Mutation Screening and Functional Study of SLC26A4 in Chinese Patients with Congenital Hypothyroidism

Objective: Defects in the human solute carrier family 26 member 4 (SLC26A4) gene are reported to be one of the causes of congenital hypothyroidism (CH). We aimed to identify SLC26A4 mutations in Chinese patients with CH and analyze the function of the mutations. Methods: Patients with primary CH were screened for 21 CH candidate genes mutations by targeted next-generation sequencing. All the exons and exon-intron boundaries of SLC26A4 were identified and analyzed. The function of six missense mutation in SLC26A4 were further investigated in vitro. Results: Among 273 patients with CH, seven distinct SLC26A4 heterozygous mutations (p.S49R, p.I363L, p.R409H, p.T485M, p.D661E, p.H723R, c.919-2A>G) were identified in 10 patients (3.66%, 10/273). In vitro experiments showed that mutation p.I363L, p.R409H, p.H723R affect the membrane location and ion transport of SLC26A4, while p.S49R did not. Mutation p.T485M and p.D661E only affected ion transport, but had no effect on the membrane location. Conclusion: The prevalence of SLC26A4 mutations was 3.66% in Chinese patients with CH. Five mutations (p.I363L, p.R409H, p.T485M, p.D661E and p.H723R) impaired the membrane location or ion transport function of SLC26A4, suggesting important roles for Ile363, Arg409, Thr485, Asp661, and His723 residues in SLC26A4 function. As all variants identified were heterozygous, the pathogenesis of these patients cannot be explained, and the pathogenesis of these patients needs further study.

17ß-estradiol regulates the expression of apolipoprotein M through estrogen receptor α-specific binding motif in its promoter.

BACKGROUND: We have previously demonstrated that estrogen could significantly enhance expression of apolipoprotein M (apoM), whereas the molecular basis of its mechanism is not fully elucidated yet. To further investigate the mechanism behind the estrogen induced up-regulation of apoM expression. RESULTS: Our results demonstrated either free 17ß-estradiol (E2) or membrane-impermeable bovine serum albumin-conjugated E2 (E2-BSA) could modulate human apoM gene expression via the estrogen receptor alpha (ER-α) pathway in the HepG2 cells. Moreover, experiments with the luciferase activity analysis of truncated apoM promoters could demonstrate that a regulatory region (from-1580 to -1575 bp (-GGTCA-)) upstream of the transcriptional start site of apoM gene was essential for the basal transcriptional activity that regulated by the ER-α. With the applications of an electrophoresis mobility shift assay and a chromatin immunoprecipitation assay, we could successfully identify a specific ER-α binding element in the apoM promoter region. CONCULSION: In summary, the present study indicates that 17ß-estradiol induced up-regulation of apoM in HepG2 cells is through an ER-α-dependent pathway involving ER-α binding element in the promoter of the apoM gene.

Increased mRNA levels of apolipoprotein M and apolipoprotein AI in the placental tissues with fetal macrosomia.

PURPOSE: The present study examined mRNA levels of apolipoprotein M (apoM) and apolipoprotein AI (apoAI) in the term placental tissues obtained from 37 women with normal birth weight neonates and from 37 women with macrosomic neonates (birth body weight ≥4,000 g), and further discussed possible clinical significance of these observations. METHODS: The mRNA levels of apoM and apoAI in the placental tissues were determined by the real time RT-PCR, which demonstrated that both apoM and apoAI mRNA levels were significantly higher in the placentas from macrosomia than those from normal birth. Moreover, we analyzed the overexpressions of apoM and apoAI with the clinical data. Meanwhile we examined several known risk factors of macrosomia including the mRNA levels of insulin-like growth factor I (IGF-I), IGF-II, insulin-like growth factor I receptor (IGF-IR) and IGF-IIR. RESULTS: It demonstrated that apoM expression was significantly positively correlated to the placental weight, fetal birth weight, pregestational body mass index (BMI), weight gain during pregnancy, maternal weight, maternal BMI and the mRNA levels of IGF-IR as well as IGF-IIR. The apoAI mRNA level was statistically significantly correlated to the placental weight, fetal birth weight, IGF-I and IGF-IR mRNA levels. CONCLUSIONS: Binary logistic regression analysis suggested that both apoM and apoAI mRNA may considered as independent risk factors for macrosomia. The clinical significance needs further investigation.

[Study on saltation of fluorescence excitation spectra of erythrosine].

The fluorescence excitation spectra and absorption spectra of six kinds of erythrosine solutions with concentrations of 10, 20, 30, 40, 50 and 60 microg x mL(-1) were experimentally measured. It was found that the fluorescence excitation peaks are both located at 530 nm significantly when the concentrations of erythrosine solutions are 10 and 20 microg x mL(-1). However, the linetype saltation of fluorescence excitation spectrum occurs as the concentration of erythrosine solution is above 30 microg x mL(-1). The valley is located at 530 nm and two new peaks appear at both flanks of the valley. Compared with fluorescence excitation spectra, the absorption spectra of erythrosine solutions are without saltation and the peaks are all located at 530 nm. According to calculations and a series of contrast experiments, it was demonstrated that the absorption characteristic of erythrosine and the spectral measurement mode conspire to cause the saltation of fluorescence excitation spectra. The results can provide guidance for further research on physical and chemical properties of erythrosine, and offer help and reference for study on saltation behavior in fluorescence excitation spectra and improvement in spectral measuring mode.

GCS induces multidrug resistance by regulating apoptosis-related genes in K562/AO2 cell line.

We have previously shown that the expression of glucosylceramide synthase (GCS) gene in drug-resistant K562/AO2 human leukemia cell was higher than that in drug-sensitive K562 cell, and the sensitivity to adriamycin of K562/AO2 cell was enhanced by inhibiting GCS. It is concluded that the overexpression of GCS gene is one of the reasons which lead to multidrug resistance (MDR) of leukemia cell. Meanwhile, we also found that higher expression of Bcl-2 gene and protein were exhibited in K562/AO2 cell compared with K562 cell. Basing on this, we hypothesized that the high expression of GCS gene which results in MDR of leukemia cell is correlated with Bcl-2 signal transduction. In order to validate the hypothesis, the inhibition of GCS gene in K562/AO2 cell was observed by using chemical suppressor PPMP and siRNA targeted at GCS, and applying RT-PCR and flow cytometry, the expression levels of apoptosis-related gene Bcl-2 and Bax were analyzed before and after inhibiting GCS gene in K562/AO2 cell. The results demonstrated that the gene and protein of Bcl-2 in K562/AO2 cell were both down-regulated significantly after GCS gene being inhibited; however, the Bax mRNA expression had no apparent change in different groups. This suggested that GCS gene may contributed to MDR of human leukemia cell K562/AO2 by Bcl-2 signal transduction.

Overexpression of glucosylceramide synthase in associated with multidrug resistance of leukemia cells.

Ceramide, as a second messenger, initiates one of the major signal transduction pathways in tumor apoptosis. Glucosylceramide synthase (GCS) catalyzes glycosylation of ceramide and produces glucosylceramide. Through GCS, ceramide glycosylation allows cellular escape from ceramide-induced programmed cell death. Here we investigated the expression of GCS in human leukemia cells and an association between GCS and multidrug resistance of leukemia cells. Using RT-PCR technique the level of GCS gene was detected in 65 clinical multidrug resistance/non-resistance cases with leukemia, and in K562 and K562/A02 cell lines. AlamarBlue Assay was applied to confirm the multidrug resistant of K562/A02 cells. PPMP, which is a chemical inhibitor for GCS, was used to determine the relationship between GCS and drug-resistance in K562/A02 cells. In addition, multidrug resistance gene (mdr1), Bcl-2 and Bax mRNA was also analyzed by RT-PCR. The expression of GCS and mdr1 mRNA in clinic multidrug resistance samples exhibited significantly increased compared with clinic drug sensitive group (P<0.05). There was the positive correlation both the expression of GCS and mdr1 genes in leukemia samples (P<0.01, gamma=0.7). AlamarBlue Assay showed that the K562/A02 cell line was 115-fold more resistant to adriamycin and 36-fold more resistant to vincristine compared with drug-sensitive K562 cell line. There also was significant expression difference of GCS and mdr1 genes between K562 and K562/A02 cells. Bcl-2 gene exhibited higher expressions whatever in clinic drug-resistance samples or K562/A02 cells, whereas the expressions of Bax gene were higher in drug-sensitive samples and K562 cells. PPMP increased sensitivity to adriamycin toxicity by inhibiting GCS in K562/A02 cells. Therefore, it is suggested that a high level of GCS in leukemia is possible contributed to multidrug resistance of leukemia cells. Abnormally expressions of the genes in associated with cell apoptosis might be one of the main molecular pathology mechanisms of multidrug resistance caused by GCS gene.