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Prevalence of WT1 mutations in a large cohort of patients with steroid-resistant and steroid-sensitive nephrotic syndrome
Abstract
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.
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).
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.
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.
... The appearance of NS may be as a result of primary (idiopathic) glomerular or secondary (systemic) disease [2]. According to the response of the patients to the steroid therapy, NS has been divided into two different categories: Steroid-Sensitive Nephrotic Syndrome (SSNS) versus Steroid-Resistant Nephrotic Syndrome (SRNS) [3]. A bout 70% of SSNS patients will have one or more relapses, and a significant percentage will go on to develop either Frequent-Relapsing NS (FRNS) or steroid-dependent NS course (SDNS) [2]. ...
... Up to date, several causative genes related to NS have been identified by either using direct DNA sequencing approaches or next-generation sequencing technology [6][7][8][9][10][11][12]. From literature, NPHS1, NPHS2 and WT1 are the most analyzed genes especially in a large cohort of patients with NS [3,9,13]. The results proposed that, NPHS1 and WT1 mutations were essentially found in children with congenital NS, while NPHS2 mutations might be more prevalent in children with idiopathic SRNS [3,13]. ...
... From literature, NPHS1, NPHS2 and WT1 are the most analyzed genes especially in a large cohort of patients with NS [3,9,13]. The results proposed that, NPHS1 and WT1 mutations were essentially found in children with congenital NS, while NPHS2 mutations might be more prevalent in children with idiopathic SRNS [3,13]. The age of onset for NPHS2 mutation is varying but commonly before 6 years of age [14]. ...
Background: Nephrotic syndrome is the predominant glomeurular disease in childhood.
Mutations in numerous genes are known to be the reason for steroid-resistant nephrotic
syndrome; however, the presence of these mutations seems to be effected by race sociocultural
differences and interethnic group. Mutations in NPHS2 and WT1 genes record for nearly
20% and 5% of all children cases with steroid-resistant nephrotic syndrome, respectively.
By contrast, mutations are absent from children with either steroid-dependent nephrotic
syndrome or frequently-relapse nephrotic syndrome.
Methods: Mutation analysis was accomplished by direct sequencing of the complete 8
exons of NPHS2 and exons 8 and 9 of WT1 in 20 patients with steroid-resistant nephrotic
syndrome, 25 with steroid-dependent nephrotic syndrome, and 13 with frequently-relapse
nephrotic syndrome.
Results: Three pathogenic mutations in NPHS2 were detected within steroid-resistant
nephrotic syndrome patients. One as a non-sense mutation in exon 1 that was reported
previously, while the second was novel and found as missense mutation in exon 7. The
third one was found in the NPHS2 promoter region. Additionally, for the frst time, one
pathogenic missense mutation in exon 8 of WT1 gene was found in one Saudi patient with
steroid-resistant nephrotic syndrome. All four mutations were documented and submitted
to the ClinVar database.
Conclusion: NPHS2 and WT1 genes mutations are risk factors for steroid-resistant nephrotic
syndrome with about 15% and 5% in Saudi pediatric patients. More molecular studies are
required to clarify other possibility genes that responsible for the development of steroidresistant nephrotic syndrome in Saudi children.
... A WT1 mutation is also a frequent cause of sporadic isolated SRNS in girls, occurring in 10.8% of girls in a worldwide cohort (Muchaet al., 2006). No WT1 mutations have been detected in boys with sporadic isolated SRNS (Ruf et al., 2004b;Aucella et al., 2006;Mucha et al., 2006;Cho et al., 2008;Li et al., 2010). Our previous study identified no mutations in exons 8 and 9 of WT1 in 38 boys with sporadic isolated SRNS (Yang et al., 2013b). ...
... No mutations in the WT1 gene have been identified in boys with sporadic isolated SRNS (Table 3) (Ruf et al., 2004b;Aucella et al., 2006;Mucha et al., 2006;Cho et al., 2008;Li et al., 2010). Mucha et al. (2006) screened a worldwide cohort (Central European, Turkish, African-American, Hispanic, or Asian backgrounds) of 84 boys with sporadic isolated SRNS for mutations in all 10 exons of WT1. ...
... Li et al. (2010) screened 43 boys with sporadic isolated SRNS for mutations in exons 8 and 9 of WT1, but no mutations were found. Ruf et al. (2004b) examined 57 boys with sporadic isolated SRNS originating from Central Europe, Turkey, or India for mutations in exons 6-9 of WT1. They were unable to identify WT1 mutations in any of these patients. ...
Mutations in the Wilms' tumor gene, WT1, can lead to syndromic steroid-resistant nephrotic syndrome and isolated steroid-resistant nephrotic syndrome. WT1 mutations have been identified in the majority of children with Denys-Drash or Frasier syndrome. WT1 mutations have not previously been identified in boys with sporadic isolated steroid-resistant nephrotic syndrome, but, recently, four boys with isolated nephrotic syndrome were identified to have WT1 mutations. However, whether boys with sporadic isolated steroid-resistant nephrotic syndrome should be routinely subjected to mutation analysis of WT1 has not been established. We examined 35 boys with sporadic isolated steroid-resistant nephrotic syndrome for mutations in WT1. Mutation analysis of all 10 exons of WT1 was performed by polymerase chain reaction and direct sequencing. Karyotype analysis or Y chromosome identification was performed for all patients. A Y chromosome or a 46, XY karyotype was demonstrated for all 35 patients. No causative WT1 mutation was identified in any of the patients. The WT1 mutation, IVS4+14T>C, which is not predicted to affect splicing, was identified in one patient who achieved complete remission after 8 weeks of oral prednisone treatment, indicating that IVS4+14T>C is not a causative mutation. Five WT1 polymorphisms were also identified in some patients and controls. Our results suggest that mutation analysis of WT1 should not be routinely performed for genetically defined boys with sporadic isolated steroid-resistant nephrotic syndrome.
... Identification of a pathogenic mutation in a patient with SRNS has direct clinical consequences, as it makes further immunosuppressive treatment unnecessary and the recurrence of NS in a kidney graft unlikely [2,4,9,[12][13][14][15]. ...
... In patients with SRNS, WT1 mutations can lead to three distinct clinical entities [2,9,[13][14][15]. 1. Denys-Drash syndrome, typically characterized by the triad of infantile SRNS with male pseudo-hermaphroditism, diffuse mesangial sclerosis and Wilms tumor; this was ruled out by the clinical manifestations (adolescent onset) and the histologically evidenced FSGS. 2. Frasier syndrome, characterized by association of SRNS with male pseudohermaphroditism, excluded by normal female karyotype (46, XX). 3. isolated NS -as we found in our case. ...
... The identified WT1 mutation is a frequent mutation in Frasier syndrome, which affects the cleavage of exon 9 [13,14]. This form of NS does not respond to immunosuppressive therapy and patients should receive symptomatic treatment only. ...
Approximately 10-20% of children and 40% of adults with idiopathic nephrotic syndrome are steroid resistant and progress to end-stage renal disease requiring dialysis or renal transplantation. In these cases, renal histology typically shows focal segmental glomerulosclerosis. Mutations in NPHS1, NPHS2, WT1, CD2AP and ACTN4 genes located on different chromosomes, expressed by glomerular podocytes, have been identified in patients with steroid-resistant nephrotic syndrome. The authors report two cases of adolescent-onset steroid-resistant nephrotic syndrome. Both cases had similar clinical and histopathological manifestations, with different prognosis and evolution due to different mechanisms leading to proteinuria: an acquired and a genetic form. The first case, a 16 year old girl presented the onset of the disease with massive, generalized edema, secondary hypothyroidism and high blood pressure. Evolution was favorable under cyclosporine therapy. The second case, a 13-years-old adolescent girl, presented an insidious onset of the disease with mild edema. Genetic testing revealed a mutation in the WT1 gene. The patient developed end-stage kidney failure eight months after the onset of the disease and following kidney transplant had a favorable evolution. Histological examination of the renal biopsy specimen showed focal segmental glomerulosclerosis in both cases. Conclusions: Genetic forms of nephrotic syndrome do not respond to immunosuppressive therapy and may progress to end-stage renal disease, but after kidney transplantation relapse is not expected, in contrast to the immune form. The early genetic diagnosis in steroid-resistant nephrotic syndrome is time-consuming, but is important for proper clinical management of the patients, prognosis and genetic counseling of the families.
... 2,3,9 The symptomatology evolves into a treatment-resistant nephrotic syndrome that progressively leads to end-stage renal failure during the first two decades of the child's life. 2,3,6,[9][10][11] We present a case of Frasier syndrome that was seen at the nephrology/dialysis unit of HUEH. ...
... years, 2,15,20 which is also the age at which the diagnosis of end-stage renal disease is often made, although the onset of renal symptoms of proteinuria, edema and hypertension typically begins between 2 and 10 years of age. 1,2,6,9,10,19,20 This patient had a similar picture; since his childhood he was often in hospital for reasons of facial oedema and alteration of the renal function. This prompted the abdominal ultrasound revealing a decrease in kidney size of kidneys, and allowed the diagnosis of preterminal renal insufficiency at age 16. ...
Objective:
Frasier syndrome is a rare genetic nephropathy characterized by the presence of progressive glomerulopathy with proteinuria associated with male pseudo hermaphroditism. This case study described a picture of a young boy where the clinical suspicion context reminded the Frasier syndrome. To our knowledge, this case is the first described in Haiti.
Case study:
This is a 19-year-old young phenotypically male, born with a genital anomaly, was seen on referral at the nephrology/dialysis unit of the internal medicine department of the State University Hospital of Haiti for evaluation and follow-up. Insidious progression of symptoms had occurred over 3 years. Over three months of outpatient follow-up, he had four sets of renal labs drawn, and all showed impaired renal function. At the ultrasound, a bilateral cryptorchidism is described in the inguinal, and presence of functional ovaries with follicles of variable size scattered in the parenchyma. So, in the light of these anamnestic, clinical and paraclinical findings, we concluded to the diagnosis of end-stage renal failure by progressive glomerulopathy in a context of Frasier's syndrome.
Conclusion:
With any clinical picture consisting of genital anomalies at birth, renal symptomatology during childhood and the diagnosis of renal failure during adolescence, rare genetic nephropathies, such as Frasier syndrome must be considered.
... (2,3,9)The symptomatology evolves into a treatment-resistant nephrotic syndrome that progressively leads to end-stage renal failure during the rst two decades of the child's life. (2,3,6,(9)(10)(11)We present a case of Frasier syndrome that was seen at the nephrology/dialysis unit of HUEH. ...
... The mean age at diagnosis of Frasier syndrome was estimated to be 16.3 ± 2.3 years (2,15,20), which is also the age at which the diagnosis of end-stage renal disease is often made, although the onset of renal symptoms of proteinuria, edema and hypertension typically begins between 2-10 years of age. (1,2,6,9,10,19,20) This patient had a similar picture; since his childhood he was often in hospital for reasons of facial oedema and alteration of the renal function. This prompted the abdominal ultrasound revealing a decrease in kidney size of kidneys, and allowed the diagnosis of preterminal renal insu ciency at age 16. ...
Objective
Frasier syndrome is a rare genetic nephropathy characterized by the presence of progressive glomerulopathy with proteinuria associated with male pseudo hermaphroditism. This case study described a picture of a young boy where the clinical suspicion context reminded the Frasier Syndrome. To our knowledge, this case is the first described in Haiti.Case studyThis is a 19 years old young phenotypically male, born with a genital anomaly, was seen on referral at the nephrology/dialysis unit of the internal medicine department of HUEH for evaluation and follow-up. Insidious progression of symptoms had occurred over 3 years. Over three months of outpatient follow-up, he had four sets of renal labs drawn, and all showed impaired renal function. At the ultrasound a bilateral cryptorchidism is described in the inguinal, and presence of functional ovaries with follicles of variable size scattered in the parenchyma. So, in the light of these anamnestic, clinical and paraclinical findings we concluded to the diagnosis of end-stage renal failure by progressive glomerulopathy in a context of Frasier's syndrome.Conclusion
With any clinical picture consisting of genital anomalies at birth, renal symptomatology during childhood and the diagnosis of renal failure during adolescence, rare genetic nephropathies, such as Frasier syndrome must be considered.
... In addition, isolated SRNS may result from a wide range of WT1 sequence variations, predominantly but not exclusively exonic point mutations. WT1-associated disorders may include proteinuria either as an initial symptom or as a later development in a patient diagnosed on the basis of extrarenal features (Lipska et al., 2014;Ruf et al., 2004). However, the clinical course is highly variable, making the outcome of an individual patient difficult to predict. ...
... Most of Wilms' tumor can be treated successfully with chemotherapy and kidney-sparing surgery. Reduction of renal function was attributed to tumor burden or relapse, and combination of chemotherapy and radiation (Ruf et al., 2004). Establishing the genetic diagnosis appears particularly important for WT1-associated disorder in view of its unique associated features and long-term complications. ...
Background:
WT1 mutations cause a wide spectrum of renal and extrarenal manifestations concerning urogenital development and the development of tumors.
Methods:
We retrospectively collected the information on the genotype and phenotype of WT1 nephropathy from the multicenter registry since 2010 to 2019. All patients were stratified by renal function decline status or by sequence timing. Rapid progressive group was defined as rapidly developing into ERSD within 12 months since disease onset. Early sequencing group was defined as gene mutation identified before ERSD.
Results:
Thirty-three (3.5%) cases were identified with a WT1 mutation in patients with steroid resistant nephrotic syndrome (SRNS), proteinuria and chronic kidney disease (CKD) 3-5 stage of unknown origin. ESRD developed in twenty patients at a median age of 4.3 years old. Comparing study between the rapid progressive group (n = 8) and non-rapid progressive group (n = 25) showed no significant difference in age of onset, gender, syndrome phenotype, genotype and proteinuria except for initial estimated glomerular filtration rate (eGFR) (p = 0.021) or sequencing timing (p = 0.003). In multivariable logistic regression analysis, the delayed sequencing was associated with rapid renal function decline, even after adjusting for established clinical factors including syndromic phenotype, genotype, age onset and eGFR at initial stage (p = 0.019). The renal survival analysis did not show a significantly better outcome in early sequencing group than in delayed sequencing group (p > 0.05).
Conclusion:
Screening for WT1 mutations should be performed in children with Wilms' tumor, proteinuria/SRNS or CKD. Early diagnosis of WT1 nephropathy through clinical and genetic findings is warranted.
... WT1-related nephropathy is generally ascribed to developmental defects in glomerular podocytes [2,3]. Several patients with DDS or FS display membranoproliferative glomerulonephritis (MPGN) that is mainly characterized by subendothelial immune deposits [3][4][5][6][7][8][9], suggesting that renal pathologies resulting from WT1 mutations are complex and affected by multiple factors. ...
... WT1 mutations can cause a broad spectrum of clinical diseases affecting urogenital development and sexual differentiation at variable severity and combinations. Mutational survey of WT1 in steroid-resistant nephrotic syndrome cohorts [2,3] revealed that an intron 9 splice mutation typically causes FSGS with a gonadal tumor, whereas missense and truncating mutations result in DMS with nephroblastoma. However, morphologic abnormalities considerably vary in histologic appearance among individuals with DDS/FS [3][4][5][6][7][8][9]. ...
Background:
Mutations in the Wilms tumor 1 gene cause a spectrum of podocytopathy ranging from diffuse mesangial sclerosis to focal segmental glomerulosclerosis. In a considerable fraction of patients with Wilms tumor 1 mutations, the distinctive histology of immune-complex-type glomerulonephritis has been reported. However, the clinical relevance and etiologic mechanisms remain unknown.
Case presentation:
A 5-year-old child presented with steroid-resistant nephrotic range proteinuria. Initial renal biopsy revealed predominant diffuse mesangial proliferation with a double-contour and coexisting milder changes of focal segmental glomerulosclerosis. Immunofluorescence and electron microscopy revealed a full-house-pattern deposition of immune complexes in the subendothelial and paramesangial areas. Serial biopsies at 6 and 8 years of age revealed that more remarkable changes of focal segmental glomerulosclerosis had developed on top of the initial proliferative glomerulonephritis. Identification of a de novo Wilms tumor 1 splice donor-site mutation in intron 9 (NM_024426.6:c.1447 + 4C > T) and 46,XY-gonadal dysgenesis led to the diagnosis of Frasier syndrome.
Conclusions:
Our findings, together with those of others, point to the importance of heterogeneity in clinicopathological phenotypes caused by Wilms tumor 1 mutations and suggest that immune-complex-mediated membranoproliferative glomerulopathy should be considered as a histological variant.
... 43 In contrast, children with SRNS in the developed world have the following histopathologic subtypes on light microscopy: FSGS (63-73%), MCN (0-15%), diffuse mesangial sclerosis (3-15%), and IgM nephropathy (3-15%). 49,50 More importantly, these causes of SRNS account for 5-20% of all pediatric patients with ESRD. 51 Again, this shows that SRNS in children, irrespective of the underlying histopathologic subtype, most likely ends up with ESKD. ...
... 53 Remarkably, subjects with HIV-associated nephropathy (HIVAN) have one or two APOL1 risk alleles. 49 Although APOL1-associated FSGS constitutes a major type of FSGS in SSA, the effect of APOL1 is mainly recessive, needing two risk alleles. But in HIV-positive South Africans, a single copy of a risk allele has a significant link with HIVAN. ...
Introduction: The prevalence rates of the common histopathologic subtypes of childhood
nephrotic syndrome associated with steroid resistance appear to be changing globally. In Sub
Saharan Africa (SSA), the trend is similar over the past few decades.
Aim: This systematic review aims to determine the current prevalence rates of the histopathologic
subtypes associated with childhood steroid-resistant nephrotic syndrome (SRNS)
in SSA.
Methods: A search of the PubMed, Google and African Journals Online databases was
conducted from January to December 2018 using PRISMA (Preferred Reporting Items for
Systematic Reviews and Meta-Analyses) flow-chart to identify relevant articles which met
the aim of the systematic review. A qualitative synthesis and descriptive analysis of the
extracted data were then conducted. The mean values for the prevalence rates of the reported
histopathologic subtypes were calculated. A meta-analysis was not done due to few numbers
of studies reviewed. The review is registered with PROSPERO, number CRD42018111916.
Results: In the West African sub-region, the currently reported histopathologic subtypes
associated with childhood nephrotic syndrome are focal segmental glomerulosclerosis
(FSGS), minimal-change nephropathy (MCN), membrano-proliferative glomerulonephritis
(MPGN), membranous nephropathy (MN) and mesangial proliferative glomerulonephritis
(MesPGN). The picture is the same in South Africa. More importantly, the predominant
histopathologic lesions associated with steroid resistance are FSGS (West Africa) and MCN/
FSGS (South Africa), with mean prevalence rates of 57.2% and 36.1% respectively.
Conclusion: The prevalence of FSGS is currently high in childhood nephrotic syndrome in
SSA. This histopathologic subtype remains the commonest lesion associated with SRNS in
this part of the globe.
Keywords: steroid-resistant nephrotic syndrome, children, histopathologic subtypes, focal
segmental glomerulosclerosis, prevalence, Sub-Saharan Africa
... Frasier syndrome is characterized by gonadal dysgenesis, gonadoblastoma, and nephrotic syndrome or isolated nephropathy in patients with 46,XY karyotype (64). WT1 mutations were also shown to be associated with isolated SRNS without tumors or gonadal abnormalities (65). Mutations in SMARCAL1, encoding a helicase, cause Schimke immuno-osseous dysplasia which is characterized by growth failure, immune deficiency, and SRNS (68). ...
... Early onset of disease and family history of nephrotic syndrome were the most important risk factors for finding pathogenic mutations in large clinical studies of adult and pediatric patients with nephrotic syndrome (8,11,106). A positive test result for certain gene mutations has been shown to be associated with SRNS and knowledge of this information will help clinicians to avoid a trial of steroid therapy in such patients, determine renal prognosis and for genetic counseling of families (65,107). Anecdotal reports have also shown certain genetic mutations causing nephrotic syndrome are responsive to non-steroidal agents such as cyclosporine, ACE inhibitors, and coenzyme Q10 supplementation (30,52,87,88,92,108,109). ...
Glomerular disease is a common cause for proteinuria and chronic kidney disease leading to end-stage renal disease requiring dialysis or kidney transplantation in children. Nephrotic syndrome in children is diagnosed by the presence of a triad of proteinuria, hypoalbuminemia, and edema. Minimal change disease is the most common histopathological finding in children and adolescents with nephrotic syndrome. Focal segmental sclerosis is also found in children and is the most common pathological finding in patients with monogenic causes of nephrotic syndrome. Current classification system for nephrotic syndrome is based on response to steroid therapy as a majority of patients develop steroid sensitive nephrotic syndrome regardless of histopathological diagnosis or the presence of genetic mutations. Recent studies investigating the genetics of nephrotic syndrome have shed light on the pathophysiology and mechanisms of proteinuria in nephrotic syndrome. Gene mutations have been identified in several subcellular compartments of the glomerular podocyte and play a critical role in mitochondrial function, actin cytoskeleton dynamics, cell–matrix interactions, slit diaphragm, and podocyte integrity. A subset of genetic mutations are known to cause nephrotic syndrome that is responsive to immunosuppressive therapy but clinical data are limited with respect to renal prognosis and disease progression in a majority of patients. To date, more than 50 genes have been identified as causative factors in nephrotic syndrome in children and adults. As genetic testing becomes more prevalent and affordable, we expect rapid advances in our understanding of mechanisms of proteinuria and genetic diagnosis will help direct future therapy for individual patients.
... Detection of NPHS2 mutations in children with SRNS is crucial, as children carrying homozygous or compound heterozygous mutations in this gene have a severe form of SRNS characterized by primary resistance to steroid treatment and rapid deterioration toward ESRD, in contrast to those without. 13 Thus, finding a mutation in an SRNS patient has clinical influences: it obviates the nonbenefit of further immunosuppression and points to the unlikely recurrence of NS in a renal graft. 14 Rood et al 5 proposed mutation screening in familial and sporadic SRNS cases to spare the patient an unnecessary long-term therapy with corticosteroids or cyclophosphamide. ...
... Ruf et al 13 reported NPHS2 mutations in as many as 26% of families with familial SRNS, and Caridi et al 16 found that 12e19% of cases of sporadic pediatric SRNS were noted in children of European descent. ...
Background/purpose:
Nephrotic syndrome is the commonest etiology of proteinuria in children. Steroid-resistant nephrotic syndrome (SRNS) is defined by resistance to standard steroid therapy, and it continues to be one of the most intractable etiologies of renal failure. Molecular studies discovered specialized molecules in podocytes that play a role in proteinuria. Mutations in NPHS2 that encodes for podocin constitute a frequent cause of SRNS worldwide. This study aimed to screen for podocin mutations in SRNS Egyptian children and their parents.
Methods:
Our study included patients from 10 unrelated Egyptian families diagnosed with SRNS. Mutational analysis of the NPHS2 gene was performed by polymerase chain reaction amplification of the whole coding region of the gene and direct sequencing.
Results:
Positive consanguinity was detected in five cases, and four of them had a positive family history of SRNS in a family member. Mutational analysis of NPHS2 revealed pathogenic mutations in four cases (40%) including a novel missense in one patient (c.1A>T; p.M1L).
Conclusion:
Our study concludes that mutations of NPHS2 gene are common among Egyptian children with SRNS. We support a model where ethnicity plays an important role in specific NPHS2 mutations, since a novel mutation was found in one patient in this study. Future study on a large number of Egyptian patients with SRNS is warranted to identify the actual genetic contribution of this gene in the development of SRNS in our population, which might help in patients' prognosis and management.
... We used the site directed mutagenesis technique to introduce a mutation in each of the exons 8 and 9 in order to be able to validate the HRM technique as described by Heish et al., 2013 [23]. The following single nucleotide change from the wild type were introduced: c.1079G > A;p.C360Y (hg19 -c.1283G > A;p.C428Y) and c.1119C > A;p.H373Q (hg19 -c.1323C > A; p.H441Q) in Exon 8 and c.1180C > T;p.R394W (hg19 -c,1384C > T;p.R462W), c.1190A > C;p.H397P (hg19 -c.1394A > C; p.H465P) and c.A1200C > T;p.H401Y (hg19 -c.1405C > T;p.H469Y) in exon 9 [24][25][26][27]. Round 1 PCR was carried out using mutagenic oligos (2 oligos per mutation; Table 1). ...
... Based on the number of children tested, the prevalence of WT-1 mutation in Indian children with SRNS with 95% confidence is ≤ 3% [32]. The prevalence of WT1 mutation in children with SRNS varies according to the population origin and is summarised in Table 3. Data from large cohort studies that included children with SRNS from various ethnic background like central and west European, Turkish, American, African-American, Hispanic, South Asian, Arabic, or African, shows that the prevalence of WT1 mutation varies between 1.7 and 9% [9,26,33]. Studies limited to specific ethnic groups also show wide variation in the prevalence of WT1 mutation with low prevalence in Japanese and Chinese population and high prevalence in Greek, Spanish and Korean population [13-16, 34, 35]. ...
Background
Mutations in Wilm’s tumor 1 (WT1) gene is one of the commonly reported genetic mutations in children with steroid resistant nephrotic syndrome (SRNS). We report the results of direct sequencing of exons 8 and 9 of WT1 gene in 100 children with SRNS from a single centre. We standardized and validated High Resolution Melt (HRM) as a rapid and cost effective screening step to identify individuals with normal sequence and distinguish it from those with a potential mutation. Since only mutation positive samples identified by HRM will be further processed for sequencing it will help in reducing the sequencing burden and speed up the screening process.
Methods
One hundred SRNS children were screened for WT1 mutations in Exon 8 and 9 using Sanger sequencing. HRM assay was standardized and validated by performing analysis for exon 8 and 9 on 3 healthy control and 5 abnormal variants created by site directed mutagenesis and verified by sequencing. To further test the clinical applicability of the assay, we screened additional 91 samples for HRM testing and performed a blinded assessment.
Results
WT1 mutations were not observed in the cohort of children with SRNS. The results of HRM analysis were concordant with the sequencing results.
Conclusion
The WT1 gene mutations were not observed in the SRNS cohort indicating it has a low prevalence. We propose applying this simple, rapid and cost effective assay using HRM technique as the first step for screening the WT1 gene hot spot region in a clinical setting.
Electronic supplementary material
The online version of this article (doi:10.1186/s12881-016-0362-7) contains supplementary material, which is available to authorized users.
... 2,3 Mutations in WT1 gene in nephrotic syndrome are restricted to exons 8 and 9, and if present, the treatment response may be affected. 2,4 Interethnic differences can influence the frequency of this gene mutation. There has been no published information so far regarding its status in the Indian population. ...
... The WT1 gene encodes a transcriptional factor of the zinc finger protein family and is involved in kidney and gonadal development and its expression is restricted to podocytes after birth. The reported incidence of WT1 gene mutations is 5.7-6.4% from different parts of the world, and is more common in female patients 1,4,5 Recently, Lipska et al 6 reported that patients with WT1 mutations more frequently presented with chronic kidney disease and hypertension at diagnosis, and had more rapid disease progression. FSGS was equally prevalent in cases with and without mutations, but diffuse mesangial sclerosis was mostly associated with WT1 disease and was present in 34% of cases. ...
... 27,28 Other genetic factors include autosomal dominant transmitted causes such as mutations in the Wilms' tumor suppressor gene (WT1), a-actinin 4, CD2AP, and TRPC6. [29][30][31][32][33] Apart from those in WT1, most of these mutations tend to result in disease at adult onset. ...
... [8][9][10]20 The importance of podocyte and slit diaphragm structure to the pathogenesis of nephrotic syndrome is further reinforced by recent observations in humans and experimental animals that mutations in genes encoding some of the slit diaphragm proteins or their transcription factors can cause SRNS and/or FSGS. 26,[29][30][31][32][33]42 These fi ndings have been the subject of many recent reviews in the literature. [43][44][45] Mutations in the gene encoding the slit diaphragm protein nephrin (NPHS1) causes CNF in infants. ...
... Mutation of WT1 is commonly reported in children with steroid-resistant nephrotic syndrome, a rare disease that affect 10-15% of patients with nephrotic syndrome (12,13). About 80% of all children with sporadic nephrotic syndrome respond to steroid treatment, however, no mutations were found in WT1 in a large cohort study (14). Reduction in expression steroid-resistant nephrotic syndrome and to a lesser extent in patients with steroid-sensitive nephrotic syndrome (15). ...
Background/aim:
Wilms' tumor 1 (WT1) is involved in the development of the urogenital system and is expressed in podocytes throughout life. Inflammation of renal glomeruli causes renal damage-induced nephrotic syndrome and steroid-resistant nephrotic syndrome have mutations in the WT1 gene. The aim of this work was to determine if the inflammatory process modulates the expression and localization of WT1 in podocytes that cause kidney damage using lipopolysaccharide (LPS)-treated mice as a sepsis model.
Materials and methods:
In investigation of renal damage, proteinuria and histology were analyzed. WT1 modulation was analyzed by indirect immunofluorescence, immunohistochemistry and western blot assays, and proinflammatory cytokines were analyzed by quantitative polymerase chain reaction assay.
Results:
WT1 expression decreased most at 24 and 36 h after the induction of inflammation and phosphorylated WT1 was mainly localized in the cytoplasm, reduced nephrin mRNA expression and increased mRNA expression of tumor necrosis factor α and interleukin 1β.
Conclusion:
These results indicate that the immune system plays an important role in the modulation of WT1, leading to kidney damage.
... SRNS patients manifest histological outcomes in 75% of FSGS and 20% of Minimal change disease (MCD). In contrast, SSNS patients exhibit renal histology in 80% of MCD and 20% of FSGS [10]. FSGS is a significant cause of proteinuria and kidney failure [11]. ...
Background
Nephrotic syndrome appears as a group of symptoms like proteinuria, edema and hyperlipidemia. Identification of monogenic forms revealed the physiology and pathogenesis of the SRNS.
Methods and Results
We performed Illumina panel sequencing of seven genes in 90 Indian patients to determine the role of these genetic mutations in nephrotic syndrome prognosis. Samtool was used for variants calling, and SnpEff and Snpsift did variants annotation. Clinical significance and variant classification were performed by the ClinVar database. In SSNS and SRNS patients, we found 0.78% pathogenic and 3.41% likely pathogenic mutations. Pathogenic mutations were found in LAMB2, LMX1B and WT1 genes, while likely pathogenic mutations were found in (6/13) LAMB2, (2/13) LMX1B, (2/13) TRPC6, (2/13) PTPRO and (1/13) PMM2 genes. Approximately 46% likely pathogenic mutations were contributed to the LAMB2 gene in SSNS and SRNS patients. We also detect 30 VUS (variants of uncertain significance), which were found (17/30) pathogenic and (13/30) likely pathogenic by different prediction tools.
Conclusions
Multigene panels were used for genetic screening of heterogeneous disorders like nephrotic syndrome in the Indian population. We found pathogenic, likely pathogenic and certain VUS, which were responsible for the pathogenesis of the disease. Therefore, mutational analysis of SSNS and SRNS is necessary to avoid adverse effects of corticosteroids, modify the intensity of immunosuppressing agents, and prevent the disease’s progression.
... The low rate of mutation frequency in WT1 gene of our study (4%) is similar to some reports by Ruf and his collogues (Ruf et al. 2004b) , cho et al (Cho et al. 2008) and Alharthi et al (Alharthi et al. 2017)( 6.9% , 5.7% and 5% respectively)but is less than reports of MUCKA study(8.9%) (Mucha et al. 2006) and more than the finding related to Indian children(1.7%) . ...
Background: Nephrotic syndrome is one of the most common kidney diseases in childhood. About 20% of children are steroid-resistant NS (SRNS) which progress to end-stage renal disease (ESRD). More than 53 genes are associated with SRNS which represent the genetic heterogeneity of SRNS. This study was aimed to screen disease causing mutations within NPHS1 and NPHS2 and evaluate new potential variants in other genes.
Method: In first phase of study, 25 patients with SRNS were analyzed for NPHS1 (exon 2, 26) and all exons of NPHS2 genes by Sanger sequencing. In the second phase, whole exome sequencing was performed on 10 patients with no mutations in NPHS1 and NPHS2.
Result: WES analysis revealed a novel mutation in FAT1 (c.10570C>A; Q3524K). We identified 4 pathogenic mutations, located in exon 4 and 5 of NPHS2 gene in 20% of patients (V180M, P118L, R168C and Leu156Phe). Also our study has contributed to the descriptions of previously known pathogenic mutations across WT1 (R205C) and SMARCAL1 (R764Q) and a novel polymorphism in CRB2.
Conclusion: Our study concludes that mutations of exon 4 and 5 NPHS2 gene are common in Iranian and some other ethnic groups. We suggest conducting WES after NPHS2 screening and further comprehensive studies to identify the most common genes in the development of SRNS, which might help in Clinical impact on management in patients with SRNS.
Detection of a novel mutation in SRNS
... Subjects with exonic variants should be monitored for Wilms tumor with abdominal US performed every 3 months until the age of 7 years [58]. After reaching ESKD, bilateral nephrectomy should be considered to prevent the development of Wilms tumor, in particular in individuals carrying truncating variants [32,59]. In subjects with a 46,XY karyotype and a female phenotype (i.e., complete gonadal dysgenesis), we recommend bilateral gonadectomy due to increased gonadoblastoma risk [32]. ...
Congenital nephrotic syndrome (CNS) is a heterogeneous group of disorders presenting with massive proteinuria within the first 3 months of life almost inevitably leading to end-stage kidney disease. The Work Group for the European Reference Network for Kidney Diseases (ERKNet) and the European Society for Pediatric Nephrology (ESPN) has developed consensus statement on genetic aspects of CNS diagnosis and management. The presented expert opinion recommends genetic diagnostics as the key diagnostic test to be ordered already during the initial evaluation of the patient, discusses which phenotyping workup should be performed and presents known genotype–phenotype correlations.
... Mutations in WT1 can also cause congenital anomalies of the kidneys and urinary tract and Wilms tumor. [97][98][99][100] The parents of BG were counseled that the variant in WT1 was the likely cause of her kidney disease and that as a de novo mutation it was very unlikely, but not impossible, to occur in a subsequent pregnancy. BG's course was further complicated by peritonitis, sepsis, and persistent respiratory failure. ...
In many cases of CKD, the cause of disease remains unknown despite a thorough nephrological workup. Genetic testing has revolutionized many areas of medicine, and promises to empower diagnosis and targeted management of such cases of kidney disease of unknown etiology. Recent studies using genetic testing have demonstrated that Mendelian etiologies account for approximately 20% of cases of kidney disease of unknown etiology. While genetic testing has significant benefits, including tailoring of therapy, informing targeted workup, detecting extrarenal disease, counseling patients and families, and redirecting care, it also has important limitations and risks that must be considered.
... NPHS2 encodes a 42 kDa membrane integral protein called Podosine. This protein is a member of Stomatin proteins family including 383 amino acids (Ruf et al. 2004;Gbadegesin et al. 2007). Podosine protein is expressed in slit podocytes of kidney diaphragm and it has a fundamental role in filtration of glomerular wall (Dong et al. 2015). ...
Nephrotic syndrome (NS) is considered as a primary disease of the kidney that represents a heterogeneous group of glomerular disorders occurring mainly in children. It is generally divided into steroid-sensitive and steroid-resistant forms, depending upon the patient’s response to steroid therapy. Among the genes involved, the NPHS2 gene has been reported as the causative gene in steroid resistant form of nephrotic syndrome. In the present study, heterozygosity rate, allelic frequency and linkage of rs2274625 and rs3829795 markers were investigated in the NPHS2 gene region. To determine the SNP alleles, tetra-primer ARMS PCR was used. After genotyping rs2274625 and rs3829795 polymorphic markers in 120 unrelated individuals and nine trios families, the data were analysed using various computer programs such as UCSC Genome Browser, dbSNP and SNPper. Based on the statistical analysis of the results, for rs2274625 marker, allele frequency for C and T alleles was 97% and 3%, respectively. For rs3829795 marker allele frequency for G and A alleles was 55% and 45%, respectively. The values of heterozygosity index for the examined markers were 5% for rs2274625 and 45/8% for rs3829795. Consequently, two informative haplotypes, CG/CA, were identified in the NPHS2 gene region through combination of these two markers. These haplotypes can serve as appropriate tools for the identification of heterozygous carriers and linkage analysis of nephrotic syndrome disease in the Iranian families with an affected child.
... However, the rate of NPHS2 mutation as a cause for SRNS is rare in certain ethnic groups, including Korean, Chinese, and Japanese populations [6,12,13]. WT1 is also commonly mutated, accounting for approximately 6% of sporadic SRNS cases in childhood [12,14,15]. WT1 mutations can manifest at any age as either isolated or syndromic forms of SRNS [12,14]. ...
Steroid-resistant nephrotic syndrome (SRNS) is a common cause of chronic kidney disease in children, and a considerable number of patients progress to end-stage renal disease. SRNS is a highly heterogeneous disorder, both clinically and genetically, and more than 50 monogenic causes of SRNS, including isolated and syndromic forms, have been identified. Recent large-cohort studies indicate that at least 30% of childhood-onset SRNS cases are genetic. The benefits of definitive molecular diagnosis by genetic testing include the avoidance of unnecessary and potentially harmful diagnostic procedures (e.g., kidney biopsy) and treatment (e.g., steroid and immunosuppressants), detection of rare and potentially treatable mutations (e.g., coenzyme Q10 biosynthesis pathway defect), prediction of prognosis (e.g., posttransplant recurrence), and providing precise genetic counseling. Furthermore, the identification of novel disease-causing genes could provide new insights into the pathogenic mechanisms of SRNS. Therefore, whenever accessible and affordable, genetic testing is recommended for all pediatric patients with SRNS, and should certainly be performed in patients with a higher probability of genetic predisposition based on genotype-phenotype correlation data. The genetic testing approach should be determined for each patient, and clinicians should, therefore, be aware of the advantages and disadvantages of methods currently available, which include Sanger sequencing, gene panel testing, and whole-exome or whole-genome sequencing. Importantly, the need for precise and thorough phenotyping by clinicians, even in the era of genomics, cannot be overemphasized. This review provides an update on recent advances in genetic studies, a suggested approach for the genetic testing of pediatric patients with SRNS.
... This mechanism of nephrotic syndrome is additional reinforced by recent observations in humans and experimental animals [4,10,11,14,[24][25][26]. The subject of many recent reviews in the literature this type of result have been discussed [27][28][29]. In infants mutations in the gene encoding the opening diaphragms protein nephrin (NPHS1) mostly causes CNF. ...
... Patients usually present with asymptomatic proteinuria in early childhood, then gradually develop steroid-resistant nephrotic syndrome that progresses to chronic kidney disease (Niaudet and Gubler 2006). Renal biopsy findings often represent focal segmental glomerulosclerosis (FSGS) (Ruf et al. 2004). Lipska et al. (2014) showed that the age at 50% kidney survival for Frasier syndrome was 13.6 years. ...
Cyclosporine A is known to be effective in some genetic podocyte injury. However, the efficacy of cyclosporine A depends on the degree of histopathological findings, and the relationship between long-term use and renal prognosis remains unknown. Frasier syndrome is a rare genetic disorder caused by intronic mutations in WT1, and is characterized by progressive glomerulopathy, a 46,XY disorder of sex development, and an increased risk of gonadoblastoma. We report here a 16-year-old phenotypically female patient with Frasier syndrome. A renal biopsy at the age of seven years showed segmentally effaced podocyte foot processes with no evidence of glomerulosclerosis. Steroid-resistant proteinuria progressed to the nephrotic range at the age of 10 years, which responded to once-daily administration of cyclosporine A with low two-hour post-dose cyclosporine A (C2) levels; she then achieved stable partial remission in combination with renin-angiotensin system (RAS) blockade. At the age of 12 years, examinations for delayed puberty confirmed the diagnosis of Frasier syndrome. The second renal biopsy showed widespread foot process effacement and a minor lesion of segmental glomerulosclerosis without findings suggestive of cyclosporine A nephropathy. She maintained partial remission and normal renal function with the continuation of once-daily low-dose cyclosporine A. The C2 levels required for the remission were between 212 and 520 ng/ml. Cyclosporine A dosages sufficient for maintaining the C2 levels were 1.1-1.2 mg/kg per day. In conclusion, the long-lasting treatment of once-daily low-dose cyclosporine A with RAS inhibition was effective for induction and maintenance of partial remission in Frasier syndrome.
... Based on their steroid responsiveness, patients are classified as steroid-sensitive and steroid-resistant. Genetic mutations that affect glomerular podocyte function, such as NPHS1, NPHS2, and WT1 [133][134][135][136][137], account for most steroid-resistant cases and patients with genetic forms of steroid-resistance are less responsive to immunotherapeutic drugs. The circulating factor, soluble urokinase receptor, has been considered a cause for the development of SRNS [138,139]. ...
Glucocorticoid receptor (GC), a founding member of the nuclear hormone receptor superfamily, is a glucocorticoid-activated transcription factor that regulates gene expression and controls the development and homeostasis of human podocytes. Synthetic glucocorticoids are the standard treatment regimens for proteinuria (protein in the urine) and nephrotic syndrome (NS) caused by kidney diseases. These include minimal change disease (MCD), focal segmental glomerulosclerosis (FSGS), membranous nephropathy (MN) and immunoglobulin A nephropathy (IgAN) or subsequent complications due to diabetes mellitus or HIV infection. However, unwanted side effects and steroid-resistance remain major issues for their long-term use. Furthermore, the mechanism by which glucocorticoids elicit their renoprotective activity in podocyte and glomeruli is poorly understood. Podocytes are highly differentiated epithelial cells that contribute to the integrity of kidney glomerular filtration barrier. Injury or loss of podocytes leads to proteinuria and nephrotic syndrome. Recent studies in multiple experimental models have begun to explore the mechanism of GC action in podocytes. This review will discuss progress in our understanding of the role of glucocorticoid receptor and glucocorticoids in podocyte physiology and their renoprotective activity in nephrotic syndrome.
... Нецелесообразность использования этих препаратов в лечении детей с нефротическим синдромом, обусловленным мутацией гена WT1, показана R. Ruf и соавт. (2004) [27]. Мутация в гене WT1 была определена у 9% больных со стероидрезистентным нефротическим синдромом и не выявлена ни у одного пациента со стероидчувствительным нефротическим синдромом. ...
This literature review is focused to change our ideas about the etiology, pathogenesis and treatment tactics of the nephrotic syndrome in recent decades. The change in the treatment outcomes of the primary nephrotic syndrome in connection with the emergence of new therapy technologies, is shown. Features of the course, examination and therapy of congenital and infantile nephrotic syndrome and the possibility of the debut of a nephrotic syndrome associated with various gene mutations and at an older age are presented. Principal differences in diagnostic and therapeutic approaches are accentuated depending on the cause of the development of the disease. Modern syndromological and pathogenetic methods of therapy of primary nephrotic syndrome are presented, and the immediate opportunities for the introduction of new treatment technologies based on the use of monoclonal antibodies, are shown.
... Conversely, reported SSNS pedigrees are scarce [12][13][14]. Several studies investigated SRNS gene mutations (mainly in NPHS1, NPHS2 and WT1) in SSNS cohorts, but no causative mutation was identified [12,[15][16][17][18]. EMP2 mutations have been identified in AR SSNS by a combination of linkage analysis and exome sequencing [19]. ...
Background:
Familial steroid-sensitive nephrotic syndrome (SSNS) is a rare condition. The disease pathophysiology remains elusive. However, bi-allelic mutations in the EMP2 gene were identified, and specific variations in HLA-DQA1 were linked to a high risk of developing the disease.
Methods:
Clinical data were analyzed in 59 SSNS families. EMP2 gene was sequenced in families with a potential autosomal recessive (AR) inheritance. Exome sequencing was performed in a subset of 13 families with potential AR inheritance. Two variations in HLA-DQA1 were genotyped in the whole cohort.
Results:
Transmission was compatible with an AR (n = 33) or autosomal dominant (AD, n = 26) inheritance, assuming that familial SSNS is a monogenic trait. Clinical features did not differ between AR and AD groups. All patients, including primary (n = 7) and secondary steroid resistant nephrotic syndrone (SRNS), (n = 13) were sensitive to additional immunosuppressive therapy. Both HLA-DQA1 variations were found to be highly linked to the disease (OR = 4.34 and OR = 4.89; p < 0.001). Exome sequencing did not reveal any pathogenic mutation, neither did EMP2 sequencing.
Conclusions:
Taken together, these results highlight the clinical and genetic heterogeneity in familial SSNS. Clinical findings sustain an immune origin in all patients, whatever the initial steroid-sensitivity. The absence of a variant shared by two families and the HLA-DQA1 variation enrichments suggest a complex mode of inheritance.
... The cause may be an imbalance between different isoforms of WT1 or decreased basal protein expression. Different research groups have observed a decrease in WT1 protein expression in children with SRNS, which was often accompanied by WT1 mutations in exons 8 and 9 [8,34,35]. However, another report suggested that reduced expression levels of WT1 cause glomerulonephritis and mesangial sclerosis, depending on the level of the protein [36]. ...
Nephrotic syndrome (NS) is a glomerular disease that is defined by the leakage of protein into the urine and is associated with hypoalbuminemia, hyperlipidemia, and edema. Steroid-resistant NS (SRNS) patients do not respond to treatment with corticosteroids and show decreased Wilms tumor 1 (WT1) expression in podocytes. Downregulation of WT1 has been shown to be affected by certain microRNAs (miRNAs). Twenty-one patients with idiopathic NS (68.75% were SSNS and 31.25% SRNS) and 10 healthy controls were enrolled in the study. Podocyte number and WT1 location were determined by immunofluorescence, and the serum levels of miR-15a, miR-16-1, and miR-193a were quantified by RT-qPCR. Low expression and delocalization of WT1 protein from the nucleus to the cytoplasm were found in kidney biopsies of patients with SRNS and both nuclear and cytoplasmic localization were found in steroid-sensitive NS (SSNS) patients. In sera from NS patients, low expression levels of miR-15a and miR-16-1 were found compared with healthy controls, but only the miR-16-1 expression levels showed statistically significant decrease ( p=0.019 ). The miR-193a expression levels only slightly increased in NS patients. We concluded that low expression and delocalization from the WT1 protein in NS patients contribute to loss of podocytes while modulation from WT1 protein is not associated with the miRNAs analyzed in sera from the patients.
... The primer sets cover the WT1 exons (gene expressed region) and introns based on a previously published report 15 , genomic DNA was amplified by polymerase chain reaction (PCR) using flanking specific sequences for confirmation of the genotype primer sets -forward 5'-CCT TTA ATG AGA TCC CCT TTT CC-3'and reverse 5'-GGG GAA ATG TGG GGT GTT TCC-3', forward 5'-CCT CAC TGT GCC CAC ATT GT-3' and reverse 5'-GCA CTA TTC CTT CTC TCA ACT GAG-3'. Amplification with the primer pairs resulted in 391 and 349 bp products. ...
Background & objectives: Clinically, nephrotic syndrome (NS) is a diverse group of symptoms; about 20 per cent of NS cases are resistant to steroid treatment, and within ten years they progress to end-stage renal disease. The present study was undertaken to identify the mutations of Wilms′ tumour 1 (WT1) gene in steroid-resistant NS (SRNS) children.
Methods: A total of 173 children with SRNS and 100 children in the control group were enrolled in the study. DNA extraction was done, screened for WT1 (exons 8 and 9) gene amplified by polymerase chain reaction and direct sequencing. Karyotype analyses were done for WT1 mutation cases.
Results: WT1 mutations were found in three of 173 SRNS cases (2 girls, 1 boy). All of them had intron 9 (IVS 9 + 4 C>T, 2; IVS + 5 G>A, 1) mutation. Of these three cases, one had familial and another two had sporadic history. Renal histology analysis showed two cases with focal segmental glomerulosclerosis (FSGS) and they had external female genitalia but 46,XY karyotype. Both of them had streak gonads. Of the three cases, one expired.
Interpretation & conclusions: The findings of the present study indicate that all females with SRNS-FSGS should be screened for WT1 gene mutation to diagnose whether they have FS for possible gonadectomy.
... The primer sets cover the WT1 exons (gene expressed region) and introns based on a previously published report 15 , genomic DNA was amplified by polymerase chain reaction (PCR) using flanking specific sequences for confirmation of the genotype primer sets -forward 5'-CCT TTA ATG AGA TCC CCT TTT CC-3'and reverse 5'-GGG GAA ATG TGG GGT GTT TCC-3', forward 5'-CCT CAC TGT GCC CAC ATT GT-3' and reverse 5'-GCA CTA TTC CTT CTC TCA ACT GAG-3'. Amplification with the primer pairs resulted in 391 and 349 bp products. ...
... Interestingly, the Arg366His mutation was reported in 11 of these cases (64.7 %), a different Arg366 missense substitution (N21) was reported in one case and distinct WT1 missense substitutions (N19, N22, N23, N24) were reported in four cases [one paper did not report the specific WT1 mutation (N20)]. All five non-Arg366His mutations associated with VEONS were within exon 8 or 9 of WT1 [6][7][8][9][10]. Oligohydramnios was apparent in five pregnancies, including two twin pregnancies, probably reflecting fetal renal impairment. ...
Steroid sensitive nephrotic syndrome (SSNS) is the most common form of nephrotic syndrome in childhood. Familial SSNS seems to be clinically homogeneous but reports on genetically informative SSNS families are lacking. Familial SSNS was found to be genetically distinct from familial steroid resistant nephrotic syndrome (SRNS). Several genes have been identified for SRNS. In general, genetic forms of nephrotic syndrome are resistant to common therapeutic approaches. But different cohort of steroid sensitive familial nephrotic syndrome were published. So precise clinical and genetic information on familial nephrotic syndrome are still lacking. Whether family history of nephrotic syndrome influences outcome is still in dilemma.
In 1990, mutations of the WT1 gene, encoding a transcription factor in embryonic kidney, were found in 10-15% of Wilms tumors; germline WT1 mutations were associated with hereditary syndromes involving glomerular and reproductive tract dysplasia. For >3 decades, these discoveries prompted investigators to explore the embryonic role of WT1 and the mechanisms by which loss of WT1 leads to malignant transformation. Here, we discuss how alternative splicing of WT1 generates isoforms that act in a context-specific manner to activate or repress target gene transcription. WT1 also regulates post-transcriptional regulation, alters the epigenetic landscape and activates miRNA expression. WT1 functions at multiple stages of kidney development, including the transition from resting stem cells to committed nephron progenitor, which it primes to respond to WNT9b signals from ureteric bud. Wt1 then drives nephrogenesis by activating WNT4 expression and directing the development of glomerular podocytes. We review the WT1 mutations that account for Denys-Drash, Frasier and WAGR syndromes. Although the WT1 story began with Wilms tumors, an understanding of the pathways that link aberrant kidney development to malignant transformation still has some important gaps. Loss of WT1 in nephrogenic rests may leave these pre-malignant clones with inadequate DNA repair enzymes and may disturb the epigenetic landscape. Yet none of these observations provide a complete picture of Wilms tumor pathogenesis. It appears that the WT1 odyssey is unfinished and still holds a great deal of untilled ground to be explored.
Le syndrome néphrotique est une néphropathie glomérulaire fréquente caractérisée par une protéinurie abondante, conséquence d'une perte de l'architecture du podocyte, une des cellules essentielles de la barrière de filtration glomérulaire. L'avènement de la génétique a permis de mettre en évidence des mutations dans une cinquantaine de gènes chez 30% des patients résistants aux corticoïdes ou aux immunosuppresseurs (SNCR). Cependant, l'origine génétique reste indéterminée chez le reste de ces patients. Par séquençage d'exome, nous avons cherché des mutations dans de nouveaux gènes dans des familles atteintes de SNCR. Une fois un gène candidat identifié, nous avons analysé la pathogénicité de la ou des mutations identifiées dans un modèle in vitro de culture cellulaire, et dans un modèle in vivo de poisson-zèbre (ZF). Nous avons d'abord mis en évidence des mutations dans le gène TBC1D8B dans deux familles de SNCR de survenue précoce. TBC1D8B a été très peu étudiée dans la littérature mais est prédite comme une Rab-GTPase Activating Protein (Rab-GAP), c'est à dire ayant la capacité d'inactiver certaines Rab-GTPases (non identifiées). Chez le ZF, l'inactivation de tbc1d8b induit un œdème péricardique, marqueur indirect des lésions rénales, associé à une perte de l'architecture des podocytes. La protéinurie a été confirmée par la détection de fluorescence dans les cellules tubulaires après l'injection d'un dextran fluorescent de 500kDa chez les poissons mutés, témoin d'un passage glomérulaire. Les Rab étant impliquées dans le trafic vésiculaire, nous avons étudié, dans les podocytes ou les fibroblastes de patients, le recyclage du récepteur de la transferrine et avons montré que les mutations p.Q246H et p.F291S étaient associées à un défaut de son recyclage. Nous avons également mis en évidence une interaction entre TBC1D8B et Rab11b, un acteur majeur du recyclage des vésicules provenant du compartiment de recyclage péri-nucléaire. Ces données confirment que les mutations de TBC1D8B sont responsables de SNCR chez nos patients par défaut du recyclage dépendant de Rab11b, et suggèrent que TBC1D8B est une Rab-GAP de Ra11b. Nous avons ensuite identifié le variant p.R28W dans le gène TRIM3 (tripartite motif containing 3) ségrégeant avec la maladie dans une famille dans laquelle 4 personnes ont présenté un SNCR autosomique dominante (AD). Dans les neurones, la protéine TRIM3 joue un rôle dans le trafic vésiculaire dans un complexe protéique comprenant la protéine a-actinine 4. Des mutations du gène ACTN4 codant l'a-actinine 4 ont été décrites dans la survenue de SNCR AD. Les patients porteurs de la mutation p.R28W étaient également porteurs d'un polymorphisme rare et non pathogène p.V801M d'ACTN4. Nous avons d'abord montré que l'injection d'ARNm muté codant pour TRIM3 p.R28W dans des embryons de poisson-zèbre, entraînait un phénotype identique à celui décrit précédemment, avec une désorganisation de l'architecture des podocytes. Dans les podocytes en culture, TRIM3 p.R28W était délocalisé au niveau des fibres d'actine. Malgré une interaction normale entre TRIM3 p.R28W et l'a-actinine 4, le trafic vésiculaire le long des fibres d'actine était altéré. La présence du polymorphisme p.V801M de l'a-actinine 4 ne modifiait pas le phénotype. Un autre variant (p.R433C) du gène TRIM3 a été identifié chez un patient ayant présenté un SNCR sporadique de survenue tardive, sans possibilité d'étudier la ségrégation du variant dans la famille. Les études chez le ZF ont montré le même phénotype après injection d'ARNm muté. Ces résultats suggèrent que des mutations dominantes du gène TRIM3 peuvent, comme celles d'ACTN4, conduire à un SNCR AD. Dans cette thèse, nous avons mis en évidence de nouveaux mécanismes moléculaires conduisant aux lésions podocytaires. En effet, ces données suggèrent que le recyclage dans le podocyte tient une place fondamentale dans le développement de certaines podocytopathies, ce qui n'avait jamais été montré auparavant.
Nephrotic syndrome (NS) defines a clinical entity of proteinuria, hypoproteinemia, and edema. Proteinuria is caused by an acquired or genetic defect in the glomerular filtration barrier of the kidney. So far, over 50 genes responsible for NS have been identified. Mutations in genes encoding for podocyte proteins nephrin (NPHS1), podocin (NPHS2), Wilms tumor suppressor 1 (WT1), phospholipase C ε1 (PLCE1), and laminin β2 (LAMB2) account for the majority of the observed disorders. Gene mutations are more probable in pediatric (especially in infants) than adult patients. Autosomal recessive inheritance is typical for NS in small children while dominant forms are more likely observed in adults. Gene analysis using next-generation sequencing is recommended in NS cases not responding to immunosuppressive medication. Renal failure often develops in a few years after diagnosis even with an optimal supportive therapy. Kidney transplantation is a successful curative treatment option for these patients.
Background
Mutations of the Wilms tumor suppressor-1 gene (WT1) are associated with life-threatening glomerulopathy, disorders of sexual development, Wilm's tumor, and gonadal malignancies. Our objectives were to describe the clinical presentations, age of progression, and onset of complications of WT1 mutation through a case series and literature review.MethodsA retrospective study included all patients followed at the University of Miami/Holtz Children's Hospital from January 2000 to December 2020 with a diagnosis of WT1 mutation. A literature review of WT1 mutation cases was analyzed for clinical manifestations, karyotype, and long-term outcomes.ResultsThe WT1 mutation was identified in 9 children, median age at presentation of 0.9 years (range 1 week to 7 years). A total of four had female phenotypes, and 5 had abnormalities of male external genitalia, while all had XY karyotypes. All progressed to end-stage kidney disease (ESKD) and received a kidney transplant at a median age of 5 years (1.5–15 years). During a median time of follow-up of 9 years (range 2–28 years), there were 2 allograft losses after 7 and 10 years and no evidence of post-transplant malignancy. From 333 cases identified from the literature review, the majority had female phenotype 66% (219/333), but the predominant karyotype was XY (55%, 183/333). Of the female phenotypes, 32% (69/219) had XY sex reversal. Wilm's tumor occurred in 24%, predominantly in males with gonadal anomalies.Conclusions
Early recognition of WT1 mutation is essential for comprehensive surveillance of potential malignancy, avoidance of immunosuppressants for glomerulopathy, and establishing long-term multidisciplinary management.
Introduction
Frasier syndrome (FS) is a rare inherited kidney disease caused by intron 9 splicing variants of WT1. For wild-type WT1, two active splice donor sites in intron 9 cause a mixture of two essential transcripts (with or without lysine–threonine–serine: +/KTS or -KTS) and imbalance of the +KTS/-KTS ratio results in the development of FS. To date, six causative intron 9 variants have been identified; however, detailed transcript analysis has not yet been conducted and the genotype–phenotype correlation also remains to be elucidated.
Methods
We conducted an in vitro minigene splicing assay for six reported causative variants and in vivo RNA sequencing to determine the +KTS/−KTS ratio using patients’ samples. We also performed a systematic review of reported FS cases with a description of the renal phenotype.
Results
The in vitro assay revealed that, while all mutant alleles produced −KTS transcripts only, the wild-type allele produced both +KTS and −KTS transcripts at a 1:1 ratio. In vivo RNA sequencing showed that patients’ samples with all heterozygous variants produced similar ratios of +KTS to −KTS (1:3.2 to 1:3.5) and wild-type kidney showed almost a 1:1 ratio (1:0.85). Systematic review of 126 cases clarified that the median age of developing ESKD was 16 years in all FS patients and there were no statistically significant differences between the genotypes or sex chromosome karyotypes in terms of the renal survival period.
Conclusion
Our study suggested no differences in splicing pattern or renal survival period among reported intron 9 variants causative of FS.
抄録
ステロイド抵抗性ネフローゼ症候群から末期腎不全に至ったFrasier症候群の１例を経験した。16歳時の原発性無月経の精査を機に性別の逆転が判明し，さらにWT1遺伝子の検索により変異を同定した。gonadoblastoma発生を予見する必要性や，腎不全期に行われるGH療法によって腫瘍発生のリスクが増加する可能性も否定できないことから，ステロイド抵抗性ネフローゼ症候群の原因の一つとして，また，ネフローゼから腎不全に至った症例においてはWT1遺伝子の検索を積極的に行う必要があると思われた。
The discovery of microRNAs (miRNAs) has opened up new avenues of research to understand the molecular basis of a number of diseases. Because of their conservative feature in evolution and important role in the physiological function, microRNAs could be treated as predictors for disease classification and clinical process based on the specific expression. The identification of novel miRNAs and their target genes can be considered as potential targets for novel drugs. Furthermore, currently, the circulatory and urinary exosomal miRNAs are gaining increasing attention as their expression profiles are often associated with specific diseases, and they exhibit great potential as noninvasive or minimally invasive biomarkers for the diagnosis of various diseases. The remarkable stability of these extracellular miRNAs circulating in the blood or excreted in the urine underscored their key importance as biomarkers of certain diseases. There is voluminous literature concerning the role of microRNAs in other diseases, such as cardiovascular diseases, diabetic nephropathy, and so forth. However, little is known about their diagnostic ability for the pediatric nephrotic syndrome (NS). The present review article highlights the recent advances in the role of miRNAs in the pathogenesis and molecular basis of NS with an aim to bring new insights into further research applications for the development of new therapeutic agents for NS.
WT1‐related disorders are a group of conditions associated with an aberrant or absent copy of the WT1 gene. WT1 encodes a zinc‐finger protein that not only acts as a tumor suppressor, but also is important for gonadal, urogenital tract, and diaphragmatic development. This group of conditions encompasses a phenotypic spectrum that includes WAGR syndrome, Denys–Drash syndrome (DDS), Frasier syndrome (FS), Meacham syndrome (MS), and isolated manifestations of these conditions. WAGR syndrome is associated with Wilms tumor, aniridia, genital anomalies, and a range of developmental delays. It is a contiguous gene deletion syndrome involving the genes WT1 and PAX6, as well as neighboring genes. DDS is classically defined as the clinical triad of incomplete male genital development, early onset nephropathy, and Wilms tumor. FS is defined as the triad of XY gonadal dysgenesis, childhood onset renal failure, and gonadoblastoma. XX females with DDS or FS usually present with the onset of nephropathy or tumor development, but have normal genitalia. Some individuals with a WT1 mutation only develop Wilms tumor, some are born with genitourinary anomalies and develop Wilms or gonadal tumors but do not go on to develop nephropathy, and yet others only develop nephrotic syndrome and end stage renal failure. Hence, affected individuals can display an “incomplete” phenotype. Meacham syndrome (MS) is a rare entity characterized by genitourinary malformations in males, cardiac and pulmonary malformations, diaphragmatic hernia, and frequently, early lethality. As we have come to understand the underlying cause of each of these conditions and have gained a better appreciation regarding their variable expressivity, we now view these conditions as a spectrum of related disorders, each requiring close monitoring for potentially life‐threatening complications.
Wilms' tumour 1 (WT1) is a transcription factor encoding a zinc finger protein that controls podocyte differentiation and is highly expressed in mature podocytes. WT1 mutations can lead to renal failure due to glomerular scarring, the underlying mechanisms, of which, are poorly understood. This project explored the mechanisms of glomerulosclerosis by using a tamoxifen-inducible Cre-LoxP system to delete Wt1 in adult mice. Following the fourth day post-induction with Tamoxifen, podocyte apoptosis was evident and increased as the disease progressed, highlighting Wt1’s key role in mature podocyte survival. At disease onset, increased podocyte Notch1 transcript and its downstream targets, including Nrarp and Hey2 were observed. Decreased expression of podocyte FoxC2 transcript at the same time-point was noted, thereby supporting previous findings in lower vertebrates for a transcriptional relationship between Wt1/FoxC2/Notch in podocyte function. Podocyte Notch1 and Hes1 protein expression was observed in mutant mouse glomeruli at the onset of glomerulosclerosis. Induced podocyte Hes1 expression was associated with an upregulation of Snai1 and Slug transcripts, genes associated with epithelial to mesenchymal transition (EMT), thus proposing a role for Hes1 in mediating podocyte EMT. Moreover, early pharmacological inhibition of Notch, with gamma secretase inhibitors, ameliorated glomerulosclerosis and albuminuria. This data provides evidence that Wt1 deletion modulates podocyte Notch signalling in mature podocytes, leading to early events in WT1-related glomerulosclerosis.
Cambridge Core - Pathology and Laboratory Science - Silva's Diagnostic Renal Pathology - edited by Xin Jin (Joseph) Zhou
Le Syndrome néphrotique idiopathique (SNI) est une maladie ayant pour siège le glomérule rénal. C’est la néphropathie glomérulaire la plus fréquente en pédiatrie bien que son épidémiologie soit encore mal connue. Son incidence est de 1 pour 20000, ce qui en fait une maladie rare. Le SNI est une pathologie des podocytes, on observe trois sous-groupes histologiques, la forme à lésions glomérulaires minimes (LGM), la hyalinose segmentaire et focale (HSF) et plus rare la néphropathie membranaire (NM). La forme LGM est la plus courante et de bon pronostic, la forme HSF est minoritaire quoiqu’en expansion et de moins bon augure. C’est aussi un trouble du système lymphocytaire T, un facteur circulant sécrété par celui-ci altèrerait les podocytes. Cliniquement, on observe des œdèmes locaux voire une anasarque dus à une rétention primaire ou secondaire de sodium. Le diagnostic repose sur une protéinurie détectable à la bandelette ou par prélèvement et une albuminémie inférieure à 30g/l. On peut aussi observer une hyperlipidémie mixte. Le traitement est principalement une corticothérapie au long cours, en cas de rechutes fréquentes ou d’intoxications stéroïdiennes des immunosuppresseurs comme les alkylants, la cyclosporine, le lévamisole ou des nouvelles molécules sont utilisés, pouvant entrainer des effets indésirables aussi graves que la pathologie traitée. Il existe une forme corticorésistante corrélée à la HSF et influencée par la race de l’enfant dont le traitement est souvent symptomatique. Auquel on rajoute des traitements préventifs des complications dues à la maladie comme au traitement. Si de nos jours la forme corticosensible est jugulée, 50% des formes corticorésistantes aboutissent à une insuffisance rénale terminale. L’avenir repose sur la recherche du facteur circulant et les nouvelles pistes de la thérapie génique.
We present a case of an infant with proximal hypospadias, penoscrotal transposition, and bilaterally descended testes found to have a clinically significantWT1gene alteration on a customized disorder of sex development genetic panel in which 62 genes associated with 46, XY disorders of sex development were evaluated. This diagnosis led to early screening for and diagnosis and treatment of Wilms tumor. Patients with proximal hypospadias are not routinely evaluated by genetic testing, and when initial hormonal analyses are within normal ranges for a typical male patient, the genital atypia is usually attributed to an isolated anatomic abnormality. There is no consensus among urologists, endocrinologists, or geneticists regarding when genetic testing is warranted in these patients or the extent of genetic testing that should be pursued. However, given advances in genetic testing and the discovery of more genetic variants, the genetic evaluation of infants with proximal hypospadias should be considered on an individual patient basis. Only with continued evaluation and the identification of further genetic variants can we establish future parameters for genetic evaluation in patients with proximal hypospadias and more appropriately counsel patients and their families regarding the implications of these variants.
Definition. Das nephrotische Syndrom (NS) ist im Kindesalter definiert durch eine große Proteinurie (>1 g/m2/Tag) und Hypalbuminämie (<25 g/l). Als Folge manifestieren sich Ödeme und Hyperlipidämie sowie häufig Oligurie und Aszites. Die Ursache ist ein Verlust von Eiweiß, überwiegend Albumin, über den Urin, dessen Ursache sehr unterschiedlich sein kann.
Renal diseases associated with nephrotic syndrome (NS) in the first year of life are uncommon and make up a heterogeneous group of disorders [73]. Congenital nephrotic syndrome (CNS) is defined as proteinuria manifesting in the first 3 months of life. NS appearing later during the first year (4–12 months) is defined infantile, and NS manifesting thereafter is called childhood NS. While this classification is used to help the clinical diagnosis, it is arbitrary in the sense that NS caused by a specific gene mutation can manifest soon after birth or later in life. However, since the management of CNS is often different from the more common forms of childhood NS, the terminology still seems warranted (Table 1)
Over the last decades, screening of large cohorts of pediatric patients presenting with steroid-resistant nephrotic syndrome (SRNS) for gene mutations has revealed the importance of genetic disorders in the pathogenesis of proteinuric glomerulopathies. Genetic forms of nephrotic syndrome have been considered as infrequent disorders; however, at least 66 % of the cases presenting with SRNS during the first year of life [1] and up to 30 % of SRNS cases with an onset below 25 years of age have an underlying monogenic defect [2]. From a clinical perspective, theoretically all patients with hereditary SRNS will be resistant to immunosuppressive agents and will not experience relapse after transplantation [3–5]. Whereas partial remission may be achieved under cyclosporin A, shown to stabilize the podocyte actin cytoskeleton in vitro through the synaptopodin pathway [6], such treatment cannot induce complete remission in SRNS related to podocyte gene mutations [4, 7–10].
Wilms’ tumor suppressor gene 1 (WT1) mutations are found in Denys-Drash syndrome, Frasier syndrome, and isolated diffuse mesangial sclerosis; these mutations lead to the occurrence of diffuse mesangial sclerosis (DMS) and focal segmental glomerulosclerosis. Nephrotic syndrome (NS) caused because of DMS is unresponsive to drug therapy and is characterized by rapid progression to end-stage renal disease. Here, we report a case of a 2-year-old girl with NS caused because of DMS who responded favorably to cyclosporin A (CsA) . CsA therapy induced a dose-dependent decrease in her urinary protein/creatinine ratio and resulted in partial remission of NS and maintenance of normal renal function for over 2 years. In addition, cases with one WT1 mutation have been reported; however, cases with two WT1 mutations have not been reported thus far. CsA may be effective for DMS with WT1 mutations, if therapy is started before creatinine levels increase. In children with WT1 mutation, CsA therapy may prevent progression to end-stage renal disease, thereby facilitating systematic preemptive kidney transplantation.
Chronic dialysis and renal transplantation are both very good treatments for end-stage renal disease (ESRD). The majority of adults with ESRD are receiving dialysis rather than undergoing renal transplantation. The number of adults seeking renal transplantation is rising, but the number performed has been limited by the lack of appropriate donors [1]. There is a survival advantage of transplantation for virtually all candidates [2]. Renal transplantation was recognized as the better form of treatment for children with ESRD almost three decades ago [3] and has repeatedly shown to provide a survival benefit for children [4, 5].
Background: Nephrotic syndrome (NS) is a common type of kidney disease in children characterized by massive proteinuria, hypoalbuminemia and edema. Response to therapy can be affected by factors like pathologic views, genetic and clinical manifestations. The incidence of genetic mutations is different in variant geographic locations and races. Response to nephrotic syndrome treatment can be influenced by some mutations in WT1 and NPHS2 genes.
Methods: 49 children suffering from nephrotic syndrome participated in two groups of steroid-sensitive (n = 14) and steroid-resistant (n = 35) nephrotic syndrome. Mutations in WT1 and NPHS2 genes analyzed by polymerase chain reaction (PCR) and direct sequencing. Clinical and pathological reviews were done too.
Findings: There was a significant relationship between both primary creatinine and hypertension in the first visit and resistance to therapy. Pthological views of focal segmental glomerulosclerosis (FSGS), glomerular fibrosis, and glomerular sclerosis were significantly related to steroid resistance group (P < 0.001). Genetic analysis for mutations of WT1 and NPHS2 genes among 29 children with idiopathic nephrotic syndrome showed 2 and 5 different mutations in WT1 and NPHS2 genes, respectively. All of the mutations were seen in steroid-resistant group.
Conclusion: This study demonstrates the importance of WT1 and NPHS2 analysis and pathological study in Iranian children with nephrotic syndrome.
Researchers have been shifting the focus of genetic testing from Mendelian disorders towards identifying susceptibility alleles that predispose individuals to developing complex traits, such as diabetic nephropathy, and incorporating pharmacogenomic testing to predict drug-genome interactions. Genetic testing involves the analysis of chromosomes, deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or specific metabolites in order to detect variants that are associated with human disease or a pharmacological response. Direct testing refers to gene-specific evaluation of a DNA and/or RNA sample. The linkage-based testing is an indirect method that examines the segregation of genetic markers located near or within the disease-causing gene with disease phenotypes within a family. Genetic testing increasingly holds the potential to inform clinical practice and impact the outcomes of patients with the disorders that disrupt the structure, function, and/or developmental patterning of the glomerulus, tubules, and urogenital tract, and those that predispose to renal cell tumors. One area where the use of genetic testing has increased rapidly is in the area of hereditary cancer, particularly in relation to hereditary breast/ovarian and hereditary bowel cancer syndromes. Genetic screening may also be of help in determining the chance of developing renal tumors in these disorders. Molecular diagnostic laboratories have developed numerous methods to scan DNA and/or RNA for mutations in specific disease genes or, in certain disorders such as cystic fibrosis, to screen for specific known mutations that cause disease in a high percentage of affected individuals. The current mainstay for molecular diagnosis involves sequence-based protocols that examine the exons and adjacent intronic regions of specific genes.
The Wilms' tumor gene WT1 plays a key role in genitourinary development and subsequent normal function. Homozygous mutations of WT1 can be found in approximately 15% of Wilms' tumors. Furthermore, somatic heterozygous loss of WT1 is known to lead to cryptorchidism and hypospadias in males. A much more severe phenotype is seen in patients with Denys-Drash syndrome which results from heterozygous dominant-negative mutations of the gene. Characteristic features are mesangial sclerosis with early kidney failure, varying degrees of gonadal dysgenesis and high risk of Wilms' tumors. Here we show that a related disease, Frasier syndrome, characterized by focal glomerular sclerosis, delayed kidney failure and complete gonadal dysgenesis, is probably caused by specific intronic point mutations of WT1 that preferentially affect a CpG dinucleotide. Disruption of alternative splicing at the exon 9 splice donor site prevents synthesis of the usually more abundant WT1 +KTS isoform from the mutant allele. In contrast to Denys-Drash syndrome, no mutant protein is produced. The splice mutation leads to an imbalance of WT1 isoforms in vivo , as detected by RT-PCR on streak gonadal tissue. Thus, WT1 isoforms must have quite different functions, and the pathology of Frasier syndrome suggests that especially gonadal development may be particularly sensitive to imbalance or relative underrepresentation of the WT1 +KTS isoform.
Sensitivity and accuracy are essential features of an assay of plasma renin activity (PRA) because the normal concentration of PRA is only 1 pmol/L, and subnormal concentrations have diagnostic relevance. Conditions for blood collection need to be standardized but the conditions are not difficult for outpatients. For routine diagnostic purposes blood should be collected from ambulatory (ideally, untreated) patients on moderate sodium intake. To avoid irreversible cryoactivation of plasma prorenin (which is present in 10-fold greater concentrations than renin), samples should be processed at room temperature and stored completely frozen. Cryoactivation occurs when plasma is liquid at temperatures <6 °C. PRA is commonly measured with an enzyme kinetic assay in which angiotensin I (Ang I) is formed by the reaction of plasma renin with endogenous renin substrate (angiotensinogen). The Ang I so formed is measured by RIA; results are expressed as an hourly rate (μg/L formed per hour). This method, which is provided by most commercial kits, has the potential for unlimited sensitivity because the step for Ang I generation can be prolonged as long as necessary, so that enough Ang I forms to be measured accurately. Unfortunately, that sensitivity is not always exploited. Dilution of plasma during pH adjustment should be kept to a minimum. The Ang I generation step should last at least 3 h. The step should last 18 h for samples with PRA <1.0 μg/L per hour, to eliminate the errors inherent in the measurement and subtraction of immunoreactive Ang I in the untreated plasma (blank subtraction). These changes actually simplify PRA measurements because they eliminate the need for ice in the clinic and reduce by almost half the number of samples to be assayed by RIA. I also describe the method for measurement of plasma prorenin, which may be an important marker for patients with diabetes mellitus who subsequently develop vascular complications.
We have isolated a series of genomic and cDNA clones mapping within the boundaries of constitutional and tumor deletions that define the Wilms' tumor locus on human chromosome 11 (band p13). The transcription unit corresponding to these clones spans approximately 50 kb and encodes an mRNA approximately 3 kb long. This mRNA is expressed in a limited range of cell types, predominantly in the kidney and a subset of hematopoietic cells. The polypeptide encoded by this locus has a number of features suggesting a potential role in transcriptional regulation. These include the presence of four zinc finger domains and a region rich in proline and glutamine. The amino acid sequence of the predicted polypeptide shows significant homology to two growth regulated mammalian polypeptides, EGR1 and EGR2. The genetic localization of this gene, its tissue-specific expression, and the function predicted from its sequence lead us to suggest that it represents the 11p13 Wilms' tumor gene.
Antisense oligodeoxynucleotides have been designed to inhibit the production of specific proteins. In models of hypertension, we have targeted the renin-angiotensin system at the level of synthesis (angiotensinogen) and the receptor (AT1 receptor). The design of antisense oligonucleotides requires choosing a site to inhibit mRNA processig or translation. The strategy we use is to make three oligonucleotides of antisense sequences, upstream and downstream from the AUG site and over the AUG site. The oligonucleotides are tested in a screening test. Antisense oligonucleotides to AT1-receptor mRNA and to angiotensinogen mRNA reduce blood pressure in spontaneously hypertensive rats when injected into the brain. They significantly reduce the concentration of the appropriate protein. The oligonucleotides are also effective when administered systemically. The decrease in blood pressure with antisense oligonucleotides delivered in blood or brain lasts 3 to 7 days. To prolong the action, direct injection of naked DNA and injection of DNA in liposome carriers have been tested. Viral vectors have been developed to deliver antisense DNA. The viral vectors available include retroviruses and adenovirus, but the adeno-associated virus (AAV) vector is the vector of choice for ultimate use in gene therapy. It offers safety because it is nonpathogenic, has longevity because it integrates into the genome, and has sufficient carrying capacity to carry up to 4.5 kb antisense or gene in a recombinant AAV. Using rAAV-antisense to AT1 mRNA, there is efficient transfection into cells and an inhibition of AT1 receptor number. In in vivo tests, rAAV-AS AT1-receptor when injected into the brains of SHR reduces blood pressure for more than 2 months. In young rats (3 weeks old), rAAV-AS AT1-receptor decreases blood pressure and slows the development of hypertension. While further experiments need to be done on dose-response relationships and on the cellular mechanisms of these effects, the results show the feasibility of AAV as a vector for antisense inhibition, which may ultimately be used in gene therapy for hypertension.
Rats exposed chronically to mild cold (5°C/4°F) develop hypertension and cardiac hypertrophy. The blood renin-angiotensin system (RAS) has been shown to play a role in both initiating and maintaining the high blood pressure in cold-induced hypertension (CIH). The mechanism also appears to involve both tissue and brain RAS as there is increased mRNA for angiotensinogen (Ao) and AT1 receptors in brain and peripheral tissues, and an increased dipsogenic response to acute administration of AII. Antisense oligodeoxynucleotides (AS-ODNs), targeted to RAS, have been shown to reduce BP in SHR. Therefore, we injected AS-ODNs in CIH to test a nongenetic environmental induced HT. Sprague Dawley rats were made hypertensive and injected icv with AS-ODNs to AT1 receptor mRNA (n=6) or AS-ODNs to Ao mRNA (n=6). BP was recorded by tail cuff 24 hr later. SBP decrease significantly to AT1 receptor-AS (41 ± 10 mmHg, p < 0.05), and to Ao-AS (40 ± 6mmHg, p < 0.001). The spontaneous dipsogenic response also decrease significantly (p < 0.05) after AT1 receptor-AS and Ao-AS icv injection. Intracardiac injection of AT1 receptor-AS (n=6) reduced SBP by 36 ± 8mmHg (p < 0.05) 24 hr after injection. These data show that AS-ODNs reduce BP in CIH and that the hypertensison involves brain RAS besides blood RAS mechanisms.
Antisense inhibition is a gene therapy strategy that offers a "knockdown" approach to produce a reduction of gene expression. The authors are studying the potential use of antisense inhibition in gene therapy for hypertension. Their first approach at modifying genes to lower hypertension was with antisense oligodeoxynucleotide directed to the AT(1) receptor and angiotensinogen mRNA. The authors made effective antisense oligomers to renin, angiotensinogen, angiotensin converting enzyme and AT(1) receptor mRNA and tested them in three different models of hypertension. In every case tested, there was consistent biological signs of gene "knockdown," Next, viral vector delivery of cDNA in the antisense direction was developed to prolong antisense effects even longer and to avoid concerns about the toxicity of liposome carriers and accumulation of sulfur from the phosphorothioated backbones of the oligos. Results using viral vector delivery in spontaneously hypertensive rats have been successful. A recombinant adeno-associated viral vector containing cDNA for the AT(1) receptor in the antisense direction with a CMV promoter and a neomycin resistance gene as a reporter gene was constructed and tested successfully. Other vectors are also being tested for hypertension gene therapy; in addition to the choice of vectors, there is a choice to be made of promoters. While a great deal more work needs to be done on the basic science of these approaches to gene therapy for hypertension before they can be applied to humans, the results are encouraging, the biology is feasible and the medical possibility plausible.
Focal and segmental glomerulosclerosis (FSGS) is a common, non-specific renal lesion. Although it is often secondary to other disorders, including HIV infection, obesity, hypertension and diabetes, FSGS also appears as an isolated, idiopathic condition. FSGS is characterized by increased urinary protein excretion and decreasing kidney function. Often, renal insufficiency in affected patients progresses to end-stage renal failure, a highly morbid state requiring either dialysis therapy or kidney transplantation. Here we present evidence implicating mutations in the gene encoding alpha-actinin-4 (ACTN4; ref. 2), an actin-filament crosslinking protein, as the cause of disease in three families with an autosomal dominant form of FSGS. In vitro, mutant alpha-actinin-4 binds filamentous actin (F-actin) more strongly than does wild-type alpha-actinin-4. Regulation of the actin cytoskeleton of glomerular podocytes may be altered in this group of patients. Our results have implications for understanding the role of the cytoskeleton in the pathophysiology of kidney disease and may lead to a better understanding of the genetic basis of susceptibility to kidney damage.
Familial idiopathic nephrotic syndromes represent a heterogeneous group of kidney disorders, and include autosomal recessive steroid-resistant nephrotic syndrome, which is characterized by early childhood onset of proteinuria, rapid progression to end-stage renal disease and focal segmental glomerulosclerosis. A causative gene for this disease, NPHS2, was mapped to 1q25-31 and we report here its identification by positional cloning. NPHS2 is almost exclusively expressed in the podocytes of fetal and mature kidney glomeruli, and encodes a new integral membrane protein, podocin, belonging to the stomatin protein family. We found ten different NPHS2 mutations, comprising nonsense, frameshift and missense mutations, to segregate with the disease, demonstrating a crucial role for podocin in the function of the glomerular filtration barrier.
We report new mutations in exon 9 of the WT1 gene that did not alter the ratio of +/– KTS splice isoforms in two unrelated patients with Frasier syndrome (FS). The mutation of intron 9 inducing defective alternative splicing was reported to be responsible for this syndrome. The mutations found in our cases occurred in the same exon of the WT1 gene as detected in Denys‐Drash syndrome (DDS) and could not be explained by the previously proposed mechanism. The results suggest that the two syndromes originate from the same WT1 gene abnormality. From a molecular biological point of view, we concluded that the two diseases were not separable, and that FS should be included as an atypical form of DDS. Hum Mutat 14:466–470, 1999. © 1999 Wiley‐Liss, Inc.
Denys‐Drash and Frasier syndromes are rare human disorders that associate nephropathy with gonadal and genital abnormalities. In DDS there is a predisposition to Wilms' tumor. Heterozygous point mutations in the Wilms' tumor, type 1 gene (WT1), particularly those altering the zinc finger (ZF) encoding exons, have been reported in most DDS patients, while mutations in intron 9 of the same gene cause FS. This paper describes two cases of DDS, one FS and one patient with Wilm's tumor and intersex genitalia, in which mutations were searched by sequencing the exons 8 and 9 of WT1 gene. Patient 1 carried a missense point mutation in exon 8 (ZF2), converting a CGA‐Arg codon to a TGA‐stop codon. Patient 2 presented a single nucleotide deletion within exon 9 (ZF3) introducing a premature chain termination at codon 398. Patients 3 and 4 had a C→T transition at position +4 of the second alternative splice donor site of exon 9 (this mutation was detected in peripheral blood and in tumor derived DNA of patient 3). However, patient 3 had previously developed a Wilms' tumor. This is the first case of Wilms' tumor development in a phenotypically and genetically confirmed case of FS. Hum Mutat 13:146–153, 1999. © 1999 Wiley‐Liss, Inc.
Mutations in the WT1 gene ere anticipated to explain the genetic basis of the childhood kidney cancer, Wilms' tumour (WT). Six years on, we revie 100 reports of intragenic WT1 mutations and examine the accompanying clinical phenotypes. While only 5% of sporadic Wilms' tumours have intragenic WT1 mutations, > 90% of patients with the Denys‐Drash syndrome (renal nephropathy, gonadal anomaly, predisposition to WT) carry constitutional intragenic WT1 mutations. WT1 mutations have also been reported in juvenile granulosa cell tumour, non‐asbestos related mesothelioma, desmoplastic small round cell tumour and, most recently, acute myeloid leukemia. Hum Mutat 9:209–225, 1997. © 1997 Wiley‐Liss, Inc.
Mutations in the WT1 gene ere anticipated to explain the genetic basis of the childhood kidney cancer, Wilms' tumour (WT). Six years on, we revie 100 reports of intragenic WT1 mutations and examine the accompanying clinical phenotypes. While only 5% of sporadic Wilms' tumours have intragenic WT1 mutations, > 90% of patients with the Denys-Drash syndrome (renal nephropathy, gonadal anomaly, predisposition to WT) carry constitutional intragenic WT1 mutations. WT1 mutations have also been reported in juvenile granulosa cell tumour, non-asbestos related mesothelioma, desmoplastic small round cell tumour and, most recently, acute myeloid leukemia. Hum Mutat 9:209–225, 1997. © 1997 Wiley-Liss, Inc.
The 521 children with the nephrotic syndrome included 389 with MCNS, of whom 219 had nil disease, 98 had focal glomerular obsolescence, 16 had mild mesangial thickening, 29 had focal tubular changes, and 27 had mild mesangial hypercellularity. There were 12 patients with diffuse mesangial hypercellularity. Complete urinary data required for the analyses in this report were available for 372 patients at 8 weeks, 358 at 6 months, 343 at 1 year, and 301 at 2 years. There were significant differences in the proportions of initial nonresponders among patients in the six groups (P<0.001). The percentages of initial nonresponders among patients with focal tubular changes (14.3), mild mesangial hypercellularity (14.8), and diffuse mesangial hypercellularity (45.5) as a group were significantly higher (P<0.02) than the group with nil disease (4.7), focal glomerular obsolescence (7.8), and mild mesangial thickening (6.3). The proportion of initial nonresponders in patients with diffuse mesangial hypercellularity was significantly higher (P<0.05) than it was in those with mild mesangial hypercellularity. Proteinuria subsided at some point in 23 (74.2%) of the total of 31 initial nonresponders, and by 52 weeks the differences in the proportions of the remaining few with persistent proteinuria were no longer statistically significant. There were no significant differences among the groups in the proportions of initial responders who became early relapsers, frequent relapsers, or late nonresponders. There also were no significant differences among the groups in the proportions of patients who died. A similar analysis of the subsequent course in initial nonresponders in the six groups would not be meaningful because the numbers in some categories were so small and because the majority of them were entered into therapeutic trials early in their course and treated in a variety of ways.
Two regimens of steroid treatment for the initial attack of idiopathic nephrotic syndrome in children were compared in a controlled multicentre study. Short-course prednisone therapy consisted of 60 mg/m2 per 24 h until proteinuria had disappeared for 3 days, followed by 40 mg/m2 per 48 h until complete remission had occurred. The standard prednisone therapy was 60 mg/m2 per 24 h for 4 weeks, followed by 40 mg/m2 per 48 h for 4 weeks. 61 children with a first attack of idiopathic nephrotic syndrome were allocated at random to these groups. Urinary remission in the short-course group was achieved after 14 days of daily prednisone, and complete remission after an additional 16 days of alternate day prednisone. The cumulative rate of patients with sustained remissions after two years was significantly lower after the short course than after standard treatment (19% versus 41%, p=0·001). The mean duration of remission in patients with a relapse was half as long after the short course (79 versus 169 days, p = 0·004). Complete initial remission of steroid responsive nephrotic syndrome can be obtained with half the standard prednisone dose, but the short course is followed by a higher rate of relapses, that require repeated prednisone administrations. In the long term, the standard regimen is preferable.
Background:
Donor splice-site de novo heterozygous mutations in intron 9 of the Wilms' tumor gene (WT1) have been reported in Frasier syndrome, which is defined by the association of focal and segmental glomerulosclerosis (FSGS), male pseudohermaphroditism, and gonadoblastoma. These splice-site mutations alter the WT1 alternative splicing leading to two WT1 isoforms, with (+) or without (-) three amino acids, lysine-threonine-serine (KTS), between zinc fingers 3 and 4. The aim of this work was to investigate the possibility that some cases of primary steroid-resistant nephrotic syndrome associated with FSGS may be caused by WT1 splice-site mutations.
Methods:
We analyzed WT1 exons 8 and 9 and the surrounding exon/intron boundary DNA sequences in 37 children with nonfamilial primary steroid-resistant nephrotic syndrome. Semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) was used to determine the relative ratio of +KTS/-KTS transcripts from immortalized lymphocyte RNA.
Results:
One boy with FSGS and associated pathologies (diaphragmatic hernia, proximal hypospadias, and unilateral testicular ectopia) was found to carry the heterozygous 1228 +4 C-->T splice-site mutation. RT-PCR quantitation of the +KTS/-KTS transcripts from immortalized lymphocyte RNA of this patient showed a diminution of the +KTS/-KTS isoform ratio (0.43), which is identical to that reported in patients with Frasier syndrome. Using the same approach, healthy control subjects have +KTS/-KTS ratios ranging from 1.50 to 2.00.
Conclusions:
This study expands the range of the phenotypic presentation of the intron 9 splice-site WT1 mutations and adds to the already reported heterogeneity of primary steroid-resistant nephrotic syndromes. We suggest that these mutations are not likely to be a common cause of isolated steroid-resistant nephrotic syndrome, and recommend a WT1 exon 9/intron 9 splice-site study in children with primary steroid-resistant nephrotic syndrome if genital or diaphragmatic anomalies are associated. The identification of such WT1 mutations has practical implications for the management of these patients.
We report new mutations in exon 9 of the WT1 gene that did not alter the ratio of +/– KTS splice isoforms in two unrelated patients with Frasier syndrome (FS). The mutation of intron 9 inducing defective alternative splicing was reported to be responsible for this syndrome. The mutations found in our cases occurred in the same exon of the WT1 gene as detected in Denys-Drash syndrome (DDS) and could not be explained by the previously proposed mechanism. The results suggest that the two syndromes originate from the same WT1 gene abnormality. From a molecular biological point of view, we concluded that the two diseases were not separable, and that FS should be included as an atypical form of DDS. Hum Mutat 14:466–470, 1999. © 1999 Wiley-Liss, Inc.
Mutations in the WT1 gene were anticipated to explain the genetic basis of the childhood kidney cancer, Wilms tumour (WT). Six years on, we review 100 reports of intragenic WT1 mutations and examine the accompanying clinical phenotypes. While only 5% of sporadic Wilms' tumours have intragenic WT1 mutations, > 90% of patients with the Denys-Drash syndrome (renal nephropathy, gonadal anomaly, predisposition to WT) carry constitutional intragenic WT1 mutations. WT1 mutations have also been reported in juvenile granulosa cell tumour, non-asbestos related mesothelioma, desmoplastic small round cell tumour and, most recently, acute myeloid leukemia.
Constitutional mutations of the WT1 gene, encoding a zinc-finger transcription factor involved in renal and gonadal development, are found in most patients with Denys-Drash syndrome (DDS), or diffuse mesangial sclerosis (DMS) associated with pseudohermaphroditism and/or Wilms tumor (WT). Most mutations in DDS patients lie in exon 8 or exon 9, encoding zinc finger 2 or zinc finger 3, respectively, with a hot spot (R394W) in exon 9. We analyzed a series of 24 patients, 10 with isolated DMS (IDMS), 10 with DDS, and 4 with urogenital abnormalities and/or WT. We report WT1 heterozygous mutations in 16 patients, 4 of whom presented with IDMS. One male and two female IDMS patients with WT1 mutations underwent normal puberty. Two mutations associated with IDMS are different from those described in DDS patients. No WT1 mutations were detected in the six other IDMS patients, suggesting genetic heterogeneity of this disease. We analyzed genotype/phenotype correlations, on the basis of the constitution of a WT1 mutation database of 84 germ-line mutations, to compare the distribution and type of mutations, according to the different symptoms. This demonstrated (1) the association between mutations in exons 8 and 9 and DMS; (2) among patients with DMS, a higher frequency of exon 8 mutations among 46, XY patients with female phenotype than among 46,XY patients with sexual ambiguity or male phenotype; and (3) statistically significant evidence that mutations in exons 8 and 9 preferentially affect amino acids with different functions.
To block renin activity, a nonapeptide converting-enzyme inhibitor was given to 65 seated hypertensive patients. Depressor responses occurred only when control plasma renin activity exceeded 2 ng of angiotensin I per milliliter per hour and correlated directly in amplitude with control plasma renin activity and with induced increments in activity (P less than 0.001 for both). Depressor responses, like renin activity, were characteristic for renin subgroups as defined by renin-sodium profiling. Before and after sodium deprivation, the nonapeptide reduced diastolic pressure in all patients with high renin (by 17.3 and 19.8 per cent) and most patients with normal renin (by 9.1 and 17.7 per cent). Low-renin patients remained unresponsive. This enzyme blockade may cause bradykinin accumulation. But if, as seems likely, depressor responses are due to blockade of angiotensin II formation, the results indicate that, irrespective of sodium balance, measurements of plasma renin activity reflect its contribution to blood-pressure maintenance. The results suggest broad participation of the renin system in common forms of hypertension.
The viral DNA sequences present in the human embryo kidney cell line (293) transformed from sheared adenovirus 5 fragments (F. L. Graham, P. S. Abrahams, C. Mulder, H. L. Heijneker, S. 0. Warnaar, F. A. deVries, W. Fiers, and A. J. van der Eb 1974, Cold Spring Harbor Symp. Quant. Biol.39, 637–650) were analyzed. By renaturation kinetics of transformed cell DNA and labeled adenovirus 5 restriction enzyme fragments obtained with HindIII we determined that there are four to five copies per cell of the left 12% of the viral genome and approximately one copy per cell of 9% of the right end of the viral genome. Analysis of the RNA sequences transcribed in nuclei isolated from this cell line indicated that only transcripts from the sequences of the left end were detected. The expression of these viral sequences was mediated by DNA-dependent RNA polymerase II. Analysis of the viral DNA from 293 cells using restriction enzymes reveals that the viral DNA is integrated and located at two sites on the cellular genome. This simple arrangement is stable through several cell passages.
A Wilms' tumor susceptibility gene (WT1) localized to 11p13 was recently isolated and shown to be altered in some sporadic Wilms' tumors. This gene encodes a DNA-binding protein with four zinc fingers (ZFs) in the carboxy-terminal region and a glutamine/proline (Gln/Pro)-rich domain near the 5' end. Two alternative splice sites were described, splice I in the Gln/Pro-rich domain (51 bp) and splice II between ZFs 3 and 4 (9 bp). Using RNA polymerase chain reaction (PCR) we show that Wilms' tumors contain all four possible transcripts, which are also identified in normal adult and embryonic kidney cells. The transcripts containing the 9-bp ZF insert were always predominant in tumors and normal cells. The presence of all four WT1 transcripts in tumors and expressing tissues suggests that each encoded protein isoform has an important role for the function of the WT1 gene.
The chromosome 11p13 Wilms tumor susceptibility gene WT1 appears to play a crucial role in regulating the proliferation and differentiation of nephroblasts and gonadal tissue. The WT1 gene consists of 10 exons, encoding a complex pattern of mRNA species: four distinct transcripts are expressed, reflecting the presence or absence of two alternative splices. Splice I consists of a separate exon, encoding 17 amino acids, which is inserted between the proline-rich amino terminus and the zinc finger domains. Splice II arises from the use of an alternative 5' splice junction and results in the insertion of 3 amino acids between zinc fingers 3 and 4. RNase protection analysis demonstrates that the most prevalent splice variant in both human and mouse is that which contains both alternative splices, whereas the least common is the transcript missing both splices. The relative distribution of splice variants is highly conserved between normal fetal kidney tissue and Wilms tumors that have intact WT1 transcripts. The ratio of these different WT1 mRNA species is also maintained as a function of development in the mouse kidney and in various mouse tissues expressing WT1. The conservation in structure and relative levels of each of the four WT1 mRNA species suggests that each encoded polypeptide makes a significant contribution to normal gene function. The control of cellular proliferation and differentiation exerted by the WT1 gene products may involve interactions between four polypeptides with distinct targets and functions.
Blockade of the renin-angiotensin system by an angiotensin converting enzyme (ACE) inhibitor or an angiotensin II (Ang II) antagonist is accompanied by a reactive rise in renin release. This rise is generally attributed to interruption of the short feedback loop between Ang II and renin release. Similarly, after the administration of a renin inhibitor, the plasma concentrations of active and total renin are increased and plasma renin activity is suppressed. The aim of the present study was to investigate if a fall in the plasma Ang II level is the unique determinant of the rise in the active renin (AR) level that follows renin inhibition. Six normal male volunteers participated in three successive 240-minute experiments at weekly intervals according to a single-blind randomized Latin square design. For experiment 1, Ang II was infused at 2 ng/kg/min from 0 to 60 minutes and at 4 ng/kg/min from 60 to 120 minutes. For experiment 2, 0.3 mg/kg of the new potent renin inhibitor Ro 42-5892 was injected at 30 minutes followed by infusion at 0.1 mg/kg/hr from 30 to 240 minutes. For experiment 3, Ang II and Ro 42-5892 were administered simultaneously at the same doses as described above. The mean +/- SEM Ang II concentration increased from 10.2 +/- 1.6 to 33.7 +/- 11.2 pg/ml after infusion of exogenous peptide. It decreased from 9.5 +/- 0.9 to 1.4 +/- 0.3 pg/ml after the injection of Ro 42-5892 and increased from 15.6 +/- 2.9 to 37.1 +/- 11.8 pg/ml after the simultaneous infusion of both compounds.(ABSTRACT TRUNCATED AT 250 WORDS)
Cytogenetic analysis has identified chromosome 11p13 as the smallest overlap region for deletions found in individuals with WAGR syndrome, which includes Wilms tumour (a recessive childhood nephroblastoma), aniridia, genito-urinary abnormalities and mental retardation. The underlying loci have since been resolved into an aniridia (AN2) locus at a telomeric position, and a locus of closely spaced genes or a single pleiotropic gene involved in genito-urinary tract abnormalities and Wilms tumour at a more centromeric position. Pulsed-field gel analysis of the 11p13 region has revealed the presence of several putative CpG islands, structures which are frequently associated with the 5' ends of expressed sequences, mainly housekeeping genes and some tissue-specific genes. Starting from a CpG island, we have now isolated four neighbouring CpG islands, all within 650 kilobases (kb), by means of two consecutive bidirectional jumps in rare-cutting restriction-enzyme jumping libraries. In two instances, flanking sequences were conserved in other species and RNA transcripts were identified. A complementary DNA clone isolated for one of them derives from an RNA highly expressed in fetal kidney, and is predicted to encode a Krüppel-like zinc-finger protein that is probably a transcription factor. The entire cDNA region is included in two partially overlapping homozygous deletions found in Wilms tumour DNA samples. Cloning of the breakpoints in one tumour revealed a deletion size of 170 kb, one-third of which is covered by the cDNA. The expression pattern and sequence of this cDNA could point to an important role for its corresponding gene in the normal development of the renal system as well as in Wilms tumour.
A 3- to 4-week exposure of rats to a cold environment (5 +/- 2 degrees C) induces hypertension, including elevation of systolic, diastolic, and mean blood pressures and cardiac (left ventricular) hypertrophy. The studies described here were designed to investigate some factors affecting both the magnitude and the time course for development of cold-induced hypertension. The objective of the first study was to determine whether there was an ambient temperature at which the cold-induced elevation of blood pressure did not occur. The objective of the second experiment was to determine whether body weight at the time of exposure to cold affected the magnitude and time course for development of hypertension. To assess the first objective, male rats were housed in a chamber whose temperature was maintained at 5 +/- 2 degrees C while others were housed in an identical chamber at 9 +/- 2 degrees C. After 7 days of exposure to cold, the rats exposed to the colder temperature had a significant elevation of blood pressure (140 +/- 2 mm Hg) compared with the group maintained at 9 degrees C (122 +/- 3 mm Hg). The rats exposed to 9 degrees C had no significant elevation of systolic blood pressure at either 27 or 40 days after initiation of exposure to cold. At the latter time, the temperature in the second chamber was reduced to 5 +/- 2 degrees C. By the 25th day of exposure to this ambient temperature, the rats had a significant increase in systolic blood pressure above their levels at 9 degrees C. Thus, there appears to be a threshold ambient temperature for elevation of blood pressure during exposure to cold. That temperature appears to lie somewhere between 5 and 9 degrees C. The second objective was assessed by placing rats varying in weight from approximately 250 to 430 g in air at 5 degrees C. There was a highly significant direct relationship (r = 0.96) between body weight at the time of introduction to cold and the number of days required to increase systolic blood pressure by 10 mm Hg above pre-cold exposure level. The third objective was to make an initial assessment of potential differences among strains of rats with respect to development of cold-induced hypertension. To this end, rats of the Fischer 344 strain were used. Systolic blood pressures of these rats also increased during chronic exposure to cold.(ABSTRACT TRUNCATED AT 400 WORDS)
Evidence concerning the clinical utility of single dose captopril in the diagnosis of renovascular hypertension was evaluated. Of 173 identified papers, 16 were specifically selected because they used single dose oral captopril and obtained pre-dose and post-dose peripheral renin levels in at least one patient with renovascular hypertension. These 16 studies were appraised independently by three reviewers using standardized forms for evaluation of diagnostic tests. The 16 studies included 805 patients. All studies involved referred hypertensive populations; detailed demographics and clinical characteristics were not provided. Captopril test procedures varied in all studies. Thirteen of 16 studies used arteriography as a gold standard for the diagnosis of renovascular hypertension, and three of 16 used surgical outcome data. In ten studies, patients with renovascular hypertension clearly had a significantly greater increase in plasma renin activity than patients with essential hypertension. In the remaining six, plasma renin activity was increased in patients with renovascular hypertension but control comparisons were not made adequately. Existing data suggest that the captopril test may be useful in identifying patients with renovascular hypertension. However, specific clinical recommendations regarding its use cannot be made until future research better defines test cutoff points and identifies which patients are most likely to benefit from the test.
Renin (EC 3.4.23.15) is an aspartyl protease that cleaves its only known substrate, angiotensinogen, to release the vasopressor hormone angiotensin. We have isolated full-length cDNAs for renin from a rat kidney cDNA library. The cDNAs are complementary to a 1434-nucleotide rat kidney mRNA that encodes preprorenin, the 402-amino acid precursor of renin. This rat cDNA was used to isolate the complete copy of a renin gene from a rat genomic library, and a comparison of this genomic clone with rat genomic DNA showed that renin is a single-copy gene in the rat. Rat renin is 85% identical to one mouse renin isozyme (renin-1) and 81% identical to the second mouse renin isozyme (renin-2), suggesting that the duplication of the mouse renin genes is a more recent event than the speciation of rats and mice. Analyses of rat, human, and mouse renin sequences revealed that the potential to form two-chain renin is apparently peculiar to mouse renin, and the expression of a tenth exon (which results in a three-amino acid insertion) is observed only in the human renin gene.
In 36 patients with unilateral renal artery stenosis and in 24 with essential hypertension the plasma levels of total immunoreactive renin, and enzymatically active renin were measured in both renal veins (V) and in the aorta (A) by direct RIA by using monoclonal renin antibodies. Active renin and trypsin-activatable inactive renin were also measured by indirect RIA with angiotensin-I antibodies. The V/A ratio for the different forms of renin calculated from the results of direct and indirect RIA were not different. The V/A ratio of active renin for the kidney with the stenotic artery was 3.04 +/- 0.28 (mean +/- sem) with direct and 3.02 +/- 0.25 with indirect RIA. The contralateral ratio was 1.04 +/- 0.02 with the direct and 1.05 +/- 0.02 with the indirect RIA. In essential hypertension it was 1.28 +/- 0.04 with direct RIA and 1.28 +/- 0.04 with indirect RIA. Chronic treatment with captopril had no influence on this ratio in both patients groups. The V/A ratio of total immunoreactive renin was lower than that of active renin and this ratio had lost discriminative power for lateralization. This ratio was significantly greater than one on the affected side in renal artery stenosis but not contralaterally and in essential hypertension. This study shows that renin activity after trypsin-activation of plasma is an accurate measure of the total renin concentration, i.e. active renin plus prorenin. It also shows that a kidney with a stenotic artery secretes inactive renin, which is immunologically related to active renin and is likely to be prorenin. Direct RIA for measuring active renin is technically more simple than indirect RIA. Direct RIA however is somewhat less sensitive. For measuring the V/A ratio for active renin in patients with renal artery stenosis this can be overcome by stimulating the renin-angiotensin system for instance by captopril.
Sar1-ala8-angiotensin II, the octapeptide competitive inhibitor of angiotensin II, was administered intravenously to twelve patients with either renovascular, advanced, or malignant hypertension, or with hypertension of pyelonephritis. In eight patients with increased plasma-renin, angiotensin blockade induced immediate striking and sustained reductions in blood-pressure to nearly normal levels. Large excesses of the drug never produced hypotension. This evidence suggests that angiotensin II was involved in sustaining the high blood-pressure. In contrast, in four patients with either low or normal plasma-renin, the angiotensin inhibitor did not alter blood-pressure. Efficacy of the drug was indicated by the striking increases in plasma-renin induced in all patients. Angiotensin blockade with its apparent lack of toxicity provides a new, safe, more specific and effective approach to diagnosis and treatment of hypertensive situations associated with high renin levels. Also, it seems useful for predicting surgical curability of renovascular hypertension.
This report concerns observations on two unrelated children originally evaluated because of sexual ambiguity. One was reared as a male, the other as a female. The first was buccal smear negative and had a unilateral intra-abdominal testis. The second had negative buccal smear, 46,XY peripheral leukocyte karyotype, and bilateral intra-abdominal testes. Their clinical courses were similar. Hypertension and proteinuria were detected early in life. Death resulted eventually from progressive renal failure in each child. A unilateral Wilms' tumor was discovered early in the course of observation of one child and as an incidental postmortem finding in the other. Deterioration in renal function was not related to the presence of the tumor. This constellation of findings, reported only once previously, probably represents an intrauterine alteration of cellular differentiation, affecting renal, gonadal, and other internal and external sexual structures.
The objective of this experiment was to determine whether bilateral renal denervation (RD) prevents the elevation of blood pressure and cardiac hypertrophy characteristically induced by chronic exposure to cold. Four groups (nine male rats each) were used. The kidneys of two groups were bilaterally denervated, while the remaining two groups were sham operated. Systolic blood pressures of the four groups, measured indirectly from the tail, did not differ significantly during the control period and following RD. At this time, 1 RD and 1 sham-operated group was exposed to cold (5 degrees C, 41 degrees F). The remaining RD and sham-operated groups were kept at 25 degrees C. Blood pressure of the cold-exposed, sham-operated group increased significantly during the 1st week of cold exposure (125 +/- 2 mmHg; 1 mmHg = 133.3 Pa), and rose to 139 +/- 4 mmHg by the 5th week, whereas the blood pressure of the RD group exposed to cold remained at the control level (116 +/- 2 mmHg). Both RD and sham-operated cold-exposed groups developed cardiac hypertrophy with significantly increased resting heart rates compared with controls kept at 25 degrees C. Plasma renin activities and renal norepinephrine content of kidneys of both RD groups at 7 weeks after RD were significantly less than those of sham-operated controls, confirming that renal nerves had been severed. Thus, RD prevented the elevation of blood pressure induced by chronic exposure to cold but had no significant effect on cardiac hypertrophy.
Two regimens of steroid treatment for the initial attack of idiopathic nephrotic syndrome (NS) in children were compared in a controlled prospective multi-centre study. Long prednisone therapy consisted of 60 mg/m2 per 24 h for 6 weeks, followed by alternate day 40 mg/m2 per 48 h for 6 weeks. The standard prednisone therapy was 60 mg/m2 per 24 h for 4 weeks, followed by 40 mg/m2 per 48 h for 4 weeks. A total of 71 children with an initial attack of idiopathic NS were allocated at random to the two groups. The cumulative rate of patients with sustained remissions after 2 years was significantly higher after the long course than after the standard treatment (49% vs 19%, P = 0.0079). The mean relapse rate per patient at intervals of 3, 6 and 12 months was lower in the long-course prednisone group than in the standard prednisone group, and the proportion of children with frequent relapses during any subsequent 6 months period was lower in the long-course group than in the standard group (29% vs 57%, P = 0.03). Mild side-effects of corticosteroid therapy were observed more frequently after long-course prednisone treatment. It is concluded that long-course prednisone therapy of the initial attack of steroid responsive NS is preferable to the standard regimen because it reduces the rate of subsequent relapses without increasing the risk for severe steroidal side-effects.
The renin-angiotensin system (RAS) is a major contributor in the regulation of blood pressure, and pharmacologic manipulation of this system has resulted in a beneficial class of therapeutic agents, which include angiotensin-converting enzyme (ACE) inhibitors. However, ACE inhibitors are not specific for RAS, and in addition, they can affect bradykinin and prostaglandin, which can also cause changes in vascular tone. Under development are renin inhibitors that are specific for angiotensinogen and act at the initial, rate-determining step of the RAS cascade. The various pharmacologic approaches to renin inhibition include specific renin antibodies, synthetic derivatives of the prosegment of renin precursor, pepstatin analogs, and angiotensinogen analogs. The last approach is the most promising for patient therapy. Multiple studies have shown the effectiveness of the renin inhibitors in both primates and human beings. Further research is now directed toward the development of an agent with good oral bioavailability for patient treatment and as a biologic probe for helping to understand the role of the RAS in control of blood pressure and blood volume.
The renin-angiotensin system plays a crucial role in the development and establishment of the hypertensive state in the spontaneously hypertensive (SH) rat. Interruption of this system's activity by pharmacological means results in the lowering of blood pressure (BP) and control of hypertension. However, such means are temporary and require the continuous use of drugs for the control of this pathophysiological state. Our objective in this investigation was to determine if a virally mediated gene-transfer approach using angiotensin type 1 receptor antisense (AT1R-AS) could be used to control hypertension on a long-term basis in the SH rat model of human essential hypertension. Injection of viral particles containing AT1R-AS (LNSV-AT1R-AS) in 5-day-old rats resulted in a lowering of BP exclusively in the SH rat and not in the Wistar Kyoto normotensive control. A maximal anti-hypertensive response of 33 +/- 5 mmHg was observed, was maintained throughout development, and still persisted 3 months after administration of LNSV-AT1R-AS. The lowering of BP was associated with the expression of AT1R-AS transcript and decreases in AT1-receptor in many peripheral angiotensin II target tissues such as mesenteric artery, adrenal gland, heart, and kidney. Attenuation of angiotensin II-stimulated physiological actions such as contraction of aortic rings and increase in BP was also observed in the LNSV-AT1R-AS-treated SH rat. These observations show that a single injection of LNSV-AT1R-AS normalizes BP in the SH rat on a long-term basis. They suggest that such a gene-transfer strategy can be successfully used to control the development of hypertension on a permanent basis.
Hypertension, tachycardia and cardiac hypertrophy develop in rats exposed to mild cold (5 degrees C, 41 degrees F) for 1 to 3 weeks. Elevation of blood pressure (BP) during cold exposure is sodium dependent, although the rats still have an elevation of BP with a minimum of NaCl in their diet. Drugs that interfere with the renin-angiotensin-aldosterone (RAA) system at various levels (propranolol, clonidine, captopril, losartan and spironolactone) are able to prevent the development of cold-induced hypertension (CIH). Plasma renin activity (PRA) increases during the first 3 weeks of exposure to cold and then gradually decreases toward control level. Increased blood pressure and dipsogenic sensitivity to administration of angiotensin II (Ang II) have been demonstrated during the first 3 weeks of exposure to cold suggesting an upregulation of Ang II receptors when PRA is elevated. Additional studies have shown greater Fos-like immunoreactivity in the diencephalon of cold-exposed compared to warm-acclimated rats after 1 hr i.v. infusion of Ang II (333 ng/kg/min). Thus, most characteristics of cold-induced hypertension mimic those of hypertension induced experimentally by chronic administration of large doses of deoxycorticosterone acetate (DOCA) and salt. The results suggest that CIH is a mineralocorticoid-induced hypertension and that various levels of the RAA system contribute.
Several mechanisms are known to participate in cold-induced hypertension, but no information exist on the role of the nitric oxide (NO). In the present study, we assessed the participation of nitric oxide in cold-induced hypertension by means of inhibition of NO synthase. Experiments were performed in rats treated with N omega-nitro-L-arginine (L-NNA) injected i.p. at a dose of 25 mg/kg body weight twice a day for four consecutive days. Control animals received saline injections of the same volume. Two days before the experiment, the femoral artery was cannulated for blood pressure recording. Arterial blood pressure was measured at 25 degrees C for 30 min (control period), followed by a 3.5 hour period at 4 degrees C (cold exposure) and, eventually, a last 3 hour period after removal from cold (back to 25 degrees C). In control animals, at 25 degrees C, mean arterial blood pressure was 112.5+/-3.6 mmHg and heart rate was 380+/-3.5 bpm. L-NNA treatment caused an increase in blood pressure to 155.0+/-3.5 mmHg (P<0.01) and in heart rate to 410+/-6.0 bpm (P<0.05). Exposure to cold caused blood pressure to increase up to 131.5+/-3.6 mmHg (P<0.05) in the control group, whereas no significant change could be measured in treated animals. Recovery from cold exposure led to a decrease in blood pressure in control animals, but not in treated animals. These results indicate that NO plays a role in the development of cold-induced elevation of blood pressure.
Calu-6 cells were characterized for studying the transcriptional regulation of the human renin gene. Analysis of cis-acting elements of the renin promoter showed the highest activity within the first 582 bp in serum-free conditions and of the 892 bp in the presence of serum. cAMP activates renin mRNA synthesis parallel to renin production (20-fold increase) as well renin promoter activity (2-fold). cAMP response element and the (-77 to -67) element are both necessary for activation of the renin promoter but do not act independently. Functional analysis of Intron A revealed the presence of a silencer specific to renin-producing cells.
Interruption of the renin-angiotensin system by pharmacological manipulations attenuates high blood pressure (BP) in the spontaneously hypertensive rat (SHR). However, these agents, such as losartan, need to be administered daily to maintain effective BP control. Therefore, we have hypothesized that a genetic intervention in the expression of angiotensin type 1 receptor (AT1R) should attenuate development of hypertension on a long-term basis in SHR. A retroviral-mediated AT1R antisense cDNA gene delivery system (LNSV-AT1R-AS) was used to test this hypothesis and to compare its BP-lowering effects with those of losartan. Introduction of LNSV-AT1R-AS into 5-day-old Wistar-Kyoto rats and SHR resulted in a robust expression of AT1R antisense (AS) within 3 days and persisted for at least 30 days. This expression was associated with a selective attenuation of high BP in SHR by 25 to 30 mm Hg. Although basal lowering of BP was exclusive to SHR, the angiotensin II (Ang II) pressor response was significantly reduced in all LNSV-AT1R-AS-treated rats. The decreased response to Ang II was associated with a similar attenuation of Ang II-induced dipsogenic responses in both strains of rats. The BP-lowering effects of LNSV-AT1R-AS treatment and losartan treatment were similar and primarily observed in SHR. However, the antihypertensive effect lasted less than 24 hours in losartan-treated SHR compared with 90 days in LNSV-AT1R-AS-treated SHR. In addition, losartan was unable to further lower BP in LNSV-AT1R-AS-treated SHR. Collectively, these results suggest that both losartan and LNSV-AT1R-AS treatment produces an antihypertensive response selectively in SHR that is mediated by interruption of AT1R function. However, a single, acute genetic treatment with LNSV-AT1R-AS can result in long-term control of high BP at a similar level of effectiveness as losartan, without altering plasma Ang II levels.
To demonstrate potential therapeutic effects of kallikrein gene delivery, we delivered adenovirus (Ad.CMV-cHK) carrying the human tissue kallikrein gene into two-kidney, one-clip Goldblatt hypertensive rats. A single intravenous injection of the recombinant adenovirus caused a delay of blood pressure increase that began 1 day after injection and continued for 24 days. A maximal blood pressure reduction was observed in rats receiving kallikrein gene delivery compared with control rats receiving Ad.CMV-LacZ (160+/-5 versus 186+/-7 mm Hg, n=6, P<.01). The expression of human tissue kallikrein mRNA was identified in the kidney, heart, aorta, and liver of rats receiving kallikrein gene delivery. Immunoreactive human kallikrein levels were measured in rat serum and urine in a time-dependent manner. Adenovirus-mediated kallikrein gene delivery caused a significant reduction in the left ventricular mass and cardiomyocyte size, as well as an increase in renal blood flow, urine flow, glomerular filtration rates, electrolyte output, and urine excretion. Enhanced renal responses were accompanied by significant increases in urinary kinin, nitrite/nitrate, and cyclic GMP levels. These findings show that the expression of human tissue kallikrein via gene delivery has protective effects against renovascular hypertension and cardiovascular and renal dysfunction.
We investigated 17 children with nephrotic syndrome (NS) of early onset (14 aged < 1 year) and rapid progression to end-stage renal disease for the presence of mutations in the Wilms' tumor suppressor gene WT1 on chromosome 11. In eight children (7 genotypic males) an association with Wilms' tumor and/or ambiguous genitalia (Denys-Drash syndrome) was observed. In these eight and two additional female patients with NS only constitutional missense mutations in the WT1 gene were detected; four children presented the so-called hot spot mutation in exon 9 (R394N) and six had different mutations in exons 8 and 9 (4 not previously described). Renal biopsy showed diffuse mesangial sclerosis in eight and focal segmental sclerosis in two cases. End-stage renal disease was reached either concomitantly or within four months after onset of NS in seven of ten patients. A unilateral Wilms' tumor was found before or concomitant with NS in four children (3 males, 1 female). From the seven genotypic males with WT1 mutations, five presented ambiguous genitalia and two a female phenotype. No mutation of the WT1 gene was found in seven other children with isolated congenital or infantile NS with or without DMS who appeared to have a slower progression than the first group. It is proposed that patients with early onset, rapidly progressive NS and diffuse mesangial or focal segmental sclerosis should be tested for WT1 mutations to identify those at risk for developing Wilms' tumor.
Congenital nephrotic syndrome of the Finnish type (NPHS1) is an autosomal-recessive disorder, characterized by massive proteinuria in utero and nephrosis at birth. In this study, the 150 kb critical region of NPHS1 was sequenced, revealing the presence of at least 11 genes, the structures of 5 of which were determined. Four different mutations segregating with the disease were found in one of the genes in NPHS1 patients. The NPHS1 gene product, termed nephrin, is a 1241-residue putative transmembrane protein of the immunoglobulin family of cell adhesion molecules, which by Northern and in situ hybridization was shown to be specifically expressed in renal glomeruli. The results demonstrate a crucial role for this protein in the development or function of the kidney filtration barrier.
The purpose of this experiment was to determine body fluid distribution during chronic cold exposure and to further understand the mechanism of cold-induced hypertension. Blood pressures, hematocrit, and the plasma, blood, and extracellular fluid volumes were measured in rats at intervals of 1, 3, and 5 weeks after exposure to cold (5 degrees C). Resting systolic, diastolic, and mean blood pressures measured by direct arterial cannula were significantly elevated in a time-dependent manner over the duration of cold exposure. The increase in diastolic blood pressure, which reflects the peripheral vascular resistance, exceeded that of systolic blood pressure after both 3 and 5 weeks of exposure to cold. Pulse pressure was significantly decreased by 3 and 5 weeks of cold exposure. The plasma, blood, and extracellular fluid volumes were significantly increased after both 1 and 3 weeks of exposure to cold, but had returned to control levels by 5 weeks of cold exposure. Cold exposure, however, did not affect the hematocrit. The 2-h water intake after the cold-exposed rats were returned to warm (25 degrees C) (thermogenic drinking) was significantly increased compared to that of warm-acclimated rats during the first, third, and fifth week of exposure to cold. The present results suggest that the development of cold-induced hypertension is associated with blood volume expansion, and that the elevated blood pressure is maintained by increased peripheral vascular resistance without blood volume expansion. The results also imply that exposure to cold induces a dehydration in rats.
Denys-Drash and Frasier syndromes are rare human disorders that associate nephropathy with gonadal and genital abnormalities. In DDS there is a predisposition to Wilms' tumor. Heterozygous point mutations in the Wilms' tumor, type1 gene (WT1), particularly those altering the zinc finger (ZF) encoding exons, have been reported in most DDS patients, while mutations in intron 9 of the same gene cause FS. This paper describes two cases of DDS, one FS and one patient with Wilm's tumor and intersex genitalia, in which mutations were searched by sequencing the exons 8 and 9 of WT1 gene. Patient 1 carried a missense point mutation in exon 8 (ZF2), converting a CGA-Arg codon to a TGA-stop codon. Patient 2 presented a single nucleotide deletion within exon 9 (ZF3) introducing a premature chain termination at codon 398. Patients 3 and 4 had a C-->T transition at position +4 of the second alternative splice donor site of exon 9 (this mutation was detected in peripheral blood and in tumor derived DNA of patient 3). However, patient 3 had previously developed a Wilms' tumor. This is the first case of Wilms' tumor development in a phenotypically and genetically confirmed case of FS.
A.B.K. is funded by a National Kidney Research Fund Twistington Higgins Fellowship. We would like to thank the renal units throughout the United Kingdom, which kindly provided patients and clinical information for this study.
Focal segmental glomerulosclerosis (FSGS) is a pathological entity characterized by proteinuria, nephrotic syndrome, and the progressive loss of renal function. It is a common cause of end-stage renal disease (ESRD). Recently, familial forms of FSGS have been identified. Two families with autosomal dominant FSGS were evaluated for linkage using 351 genomic microsatellite markers. Linkage, multipoint analysis, and tests for heterogeneity were performed on the subsequent results. In addition, three small families were used for haplotype analysis. Evidence for linkage was found on chromosome 11q21-q22 for the largest family, with a maximum lod score of 9.89. The gene is currently localized to an 18-cM area between flanking markers D11S2002 and D11S1986. The disease in a second family was not linked to this locus or to a previously described locus on chromosome 19q13. There were no shared haplotypes among affected individuals in the three smaller families. Our findings demonstrate that genetic heterogeneity is prevalent in FSGS in that at least three genes cause the FSGS phenotype. Identification of the genes that cause familial FSGS will provide valuable insights into the molecular basis and pathophysiology of FSGS.
The podocyte is a highly specialized kidney cell that is essential to the ultrafiltration of blood, resulting in the extraction of urine and the retention of protein. Four studies, the most recent of which is published in this issue of Nature Genetics, indicate its critical role in the pathology of nephrotic syndrome.