Background  Peutz-Jeghers syndrome (PJS) is an autosomal dominantly inherited disease. STK11/LKB1 gene germline mutations have been identified as responsible for PJS. In our study, we investigated the molecular basis of PJS and evaluated correlation between the STK11 mutations and the Chinese population.
Methods  We collected three pedigrees of PJS and screened the 9 exons and their flanking intronic sequences of STK11/LKB1 gene in the probands and normal individuals in the families using polymerase chain reaction (PCR) and direct sequencing.
Results  Sequencing of the STK11 gene in the probands of 3 families revealed two novel mutations (c180C→G and c998-1002delGCAGC) in exon 1 and exon 8, respectively. The mutation of c180C→G resulted in a premature termination codon. The other mutation, a deletion of five nucleotides (998-1002delGCAGC) in exon 8, predicted to generate a translational frameshift and a termination at codon 1070.
Conclusions  The growing number of mutations in PJS pedigrees suggests the molecular basis of PJS. STK11 gene mutation can be detected in most patients with PJS.

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Peutz-Jeghers syndrome (PJS; OMIM#175200) is an autosomal dominantly inherited disease characterized by hamartomatous gastrointestinal polyposis and melanin spots on the buccal mucosa, hands, feet and lips. Over 90% of patients with PJS develop significant harmartomatous polyps of the small bowel, and polyps commonly arise within the stomach and colorectum.¹ About 48% of patients had a history of intussusception with a median age of 15.0 years.¹ PJS has been reported to be a risk factor of benign and malignant neoplasms of the intestinal tract, pancreas, ovaries, testes, breasts, and uterus.² The risk for developing cancer at 20, 30, 40, 50, 60 and 70 years of age was estimated to be 1%, 3%, 19%, 32%, 63%, and 81%, respectively.²

The STK11/LKB1 gene germline mutation has been identified as being responsible for PJS, and the detection rate of germline mutations in PJS is 30%−80% of PJS patients.^1,3-7 To date, 132 germline mutations of the STK11 gene have been reported in PJS at the Human Gene Mutation database (HGMD) website (http://archive.uwcm.ac.uk/uwcm/mg/search/9732383.html). Most mutations are single base substitutions/insertions or small deletions resulting in an abnormal truncated protein. In the present study, we performed a mutation analysis of STK11 in three Chinese PJS families.

Subjects
The proband (III-1, Fig. 1A) was a 7-year-old Han Chinese boy from Henan Province, China. He was diagnosed as PJS at 5 years of age based on the presence of hamartomatous polys in the gastrointestinal tract and mucocutaneous melanin pigmentation on his hands, feet and lips. At age 5, he visited the local hospital due to persistent abdominal pain. An endoscopic examination showed multiple hamartomatous polyposis in the duodenum and colon. His mother and younger brother were also diagnosed as PJS without gastric cancer findings based on a histopathological examination of the polyp's samples. The age of onset ranged from 5 to 8 years old. After obtaining informed consent for clinical and genetic investigations, we collected blood samples from the affected individuals and two unaffected individuals (I-2, II-3).

The other two families with PJS, altogether containing five affected and nine unaffected individuals (Fig. 1 B, C), were identified through probands from Jiangsu Province and Hebei Province, China. The age of onset ranged from 5 to 10 years. Relatives who were diagnoised with PJS did not have evidence of gastric cancer in polyp samples through histopathological examinations. After obtaining informed consent, blood samples were collected from all the patients and several healthy family members. The study protocol was approved by Peking Union Medical College Hospital Ethic Committee.

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Fig. 1. Pedigrees of the families of the patient of PJS. Arrow represents proband.

Direct sequencing
Direct sequencing was performed using a DNA sequencing system (model 377; ABI, USA) by TaKaRa gene company, Dalian, China. All detected mutation was verified in both sense and antisense directions using PCR products from independent reactions to exclude the mispairing resulting from Taq DNA polymerase. Exon 1 and exon 8 were simultaneous screened in all individuals from the families and 50 unrelated population-matched healthy individuals.

A survey of all coding sequences and its boundary regions of the STK11 gene revealed two heterozygous germline mutations (c180C→G and c998-1002delGCA GC) in exon 1 and exon 8, respectively. The mutation of c180C→G in exon 1 in family 1 resulted in a premature termination codon (Fig. 2). In family 2, we identified a deletion of five nucleotides (998-1002delGCAGC) in exon 8 (Fig. 3) which results in a translational frameshift and termination at codon 1070. The mutations were found in all patients in family 1 and 2, but not in healthy individuals in the two families and 50 unrelated healthy individuals.

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Fig. 2. A DNA sequence analysis of exon 1 of the STK11 gene, using genomic DNA from peripheral leucocytes. A: A heterozygous germline mutation (C180G) was detected in the proband in family 1, resulting in a premature termination codon. B: The sequence of exon 1 from a healthy individual in family 1.

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Fig. 3. Mutation analysis of exon 8 of the STK11 gene using genomic DNA from peripheral leucocytes. A: A deletion of five nucleotides (998-1002delGCAGC) in exon 8 of the STK11 gene. This mutation results in a translational frameshift and a termination at codon 1070. B: The sequence of exon 8 from a healthy individual in family 2.

DISCUSSION

All probands recruited in this study fulfilled the established criteria for diagnosis of PJS.⁸ The criteria include having more than three histopathologically proven PJS polyps, with classical mucocutaneous pigmentation and a positive family history.⁸ Since 1998, mutations in STK11 gene on chromosome 19p13.3 have been identified as the cause of PJS.⁹ STK11 is a highly conserved gene that extends over 23 kb and consists of nine coding exons, coding for a 433-amino acid coding sequence and one non-coding exon.¹⁰ STK11 protein is mainly composed of three major domains, including an N-terminal non-catalytic domain, a catalytic kinase domain and a C-terminal regulatory domain.¹⁰ Although the exact function of STK11 remains unclear, many studies have demonstrated mutation of STK11 gene leads to a loss of kinase activity and that loss of its activity is likely responsible for development of the PJS phenotype.¹¹ Codons 50-337 encode the catalytic kinase domain. STK11 gene supposedly acts as a tumor suppressor gene and might be involved in the very early development of the pathogenesis of hamartomas into adenocarcinoma.¹²

In our study, we identified two novel heterozygous germline mutations in STK11 gene in the probands and all the other patients, but not in healthy members of these families or healthy unrelated individuals. These mutations have not been reported in any database or articles, indicating that they are novel mutations. The mutation of c180C→G in exon 1 in family 1 resulted in a premature termination codon. This mutation affects codons that encode for the catalytic kinase domain. We hypothesize that the mutation reported here may lead to an altered kinase activity of the protein, which would cause a partial or complete loss of the kinase domain. The other mutation, a deletion of five nucleotides (998-1002delGCAGC) in exon 8 identified in family 2, generates a translational frameshift and a termination at codon 1070. This mutation results in a dysfunctional protein,namely a truncated protein that is non-functional due to the loss of activity of critical functional domains. A few studies have shown that partial or complete loss of the C-terminal domain of STK11 can lead to loss of cell polarity and hamartoma formation, resulting inappropriate overgrowth of differentiated cells.¹³ Based on these results, we hypothesize that the mutation in exon 8 of STK11 gene in this study may contribute to polyp formation through suppression of growth arrest, apoptosis and dysregulation of AMP-activated protein kinase (AMPK) pathway.^14,15

We did not detect any mutations in family 3. The reason for absence of STK11 in some PJS patients is unclear. The original report of STK11 mutations in PJS showed that only 18% of PJS patients contained STK11.⁷ Recent studies using the multiplex ligation dependent probe amplification (MLPA) analysis screened for gene and exon scale mutations in a set of PJS patients without detected STK11 mutations. These studies detected rates of germline mutations in PJS patients to be very high at approximately 80%.⁴ The results demonstrated that large deletions, causing the absence of detectable STK11 mutation, were very common in PJS and cannot be detected using routine mutation screening methods. Other studies reached similar conclusion that germline exonic deletions of STK11 were a common cause of PJS, as the overall mutation detection rate was 94% when patients who met the clinical criteria for PJS were considered.^16,17 Our research identified two novel mutations in two families with PJS, but we were unable to detect any mutations in the third family based on the routine PCR and sequencing method. Based on these previous studies,it is very possible that germline STK11 exonic deletions of affected members of family 3 exist, but can not be detected via PCR.

Some evidence exists and indicates mutation in exon 3 may be associated with a higher cancer risk.⁶ However, since we were unable to find any mutations in exon 3 in our families and none of our patients had any cancers, this result could not be confirmed by our study. After review of all published Chinese journals, we were also unable to make the conclusions that mutations in exon 3 could be associated with cancer. Recently, Massa et al¹⁸ reported an STK germline mutation in exon 1 (C130A→T) in a girl with an ovarian stertoli cell tumor. It is still unclear whether this particular mutation predisposes the patient to development of ovarian tumours. Restricted by the few samples collected in the reported studies and the few published papers on this topic, the correlation between mutation and cancer risk needs to be further researched in future studies.

In summary, we have reported two novel mutations of STK11 involved in PJS. The results suggest the molecular basis of PJS in these Han Chinese families. STK11 gene plays a great role in PJS and STK11 mutation can be detected in most patients with PJS.

Acknowledgement: We gratefully thank Liu XR and Du RX for collecting blood samples and thank the patients and healthy controls for their cooperation in the study.

1. Hearle N, Schumacher V, Menko FH, Olschwang S, Boardman LA, Gille JJ, et al. STK11 status and intussusception risk in Peutz-Jeghers syndrome. J Med Genet 2006; 43: e41.[PubMed]

2. Launonen V. Mutations in the human LKB1/STK11 gene. Hum Mutat 2005; 26: 291-297.[PubMed]

3. Shinmura K, Goto M, Tao H, Shimizu S, Otsuki Y, Kobayashi H, et al. A novel STK11 germline mutation in two siblings with Peutz-Jeghers syndrome complicated by primary gastric cancer. Clin Genet 2005; 67: 81-86. [PubMed]

4. Volikos E, Robinson J, Aittomaki K, Mecklin JP, Jarvinen H, Westerman AM, et al. LKB1 exonic and whole gene deletions are a common cause of Peutz-Jeghers syndrome. J Med Genet 2006; 43: e18. [PubMed]

5. Scott RJ, Crooks R, Meldrum CJ, Thomas L, Smith CJ, Mowat D, et al. Mutation analysis of the STK11/LKB1 gene and clinical characteristics of an Australian series of Peutz-Jeghers syndrome patients. Clin Genet 2002; 62: 282-287. [PubMed]

6. Schumacher V, Vogel T, Leube B, Driemel C, Goecke T, Moslein G, et al. STK11 genotyping and cancer risk in Peutz-Jeghers syndrome. J Med Genet 2005; 42: 428-435.[PubMed]

7. Boardman LA, Couch FJ, Burgart LJ, Schwartz D, Berry R, McDonnell SK, et al. Genetic heterogeneity in Peutz-Jeghers syndrome. Hum Mutat 2000; 16: 23-30. [PubMed]

8. Thakur N, Reddy DN, Rao GV, Mohankrishna P, Singh L, Chandak GR. A novel mutation in STK11 gene is associated with Peutz-Jeghers Syndrome in Indian patients. BMC Med Genet 2006; 7: 73. [PubMed]

9. Hemminki A, Markie D, Tomlinson I, Avizienyte E, Roth S, Loukola A, et al. A serine/threonine kinase gene defective in Peutz-Jeghers syndrome. Nature 1998; 391: 184-187.[PubMed]

10. Hanks SK, Quinn AM, Hunter T. The protein kinase family: conserved features and deduced phylogeny of the catalytic domains. Science 1988; 1: 42-52. [PubMed]

11. Mehenni H, Gehrig C, Nezu J, Oku A, Shimane M, Rossier C, et al. Loss of LKB1 Kinase Activity in Peutz-Jeghers Syndrome, and Evidence for Allelic and Locus Heterogeneity. Am J Hum Genet 1998; 63: 1641-1650. [PubMed]

12. Nakanishi C, Yamaguchi T, Iijima T, Saji S, Toi M, Mori T, et al. Germline mutation of the LKB1/STK11 gene with loss of the normal allele in an aggressive breast cancer of Peutz-Jeghers syndrome. Oncology 2004; 67: 476-479. [PubMed]

13. Forcet C, Etienne-Manneville S, Gaude H, Fournier L, Debilly S, SalmiM, et al. Functional analysis of Peutz-Jeghers mutations reveals that the LKB1 C-terminal region exerts a crucial role in regulating both the AMPK pathway and the cell polarity. Hum Mol Genet 2005; 15: 1283-1292.[PubMed]

14. Hawley SA, Boudeau J, Reid JL, Mustard KJ, Udd L, Makela TP, et al. Complexes between the LKB1 tumor suppressor, STRAD alpha/beta and MO25 alpha/beta are upstream kinases in the AMP-activated protein kinase cascade. J Biol 2003; 2: 28. [PubMed]

15. Shaw RJ, Kosmatka M, Bardeesy N, Hurley RL, Witters LA, DePinho RA, et al. The tumor suppressor LKB1 kinase directly activates AMP-activated kinase and regulates apoptosis in response to energy stress. Proc Natl Acad Sci USA 2004; 101: 3329-3335. [PubMed]

16. Hearle NC, Rudd MF, Lim W, Murday V, Lim AG, Phillips RK, et al. Exonic STK11 deletions are not a rare cause of Peutz-Jeghers syndrome. J Med Genet 2006; 43: e15. [PubMed]

17. Aretz S, Stienen D, Uhlhaas S, Loff S, Back W, Pagenstecher C, et al. High proportion of large genomic STK11 deletions in Peutz-Jeghers syndrome. Hum Mutat 2005; 26: 513-519.[PubMed]

18. Massa G, Roggen N, Renard M, Gille JJ. Germline mutation in the STK11 gene in a girl with an ovarian Sertoli cell tumour. Eur J Pediatr 2006; [Epub ahead of print]. [PubMed]