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. 2000 Mar;66(3):778-89.
doi: 10.1086/302819.

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

S Saunier  1 , J Calado, F Benessy, F Silbermann, R Heilig, J Weissenbach, C Antignac
Affiliations

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

S Saunier et al. Am J Hum Genet. 2000 Mar.

Abstract

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.

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Figures

Figure 1
Figure 1
Characterization of the genomic region surrounding the NPHP1 gene. A, Diagram of the sequenced region. The thick line represents the entirely sequenced interval, whereas the dashed thick line represents the partially sequenced region. BAC clones are represented as solid lines, and the BAC ends mapped to the region are shown as open rectangles. B, Restriction map of the NPHP1 region and YAC contig spanning the region. STS positions and restriction sites (F = SfiI; C = ClaI; S = SalI; M = MluI; Sc = SacII; N = NotI) are indicated by vertical lines. NotI sites used to define the extent of the deletion are shown in red. The markers in green and blue are localized within the 45-kb direct and the 330-kb inverted repeats, respectively, and the markers in red are those initially found to be deleted by PCR analysis of individuals with nephronophthisis. C, Schematic representation of the genomic region surrounding the NPHP1 gene. The shaded blue and green arrows represent, respectively, the inverted 330-kb repeats (named “330RI” and “330RII”) and the direct 45-kb repeats (named “45RI” and “45RII”). The NPHP1 gene is represented by an open box, and the direction of transcription is indicated by the small arrow above it. The red rectangle indicates the region around 804H10R in 330RI, deleted by the distal copy of the direct 45-kb repeat in 330RII.
Figure 2
Figure 2
Southern blot hybridization of SalI (a, b) and SfiI (c, d) digested DNA from five YACs spanning the NPHP1 region with 96G18BD (a, c) and 804H10R (b, d). Only a 165-kb SalI fragment and a 68-kb SfiI fragment were detected with the 804H10R probe in the two YACs covering the whole region (879D3 and 876H12), and no fragment was detected in the YACs covering only the distal copy of the inverted repeats (765F2 and 769G7), demonstrating that 804H10R escapes duplication.
Figure 3
Figure 3
SfiI-digested DNA of four YACs spanning the NPHP1 region successively hybridized with probes 57A (a), 183K24BD (b), R30A (c), and N123 (d). All markers are duplicated; two SfiI fragments are present in YACs covering the whole NPHP1 region (879D3 and 876H12), whereas only one fragment is detected in YACs covering only the telomeric part of the region (765F2 and 769G7), allowing us to distinguish both copies. Note that R30A and 183K24BD detect the same 80-kb telomeric fragment, and that 57A and N123 detect the same 85-kb telomeric fragment.
Figure 4
Figure 4
A, Schematic representation of the common NPHP1 deletion involving an homologous recombination between the 45-kb direct repeats and leading to a deletion of 290 kb; top, normal chromosome; bottom, deleted chromosome. B, Model of unequal recombination by chromosome misalignment followed by formation of a loop structure. C, Model of unequal recombination by unequal crossing-over between two different chromosomes, leading in theory to either a deletion (middle) or duplication (bottom) of the NPHP1 gene.
Figure 5
Figure 5
PFGE of NotI-digested DNA of 12 patients and a control individual hybridized with 183K24BD. Lane 1, control individual showing a normal ∼800-kb NotI fragment. Lanes 2–10, patients from group A with homozygous deletion of makers 765F2L and 804/6, as detected by PCR. An abnormal ∼510 kb NotI fragment is detected. Lane 11, patient from group C only deleted for 804H10R by PCR, with an apparently normal 800-kb NotI fragment. Lanes 12 and 13, patients from group A deleted for 765F2L and 804/6 with two distinct NotI fragments (∼510 kb and ∼550 kb).
Figure 6
Figure 6
PFGE of SfiI-digested DNA of 11 patients and a control individual successively hybridized with 96G18BD and 183K24BD. Hybridization with probes 96G18BD (a) and 183K24BD (b) reveals two SfiI fragments in the control individual (lane 1), 95-kb and 45-kb proximal fragments, and 80- and 65-kb telomeric fragments, respectively. Lanes 2–10, patients from group A with deletion of markers 765F2L and 804/6 by PCR. The normal proximal fragments detected by both probes are missing. Lanes 11 and 12, patients from group A with two distinct abnormal NotI fragments (fig. 5). Hybridization with 183K24BD reveals the normal 80-kb telomeric SfiI fragment.
Figure 7
Figure 7
A, Genomic SfiI map of the NPHP1 region when 804H10R is missing and replaced by an additional copy of 45RII. B, Reciprocal rearrangement leading to the duplication of 804H10R. C, Models for nonpathogenic DNA rearrangements mediated by mispairing of the inverted repeats and double homologous recombination (C) or by a crossing-over between the inverted repeats leading to an inversion of the whole NPHP1 region (C′), followed by a crossing-over, localized telomeric of the 804H10R marker (C″). Centromeric and telomeric copies of 57A and 183K24BD are indicated in orange and blue, respectively.
Figure 8
Figure 8
Southern blot analysis of PstI-digested DNA of control individuals hybridized with probe C40. Lanes 3, 5, and 7 show that the 7-kb fragment is more intense than the 8.7-kb fragment, suggesting that each individual has only one copy of 804H10R and three copies of 45RII. Lanes 8–10 show that the 8.7-kb fragment is more intense than the 7-kb fragment, suggesting the occurrence of the reciprocal event (i.e., three copies of 804H10R and one copy of 45RII) in these individuals. Lane 4 shows a control individual carrying a homozygous deletion of 804H10R as detected by PCR. Lanes 1, 2, and 6 depict control individuals showing two bands of the same intensity; therefore, they are not carrying a nonpathogenic rearrangement.
Figure 9
Figure 9
PFGE of NotI-digested DNA of a patient and her parents successively hybridized with 183K24BD (a) and 804H10R (b). Lane 3 depicts an affected individual. Lanes 1 and 2 depict the father and the mother of the patient, respectively. Each parent is heterozygous for the NPHP1 deletion, as shown by hybridization with 183K24BD, but the father lacks the 804H10R marker on the deleted allele. Furthermore, the abnormal NotI fragment detected by 183K24BD in the father (∼550 kb) is slightly larger than the abnormal fragment detected in the mother (∼510 kb), in agreement with the occurrence of a 290-kb deletion on a chromosome carrying the nonpathogenic rearrangement and, thus, two copies of the 45-kb repeat. This proves that the child bears the nonpathogenic rearrangement on her paternal allele in addition to the 290-kb deletion on both alleles, as was suggested by the SfiI pattern.
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References

Electronic-Database Information

    1. Online Mendelian Inheritance in Man (OMIM), http://www.ncbi.nlm.nih.gov/Omim (for familial juvenile nephronophthisis [MIM 256100])
    1. RepeatMasker program, http://ftp.genome.washington.edu/cgi-bin/RepeatMasker

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