A systematic approach to mapping recessive disease genes in individuals from outbred populations.

The identification of recessive disease-causing genes by homozygosity mapping is often restricted by lack of suitable consanguineous families. To overcome these limitations, we apply homozygosity mapping to single affected individuals from outbred populations. In 72 individuals of 54 kindred ascertained worldwide with known homozygous mutations in 13 different recessive disease genes, we performed total genome homozygosity mapping using 250,000 SNP arrays. Likelihood ratio Z-scores (ZLR) were plotted across the genome to detect ZLR peaks that reflect segments of homozygosity by descent, which may harbor the mutated gene. In 93% of cases, the causative gene was positioned within a consistent ZLR peak of homozygosity. The number of peaks reflected the degree of inbreeding. We demonstrate that disease-causing homozygous mutations can be detected in single cases from outbred populations within a single ZLR peak of homozygosity as short as 2 Mb, containing an average of only 16 candidate genes. As many specialty clinics have access to cohorts of individuals from outbred populations, and as our approach will result in smaller genetic candidate regions, the new strategy of homozygosity mapping in single outbred individuals will strongly accelerate the discovery of novel recessive disease genes.

Mutations in PLCE1 are a major cause of isolated diffuse mesangial sclerosis (IDMS).

BACKGROUND AND OBJECTIVES: Diffuse mesangial sclerosis (DMS) is a histologically distinct variant of nephrotic syndrome (NS) that is characterized by early onset and by progression to end-stage kidney disease (ESKD). Besides syndromic DMS, isolated (non-syndromic) DMS (IDMS) has been described. The etiology and pathogenesis of DMS is not understood. We recently identified by positional cloning recessive mutations in the gene PLCE1/NPHS3 as a novel cause of IDMS. We demonstrated a role of PLCE1 in glomerulogenesis. Mutations in two other genes WT1 and LAMB2 may also cause IDMS. We therefore determine in this study the relative frequency of mutations in PLCE1, WT1 or LAMB2 as the cause of IDMS in a worldwide cohort. METHODS: We identified 40 children from 35 families with IDMS from a worldwide cohort of 1368 children with NS. All the subjects were analyzed for mutations in all exons of PLCE1 by multiplex capillary heteroduplex analysis and direct sequencing, by direct sequencing of exons 8 and 9 of WT1, and all the exons of LAMB2. RESULTS: The median (range) age at onset of NS was 11 (1-72) months. We detected truncating mutations in PLCE1 in 10/35 (28.6%) families and WT1 mutations in 3/35 (8.5%) families. We found no mutations in LAMB2. CONCLUSIONS: PLCE1 mutation is the most common cause of IDMS in this cohort. We previously reported that one child with truncating mutation in PLCE1 responded to cyclosporine therapy. If this observation is confirmed in a larger study, mutations in PLCE1 may serve as a biomarker for selecting patients with IDMS who may benefit from treatment.

NPHS3: new clues for understanding idiopathic nephrotic syndrome.

Hereditary forms of childhood nephrotic syndrome (H-CHNS) have long been counted as rare variants of steroid-resistant nephrotic syndrome (SRNS). This concept must be specified by two new findings: First, a study on nephrotic syndrome manifesting in the first year of life documents that H-CHNS are actually the predominant cause of nephrotic syndrome in infants. Second, the recent identification of autosomal recessive nephrotic syndrome type 3 (NPHS3) caused by mutations in the phospholipase PLCE1 gene has, for the first time, shown steroid responsiveness in H-CHNS. NPHS3 is a severe form of isolated nephrotic syndrome with rapid progression to terminal renal failure. NPHS3 is caused by a developmental rather than structural podocyte dysfunction and is a major cause of diffuse mesangial sclerosis. Therapy response in NPHS3 is documented and could open insights into direct genomic and nongenomic effects of glucocorticoids on podocytes. The findings on NPHS3 support the idea that both clinical course and histology in H-CHNS are subject to genotypic variability and that mutational analysis is the most reliable diagnostic tool. Future studies are needed to determine the clinical implications of NPHS3. Identification of further variants of H-CHNS can be anticipated and may include steroid-responsive hereditary diseases.

Low prevalence of NPHS2 mutations in African American children with steroid-resistant nephrotic syndrome.

In African American (AA) children, focal segmental glomerulosclerosis (FSGS) is the leading cause of nephrotic syndrome (NS). It has been shown that AA children suffer from FSGS and steroid-resistant nephrotic syndrome (SRNS) at a higher frequency and with a more severe renal outcome in comparison with Caucasian children. Previous mutation analysis of large cohorts revealed that a high percentage of childhood SRNS is monogenic and that mutations in podocin (NPHS2) and Wilms' tumor gene 1 (WT1) account for approximately 30% of SRNS in children. To test whether AA children with SRNS have a similar or a higher mutation rate, we performed mutation analysis of NPHS2 and WT1 in a cohort of AA children with SRNS. Direct sequencing was carried out for all exons of NPHS2 and for exons 8 and 9 of WT1. We ascertained 18 children of AA descent in whom renal biopsy findings showed FSGS in 13 patients (72%) and minimal-change disease in five patients (28%). In both NPHS2 and WT1, no disease-causing mutations were detected. Our data strongly suggest that in AA children with SRNS, the frequency of NPHS2 mutations is much lower than in large cohorts of pediatric SRNS patients in the general population. Knowledge of mutation rate of NPHS2 in different populations of SRNS patients facilitates the physician in planning a suitable genetic screening strategy for patients.

Nephrotic syndrome in the first year of life: two thirds of cases are caused by mutations in 4 genes (NPHS1, NPHS2, WT1, and LAMB2).

OBJECTIVES: Mutations in each of the NPHS1, NPHS2, WT1, and LAMB2 genes have been implicated in nephrotic syndrome, manifesting in the first year of life. The relative frequency of causative mutations in these genes in children with nephrotic syndrome manifesting in the first year of life is unknown. Therefore, we analyzed all 4 of the genes jointly in a large European cohort of 89 children from 80 families with nephrotic syndrome manifesting in the first year of life and characterized genotype/phenotype correlations. METHODS: We performed direct exon sequencing of NPHS1, NPHS2, and the relevant exons 8 and 9 of WT1, whereas the LAMB2 gene was screened by enzymatic mismatches cleavage. RESULTS: We detected disease-causing mutations in 66.3% (53 of 80) families (NPHS1, NPHS2, WT1, and LAMB2: 22.5%, 37.5%, 3.8%, and 2.5%, respectively). As many as 84.8% of families with congenital onset (0-3 months) and 44.1% with infantile onset (4-12 months) of nephrotic syndrome were explained by mutations. NPHS2 mutations were the most frequent cause of nephrotic syndrome among both families with congenital nephrotic syndrome (39.1%) and infantile nephrotic syndrome (35.3%), whereas NPHS1 mutations were solely found in patients with congenital onset. Of 45 children in whom steroid treatment was attempted, only 1 patient achieved a lasting response. Of these 45 treated children, 28 had causative mutations, and none of the 28 responded to treatment. CONCLUSIONS: First, two thirds of nephrotic syndrome manifesting in the first year of life can be explained by mutations in 4 genes only (NPHS1, NPHS2, WT1, or LAMB2). Second, NPHS1 mutations occur in congenital nephrotic syndrome only. Third, infants with causative mutations in any of the 4 genes do not respond to steroid treatment; therefore, unnecessary treatment attempts can be avoided. Fourth, there are most likely additional unknown genes mutated in early-onset nephrotic syndrome.

Childhood pineoblastoma: experiences from the prospective multicenter trials HIT-SKK87, HIT-SKK92 and HIT91.

OBJECTIVE: To analyze the outcome of children with pineoblastoma (PB), treated within the prospective multicenter trials HIT-SKK87, HIT-SKK92 and HIT91 of German-speaking countries. PATIENTS: We report on 11 children suffering from PB. Five children younger than 3 years of age received chemotherapy after surgery until eligible for radiotherapy (HIT-SKK87 and HIT-SKK92). Five of six children older than 3 years were treated after surgery with immediate chemotherapy and craniospinal irradiation, and one child received maintenance chemotherapy after postoperative radiotherapy (HIT91). RESULTS: Five of the six older children are still alive in continuous complete remission (CCR) with a median overall survival (OS) and progression free survival (PFS) of 7.9 years. Five of these six HIT91 patients responded to postoperative chemotherapy and radiotherapy. The only patient with tumor progression during initial chemotherapy achieved complete remission with radiotherapy and is alive. In contrast, all five young children died of tumor progression after a median OS of 0.9 years (PFS 0.6 years). They had either metastatic disease (M1) and/or postoperative residual tumor. Response to postoperative chemotherapy was lower than in the older age group, and only one of these children received radiotherapy. CONCLUSIONS: Combined chemotherapy and radiotherapy were feasible and effective in the older age group, leading to prolonged remissions in five of six children. Tumor biology may be more aggressive in younger children with PB, who presented more frequently with high-risk features at diagnosis and had poorer response rates to neoadjuvant postoperative chemotherapy. More intensified treatment regimens may be needed for young children with PB.

Mutations in the Wilms' tumor 1 gene cause isolated steroid resistant nephrotic syndrome and occur in exons 8 and 9.

Primary steroid-resistant nephrotic syndrome (SRNS) is characterized by childhood onset of proteinuria and progression to end-stage renal disease. Approximately 10-25% of familial and sporadic cases are caused by mutations in NPHS2 (podocin). Mutations in exons 8 and 9 of the WT1 gene have been found in patients with isolated SRNS and in SRNS associated with Wilms' tumor (WT) or urogenital malformations. However, no large studies have been performed to date to examine whether WT1 mutations in isolated SRNS are restricted to exons 8 and 9. To address this question, we screened a worldwide cohort of 164 cases of sporadic SRNS for mutations in all 10 exons of the WT1 gene by multiplex capillary heteroduplex analysis and direct sequencing. NPHS2 mutations had been excluded by direct sequencing. Fifteen patients exhibited seven different mutations exclusively in exons 8 and 9 of WT1. Although it is possible that pathogenic mutations of WT1 may also reside in the introns, regions of the gene that were not able to be screened in this study, these data together with our previous results (Ruf et al.: Kidney Int 66: 564-570, 2004) indicate that screening of WT1 exons 8 and 9 in patients with sporadic SRNS is sufficient to detect pathogenic WT1 mutations and may open inroads into differential therapy of SRNS.