Study On Clinical Diagnosis And Treatment Of Peutz-Jeghers Syndrome And The Role Of STK11/LKB1 In Peutz-Jeghers Syndrome

Part ?The detection of mutation rate of STK11/LKB1 andcolorectal cancer related genesBackground and Aims: The colorectal cancer risk of patients with Peutz-Jeghers syndrome is significantly increased.The germline mutation of STK11 is the major factor of PJ syndrome.The aim of this study was to detect the mutation rate of colorectal cancer related genes.Methods: Peripheral blood of 65 Chinese PJS patients and 85 healthy controls in our department from June 2013 to December 2018 was collected.Genetic screening was carried out using a combination of next generation sequencing with a panel including 16 genes related colorectal cancer and multiplex ligation-dependent probe amplification(MLPA)assay.Results: In all 65 cases,no gene mutation was detected in 7 cases.28 mutational sites of STK11,5 mutational sites of PTEN,9 mutational sites of MSH2,4 mutational sites of EPCAM,6 mutational sites of MLH3 and 1 mutational sites of MLH1 were respectively detected in 42(65%),18(28%),17(25%),12(18%),8(12%)and 7(11%)cases with PJ syndrome.KEGG enrichment analysis showed the signal pathways associated with DNA mismatch repair,endometrial cancer and colorectal cancer were influenced.Among the 28 mutation sites of STK11,23 were located in exon and 5 in splicing area.26.7% cases was accompanied the gene mutation in exon 6 and the mutation sites were mostly located in exon 4 and exon 6.A total of 9 following novel mutation sites were detected: c.464+1G>A,c.862+1G>A,c.T482 G,c.G716 C c.T922 C,c.C783 G,c.C816 A,c.385 dup A,c.397 del G.The overall frequency of large deletions was 31.8%(7/22).Large fragment deletion was detected in 7 cases by MLPA.Conclusion: The mutation rate of colorectal cancer related genes including STK11,PTEN,MSH2,EPCAM,MLH3,MLH1 was significantly increased,which mainly related to the DNA mismatch repair and the pathways of endometrial cancer and colorectal cancer.The total germline mutation rate of STK11 was 76.9%,among which 66.2% was snp and 10.8% was large fragment deletion.Part? Study on the role of LKBl/AMPK/m TOR signalpathway in Peutz-Jeghers syndromeBackground and aims: To explore the role of LKB 1/AMPK/m TOR signal pathway in the occurrence of PJ syndrome and clarify the pathogenesis of PJ syndrome.Methods: All tissue specimens were collected in gastroenterology and general surgery department from June 2013 to December 2018 in Changhai Hospital.In 30 cases of normal para-carcinoma tissue of intestinal or colon as control group and 30 cases of PJ type polys were collected respectively as experimental group.LKB1,p AMPK,pm TOR expression was detected in the tissue with immunohistochemistry and western blot method.Results: The positive rate of LKB1,p AMPK in normal tissues were significantly higher than those in PJ-type polys tissues(83.3% vs 13.3%,P<0.001)and(76.7% vs 20%,P<0.001).While the pm TOR in normal tissues was significantly lower(33.3% vs 76.7,P=0.001).The results of WB also showed LKB1,p AMPK were remarkable higher and pm TOR remarkable lower in in normal tissues than PJ-type polys.The p AMPK and pm TOR in the 293 T cells transfected with LKB1 significantly decreased.Conclusion: The results of this study verified that LKB1/AMPK/m TOR signal pathway was crucial in the occurrence and development of PJ syndrome and it may become a new point or treatment of PJ syndrome..Part ? Altered Gut Microbiome in Peutz-Jeghers SyndromePatients with or without Germline Mutation in STK11Background and aims: Little is known about the potential link between gut microbiota and gastrointestinal polys of Peutz-Jeghers Syndrome(PJS).The aim of this case control study was to investigate the change of intestinal microbiota in PJS patients and explore the association between gut microbiota and germline mutations in STK11.Methods: 32 PJS patients(P group)and 35 age matched healthy controls(H group)from January 2017 to June 2018 were enrolled.Spouse was preferred to be selected as controls.Fecal samples of all participants were collected for gut microbiota analysis via 16 s r RNA gene(regions V3-V4)sequencing.Blood specimen of PJS patients were collected and next generation gene sequencing was used to detect STK11 mutations.Then the community of intestinal microbiota of PJS patients between STK11 mutation group(Y group)and no mutation(N group)group was furtherly investigated.Results: The mean community diversity indexes(?-diversity,including the Chao,Observed species,Shannon,and Simpson indexes)of the P group were comparable to those of the H group.There were significant differences of ?-diversity based on the weighted Uni Frac(P=0.001)and the unweighted Uni Frac(P=0.004)via Anosim analysis between the P and H group.Principal coordinate analysis(PCo A)demonstrated major clustering occurred at the individual level.The proteobacteria(LDA=4.84,P<0.001),gammaproteobacteria(LDA=4.91,P < 0.001),Aeromonadaceae(LDA=3.54,P=0.026),Actinomycetaceae(LDA=3.94,P=0.04),Morganella(LDA=3.71,P <0.001)and Proteus(LDA=2.83,P=0.003)were enriched in P group.Bacteroidetes(LDA=5.01,P < 0.001),Betaproteobacteria,Bacteroidales,Clostridium XI(LDA=3.52,P<0.001),Clostridium XVIII(LDA=2.97,P<0.001)were enriched in H group.29 significant pathways were identified in the level 2 of KEGG between P and H group.Comparied with the N group,Chao,Observed species,Shannon,and Simpson indexes were significantly lower.There were no?-diversity based on the weighted Uni Frac and the unweighted Uni Frac via Anosim analysis between the Yand N group.The following species were enriched in N group: Firmicutes(LDA=4.90,P=0.007),Clostridia(LDA=4.82,P=0.013),Clostridiales(LDA=4.82,P=0.013),Coriobacteriales(LDA=4.13,P=0.003)Coriobacteriaceae(LDA=4.13,P=0.003),Lachnospiraceae(LDA=4.71,P=0.004),Megamonas(LDA=4.18,P=0.004),Ruminococcus2(LDA=3.73,P=0.002),Clostridium Xl Va(LDA=4.20,P,=0.03),and Clostridium Xl Vb(LDA=3.51,P<0.001).8 significant pathways were identified in the level 2 of KEGG between Y and N group.Morganella was positively associated with JPN(r=0.96,P<0.001)and JPNG(r=0.78,P=0.04).Desulfovibrio was positively associated with JPNG(r=0.87,P=0.01).Klebsiella and Aeromonas were positively associated with BMI(r=0.93,P<0.001 and r=0.87,P=0.01).Blautia was negatively associated with JPS(r=-0.85,P=0.02).Anaerostipes was negatively associated with JPN(r=-0.77,P<0.04),JPNG(r=-0.88,P < 0.001)and JPS(r=-0.47,P < 0.04).Clostridium XVIII and Fusicatenibacter were negatively associated with JPN(r=-0.53,P=0.002)and JPS(r=-0.50,P=0.002)respectively.Conclusion: This study presents the remarkable alteration of gut microbiome in patients with PJS and elucidating the effect of STK11 gene mutation to gut microbiome.The changed gut microbiome can cause variation of metabolic pathways and furtherly lead to different clinical manifestion of PJ syndrome.Part ? Diagnosis and treatment of Peutz-Jeghers Syndromeand clinical relevance of the STK11 gene mutation with Peutz-Jeghers SyndromeBackground and Aims: The aim of this study was to summarize the clinical characteristics of PJ syndrome and evaluate the efficacy and safety of management of small-bowel polyps in patients with PJS under DBE,and furtherly clarify clinical relevance of the STK11 gene mutation in Peutz-Jeghers syndromeMethods: All consecutive patients diagnosed with PJS who accepted polypectomy under DBE in our department from January 2006 to December 2018 were enrolled.We retrospectively reviewed the records of all patients.Data include gender,age,history of surgery,the way of DBE insertion,the number of DBE procedures,the maximum size of resected polys,et al.According to if the patients were accompanied with germline mutation of STK11,all patients were assigned into mutation group(M group)and no mutation group(NM group).Then we analyze the difference of surgery history,age of first surgery,age of onset and definitive diagnosis,the number and maximum size of resected polys,et al between two groups.Results: A total of 97 patients with PJS were enrolled in this study.For both antegrade and retrograde DBE,the median maximum size of the resected polys during second hospitalization was significantly decreased compared to that during the first hospitalization(antegrade DBE: 2.13±1.51 cm vs 3.63±0.92 cm,P=0.012;retrograde DBE: 1.20±1.10 cm vs 2.95±1.95 cm,P=0.03),while remarkably larger than that during the third hospitalization sessions(antegrade DBE: 2.13±1.51 cm vs 0.88±0.70 cm,P=0.012;retrograde DBE: 1.20±1.10 cm vs 0.46±0.40 cm,P=0.048).The interval between the second and third hospitalization was significantly longer than that between the first and second period(899.00±382.12 vs 537.27±326.28,P=0.027).The success rate of total enteroscopy was 58.3%(28/48),which was not affected by a history of laparotomy(61.5% vs 52.4%,P=0.528).Among 64 cases,53 cases accepted poly resection under double ballon endoscopy and 40 cases were accompanied with STK11 gene mutation.Moreover,the first symptom occurs in M group was at a younger age than those in NM group(17.70±9.34 years vs 24.75±12.93 years,P=0.045)and the patients in M group were diagnosed at younger age than those in NM group(18.72±10.37 years vs 26.05±14.68 years,P=0.041).The family history has no significance between two groups.The patients in M group was more likely to have abdominal pain than those in NM group(86.8% vs 53.8%,P=0.013)and gastrointestinal bleeding(47.4% vs 15.4%,P=0.041).The insertion depth via antegrade DBE was 150(120,250)cm in M group,which was significantly smaller than NM group[250(200,250)cm,P=0.019],while there was no significant difference of insertion depth via retrograde DBE between two groups [150(115,200)cm vs 150(100,200)cm,P=0.830)].The number of admission and treatment between two groups were similar.For the first hospitalization,the number and the maximum size of resected polys via antegrate DBE in M group were remarkably larger than NM group[6(4,10)vs 4.5(2,7.7),P=0.042] and [4(3,5)cm vs 3(2,3)cm,P=0.014].For the second hospitalization,the number and the maximum size of resected polys via antegrate DBE in M group were larger than NM group,though with no significant difference [6(2.75,10)vs 6(5,16),P=0.415] and [3(1.5,4)cm vs 3(0.80,3.5)cm,P=0.823].While via retrograde DBE,no matter the during the first and the second session,the maximum size of resected polys were parallel in two groups.However,the maximum size of resected polys in M group was significantly larger than that in NM group[3(1.125,5)cm vs 1.5(0.65,3)cm,P=0.036].The first surgery of patients with deletion mutation at a remarkable younger age than those with splicing mutation[(24.0±5.16)vs(10.5±4.20)P=0.035].The times of hospitalation in Stop Mutation group was remarkable more than Nonsynomous(2.50±0.84 vs 1.67±0.78,P=0.032),Insertion(2.50±0.84 vs 1.00±0.00,P=0.008)and Deletion Mutation group(2.50±0.84 vs 1.50±0.55,P=0.026).During the first insertion via oral,the number of resected polys in Stopgain and Deletion Mutation group was significantly more than Nonsynomous[(12.00±8.83)vs(6.18±3.37,P=0.022],[(12.20±2.28 vs(6.18±3.37),P=0.025)],Splicing Mutation group[(12.00±8.83)vs(4.86±4.34),P=0.011],[(12.2±2.28 vs(4.86±4.34),P=0.013)] and Large fragment deletion group [(12.00±8.83)vs(3.67±1.53),P=0.019],[(12.2±2.28 vs(3.67±1.53),P=0.019)].The maximum size of resected polys in Deletion Mutation group was larger than Nonsynomous Mutation group [(4.60±0.55)cm vs(3.08±1.32)cm P=0.037].During first insertion via anal,the number of resected polys in Insertion Mutation group was more than Splicing Mutation group[(5.33±4.51)vs(1.73±1.73)P=0.038].Conclusion: DBE is clinically effective and safe for the diagnosis and therapy of small-bowel polyps in patients with PJS.The germline mutation of STK11 can affect the age of first surgery,onset and diagnosis,the insertion depth,the number and the maximum size of resected polys under DBE.