Correlation of interleukin-10 gene haplotype with hepatocellular carcinoma in Taiwan.

Polymorphisms in cytokine genes can influence immune responses, inflammation and tissue injury, and may affect the outcome of hepatitis B virus (HBV) infection. We analyzed single nucleotide polymorphisms (SNP) in the interleukin (IL)-10 gene among 344 HBV carriers and 208 patients with hepatocellular carcinoma (HCC). Genotypes and haplotypes were tested for association with HCC. IL-10/-592 C/C genotype was associated with a higher risk for HCC compared with IL-10/-592 A/C and A/A genotypes [odds ratio (OR): 2.1, 95% confidence interval (CI): 1.2-3.6]. IL-10/1927 A/A genotype was also associated with a higher risk for HCC compared with IL-10/1927 A/C and C/C genotypes (OR: 1.5, 95% CI: 1.0-2.2). Haplotype analysis revealed that the homozygosity of the C-A haplotype (defined by SNPs at positions -592 and 1927) of IL-10 gene conveys the highest risk for HCC among HBV carriers compared with the homozygosity for the A-C haplotype (OR: 2.6, 95% CI: 1.3-4.9). The results demonstrate that IL-10 gene polymorphism can affect the outcome of chronic HBV infection. Further studies are necessary to clarify how variation in the IL-10 gene affects IL-10 function and risk of HCC.

Simultaneous genotyping of single nucleotide polymorphisms in the IL-6, IL-10, TNFalpha and TNFbeta genes.

Single nucleotide polymorphisms (SNP) in the human IL-6, IL-10, TNFalpha and TNFbeta genes have been associated with gene function and susceptibility to disease. In this study, primers containing mismatches at 1-3 nucleotide positions were designed to incorporate a new restriction site recognized by endonucleases AlwNI, BcgI, BglI, BsaBI, BslI, BstXI, EcoNI or XcmI for genotyping SNPs in the IL-6 gene (position - 174), IL-10 gene (positions -592 and -1082), TNFalpha gene (positions -238, - 308 and -863) and TNFbeta gene (position + 249) by mismatched polymerase chain reaction and restriction fragment length polymorphism (PCR/RFLP). Our results show that appropriately designed BslI-based mismatched PCR/RFLP assays can be successfully used to determine the genotypes for approximately 40% of SNPs. The mismatched PCR strategy can be coupled with multiplex-amplification to enable simple and rapid determination of several SNP genotypes in a single reaction.

The effect of epidural analgesia on postpartum urinary retention in women who deliver vaginally.

There have been several investigations reporting on urinary retention in postpartum women who delivered vaginally with epidural blockade. The mechanism and incidence of urinary retention in relation to epidural analgesia, however, are not established. The objectives of this study were to investigate the association between various obstetric parameters and urinary retention and to determine whether those women with postpartum urinary retention subsequently develop urinary problems. From December 1999 to September 2000, 110 primiparas who delivered vaginally with epidural analgesia for labor pain relief were recruited prospectively. One hundred primiparas delivering under similar conditions without epidural analgesia were selected as the control group. Residual urine volume was calculated by trans-abdominal sonogram. A computerized obstetric database was analysed to compare the two groups. Women with epidural analgesia, especially those with residual volume exceeding 500 ml, had significantly longer labor course, a higher percentage of instrumental deliveries and more extensive vaginal or perineal lacerations than the control group. Only a few women had persistent problems with micturition six months after delivery. Epidural analgesia provides valuable pain relief but may be associated with greater residual urine. Postpartum urinary retention is, however, more related to prolonged labor than to the effect of epidural analgesia itself. Close monitoring of the progress of labor and avoiding urine retention are essential.

Influence of pressure upon coupling pressurized capillary electrochromatography with nuclear magnetic resonance spectroscopy.

In this work, the influence of supplementary pressure on the separation efficiency of pressurized capillary electrochromatography (pCEC) was examined. At low pressures of up to 30 bar, which is more than sufficient to prevent bubble formation, no significant loss in separation efficiency is observed. Even at 100 bar, the efficiency of pCEC is still significantly better than without application of an electric field. In addition, analysis times are drastically reduced compared to both capillary electrochromatography (CEC) and capillary HPLC. On the basis of these results, an improved interface for capillary NMR coupling is described and used for the separation and identification of a mixture of unsaturated fatty acid methyl esters. Under these conditions, the analysis time could be shortened by up to a factor of 10 when pCEC is coupled to NMR spectroscopy.

Single nucleotide polymorphisms in intron 2 of the human interleukin-1 receptor antagonist (IL-1Ra) gene: further definition of the IL-1 beta and IL-1Ra polymorphisms in North American Caucasians and Taiwanese Chinese.

Previous studies have suggested that a variable number tandem repeat (VNTR) polymorphism in the second intron of the interleukin-1 receptor antagonist (IL-1Ra) gene and the single nucleotide polymorphisms at positions -511 and +3954 of the IL-1beta gene might be associated with increased risks of chronic inflammatory diseases, autoimmune diseases and gastric cancer. In the present study, IL-1beta and IL-1Ra genotypes were analyzed among Asians in Taiwan and Caucasians in North America. We identified a novel polymorphism with 3 nucleotide substitutions in the IL-1Ra VNTR 2-repeat allele. One of the substitutions corresponds with the fourth 3' end nucleotide of the reverse primer that is often used for analysis of the IL-1Ra-associated VNTR locus. Mismatching between this primer and the 2-repeat allele can cause misleading amplification results when stringent conditions are used for annealing. The estimated haplotype frequencies of the variant IL-1 genes were significantly different between Taiwanese and Caucasians. The frequency of the pro-inflammatory IL-1Ra 2-repeat allele was significantly lower in Taiwanese than in Caucasians. In contrast, the frequencies of the pro-inflammatory IL-1beta -511T allele and +3954C allele were significantly higher among Taiwanese compared with Caucasians.

Increased apoptosis of peripheral blood T cells following allogeneic hematopoietic cell transplantation.

Lymphopenia and immune deficiency are significant problems following allogeneic hematopoietic cell transplantation (HCT). It is largely assumed that delayed immune reconstruction is due to a profound decrease in thymus-dependent lymphopoiesis, especially in older patients, but apoptosis is also known to play a significant role in lymphocyte homeostasis. Peripheral T cells from patients who received HCT were studied for evidence of increased cell death. Spontaneous apoptosis was measured in CD3(+) T cells following a 24-hour incubation using 7-amino-actinomycin D in conjunction with the dual staining of cell surface antigens. Apoptosis was significantly greater among CD3(+) T cells taken from patients 19-23 days after transplantation (30.4% +/- 12.5%, P <.05), and 1 year after transplantation (9.7% +/- 2.8%, P <.05) compared with healthy controls (4.0% +/- 1.5%). Increased apoptosis occurred preferentially in HLA (human leukocyte antigen)-DR positive cells and in both CD3(+)/CD4(+) and CD3(+)/CD8(+) T-cell subsets, while CD56(+)/CD3(-) natural killer cells were relatively resistant to apoptosis. The extent of CD4(+) T-cell apoptosis was greater in patients with grade II-IV acute graft-versus-host disease (GVHD) (33. 9% +/- 11.3%) compared with grade 0-I GVHD (14.6 +/- 6.5%, P <.05). T-cell apoptosis was also greater in patients who received transplantations from HLA-mismatched donors (39.5% +/- 10.4%, P <. 05) or HLA-matched unrelated donors (32.1% +/- 11.4%, P <.05) compared with patients who received transplantations from HLA-identical siblings (19.6% +/- 6.7%). The intensity of apoptosis among CD4(+) T cells was significantly correlated with a lower CD4(+) T-cell count. Together, these observations suggest that activation of T cells in vivo, presumably by alloantigens, predisposes the cells to spontaneous apoptosis, and this phenomenon is associated with lymphopenia. Activation-induced T-cell apoptosis may contribute to delayed immune reconstitution following HCT. (Blood. 2000;95:3832-3839)

Molecular modeling of the minor histocompatibility antigen HA-1 peptides binding to HLA-A alleles.

Mismatch of the minor histocompatibility antigen HA-1 has been shown to correlate with graft-versus-host disease in HLA-matched sibling marrow transplants. The HA-1H peptide (VLHDDLLEA) that generates this response is known to be presented by HLA-A*0201. In order to understand the interaction of HA-1 peptides with other HLA-A alleles, we have used the LOOK interface to construct molecular models of both HA-1H peptide (VLHDDLLEA) and HA-1R peptide (VLRDDLLEA) binding with 103 HLA-A alleles. The results show that in addition to A*0201, 21/103 other HLA-A alleles should be able to bind HA-1H peptide but not HA-1R peptide. Based on the modeled predictions, HLA alleles can be categorised into 4 groups with respect to their interaction with HA-1 peptides: Group 1 - bind HA-1H peptide but not HA-1R peptide; Group 2 - bind HA-1R peptide but not HA-1H peptide; Group 3 - bind both HA-1H and HA-1R peptides; Group 4 - bind neither peptide. These predicted binding patterns of HA-1 peptides will be useful as an aid for defining a wider pool of HLA-A alleles in which HA-1 disparities among donor-recipient pairs can be investigated.

Correlation between disparity for the minor histocompatibility antigen HA-1 and the development of acute graft-versus-host disease after allogeneic marrow transplantation.

Results of a previous study suggested that recipient mismatching for the minor histocompatibility antigen HA-1 is associated with acute graft-versus-host disease (GVHD) after allogeneic marrow transplantation. In that study, most patients received either cyclosporine or methotrexate for GVHD prophylaxis, and a cytotoxic T-cell clone was used to test for HA-1 disparity. To facilitate large-scale testing, we developed a method that uses genomic DNA to identify HA-1 alleles. A retrospective study was conducted to correlate HA-1 disparity and the occurrence of acute GVHD in 237 HLA-A2-positive white patients who had received a marrow or peripheral blood stem cell transplant from an HLA-identical sibling. All patients received both methotrexate and cyclosporine for GVHD prophylaxis. The presence of HLA-A*0201 was confirmed in 34 of the 36 HA-1 disparate pairs by sequencing the HLA-A locus. Grades II-IV GVHD occurred in 22 (64.7%) of these 34 patients, compared with 86 (42.8%) of the 201 patients without HA-1 disparity (odds ratio, 2. 45; 95% confidence interval [CI], 1.15 to 5.23; P =.02). Recipient HA-1 disparity showed a trend for association with acute GVHD (odds ratio, 2.1; 95% CI, 0.91 to 4.68; P =.08) when a multivariable logistic regression model was used to include additional risk factors. These data are consistent with results of the previous study, suggesting an association between HA-1 disparity and risk of acute GVHD, but the strength of this association may be lower in patients who received both methotrexate and cyclosporine than in those who received methotrexate or cyclosporine alone.

Molecular genetic study of Pompe disease in Chinese patients in Taiwan.

Pompe disease is caused by mutations in the acid alpha-glucosidase (GAA) gene. Multiple kinds of mutations in the GAA gene have been reported worldwide. In order to elucidate the molecular basis of the disease in Taiwanese patients of Chinese origin, we have recruited 11 unrelated families who had at least one member with Pompe disease for study. We used 16 pairs of oligonucleotide primers to amplify all the coding regions from exon 2 to 20 in the family members. The coding regions were sequenced on both the sense and antisense strands. We identified 7 different mutations in 17 alleles but failed to identify the defects in the other 5 alleles. The most common defect was D645E (Asp645Glu), accounting for 36% (8/22 alleles) of mutations, followed by G615R (Gly615Arg) (3 alleles); 1411del4 (Glu471-shift) (2 alleles); and one allele each of R600H (Arg600His); deltaN675 (deltaAsn675); 2380delC (Arg794-shift) and 2815delGT (Val939-shift). The molecular defects of Pompe disease are highly heterogeneous in Chinese. Characterization of the molecular defects of the disease is useful for a genotype-phenotype correlation and for genetic counseling and prenatal diagnosis.

Gradient elution capillary electrochromatography and hyphenation with nuclear magnetic resonance.

Coupling of gradient capillary electrochromatography (gradient CEC) and capillary zone electrophoresis (CZE) with nuclear magnetic resonance spectroscopy (NMR) was performed using a recently developed capillary NMR interface. This technique was applied for the analysis of pharmaceuticals and food. An analgesic was investigated using isocratic and gradient continuous-flow CEC-NMR. Comparison of the results demonstrated the superiority of gradient CEC over isocratic CEC. Aspartame and caffeine, both ingredients of soft beverages, were separated and analyzed by continuous flow CZE-NMR. The order of elution could be reversed by altering the pH. This reversal led to an increased sample concentration in the NMR detection cell, thus allowing the acquisition of a totally correlated spectroscopy (TOCSY) two-dimensional (2-D) spectrum of the synthetic peptide aspartame.

Definition of the gene encoding the minor histocompatibility antigen HA-1 and typing for HA-1 from genomic DNA.

Recipient mismatching for the minor histocompatibility antigen HA-1 has been associated with acute graft-versus-host disease after allogeneic marrow transplantation. Two polymorphic nucleotides near an exon-intron junction of the gene encoding this minor histocompatibility antigen have been identified. In this study, we determined the genomic DNA sequence of the intron downstream from this polymorphic exon. Based on this sequence, primers were designed to amplify the genomic HA-1 gene sequence, and analysis of restriction fragment length polymorphisms was used to assign the HA-1 genotypes of 160 unrelated probands and a paired sibling for each proband. Among probands, the HA-1H allele frequency was 0.441, and the HA-1R allele frequency was 0.559. The distribution of HA-1 genotypes showed close fit with Hardy-Weinberg equilibrium. Likewise, the number of sibling pairs with disparity for HA-1 alleles showed close fit with predictions based on Hardy-Weinberg equilibrium. These results provide a simple and well validated method for future studies correlating HA-1 disparity with clinical outcome after allogeneic marrow transplantation.

Evidence of Epstein-Barr virus in lymphoepithelioma-like carcinoma of the uterine cervix: a case report.

Lymphoepithelioma-like carcinoma of the uterine cervix is very rare. A 71-year-old woman, who had cervical cancer stage IB, presented lymphoepithelioma-like carcinoma pathologically. The neoplasm revealed an undifferentiated, nonkeratinizing carcinoma, which contained prominent vesicular nucleoli histologically. Epstein-Barr virus (EBV) DNA and HPV-16 DNA were demonstrated in cancer cells when using polymerase chain reaction. The patient underwent a radical hysterectomy with bilateral pelvic lymph node dissection and bilateral salpingooophorectomy. Her postoperative course was uneventful. The literature and pathologic findings are reviewed and discussed.

Fluorescence microsatellite analysis to study the parental origin of the supernumerary chromosome in Down's syndrome.

OBJECTIVE: Down's syndrome (DS) is an important cause of mental retardation. This study investigated the parental origin of the extra chromosome 21 in DS patients. METHODS: Fourteen families each with a DS patient were recruited for analysis of nine microsatellite markers on chromosome 21. We collected DNA from both parents and the patient and used polymerase chain reaction to amplify nine segments on chromosome 21: D21S1435, D21S1436, D21S1437, D21S1446, D21S156, D21S258, D21S263, D21S265 and D21S270. One of each pair of DNA primers was labeled with a fluorescence dye. The amplified products were subjected to electrophoresis in a semi-automated DNA sequencer and then analyzed with Genescan software to determine the origin of the extra chromosome 21. RESULTS: The extra chromosome 21 originated from the mother in 13 (93%) patients and from the father in one (7%) patient. CONCLUSIONS: Our findings were compatible with those from Caucasian patients. A great majority of Down's syndrome cases resulted from meiotic errors in the eggs.

Congenital adrenal hyperplasia. Molecular characterization.

OBJECTIVE: To study the molecular defects of congenital adrenal hyperplasia (CAH). STUDY DESIGN: Twenty Chinese patients, including 8 with salt-wasting (SW) type CAH, 11 with simple virilizing (SV) type CAH and 1 with nonclassical (NC) type CAH, were recruited. Two rounds of the polymerase chain reaction (PCR) were used to study the 21-hydroxylase gene (CYP21). The primary PCR amplified CYP21-specific DNA fragments, and the secondary PCR used products from the primary PCR for analysis of amplification-created restriction sites (ACRS) and direct DNA sequencing. In all patients, ACRS analysis was done at 12 possible mutation sites, and then direct DNA sequencing was performed to confirm or define the molecular defects. RESULTS: Ten different mutations, including nine point mutations and gross gene deletion or conversion, were found in this study. Of the nine point mutations, eight could be easily detected by ACRS analysis. The three most common mutations were codon (CD)172 t-->a (I172N), IVS-II 656 c/a-->g, and gross gene deletion or conversion, accounting for 27.5% (11/40 alleles), 25% (10/40) and 20% (8/40) of all identified mutations, respectively. All SW patients were compound heterozygotes of IVS-II 656, gross gene deletion or conversion, or other severe defects, including CDs236 (t-->a) (I236N)+ 237 (t-->a) (V237E)+ 239 (t-->a) (M239K), CD306 (+t), CD318 (c-->t) (Q318X) and CD356 (c-->t) (R356W) mutations. All SV patients had one allele with a CD172 (I172N) mutation. One allele of an NC patient had a CD183 (c-->g) (D183E) mutation, and the other allele was not defined. In the whole series, four alleles (10%) had more than one mutation. CONCLUSION: We found 10 different mutations in this study. The correlation between genotypes and phenotypes was compatible with the reported data. Two rounds of PCR and ACRS analysis may provide important information for genetic counseling, prenatal diagnosis and management of families at risk for CAH.

Familial transmission of human T-lymphotropic virus type 1 (HTLV-1) in patients with adult T-cell leukemia/lymphoma or HTLV-1-associated myelopathy.

The seroprevalence of human T-lymphotropic virus type 1 (HTLV-1) in Taiwan is 0.48%. In this study, we investigated the patterns of intrafamilial transmission of HTLV-1 in Taiwanese patients with adult T-cell leukemia/lymphoma (ATL/L) or tropical spastic paraparesis/HTLV-1-associated myelopathy (TSP/HAM). Fifteen index patients (9 men, 6 women, aged 31-71 yr), 13 with ATL/L, and two with TSP/HAM, and 98 relatives were included. Of the 98 relatives, 23 were seropositive for HTLV-1. Spouses of 11 patients were studied. Seven of eight wives of male patients but none of the three husbands of female patients were HTLV-1 carriers. Mother-to-child transmission was found in seven of 13 families and in 15 of 75 children tested. The correlation of breast-feeding with seropositivity in two families with seropositive mothers indicates its important role in vertical transmission of HTLV-1. Our findings suggest that husband-to-wife and mother-to-child transmission are the main forms of intrafamilial transmission of HTLV-1 in Taiwan, a nonendemic area. Screening for HTLV-1 in family members of patients with ATL/L or TSP/HAM, and seropositive blood donors, may be warranted. Seropositive individuals should be educated to prevent the spread of the virus through sexual contact and breast feeding.

Prenatal diagnosis of monosomy 10q25 associated with single umbilical artery and sex reversal: report of a case.

The association of rare chromosomal rearrangements involving a specific 10q breakpoint with a single umbilical artery (SUA) and sex reversal has never been reported. This report describes the case of a fetus with prenatal ultrasound features of severe intrauterine growth retardation (IUGR), congenital heart disease, and SUA. Fetal blood study revealed de novo deletion of 10q25 and a 46,XY karyotype, while ultrasound demonstrated female genitalia. Based on these findings, sex reversal was diagnosed. Polymerase chain reaction (PCR) amplification revealed the presence of the sex-determining region of the Y (SRY) gene. The pregnancy was terminated at 36 weeks and the newborn weighed 1908 g with marked facial dysmorphism and abnormal genitalia. Because the parents refused autopsy for this case, histopathological examination of gonads was not performed. Breakpoint of the long arm of chromosome 10 may be responsible for sex reversal in the present case and it could thus confirm the concept of autosomal sex reversal proposed in previous reports.