OBJECTIVE: To explore the genetic etiology in four patients with hyperbilirubinemia, and discuss the correlation between clinical characteristics and molecular basis. METHODS: The data of clinical manifestation and auxiliary examinations were collected. Genomic DNA of the four patients was extracted and analyzed by next-generation sequencing using the panel including genes involved in hereditary metabolic liver diseases. Suspected variants were verified by Sanger sequencing. RESULTS: All of the four patients were males with normal liver enzymes. It was revealed that all the patients had heterozygous variants, among which c.3011C>T, c.2443C>T and c.2556del were the variants which have not been reported previously. CONCLUSION All of the patients were diagnosed as Dubin-Johnson syndrome (DJS) caused by ABCC2 gene variants. The novel variants add to the spectrum of genetic variants of the disease. Because of the favorite prognosis, precise diagnosis can greatly reduce the psychological pressure of patients and avoid excessive treatments. At the same time, it could provide pertinent genetic counseling for the families.
DNA ; Female ; Heterozygote ; Humans ; Jaundice, Chronic Idiopathic/genetics* ; Male ; Multidrug Resistance-Associated Protein 2 ; Multidrug Resistance-Associated Proteins/genetics* ; Phenotype

ABCC1 gene is expressed in various tissues and organs of the human body, and can transport substrates including drugs, heavy metals, toxic substances and organic anions. Previous research on ABCC1 gene has mostly focused on tumor multidrug resistance. Recently, ABCC1 has been proposed as a candidate gene for hereditary hearing impairment, which has attracted much attention. ABCC1-associated deafness may be related to its role in biological barriers. This article has summarized recent progress in the study of the role of ABCC1 in the blood-testis barrier, placental barrier, blood-brain barrier, blood-labyrinth barrier, which may provide insight into its biological functions.
Biological Transport ; Deafness/genetics* ; Female ; Humans ; Male ; Multidrug Resistance-Associated Proteins/genetics* ; Placenta ; Pregnancy

OBJECTIVE: To explore the genetic etiology and differential diagnosis for a patient with jaundice. METHODS: Clinical data of the patient and his parents were collected. Genes associated with metabolic liver diseases were subjected to high-throughput sequencing. The pathogenicity of the candidate variants was predicted by using bioinformatics software. RESULTS: High-throughput sequencing revealed that the proband has harbored two variants of the ABCC2 gene (NM_000392) including c.3011C>T (p.T1004I) and c.3541C>T (p.R1181X), which were respectively inherited from his father and mother. Both variants have been previously unreported and predicted to be pathogenic by bioinformatics analysis. CONCLUSION The proband was diagnosed with Dubin-Johnson syndrome due to the compound heterozygous variants of the ABCC2 gene. Genetic testing has enabled accurate differential diagnosis of Dubin-Johnson syndrome in this patient.
Genetic Testing ; High-Throughput Nucleotide Sequencing ; Humans ; Jaundice, Chronic Idiopathic/pathology* ; Multidrug Resistance-Associated Protein 2 ; Multidrug Resistance-Associated Proteins/genetics* ; Mutation

OBJECTIVETo investigate the effect of multidrug resistance-associated protein 4 (MRP4) expression on the radiosensitivity of colorectal carcinoma cell lines in vitro.

METHODSThe vector of shRNA for RNA interference was constructed and then transfected into HCT116 cell line to steadily down-regulate the expression of MRP4. HCT116 cells were divided into 3 groups including the CON group(non-transfected), NC group (negative control virus was added), and KD group (RNAi target was added for transfection). To test the effectiveness of RNA interference, real-time polymerase chain reaction and Western blot were used to measure the expression pattern of MRP4 at both mRNA and protein levels, respectively. For the examination of the effect of RNA interference of MRP4 on the radiosensitivity, flow cytometry was used to calculate the rate of apoptotic cells 24 h after 4 Gy radiation. Proliferation of the cells was measured via MTT assay at different time points.

RESULTSShRNA plasmid was successfully constructed. Transfection of this constructed vector into HCT116 cell line caused steady silencing of MRP4 expression (HCT116-KD). MRP4 mRNA and protein expression were significantly down-regulated following RNA interference(P<0.05). Twenty-four hours after radiation, the apoptosis rate of KD cell line was (71.7±0.8)%, significantly higher than that in the CON group [(56.1±0.9)%] and NC group[(59.8±0.8)%](P<0.05). Fourty-eight hours and 72 hours after radiation, the proliferation was significantly inhibited in KD cells compared to the control groups(P<0.05).

CONCLUSIONSExpression of MRP4 is closely related to radio-tolerance of colorectal carcinoma. Down-regulation of MRP4 expression by RNA interference enhances radiosensitivity of colorectal carcinoma cell lines in vitro. MRP4 may be an effective molecular marker for predicting the radiosensitivity of colorectal carcinoma.

Colorectal Neoplasms ; genetics ; metabolism ; Down-Regulation ; HCT116 Cells ; Humans ; Multidrug Resistance-Associated Proteins ; genetics ; RNA Interference ; Radiation Tolerance ; genetics

Dubin-Johnson syndrome (DJS) is an autosomal recessive disorder resulting from biallelic mutations of ABCC2 gene, with long-term or intermittent conjugated hyperbilirubinemia being the main clinical manifestation. This paper aims to report the clinical features and ABCC2 genotypes of an infant with DJS. A 9.5-month-old male infant was referred to the hospital due to abnormal liver function discovered over 9 months. The major clinical presentation was prolonged jaundice since neonatal period. A series of biochemistry analysis revealed markedly elevated total bilirubin, conjugated bilirubin and total bile acids. The patient had been managed in different hospitals, but the therapeutic effects were unsatisfactory due to undetermined etiology. Physical examination revealed jaundiced skin and sclera, and a palpable liver 3 cm below the right subcostal margin with medium texture. The spleen was not enlarged. Genetic analysis revealed a splice-site variant c.3988-2A>T and a nonsense variant c.3825C>G (p.Y1275X) in the ABCC2 gene of the infant, which were inherited from his mother and father respectively. The former had not been previously reported. Then ursodeoxycholic acid and phenobarbital were given orally. Half a month later, as a result, his jaundice disappeared and the biochemistry indices improved. However, the long-term outcome needs to be observed. Literature review revealed that neonates/infants with DJS presented with cholestatic jaundice soon after birth as the major clinical feature, and the ABCC2 variants exhibited marked heterogeneity.
Bile Acids and Salts ; Bilirubin ; Humans ; Infant ; Jaundice ; Jaundice, Chronic Idiopathic ; genetics ; Male ; Multidrug Resistance-Associated Proteins ; genetics ; Ursodeoxycholic Acid

Breast Neoplasms ; metabolism ; Female ; Humans ; Multidrug Resistance-Associated Proteins ; genetics ; metabolism ; Receptor, Epidermal Growth Factor ; metabolism ; Receptors, Progesterone ; metabolism

OBJECTIVETo study the reverse effect and its mechanism of tetrandrine (Tet) on human breast cancer cells resistant to tamoxifen (MCF-7/TAM).

METHODSThe drug toxicity and the reverse effect of Tet on MCF-7/TAM cells were detected by MTT assay. The effects of multidrug resistance protein 1 (MRP1) gene of Tet on MCF-7iTAM cells were detected by real-time fluorescence quantitative PCR. The changes of MRP1 protein on MCF-7JTAM cells were detected by Western blot.

RESULTSTet had a significant reversal of drug resistance on MCF-7/TAM cells. The non-cytotoxic dose (0. 625 microg/mL) reversed the resistance by 2.0 folds. MRP1 was reduced at gene (P <0.05) and protein levels when Tet effected on MCF-7ITAM cells.

CONCLUSIONTet could reverse the drug resistance of MCF-7/TAM cells, and the reverse mechanism may be related to down-regulating MRP1 expression.

Benzylisoquinolines ; pharmacology ; Drug Resistance, Neoplasm ; drug effects ; genetics ; Female ; Humans ; MCF-7 Cells ; drug effects ; Multidrug Resistance-Associated Proteins ; metabolism ; Tamoxifen ; pharmacology

This study was aimed to explore the effect of vascular endothelial growth factor (VEGF) on sensitivity of leukemia cell line K562/A02 to doxorubicin by using RNA interference, and to investigate its mechanism. The 3 shRNA targeting human vegf gene were synthesized, then transfected into K562/A02 cells by lipofectamine 2000 reagent. RT-PCR was used to detect the expression of vegf and mrp1 at the mRNA level;Western blot was used to analyze the expression of VEGF, MRP1, AKT, P-AKT at the protein level; MTT was used to determine the IC(50) value of transfected cells to doxorubicin (DOX); flow cytometry was used to detect cell apoptosis and intracellular Rho123 retention. The results showed that after vegf shRNA were transfected into K562/A02 cells, the expression of vegf at the mRNA level decreased, and the difference between vegf shRNA2 group or vegf shRNA3 group and HK group was statistically significant (p < 0.05), the greatest decrease was observed in the cells transfected with vegf shRNA3; and the protein level of VEGF was also down-regulated. The IC(50) value of positively transfected group was lower than that of control groups, and the difference between vegf shRNA2 group or vegf shRNA3 group and HK group was significant (p < 0.05). The retention of intracellular Rho123 was enhanced in three positively transfected groups (p < 0.05). Cell apoptosis increased in positively transfected groups, and there was statistically difference between vegf shRNA2 group or vegf shRNA3 group and HK group (p < 0.05). The expression of mrp1 at the mRNA level were decreased, and there were statistical difference between vegf shRNA3 group and HK group (p < 0.05), and the protein level of mrp1 was also down-regulated; the expression of P-AKT at protein level decreased in positively transfected groups, and the greatest decrease was seen in vegf shRNA3 group. It is concluded that the transfection with exogenous vegf shRNA can inhibit the expression of vegf at both mRNA and protein levels, and enhance the sensitivity of K562/A02 cell to doxorubicin, the mechanism of which may be the inhibition of apoptosis and down-regulation of MRP1 by inactivating PI3K/AKT signaling pathway.
Apoptosis ; Doxorubicin ; pharmacology ; Drug Resistance, Multiple ; genetics ; Drug Resistance, Neoplasm ; genetics ; Humans ; K562 Cells ; Multidrug Resistance-Associated Proteins ; genetics ; RNA Interference ; RNA, Messenger ; genetics ; RNA, Small Interfering ; genetics ; Transfection ; Vascular Endothelial Growth Factor A ; genetics

In the present study, the specifically knockdown models of P-gp or MRP2 were constructed by using a series of chemically synthesized small interfering RNA (siRNA) in vitro. The expression of P-gp and MRP2 was measured by real-time PCR and Western blot, and the function was evaluated by applying P-gp and MRP2 substrate, rhodamine and methotrexate. The results showed that MRP2 siRNA-3 or P-gp siRNA-2 significantly decreased the mRNA expression of MRP2 or P-gp, the inhibition ratio was 68% or 84%; MRP2 siRNA-3 or P-gp siRNA-2 at a dose of 80 nmol x L(-1) significantly reduced the protein expression of MRP2 or P-gp at 48 h after treatment, the inhibition ratio was 62% or 70%. Meanwhile, other transporters were not influenced by siRNA. When pretreatment with MRP2 siRNA-3 or P-gp siRNA-2, the efflux of methotrexate or rhodamine decreased significantly and the intra-cellular concentration increased. The results suggested that chemically synthesized siRNA could significantly inhibit the expression and function of MRP2 and P-gp, and the model of RNAi in vitro could be used to evaluate the role of efflux transporters in transportation of drugs.
ATP-Binding Cassette, Sub-Family B, Member 1 ; genetics ; Gene Knockdown Techniques ; Multidrug Resistance-Associated Proteins ; genetics ; RNA Interference ; RNA, Small Interfering ; Real-Time Polymerase Chain Reaction

The aim of the study was to explore the possible molecular markers of chloroquine resistance in Plasmodium vivax isolates in Thailand. A total of 30 P. vivax isolates were collected from a malaria endemic area along the Thai-Myanmar border in Mae Sot district of Thailand. Dried blood spot samples were collected for analysis of Pvmdr1 and Pvcrt-o polymorphisms. Blood samples (100 mul) were collected by finger-prick for in vitro chloroquine susceptibility testing by schizont maturation inhibition assay. Based on the cut-off IC50 of 100 nM, 19 (63.3%) isolates were classified as chloroquine resistant P. vivax isolates. Seven non-synonymous mutations and 2 synonymous were identified in Pvmdr1 gene. Y976F and F1076L mutations were detected in 7 (23.3%) and 16 isolates (53.3%), respectively. Analysis of Pvcrt-o gene revealed that all isolates were wild-type. Our results suggest that chloroquine resistance gene is now spreading in this area. Monitoring of chloroquine resistant molecular markers provide a useful tool for future control of P. vivax malaria.
Amino Acid Substitution ; Antimalarials/*pharmacology ; Chloroquine/*pharmacology ; *Drug Resistance ; Humans ; Inhibitory Concentration 50 ; Malaria, Vivax/*parasitology ; Membrane Transport Proteins/*genetics ; Multidrug Resistance-Associated Proteins/*genetics ; Mutation, Missense ; Myanmar ; Parasitic Sensitivity Tests ; Plasmodium vivax/*drug effects/genetics ; Protozoan Proteins/*genetics ; Thailand