OBJECTIVETo investigate the mutations of SLC22A5 gene in patients with systemic primary carnitine deficiency (CDSP).

METHODSHigh liquid chromatography tandem mass spectrometry (HPLC/MS/MS) was applied to screen congenital genetic metabolic disease and eight patients with CDSP were diagnosed among 77 511 samples. The SLC22A5 gene mutation was detected using massarray technology and sanger sequencing. Using SIFT and PolyPhen-2 to predict the function of protein for novel variations.

RESULTSTotal detection rate of gene mutation is 100% in the eight patients with CDSP. Seven patients had compound heterozygous mutations and one patient had homozygous mutations. Six different mutations were identified, including one nonsense mutation [c.760C>T(p.R254X)] and five missense mutations[c.51C>G(p.F17L), c.250T>A(p.Y84N), c.1195C>T(p.R399W), c.1196G>A(p.R399Q), c.1400C>G(p.S467C)]. The c.250T>A(p.Y84N) was a novel variation, the novel variation was predicted to have affected protein structure and function. The c.760C>T (p.R254X)was the most frequently seen mutation, which was followed by the c.1400C>G(p.S467C).

CONCLUSIONThis study confirmed the diagnosis of eight patients with CDSP on the gene level. Six mutations were found in the SLC22A5 gene, including one novel mutation which expanded the mutational spectrum of the SLC22A5 gene.

Adult ; Amino Acid Sequence ; Base Sequence ; Cardiomyopathies ; diagnosis ; genetics ; metabolism ; Carnitine ; deficiency ; genetics ; metabolism ; DNA Mutational Analysis ; methods ; Female ; Gene Frequency ; Genotype ; Humans ; Hyperammonemia ; diagnosis ; genetics ; metabolism ; Infant, Newborn ; Male ; Muscular Diseases ; diagnosis ; genetics ; metabolism ; Mutation ; Organic Cation Transport Proteins ; genetics ; metabolism ; Reproducibility of Results ; Sensitivity and Specificity ; Sequence Homology, Amino Acid ; Solute Carrier Family 22 Member 5 ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

Objective: To investigate the expressions of solute carrier family 22 member 14 (SLC22A14) and sperm-associated antigen 6 (SPAG6) in the sperm of idiopathic asthenospermia men. METHODS: We collected semen samples from 50 idiopathic asthenozoospermia patients and another 50 normal sperm donors, purified the sperm by discontinuous density centrifugation on Percoll gradients, and then determined the mRNA and protein expressions of SLC22A14 and SPAG6 by RT-PCR and Western blot, respectively. RESULTS: Compared with the normal controls, the idiopathic asthenozoospermia patients showed significantly decreased mRNA expressions of SLC22A14 (0.77 ± 0.08 vs 0.53 ± 0.10, P<0.01) and SPAG6 (0.78 ± 0.09 vs0.52 ± 0.10 , P<0.01) and protein expressions of SLC22A14 (0.80 ± 0.09 vs 0.55 ± 0.10 , P<0.01) and SPAG6 (0.78 ± 0.09 vs 0.56 ± 0.09, P<0.01). CONCLUSIONS T The expressions of SLC22A14 and SPAG6 are reduced in the sperm of the patients with idiopathic asthenospermia, which may be one of the important causes of asthenospermia.
Asthenozoospermia ; metabolism ; Blotting, Western ; Ejaculation ; Humans ; Male ; Microtubule Proteins ; genetics ; metabolism ; Organic Cation Transport Proteins ; genetics ; metabolism ; Proteomics ; RNA, Messenger ; metabolism ; Sperm Motility ; Spermatozoa ; metabolism

To establish single- and double-transfected transgenic cells stably expressing hMATE1, hMATE1 cDNA was cloned by RT-PCR from human cryopreserved kidney tissue, and subcloned into pcDNA3.1(+) plasmid by virtue of both HindIII and Kpn I restriction enzyme sites. Subsequently, the recombined pcDNA3.1(+)- hMATE1 plasmid was transfected into MDCK, MDCK-hOCT1 or MDCK-hOCT2 cells using Lipofectamine 2000 Reagent. After a 14-day-cultivation with hygromycin B at the concentration of 400 µg · mL(-1), all clones were screened with DAPI and MPP+ as substrates to identify the best candidate. The mRNA content of hMATE1, the cellular accumulation of metformin with or without cimetidine as inhibitor, or transportation of cimetidine was further valuated. The results showed that all of the three cell models over expressed hMATE1 mRNA. The cellular accumulation of metformin in MDCK-hMATE1 was 17.6 folds of the control cell, which was significantly inhibited by 100 µmol · L(-1) cimetidine. The transcellular transport parameter net efflux ratios of cimetidine across MDCK-hOCT1/hMATE1 and MDCK-hOCT2/hMATE1 monolayer were 17.5 and 3.65, respectively. In conclusion, cell models with good hMATE1 function have been established successfully, which can be applied to study the drug transport or drug-drug interaction involving hMATE1 alone or together with hOCT1/2 in vitro.
Animals ; Biological Transport ; Cimetidine ; pharmacology ; DNA, Complementary ; Dogs ; Drug Interactions ; Humans ; Madin Darby Canine Kidney Cells ; Metformin ; pharmacology ; Organic Cation Transport Proteins ; genetics ; metabolism ; Transfection

OBJECTIVETo explore the correlation between Pi and Shen by observing the relationship between the metabolism of aristolochic acid (AA) and mRNA and protein expression levels of organic anion transporting polypeptide (oatp) superfamily member 2a1 and 2 b1 (oatp2al and oatp2bl) in renal, small intestinal, and large intestinal tissues of Pi deficiency syndrome (PDS) model rats.

METHODSTotally 46 Sprague-Dawley (SD) rats were randomly divided into four groups, i.e., the blank group (n = 12), the PDS group (n = 22), the AA-I group (n = 6), and the PDS AA-I group (n = 6). PDS model was established by subcutaneously injecting Reserpine at the daily dose of 5 mg/kg for 16 successive days. Carotid intubation was performed in 6 rats selected from the blank group and the PDS group. Pharmacokinetics of AA-I were detected at 5, 15, 30, 45, and 60 min after gastrogavage of AA-I. AA-I concentrations in renal, small intestinal, and large intestinal tissues of 10 rats selected from the PDS group were determined. Normal saline was administered to 6 rats selected from the PDS group and the blank group by gastrogavage. Renal, small intestinal, and large intestinal tissues were collected in the AA-I group and the PDS AA-I group at 60 min after gastrogavage of AA-I. mRNA and protein expression levels of oatp2a1 and oatp2b1 in each tissue were detected using real-time polymerase chain reaction (RT- PCR) and Western blot.

RESULTSCompared with the blank group, plasma concentrations of in vivo AA-I were obviously higher in the PDS group at 15, 30, 45, and 60 min after gastrogavage of AA-I with statistical difference (P < 0.05). Plasma concentrations of AA-I were obviously decreased at 60 min after gastrogavage of AA-I; AA-I concentrations in renal and large intestinal tissues were elevated; AA-I concentrations in small intestinal tissues were obviously reduced in the PDS group. There was no statistical difference in mRNA expression levels of oatp2a1 and oatp2b1 in the aforesaid three tissues of rats between the blank group and the PDS group. Compared with the blank group, mRNA expression levels of oatp2a1 and oatp2b1 decreased in small intestinal tissues of the AA-I group, and the mRNA expression level of oatp2a1 in large intestinal tissues significantly decreased (P < 0.05, P < 0.01). Compared with the PDS group, mRNA expression levels of oatp2a1 and oatp2b1 increased in renal tissues of the PDS AA-I group (P < 0.05); mRNA expression levels of oatp2b1 increased in large intestinal tissues of the PDS AA-I group (P < 0.05).

CONCLUSIONSThe difference in AA-I metabolism might be associated with changed expression levels of oatp2a1 and oatp2b1 in renal, small intestinal, and large intestinal tissues under Pi deficiency induced loss of transportation. Shen and Dachang played important roles in substance metabolism under Pi deficiency state, which proved Pi-Shen correlated in Chinese medical theories.

Animals ; Anions ; Aristolochic Acids ; metabolism ; Drugs, Chinese Herbal ; Kidney ; Medicine, Chinese Traditional ; Organic Cation Transport Proteins ; metabolism ; Peptides ; RNA, Messenger ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo identify potential mutation of SLC22A5 gene in a 5-month-old boy affected with primary carnitine deficiency and provide genetic counseling and prenatal diagnosis for the members of his family.

METHODSDNA was extracted from peripheral blood samples derived from the proband, his parents and elder sister, as well as amniotic fluid from his pregnant mother. All of the 10 exons of the SLC22A5 gene were amplified by PCR and subjected to Sanger sequencing. The amniotic fluid sample was also subjected to G-banded karyotyping and multiplex ligation-dependent probe amplification (MLPA).

RESULTSA homozygous mutation c.760C>T (p.R254X) of the SLC22A5 gene was detected in the proband. Heterozygous mutation c.760C>T (p.R254X) was also found in other family members including the fetus. The karyotyping and chromosomal microdeletion testing for the amniotic fluid sample were both normal.

CONCLUSIONThe newly identified homozygous nonsense c.760C>T (p.R254X) mutation of the SLC22A5 gene probably underlies the primary carnitine deficiency of the proband. Genetic counseling and prenatal diagnosis have been provided for this family.

Adult ; Asian Continental Ancestry Group ; genetics ; Base Sequence ; Cardiomyopathies ; embryology ; genetics ; Carnitine ; deficiency ; genetics ; China ; Exons ; Female ; Genotype ; Humans ; Hyperammonemia ; embryology ; genetics ; Infant ; Male ; Molecular Sequence Data ; Muscular Diseases ; embryology ; genetics ; Organic Cation Transport Proteins ; genetics ; Pedigree ; Pregnancy ; Prenatal Diagnosis ; Solute Carrier Family 22 Member 5

OBJECTIVETo evaluate the association between SLC22A1 expression and the outcomes of hepatocellular carcinoma (HCC) patients.

METHODSA tissue microarray of 303 HCC and matched adjacent noncancerous liver tissues (ANLTs) were constructed. The expression of SLC22A1 was tested by immunohistochemistry (IHC) and scored by two pathologists according to a 12-score scale (a score>6 was defined as high expression, and a score≤6 as low expression). The correlation of SLC22A1 expression with the clinicopathological features and the patients' outcome was analyzed.

RESULTSAll the ANLTs had a IHC score of 12, as compared to only 29 (9.6%) of the HCC tissues. The patients were divided into 2 groups based on the IHC scores: 59% (180/303) in low expression group and 41% (123/303) in high expression group. The disease-free survival (DFS) rates and overall survival (OS) rates were significantly lower in low SLC22A1 expression group than in the high expression group. The 1-, 3-, and 5-year DFS rates were 43%, 31% and 27% in the low expression group, and were 58%, 47% and 43% in the high expression group, respectively. The 1-, 3-, and 5-year OS rates were 66%, 38% and 32% in low expression group, and were 80%, 57% and 50% in the high expression group, respectively. A low expression of SLC22A1 was positively correlated with the tumor diameter, BCLC stage, tumor differentiation, and AFP levels (P<0.05), and was an independent predictor of poor overall survival (HR=1.454; 95% CI, 1.050-2.013).

CONCLUSIONSDown-regulation of SLC22A1 is a malignant feature and a potential prognostic marker of HCC.

Carcinoma, Hepatocellular ; diagnosis ; metabolism ; Disease-Free Survival ; Down-Regulation ; Humans ; Immunohistochemistry ; Liver Neoplasms ; diagnosis ; metabolism ; Organic Cation Transporter 1 ; metabolism ; Prognosis ; Survival Rate ; Tissue Array Analysis

OBJECTIVETo identify pathogenic mutation in a boy affected with riboflavin responsive-multiple acyl-CoA dehydrogenase deficiency (RR-MADD).

METHODSThe patient was initially diagnosed as primary carnitine deficiency (PCD) and has been treated with carnitine supplementation for 7 years. Clinical manifestations and characteristics of fibula muscle specimen were analyzed. Potential mutation in electron transfer flavoprotein dehydrogenase (ETFDH) gene (for the patient and his parents) and carnitine transfer protein gene (SLC22A5) (for the patient) was screened.

RESULTSElectronic microscopy of the muscle specimen has suggested lipid storage myopathy. Mutation analysis has found that the patient carried compound heterozygous mutations, c.250G>A and c.380T>C, in exon 3 of the ETFDH gene, whilst his father and mother were heterozygous for the c.380T>C and c.250G>A mutations, respectively. Screening of the SLC22A5 gene has yielded no clinically meaningful result. After the establishment of diagnosis of RR-MADD, the condition of the patient has improved greatly with supplementation of high doses of riboflavin along with continuous carnitine supplement.

CONCLUSIONThe c.250G>A (p.Ala84Thr) mutation of exon 3 of the ETFDH gene has been a hot spot in Southern Chinese population, whilst the c.380T>C (p.Leu127Pro) is rarely reported. Our case has suggested that therapeutic diagnosis cannot substitute genetic testing. The mechanism for having stabilized the patient with only carnitine supplementation for 7 years needs further investigation.

Adolescent ; Adult ; Base Sequence ; Child ; DNA Mutational Analysis ; Electron-Transferring Flavoproteins ; genetics ; metabolism ; Female ; Humans ; Iron-Sulfur Proteins ; genetics ; metabolism ; Male ; Molecular Sequence Data ; Multiple Acyl Coenzyme A Dehydrogenase Deficiency ; enzymology ; genetics ; metabolism ; Muscle, Skeletal ; metabolism ; Organic Cation Transport Proteins ; genetics ; metabolism ; Oxidoreductases Acting on CH-NH Group Donors ; genetics ; metabolism ; Riboflavin ; metabolism ; Solute Carrier Family 22 Member 5

Mammal multidrug and toxin extrusion proteins (MATEs) play an important role in the transport of organic cations in the body. MATEs mediate the final excretion step for multiple organic cation drug used clinically and important endogenous substances. This article reviews the discovery, type, gene coding and polymorphism, body distribution, classification of substrates and inhibitors and their research method of MATEs. The article also discusses the major research significance of MATEs with examples.
Animals ; Biological Transport ; Cations ; Organic Cation Transport Proteins ; metabolism ; Polymorphism, Genetic

OBJECTIVETo investigate the mutation and background of SLC22A5 in 6 patients with primary carnitine deficiency (PCD) who only presented as cardiomyopathy.

METHODGenomic DNA were abstracted from the blood of the patients and their parents. Using high-throughput sequencing to determine the mutation site.Using Sanger method to confirm the mutated alleles in PCD patients and detect the corresponding sequences in their patients. Using SIFT and PolyPhen to predict the function of protein for detected missense mutations.

RESULTThree different mutations were identified, including 2 nonsense mutations (R254X and R289X), 1 missense mutation (C113Y), R254X was the most frequently seen mutation. Four patients had compound heterozygous mutations and 2 patients had homozygous mutations. Their parents were found to have heterozygous mutations in corresponding alleles.

CONCLUSIONR254X, R289X and C113Y might be associated with primary carnitine deficiency.

Adolescent ; Base Sequence ; Cardiomyopathies ; genetics ; Carnitine ; deficiency ; genetics ; Child ; Child, Preschool ; DNA Mutational Analysis ; Female ; Genotype ; Heterozygote ; High-Throughput Nucleotide Sequencing ; Humans ; Hyperammonemia ; genetics ; Infant ; Male ; Muscular Diseases ; genetics ; Mutation ; Organic Cation Transport Proteins ; genetics ; Pedigree ; Solute Carrier Family 22 Member 5

Liver is regarded as one of the most important organs for drug clearance in the body, which mediates both the metabolism and biliary excretion of drugs. Transporters are a class of functional membrane proteins and control the movement of substances into or out of cells. Transporters, which are extensively expressed in the liver, play important roles in the drug hepatic disposition by regulating the uptake of drugs from blood into hepatocytes or the efflux of drugs and their metabolites into bile. In this review, the localization, functions and substrate selectivity of the major transporters in the liver will be summarized, and the impacts of these transporters on drug hepatic disposition, the potential drug-drug interactions as well as their genetic polymorphisms will also be reviewed.
ATP Binding Cassette Transporter, Sub-Family G, Member 2 ; ATP-Binding Cassette Transporters ; genetics ; metabolism ; ATP-Binding Cassette, Sub-Family B, Member 1 ; genetics ; metabolism ; Bile ; metabolism ; Biological Transport ; Drug Interactions ; Humans ; Liver ; metabolism ; Membrane Transport Proteins ; genetics ; metabolism ; Metabolic Clearance Rate ; Multidrug Resistance-Associated Proteins ; genetics ; metabolism ; Neoplasm Proteins ; genetics ; metabolism ; Organic Anion Transporters ; genetics ; metabolism ; Organic Anion Transporters, Sodium-Dependent ; metabolism ; Organic Anion Transporters, Sodium-Independent ; genetics ; metabolism ; Organic Cation Transport Proteins ; genetics ; metabolism ; Pharmacokinetics ; Polymorphism, Genetic ; Symporters ; metabolism