Original article
A novel de novo mutation in LKB1 gene in a Chinese Peutz Jeghers syndrome patient significantly diminished p53 activity

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Summary

Peutz Jeghers syndrome (PJS) is an autosomal dominant disease caused by mutations in the LKB1 gene. We screened for the LKB1 gene mutation in a Chinese PJS patient. Sequence analysis of LKB1 exons showed that there was a novel de novo mis-sense mutation of codon 16 (GAG to GGG) in exon 1 in LKB1 gene in the Chinese PJS patient. Furthermore, we observed that wild type LKB1 expression increased p53 activity, while LKB1 A47G mutation had no such effect on p53 activity, indicating that the mis-sense variant of LKB1 influenced the p53 activation function of LKB1 protein. In addition, real-time RT-PCR analysis revealed that the expression levels of p53 downstream targets were significantly diminished in affected PJS patient compared with those in unaffected parents, further confirming the roles of LKB1 and p53 in PJS pathogenesis. Therefore, a novel PJS associated LKB1 gene mutation is provided, and the roles of LKB1 and p53 in PJS pathogenesis is validated in this research.

Introduction

Peutz Jeghers syndrome (PJS) is an autosomal dominant inherited disorder occurring in 1 of every 120,000 births [1]. It is characterized by the presence of hamartomatous polyps in the gastro-intestinal tract and by melanin spots on the lips, buccal mucosa, and digits [2]. In addition to these typical symptoms, patients often have an increased risk of gastro-intestinal malignancies and cancers at other sites, such as in breast, pancreas, ovary, uterus, lung and testicle [3], [4]. Genetic linkage studies have mapped the disease to genomic region 19p 13.3 and germ-line mutations in the LKB1 gene at this locus have been identified as a major reseason of PJS [5], [6], [7]. Human LKB1 (also known as STK11) gene encodes a 433-amino acid serine/threonine protein kinase [5], [6], consisting of 10 exons (nine coding exons), and codons 50-337 encode the catalytic kinase domain. Appromitely 70–80% of the PJS patients harbor germ-line mutations or deletions in the LKB1 gene. Studies of LKB1 mouse models show that Lkb1+/- mice develop gastro-intestinal polyposis and hepatocellular carcinomas, while Lkb1-/- embryos die during midgestation [8], [9].

The p53 gene is one of the most important and widely studied tumor suppressor genes. P53 controls the cell proliferation and apoptosis by regulating of the expression of various genes that are implicated in cell cycle arrest, DNA damage repair and apoptosis [10]. About 50% of human cancers have p53 gene mutations, suggesting that p53 inactivation is crucial for tumorgenesis [11]. It has been reported that LKB1 plays a critical role in p53-mediated cell apoptosis [12]. Gene knock-out studies have revealed that the Lkb1+/-/p53+/- mice showed a dramatically reduced life span and increased tumor incidence compared to the mice with either Lkb1 or p53 single gene knockout, indicating that p53 and Lkb1 gene mutations cooperate in tumor progression [13].

In this study, we identified a novel de novo mis-sense mutation in LKB1 gene from a Chinese PJS patient, and observed that the loss of LKB1 function in PJS patient resulted in diminished in p53 activity.

Section snippets

Patient

A 26-year-old woman was diagnosed clinically with PJS at the age of 18 years old. The patient had no relations to be affected. Diagnosis was based on histologically confirmed gastro-intestinal hamartomatous polyps after polypectomy and the presence of characteristic mucocutaneous pigmentation of the lips, buccal mucosa and fingers.

DNA and RNA extraction

Total genomic DNA was extracted from peripheral blood using the genomic DNA extraction Kit (Takara) according to the manufacturer's instructions. Total RNA was

A novel de novo mutation of LKB1 gene was identified in a Chinese PJS patient

In order to investigate the genetic characteristics of this Chinese PJS patient, the total genomic DNA was extracted from peripheral blood and the entire nine encoding exons of LKB1 gene were analyzed by PCR amplification. PCR samples were subjected to ligate into pGEM-T easy vectors and subcloned using the TA cloning system. A minimum of eight individual clonies were selected. Plasmid DNAs were isolated and used for DNA sequencing analysis. Sequencing analysis of the LKB1 gene revealed a

Discussion

LKB1 was initially characterized as a key determinant in Peutz-Jeghers syndrome, an inherited predisposition to gastro-intestinal and other cancers [2], [3], [4]. Till now, up to 200 kinds of mutations in LKB1 have been reported [15], [16], [17], [18]. However, the LKB1 mutation manners of Chinese PJS patients were limited. Here, we sequenced the entire nine encoding exons of LKB1 from a Chinese PJS sample. Unexpectedly, we observed a novel de novo mis-sense mutation of LKB1 in exon 1.

Previous

Conclusion

Our study presents the molecular characteristics of a Chinese PJS patient, increases the mutational spectrum of LKB1 gene, and provides a new insight useful for clinical diagnosis and genetic counseling of PJS families.

Conflicts of interest statement

No potential conflict of interest relevant to this article exists.

Acknowledgements

The authors thank all the patients and their families for participation in the study.

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    Supported by National Natural Science Foundation of China, NO. 60601018.

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