Purpose To investigate the relationship between mitochondrial DNA 11778 mutation and clinical characteristics of patients with Laber is hereditary optic neuropathy(LHON). Methods PCR RFLPs (MaeⅢ) and mutation specific primer PCR(MSP-PCR) were used simultaneously to detect mitochondrial DNA 11778 mutation. Results Among 10 subjects who habored 11778 mutation,one was a carrier and nine were patients with LHON.Of the nine patients,six were males and three were females.The age of onset ranged from 12 to 25 years old and the onset interval of the two eyed varied between 0 to 6 months. The visual acuity was CF/10cm-0.1 except one who lost her vision after delivery but recovered gradually.The results of visual field,VEP and color vision were abnormal but ERG and systemic status were all normal. Conclusion Molecular biological detection of the ten subjects showed that they all habored mtDNA 11778 mutation.The existence of carrier and visual recovery imlied that mtDNA mutation was a primary cause of LHON,but other factors such as endocrine disorder might influence the pathogenesis of LHON. (Chin J Ocul Fundus Dis,1998,14:156-158)
Objective To evaluate the potential of specific mRNA marker keratin 19(K19) to detect micrometastasis by reverse transcriptase polymerase chain reaction (RT-PCR) .Methods One hundred and ninty four regional lymph nodes harvested from 6 cases of benign diseases, 4 cases of breast carcinoma, 5 cases of gastric carcinoma and 12 cases of colorectal carcinoma patients were examined by conventional pathology and amplifying tissue specific K19 mRNA by RT-PCR separately, then the two methods were compared with each other. Results None of the 34 lymph nodes which were pathological metastasis-negative from benign diseases expressed K19 mRNA by RT-PCR, all of the 28 regional lymph nodes which were pathological metastasis-positive from malignant cases showed trains of K19 mRNA by RT-PCR. Of the 132 lymph nodes which were pathological metastasis-negative from malignant cases, 11 lymph nodes were detected with micrometastasis by genetic diagnosis.Conclusion Genetic diagnosis of lymph node micrometastasis is more sensitive than conventional pathology and has diagnostic value and merits further study.
To investigate the mRNA expression of nm23-H1 gene in human liver tumor. In tumor and corresponding nontumoral liver specimens from 20 patients, nm23-H1 mRNA were examined by reverse transcriptionpolymerase chain reaction (RT-PCR) method with specific primers. Results: The primers designed in this study could amplified nearly entire coding sequence of nm23-H1 gene. All the samples showed positive expression of nm23-H1 mRNA, indicating there was no expression loss or obvious alteration. Conclusions: The achievement of RT-PCR method lays foundation for quantitative gaugement of nm23-H1 mRNA in liver tumor.
Objective To investigate the expression of T cell receptor (TCR) Vβ8.3 gene on CD4+ T lymphocytes in the rats with experimental autoimmune uveoretinitis (EAU). Methods Eighteen Lewis rats were divided into EAU, complete Freund′s adjuvant, and the control group. Inter photoreceptor retinoid-binding protein (IRBP) R16 peptide was synthesized using Fmoc procedure for induction of EAU. Magnetic absorption cell sorting (MACS) me thod was used to isolate the CD4+T lymphocytes from the spleen of the rats. Flow cytometry was used to monitor the efficiency of isolation. The expression of TCR Vβ8.3 gene segment on CD4+T lymphocytes was determined by fluorescent quantitative polymerase chain reaction. Results EAU was successfully induced in the Lewis rats immunized with IRBP R16 peptide. The proportion of CD4+T lymphocytes isolated by means of MACS was statistically higher than that before isolation (P＜0.001). The expression of TCR Vβ8.3 gene segment on CD4+ T lymphocytes in EAU rats was significantly higher than that in the control (P＜0.05). Conclusions There is a predominant usage of antigen-specific TCR Vβ 8.3 gene in EAU rats induced by IR BP R16 peptide, which may serve as a target for immunotherapy of EAU. (Chin J Ocul Fundus Dis，2004，20：165-167)
ObjectiveTo analyze the sensitivity and specificity of polymerase chain reaction (PCR) tests in the detection of cytomegalovirus (CMV) in the diagnosis of patients with acquired immune deficiency syndrome (AIDS), using aqueous humor samples. Methods25 AIDS patients (including 21 men and 4 women) were studied. The age of the patients varied from 24 to 59 years, with an average of (39.2±9.3) years. The CD4+ T cell count was from 1 to 523 cells/μl, with a medium of 40 cells/μl. They were infected with human immunodeficiency virus(HIV)for a period from 15 days to 9 years with a median of 10 months. They were divided into three groups according to the fundus and treatment, including untreated cytomegalovirus retinitis (CMVR), treated CMVR and control group. There were 10 patients without anti-CMV treatment and 7 patients treated previously with foscarnet or ganciclovir whose eyes were diagnosed CMVR. Control group has 8 patients who had normal fundus or minor retinopathy excluded from CMVR. Approximately 100 μl of aqueous humor was obtained by anterior-chamber paracentesis and PCR was performed in all cases. ResultsThere were CMV DNA in 9 of 10 eyes with untreated CMVR (90.0% sensitivity). Of 7 specimens from eyes with treated CMVR, 3 were CMV PCR positive (42.9% sensitivity). All 8 samples of the control group were negative for CMV DNA, indicating the clinical specificity of our PCR was greater than 99.9% for CMVR. The anterior chamber paracentesis did not cause any complications in our patients except for a patient with subconjunctival hemorrhage. ConclusionsThe assay had an estimated sensitivity of 90.0% in detecting untreated CMVR and a sensitivity of 42.9% in detecting CMVR that had been treated. The specificity of this assay was greater than 99.9%.
PURPOSE:To investigate mitochondrial DNA(mtDNA) of Leber's hereditary optic neuropathy(LHON). METHODS:Polymerase chain reaction(PCR)method was used to analyse mtDNA of 11 patients in a pedigree with LHON and 4 control subjects from none LHON pedigree. RESULTS:There was a loss of a restriction site for the restriction endonuclease SfaN.Ⅰin Ihe Patients with LHON. In this pedigree,maternal lineage was regarded a carrier of the pathogenic gene. CONCLUSIONS:The patients with Leber's hereditary optic neuropathy have a point mutation in mtDNA,which results in loss ol SfaN I endonuclease restriction site .and this change is one of mechanisms inducing this disaese. （Chin J Ocul Fundus Dis,1997,13: 27-29）
Objective To investigate the role of β-catenin gene in breast tumorigenesis by detecting mutation and expression of β-catenin gene in breast hyperplasia and breast cancer. Methods Mutation and expression of β-catenin gene in 42 breast cancer, 15 simple hyperplasia and 15 atypical hyperplasia were detected by polymerase chain reaction-single strand conformation polymorphism and immunohistochemistry. Results Normal expression of β-catenin occurred in tissue of breast simple hyperplasia. The rate of abnormal expression of β-catenin in tissue of breast atypical hyperplasia and breast cancer were 26.7% （4/15） and 59.5% （25/42）, respectively, which were higher than that of simple hyherplasia tissue (P＜0.05). And there was a markedly difference between the atypical hyperplasia tissue and breast cancer tissue (P＜0.05). Mutation of β-catenin gene wasn’t detected in this three kinds of tissues. Conclusion Abnormal expression of β-catenin plays an important role in human breast tumorigenesis, reason of abnormal expression of β-catenin isn’t mutation of β-catenin gene. Expression of β-catenin can be regulated by other mechanisms.
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)
Objective To investigate whether mutations exist in codon 58 and codon 347 of the rhodopsin gene in patients with autosomal dominant retinitis pigmentosa(ADRP)． Methods Point mutations at codons 58 and 347 were detected by restriction endonuclease digestion of exons 1 and 5 amplified by polymerase chain reaction(PCR)．This method was applied to screen genomic DNAs from 57 patients of 38 families with ADRP and 60 normal controls． Results Four patients from one family of ADRP were confirmed to have a point mutation at the second nucleotide of codon 58,and 6 patients from two families of ADRP were found to have a mutation at codon 347．None of these mutations were found in 60 normal subjects． Conclusion It is suggested that molecular genetic heterogeneity exists within ADRP and some subtypes of ADRP are caused by points mutations of the rhodopsin gene． (Chin J Ocul Fundus Dis,1998,14:108-110)
Objective To investigate the genetic interaction of HLA-DQB1 promoter and coding alleles in the pathogenesis of Vogt-Koyanagi-Harada syndrome (VKH). Methods Eighty-eight Chinese Han patients with VKH and eighty-eight non-VKH normal controls were enrolled in this study. DNA was extracted from white blood cells of the subjects by phenolchloroform method. Thirteen alleles were genotyped by polymerase chain reaction-sequence-specific primers (PCR-SSP), polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and clone-sequencing was applied to determine the polymorphisms of the promoter and coding regions of HLA-DQB1 gene. Chromas and Bioedit software were used to analyze the sequences of the promoter of HLA-DQB1. Chi-square test and Fisher exact test were the statistical methods. Relationships among single nucleotide polymorphism (SNP) in the promoter and coding region were analyzed. Results Twelve of thirteen already known HLA-DQB1 alleles were genotyped by PCR-SSP in VKH patients. The most frequent allele in VKH patients was HLA-DQB10401 (0.318, 56∶176) which was significantly higher in patients than that in normal controls (0.045, 8∶176) (chi;2＝44.00, P＝0.000, OR＝9.8). So was for HLA-DQB10303 (0.068 vs. 0.006, chi;2＝9.67, P＝0.002, OR＝12.81). In contrast, the frequency of HLA-DQB10601 (0.017 vs.0.096, chi;2＝10.39, P＝0.001, OR＝0.16) and HLA-DQB10302 (0.062 vs. 0.193,chi;2＝13.48, P＝0.000, OR＝0.28) in VKH patients were significantly lower than normal controls. Twelve SNP were found in all subjects. The frequency of C allele at position -189C/A in VKH patients was significantly higher than that in controls (0.324 vs. 0.074, chi;2＝45.92, P＝0.000). However, the frequency of G allele at position -227G/A in VKH patients was significantly lower than that in the normal controls (0.011 vs. 0.108, chi;2＝15.63,P＝0.000). The frequency of combination of susceptible alleles in promoter and coding area （-189C and HLA-DQB10401） in VKH patients was statistically higher than that in controls, the frequency of combination of resistant alleles in control (-227G and HLA-DQB10601) was higher than that in VKH patients. Conclusions The specific interactions of SNP in the promoter and coding alleles of HLA-DQB1 are associated with the pathogenesis of VKH.