-induced p38 activation significantly increased MMP2 and MMP9 mRNA and protein expression and activity. Luciferase reporter assays demonstrated that the activator protein-1 (AP-1) and the AP-1 binding sites of the MMP9 promoter (-670/MMP9) were activated by IL-1ß-induced p38 activation. Phospho-p38 was significantly upregulated in human GA tissues (compared to matched non-neoplastic tissues), and significantly associated with lymph node metastasis, and invasion beyond the serosa. Expression of phospho-p38 significantly correlated with IL-1ß, MMP2, MMP9, and c-fos expression in both human GA tissues and GA cell metastases in the lungs of nude mice. IL-1ß was also capable of activating JNK in GA cells, but activation of JNK was not associated with GA cell migration and invasion. Therefore, IL-1ß-induced the migration and invasion in GA cells were regulated by p38, but not by JNK. CONCLUSIONS: IL-1ß-induced p38 activation and the IL-1ß/p38/AP-1(c-fos)/MMP2 & MMP9 pathway play an important role in metastasis in GA; this pathway may provide a novel therapeutic target for GA.

Epidermal growth factor (EGF) and interleukin (IL)-1ß synergistically promote ERK1/2-mediated invasive breast ductal cancer cell migration and invasion.

BACKGROUND: Patients with invasive breast ductal carcinoma (IBDC) with metastasis have a very poor prognosis. Little is known about the synergistic action of growth and inflammatory factors in IBDC metastases. METHODS: The expression of activated extracellular signal-regulated kinase1/2 (phosphorylated or p-ERK1/2) was analyzed by immunohistochemistry in IBDC tissue samples from 80 cases. BT474 IBDC cell migration and invasion were quantified using the Transwell assay. Matrix metalloproteinase (MMP)-9 expression and activity were analyzed by RT-PCR, Western blotting and zymography. Activator protein (AP)-1 activity was measured with a luciferase reporter gene assay. The Wilcoxon signed-rank test, Chi-square test, the partition of Chi-square test, independent t-test, and Spearman's method were used for the statistical analysis. RESULTS: Phosphorylated ERK1/2 was detected in 58/80 (72.5%) IBDC tissues, and was associated with higher TNM stage and lymph node metastasis, but not patient age or tumor size. Individually, epidermal growth factor (EGF), and interleukin (IL)-1ß activated ERK1/2, increased cell migration and invasion, MMP-9 expression and activity, AP-1 activation in vitro and the expression of p-ERK1/2 was positively correlated with EGF expression levels, as well as IL-1ß, MMP-9 and c-fos in IBDC tissue samples. Co-stimulation with EGF and IL-1ß synergistically increased ERK1/2 and AP-1 activation, cell migration and invasion, and MMP-9 expression and activity. Inhibition of ERK1/2 using U0126 or siRNA abolished EGF and/or IL-1ß-induced cell migration and invasion in a dose-dependent manner. CONCLUSION: Activated ERK1/2 was associated with higher TNM stage and lymph node metastasis in IBDC. Both in vitro and in vivo studies indicated that ERK-1/2 activation may increase the metastatic ability of IBDC cells. Growth and inflammatory factors synergistically induced IBDC cell migration and invasion via ERK1/2 signaling, AP-1 activation and MMP-9 upregulation.

Akt2 kinase suppresses glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-mediated apoptosis in ovarian cancer cells via phosphorylating GAPDH at threonine 237 and decreasing its nuclear translocation.

Protein kinase B (Akt) plays important roles in regulation of cell growth and survival, but while many aspects of its mechanism of action are known, there are potentially additional regulatory events that remain to be discovered. Here we detected a 36-kDa protein that was co-immunoprecipitated with protein kinase Bß (Akt2) in OVCAR-3 ovarian cancer cells. The protein was identified to be glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by MALDI-TOF/TOF MS, and the interaction of Akt2 and GAPDH was verified by reverse immunoprecipitation. Our further study showed that Akt2 may suppress GAPDH-mediated apoptosis in ovarian cancer cells. Overexpression of GAPDH increased ovarian cancer cell apoptosis induced by H(2)O(2), which was inhibited by Akt2 overexpression and restored by the PI3K/Akt inhibitor wortmannin or Akt2 siRNA. Akt2 phosphorylated Thr-237 of GAPDH and decreased its nuclear translocation, an essential step for GAPDH-mediated apoptosis. The interaction between Akt2 and GAPDH may be important in ovarian cancer as immunohistochemical analysis of 10 normal and 30 cancerous ovarian tissues revealed that decreased nuclear expression of GAPDH correlated with activation (phosphorylation) of Akt2. In conclusion, our study suggests that activated Akt2 may increase ovarian cancer cell survival via inhibition of GAPDH-induced apoptosis. This effect of Akt2 is partly mediated by its phosphorylation of GAPDH at Thr-237, which results in the inhibition of GAPDH nuclear translocation.