Alu-Mediated Genomic Deletion of the Serine/Threonine Protein Kinase 11 (STK11) Gene in Peutz–Jeghers Syndrome

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Dear Sir:

The key feature of Peutz–Jeghers syndrome (PJS) is a greatly increased risk of developing malignant tumors in multiple tissues. In addition to an elevated risk of gastrointestinal malignancies, such as gastroesophageal, small bowel, and colorectal cancers, an increased risk of cancers at other sites, particularly in breast, pancreas, ovary, uterus, cervix, lung, and testicle, has been reported.¹, ² Thus, an appropriate diagnosis is crucial for cancer prevention.

PJS (MIM 175200), which has an incidence of 1 in 150,000 in North America and Western Europe, is a rare autosomal-dominant inherited precancerous condition characterized by multiple polyps in the gastrointestinal tract and distinctive mucocutaneous pigmentation.

Germline mutations in the serine/threonine kinase 11 gene (STK11/LKB1) (OMIM*602216) are documented in up to 70%–80% of the PJS patients; of these, about 15% have germline deletions of all or part of the gene.³, ⁴, ⁵ In the remaining 20%–30% of PJS patients, defects in other genes or not yet identified ways of LKB1 inactivation might be responsible for PJS.

The aim of this work is to clarify the molecular basis of the disease in Italian PJS patients. We investigated the STK11/LKB1 gene mutations in a well-characterized series of 9 unrelated Italian PJS patients, by using a combination of polymerase chain reaction (PCR), reverse transcriptase (RT)-PCR, DNA sequencing, Southern blot analysis, and real-time PCR techniques.

As shown in Table 1, we have characterized the specific STK11 mutation in 6 probands, consisting of: 2 truncating mutations (1 novel and 1 known mutation), 1 missense known mutation in the exon 4, and 2 novel small in-frame deletions in exon 6. Finally, we have found an intra-exonic in-frame deletion encompassing exons 2 and 3 and we have characterized the breakpoints of this LKB1/STK11 intragenic deletion. This rearrangement, that deletes about 7 kb of the LKB1 genomic region encompassing exons 2 and 3 (Figure 1A), is most likely an Alu–Alu homologous recombination event. Two 26-bp core sequences of 2 Alu elements (both AluY sequences), showing a 96% homology, are indeed localized at the 5′ and 3′ end of the breakpoints, respectively. This sequence, could itself act as a recombinase (Figure 1).

Table 1. Clinical Phenotype and Mutation Spectrum of Italian PJS Patients Studied

F, familial cases; S, sporadic cases; N.F., mutation not found; the bold character indicates the novel mutations.

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• Figure 1. 

  Molecular characterization of the STK11/LKB1 intragenic deletion. (A) Repetitive elements present in the genomic region from exons 1–4 of the STK11/LKB1 gene and a diagram (below) of the deletion are shown. The arrow indicates the novel genomic junction; 5′ BP and 3′ BP: 5′ and 3′ breakpoints. (B) Sequence analysis of the amplified product obtained from deleted allele. Boxed sequence corresponds to the twelve nucleotides repeated both at the 5′ and 3′ breakpoints sharing 100% of homology and representing the novel genomic connection produced by the deletion event. Bold arrows indicate the genomic region involved in the deletion and the genomic nucleotides position involved in the novel genomic junction, referred to GenBank sequence with accession number: NC_000019.9. (C) Sequence of Alu elements present at the 5′ and 3′ breakpoints (5′ BP, 3′ BP, of A); boxed nucleotides represent the core sequence of Alu elements.

Alu-mediated homologous recombination is a mechanism well documented so far; however, it is the first evidence that this mechanism is involved in the STK11/LKB1 gene rearrangements. Because the average frequency of Alu elements along the human genome is estimated to be 1 element every 4 kb,⁶ the region spanning the 7-kb deletion (including introns from 1 to 3 of the LKB1 gene), shows an over-representation of complete Alu elements. Seventeen Alu elements are detected in this 14.5 Kb region by using in silico analysis with the RepeatMasker program, which corresponds to >1 element every 1 kb. The over-representation of Alu elements indicates that this is a region of DNA instability; thus, it is not surprising that several intragenic rearrangements involving this region have been reported so far. Intriguingly, genomic sequence of chromosome 19 from GeneBank position 1203099 to 1230861 shows the presence of Alu elements at the genomic regions involved in the intragenic deletions in most of the cases so far described.

Our strategy to screen the entire coding region of the LKB1 gene, both at DNA and RNA level, has allowed the identification of the disease causing mutations in about 67% of PJS patients. Other gene inactivating mechanisms might be responsible for PJS in mutations negative population subset. However, the existence of genetic heterogeneity cannot be excluded.

Because of an over-representation of Alu elements in this region, in our opinion, is reasonable to hypothesize that Alu-mediated homologous recombination could give rise to several intragenic rearrangements, as translocations or inversions, not always detectable using classical molecular biology techniques. These rearrangements could in part explain the PJS cases without identified molecular alterations.

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