Large homozygous deletions of the 2q13 region are a major cause of juvenile nephronophthisis
Large homozygous deletions of the 2q13 region are a major cause of juvenile nephronophthisisMartin Konrad+, Sophie Saunier+, Laurence Heidet, Flora Silbermann, France Benessy, Joaquim Calado, Denis Le Paslier1, Michel Broyer2, Marie-Claire Gublerand Corinne Antignac*
INSERM U 423, Hôpital Necker-Enfant Malades, 75743 Paris Cedex 15, France, 1CEPH, 27 rue Juliette Dodu, 75010 Paris, France and 2Service de Néphrologie, Hôpital Necker-Enfants Malades, Université René Descartes, 75743 Paris Cedex 15, France
Received November 3, 1995;Revised and Accepted January 4, 1996
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.
Familial juvenile nephronophthisis (NPH) or recessive medullary cystic kidney disease is an autosomal recessive kidney disorder leading to end-stage renal disease during childhood or adolescence. The underlying pathology is a chronic tubulo-interstitial nephropathy with characteristic tubular basement membrane thickening and medullary cyst formation (1 ). NPH represents the most frequent inherited cause of chronic renal failure in children (2 ). Associations with various extrarenal symptoms, especially ocular lesions, are frequently observed (reviewed in ref. 3 ). By means of linkage analysis, it has been shown that a gene (NPH1), responsible for the vast majority (approximately 85%) of the purely renal form of NPH, maps to chromosome 2q13 (4 -6 ). By haplotype analysis we have identified two DNA markers flanking the NPH1 gene at loci D2S1890 and D2S1888 (7 ). This interval of approximately 2 cM and the surrounding region was cloned in a yeast artificial chromosome (YAC) contig. The region turned out to be partially duplicated on chromosome 2p12. Furthermore, several markers mapped to more than one locus within the NPH1 region on 2q13 suggesting the presence of low copy repeats (Fig. 1 A).
No abnormal patterns were observed in any of the patients for the polymorphic markers which were tested initially (D2S1890, D2S340, D2S1889, D2S1891, D2S1893 and D2S1888) (7 ). In a second attempt, we tested probands from 13 inbred families for 10 non-polymorphic markers mapping to the critical NPH1 interval between D2S1890 and D2S1888 (Fig. 1 A). These markers were obtained mainly by YAC end sequencing during the contig construction (7 ) or by screening DNA sequence databases for D2S1735 and WI5972. No amplification product was detected using primers generated from the left insert end of YAC 765F2 (765F2L) (Table 1 ) in affected individuals from 11 of the 13 consanguineous families. This suggested a rearrangement of both mutant chromosomes, encompassing, at least, part of the NPH1 gene. We subsequently tested individuals from 13 non-consanguineous NPH1 multiplex-families and 23 sporadic cases. In 10 of 13 and 15 of 23 of these sets, respectively, no PCR amplification product was obtained with the 765F2L primers in affected children (Fig. 2 ). This is consistent with the expected frequency of rearrangements deduced from the results obtained in consanguineous families. The lower proportion of rearrangements in sporadic cases is in agreement with the 10-15% of NPH patients unlinked to NPH1. In contrast, an amplification product was detected in all parents (n = 61), all healthy sibs (n = 41), in a large cohort of control subjects (n = 203), and in the NPH patients belonging to the four families unlinked to NPH1. To study the extent of these rearrangements, we tested flanking markers already mapped to the region (876H12L, 921H4R, WI5972, 851H5R) (7 ) as well as additional markers generated by vector-arm PCR (804H10R) or inter-Alu PCR (804/6) (Table 1 ) by PCR on patients' DNA. In all patients lacking the 765F2L amplification product, the 804/6 band was also absent. In only one patient did the rearrangement also encompass the marker 804H10R. All other markers revealed normal amplification products.
Using the purified 765F2L amplification product from normal individuals as a probe for Southern blot analysis, 7.0 and 4.8 kb EcoRI fragments were detected in the genomic DNA in all healthy controls (n = 50) and unaffected family members (n = 45) tested. In contrast, the 4.8 kb band was found to be absent in all affected individuals from 15 families who had no amplification product for 765F2L (Fig. 3 ). As hybridization to a cell hybrid panel revealed that the 7.0 kb fragment is located on chromosome 1 and the 4.8 kb on chromosome 2 (data not shown), a simple polymorphism could be ruled out. Thus, the absent band demonstrates the presence of a homozygous deletion.
To elucidate the physical organization of NPH1 candidate region, we performed long-range restriction mapping of the YACs covering the region using PFGE and field inversion gel electrophoresis (FIGE). We used the restriction enzymes SalI, SacII, SfiI and ClaI for single and double digestions. The markers 921H4R and WI5972 which were thought to lie telomeric to 765F2L after STS content mapping, turned out to flank the deleted region on both sides indicating a duplication. The repeats were distinguished by the detection of two restriction fragments of different sizes for markers WI5972, 921H4R and 769G7R in YACs covering the whole region (876H12, 879D3 and 804H10), whereas only one fragment was detected in YACs distal to the 804/6 marker (765F2, 769G7 and 921H4). The positions of the restriction sites and markers provided evidence for a large inverted duplication of approximately 100 kb (Fig. 1 B).
To determine the size of the deletions, genomic DNA from 18 unrelated patients (13 of whom carry the homozygous deletion) and six controls was digested with NotI and separated by PFGE. A 790 kb fragment was detected, containing 804/6 and 765F2L (which are deleted in the patients) as well as the flanking markers 804H10R and 921H4R, in normal controls (Fig. 4 ). This enabled us to characterize the size of the deletion. A fragment length difference of approximately 250 kb was observed between the 13 patients already known to be deleted by PCR or Southern blot analysis versus controls and patients who were not thought to be deleted (Fig. 4 ). In addition, three patients were found to be heterozygous for the deletion. Even though a larger deletion of one of the alleles could not be completely ruled out, this possibility seems unlikely, as the PCR analysis did not detect a larger deletion in the homozygous state for the flanking markers in any of the patients. Hybridization of patients' DNA after SfiI digestion with the 921H4R probe revealed the absence of the normal 65 kb fragment in all the 11 deleted patients tested. In contrast, the normal 95 kb fragment was detected in all patients (data not shown). This indicates that the telomeric copy of the duplication is at least partially missing.
This study demonstrates large homozygous deletions to be responsible for the majority of cases of juvenile NPH without extrarenal manifestations. The sizes of the deletions seem to be very similar in most patients (approximately 250 kb). Additionally, the deletions involve, at least partially, the telomeric copy of an inverted duplication. Assuming that the breakpoints of these deletions are identical (they have not yet been cloned), this unusual high deletion rate might be due to a founder effect. A common haplotype did not emerge significantly from haplotype analysis of the flanking markers in the NPH outbred population (in 26 NPH1 families; data not shown). However, the possibility of a common ancestor cannot be definitely ruled out, as the two closest polymorphic markers flanking the deletion are, at least, 1 Mb apart. It has been shown that, even in a homogeneous population, no significant linkage disequilibrium could be demonstrated for loci located, respectively, 900 and 1600 kb away from the disease gene (9 ). It is more likely that these rearrangements occurred independently, due to a common mechanism. There are examples of human chromosomal rearrangements in which low copy repeats or duplicated regions predispose to deletion and/or duplication events (8 ). However, these reports almost always concern autosomal dominant or X-linked disorders. Large rearrangements as a common disease-causing mechanism, have been reported for instance on chromosome 17p where a duplication results in Charcot-Marie-Tooth disease type 1A (10 ); whereas patients with a deletion of the same region have hereditary neuropathy with liability to pressure palsies (11 ). Large molecular deletions have also been identified in more than 85% of patients with X-linked ichthyosis (12 ). Large-scale rearrangements have rarely been shown to be a common cause of autosomal recessive diseases. In familial growth hormone deficiency type 1A homozygous deletions have been described (13 ) and `hot spots' for recombination leading to these deletions have been identified (14 ). However, these rearrangements (deletions of 6.7 or 7.6 kb) were found in only 10 of 78 patients (15 ). Furthermore, all the deletions detected in that study were in consanguineous families. In spinal muscular atrophy (SMA), large deletions have been described in a small proportion (seven of 201) of families (16 ), but a marked heterozygosity deficiency indicates deletions in at least 18% of patients with Werdnig-Hoffmann disease (SMA type I). These findings contributed considerably to the identification of two genes, implicated in SMA (17 ,18 ). These genes were subsequently found to be at least partially deleted in a homozygous state in most of the patients studied.
Within the context of repeated sequences triggering rearrangements, it has to be noted, that the telomere-telomere fusion point of the two ancestral ape chromosomes, from which the human chromosome 2 arose (19 ), has been located to the band 2q13 (20 ). This fusion point is organized as an inverted array of TTAGGG repeats, which are typical of human telomeres (21 ). As these telomere-like repeats are extremely unstable and therefore probably prone to breakage, fragility and recombination (22 ) there could be a link between this ancestral fusion and the instability of the NPH1 region. A fine physical map of this region should allow the mapping of this fusion point with respect to the NPH1 locus.
To date, the mechanism for the deletions in NPH is difficult to explain. High-frequency large deletions clustered between low copy repetitive sequences and involving parts of the repeats have been described in the human steroid sulfatase gene, but they result from recombination between repeats oriented in the same direction (12 ). In NPH, the inverted repeats flanking the deletion region might potentiate the formation of a stem-loop structure on the same DNA strand, as suspected in deletions occurring between inverted Alu sequences (23 ). In this model, staggered breakpoints could explain why the distal copy of the repeat is deleted, at least partially. More detailed mapping and sequencing will help in the understanding of the mechanism for these deletions.
This study underlines the usefulness of a systematic search for large scale rearrangements in regions containing low copy repeats, even in recessive traits. The further characterization of these rearrangements will contribute to the isolation of the disease-causing gene or genes in NPH.
The detection of these deletions is also of great clinical interest, as they will contribute to the diagnosis of the most frequent cause of chronic renal failure in children, without invasive diagnostic procedures such as kidney biopsy in sporadic cases, and enable the unequivocal identification of children at risk in NPH1 families.
The polymorphic microsatellite markers mentioned in Figure 1 A were amplified using conditions as described in ref. 7 . Southern blots were used according to standard laboratory protocols.
YAC end sequences were amplified by anchored PCR using the primers described by Kere et al. (24 ). PCR products were directly sequenced after purification or cloned using the TA cloning kit (Invitrogen, San Diego, CA, USA) and subsequently sequenced on an automated sequencer (Applied Biosystems, Foster City, CA, USA) after plasmid DNA isolation (RPM-Kit, Vista, CA, USA). Primer sequences and annealing temperatures for the STSs shown in Figure 1 B are taken from ref. 7 or are listed in Table 1 for new markers.
Inter-Alu PCR was performed with the primers as described (25 ) following DNA preparation and amplification conditions proposed by Lengauer et al. (26 ). PCR products were sequenced after cloning in the TA cloning kit.
Agarose embedded high molecular weight yeast or human DNA was digested with SalI, SacII, SfiI, ClaI and NotI. Double digestions of DNA from YAC clones with SacII/ClaI, SacII/SalI, ClaI/SfiI, SalI/SfiI were also performed. Fragments were separated through 1% agarose gels in 0.5*TBE buffer using a CHEF DRII electrophoresis system (Biorad, Richmond, CA, USA) for large fragments at the following conditions: Fragments >1 Mb: 200 V with a 60-120 s switch time for 24 h; fragments with a size of 0.1-1 Mb: 200 V with 10-40 s switch time (24 h). For details see ref. 27 . Small fragments (5-100 kb) were separated by field inversion gel electrophoresis (FIGE Mapper, Biorad, Richmond, CA, USA) under the following conditions: forward 180 V; reverse 120 V with 0.1-2 s switch time for 16 h at 20oC. Filters were hybridized as described (27 ) either with the purified STS amplification products (804H10R, 804/6, 765F2L, 921H4R and WI5972), or with the cloned YAC end product, when primers were not available (769G7R).
We greatfully acknowledge J. Melki for providing the cell hybrid panel and for helpful discussion. We thank E. Boye for the critical review of the manuscript. This study was supported by INSERM, the Association Française contre les Myopathies, the Groupement de Recherches et d'Etudes sur les Génomes (GREG), the Assistance Publique-Hôpitaux de Paris, the Association Française Retinitis Pigmentosa, and the Association pour l'Utilisation de Rein Artificiels.
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*To whom correspondence should be addressed
+The two first authors contributed equally to this work
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