Characterization of the NPHP1 locus: mutational mechanism involved in deletions in familial juvenile nephronophthisis.

Familial juvenile nephronophthisis is an autosomal recessive, genetically heterogeneous kidney disorder representing the most frequent inherited cause of chronic renal failure in children. A gene, NPHP1, responsible for approximately 85% of the purely renal form of nephronophthisis, has been mapped to 2q13 and characterized. The major NPHP1 gene defect is a large homozygous deletion found in approximately 80% of the patients. In this study, by large-scale genomic sequencing and pulsed-field gel electrophoresis analysis, we characterized the complex organization of the NPHP1 locus and determined the mutational mechanism that results in the large deletion observed in most patients. We showed that the deletion is 290 kb in size and that NPHP1 is flanked by two large inverted repeats of approximately 330 kb. In addition, a second sequence of 45 kb located adjacent to the proximal 330-kb repeat was shown to be directly repeated 250 kb away within the distal 330-kb repeat deleting the sequence tag site (STS) 804H10R present in the proximal copy. The patients' deletion breakpoints appear to be located within the 45-kb repeat, suggesting an unequal recombination between the two homologous copies of this smaller repeat. Moreover, we demonstrated a nonpathologic rearrangement involving the two 330-kb inverted repeats found in 11 patients and, in the homozygous state, in 2 (1.3%) control individuals. This could be explained by interchromosomal mispairing of the 330-kb inverted repeat, followed by double recombination or by a prior intrachromosomal mispairing of these repeats, leading to an inversion of the NPHP1 region, followed by an interchromosomal unequal crossover event. This complex rearrangement, as well as the common deletion found in most patients, illustrates the high level of rearrangements occurring in the centromeric region of chromosome 2.

A novel gene that encodes a protein with a putative src homology 3 domain is a candidate gene for familial juvenile nephronophthisis.

Familial juvenile nephronophthisis (NPH) is an autosomal recessive, genetically heterogeneous disorder, representing the most frequent inherited cause of chronic renal failure in children. One of the responsible loci, NPH1 , has been mapped to 2q13. The presence of large homozygous deletions of approximately 250 kb in the majority of affected patients allowed us to define a minimal deletion interval for NPH1 . A BAC contig covering this interval was established. Combination of large scale genomic sequencing, cDNA selection and computer-aided analysis led to the characterization of two transcriptional units. One encodes the already known BENE protein, and the other encodes a novel protein of at least 732 amino acids containing a putative src homology 3 domain. In two patients carrying the large deletion of the NPH1 region on only one allele, two mutations were detected in two independent exons of the novel gene. One consists of a single base deletion, causing a frameshift, and the other is a G-->A substitution in the consensus 5' splice donor site. Both mutations thus potentially generate null mutants. One of these mutations was found to segregate with the disease in the family, and the second appeared to be a de novo mutation. We therefore conclude that this novel gene is a strong candidate for NPH.

Large homozygous deletions of the 2q13 region are a major cause of juvenile nephronophthisis.

Juvenile nephronophthisis (NPH) is a genetically heterogeneous disorder representing the most frequent inherited cause of chronic renal failure in children. We recently assigned a gene (NPH1) to the 2q13 region which is responsible for approximately 85% of cases. Cloning this region in a yeast artificial chromosome contig revealed the presence of low copy repeats. Large-scale rearrangements were detected in 80% of the patients belonging to inbred or multiplex NPH1 families and in 65% of the sporadic cases. Surprisingly, these rearrangements seem to be, in most cases, large homozygous deletions of approximately 250 kb involving an 100 kb inverted duplication. This suggests a common genetic disease-causing mechanism, which could be responsible for the highest frequency of large rearrangements reported in an autosomal recessive trait. Our findings are also of major clinical interest, as they permit the diagnosis in the majority of sporadic cases without the need for kidney biopsy.

A 11 Mb YAC-based contig spanning the familial juvenile nephronophthisis region (NPH1) located on chromosome 2q.

A gene (NPH1) responsible for approximately 90% of the purely renal form of familial juvenile nephronophthisis, a progressive tubulo-interstitial kidney disorder, maps to human chromosome 2. We report the construction of a YAC-based contig spanning the critical NPH1 region and the flanking genetic markers. This physical map was integrated with a refined genetic map that restricted the NPH1 interval to about 2 cM; this interval corresponds to a maximum physical distance of 3.5 Mb. The entire contig covers 9 cM between the loci D2S135 and D2S121. The maximum physical distance between these two markers is approximately 11.3 Mb. Forty-five sequence-tagged sites, including six genes, have been located within this contig. PAX8, a member of the human paired box gene family, that is expressed in the developing kidney, was assigned outside the restricted NPH1 critical region and cannot therefore be regarded as a candidate gene. This set of overlapping clones represents a useful resource for further targeted development of genetic markers and for the characterization of candidate genes responsible for juvenile nephronophthisis.

Refined mapping of a gene (NPH1) causing familial juvenile nephronophthisis and evidence for genetic heterogeneity.

Familial juvenile nephronophthisis (NPH) is an autosomal recessive progressive tubulo-interstitial kidney disorder, responsible for 6-10% of end-stage renal failure in children, and is frequently associated with Leber amaurosis (termed Senior-Løken syndrome). The biochemical basis of NPH is unknown. We recently reported linkage of the purely renal form of NPH to three markers on chromosome 2. Our results also suggested the existence of genetic heterogeneity between NPH and SLS. To map this NPH gene more precisely, we have now tested the segregation of six new microsatellite markers and five additional families. Haplotype analyses show unequivocally that four NPH families are not linked to the chromosome 2 markers, although there are no clinical or pathological features discernible in these families that could separate them from the families linked to the chromosome 2 NPH locus (NPH1). This reveals genetic heterogeneity in the purely renal form of NPH. In situ hybridization of YAC clones isolated with two closely linked markers assigned the NPH1 region to 2q13. Furthermore, based on haplotype analysis and specific recombination events, the NPH1 gene has been placed between D2S293/D2S340 and D2S121, a genetic interval of about 5-7 cM.

Post-transcriptional regulation of transferrin receptor mRNA by IFN gamma.

IFN gamma inhibits the rise in transferrin receptor mRNA level which is normally observed when stationary WISH cells are stimulated to proliferate. This effect is not attributable to a change in the transcription rate of the transferrin receptor gene or in the cytoplasmic stability of the mRNA. The IFN gamma-induced reduction of the transferrin receptor mRNA content is already present at the nuclear level to an extent comparable to that observed in whole cells. Thus, IFN gamma does not impair the passage of this mRNA from the nuclear to the cytoplasmic compartment but probably interferes with a nuclear post-transcriptional event during the processing of the immature transferrin receptor mRNA. Two different levels of regulation of transferrin receptor mRNA have been previously reported. Iron modulates the cytoplasmic stability of this mRNA through the binding of a specific cytoplasmic factor, whereas cell growth variation influences the transcription of this gene. Our results suggest the existence of another mechanism of regulation for transferrin receptor gene expression not so far considered. Furthermore, the distinction between the mechanism of regulation exerted by IFN gamma and that exerted by cell proliferation on transferrin receptor gene expression suggests that, in WISH cells, the IFN-induced transferrin receptor decay is not a consequence of cell growth arrest but rather one of the causes of the antiproliferative effect of IFN through iron deprivation.

Reduction of transferrin receptor expression by interferon gamma in a human cell line sensitive to its antiproliferative effect.

Interferon gamma (IFN gamma) reduced 125I-transferrin binding to WISH cells which are sensitive to its antiproliferative effect. IFN gamma did not affect transferrin binding to Daudi cells or phytohemagglutinin-stimulated human lymphocytes, neither of which respond to its antigrowth action. Scatchard analyses of the equilibrium binding of 125I-transferrin to WISH cells exposed to IFN gamma revealed a decrease in the number of cell surface receptors but no change in the apparent association constant compared with control cells. When 125I-transferrin binding was measured using detergent-extracted cells, the IFN-induced reduction of binding was smaller than with intact cells. This suggests that in WISH cells, IFN gamma not only reduced the total number of transferrin receptors, but also modified the process of receptor internalization and recycling. Labeling of newly synthesized receptors with [35S]-methionine indicated that a reduction in the biosynthesis might account for the decrease in the total number of transferrin receptors in IFN gamma-treated cells. Our results suggest that the antigrowth effect of IFN gamma is at least partly due to its inhibitory action on transferrin receptor expression leading to iron starvation.

Relationship between inhibition of cell growth and of transferrin receptor expression by interferon (IFN) alpha: studies in IFN-sensitive and IFN-resistant Daudi cells.

We previously showed that treatment of different cell lines with interferon-alpha (IFN-alpha) concurrently inhibited both cell growth and the rise observed in 125I-labelled transferrin binding when cells are exposed to culture conditions that stimulate proliferation. To gain insight into the relationship between these two IFN-induced inhibitory processes, we investigated the effect of IFN-alpha on the binding of 125I-labelled transferrin to Daudi cells sensitive or resistant to its antiproliferative action. We found a close correlation between the ability of IFN-alpha to inhibit cell growth and to inhibit transferrin receptor expression. Since growth inhibition induced by other agents is not always accompanied by an inhibition of transferrin receptor expression, the previous and present observations suggest that the inhibitory effect of IFN on this expression is at least one of the mechanisms by which IFN inhibits cell proliferation. We also observed that IFN-alpha did not modify transferrin receptor biosynthesis in IFN-sensitive Daudi cells, suggesting that IFN-alpha may change the processing of the transferrin receptor molecules, making them unable to bind transferrin.