Comparative analysis of dideoxy sequencing,the KRAS StripAssay and pyrosequencing for detection of KRAS mutation

AIM:To compare the differences between dideoxy sequencing/KRAS StripAssay/pyrosequencing for detection of KRAS mutation in Chinese colorectal cancer (CRC) patients.METHODS:Formalin-f ixed, paraff in-embedded (FFPE) samples with tumor cells ≥ 50% were collected from 100 Chinese CRC patients at Beijing Cancer Hospital. After the extraction of genome DNA from FFPE samples, fragments contained codons 12 and 13 of KRAS exon 2 were amplified by polymerase chain reaction and analyzed by dideoxy sequencing, the KRAS Strip Assay and pyrosequencing. In addition, the sensitivities of the 3 methods were compared on serial dilutions (contents of mutant DNA: 100%,50%,20%, 5%,10%, 5%,1%,0%) of A549 cell line DNA (carrying the codon 12 Gly>Ser mutation) into wild-type DNA (human normal intestinal mucosa). The results of dideoxy sequencing,the KRAS StripAssay and pyrosequencing were analyzed by Chromas Software, Collector forKRAS Strip Assay and the pyrosequencing PyroMarkTM Q24 system, respectively.RESULTS: Among 100 patients, KRAS mutations were identif ied in 34%, 37% and 37% of patients by dideoxy sequencing, the KRAS StripAssay and pyrosequencing, respectively. The sensitivity was highest with the KRAS Strip Assay (1%), followed by pyrosequencing (5%), and dideoxy sequencing was lowest (15%). Six different mutation types were found in this study with 3 main mutations Gly12 Asp (GGT>GAT), Gly12 Val (GGT>GTT) and Gly13 Asp (GGC>GAC). Thirty-three patients were identifi ed to have KRAS mutations by the 3 methods, and a total of 8 patients had conflicting results between 3 methods: 4 mutations not detected by dideoxy sequencing and the KRAS StripAssay were identified by pyrosequencing; 3 mutations not detected by dideoxy sequencing and pyrosequencing were identif ied by the KRAS StripAssay; and 1 mutation not detected by pyrosequencing was conf irmed by dideoxy sequencing and the KRAS StripAssay. Among these discordant results, the results identif ied by dideoxy sequencing were consistent either with the KRAS StripAssay or with pyrosequencing, which indicated that the accuracy of dideoxy sequencing was high. CONCLUSION: Taking a worldwide view of reports and our results,dideoxy sequencing remains the most popular method because of its low cost and high accuracy.

APC and K-ras gene mutation in aberrant crypt foci of human colon

AIM:To study the genetic alteration in ACF and to define the possibility that ACF may be a very early morphological lesion with molecular changes,and to explore the relationship between ACF and colorectal adenoma even carcinoma. METHODS: DNA from 35 CRC, 15 adenomas, 34 ACF and 10 normal mucus was isolated by means of microdissection. Direct gene sequencing of K-ras gene including codon 12, 13 and 61 as well as the mutation cluster region (MCR) of APC gene was performed. RESULTS: K-ras gene mutation frequency in ACF, adenoma and carcinoma was 17.6% (6/34), 13.3% (2/15), and 14.3% (5/35) respectively, showing no difference (P 】 0.05) in K-ras gene mutation among three pathologic procedures. The K-ras gene mutation in adenoma, carcinoma and 4 ACF restricted in codon 12 (GGT GAT), but the other 2 mutations from ACF located in codon 13 (GGC GAC). K-ras gene mutation was found more frequently in older patients and patients with polypoid cancer. No mutation in codon 61 was found in the three tissue types. Mutation rate of APC gene in adenoma and carcinoma was 22.9% (8/35) and 26.7% (4/15), which was higher than ACF (2.9%) (P 【0.05). APC gene mutation in carcinoma was not correlated with age of patients, location, size and differentiation of tumor. CONCLUSION: ACF might be a very early morphological lesion in the tumorogenesis of colorectal tumor. The morphological feature and gene mutation status was different in ACF and adenoma. ACF is possibly putative microadenoma that might be the precursor of adenoma. In addition, the development of a subgroup of colorectal carcinomas might undergo a way of normal epithelium ACF carcinomas .

Clinical and genetic characteristics of Chinese hereditary nonpolyposis colorectal cancer families

AIM： To analyze the clinical characteristics of Chinese hereditary nonpolyposis colorectal cancer （HNPCC） families and to screen the germline mutations of human mismatch repair genes hMLH1 and hMSH2 in the probands. METHODS： Thirty-one independent Chinese HNPCC families were collected in Zhejiang Province. All of them met Chinese HNPCC criteria. Clinical data about patient gender, site of colorectal cancer, age of onset, history of multiple colorectal cancer, associated extracolonic cancer were recorded. PCR and denaturing high performance liquid chromatography （DHPLC） were employed to screen the mutations. Sequencing analysis was used to find out the exact mutation site and characteristics of the samples showing abnormal DHPLC profiles. RESULTS： One hundred and thirty-six malignant neoplasms were found in 107 patients including 14 multiple cancers. One hundred and six of the 136 neoplasms （77.9%） were diagnosed as colorectal cancer, with an average age of onset at 48.57 ±29.00 years. Gastric cancer was the most common extracolonic cancer （10.3%） in these families. Twenty-three different sequence variations in hMLHI and hMSH2 genes were detected in these 17 families. Fifteen sequence variations were located in the exons, including 5 SNPs, 3 silent mutations, 3 missense mutations, 2 nonsense mutations and 2 frameshift mutations. The latter seven mutations seemed to be pathogenic. CONCLUSION： Germline mutations of hMLH1 and hMSH2 genes are identified in about one-third HNPCC kindreds fulfilling Chinese HNPCC criteria. Chinese HNPCC families have some particular clinical characteristics, such as a left-sided predominance, less synchronous or metachronous colorectal cancer, and frequent occurrence of gastric cancer.

Relationship between a novel polymorphism of lipoprotein lipase gene and coronary heart disease

OBJECTIVE: To investigate polymorphisms in the gene for lipoprotein lipase (LPL) in Chinese populations with coronary heart disease (CHD) and to inquire into the relationship between these polymorphisms in LPL gene and CHD. METHODS: Genomic DNA was extracted from patients with CHD and normal control subjects using a salting out method. The entire coding region and flanking sequences of all coding exons of the LPL gene were amplified by PCR technique and PCR products were detected by denaturing high-performance liquid chromatography (DHPLC) and sequenced with a dideoxy terminal termination method. RESULTS: A novel polymorphic site, G830A, that is within the fifth exon of the LPL gene was found. The 192 codon CGA was changed into CAA and resulted in the substitution of glutamine for arginine. Between the control and CHD groups, chi-square test showed no significant difference in the frequencies of the A/A genotype and A allele (P > 0.05). However, the frequencies of A/A genotype and A allele (0.653 and 0.786) in CHD patients with high plasma triglyceride/lowed plasma high density lipoprotein cholesterol were higher than those (0.415 and 0.642) in CHD patients without hyperlipidemia (P Gln substitution polymorphism and CHD, but there is a significant positive correlation between the A/A genotype of the LPL gene and CHD associated with high triglyceride/lowed high density lipoprotein cholesterol. This study may provide new data for exploring the molecular mechanism of CHD.

Identification of Seven Novel Mutations in the Acid Alpha-glucosidase Gene in Five Chinese Patients with Late-onset Pompe Disease

Background： Pompe disease is a rare lysosomal glycogen storage disorder linked to the acid alpha-glucosidase gene （GAA）. A wide clinical and genetic variability exists between patients from different ethnic populations, and the genotype-phenotype correlations are still not well understood. The aim of this study was to report the clinicopathological and genetic characteristics of five Chinese patients with late-onset Pompe disease （LOPD） who carried novel GAA gene mutations. Methods： Clinical and pathological data of patients diagnosed with glycogen storage disease at our institution from April 1986 to August 2017 were collected, and next-generation sequencing of frozen muscle specimens was conducted. Results： Of the five patients included in the study, the median disease onset age was 13 years, with a median 5 years delay in diagnosis. The patients mainly manifested as progressive weakness in the proximal and axial muscles, while one patient developed respiratory insufficiency that required artificial ventilation. In muscle biopsies, vacuoles with variable sizes and shapes appeared inside muscle fibers, and they stained positive for both periodic acid-Schiff and acid phosphatase staining. Ten GAA gene mutations, including seven novel ones （c.796C〉A, c. 1057C〉T, c. 1201C〉A, c. 1780C〉T, c. 1799G〉C, c.2051C〉A, c.2235dupG）, were identified by genetic tests. Conclusions： The seven novel GAA gene mutations revealed in this study broaden the genetic spectrum of LOPD and highlight the genetic heterogeneity in Chinese LOPD patients.

Receptor expression-enhancing protein 1 gene （SPG31） mutations are rare in Chinese Han patients with hereditary spastic paraplegia

Hereditary spastic paraplegia （HSP）, also known as familial spastic paraparesis or Stumpell-Lorrain disease, is a large group of inherited, heterogeneous neurologic disorders caused by the degeneration of corticospinal axons. The prevalence is estimated at 3-10 cases per 100000 people in Europe, and is uncertain in other continents. Most patients have the same core features, which are characterized by spastic gait, lower limb hypertonicity, hyperreflexia, extensor-plantar responses, muscle weakness, and occasionally decreased vibration sense at the ankles, bladder dysfunction, pes cavus, or scoliosis.