APC gene mutations in Chinese familial adenomatous polyposis patients

AIM:To study the characteristics of APC(adenomatous polyposis coli)gene germline mutation in Chinese patients with familial adenomatous polyposis(FAP).METHODS:APC gene from 14 FAP families was amplified by polymerase chain reaction(PCR)and underwent direct sequencing to determine the micromutation type.For the samples without micromutation,the large fragment deletion of APC gene was examined by multiplex ligation-dependent probe amplification(MLPA).RESULTS:There were gene micromutations in 9 families with a micromutation detection rate of 64.3%(9/14),including 6 frameshift mutations(66.7%),1 nonsense mutation(11.1%)and 2 splicing mutations(22.2%).Large fragment deletions were detected by MLPA in 2 families.The total mutation detection rate of micromutations and large fragment deletions was 78.6%(11/14).CONCLUSION:The detection rate of APC gene germline mutation can be improved by direct sequencing combined with MLPA large fragment deletion detection.

Genetic diagnosis strategy of hereditary non-polyposis colorectal cancer

AIM: To study the characteristics of mismatch repair gene mutation of Chinese hereditary non-polyposis colorectal cancer (HNPCC) and hMLH1 gene promoter methylation, and to improve the screening strategy and explore the pertinent test methods. METHODS: A systematic analysis of 30 probands from HNPCC families in the north of China was performed by immunohistochemistry, microsatellite instability (MSI), gene mutation and methylation detection. RESULTS: High frequency microsatellite instability occurred in 25 probands (83.3%) of HNPCC family. Loss of hMLH1 and hMSH2 protein expression accounted for 88% of all microsatellite instability. Pathogenic muta-tion occurred in 14 samples and 3 novel mutational sites were discovered. Deletion of exons 1-6, 1-7 and 8 of hMSH2 was detected in 3 samples and no large fragment deletion was found in hMLH1. Of the 30 probands, hMLH1 gene promoter methylation occurred in 3 probands. The rate of gene micromutation detection combined with large fragment deletion detection was 46.7%-56.7%. The rate of the two methods in combination with methylation detection was 63.3%. CONCLUSION: Scientific and rational detection strategy can improve the detection rate of HNPCC. Based on traditional molecular genetics and combined with epigenetics, multiple detection methods can accurately diagnose HNPCC.

Is the human dystrophin gene's intron structure related to its intron instability?

Objective To study the human dystrophin gene molecular deletion mechanism, we analyzed breakpoint regions within junction fragments of deletion-type patients and investigated whether the dystrophin gene's intron structure might be related to intron instability.Methods Junction fragments corresponding to exon 46 and 51 deletions were cloned. The breakpoint regions were sequenced, and the features of introns with available Genebank sequences were analyzed.Results An analysis of junction fragment sequences corresponding to exon 46 and 51 deletions showed that all 5' and 3' breakpoints are located within repeat sequences. No small insertions, small deletions, or point mutations are located near the breakpoint junctions. By analyzing the secondary structure of the junction fragments, we demonstrated that all junction fragment breakpoints are located in non-matching regions of single-stranded hairpin loops. A high concentration of repetitive elements is found to be a key feature of many dystrophin introns. In total, 34. 8% of the overall dystrophin intron sequences is composed of repeat sequences.Conclusion Repeat elements in many dystrophin gene introns are the key to their structural bases and reflect intron instability. As a result of the primary DNA sequences, single-stranded hairpin loops form, increasing the instability of the gene, and forming the base for breaks in the DNA. The formation of the single-stranded hairpins can result in reattachment of two different breakpoints, producing a deletion.