Characterization of large rearrangements in autosomal dominant polycystic kidney disease and the PKD1/TSC2 contiguous gene syndrome

Abstract

Large DNA rearrangements account for about 8% of disease mutations and are more common in duplicated genomic regions, where they are difficult to detect. Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in either PKD1 or PKD2. PKD1 is located in an intrachromosomally duplicated region. A tuberous sclerosis gene, TSC2, lies immediately adjacent to PKD1 and large deletions can result in the PKD1/TSC2 contiguous gene deletion syndrome. To rapidly identify large rearrangements, a multiplex ligation-dependent probe amplification assay was developed employing base-pair differences between PKD1 and the six pseudogenes to generate PKD1-specific probes. All changes in a set of 25 previously defined deletions in PKD1, PKD2 and PKD1/TSC2 were detected by this assay and we also found 14 new mutations at these loci. About 4% of the ADPKD patients in the CRISP study were found to have gross rearrangements, and these accounted for about a third of base-pair mutation negative families. Sensitivity of the assay showed that about 40% of PKD1/TSC contiguous gene deletion syndrome families contained mosaic cases. Characterization of a family found to be mosaic for a PKD1 deletion is discussed here to illustrate family risk and donor selection considerations. Our assay improves detection levels and the reliability of molecular testing of patients with ADPKD.

Figure 1. Diagram showing the defined regions deleted in patients with the PKD1/TSC2-CGS

Upper part of the map shows: hybridization probes (open boxes); the SM6 microsatellite; relevant restriction sites for EcoRI (E), BamHI (B) and NruI (N); and centromeric (cen) and telomeric (tel) directions. Lower map shows the positions of PKD1, TSC2, and flanking genes in 16p13.3; transcribed telomeric above the line, and centromeric below; plus positions of MLPA probes (red arrowheads; tagged genes are in red). The intron/exon structures of PKD1 and TSC2 are shown at the bottom with positions of MLPA probes and the duplicated region of PKD1 indicated with a light blue bar. The deleted region detected by MLPA for each subject is shown in red (or green for the newly described cases) and in black for previous gel-based analyses (Table 1). The family identifier and the best estimate of the size of the deleted region is indicated. Mosaic cases are italicized. The large deletion in M29 that just has an ADPKD phenotype is shown at the top (blue for the MLPA analysis).

Figure 2. Examples of MLPA results from PKD1/TSC2-CGS cases

MLPA data for PKD1 (blue), TSC2 (orange), flanking probes (red), and control markers (green) is indicated, with details of specific probes at the top. The y axis shows copy number at the diploid (2) and haploid (heterozygous deleted; 1) level. The pedigree identifier is indicated and the deleted region shown with a black bar. Cases 8, 11, 12, 14, and 16 have previously been described (Table 1).

Figure 3. Analysis 2 PKD1 deletion mutations

(a) MLPA data showing PKD1 deletion cases (colors and markings as indicated for Figure 2). Further probes within and immediately 5′ to PKD1 were used to provide higher resolution analysis for these cases. P98, P95, 100001, 120395, and 393936 have been previously described (Table 1). (b) Family 301157 is a mosaic for a PKD1 deletion. (Top): MLPA showing deletion in two siblings, R296 and R1502, but not visible in the affected father, F301157. (Middle, left): PCR with primers in PKD1 exons 25 and 41 generated a 1.7 kb breakpoint fragment in R296, which is at a reduced level in F301157, and not in the wild type (WT). (Middle, right): Hybridization of a BamH1 digest with PKD1 cDNA probe 3A3 (exons 43–46). The wild-type fragment (14 kb) is reduced to 11kb due to the deletion (R296; Figure 4). The deleted product is seen at a low level in the father (F301157; arrowhead), indicating mosaicism. (Bottom): Pedigree showing no known PKD in the grandparents and seven siblings (two brothers and five sisters), and mosaicism indicated by shading in the father.

Figure 4. Diagram showing the regions deleted or duplicated in the PKD1 cases

Restriction sites for EcoRI (E) and BamHI (B), plus hybridization probes (open boxes) and centromeric (cen) and telomeric (tel) directions are indicated. The intron/exon structure of PKD1 (light blue bar indicates the duplicated area) and 3′ end of TSC2 are shown at the bottom, plus PKD1 flanking genes and positions of MLPA probes (red arrowheads). MLPA detected rearrangements are in red (or blue for novel changes) and black bars indicate disrupted regions determined by FIGE, PCR, and sequencing. Pedigree identifiers, plus deletion (duplication; dup) sizes, are indicated, with the mosaic case (F301157) italicized.

Figure 5. Analysis of PKD2 deletions

(a)MLPA analysis of patient with large deletion of PKD2 region (P96412) and case with deletion of exon 5 (M363). (b) LR-PCR with primers in exons 4 and 6 showed a ~2.8kb breakpoint fragment in M363 compared to 8.5kb in the wild type (WT).

Figure 6. Characterizations of mosaic deletions associated with the PKD1/TSC2-CGS

(a) MLPA analysis of mosaic PKD1/TSC2-CGS cases. Reduction in copy number appears less than 50% as all cells do not have the deletion (see Table 1 for estimate of level of mosaicism). Full deletion is indicated by a black bar and mosaic deletions with a blue bar. M7 and F13 are mosaic parental samples of cases with deletions (7 and 13, respectively). The M7 deletion is evident but the F13 deletion (previously estimated as present in 15% of alleles) is not seen by MLPA. Subjects 7, M7, 13, F13, 19, 20, and 22 have previously been described (Table 1). Cases 4 and 6 were described in the same publication, but not identified as mosaics. (b) PFGE of NruI-digested P974 DNA hybridized with CW9 showing a deleted fragment, ~60 kb smaller than normal present at a low level (arrowhead), indicating mosaicism.

Figure 7. Pedigree analysis of PKD1 rearrangement cases

(a) Family (100009) with deletion of PKD1 exon 1. (Top): MLPA analysis showing deletion of two probes located 5′ to exon 1 in three affected family members. (Bottom): EcoR1-digested genomic DNA hybridized with N54 shows a ~ 2.5 kb smaller deletion fragment in these cases. (b) Family 259940 has a duplication: (top) MLPA shows increased signal with the exon 18 probe and (bottom) LR-PCR from exons 15–21 reveals a ~250bp larger product in the patient. (c) MLPA in M143 showing deletion of exons 18–21 in two affected siblings. (d) Family M29 with large PKD1 deletion. (Top): MLPA of four siblings showed a deletion of 120–175 kb extending centromeric to PKD1 and including E4F1 but not ABCA3. Bottom: partial pedigree of this large family indicating a total of 15 siblings, 7 affected.