[Analysis on association of glucocorticoid receptor gene polymorphism with steroid-resistance in idiopathic nephrotic syndrome of children].
OBJECTIVE
The nephrotic syndrome is defined by heavy proteinuria, edema, hypoalbuminemia, and hyperlipidemia. Idiopathic nephrotic syndrome (INS) mainly occurs in children, which is generally treated with glucocorticoids. The majority of patients are steroid-sensitive (SSINS) while steroid-resistance occurs in a subset of NS children (SRINS). Although intensive efforts have been undertaken to study the associations between SRINS and renal pathological changes, pharmacokinetics, and the GR density and binding affinity, the mechanisms underlying steroid-resistance are still not elucidated entirely. The authors hypothesized that it might be associated with polymorphisms in the glucocorticoid receptor gene (NR3C1). The study aimed to screen the NR3C1 gene for polymorphisms in genomic DNA samples from SRINS, SSINS children and control group, and to analyze the association of the polymorphisms in the NR3C1 gene and SRINS of children.


METHODS
Totally 39 SRINS and 67 SSINS children (81 males and 25 females with the mean age of 7 years) were involved in the study. Umbilical cord blood of 62 normal neonates and peripheral blood of 2 healthy volunteers were selected as controls. Genomic DNA was isolated from peripheral blood lymphocytes of all subjects. All the NR3C1-coding exons and intron-flanking portions were amplified by polymerase chain reaction (PCR). For polymorphism screen, PCR products were analyzed by denaturing high performance liquid chromatography (DHPLC). DNA fragments with aberrant elution profiles were re-amplified and sequenced directly.


RESULTS
Twelve aberrant elution profiles were identified with DHPLC in SRINS, SSINS and controls. Among them, 6 previously reported polymorphisms and 6 novel polymorphisms were confirmed by sequencing (198G > A, 200G > A, IVSD-16G > T, 1896C > T, 2166C > T, 2430T > C; novel, 1206C > T, 1374A > G, IVSG-68_IVSG-63delAAAAAA, 2193T > G, IVSH-9C > G, 2382C > T), and 3 groups of SNPs were in complete linkage disequilibrium, which resulted in 3 different haplotypes ([198G > A + 200G > A], [1374A > G + IVSG-68_IVSG-63delAAAAAA + IVSH-9C > G + 2382C > T], [1896C > T + 2166C > T + 2430T > C]). The last two genotypes were first reported. The genotype frequencies of the 2 novel haplotypes were 10.3% vs 1.5% in SRINS and SSINS, and 15.4% vs 7.5% in SRINS and SSINS, respectively. Other polymorphisms were relatively rare detectable both in patients and controls.


CONCLUSIONS
Twelve polymorphisms in the NR3C1 gene were detected with the technique of DHPLC, of which six polymorphisms were identified at the first time. Two types of newly found haplotypes were associated with steroid-resistant idiopathic nephrotic syndrome of children, which might be responsible for steroid-resistance in partial idiopathic nephrotic syndrome of children.
Ye
Jian-wei
JW
Department of Pediatrics, First Hospital, Peking University, Beijing 100034, China.
Ding
Jie
J
Huang
Jian-ping
JP
Chen
Yan
Y
Yao
Yong
Y
Xiao
Hui-jie
HJ
Yang
Ji-yun
JY
Shen
Ying
Y
Meng
Qun
Q
D000293:Adolescent; D002648:Child; D002675:Child, Preschool; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004351:Drug Resistance / Q000235:genetics; D005260:Female; D006801:Humans; D008297:Male; D009404:Nephrotic Syndrome / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic; D011965:Receptors, Glucocorticoid / Q000235:genetics*
D011965:Receptors, Glucocorticoid