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Chien-Nan Lee

Publications and source records attributed to Chien-Nan Lee.

At least 19 recordsLinked to original sources
PubMed2006

Quantitative assay of deletion or duplication genotype by capillary electrophoresis system: Application in Prader-Willi syndrome and Duchenne muscular dystrophy.

BACKGROUND: Deletions and duplications involving large DNA segments result in underexpression or overexpression, depending on the changes in allele dose, and are known to cause many common disorders. Detection of allele dose variations in the human genome is increasingly important in medical genetic diagnosis. METHODS: We used multiplex quantitative PCR coupled with capillary electrophoresis for accurate allele dose determination. In cases of Prader-Willi syndrome (PWS), a total of 24 patients with PWS, as well as 205 control individuals from the general population, were analyzed by use of multiplex quantitative PCR to amplify the FGFR2 gene, the KRIT1 gene, and the SNRPN gene simultaneously. In cases of Duchenne muscular dystrophy (DMD), we optimized the multiplex quantitative PCR to amplify 38 exons to analyze the DMD gene for rapid diagnosis of 12 DMD-affected males, 12 obligate carriers from families, and 50 unaffected female controls. RESULTS: We were able to unambiguously diagnose the deletion genotype in PWS patients and identify all deletion or duplication genotypes and carrier status in DMD-affected cases with 100% sensitivity and specificity. CONCLUSIONS: This report describes a novel single assay that can rapidly quantify allele dose to provide accurate clinical genetic diagnosis. This technique offers a valuable alternative for the rapid detection of genomic deletions or duplications and decreases costs because it does not require expensive fluorescent reagents.

Autoantigens↗
PubMed2006

Molecular and clinical analyses of 84 patients with tuberous sclerosis complex.

BACKGROUND: Tuberous sclerosis complex (TSC) is an autosomal dominant disease characterized by the development of multiple hamartomas in many internal organs. Mutations in either one of 2 genes, TSC1 and TSC2, have been attributed to the development of TSC. More than two-thirds of TSC patients are sporadic cases, and a wide variety of mutations in the coding region of the TSC1 and TSC2 genes have been reported. METHODS: Mutational analysis of TSC1 and TSC2 genes was performed in 84 Taiwanese TSC families using denaturing high-performance liquid chromatography (DHPLC) and direct sequencing. RESULTS: Mutations were identified in a total of 64 (76 %) cases, including 9 TSC1 mutations (7 sporadic and 2 familial cases) and 55 TSC2 mutations (47 sporadic and 8 familial cases). Thirty-one of the 64 mutations found have not been described previously. The phenotype association is consistent with findings from other large studies, showing that disease resulting from mutations to TSC1 is less severe than disease due to TSC2 mutation. CONCLUSION: This study provides a representative picture of the distribution of mutations of the TSC1 and TSC2 genes in clinically ascertained TSC cases in the Taiwanese population. Although nearly half of the mutations identified were novel, the kinds and distribution of mutation were not different in this population compared to that seen in larger European and American studies.

Adult↗
PubMed2006

A rapid and reliable detection system for the analysis of PMP22 gene dosage by MP/DHPLC assay.

Charcot-Marie-Tooth disease type 1A (CMT1A) and hereditary neuropathy with liability to pressure palsies (HNPP) are caused by a 1.5-Mb duplication and a deletion at chromosome 17p11.2-12 encompassing the peripheral myelin protein 22 gene (PMP22), respectively. We developed a rapid and reliable detection system for duplications/deletions of the PMP22 gene based on measurement of gene copy number. The method involves amplification of a test locus with unknown copy number and a reference locus of known copy number by multiplex PCR (MP), followed by denaturing high-performance liquid chromatography (DHPLC) or capillary electrophoresis detection to identify single copy changes. Thirty-two patients with CMT1A, 17 patients with HNPP, and 61 unaffected individuals were analyzed. Using the same competitive MP protocol, the measured PMP22 gene dosage revealed concordant results between DHPLC and capillary electrophoresis analysis. The results of the MP/DHPLC or the MP/capillary electrophoresis assay were all confirmed by PCR-restriction fragment length polymorphism analysis. We concluded that the MP/DHPLC assay is an efficient, accurate, and reliable technique for gene dosage determination of the PMP22 gene for CMT1A duplication and HNPP deletion. This technique further extends the application of DHPLC as an alternative method for the measurement of gene amplifications and heterozygous deletions in different genetic diseases.

Base Sequence↗
PubMed2006

Rapid genetic analysis of oculocutaneous albinism (OCA1) using denaturing high performance liquid chromatography (DHPLC) system.

OBJECTIVES: To present the prenatal genetic diagnoses and counseling for two cases of oculocutaneous albinism (OCA) type I family by detection of mutations in the OCA1 gene by denaturing high performance liquid chromatography (DHPLC) system and a review of the literature. METHODS: All DNA samples were extracted from peripheral whole blood and amniocentesis-derived cells. Mutation analysis was performed for all five coding exons of the TYR gene, which were amplified by PCR. DHPLC was used for heteroduplex detection and sequence analysis was performed to demonstrate the mutation loci. RESULTS: Case 1: After sampling of blood from the family members and performing amniocentesis of the fetus, it was demonstrated that the affected boy and the female fetus were shown to be compound heterozygotes for mutations in the TYR gene. In addition, it was shown that the parents were carriers of the two mutations. However, the couple chose to keep the baby. Case 2: Mutation analysis of the DNA of the siblings revealed two heterozygous mutations in the TYR gene. Her husband is free of the disease. According to the principles of autosomal recessive inheritance, the incidence of affected offspring is very low. CONCLUSIONS: Herein we introduce a novel application for molecular diagnostic of DHPLC coupled with direct sequencing, which can provide an effective and exact diagnosis in patients with albinism. Clinicians should be cognizant of the risk of OCA inheritance by the offspring through careful identification of genetic mutations and the inheritance mode, both important to ensure comprehensive genetic counseling.

Adult↗
PubMed2006

Effect of availability of a parturient-elective regional labor pain relief service on the mode of delivery.

BACKGROUND/PURPOSE: Regional analgesia for labor pain relief is effective and widely used. This study evaluated the controversial association between mode of operative delivery and patient-elective labor regional analgesia. METHODS: We retrospectively compared the rates of instrumental vaginal and cesarean deliveries in parturients before the introduction, in the first 15 months after, and in the subsequent 36 months after the implementation of an elective labor regional analgesia service. A total of 9779 low-risk singleton cephalic pregnancies above 36 weeks of gestation were included. The maternal and fetal outcomes for parturients before the service was implemented and in those with or without pain relief service in the two postimplementation periods were analyzed. Multivariate logistic regression analyses were used to investigate the effects of maternal age, gestational weeks and newborn weight, in addition to regional analgesia, on the mode of delivery in nulliparous women. RESULTS: After adjusting for maternal age, gestational weeks, and newborn weight, no significant association was found between regional analgesia and cesarean delivery in nulliparas. Further, this lack of association was not affected by the receipt of regional analgesia in the early period of program implementation or in the period after staff had become familiar with the service. A higher rate of instrumental vaginal delivery was noted in nulliparas given regional analgesia. CONCLUSION: Regional analgesia for pain relief increased the likelihood of instrumental vaginal delivery, but did not increase the likelihood of cesarean delivery.

Adult↗
PubMed2006

Submucous myoma induces uterine inversion.

OBJECTIVE: Inversion of the uterus is indeed a rarity for a gynecologist. The puerperal variety is associated with pregnancy, including term pregnancy and abortion. The nonpuerperal condition may be tumor-induced or idiopathic. We present a nonpuerperal uterine inversion and discuss a reasonable plan for its management. CASE REPORT: A 42-year-old, unmarried woman without sexual experience and any systemic diseases noted a mass outside of the vaginal introitus combined with massive vaginal bleeding and abdominal pain of sudden onset after taking laxative agents for colonoscopic preparation. Then she had voiding difficulty with distended bladder. A suprapubic urinary catheter was inserted and 800 mL urine was drained out. The patient received emergency tumor resection and subtotal hysterectomy. The diagnosis of uterine inversion was confirmed during operation. The postoperative course was uneventful and she was discharged without complication. CONCLUSION: Nonpuerperal inversion of the uterus is rarely encountered by gynecologists. Diagnosis of uterine inversion is often not easy and imaging studies might be helpful. Surgical treatment is the method of choice in nonpuerperal uterine inversion.

Adult↗
PubMed2006

Preservation of uterine integrity via transarterial embolization under postoperative massive vaginal bleeding due to cesarean scar pregnancy.

OBJECTIVE: Cesarean scar pregnancy (CSP) is an uncommon type of ectopic pregnancy. It results in uterine rupture and severe hemorrhage during the proceeding gestation. Whether diagnosed early or not, it may cause maternal mortality or morbidity during emergency management. Life-saving emergency hysterectomy is usually the treatment of choice when there is profuse bleeding intraoperatively or after initial management. CASE REPORT: A 38-year-old woman with a history of two previous cesarean deliveries was referred to our clinic under the impression of CSP at 11 weeks' gestation. A viable embryo with a crown-rump length of 4.8 cm in the anterior wall of the uterus at the cervico-isthmic region was detected. Under the confirmation of CSP via ultrasonography, she was admitted for management. During hysterotomy, profuse bleeding with 1,000 mL blood loss was noted. After conservative procedure for hemostasis, however, massive vaginal bleeding persisted. As a result, we immediately transferred the patient to receive transarterial embolization (TAE) for bleeding control. The patient was discharged 4 days after the operation and TAE and her period resumed 1 month later. CONCLUSION: Management of CSP is usually accompanied by profuse blood loss. Hysterectomy is inevitable if massive blood loss occurs during surgical intervention. For preservation of fertility and avoidance of mortality, our physicians offered an alternative life-saving policy even under catastrophic blood loss.

Adult↗
PubMed2005

Quantification of relative gene dosage by single-base extension and high-performance liquid chromatography: application to the SMN1/SMN2 gene.

One of the most commonly used techniques for genotyping of single-nucleotide polymorphism (SNP) is detection of single-base extensions (SBEs). We present a new, rapid, simple, and highly reliable method for accurate quantification of SNP variants in a single reaction. Our approach is based on SBE detection coupled with high-performance liquid chromatography (HPLC) quantification. To demonstrate the utility of our approach, we report data to determine the gene dosage for relative amounts of alleles in a homologous gene, allowing detection of mutation causing exon skipping in human SMN genes to determine the ratio between the copy numbers of the SMN1/SMN2 gene. We successfully determined the relative ratio of the SMN1 and SMN2 genes and showed assay characteristics using the SBE reaction coupled with HPLC. This assay approach readily scaled to high parallelization with multiplex SBE reactions in a single sample screened in one analysis. By screening for particular SNP genotypes, this assay can be used to determine the relative gene dosage that correlates highly with the patient's disease state. The next challenge is to apply this novel methodology in a clinical screening and quantification setting for special gene regions within highly homologous genes.

Base Sequence↗
PubMed2005

Fusion protein vaccine by domains of bacterial exotoxin linked with a tumor antigen generates potent immunologic responses and antitumor effects.

Antigen-specific immunotherapy represents an attractive approach for cancer treatment because of the capacity to eradicate systemic tumors at multiple sites in the body while retaining the requisite specificity to discriminate between neoplastic and nonneoplastic cells. It has been shown that certain domains of bacterial exotoxins facilitate translocation from extracellular and vesicular compartments into the cytoplasm. This feature provides an opportunity to enhance class I and/or II presentation of exogenous antigen to T lymphocytes. We investigated previously whether the translocation domain (domain II) of Pseudomonas aeruginosa exotoxin A with a model tumor antigen, human papillomavirus type 16 E7, in the context of a DNA vaccine could enhance vaccine potency. We then attempted to determine whether this chimeric molecule could also generate strong antigen-specific immunologic responses and enhance the potency of cancer vaccine in the protein format. Our results show that vaccination with the PE(DeltaIII)-E7-KDEL3 fusion protein enhances MHC class I and II presentation of E7, leading to dramatic increases in the number of E7-specific CD8+ and CD4+ T-cell precursors and markedly raised titers of E7-specific antibodies. Furthermore, the PE(DeltaIII)-E7-KDEL3 protein generates potent antitumor effects against s.c. E7-expressing tumors and preestablished E7-expressing metastatic lung tumors. Further, mice immunized with PE(DeltaIII)-E7-KDEL3 protein vaccine also retained long-term immunologic responses and antitumor effects. Our results indicate that retrograde-fusion protein via the delivery domains of exotoxins with an antigen greatly enhances in vivo antigen-specific immunologic responses and represents a novel strategy to improve cancer vaccine potency.

ADP Ribose Transferases↗
PubMed2005

Chimeric DNA vaccine reverses morphine-induced immunosuppression and tumorigenesis.

Although long-term use of morphine has been shown to promote tumor growth, the question whether tumorigenesis occurs as a result of an immunosuppressive effect remains to be investigated. In mice rendered tolerant to morphine, the efficacy and mechanism of a vaccination to rescue morphine-induced immunosuppression and prevent tumor growth was assessed both in vitro and in vivo. Herein, we found that morphine-injected mice exhibited higher tumor growth rates and lower percentages of CD8+ T lymphocytes. The mechanism of morphine suppression of immunity might be through the suppression of E7-specific CD8+ T lymphocyte proliferation and the promotion of apoptosis of these cells by the Bcl-2 and Bax pathways. The suppressive effect of E7-specific CD8+ T lymphocytes by morphine could be reversed by naloxone. We have previously shown that calreticulin linked with E7 (CRT/E7) could enhance the CD8+ T cell response and the anti-tumor effects (W. F. Cheng et al. (2001) J. Clin. Invest. 108, 669-678). CRT/E7 DNA vaccine could overcome the immunosuppressive effect of morphine and suppress tumor growth. Our findings reveal that long-term morphine treatment dose-dependently promotes tumor growth and a DNA vaccine may serve as a useful approach to treat the profound immunosuppressive function and prevent tumorigenesis after long-term morphine treatment.

Analgesics, Opioid↗
PubMed2005

Antigen-specific CD8+ T lymphocytes generated from a DNA vaccine control tumors through the Fas-FasL pathway.

Human papillomavirus, particularly type 16, and its oncogenic proteins, E6 and E7, are consistently expressed in most cervical cancers. One of the major issues facing cancer immunotherapy is that many human cancers evade the immune system by downregulating the expression of Fas molecules. An E7-expressing murine tumor model with a downregulated Fas expression--TC-1 P3(A15) tumors--was created. A DNA vaccine encoding calreticulin linked to E7 (CRT/E7) was able to generate protective and therapeutic antitumor effects against TC-1 P3(A15) tumors. In vitro Ab depletion and in vivo adoptive experiments showed that the antitumor effect of E7-specific CD8+ T lymphocytes against the TC-1 P3(A15) tumor cells was through the Fas-FasL-dependent CTL effector mechanism, and the TC-1 P3(A15) tumor cells needed higher numbers of antigen-specific CD8+ T lymphocytes for in vivo elimination. Our results demonstrated that chimeric CRT/E7 DNA vaccine resulted in control of tumors with downregulated Fas expression, highlighting the importance of the Fas-FasL pathway in the potent antitumor effect of antigen-specific CD8+ cytotoxic T lymphocytes and the role of Fas as part of in vivo tumor evasion.

Animals↗
PubMed2005

Quantitative analysis of SMN1 and SMN2 genes based on DHPLC: a highly efficient and reliable carrier-screening test.

Autosomal recessive spinal muscular atrophy (SMA) is a common, fatal neuromuscular disease caused by homozygous absence of the SMN1 gene in approximately 94% of patients. However, a highly homologous SMN2 gene exists in the same chromosome interval, centromeric to SMN1, and hampers detection of SMN1. We present a new, rapid, simple, and highly reliable method for detecting the SMN1 deletion/conversion and for determining the copy numbers of the SMN1 and SMN2 genes by DHPLC. We analyzed SMN1/SMN2 gene exon 7 deletion/conversion by DHPLC. A total of 25 patients with spinal muscular atrophy lacking the SMN1 gene as well as 309 control individuals from the general population and the family members of patients with SMA were analyzed. By DHPLC analysis, we could detect the SMA-affected cases efficiently just by recognizing an SMN2-only peak. Furthermore, after specific primer amplification and adjustment of the oven temperature, all of the SMA carriers with an SMN1/SMN2 ratio not equal to 1 could be identified unambiguously by this simple and efficient detection system. To calculate the total SMN1/SMN2 gene dosages further, we developed a specific multiplex competitive PCR protocol by simultaneously amplifying the CYBB gene (X-linked), the KRIT1 gene (on chromosome arm 7q), and the SMN1/SMN2 gene ratio by DHPLC. By applying this technique, we could successfully designate all of the genotypes with different SMN1/SMN2 gene copy numbers, including equal and unequal amounts of SMN1 and SMN2. We demonstrated that DHPLC is a fast and reliable tool for detection of carriers of SMA.

Chromatography, High Pressure Liquid↗
PubMed2005

Association of the C677T methylenetetrahydrofolate reductase mutation with congenital heart diseases.

BACKGROUND: To investigate whether the cytosine-to-thymine mutation at base 677 of the gene for methylenetetrahydrofolate reductase (MTHFR) is associated with congenital heart diseases (CHD), using high throughput heteroduplex analysis based upon the powerful technique of denaturing high-performance liquid chromatography. METHODS: We investigated the MTHFR genotype of a cytosine-to-thymine mutation at base 677 for 213 patients of CHDs as confirmed by cardiac catheterization and also for 195 healthy controls. RESULTS: The overall genotype frequencies of the MTHFR C677T polymorphism were not significantly different between the CHD patients and the healthy control (P = 0.345). Furthermore, taking various subgroups of CHD patients into consideration, we noted a significantly increased proportion of homozygous TT genotypes for patients suffering from valvular pulmonary stenosis (PS) or pulmonary atresia with an intact ventricular septum (PA + IVS) (p = 0.0005). For patients revealing heterotaxy syndrome, a conotruncal anomaly including tetralogy of Fallot, an interruption of the aortic arch, persistent truncus arteriosus, and aortopulmonary window, no statistically significant difference existed. CONCLUSIONS: The discrepancy in the distribution of MTHFR genotypes amongst various subtypes of CHD reflects some heterogeneity in the developmental mechanism of CHD. The increased percentage of homozygous TT genotypes might contribute to the pathogenesis of valvular PS and PA + IVS.

Asian People↗
PubMed2005

Induction of human papillomavirus type 16-specific immunologic responses in a normal and an human papillomavirus-infected populations.

Human papillomavirus (HPV) infection, especially with the oncogenic genotypes, is the most important risk factor for developing cervical cancer. We focused on generating HPV16 E7-specific cytotoxic CD8(+) T lymphocytes and evaluating HPV16 E7-specific immune responses in HPV16-infected and uninfected populations. Peripheral blood mononuclear cells (PBMCs) were first collected from an uninfected group with an human lymphocyte antigen (HLA) A2 haplotype (four volunteers). Mature monocyte-derived dendritic cells (DCs) were generated from the PBMCs and pulsed with one of two HLA-A2-restricted E7 peptides, aa 11-20 [YMLDLQPETT] and aa 86-93 [TLGIVCPI], as antigen presenting cells. The autologous naive or cultured PBMCs were then cultured with peptide-pulsed DCs to detect the HPV16 E7-specific immune responses by a variety of techniques such as enzyme-linked immunosorbent assay (ELISA), enzyme-linked immunospot (ELISPOT) assay and cytotoxic T lymphocyte assay. Interferon-gamma (IFN-gamma) from E7-specific cytotoxic CD8(+) T lymphocytes stimulated with the respective peptide was detected by ELISA. Using ELISPOT analysis, a marked increase in the number of IFN-gamma-secreting CD8(+) E7-specific lymphocytes was observed following peptide stimulation. Cultured CD8(+) T lymphocytes were highly cytotoxic against the CaSki cells. PBMCs were then collected from an HPV16-infected population of the HLA-A2 haplotype, including four persons of HPV16 infection only, four with cervical intraepithelial neoplasia (CIN) lesions, and four cervical cancer patients. We then compared the immunologic responses to E7 between HPV16-infected and uninfected populations by ELISA and ELISPOT assay. The E7-specific immunologic responses of the HPV16-infected populations were significantly higher than those of the uninfected population. In addition, persons with an HPV16 infection only or those with CIN lesions generated higher E7-specific immunologic responses than cervical cancer patients. Our results demonstrate methods for evaluating E7-specific immunologic responses and reflect the biological responses of HPV16-infected people during different periods of cervical disease.

CD8-Positive T-Lymphocytes↗