Results and discussion

Of the 12 PJS patients, three were sporadic and nine were familial cases. Each patient had a personal or family history of both pigmentation and polyps which were of a type characteristic of PJS. Germline LKB1 mutations were found in seven (58%) of the 12 cases (table 2), in exons 1, 2, 4, 6, and 9. No bias towards location of mutations in exons 1 or 6 was found. Examples of mutations are shown in fig 1. None has been reported previously. Five of the mutations are predicted to lead to a truncated protein (two nonsense changes and three frameshifts). Of these five mutations, two are identical frameshifts. A sixth mutation is an in frame deletion of 6 bp and a seventh mutation is a missense change (lysine→arginine), both of these occurring within the LKB1 kinase core. Linkage of disease to the D19S886 marker (less than 0.2 Mb from LKB1, http://www-bio.llnl.gov/) and cosegregation of mutations with disease were observed in those families (of patients 16, 20, and 21) from which suitable samples were available.

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Figure 1

Examples of germline LKB1 mutations in Peutz-Jeghers patients. Mutations are arrowed. See table 2 for details of mutations shown. As per standard ABI sequencing analysis format, blue=C, black=G, red=T, green=A. (A) Patient 20 (forward sequence). CCCCCCGCTCTCTG (wt), CCCCCGCTCTCTGA (mutant). (B) Patient 2 (forward sequence). CGGCACAA (wt), CGGCATGT (mutant). (C) Patient 16 (forward sequence). Note the visible, but weak “wild type” C peak at the mutation site. AAGGACAT (wt), AAGGAATC (mutant). (D) Patient 6 (forward (left) and reverse (right) sequences are shown). TTACG (wt, forward), CGTAA (wt, reverse). TTAAG (mutant, forward), CTTAA (mutant, reverse).

Five of the eight mutations are worthy of particular comment. The identical frameshift mutations found in two families (patients 20 and 21) produced a stop at codon 283 and occurred in a poly(C) tract at a site of potential slippage during DNA replication (table 2). These two families are not known to have any common ancestry. Nevertheless, both kindreds are from northern China and the disease is associated with the same D19S886 allele in both families (not shown). It is most likely, therefore, that these mutations have a common origin, although identical LKB1 mutations without evidence of a common origin have been reported previously.5 6

Another mutation (patient 2) occurred in a poly(A) tract, although this change results in a deletion of six bases, rather than base slippage. The mechanism underlying the origin of this mutation is unclear. The mutation results in the removal of amino acids lysine and asparagine at codons 108 and 109 in the protein. The asparagine residue is conserved among the human, mouse, and Xenopushomologues of LKB1.

A further mutation (patient 4) produces a premature stop codon in exon 9, removing just 56 residues from the protein of 434 amino acids. Clearly, there is some doubt as to the pathogenic effect of a mutation which occurs so late in the gene and which lies outside the LKB1 protein kinase core.5 This case is familial, but material from other family members is not available and cosegregation of the variant with disease cannot therefore be determined. The mutation has not, however, been observed in any of more than 65 other PJS and normal subjects sequenced for exon 9 to date. Other germline mutations in LKB1 have been found in exon 8,4 although no exon 9 mutation has been reported previously.

The missense mutation in patient 3 which converts lysine to arginine substitutes one amino acid for another with a similar basic side chain. The cosegregation of the mutant with disease cannot be tested, since the case is sporadic. This amino acid is neither conserved among human, mouse, and Xenopus, nor between LKB1 and other human protein kinases. Thus, although this mutation has not been observed in more than 70 other PJS and normal subjects sequenced for exon 2 to date, and the mutation in patient 2 affects the same amino acid, the pathogenic effects of this sequence change must remain uncertain and we cannot exclude it as a very rare polymorphism.

Associations between the molecular data and the clinicopathological data (table 1) are difficult to assess formally for a rare disease such as PJS with a necessarily limited number of cases. In our sample, every case had clinical features typical of PJS, since these were the referral criteria specified to clinicians. The existence of intrafamilial variation in PJS was shown by the absence of pigmentation in a relative of case 12. As in previous studies,4 5there is no clear association in our sample between a detectable LKB1 mutation and a family history of PJS. Thus, using molecular criteria to define disease, the presence of typical PJS pigmentation and polyps is a much stronger indication of PJS than having a positive family history. It is not clear whether most classical PJS cases without a family history result from new mutations, from variable penetrance, or from under-reporting of family history. To the date of study, subjects in two families had developed carcinomas at early ages and at sites which are at increased risk of cancer in PJS: one of these came from a family with a mutation in exon 9 and the other had no detectable mutation.

DNA sequencing is a viable method of genetic testing for LKB1 mutation in the diagnostic laboratory. In the few studies published previously,4 5 and in our study, germline mutations can be detected using DNA sequencing in up to two thirds of patients with typical features of PJS, irrespective of whether or not these cases have a family history. Similar success rates are achieved using SSCP analysis.6 The reason for the failure to detect LKB1 mutations in more than one third of all PJS patients is unclear. In our sample, we found no mutation in four families (of patients 8, 11, 12, and 22) which were suitable for linkage analysis, yet each family was compatible with linkage to LKB1.14 We suspect that a combination of the imperfect (although high) sensitivity of DNA sequencing and SSCP analysis, some large scale mutations, promoter changes, and, perhaps, an uncharacterised minor PJS locus13 14 combine to reduce the proportion of PJS patients in whom LKB1 mutations can be easily found. We suspect, however, that alternatives to sequencing or SSCP, such as Southern analysis, will not be of sufficient yield5 to warrant their routine use in the diagnostic genetics laboratory, although about 5% of LKB1 variants are detectable by this method.5 15

Our results and those of other studies suggest the following characteristics for LKB1 mutations in PJS. Most germline mutations result in a truncated protein, but a minority of changes are missense or small in frame deletions; in sporadic tumours, most mutations are missense, even though they are generally accompanied by loss of the other allele. Germline mutations in LKB1 can occur throughout the gene, with a possible bias towards exons 1 and 6.4-6 Predictive testing for PJS is likely to find a clinical application in selected relatives of PJS patients. To date, however, no study has examined the prevalence of LKB1 mutations in patients with atypical features of PJS (such as isolated hamartomas or pigmentation without family history) and it is unclear as to how many of these patients are truly variants of PJS. Diagnostic testing for LKB1 mutations in these patients may also be of importance, not least for the subject’s risk of cancer and their relatives’ risk of disease.