Analysis of PKD1 for genomic deletion by multiplex ligation-dependent probe assay: absence of hot spots

Abstract

Autosomal dominant polycystic kidney disease is largely due to mutations in PKD1. PKD1 has an unusual genomic structure, including a 2.5-kb polypyrimidine sequence in intron 21, which has been postulated to lead to a high rate of spontaneous genomic mutation events. In addition, the majority of the gene is duplicated three to six times at 97-99% identity elsewhere in the genome. To identify genomic mutations in PKD1, we developed a multiplex ligation-dependent probe assay (MLPA) in which sites of variation between PKD1 and its copies were positioned at the ligation sites of the MLPA probe sets. Thirteen probe sets covered PKD1 exons 2 through 46, at an average spacing of 2.5 kb. Analysis of 27 independent PKD patient samples showed no evidence for genomic deletions confined to PKD1. Analysis of 15 tuberous sclerosis patient samples in which deletions in TSC2 extended into PKD1 showed no evidence of clustering of breakpoints near the polypyrimidine tract.

Figure 1. Genomic structure of PKD1 and strategy for probe design in duplicated regions

A. Genomic structure of PKD1, according to UCSC genome browser (Mar 2006), with 1^st (e1) and last (e46) exons indicated. Black and yellow bars indicate PKD1 fragments duplicated elsewhere in the genome at 97-98% and 98-99% identity, respectively. B. Sequence alignment for PKD1 exon 25 and 6 duplicated copies of that exon located elsewhere on chromosome 16. Blue and red colors highlight matching and discordant nucleotides. Top, 5′ and 3′ half-probes are shown. Note that one of the nucleotides at the ligation position (position - 1 in the 5′ half-probe, A) is present only in the PKD1 copy, not in the duplicates, by our design (additionally also nucleotide at position -9 is PKD1 specific).

Figure 2. Map of all PKD1 deletions

A. Map of PKD1 and flanking regions. Exons are indicated by black vertical lines of width proportional to exon size. Thin blue lines indicate the position of PKD1 MLPA probes. The duplicated regions are indicated by the red line. B. Deletion map. Each deletion mutation in PKD1 is represented by a horizontal black/gray line indicating its extent, as mapped previously in TSC2 (18) and in PKD1, the current work. The black line indicates the minimum deleted region, and gray the possible extent to the next undeleted probe. For two mutations the breakpoints were identified by LRPCR/sequencing. C. Cumulative frequency of breakpoints within PKD1 as a function of linear distance. The position of mutations was assigned to the midpoint of the distance between the last probe showing deletion and the first probe not showing deletion. Note that breakpoints are distributed fairly evenly across PKD1 with none in introns 21 or 22, highlighted in yellow background. Intron 21 contains the polypurine-polypyrimidine tract.

Figure 3. Electropherograms and normalized peak height graphs for PKD1 MLPA

A. Electropherograms of PKD1 MLPA. A control sample is shown at top. The sample below has a deletion encompassing the entire PKD1 gene, with all peaks of reduced height indicated with *. The location of each probe within the gene is shown: P1, PKD1; T2, TSC2; P2, PKD2; e, exons; control, control probes; i, intron; 5′flank, 5′ flanking probe. The size of the amplified products are 90 - 154 nucleotides. B. Normalized peak height graphs for 8 DNA samples. Each bar represents the normalized peak height for the probe indicated on the x axis, with control probes first, then PKD1 probes, then a TSC2 probe, then two PDK2 probes. The heavy black lines indicate probes with reduced signal. Upper left is a control sample; next is an apparent single exon deletion, that proved to be a single nucleotide deletion under the probe. The remainder are all deletions extending from TSC2 variable distances into the PKD1 gene.