Eleven of the 28 PJS patients tested (39%) had germline LKB1 mutations detected by MLPA (table 1). Five patients had whole gene deletions, two had the promoter region and exon 1 deleted, and in one patient exon 8 was deleted. The remaining detected deletions, each in a single patient, involved the following: loss of exons 2–10; deletion of the promoter region and exons 1–3; and loss of part of the promoter region. No duplications of any part of the gene were detected. Twenty two healthy individuals were screened by MLPA and no copy number changes detected.
Table 1MLPA results for patients with LKB1 changes
For nine samples with deletions (75%), the appropriate region of the gene was sequenced in the patients concerned, so as to confirm the MLPA data by excluding heterozygosity at all reported SNPs. Two SNPs (rs3764640, rs3764641) lying near to exon 1 of LKB1 were found to be homozygous in the three patients with the promoter region and exon 1 deletions. Five SNPs (rs3795063, rs2075604, rs2075606, rs741764, and rs2075608), spread through LKB1 from intron one to intron eight, were homozygous in the five patients with loss of the entire copy of LKB1. Absence of polymorphic sites underlying the MLPA detection oligonucleotides was additionally confirmed using the latest version of dbSNP (BUILD 125, 29 September 2005). Given the original sample of 76 patients, we estimate the proportion of PJS cases with large deletions at 14% (11/76) These patients had no clinicopathological features which distinguished them from the other patients with or without LKB1 mutations in terms of family history, number of polyps, presence of pigmentation, or development of cancer (details not shown).
LKB1 is a highly conserved protein, especially within the kinase domain (codons 48–309), and deletion of the promoter region or single or multiple exons are all predicted to have profound effects on kinase activity. Exon 8 encodes amino acids 308–369, distal to the kinase domain, but functional effects of such C‐terminal mutations have been shown using in vitro assays. Sapkota et al20
found that phosphorylation of residue 431 was essential for cell growth suppression by LKB1 and Forcet et al21
showed that C‐terminal LKB1
mutations reduced activation of AMPK and diminished the ability of LKB1 to induce and maintain cell polarity.
Large scale germline deletions have very rarely been reported in PJS, probably because methods suitable for their detection have not been used. Jiang et al6
found one PJS patient with a deletion spanning exons 2–7 of LKB1
. One whole gene deletion and one genomic rearrangement (a ~2 kb deletion) has been reported in two PJS patients.19,22
used long range polymerase chain reaction to amplify from exon 3 to exon 8 of LKB1
in order to search for products of novel sizes, but found no changes in their PJS patients. However, this method would not have detected any of the cases in our study with whole gene deletions or deletion of the promoter region and exon 1.
The finding of relatively frequent, large scale germline LKB1
mutations in PJS patients strongly suggests that MLPA testing (or some suitable alternative) should be introduced into the diagnostic genetics laboratory. Our findings also have consequences for the likelihood that there exists a second uncharacterised PJS locus. Analysis of published LKB1
mutations shows that a mean of 59% of PJS cases (median
10%–100%) have harboured a germline mutation, which was detectable using standard DNA based screening methods such as single stranded conformational polymorphism (SSCP) analysis, conformation specific gel electrophoresis (CSGE), dHPLC, and direct sequencing of coding regions and intron–exon boundaries.1,6,7,9,10,18,23,24,25,26,27,28,29,30
In addition, we estimate that approximately 10% of cases appear to have mutations which are detectable using RNA based methods only.1,23,30
Therefore, allowing for a mutation detection rate of about 40% by MLPA in the remaining 30% of PJS cases, the frequency of PJS patients with detectable mutations becomes about 80–85%. Given that the mutation detection rate in familial PJS cases has been consistently higher in families than in isolated cases, it is likely that a proportion of the latter have only had a presumptive or possible diagnosis of PJS.
In our view, therefore, the proportion of unambiguous PJS cases with LKB1
mutations is sufficiently high for there to be no good case for a second PJS locus based on these data alone. During the preparation of this paper, another study on germline LKB1
reached identical conclusions. Seventeen of 34 PJS patients without small scale LKB1
mutations were found to have germline deletions by MLPA. The authors found that when only patients who met the clinical criteria for PJS were considered, the overall mutation detection rate was 94% (64% point mutations and 30% large deletions), very close to our estimate from this study. We therefore conclude that the remaining evidence for the existence of an uncharacterised minor PJS locus therefore comes solely from families whose disease is unlinked to LKB1