Analysis of Small Fragment Deletions of the APC gene in Chinese Patients with Familial Adenomatous Polyposis, a Precancerous Condition.

BACKGROUND: Familial adenomatous polyposis (FAP) is an autosomal dominant inherited disease mainly caused by mutations of the adenomatous polyposis coli (APC) gene with almost complete penetrance. These colorectal polyps are precancerous lesions that will inevitable develop into colorectal cancer at the median age of 40-year old if total proctocolectomy is not performed. So identification of APC germline mutations has great implications for genetic counseling and management of FAP patients. In this study, we screened APC germline mutations in Chinese FAP patients, in order to find novel mutations and the APC gene germline mutation characteristics of Chinese FAP patients. MATERIALS AND METHODS: The FAP patients were diagnosed by clinical manifestations, family histories, endoscope and biopsy. Then patients peripheral blood samples were collected, afterwards, genomic DNA was extracted. The mutation analysis of the APC gene was conducted by direct polymerase chain reaction (PCR) sequencing for micromutations and multiplex ligation-dependent probe amplification (MLPA) for large duplications and/or deletions. RESULTS: We found 6 micromutations out of 14 FAP pedigrees, while there were no large duplications and/or deletions found. These germline mutations are c.5432C>T(p. Ser1811Leu), two c.3926_3930delAAAAG (p.Glu1309AspfsX4), c.3921_3924delAAAA (p.Ile1307MetfsX13), c3184_3187delCAAA(p.Gln1061AspfsX59) and c4127_4126delAT (p.Tyr1376LysfsX9), respectively, and all deletion mutations resulted in a premature stop codon. At the same time, we found c.3921_3924delAAAA and two c.3926_3930delAAAAG are located in AAAAG short tandem repeats, c3184_3187delCAAA is located in the CAAA interrupted direct repeats, and c4127_4128 del AT is located in the 5'-CCTGAACA-3' ,3'-ACAAGTCC-5 palindromes (inverted repeats) of the APC gene. Furthermore, deletion mutations are mostly located at condon 1309. CONCLUSIONS: Though there were no novel mutations found as the pathogenic gene of FAP in this study, we found nucleotide sequence containing short tandem repeats and palindromes (inverted repeats), especially the 5 bp base deletion at codon 1309, are mutations in high incidence area in APC gene.

Plasma post-operative miR-21 expression in the prognosis of gastric cancers.

Tumor-associated microRNAs have been detected in serum or plasma, but whether plasma microRNA-21 (miR-21) could be a potential circulating biomarker for gastric cancer (GC) prognosis in Chinese is still uncertain. Real-time quantitative reverse transcription PCR (qRT-PCR) was employed in this study to compare the relative expression of miR-21 between pre-operative and post-operative paired plasmas from 42 patients with primary GCs. The results showed that the expression levels of miR-21 in the post-operative plasmas were significantly reduced by an average of 18.2 times in all patients when compared to the pre-operative plasmas, and by 22.1 times in the subgroup of patients without family history, while only 1.76 times in the subgroup of patients with a family history. With respect of clinicopathological characteristics, the plasma miR-21 expression was highly associated with differentiation degree and lymph node metastasis rate. The results suggested plasma miR-21 could be a novel potential biomarker for GC prognosis and evaluation of surgery outcomes, especially in patients without a family history.

[Mutation screening of LKB1 gene in familial Peutz-Jeghers syndrome patients].

OBJECTIVE: To screen for potential mutations of LKB1 gene in Chinese familial Peutz-Jeghers syndrome (PJS) patients and analyze their clinical manifestations. METHODS: Eleven PJS families were collected and genomic DNA of peripheral blood was extracted. Typically mucosal pigmentation and hamartomatous polyps were present in all 11 probands. Mutation screening of the probands were carried out by PCR and direct sequencing. Two hundred and fifty healthy adults were enrolled as normal controls, for whom genomic DNA of peripheral blood was also extracted. PCR-denaturing high performance liquid chromatography was carried out to verify the mutation identified in the patients. RESULTS: Nine germline mutations were identified in eight PJS patients, which included 7 point mutations, 1 deletion and 1 insertion. Among these, 4 were considered to be pathogenic, of which 2 were de novel, 4 were considered to be polymorphism, and 1 was uncertain. CONCLUSION: LKB1 gene mutations with pathogenic effect are a common cause of familial PJS in Chinese patients. Most mutations are point mutations.

[Clinical therapeutic effect and biological monitoring of p53 gene in advanced hepatocellular carcinoma].

OBJECTIVE: To investigate the therapeutic effect and biological changes of hepatic arterial perfusion of p53 gene via port catheter system (PCS) on advanced hepatocellular carcinoma. METHODS: A total of 48 cases of advanced hepatocellular carcinoma were divided into the experimental group (30) and the control group (18). Transiliac external artery PCS implantation was performed in all cases. p53 gene was perfused into target artery confirmed by angiography. In the experimental group, 10(12) VP of p53 gene and 20 mg OPT were employed every week as a course for 21 days. In the control group, only 20 mg OPT was used. KPS, AFP and the survival period, RECIST (response evaluation criteria in solid) tumor were analyzed. Flow cytometry (FCM) and micronucleus (MN) assay in vivo were used to detect p53 gene mutation and spontaneous micronucleus formation in peripheral blood of the experimental group. RESULTS: The experimental group were performed 1 to 8 courses. There was a significant difference with AFP level and KPS in the experimental group(P < 0.05). However there was no significant difference (P > 0.05) in the control group. After one month, survival rate of the the experimental group and the control group was 96.6% and 94.4%, there was a significant difference between the two groups in objective tumor relieve (P < 0.05). After three months, survival rate of the the experimental group and the control group was 83.3% and 55.6%, there was a significant difference between the two groups in objective tumor relieve (P < 0.05). After six months, survival rate of the the experimental group and the control group was 50.0% and 11.1%, there was a significant difference between the two groups in objective tumor relieve (P < 0.05). After nine months, survival rate of the the experimental group and the control group was 23.3% and 0%, there was a significant difference between the two groups in objective tumor relieve (P < 0.05). After twelve months, survival rate of the the experimental group and the control group was 6.67% and 0%, there was a significant difference between the two groups in objective tumor relieve (P < 0.05). The depression of p53 expression was observed in the HCC patients who were employed four times of intervention operations. The difference of p53 expression between before and after interventional rAd-p53 therapy were statistically significant (P < 0.01). The frequency of MN depressed by the rAd-p53 was seen in the patients, and the differences of the frequency of MN between before and after interventional rAd-p53 therapy were statistically significant (P < 0.05). CONCLUSION: p53 gene sequential infusion via hepatic artery is effective for advanced hepatocellular carcinoma. The biological study will play a important role in selecting the therapeutic dose and judging therapeutic efficacy by means of guiding and monitoring.

[Association of IVS10+12G>A polymorphism in hMSH2 gene with colorectal cancer in Chinese].

OBJECTIVE: To investigate the association of the single-nucleotide polymorphism (SNP) IVS10+12 G>A in hMSH2 gene with colorectal cancer in a Chinese population of Jiangsu province. METHODS: A case-control study to investigate whether this SNP affects the risk of developing colorectal cancer was conducted. Subjects included 108 colorectal cancer patients and 180 healthy individuals. Peripheral white blood cell DNA was obtained from all subjects. The hMSH2 gene IVS10+12 G>A was genotyped using a PCR-based DHPLC, the existence of IVS10+12 G>A was verified by DNA sequencing. RESULTS: The allele frequency of the IVS10+12 G>A in the hMSH2 gene in the healthy individuals was 51.7%. There was significant difference in the frequency of the IVS10+12 G>A between patients and healthy controls (P<0.05), and between familial patients and healthy controls (P<0.05). There was also significant difference of the frequency of the IVS10+12 G>A between patients younger than 50 years, and patients with high consumption of fried food and pickled vegetable and healthy controls respectively (P<0.05). CONCLUSION: This SNP may be associated with colorectal cancers in Chinese. Further investigation with larger sample size is needed.

[Mismatch repair gene hMLH1 A655G/A polymorphism and colorectal cancer].

OBJECTIVE: To investigate the etiological role of hMLH1 gene A655 polymorphism in colorectal cancer. METHODS: A case-control study was carried out, including 115 colorectal cancer patients and 135 healthy people as control. Genomic DNA was extracted from peripheral white blood cell from all the subjects. Polymorphism was detected by PCR-based DHPLC analysis and verified by DNA sequencing. RESULTS: The hMLH1 gene A655G polymorphism was detected in 3.0% of healthy people and 11.3% of colorectal cancer patients (P<0.01), and the difference was significant (P<0.01). The hMLH1 gene A655G polymorphism was detected in 8.2% of tubular adenocarcinoma or tubular-papillary adenocarcinoma and 27.8% of mucinous adenocarcinoma, which was also significant (P<0.05).Meanwhile, hMLH1 gene A655G polymorphism was not associated with age, gender and lymphatic metastasis (all P>0.05). CONCLUSIONS: The hMLH1 gene A655G polymorphism may play a role in the pathogenesis of colorectal cancer. Determination of the polymorphism may be a potential marker to predict the prognosis of colorectal cancer patients.

[Promoter hypermethylation and loss of heterozygosity of the APC gene in patients with familial adenomatous polyposis].

OBJECTIVE: To investigate the status of hypermethylation in the promoter 1A region of the adenomatus polyposis coli (APC) gene in 3 familial adenomatous polyposis (FAP) pedigrees and to screen large fragment deletions in the APC gene. METHODS: DNA from tumor tissues and corresponding normal tissues of 5 FAP patients was modified by sodium bisulfite. Then the methylation status of the APC gene was analyzed by methylation specific-PCR (MSP) and DNA sequencing. Multiplex ligation-dependent probe amplification (MLPA) was used to screen aberrations involving large fragments from all the 15 exons and promoter region of APC gene. RESULTS: No methylation was present in normal tissues. Hypermethylation was found in tumor tissues of one proband and her son. Loss of heterozygosity was observed in another patient from the same FAP family. CONCLUSION: Aberrant methylation of the APC promoter region provides an important mechanism for impairing APC function and may occur early during colon neoplasia progression. Loss of heterozygosity may play a role in patients with classical polyposis.

[Simultaneous detection of polymorphisms in the 3'- and 5'-UTR of thymidylate synthase gene using denaturing high-performance liquid chromatography].

Polymorphism which is either in the thymidylate synthase (TS)enhancer region (TSER) of the 5-primer untranslation region (5' UTR) or in the 3-primer untranslation region (3' UTR) has been reported to be associated with the alterations in TS mRNA and protein levels. The TSER is characteristic of the presence of variable double (2R) and triple (3R) number tandem repeats (VNTRs). In addition to VNTRs, single nucleotide polymorphism (SNPs) in 3R, as well as the polymorphism of the fragment length(FLP) in the TS 3'-UTR which is characteristic of the presence or absence of a 6 bp- nucleotide fragment, has recently been reported to be associated with the response to 5-fuorouracil (5-FU)-based chemo-therapy. The aim of the present study was to develop a specifc denaturing high-performance liquid chromatography (DHPLC) method for the rapid and simultaneous detection of these variations in clinical samples. Multi-PCR primers were designed to amplify the two regions simultaneously, The 8.6 min DHPLC gradient was optimized to include the analysis of multiplexed TSER/3' UTR chromatogram peaks, allowing for the simultaneous detection of 28 bp VNTRs and 6 bp FLP under nondenaturing conditions (50). The optimal melting temperature was determined experimentally for the detection of SNP in the TSER VNTRs. Finally, the DHPLC analysis was verified in parallel with PCR-RFLP and sequencing. The optimized DHPLC method resolved 100% of the known TS variations, discriminated between homozygous and heterozy-gous genotypes. This developed DHPLC method could permit the rapid, sensitive, and accurate identification of the TS genotypes (VNTRs, SNPs, and FLP).

[A novel APC gene germline mutation in a familial adenomatous polyposis pedigree].

OBJECTIVE: To detect the adenomatous polyposis coli (APC) gene germline mutation in the proband and her family members with familial adenomatous polyposis (FAP). METHODS: The diagnosis of a patient with FAP was validated by colonoscopy, pathology and the family history. The systematic screening with multiplex ligation-dependent probe amplification (MLPA), denaturing high-performance liquid chromatography (DHPLC) and DNA sequencing were carried out to detect APC gene germline mutations. RESULTS: A novel mutation c.1999 C >T (Q667X) of APC, which leads to premature termination of the protein, was identified in this family. This mutation manifested an aggressive form of FAP with early onset of colorectal adenocarcinoma and colonic adenoma. CONCLUSION: The mutation of APC Q667X is the cause of clinical phenotype of this family with FAP, and the prophylactic colectomy for the affected family members should be considered.

[The expression of mutation p53 gene from circulating cancer cells of peripheral blood and the clinical significance in detecting the patients with colorectal cancer].

OBJECTIVE: To study the clinical significance of detecting p53 gene mutation expression in colorectal cancer cells of peripheral blood. METHODS: Flow cytometry (FCM) was used to detect p53 gene mutation expression in peripheral blood cancer cells of 128 patients with colorectal cancer. Experimental data were analyzed by SPSS (v.11.0) software. RESULTS: The lymph node metastasis showed the significant difference statistically (P<0.01) between p53 positive and negative expression in the colorectal cancer patients. The mutation p53 expression associated with existing histological differentiation (r=0.8476, P<0.05). A lymph node metastasis difference was observed between left and right colorectal cancers of mutation p53 positive expression. CONCLUSION: Detecting the mutation p53 expression in cancer cells of peripheral blood might be helpful to the early diagnosis of colorectal cancer.

[Study of in vivo micronucleus formation in lymphocytes from the patients and its relation to malignant degrees of colorectal cancer].

OBJECTIVE: To investigate the association of the micronucleus (MN) formation in lymphocytes from patients with the malignant degrees of colorectal cancer. METHODS: The MN test in capillary blood lymphocytes was conducted in 112 patients randomly selected from in-hospital patients before therapy. Experimental data were analyzed by SPSS (v.10.1) software. RESULTS: The differences in the frequency of MN between 7 pathological types of colorectal cancers and controls were statistically significant (P<0.01). The frequency of MN increased with the decrease of the histological differentiation in colorectal cancer, and the statistically significant differences were seen between low differentiation group and the other differentiation groups in colorectal cancers. CONCLUSION: There is a significant correlation between MN formation and the malignant degrees of colorectal cancer, and MN formation will be a useful biomarker for the identification of malignant degrees of colorectal cancer before operation or for the screening of high risk subgroup.