Clinical features and an atypical WT1 mutant site in a child with incomplete Denys-Drash syndrome

Dear Editor, Denys-Drash syndrome （DDS） is characterized by the triad of progressive nephropathy, urogenital malformation and Wilms＇ tumor. Incomplete variants present with nephropathy with either intersex disorders or Wilms＇ tumor.1 Most DDS patients carry WTI mutations in exon 8 or 9.1 Here, we report an incomplete DDS child who carried a novel WT1 missense mutation in exon 6 and had unique clinical manifestations.     

A mutant form of the Wilms' tumor suppressor gene WT1 observed in Denys-Drash syndrome interferes with glomerular capillary development

The Wilms' tumor suppressor gene WT1 encodes a zinc finger protein that is required for urogenital development. In the kidney, WT1 is most highly expressed in glomerular epithelial cells or podocytes, which are an essential component of the filtering system. Human subjects heterozygous for point mutations in the WT1 gene develop renal failure because of the formation of scar tissue within glomeruli. The relationship between WT1 expression in podocytes during development and glomerular scarring is not well understood. In this study, transgenic mice that expressed a mutant form of WT1 in podocytes were derived. The capillaries within transgenic glomeruli were dilated, indicating that WT1 might regulate the expression of growth factors that affect capillary development. Platelet endothelial cell adhesion molecule-1 expression was greatly reduced on glomerular endothelial cells of transgenic kidneys. These results suggest that WT1 controls the expression of growth factors that regulate glomerular capillary development and that abnormal capillary development might lead to glomerular disease.     

Novel WT1 mutation (C388Y) in a female child with Denys-Drash syndrome

We report the identification of a novel Wilms tumor suppressor gene mutation in a 5-month-old girl who presented with unilateral Wilms tumor (WT) and renal diffuse mesangial sclerosis typical of Denys-Drash syndrome (DDS). The patient did not have ambiguous genitalia and the karyotype (by amniocentesis) was 46, XX. A de novo constitutional heterozygous mutation in WT1 gene exon 9 coding for the third zinc-finger (1163G→A, C388Y) was identified. This mutation affects a cysteine residue involved in the coordination of the zinc atom, confirming the importance of these residues in the biological function of WT1 protein. Received: 10 November 2000 / Revised: 26 March 2001 / Accepted: 29 March 2001     

Denys-Drash综合征及其致病基因突变鉴定二例报道

目的 研究中国Denys-Drash综合征(DDS)患者Wilms瘤基因1(WT1)的基因突变。 方法 应用PCR方法扩增出WT1基因全部10个外显子及其相邻内含子序列，经纯化后进行PCR产物直接测序。 结果 2例患者WT1基因分别存在1个杂合错义突变。例1的X外显子9第1180位碱基C→T突变，造成第394位精氨酸改变为色氨酸，即p.R394W(c.1180C>T)。例号2的S外显子9第1203位碱基C→A突变，造成第401位组氨酸改变为谷氨酰胺，即p.H401Q(c.1203C>A)。其中第1203位碱基C→A突变，p.H401Q (c.1203C>A)，在国内外文献及突变数据库中均未见报道，属新发现的突变。 结论 DDS综合征WT1基因中外显子9为突变热点，并发现一种新的WT1基因突变。     

Clinical pictures and novel mutations of WT1-associated Denys-Drash syndrome in two Chinese children

Denys-Drash syndrome (DDS) is characterized by early onset of nephropathy, genitalia malformation, and Wilms' tumor, where WT1 is the gene that is mutated in most patients. We report two de novo mutations in WT1 found in two Chinese DDS children. Patient 1 was a boy with complete DDS who was presented with progressive nephropathy, unilateral Wilms' tumor, bilateral cryptorchidism, and renal histology showing diffuse mesangial sclerosis (DMS). When the patient was 24 months old, a liver ultrasound showed multiple nodules, and the patient died of pneumonia 1 month later. The de novo novel mutation, c.1130A>T (p.His377Leu), was identified; the mutation replaces histidine with leucine in the zinc finger (Znf) structure and is predicted to change the local spatial structure of the protein. Patient 2 had 46 XX with incomplete DDS and presented with normal genitalia, proteinuria, unilateral Wilms' tumor with renal pedicle lymph node metastasis, and renal histology showing DMS. Her renal function remains normal after 48 months. A de novo mutation, c.1168C>T (p.Arg390Term), was identified; it truncates 60 amino acids at the C terminus, and it is predicted to result in loss of the DNA-binding capacities of the WT1 protein.     

A novel mutation of<Emphasis Type="Italic"> WT1</Emphasis> exon 9 in a patient with Denys-Drash syndrome and pyloric stenosis

We report a novel mutation in WT1 exon 9 (1214 A>G) resulting in an amino acid change from H to R at codon 405 in a 46 XY female patient who had congenital hypertrophic pyloric stenosis, pseudohermaphroditism masculinus, renal failure, and Wilms tumor, and died at the age of 22 months. The patient demonstrated the difficulty in diagnosing a patient with intersex before conclusive genetic characterization.     

Inhibition of prostasin-induced ENaC activities by PN-1 and regulation of PN-1 expression by TGF-beta1 and aldosterone

Prostasin has been shown to regulate sodium handling in the kidney. Recently, a serine protease inhibitor, protease nexin-1 (PN-1), was identified as an endogenous inhibitor for prostasin. Therefore, we hypothesized that PN-1 may regulate sodium reabsorption by reducing prostasin activity, and that expression of PN-1 was regulated by transforming growth factor-beta1 (TGF-beta1) or aldosterone, like prostasin. cRNAs for epithelial sodium channel (ENaC), prostasin, and PN-1 were expressed in Xenopus oocytes, and the amiloride-sensitive sodium currents (I(Na)) were measured. The effect of TGF-beta1 and aldosterone on the mRNA and protein abundance of PN-1 and ENaC was detected by real-time polymerase chain reaction and immunoblotting in M-1 cells. Expression of PN-1 substantially decreased prostasin-induced I(Na) by approximately 68% in oocytes. Treatment of M-1 cells with 20 ng/ml TGF-beta1 significantly increased protein expression of PN-1 by 3.8+/-0.5-fold, whereas administration of 10(-6) M aldosterone markedly decreased protein expression of PN-1 to 53.7+/-6.7%. Basolateral, but not apical, application of TGF-beta1 significantly reduced I(eq). To elucidate the involvement of PN-1 in basal ENaC activity, we silenced the expression of PN-1 by using short-interfering RNA. This increased I(eq) by 1.6+/-0.1-fold. Our study indicates that PN-1 could have a natriuretic role by inhibiting prostasin activity and suggests the possibility that aldosterone and TGF-beta reciprocally regulate the expression of PN-1 in renal epithelial cells contributing to salt retention or natriuresis, respectively by an additional mechanism. PN-1 could represent a new factor that contributes to regulation of ENaC activity in the kidney.     

斑马鱼notch1a对pka报告基因转录调控作用

目的 通过双荧光素酶报告基因研究斑马鱼notch1a对pka的转录调控作用.方法 利用NCBI数据库获得斑马鱼notch1a基因序列,克隆notch1a基因的胞内段NICD(Notch intracellular domain),构建pCMV-N1aICD表达载体,利用蛋白质印迹法检测N1aICD蛋白的表达水平;利用加...     

WT1 mutation and podocyte molecular expression in a Chinese Frasier syndrome patient

We report on a Chinese girl with Frasier syndrome (FS). She presented with steroid-resistant focal segmental glomerulosclerosis (FSGS) and male pseudohermaphroditism. The WT1 IVS 9  +  5 G>A mutation was detected in one allele in the proband. The ratio of +KTS/−KTS was 0.67 in the proband’s cDNA. The expression of podocyte molecules (WT1, nephrin, podocin, α-actinin 4 and CD2AP) were also investigated in a renal specimen of this FS patient. WT1 expression showed diffuse nuclear staining, with less obvious speckles in the patient’s glomeruli than in those of controls. The distribution and intensity of podocyte molecules were altered both in normal- and abnormal-appearing glomeruli. In conclusion, the study presented a case of FS by clinical manifestation, renal pathology, karyotype analysis and genetic testing. A lower ratio of +KTS/−KTS and an abnormal distribution of WT1, as well as abnormal expressions of other podocyte molecules, were also revealed. The mechanisms of WT1 mutation causing FS still need to be investigated. Jianguo Li and Dan Zhao contributed equally to this work.     

Differential regulation of proteoglycan 4 metabolism in cartilage by IL-1alpha, IGF-I, and TGF-beta1

OBJECTIVES: To determine (1) if interleukin-1 alpha (IL-1alpha), insulin like growth factor I (IGF-I), and transforming growth factor-beta 1 (TGF-beta1) regulate proteoglycan 4 (PRG4) metabolism in articular cartilage, in terms of chondrocytes expressing PRG4 and PRG4 bound at the articular surface, and (2) if these features of cartilage PRG4 metabolism correlate with its secretion. METHODS: Articular cartilage explants were harvested and cultured for 6 days with or without 10% fetal bovine serum (FBS), alone, or with the addition of 10ng/ml IL-1alpha, 300ng/ml IGF-I, or 10ng/ml TGF-beta1. PRG4 expression by chondrocytes in the cartilage disks was assessed by immunohistochemistry (IHC). PRG4 bound to the articular surface of disks was quantified by extraction and enzyme-linked immunosorbent assay (ELISA). PRG4 secreted into culture medium was quantified by ELISA and characterized by Western Blot. RESULTS: PRG4 expression by chondrocytes near the articular surface was markedly decreased by IL-1alpha, stimulated by TGF-beta1, and not affected by IGF-I. The level of PRG4 accumulation in the culture medium was correlated with the number of chondrocytes expressing PRG4. The amount of PRG4 bound at the articular surface was modulated by incubation in medium including FBS, but did not correlate with levels of PRG4 secretion. CONCLUSIONS: Cartilage secretion of PRG4 is highly regulated by certain cytokines and growth factors, in part through alteration of the number of PRG4-secreting chondrocytes near the articular surface. The biochemical milieu may regulate the PRG4 content of synovial fluid during cartilage injury or repair.     

MAPK/AP-1-dependent regulation of PAI-1 gene expression by TGF-beta in rat mesangial cells

BACKGROUND: Receptor-regulated Smads and/or mitogen-activated protein kinases (MAPKs) are involved in transforming growth factor-beta (TGF-beta)-induced expression of various genes, including plasminogen activator inhibitor-1 (PAI-1). Because the sequence of the promoter region in rat PAI-1 gene differs from that in the human gene, we examined the mechanisms of TGF-beta-induced rat PAI-1 expression in rat mesangial cells. METHODS: TGF-beta1-induced PAI-1 and c-fos mRNA expressions were determined by Northern blot analysis. Activation of MAPKs and Smad proteins was evaluated by an immunoblot analysis. DNA binding activities of nuclear protein were examined by using an electrophoretic mobility shift assay (EMSA). The activities of PAI-1 promoter were measured by a luciferase reporter assay. RESULTS: Extracellular-regulated kinase (ERK) and c-Jun NH-terminal kinase (JNK) phosphorylation, c-fos mRNA expression, and activator protein-1 (AP-1) DNA binding activity stimulated by TGF-beta1 were completely suppressed by the ERK kinase (MEK) inhibitors. EMSA and reporter analysis revealed that an AP-1-like sequence located in the proximal region of the rat PAI-1 promoter was the target for TGF-beta1, and the disruption of this AP-1-like sequence suppressed basal and TGF-beta1-induced promoter activation. TGF-beta1 also stimulated nuclear translocation of Smads and binding to palindromic Smad binding element (SBE) located in the rat PAI-1 promoter, without being affected by MEK inhibitor. Point mutation and deletion of palindromic SBE did not affect TGF-beta1-induced rat PAI-1 promoter activity. Moreover, interferon-gamma (IFN-gamma) inhibited TGF-beta1-induced PAI-1 expression through selectively suppressing the ERK-AP-1 pathway. CONCLUSION: These results suggest that the essential requirement of MAPK/AP-1 activation for TGF-beta1-induced PAI-1 expression is unique to rat mesangial cells.     

Transcriptional regulation of a BMP-6 promoter by estrogen receptor alpha.

The effects of 17beta-estradiol (E2) and ICI 182,780 (ICI) on activity of a BMP-6 promoter were compared in osteoblast-like and breast cancer cells transiently transfected with ERalpha. E2 but not ICI stimulated BMP-6 reporter activity in breast cancer cells, whereas the opposite was observed in osteoblast-like cells, associated with lack of AF-2 dependence of the response, and absent intranuclear localization of ERalpha, suggesting the involvement of a distinct ERalpha-dependent response mechanism in osteoblasts. INTRODUCTION: Previous studies suggest that the tissue-selective effect of antiestrogens on bone reflects the ability of these compounds to target certain osteoblast regulatory genes. To explore this hypothesis, we examined whether antiestrogens preferentially stimulate the bone morphogenetic protein 6 (BMP-6) promoter in bone cells, and if so, whether this activity is associated with a distinct estrogen receptor (ER)alpha-dependent response mechanism to that in other cell types. MATERIALS AND METHODS: We compared the effects of 17beta-estradiol (E2) and ICI 182,780 (ICI) on activity of a 4.3-kb BMP-6 reporter construct in osteoblast-like cells (human MG63 and SaOS-2 cells and rat ROS 17/2.8 cells), human MCF-7 and T47-D breast cancer cell lines, and HepG2 hepatoma cells, after transient transfection with ERalpha, ERbeta, and mutant ER constructs. RESULTS: E2, but not ICI, stimulated BMP-6 reporter activity by approximately 100% in MCF-7, T47-D cells, and HepG2 cells when transfected with ERalpha. In contrast, in ERalpha-transfected osteoblast-like cells, an increase in reporter activity of approximately 75% was observed after treatment with ICI but not E2. The response of MG63 cells to ICI and MCF-7 cells to E2 both required ERalpha as opposed to ERbeta and the ERalpha activation function (AF)-1 activation domain. However, whereas the AF-2 domain was also required for E2 to stimulate reporter activity in MCF-7 cells, the response to ICI in MG63 cells was AF-2 independent. In further studies where we compared the intracellular distribution of ERalpha associated with these responses, E2-dependent stimulation of the BMP-6 reporter in MCF-7 cells was associated with intranuclear localization of ERalpha, whereas extranuclear localization was seen in rat osteosarcoma cells (ROS) cells treated with ICI. CONCLUSIONS: Antiestrogens selectively stimulate BMP-6 reporter activity in osteoblast-like cells through a distinct ERalpha-dependent mechanism characterized by independence of the AF-2 domain and extranuclear localization of ERalpha.     

一例NPHP1纯合缺失所致的Joubert综合征

患儿,男,因"6月龄竖头欠稳"于浙江大学医学院附属儿童医院康复科就诊。查体发现双眼追视欠灵活,竖头欠稳,不会翻身,双手握拳,双下肢负重差,四肢肌张力低。磁共振结果显示小脑蚓部小,中脑呈"磨牙状"改变,诊断为Joubert综合征。全外显子组测序及Sanger测序发现患儿存在AHI1基因的杂合移码突变(c.533_534delAA),该突变位点遗传自母亲;而针对已知致病基因的拷贝数变异分析提示NPHP1基因可能存在纯合型缺失。经多重连接探针扩增(multiplex ligation-dependent probe amplification,MLPA)方法进行验证,结果表明患儿的NPHP1基因确实存在纯合型缺失,且父母均为NPHP1杂合缺失的携带者。这是国内首次报道的由NPHP1纯合缺失所导致的Joubert综合征病例。由于NPHP1基因突变与肾脏损害相关性较高,应注意对此类突变患儿的密切随访。     

Prevalence of WT1 mutations in a large cohort of patients with steroid-resistant and steroid-sensitive nephrotic syndrome

BACKGROUND: Nephrotic syndrome (NS) represents the association of proteinuria, hypoalbuminemia, edema, and hyperlipidemia. Steroid-resistant nephrotic syndrome (SRNS) is defined by primary resistance to standard steroid therapy. It remains one of the most intractable causes for end-stage renal disease (ESRD) in the first two decades of life. Sporadic mutations in the Wilms' tumor suppressor gene WT1 have been found to be present in patients with SRNS in association with Wilms' tumor (WT) and urinary or genital malformations, as well as in patients with isolated SRNS. METHODS: To further evaluate the incidence of WT1 mutations in patients with NS we performed mutational analysis in 115 sporadic cases of SRNS and in 110 sporadic cases of steroid-sensitive nephrotic syndrome (SSNS) as a control group. Sixty out of 115 (52%) patients with sporadic SRNS were male, 55/115 (48%) were female. Sex genotype was verified by haplotype analysis. Mutational analysis was performed by direct sequencing and by denaturing high-performance liquid chromatography (DHPLC). RESULTS: Mutations in WT1 were found in 3/60 (5%) male (sex genotype) cases and 5/55 (9%) female (sex genotype) cases of sporadic SRNS, and 0/110 (0%) sporadic cases of SSNS. One out of five female patients with mutations in WT1 developed a WT, 2/3 male patients presented with the association of urinary and genital malformations, 1/3 male patients presented with sexual reversal (female phenotype) and bilateral gonadoblastoma, and 4/5 female patients presented with isolated SRNS. CONCLUSION: According to the data acquired in this study, patients presenting with a female phenotype and SRNS and male patients presenting with genital abnormalities should especially be screened to take advantage of the important genetic information on potential Wilms' tumor risk and differential therapy. This will also help to provide more data on the phenotype/genotype correlation in this patient population.     

中国汉族人3个家族性激素耐药型肾病综合征家系WT1和PLCE1基因突变分析

目的 分析中国汉族人家族性激素耐药型肾病综合征（SRNS）家系WT1和PLCE1基因突变及其特点。 方法 研究对象为A、B、C 3个汉族人SRNS家系的先证者（已除外NPHS2基因突变）及其父母,A、B 2个家系先证者的姐姐,50例尿检正常的汉族成年人作为对照人群。取所有研究对象外周静脉血3 ml,提取基因组DNA,PCR扩增WT1基因全部10个外显子和PLCE1基因全部31个编码外显子及其周围的部分内含子,应用直接DNA序列测定法和限制性片段长度多态性PCR（RFLP-PCR）分析法检测WT1和PLCE1基因变异。 结果 未发现WT1和PLCE1基因的致病突变。但是,在3个SRNS家系的先证者检测到3个WT1基因多态性：126C>T（P42P）、IVS5－64A>G和903A>G（R300R）,其中IVS5－64A>G为新发现的WT1基因多态性,126C>T和903A>G已见文献报道;还检测到13个PLCE1基因多态性 －134A>G、810T>C（C270C）、960G>A（E320E）、IVS11－28C>G、IVS15+26A>C、4724G>C（R1575P）、IVS20+40C>T、IVS21+64G>A、IVS22－26T>A、5320C>T（T1777I）、5780A>G（H1927R）、IVS27+24A>G和IVS31+48_49insT,其中IVS22－26T>A为新发现的PLCE1基因多态性,其余12个PLCE1基因多态性已见公布。 结论 WT1和PLCE1基因突变不是本研究3个中国汉族人家族性SRNS家系的主要致病原因。