Lauri A Aaltonen and Egle Avizienyte 1 Introduction

While Peutz-Jeghers syndrome (PJS) has been acknowledged as a clinical entity for decades (1,2), the molecular background for the disease has been unraveled only very recently. PJS has two cardinal features: First, many but not all patients display mucocutaneous melanin pigmentation that is most prominently seen around the mouth, but can also be present for example in the buccal mucosa, lips, palms, feet, and in the anal region. Second, the patients have a predisposition to hamartomatous intestinal polyps. These lesions can occur anywhere in the gastrointestinal tract, but are most commonly seen in the small intestine (3). Tumor predisposition is not limited to intestinal hamarto-mas. The patients have a relatively unfocused increased risk of cancer, which has been reported to be 10- to 18-fold of that of the general population. Especially the relative risk for breast and gynecologic cancers is high (4,5). Other sites possibly involved include at least small and large intestine, and pancreas (3). Benign testicular tumors also occur commonly in the syndrome (6). Some of the malignant tumors may arise from the benign hamartomatous lesions, which appear to have some malignant potential at least in the context of PJS (7-10).

Because of the predisposition to tumors, PJS diagnosis is of clinical relevance. Intussusception due to benign intestinal hamartomas occurs frequently at young age, and at older age risk of malignant transformation increases (11). While endoscopic cancer screening along the lines shown in hereditary nonpolyposis colorectal cancer (12) may be feasible in preventing Peutz Jeghers intestinal cancers occurring in regions that can be reached, the wide spectrum of involved organ systems is a major clinical challenge. Prominent

From: Methods in Molecular Medicine, vol. 50: Colorectal Cancer: Methods and Protocols Edited by: S. M. Powell © Humana Press Inc., Totowa, NJ

mucocutaneous pigmentation is a useful sign of the syndrome, but it must be emphasized that pigment spots around the mouth are common in the general population, and these lesions are not always present in PJS individuals (the pigmentation tends to be most prominent in adolescence, and is often absent at very young or older age). Histopathological features of the polyps give useful clues: PJS polyps display a pathognomonic tree-like smooth muscle cell core (3). For unambiguous uninvasive diagnosis, molecular methods must be used.

The gene for PJS was recently identified as LKB1 (STK11) (13). First clues to the location and function of the gene came from studies showing allelic loss in 19p in PJS polyps (14), suggesting that this locus harbors a PJS predisposition gene and that the wild type allele is somatically inactivated as proposed in Knudson's two-hit hypothesis (15). Subsequent linkage and physical mapping efforts demonstrated that indeed chromosome 19p harbors the PJS gene (16-19), and that the disease is caused by inactivating mutations of the LKB1 serine/threonine kinase (13,20). This was the first example of inactivating mutations in a kinase in hereditary cancer.

Before blood samples are drawn from at risk individuals for the purpose of genetic PJS testing the individuals should undergo genetic counseling, and give an informed consent. We recommend direct genomic sequencing of LKB1 as the method of choice in PJS diagnostics. The gene is relatively small, and no close human homologues are known (13); the 9 coding exons contain a 1302 base pair open reading frame corresponding to 433 amino acids. Thus genomic sequencing of the gene in nine fragments is not an extensive task. The interpretation of the results is facilitated by frequent occurrence of truncating mutations (13,21-24) which are likely to be disease-causing. In the case of missense mutations interpretation is facilitated by sequence comparisons (25) to evaluate whether the variant is in a conserved position. In research environment mutations affecting the kinase domain can be evaluated functionally, through an autophosphorylation assay (23,24). This way common polymorphisms can often be excluded as a cause of the disease. While the present notion is that most if not all PJS cases arise from the background of an LKB1 mutation, mutation detection rate by genomic sequencing is roughly two thirds (21,22,24). A proportion of the remaining one third may be due to other predisposing genes (17,18,23), but another obvious explanation is occurrence of mutations that cannot be detected by sequencing. Large deletions and other rearrangements are usually impossible to detect in direct genomic sequencing, and the promotor area is not evaluated.

LKB1 somatic mutations appear to be rare in most tumor types, but have been described, e.g., in colorectal, endometrial, testicular and pancreatic malignancies, as well as malignant melanomas (26-32). Direct genomic sequencing of LKB1 is one option for analysis of somatic mutations especially if samples of particular interest are to be scrutinized.

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