Genetic polymorphism of NPHS1 modifies the clinical manifestations of Ig A nephropathy

Nephrin, the molecule responsible for congenital nephrotic syndrome of Finnish type, is crucial in maintaining the glomerular filtration barrier. Recently, its complete gene structure and common gene polymorphisms in its exons have been reported, although the functional and clinical significance of these polymorphisms has not yet been elucidated. We investigated a possible association of the NPHS1 polymorphisms with the development of Ig A nephropathy (IgAN), as well as the clinical and histologic manifestations in IgAN. A total of 464 Japanese subjects, including 267 patients with histologically proven IgAN and 197 healthy controls with normal urinalysis, were genotyped for the NPHS1 G349A, G2289A, and T3315C polymorphisms. The frequencies of the genotypes, alleles, and estimated haplotypes of NPHS1 polymorphisms were no different between patients with IgAN and the controls. Within the IgAN group, patients carrying at least one G allele of G349A tended to present with more proteinuria, lower renal function, and more severe histopathologic injury than those with the AA genotype, although the time from the first urinary abnormality to the renal biopsy was no different between both groups. The logistic regression analysis indicated that even after adjusting for the effect of proteinuria and hypertension the GG genotype of NPHS1 G349A was an independent risk factor for the deteriorated renal function at the time of diagnosis. This study suggests that the NPHS1 G349A polymorphism may be associated with heavy proteinuria and a decline in renal function in patients with IgAN.     

CD38 polymorphisms in Spanish patients with systemic lupus erythematosus

The ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase (CD38) gene is a positional and functional candidate gene for the susceptibility to systemic lupus erythematosus (SLE) because CD38 gene maps in the described SLE risk region 4p15 and CD38 molecule is a leukocyte activation antigen and ectoenzyme involved in numerous immune functions. The aim of this study was to investigate the possible association of the polymorphisms located at positions 182 of intron 1 (C/G) and 418 (C/T, located in exon 3) of the CD38 gene with the susceptibility and clinical features of SLE. Genotyping of 276 Spanish patients with SLE and 194 controls was performed by polymerase chain reaction amplification-refractory mutation system techniques. No association between the polymorphisms studied and the susceptibility to SLE was found. However, when patients were stratified according to their clinical manifestations, a significant increase of CC individuals and a significant decrease of CG individuals among patients with discoid rash (67.9% vs. 53.1% in controls p = 0.02, pc > 0.05, odds ratio [OR] = 1.87, 95% confidence interval [95% CI] 1.05-3.35; and 23.5% vs. 40.2% in controls, p = 0.008, pc = 0.024, OR = 0.46 95% CI 0.24-0.85) were found. Logistic regression analysis identified CC genotype as an independent risk factor for discoid rash among patients with SLE (p = 0.01, OR = 2.23, 95% CI 1.19-4.18). In conclusion, a slight contribution of the polymorphism located in intron 1 of the CD38 gene in the clinical features of SLE could be postulated.     

Association between lymphotoxin beta receptor gene polymorphisms and IgA nephropathy in Korean children

Lymphotoxin beta receptor (LTBR) is essential for development and organization of the secondary lymphoid tissues. To investigate whether LTBR polymorphisms are associated with IgA nephropathy (IgAN) in Korean children, One hundred ninety nine patients with IgAN and 289 controls were recruited. Two promoter single nucleotide polymorphisms (SNPs) (rs3759333, -1387C/T and rs3759334, -1326A/G) and one coding SNP (rs2364480, Ala172Ala) in LTBR gene were selected and genotyped by direct sequencing. For analysis of data, SNPStats, SPSS 18.0, and Haploview version 4.2 were used. Multiple logistic regression models (codominant 1, codominant 2, dominant, and recessive models) were performed for odds ratio (OR), 95% confidence interval (CI), and p value. The rs3759334 was significantly associated with IgAN in codominant 1 (G/G vs. A/G, p = 0.025) and dominant (p = 0.017) models. The A alleles of rs3759334 and rs2364480 were related to risk of developing IgAN, respectively (rs3759334, p = 0.015; rs2364480, p = 0.041). Haplotypes CGC and TAA in LTBR gene were also associated with IgAN, respectively (CGC, p = 0.032 in codominant; TAA, p = 0.008 in codominant, p = 0.009 in dominant models). In conclusion, results suggest that LTBR gene polymorphisms may be associated with risk of IgAN in Korean children.     

Identification of mannose-binding lectin as a mechanism in progressive immunoglobulin A nephropathy

Immunoglobulin A nephropathy (IgAN), the pathogenesis of which remained still unclear is one of the leading courses of end-stage renal disease in approximately 50% affected patients. On the basis of several researches, the activation of complement mannose-binding lectin (MBL) pathway might be the underlying mechanism in disease progress. In order to investigate the relationship between MBL pathway and IgAN, we discussed the MBL gene polymorphism as well as its expressed level in serum, urine and renal parenchymal, with renal outcome in IgAN patients. The significantly down-regulated expression of MBL was discovered, which may serve as a potential urinary biomarker in progressive IgAN according to the results of difference in gel electrophoresis and matrix-assisted laser desorption/ionization time of flight mass spectrometry. The single nucleotide polymorphisms of MBL gene in promoter and exon region were found and confirmed relating with the poor prognosis of progressive IgAN patients. As a result, the deficient activation of MBL pathway caused by the mutation of MBL accompanied with low expressed level of MBL in serum might be the potential inspiring regulation in IgAN, and will attract a promising insight in remedy of IgAN to inhibit further progress.     

Sequence Analysis of TNFRSF13b, Encoding TACI, in Patients with Systemic Lupus Erythematosus

B cell activating factor belonging to the TNF family (BAFF) and a proliferation inducing ligand (APRIL), and their receptors BAFF receptor (BAFFR), B cell maturation antigen (BCMA), and transmembrane activator and CAML interactor (TACI) are involved in the regulation of B cell homeostasis and differentiation. BAFF overexpression leads to systemic lupus erythematosus (SLE) in mice and elevated BAFF levels have been observed in human SLE and mouse models for SLE. Furthermore, genetic inactivation of TACI in mice results in a SLE-like phenotype. Based on our recent finding that TACI is mutated in patients with common variable immunodeficiency, of whom more than 30% suffer from autoimmune conditions, we analyzed TACI in humans with SLE. Sequence analysis of TNFRSF13b/TACI in 119 unrelated SLE patients revealed four variants: R20C in exon 1, R72H in exon 3, the silent variation c.327 G > A in exon 3, and A181E in exon 4. No significant association with any of these variants was found, when compared to the frequencies of the variants in a healthy control cohort. Furthermore, the mutated alleles R20C and R72H did not segregate with the SLE phenotype in familial cases of SLE. Thus, our evaluation of the coding region of TNFRSF13b/TACI did not reveal any deleterious or disease-associated mutations. Inga Melchers and Bodo Grimbacher contributed equally to this work.     

Increased frequency of the mannose-binding lectin LX haplotype in Chinese systemic lupus erythematosus patients.

Mannose-binding lectin (MBL) is an important complement-activating protein of the human immune system. As a result of one of three structural gene mutations in exon 1 (variants B, C and D) and/or the presence of a low-efficiency promoter polymorphism, MBL deficiency may be associated with increased susceptibility to infectious diseases and to autoimmune disorders, including systemic lupus erythematosus (SLE). Using a combined approach of heteroduplex generator and polymerase chain reaction, a systematic search for mutations in exon 1 and the promoter region of the MBL gene was performed in a Chinese study population comprising 41 SLE patients and 111 healthy controls. Two alleles, a wild-type allele A and a variant allele B (a previously reported mutation of GGC to GAC at codon 54), were identified in MBL exon 1. The frequency of the B allele (0.15) was higher in the SLE patients than in the healthy controls (0.09), but the difference did not attain statistical significance (P > 0.05). However, for two polymorphisms at positions -550 and -221 in the promoter region, the frequency of the low-MBL-producing haplotype (LX) in the patients (0.2073) was significantly higher than that in the controls (0.0855) (P = 0.003, relative risk = 2.79). Our results suggest that the LX haplotype represents a strong risk factor among Chinese SLE patients. Although of lesser importance, the MBL B allele also may be a risk component in the developing process of SLE in Chinese patients.     

Increased serum levels of IgA1-IgG immune complexes and anti-F(ab')2 antibodies in patients with primary IgA nephropathy

A solid-phase ELISA was used to detect IgA1 immune complexes (IgA1 ICs) containing IgG and IgM in 38 serum samples from 30 patients with primary IgA nephropathy (IgAN) and 14 subjects with non-IgA chronic glomerulonephritis. A jackfruit lectin, jacalin, was used as the substrate for the selective binding of human IgA1 ICs in serum PEG precipitate (7%). The presence of IgG, A and M antibodies against the F(ab')2 region of IgG was also investigated by the solid-phase ELISA. Six patients were studied during remission and relapse (fever, upper respiratory tract infection and macroheamaturia). The results showed significant increases in serum levels of IgA1 ICs (P less than 0.001) in 39.4% of the IgAN patients, IgA1-IgG ICs (P less than 0.001) in 68.4%, and IgA1-IgM ICs (P less than 0.002) in 10.5% of the patients. A significant increase in IgA1-IgG ICs was observed during relapse (P less than 0.02). Significantly high values of IgG (P less than 0.003) and IgA (P less than 0.001) antibodies directed at the F(ab')2 region of IgG were found. A significant increase in anti F(ab')2 antibodies (class IgA and IgM) was seen in the acute phase of the disease. The data suggest that an increased production of IgA1 ICs occurs in IgAN patients; ICs are mainly IgA1-IgG ICs during relapse. The presence of high serum levels of IgG and IgA antibodies against the F(ab')2 region of IgG indicates that in addition to the multiple anomalies of IgA regulation described in IgAN patients there may be further aberrances.     

PAI-1基因4G/5G多态性与内蒙地区IgA肾病的相关性研究

目的：探讨纤溶酶原活化物抑制剂-1（plasminogen activator inhibitor-1,PAI-1）基因启动子区4G/5G多态性与内蒙地区IgA肾病的相关性。方法：随机收集包头医学院第一附属医院肾内科IgA肾病患者100例,另设100名健康体检者为对照组。采用等位基因特异聚合酶链反应（ASPCR）法进行PAI-14G/5G基因型分析。结果：IgA肾病组的4G/4G基因型发生频率（41%）显著高于对照组（23%,P值〈0.01）。4G、5G等位基因频率比较无统计学差异（χ2=1.818,P〉0.05）。结论：PAI-14G/4G基因型是内蒙地区IgA肾病发病的易感基因型,但PAI-14G等位基因不是内蒙地区IgA肾病的易感基因。     

Peroxisome proliferator-activated receptor gamma C161T polymorphisms and survival of Japanese patients with immunoglobulin A nephropathy

Peroxisome proliferator-activated receptor gamma (PPAR gamma) plays an important role in lipid metabolism, insulin sensitivity, atherogenesis, and immune regulation. A genetic polymorphism (C161T) at exon 6 of PPAR gamma gene (PPARG) was reported to be associated with the onset of coronary artery disease. However, there has been no report of an association with renal disease. Genomic DNAs were isolated from 225 Japanese patients with histologically confirmed immunoglobulin A nephropathy (IgAN). The PPARG C161T genotype was determined by polymerase chain reaction-restriction fragment length polymorphism. The association of the polymorphism with renal prognosis in IgAN patients was analyzed using the Kaplan-Meier method and Cox proportional hazard regression model. The PPARG polymorphism was not associated with the renal survival rate. However, when patients were stratified into those either with or without hypertension at the time of diagnosis, the renal survival of the CT/TT genotypes was significantly better in those without hypertension than those with the CC genotype. We report that the PPARG C161T polymorphism is associated with the survival of IgAN patients without hypertension. The T allele of the polymorphism might have a protective effect on the progression of IgAN.     

Polyethylene Glycol Precipitates of Serum Contain a Large Proportion of Uncomplexed Immunoglobulins and C3

High pressure liquid chromatography (HPLC) was used to fractionate redissolved polyethylene glycol (PEG) precipitates isolated from the sera of normal volunteers and from patients with IgA nephropathy (IgAN) and systemic lupus erythematosus (SLE), 2 diseases characterized by elevated levels of circulating immune complexes. The individual fractions were analyzed by solid phase ELISA for IgA, IgM, C3, IgG, and complexes of IgG-IgA and IgG-C3. Although PEG precipitates were enriched for high molecular weight IgA and IgG (presumably bound within CIC), significant amounts of IgM, unbound IgG and C3 were also present. The quantities of the PEG-precipitable proteins did not correlate with their serum concentrations. IgG-IgA and IgG-C3 complexes were found in all precipitates examined, but the levels of complexes were higher in both patient groups. These results indicate that PEG precipitates a considerable quantity of proteins not bound in immune complexes. There appeared to be greater protein precipitation from sera of the patient groups compared to the amount precipitated from the normal sera. These results suggest that an understanding of the mechanism of PEG precipitation may be important in defining abnormalities in IgAN, SLE and perhaps other diseases characterized by elevated levels of CIC. In addition, the possibility of undetected CIC in PEG precipitable material must be considered.     

Polyethylene Glycol Precipitates of Serum Contain a Large Proportion of Uncomplexed Immunoglobulins and C3

High pressure liquid chromatography (HPLC) was used to fractionate redissolved polyethylene glycol (PEG) precipitates isolated from the sera of normal volunteers and from patients with IgA nephropathy (IgAN) and systemic lupus erythematosus (SLE), 2 diseases characterized by elevated levels of circulating immune complexes. The individual fractions were analyzed by solid phase ELISA for IgA, IgM, C3, IgG, and complexes of IgG-IgA and IgG-C3. Although PEG precipitates were enriched for high molecular weight IgA and IgG (presumably bound within CIC), significant amounts of IgM, unbound IgG and C3 were also present. The quantities of the PEG-precipitable proteins did not correlate with their serum concentrations. IgG-IgA and IgG-C3 complexes were found in all precipitates examined, but the levels of complexes were higher in both patient groups. These results indicate that PEG precipitates a considerable quantity of proteins not bound in immune complexes. There appeared to be greater protein precipitation from sera of the patient groups compared to the amount precipitated from the normal sera. These results suggest that an understanding of the mechanism of PEG precipitation may be important in defining abnormalities in IgAN, SLE and perhaps other diseases characterized by elevated levels of CIC. In addition, the possibility of undetected CIC in PEG precipitable material must be considered.     

Increased frequency of the mannose‐binding lectin LX haplotype in Chinese systemic lupus erythematosus patients

Mannose‐binding lectin (MBL) is an important complement‐activating protein of the human immune system. As a result of one of three structural gene mutations in exon 1 (variants B, C and D) and/or the presence of a low‐efficiency promoter polymorphism, MBL deficiency may be associated with increased susceptibility to infectious diseases and to autoimmune disorders, including systemic lupus erythematosus (SLE). Using a combined approach of heteroduplex generator and polymerase chain reaction, a systematic search for mutations in exon 1 and the promoter region of the MBL gene was performed in a Chinese study population comprising 41 SLE patients and 111 healthy controls. Two alleles, a wild‐type allele A and a variant allele B (a previously reported mutation of GGC to GAC at codon 54), were identified in MBL exon 1. The frequency of the B allele (0.15) was higher in the SLE patients than in the healthy controls (0.09), but the difference did not attain statistical significance (P > 0.05). However, for two polymorphisms at positions ?550 and ?221 in the promoter region, the frequency of the low‐MBL‐producing haplotype (LX) in the patients (0.2073) was significantly higher than that in the controls (0.0855) (P = 0.003, relative risk = 2.79). Our results suggest that the LX haplotype represents a strong risk factor among Chinese SLE patients. Although of lesser importance, the MBL B allele also may be a risk component in the developing process of SLE in Chinese patients.     

Autoantibodies in vitiligo patients are not directed to the melanocyte differentiation antigen MelanA/MART1

IgA nephropathy (IgAN) is characterized by mesangial deposition of polymeric IgA (pIgA). Abnormalities of the IgA system include reduced mucosal and increased bone marrow (BM) pIgA production. Gammadelta T cells are regulators of mucosal IgA production and oral tolerance. We have described previously a deficiency of gammadelta T cells expressing Vgamma3 and Vdelta3 from the duodenal mucosa in IgAN. Since pIgA production is displaced to the BM, we have now studied BM gammadelta T cells in IgAN. Peripheral blood and BM aspirates were obtained from 14 patients with IgAN and 15 controls. Expression of TCR gamma and delta V region families was analysed by semiquantitative RT-PCR, and CDR3 spectratyping of Vgamma1-4 and Vdelta3 genes was performed. We found no difference between IgAN and controls in the V region usage of blood gammadelta T cells. However, in the BM of patients with IgAN, there was significantly reduced expression of the V region families Vgamma3 and Vdelta3, with the decrease in Vdelta3 being particularly striking. CDR3 spectratyping showed no abnormalities in blood or BM samples. Vgamma3 and Vdelta3 are underexpressed in the duodenum and the BM in IgAN. The combination of imbalanced mucosal and systemic pIgA production with deficient expression of gammadelta T cells using Vgamma3 and Vdelta3 in both sites may imply a role for these gammadelta T cells in the normal regulation of IgA immune responses, and in the complex immunopathogenesis of IgAN.     

68例维吾尔族IgA肾病患者MBL基因多态性与临床病理相关性分析

目的 探讨甘露糖结合凝集素(mannose-binding lectin,MBL)基因第54位密码子多态性与维吾尔族IgA肾病患者临床和病理的关系.方法 应用PCR-RFLP方法对68例维吾尔族IgAN患者进行MBL多态性检测,并与患者临床和病理特点进行相关性分析.结果 ①维吾尔族IgAN中表现为蛋白尿的患者突变型等位基因GAC的发生频率显著高于表现为单纯血尿的患者(P＜0.05);②维吾尔族IgAN中表现为复合性免疫沉积的患者等位基因GAC的发生频率显著高于表现为单纯免疫沉积的患者(P＜0.05).结论 MBL突变型等位基因GAC与维吾尔族IgAN 蛋白尿发生和免疫复合沉积相关.     

Defective immunoglobulin A (IgA) glycosylation and IgA deposits in patients with IgA nephropathy

Defective glycosylation and immune complex (IC) formation may be of primary importance in immunoglobulin A nephropathy (IgAN) pathogenesis. The aim of this study was to determine whether defective IgA1 glycosylation might support renal deposition of IgA and disease activity. IgA was isolated from the serum of 44 IgAN patients and 46 controls and glycosylation analysed by ELISA using glycan‐specific lectins. IgA was measured by immunodiffusion and immune complexes by ELISA. IgA subclasses in IC deposits in kidney glomeruli were identified by immunohistochemical methods. A significant increase in N‐acetylgalactosamine (GalNAc) in terminal position (p = 0.02) observed in some of the IgAN patients, became more pronounced when sialic acid was removed from IgA1, indicating enhanced expression of α‐2,6‐sialyltransferase in patients compared with controls (p < 0.0001). Patients with defective galactosylation had lower serum IgA than other IgAN patients (p = 0.003). IgAN patients with both IgA1 and IgA2 glomerular deposits (21.7%) had increased GalNAc in terminal position (p = 0.003). Taken together, our results show that increased IgA glycosylation in IgAN associates with low levels of IgA, concomitant IgA1 and IgA2 glomerular deposits and poor clinical outcome.     

中国甘肃汉/ 回族人群C1GALT1、DEFA 基因多态性与IgA 肾病易感性的关系

目的:探讨β-1,3鄄半乳糖基转移酶(C1GALT1)基因rs1008898 位点及琢防御素(α-defensin,DEFA) 基因rs2738081 位点多态性与中国甘肃省汉族人群和回族人群IgA 肾病易感性的关系,筛查预测IgA 肾病的分子标记。方法:本研究选取甘肃地区患IgA 肾病的汉族个体146 例、回族个体83 例作为实验组,另选取健康汉族个体180 例、健康回族个体100例作为对照组。采用聚合酶链反应鄄限制性片段长度多态性(Polymerase chain reaction-restriction fragment length polymorphism,PCR-RFLP) 方法以及PCR 产物基因测序法对单核苷酸多态性基因位点进行基因分型,采用卡方检验比较各基因型、等位基因在病例组和对照组中的分布差异,评价各基因型、等位基因与IgA 肾病发病风险的关系。结果: 汉族IgA 肾病患者rs1008898 位点的GG 基因型频率与健康对照组之间有统计学差异(OR=2.489,95CI:1.259 ~4.922,字2 =7.037,P =0.008),且G 等位基因可能增加患IgA 肾病的风险(OR = 1.268,95% CI:1.056 ~ 1.523,字2 = 6.709,P = 0.01)。回族IgA 肾病患者rs1008898 位点的多态性与健康对照组之间差异无统计学意义(P>0.05)。(3)rs2738081 位点多态性在汉族、回族IgA 肾病患者及其健康对照组之间的分布差异无统计学意义(P>0.05)。结论:rs1008898 位点G 等位基因可能增加甘肃地区汉族人群患IgA 肾病的风险。     

Study of the CTLA-4 gene polymorphisms in systemic lupus erythematosus (SLE) samples from Malaysia

Abstract

In this study, we investigated the polymorphisms of the exon 1 (+49A/G), promoter sites (–1722T/C, –1661A/G, –318C/T), and 3′-untranslated region (3′-UTR) (+6230 A/G) of the CTLA-4 gene in systemic lupus erythematosus (SLE) affected patients. Polymerase chain reaction-restriction fragment length polymorphism was used to determine genotypes of these five markers in 130 SLE patients and 130 healthy controls. Of the five tested polymorphisms, there was no statistical significant difference between the genotypic and allelic frequencies of patients with SLE and controls. Hence, we propose that the CTLA-4 gene does not play a major role in the genetic susceptibility to the development of SLE in the Malaysian population.