ObjectiveTo determine the frequency and type of p16 gene homozygous deletion and mutation with the progression of human gastric carcinogenesis.MethodsPCR （polymerase chain reaction），SSCP (single strand conformation polymorphism) and DNA sequencing were performed on 50 operative tissues, which included 7 early gastric cancer and 43 advanced gastric cancer. In this group there were 36 male and 14 female patients aged 35 to 68,which included 30 welldifferetiated adenocarcinoma and 20 poordifferentiated carcinoma; 19 patients with lymph node metastases.ResultsTotal mutation frequency of p16 gene was 8.00%(4/50), including 14.29%(1/7) of early cancer and 7.00%(3/43) of advanced cancer，there was no significant difference between early and advanced cancer （P＞0.05）. In addition, p16 gene mutation was not related to site，location，grade of tumor differentiation and lymph node metastases, respectively. Total deletion frequency of p16 gene was 16.00%(8/50), of which 18.60%(8/43) was detected in advanced cancer, but no cases in early cancers. There was a significant difference between early and advanced cancer （P＜0.05）, and frequency p16 gene homozygous deletion was related to grade of tumor differentiation and lymph node metastases, respectively. Direct DNA sequencing displayed that 4 point mutations detected in 99,125,433,441 base, there were 3 missense mutation and 1 samesense mutation, no deletion or insertion was found. Conclusionp16 gene mutation is an early event in gastric carcinogenesis，it helps early diagnosis of human gastric carcinoma. p16 gene homozygous deletion is a late event in gastric carcinogenesis，it might relate to metastases and recurrence of gastric cancer.
Objective To investigate the feasibility of detection of epidermal growth factor receptor ( EGFR) exon 19 deletions and exon 21 L858R mutations in pleural effusion fromnon-small-cell lung cancer ( NSCLC) patients by mutant enriched PCR assay. Methods The mutations of exon 19 and 21 of EGFR gene in pleural samples fromthirty NSCLC patients were analyzed using both the mutant-enriched PCR assay and the non-enriched PCR assay. Results Ten ( 33. 3% , 10/ 30) exon 19 deletions and five ( 16. 7% , 5/30) exon 21 L858R mutation were detected by the mutant-enriched PCR assay, while only 6 cases ( 20. 0% ) and 1 case ( 3. 3% ) were detected by the non-enriched PCR assay respectively. The difference of mutation detection rate of EGFR gene between the two methods was statistically significant ( P = 0. 032) . Mutations were detected in all of partial responders ( 2 /4) among the four patients who received gefitinib therapy. Conclusions Mutant-enriched PCR assay can detect EGFR exon 19 deletions and exon 21 L858R mutation in pleural effusion from NSCLC patients effectively, economically and accurately. It may be a valuable biomarker for gefitinib therapy in advanced NSCLC.
Objective To evaluate the clinical significance of epidermal growth factor receptor EGFR) mutations in the treatment of non-small cell lung cancer ( NSCLC) . Methods Plasma DNAs solated fromblood specimens of 170 NSCLC patients, who were admitted in the First Affiliated Hospital of uangzhou Medical College from December 2005 to December 2007, were subjected to the test of EGFR utant-enriched PCR. The correlation of mutant detection with clinical characteristics was analyzed as well.Results Out of the total 170 patients, EGFR mutations were identified in 77 cases ( 77 /170, 45. 3% ) .EGFR mutations were more frequent in the patients with adenocarcinoma ( P lt; 0. 001) and in the nonsmokers P =0. 001) . In the 33 patients treated with gefitinib, those with mutations ( + ) showed a higher esponse rate and prolonged progression-free survival after the treatment compared with those with mutations( - ) ( P =0. 001 and 0. 001, respectively) . Conclusions EGFR active mutations can be specifically and ensitively detected by EGFR mutant enriched PCR assay. Plasma EGFR mutants detection is valuable in uiding clinical decision.
Objective To observe the molecular genetic characteristics of seven Chinese families with Leberprime;s hereditary optic neuropathy (LHON). Methods Ophthalmologic examinations were performed on seven probands, maternal members from seven Chinese families and 134 healthy controls. There were two LHON patients in seven Chinese families except probands. The entire mitochondrial genome was amplified using 24 pairs of oligonucleotide primers with overlapping fragments.The mutational site was analyzed through comparison of the Results and Cambridge reference sequence. The penetrance of mutation site was calculated and the haplotype was analyzed. Results Molecular analysis of mitochondrial DNA (mtDNA) in these pedigrees revealed the absence of three common LHON associated with ND4 G11778A, ND1 G3460A and ND6 T14484C mutations. The ND1 T3394C mutation in probands and other matrilineal relatives was present in four out of 134 Chinese healthy controls. Strikingly, these families exhibited very low penetrance of visual impairment. The penetrance was 12.50%, 22.22%, 16.76%, 6.25%, 9.09%, 11.11% and 28.57%. The Results of phylogenetic tree analysis of submitochondrial haplotype showed that these mtDNA polymorphism sites belong to the Asian haplogroups M9, M9, M, D4, M, M9 and M9. Conclusions T3394C mutation exists in seven Chinese LHON pedigrees, and the penetrance was ranged from 6.25% to 28.57%. The patients have different clinical manifestations.
Objective To find the new mutations of Leber's hereditary optic neuropathy (LHON). Methods Two LHON families were enrolled in this study. The probands and all maternal members in this two families were underwent ophthalmologic examinations. The ages of probands were seven and 14 years old respectively. A total of 358 healthy adults were enrolled in this study as control group. The genomic DNA from whole blood of participants were extracted. The entire mitochondrial genome of probands were PCR amplified and sequenced in 24 overlapping fragments using primers as designed. At the same time, the mtDNA of maternal relatives and 358 controls were also detected. Fourteen primate species were selected from GenBank to analyzed the phylogenetics of mitochondrial sequence. Results There was no ND4 G11778A, ND1 G3460A, ND6 T14484C mutational site in all maternal members. Molecular analysis of mtDNA in this two families identified the homoplasmic tRNAGluA14683G mutation and distinct set of variants belonging to the Asian haplogroup F1a1 and G2. The site was at theTpsi;C stem oftRNAGlu and extremely conserved among 14 primate species. It was anticipated that the A14683G increased the highly conserved C-G basepairing. Furthermore, the A14683G was absence in control group. Conclusion The tRNAGluA14683G mutation is likely a new mutation associated with LHON.
Objective To investigate the correlation between mutation genotypes and phenotypes of X-linked retinoschisis (XLRS) patients. Methods 33 male XLRS patients, 26 female carriers and 100 normal subjects were enrolled in this study. All 33 XLRS patients were bilateral, which included 18 patients from 8 families and 15 sporadic patients. Among 66 XLRS eyes, there are microcystis-like foveal splitting in 49 eyes (74.2%), lamellar macular splitting in 43 eyes (65.2%), peripheral splitting in 32 eyes (48.5%), retinal detachment in 17 eyes (25.8%), and vitreous hemorrhage in 8 eyes (13.6%). Electroretinogram was performed on 42 eyes which showed decreased amplitude of b-wave. The 6 exons of RS1 gene were amplified by polymerase chain reaction and then directly sequenced.The correlation analysis was performed between mutation genotypes and phenotypes. Results There were 19 RS1 gene mutations including 6 novel mutations (p.Gly70Cys, p.Trp112Arg, p.Arg156Trp, p.His207ProfsX56, p.Arg209AlafsX28, p.Cys223Tyr). There was no correlation between mutation genotypes and phenotypes (chi;2=0.731, 3.438, 0.820, 3.208, 1.992; P＞0.05 ).Conclusions RS1 gene mutation is a major cause of XLRS. The RS1 mutation genotype is not correlated with phenotype, so that the prognosis cannot be predicted by the genotypes.
Objective To screen and analyze NR2E3 gene mutations in rentinitis pigmentosa (RP) patients from Ningxia area of China. Method 120 RP patients were enrolled in this study. The patients include 33 autosomal dominant RP (ADRP) patients from 18 families, 20 autosomal recessive RP (ARRP) patients from 15 families, and 67 simplex RP (SRP) patients.100 healthy people were collected as the control group. PCR and direct DNA sequencing were used to screen the entire coding region and splice sites of NR2E3 gene. Multiple analysis was used to study the effects of NR2E3 gene on RP. ResultsA total of 12 different sequence variants in the NR2E3 gene were identified, including 6 novel sequence variants. 5 variants were detected in non-coding regions; 7 variants were detected on the 4th, 6th, 7th exon which including 3 synonymous mutations and 4 missense mutations. All of them were NR2E3 gene polymorphisms and showed no positive correlation with the RP confirmed by the multivariate logistic regression analysis. The missense mutation of p.Glu121Lys was first found in 1 ADRP proband, 2 SRP patients and 2 control subjects. Among other 8 affected individuals in this ADRP family, 5 patients also had the p.Glu121Lys variant. Notably, the 6 affected individuals with p.Glu121Lys showed more serious ophthalmic findings (early onset and early central visual impairment) than other 3 affected individuals without p.Glu121Lys.Conclusion The mutation frequency of NR2E3 and p.Glu121Lys variant in NR2E3 gene in Ningxia RP patients were lower than previous reports in other populations.
Objective To screen mitochondrial DNA mutations in 3 Chinese pedigrees with Leberprime;s hereditary optic neuropathy (LHON) carrying the ND1 G3635A mutation. Methods 88 members（53 maternal relatives and 35 paternal relatives）in 3 pedigrees were enrolled. The ophthalmologic examinations were performed for all members, including visual acuity (standard logarithmic visual acuity charts), fundus photography (Canon fundus camera),visual field (Humphrey Visual Field Analyzer), color vision (Yu zhiping color vision plate), and visual evoked potentials (Roland Consult RETI port gamma, flash VEP). 16 members had LHON, 72 members did not have LHON. 135 healthy people from Wenzhou were included as the control group. Genomic DNA was extracted from peripheral blood leukocytes of all subjects. G3635A mutation was screened by PCR mplification of mitochondrial DNA for all subjects. Mitochondrial haplotypes and other mutations in the entire mitochondrial genome were also determined by PCR using 24 pairs of primers for the probands. Results Analysis of mitochondrial DNA (mtDNA) in 3 pedigrees revealed the presence of ND1 G3635A mutation in 3 probands and all maternal relatives, but not in paternal relatives and healthy controls. Probandprime;s haplogroup belong to East Asia group N9a3, D4, and R11a. In addition to the G3635A mutation, probands also had other variants including 12 variants in D-loop region, 6 variants in RNA gene, and 36 variants in protein-encoding gene. Conclusions G3635A mutation was identified in probands and maternal relatives of 3 pedigrees of LHON. It showed that G3635A mutation was the pathogenic molecular basis for those patients.
Objective To make the diagnosis of a pedigree of X-linked congenital stational night blindness(CSNB) and to identify the disease-causing gene. Methods Clinical examination and family analysis were made. Venous blood was drawn from 5 affected and 16 unaffected individuals from the family. Genomic DNA was extracted. The locus of the candidate gene was mapped by linkage study. Mutation was screened by polymerase chain reaction (PCR) of the candidate gene exons and flanked introns. The PCR products are directly sequenced. The healthy people in and out of the family who were selected according to certain standards were as the control. Results A Chinese family with X-linked complete congenital stationary night blindness (CSNB1) was diagnosed. A missense mutation A772C (T258P) in exon 2 of NYX gene was identified in all affected patients and all female carriers were heterozygous. This mutation was neither found in normal family members nor among 110 unrelated normal controls. Conclusion A novel mutation of NYX gene with threonine to proline change is responsible for this Chinese CSNB1 family. (Chin J Ocul Fundus Dis, 2007, 23: 184-188)
Objective To investigate the mutations of the gene in Chinese patients with X linked juvenile retinoschisis (XLRS), and to provide the genetic diagnosis and consultation of heredity for the patients and their families. Methods Genomic DNA was isolated from leukocytes of 29 male patients with XLRS, 38 female carriers and 100 normal controls (the patients and the carriers were from 12 families). All 6 exons of XLRS1 gene were amplified by polhism (SSCP) assay. The positions and types of XLRS1 gene mutations were determined by direct sequencing. Results Eleven different XLRS1 mutations were identified in these 12 families, including one frameshift mutation due to base loss of the first exon: c.22delT（L9CfsX20）, one nonsense mutation due to base loss of the first exon (Trp163X), one splice donor site mutation(c.52+2 Trarr;C; IVS1+2T to C), and eight missense mutation due to base replacement（Ser73Pro, Arg102Gln, Asp145His, Arg156Gly, Arg200Cys, Arg209His, Arg213Gln, and Cys223Arg). No gene mutation was detected in the control group. Four new mutations included frmaeshift mutation（L9CfsX20）and mutations of Asp145His, Arg156Gly, and Trp163X at the fifth exon. A newly discovered non-disease-related polymorphism (NSP) was the c.576C to T (Pro192Pro) change at the sixth exon. Conclusion Eleven different XLRS1 mutations were detected, which is the cause of XLRS in Chinese people. The detection of gene mutations may provide the guidance of genetic diagnosis and the consultation of family heredity for the patients and their families. (Chin J Ocul Fundus Dis, 2006, 22: 77-81)