Genetic defects underlying Peutz–Jeghers syndrome (PJS) and exclusion of the polarity-associated MARK/Par1 gene family as potential PJS candidates

Peutz–Jeghers syndrome (PJS) is an autosomal dominant disorder characterized by mucocutaneous melanin pigmentation, hamartomatous polyps and an increased cancer risk (1, 2). The discovery of underlying mutations in the tumor suppressor gene LKB1/STK11, has provided further insight into this disorder. However, the precise function of LKB1 remains elusive as is the exact molecular mechanism responsible for the phenotypic characteristics of PJS. We recently hypothesized that loss of the polarity function of LKB1 results in mucosal prolapse, ultimately leading to PJS polyp formation, and tumor growth (3). Although LKB1 was identified as the PJS gene, germline mutations were found in only 30–70% of patients using conventional mutation analyses (4). LKB1 might, however, be alternatively inactivated and recently exonic deletions have been described, resulting in 66–94% of PJS patients with LKB1 inactivation (5–7). A subset of PJS patients remains with seemingly no LKB1 inactivating mutation and consequently a second PJS gene may exist. Several possible candidates have been studied, including genes encoding LKB1 interacting proteins LIP1, BRG1, STRAD and its co-activator MO25, but to date no second PJS gene has been identified (8–11). In search of a second PJS locus, linkage to chromosome 19q13.4 was found in one Indian PJS family (12) and a 6-day-old patient presenting with a hamartoma with the histology of a PJS polyp had a translocation in the same region (11). Several genes within 0.5 Mb of the breakpoint were sequenced (including BRSK1/KIAA1811), but none was mutated in PJS patients without LKB1 mutation. As the region on chromosome 19q13.4 was implicated in two different PJS families, it may harbor a second PJS gene. One of the genes located in the 19q13 region is the MARK4 gene. The MARK proteins are part of the family of AMPK-related kinases of which LKB1 is an upstream activator (13, 14). These four microtubule affinity-regulating kinases play a role in microtubule dynamics during polarization of cells (15). MARK2 knockout and heterozygous mice were also described to present with a phenotype of colorectal prolapse (16). Interestingly, PJS polyps histologically resemble mucosal prolapse (3). However, the MARK2 knockout mice also develop characteristics not linked to PJS-like immune system dysfunction, overall proportionate dwarfism and a peculiar hypofertility (16, 17). The MARK genes have also been implicated in tumorigenesis since in two colorectal tumors a mutation and 1 bp insertion were reported in MARK3 (18). The MARK proteins are the human homologues of Par1, which is, like LKB1/Par4, a member of the par family of polarity proteins. This family is conserved during evolution and the six members of the family regulate epithelial polarity in Drosophila melanogaster, Caenorhabditis elegans and vertebrates, by involvement in cell migration and the establishment of the anterior–posterior axis. Due to their relation to LKB1 and their role in polarity, the MARK genes make interesting PJS candidates. To further investigate germline alterations related to PJS, we screened for LKB1 defects and for mutations in the MARK1, MARK2, MARK3 and MARK4 genes. LKB1 mutation analysis was performed on 23 PJS patients from different families using sequence analysis and multiplex ligation-dependent probe amplification (MLPA) to detect point mutations and exon deletions. Furthermore, a mutation analysis of the four MARK genes and an exon deletion screen of MARK4 on eight PJS patients from families without LKB1 germline mutations was performed including the Indian PJS family where linkage was found at chromosomal region 19q13.4 (19).