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.

[Clinical characteristics and mutation analysis of the LKB1 gene in a Peutz-Jeghers syndrome pedigree].

OBJECTIVE: To investigate clinical characteristics and mutation of the LKB1 gene in a Peutz-Jeghers syndrome (PJS) pedigree. METHOD: Clinical data of a PJS family were analyzed and LKB1 gene mutation was detected by systematic screening with multiplex ligation-dependent probe amplification (MLPA) and DNA sequencing. Meanwhile, two hundred and fifty healthy adults were enrolled in this study and denaturing high performance liquid chromatography (PCR-DHPLC) was carried out to verify the mutation excluding polymorphism sites found in this family. Changes in protein structure and function caused by the mutated coding sequence was analyzed by SWISS-MODEL software. RESULT: The proband had pigmented mucocutaneous lesions and multiple hamartomatous polyps in the gastrointestinal tract. There was no fragment deletion of LKB1 gene detected by MLPA. Among PJS family and 250 healthy adults, germline mutation c. 924G > C of LKB1 which cause Trp308Cys in protein sequence was identified only in the proband and another affected member. LKB1 protein activity could be reduced due to changes in LKB1 protein conformation structure by Trp308Cys. CONCLUSION: Peutz-Jeghers syndrome (PJS) is an autosomal dominant disorder characterised by mucocutaneous pigmentation, multiple gastrointestinal hamartomatous polyps and heredofamilial nature. Gene identification and mutagen screening of LKB1 gene in all PJS patients and first degree relatives will contribute to a definite diagnosis and improve the life span of the family.

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.

[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.

[Germline mutation of adenomatous polyposis coli gene in Chinese patients with familial adenomatous polyposis].

OBJECTIVE: To explore the characteristics of adenomatous polyposis coli (APC) gene germline mutations in Chinese patients with familial adenomatous polyposis (FAP). METHODS: Eighteen members from nine FAP pedigrees were studied by using multiplex ligation-dependent probe amplification(MLPA) to detect large fragment deletion of APC gene. Then, PCR were performed to amplify all exons of APC gene for mutation screening by denaturing high performance liquid chromatography (DHPLC). When abnormal elution profile of DHPLC was found, DNA sequencing was performed to determine the mutations. RESULTS: Mutations were identified in three pedigrees among the nine pedigrees. They were c.3184_3187 del CAAA in pedigree 2, c.5432C to T in pedigree 4 and c.3925_3929 del AAAAG in pedigree 9 respectively. Among them, c.5432C to T was novel. CONCLUSION: APC gene germline mutations can cause the development of FAP. The FAP patients without APC gene germline mutations could be caused by other mechanisms.

[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.