STK11/LKB1Gene Mutation Screening And Proteomic Analysis Of Chinese PJS Patients

BackgroundPeutz-Jeghers Syndrome (PJS, MIM#175200) is a rare autosomal dominant disorder characterized by gastrointestinal hamartomatous polyps and mucocutaneous pigmentation. Multiple gastrointestinal polyps of the PJS patients often caused intussusception, intestinal obstruction and gastrointestinal bleeding in adolescence. PJS causes not only higher cancer risk in digestive tract, but there is also an age-dependent increased risk for development of extra-intestinal malignancies compared to the general population. There is no intervention to prevent the occurrence of PJS polyps development or their malignant transformation. Therefore, exploration of PJS related genes, pathways and the mechanisms of PJS polyp development and malignant transformation has great significance.The major cause of PJS is mutations in the serine/threonine protein kinase STK11/LKB1,a family member of AMPKK and a tumor suppressor gene. It plays an important role in energy metabolism regulated by the AMPK allosteric modulator. STK11/LKB1could negatively regulate the transcription factor SPI to alter VEGF expression and thereby inhibit cell proliferation. Mutated STK11/LKB1could also regulate cell cycle arrest in G1to promote apoptosis. Nerve cells polarity is controlled by formation of the complex of STK11/LKB1and STRAD proteins leading to overexpression of KB1S431A (a serine to alanine mutation) to inhibit axis differentiation. STK11/LKB1controls cell polarity of non-small lung cancer cells by regulating JNK signaling pathways, which could regulate the transcription factor AP-1, c-Jun, JunD and ATF2. Despite evidence of the role of STK11/LKB1in energy metabolism, cell cycle and cell polarity, the mechanism of PJS polyp formation and malignant transformation is still not clear.Recently, it was found that some PJS polyps were adenomatoid polyps and may have higher risk of carcinogenesis. However, there is no conclusion whether the adenomatous polyps are evolved from hamartomas, or if they are two distinct clinical subtypes. Some scholars speculated that two mechanisms resulted in PJS malignancy, including the pathway of hamartomas-adenoma-adenocarcinoma and de novel malignant transformation. However, given the very low frequency of dysplasia, the exact role, if any, of the PJS polyp in cancer development is unclear.The attempt to correlate both the type and site of STK11/LKB1genetic mutations with the risk of malignancy has been the focus of many studies and progress in this area would represent an important research and clinical advance. Previous evidence suggested that mutations in exon3were associated with a higher cancer risk whereas in another study, statistically significant evidence correlated mutations in exon6with higher cancer risk. Schumacher et al found that missense mutations in the C terminus and regions VIB-VIII of the protein were more frequently associated with malignancies. On the other hand, in-frame deletions and splice site mutations have been found to be only rarely associated with malignancies while PJS patients with breast cancer were found to predominantly have truncating mutations. Unfortunately the analysis of additional sample sets by other groups seeking to validate these findings has not yet provided a clear genotype-phenotype association with malignancy.To date, the rate of STK11/LKB1gene mutations showed variation by geographical region (from50%to90%), and the relationship between genotype and phenotype was inconclusive. The malignant risk and pathways in PJS polyps were still controversial. Lack of concurrence between groups may be due to the different genetic backgrounds of the populations studied, but also the limited sample size. Thus, it is of great importance to establish a library of PJS pedigrees and biological specimens for further study of this rare genetic disease. In the present study, we established a biological specimen bank of Chinese PJS pedigrees according to the standardized procedures. We analyzed the clinical and pathological characteristics and assessed the malignant risk in this cohort of PJS patients. Then, we directly interrogated STK11/LKB1gene mutations in a subset of (116) these patients representing52index cases and analyzed the genotype and phenotype corelation. In addition, Protein Pathway Array analysis was used to identify proteins with altered expression in PJ polyps.Methods1. Reference standardized procedures for establishing a PJS biological specimen bank and use these to develop and standardize an operational process in PJS bio-sample collection. Collection PJS polyps tissue and blood samples nationwide began under implemented protocol. Using of the Collect-Feedback-Modify mode to review and improve the specimens collection operational processes and strengthen management and quality control measures.2. Twenty-eight PJS families and33sporadic cases were identified from a regional Chinese population for a total of133patients for clinical characteristic and malignant risk evaluation.3. Peripheral blood genomic DNA samples from116Chinese PJS "patients from52unrelated families (25PJS families and27sporadic cases) were investigated for STK11mutations using a combination of conventional direct DNA sequencing and the multiplex ligation-dependent probe amplification (MLPA) assay. Phenotypic correlations were investigated.4. To identify the PJS polyps protein expression profiles,28fresh PJS polyps (with clearly STK11/LKB1mutations) and35normal tissues (without STK11/LKB1gene mutation) were used for Protein Pathway Array screening.5. Statistical methods:Differences between groups were determined using chi-squared (x2) test or Fisher’s exact test. The statistical analyses were assessed using SPSS17.0software (SPSS Inc, Chicago, IL). The differentially expressed proteins were analyzed using the Student’s t-test and Significant Analysis of Microarray (SAM) screening. A value of P<0.05or Q<5was considered statistically significant.Results1. Establishment of the PJS pedigrees biological specimen bank.After the development of standardized specimen collection process, we collected61Chinese PJS families, including133PJS patients and105normal family members in the biological specimen bank. Of which,28PJS pedigrees have family history and33cases were sporadic.2.Clinical characteristics of patients with Peutz-Jeghers syndrome.In all,85patients (85/133,64%) had received at least one laparotomy (range,1-5) and the average age at first laparotomy was17.29years (range,3-68years). Polyp-induced complications, primarily intussusception of the small bowl, were the major referring cause for laparotomies. The overall cancer frequency in our study population was25/133(18.8%) and the mean age at cancer onset was37.4years (n=27). Gastrointestinal cancer was noted in18cases (18/27,66.7%). Breast and gynecological cancers were noted in25.9%(7/27). Of particular interest, gastrointestinal dysplastic hamartomas were identified in16patients (16/133,12%) and these were detected at a mean age of30.4years.3. STK11/LKB1gene point mutations.We identified germline point mutations in27of52(51.9%) of the index cases. Of these,15were found in familial (15/25,60%) and12in sporadic (12/27.44.4%) cases. We detected8missense mutations and17different truncating mutations. Ten mutations (10/25,40%) were associated with cancer in the index patient and/or in relatives with PJS. To our knowledge,14(14/25,56%) of these mutations are novel. Intriguingly, nearly one-third (8/27,29.6%) of all point mutations clustered in exon7, the shortest of the9exons.4. STK11/LKB1gene Large Genomic DeletionsWe next tested for the presence of exonic rearrangements by using MLPA in the25PJS probands in whom no mutation was identified by Sanger sequencing. The overall frequency of large deletions was8/52(15.4%). Three (3/8,37.5%) deletions were associated with cancer in the index patient and/or in relatives.5. Genotype-phenotype correlations.Analysis of the genotype-phenotype correlations showed that a higher frequency of mutations was identified in our patients with a family history of PJS (20/25,80%) when compared to our sporadic cases (15/27,55.6%); however, the difference was not statistically significant (p=0.06, x2=3.525). Eighteen (18/83,21.7%) patients with an STK11mutation developed a malignancy, which was not statistically different than the cancer frequency in our patients without STK11mutations (5/33,15.2%)(p=0.426, x2=0.634). None of the five splice site mutations or four small in-frame deletions was associated with malignancies in the index patient or their affected relatives. In contrast, all five insertion mutations and three out of four nonsense mutations were associated with malignancies.6. A novel correlation between kinase domain XI mutations with the development of dysplastic GI polyps.Ten of the27germline mutations (37%) that we identified were present in kinase domain XI (amino acids277-309). Strikingly, nine of the ten mutations (90%) were associated with gastrointestinal tract hamartomatous polyp dysplasia.Conversely, only two of the remaining17(11.8%) mutations were associated with dysplastic changes in polyps (p=0.0001).7. p-p38MAPK protein expression significantly increased in XI domain mutation-polyps compared to I-X domain mutation-polyps.Analysis of the differently expressed proteins between13PJS polyps with mutations in STK11/LKB1I-X functional domain and four polyps carrying XI domain mutations, we found that p-p38MAPK protein was upregulated1.83times in I-X domain mutation-polyps than the normal tissue. However, in XI domain mutation carrying polyps this upregulation was more pronounced at3.23times higher than the normal tissue (p=0.003, I-X vs. XI). The results were verified by Western Blot.8. Galectin-3protein a potential molecular therapeutic target for inhibition of PJ polyps. In addition to the p-p38MAPK protein, we found39differentially expressed proteins between PJS polyps and normal tissues by Protein Pathway Array screening. Of these,13proteins had increased expression in PJS polyps and26proteins lower expression compared to normal tissues. The expressions of proteins Galectin-3, GSTP1, NQO1, COX-2and ICAM-1were verified by Western Blot and IHC.Most notably, Galectin-3protein expression in PJ polyps was7times higher than the normal mucosa. IHC showed PJS hamartoma and the dysplasia polyps had strong cytoplasmic expression, but expression was negative in normal tissue. The inhibitor of Galectin-3protein, low molecular weight citrus pectin (MCP), could be a molecular therapeutic target for inhibition of PJ polyps given that it has minimal side effects.9. Prediction of the mechanism of PJ polyps development by IPA (Ingenuity Pathway Analysis).After IPA analysis, we speculated that STK11/LKB1mutations could activate p38MAPK, which would cause the formation of PJS polyps through the inflammatory response (COX-2, ICAM-1) and metabolic pathways (GSTP1, Galectin-3, NQO1).Conclusion1. The establishment of standardized operation process is conducive to improve the efficiency of collecting PJS pedigrees biological specimens and to improve the quality of specimen bank (including biological samples and clinical follow-up data).2. Our study is the first to report the mutation discovery rate of67.3%in Chinese PJS patients. Exon7could be the mutation hot-spot in this cohort. And we also identified14novel STK11/LKB1mutations.3. The present study is the first to identify a novel correlation between STK11kinase domain Ⅺ mutations with the development of PJS dysplastic GI polyps. The extent of the association between dysplasia and the development of G1-related cancers is currently unknown but our results highlight a novel STK11genotype-phenotype association as the basis for future studies. In addition, we found p-p38MAPK protein expression was significantly higher in XI domain mutation-polyps than I-X domains.4. Galectin-3protein could be a new molecular therapeutic target for inhibition of PJ polyps. The natural inhibitor of Galectin-3,low molecular weight citrus pectin (MCP) has potential to be used in the clinical treatment of PJS patients.5. In our cohort, the average ages of the first laparotomy (64%), the gastrointestinal dysplastic hamartomas (12%) and malignancies were17.29,30.4and37.4years separately. These data provide an important theoretical basis for the development of monitoring programs to prevent the occurrence of intestinal obstruction and malignancies.