Amplification-created Restriction Site Method Research Articles

FAS −670 A/G polymorphism may be associated with the depletion of CD4+ T lymphocytes in HIV-1 infection

In this study, the polymorphisms in the FAS and FASL genes was investigated in a sample of 198 HIV-1-seropositive individuals and 191 seronegative controls to evaluate a possible association between polymorphisms and the infection. The identification of the A and G alleles of the FAS −670 polymorphism was accomplished through polymerase chain reaction assays followed by digestion with the restriction enzyme MvaI. The identification of the A and G alleles of the FAS −124 polymorphism and the T and delT alleles of the FAS −169 polymorphism were performed using the amplification-created restriction site method followed by restriction fragment length polymorphism reactions. The comparative analysis of allelic and genotypic frequencies between the groups did not reveal any significant differences. However, the quantitative analysis of CD4+ T lymphocytes suggests that the G allele of the FAS −670 A/G polymorphism can be a protective factor against the depletion of these cells in the course of an HIV-1 infection. Polymorphisms in the FAS and FASL genes were not associated with the number of CD8+ T lymphocytes or the plasma viral load. Our findings suggest that the FAS −670 polymorphism may be associated with apoptosis of CD4+ T lymphocytes after infection by HIV-1.

Low frequency of the CYP21A2 deletion in ethnic Chinese (Taiwanese) patients with 21-hydroxylase deficiency

Congenital adrenal hyperplasia (CAH) is a common autosomal recessive disorder which causes more than 90% of CAH cases due to defects in the steroid 21-hydroxylase gene ( CYP21A2). The frequency of large mutations was determined in 200 ethnic Chinese (i.e., Taiwanese) CAH patients belonging to 200 families with different clinical forms of CYP21A2 deficiency over 10 years of molecular diagnoses. For a large-gene deletion (or conversion) and the CYP21A2 deletion identification, a PCR product covering the TNXB gene and the 5′-end of the CYP21A2 gene with TaqI endonuclease digestion was analyzed by electrophoresis on agarose gels. For CYP21A2 mutational analysis, secondary PCR amplification of the amplification-created restriction site method was applied. From the results of the analysis, we found that large-gene deletions (or conversions) occurred in 7.5% of the alleles including three different types of the chimeric CYP21A1P/ CYP21A2 genes and the haplotype of IVS2-12A/C>G in combination with the 707-714del mutation (without the P30L mutation). The CYP21A2 deletion occurred in 2.0% of the alleles which contained three types of the chimeric TNXA/TNXB genes with two novel ones. We concluded that the CYP21A2 deletion in the ethnic Chinese (Taiwanese) patients exhibits a low occurrence, with the haplotype of the IVS2-12A/C>G in combination with the 707-714del mutation (without the P30L mutation) being prevalent among large gene deletions or conversions.

Distribution of signal transducer and activator of transcription 6 gene G2964A polymorphism in Chinese patients with ulcerative colitis

The signal transducer and activator of transcription 6 (STAT6) gene is located on chromosome 12q13.3-14.1 just within the IBD2 region and is a key transcription factor involved in interleukin (IL)-4 and IL-13-mediated Th2 response. The aim of the present study was to determine distribution of the STAT6 gene polymorphism in Chinese patients with ulcerative colitis. The G2964A polymorphism in the 3' untranslated region of the STAT6 gene was studied in 84 unrelated Chinese patients with ulcerative colitis and 176 healthy controls by PCR and the amplification created restriction site method. The results were then compared with those from a Dutch study published previously. Significant differences in genotype and allele frequencies of the STAT6 G2964A polymorphism were not found between patients with ulcerative colitis and healthy controls. Subgroups of the patients with ulcerative colitis classified according to the age at onset, sex and location of disease did not differ significantly in the distribution of this polymorphism. However, the genotypes (P < 0.0001, chi-squared = 75.332) and allele frequencies (P < 0.0001, odds ratio = 4.298, 95% confidence interval = 3.070-6.018) were significantly different between the Chinese and Dutch populations. The STAT6 G2964A polymorphism is not involved in the genetic susceptibility to ulcerative colitis in Chinese patients.

Hepatitis B virus genotyping, core promoter, and precore/core mutations among Afghan patients infected with hepatitis B: A preliminary report

In spite of hepatitis B virus (HBV) vaccination, HBV infection remains an important public health problem worldwide. Although the HBV genotype distribution has been determined in some parts of South Central Asia, no survey has been conducted to determine the HBV genotype in Afghanistan. Twelve Afghan patients infected with HBV living in Afghanistan were enrolled in this study. Partial HBsAg and basic core promoter, precore, and core (BCP/preC/C) regions were amplified and subjected for direct sequencing. In parallel, precore G1896A mutation was also determined by an amplification-created restriction site method. Results revealed HBV genotype D (95% bootstrap value), sub-genotype D1 (98% bootstrap value), and subtype ayw2 in all Afghan isolates. Afghan isolates clustered in a separate branch in the D1 sub-genotype called D1', while supported by 82% bootstrap value. The percentage of intra-genotypic distance among Afghan isolates was 1.05% and inter-genotypic distance with the other genotype D was 2.87% and with other genotypes was 7.50%-11.1%. The wild-type, mixed infection, and precore mutant were found in six, two, and four HBV isolates, respectively. The A1762T/G1764A BCP dual mutation was found in one isolate. Three isolates presented single mutation in the BCP dual mutation region, whereas two showed a novel G1764T mutation. In conclusion, this preliminary study revealed HBV genotype D, sub-genotype D1, and subtype ayw2 of HBV among hepatitis B infected patients from Afghanistan. Further investigation should be carried out.

Emergence and takeover of precore-stop mutant prior to exacerbation of e antigen-negative chronic hepatitis B after withdrawal of lamivudine therapy

Although hepatitis B virus (HBV) precore-stop mutant (nonsense mutation at codon 28) is usually isolated from patients with hepatitis B e antigen (HBeAg)-negative hepatitis, it is unclear whether the mutant is the cause of the hepatitis flare or it is a consequence of necroinflammation. Using the amplification-created restriction site method, the ratios of precore-stop mutant were measured in serial serum samples obtained from three patients experiencing HBeAg-negative hepatitis after withdrawal of lamivudine therapy. In all three patients, the precore-stop mutant emerged and took over the viral population at least 2 months prior to the hepatitis exacerbations. After the onset of exacerbation, transient elevation of serum HBV-DNA was observed, which was followed by progressive decrease of the ratio of precore-stop mutant as well as normalization of serum transaminases. The present data indicate that at least in some patients, the precore-stop mutant is the cause but not the consequence of the flare of HBeAg-negative hepatitis.

Untreated hypertension can lead to memory loss by cutting down on blood flow to the brain

Aim: Breast cancer is the most common cancer in women. In recent years, mounting evidence has identified the possibility that 2q35, 3p24, 17q23 and fibroblast growth factor receptor 2 (FGFR2) may be genetic susceptibility loci for breast cancer. This study aimed to evaluate the association of four polymorphic genotypes in these loci with breast cancer in Taiwanese women. Patients and Methods: Eighty-eight patients with breast cancer and 70 controls without breast cancer were selected. Polymorphic variants of 2q35-rs13387042, 3p24-rs4973768, 17q23-rs650490 and FGFR2-rs2981578 were analyzed to test for their association with breast cancer susceptibility. The 2q35, 17q23 and FGFR2 polymorphisms were detected using polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) and the 3p24 polymorphism was detected using an amplification-created restriction site method. Results: The distribution of genotypes of 2q35 were significantly different between the breast cancer group and the control group (p=0.035), while the distributions for 3p24, 17q23, and FGFR2 did not produce statistically significant differences (p&gt;0.05). In addition, allele A of 2q35 conferred a higher risk for breast cancer risk than allele G (odds ratio, OR=2.95, 95% confidence interval, CI=1.29-6.71, p=0.008). Furthermore, the genotypic distribution of 2q35 was not significantly different among patients with different tumor stages, or from different specimen type. Conclusion: The 2q35 allele A may be a potential biomarker for breast cancer risk, but further confirmation is required to determine its role in breast carcinogenesis. Blood samples can be used for determining the genotypes for 2q35-rs13387042 in patients for risk of breast cancer.

A novel accurate amplification created restriction site method for determination of the wild type and the precore mutant hepatitis B virus variants

The most commonly occurring hepatitis B virus (HBV) mutation is the G to A mutation at nucleotide 1896 in the precore region. The aim of this study was to develop a novel accurate amplification created restriction site (ACRS) method for determination of the TGG wild type and the TAG precore mutant HBV variants. Two conserved and consensus specific and diagnostic primers introducing BstXI and XagI cleavage sites were designed in order to determine the G1896 wild type and the A1896 precore mutant HBV variants in all HBV genotypes. The results of the ACRS method were compared with sequencing data. With the ACRS method, three different patterns could be distinguished for the wild type, the precore mutant and mixed infection HBV variants. The results of the ACRS method on 30 HBV isolates revealed the TAG precore mutant in 50% (15/30), the TGG wild type variant in 30% (9/30) and the mixed infection in 20% (6/30). The sequencing data of these samples were in agreement with the ACRS results. The ACRS method is a rapid and cost-effective technique for detecting both the TGG wild type and the TAG HBV precore mutant variants. It can be carried out for follow-up of G1896A precore mutant variant in hepatitis B virus infected subjects at routine molecular diagnostic laboratories.

Signal transducer and activator of transcription 6 gene G2964A polymorphism and inflammatory bowel disease.

Signal transducer and activator of transcription 6 (STAT6) is a key transcription factor involved in interleukin 4 (IL-4) and IL-13-mediated Th2 response. The STAT6 gene is located on chromosome 12q13.3-14.1 (IBD2 region) and is therefore a positional and functional candidate gene for study in inflammatory bowel disease. We investigated the G2964A polymorphism in the 3' untranslated region of the STAT6 gene in Dutch patients with inflammatory bowel disease and healthy controls. The G2964A polymorphism in the STAT6 gene was genotyped in 141 unrelated Dutch Caucasian patients with ulcerative colitis, 183 patients with Crohn's disease and 173 healthy individuals by PCR and the amplification-created restriction site method. Patients with Crohn's disease were classified according to the Vienna classification and the patients with ulcerative colitis were classified with the age at onset, extent of disease and colectomy. We did not find significant differences in genotype and allele frequencies of the G2964A polymorphism in the STAT6 gene between ulcerative colitis, Crohn's disease and healthy controls. Subgroups of the patients with Crohn's disease classified according to the Vienna classification and those with ulcerative colitis classified according to age of onset, disease extension and colectomy did not differ in the distribution of this polymorphism. The STAT6 G2964A gene polymorphism is not involved in the overall susceptibility or in determining the phenotype of IBD.

Frequency of CYP21 Gene Mutations in Czech Patients with Steroid 21-Hydroxylase Deficiency and Statistical Comparison with Other Populations

Objective: To undertake mutational analysis in patients with different forms of steroid 21-hydroxylase deficiency. Subjects and Methods: CYP21 gene molecular analysis was performed in 76 Czech patients diagnosed with steroid 21-hydroxylase deficiency. Eight of the most common point mutations (intron 2 splice, P30L, 8bp deletion in exon 3, I172N, V281L, Q318X, R356W, and P453S) were analyzed using an amplification-created restriction site method, and 5 additional mutations (intron 7 splice, F307insT, cluster in exon 6, R484P, and R484X) were analyzed using dot-blot hybridization with 5′-biotin-labeled oligonucleotides. Deletions and conversions were screened using a sequence-specific oligonucleotide hybridization method. Comparison of common mutation frequencies in CYP21 reported for different regions, both within Europe and worldwide (North and South America, Asia, and North Africa), was undertaken and the significance of the differences was determined by statistical analysis (Fisher’s F test, Student’s t test, paired t test, and confidence intervals) using a value of p < 0.05. Results: The most frequent genetic defect found in this group of Czech patients was intron 2 splice mutation (46.7%). Comparison of mutation frequencies between Czech and other European populations showed that the Czech patients had a lower frequency of deletions/large gene conversions, R356W, and cluster mutations in exon 6, together with a higher frequency of intron 2 splice mutation, 8-bp deletion and F307insT compared with other populations. Conclusion: A high prevalence of P30L mutations, mostly associated with nonclassical forms of congenital adrenal hyperplasia, was found in Czech patients with classic simple virilizing forms of steroid 21-hydroxylase deficiency.

Identification and characterization of a prevalent hepatitis B virus X protein mutant in Taiwanese patients with hepatocellular carcinoma.

The aim of this study was to investigate whether there was a particular hepatitis B virus (HBV) X protein (HBx) mutant associated with Taiwanese patients with hepatocellular carcinoma (HCC). Initially, the entire coding region of HBx gene from the serum samples of 14 Taiwanese patients were sequenced. A novel mutant, HBx-A31, was preferentially found in patients with HCC. Sera from 67 patients with HCC and 100 patients with chronic hepatitis B were thus subjected for codon 31 analysis using a dual amplification created restriction site method. HBx-A31 was detected more frequently in patients with HCC (52% versus 12%; P<0.001) and in patients with liver cirrhosis (44% versus 6%; P<0.001). Site directed mutagenesis experiment revealed that HBx-A31 was less effective in transactivating HBV enhancer I-X promoter complex, less efficient in supporting HBV replication, and less potent in enhancing TNF-alpha induced increment of CPP32/caspase 3 activities in HepG2 cells. In conclusion, a prevalent HBx mutant was identified in Taiwanese patients with hepatocellular carcinoma. Development of this mutant might represent a strategy of the virus to escape immune surveillance and thus contribute to the process of multiple-step hepatocarcinogenesis.

Prednisolone modulates the therapeutic effect of interferon to eliminate preferentially the hepatitis B virus precore stop mutant

Background/Aims: The aim of this study was to understand the changes in the proportion of hepatitis B virus precore stop mutant during the course of prednisolone primed interferon (IFN) therapy. Methods: Three groups of patients were included: patients receiving prednisolone-primed IFN treatment (Group I, n=31), IFN treatment only (Group II, n=29), and placebo (Group III, n=25). The proportion of precore stop mutant was measured by a quantitative amplification-created restriction site method. Results: Distinct patterns of the progression of the proportion of mutant were found among these three groups. A steady increase in the proportion of mutant was observed only in Group III patients. In Group II patients, the presence of a higher percentage of mutant (>25%) immediately before IFN treatment was predictive for the subsequent clearance of hepatitis B e antigen (HBeAg) ( p<0.01), but not for complete anti-viral response ( p>0.05). Prednisolone pretreatment resulted in an increase in the proportion of mutant in patients with initially low percentages (≤25%) of mutant. During the period of IFN treatment, both the relative and absolute amount of the precore stop mutant decreased significantly in Group I patients who cleared HBeAg. The presence of such a decrease in this group of patients was predictive for both HBeAg clearance and complete anti-viral response. Conclusions: Our data suggest that prednisolone serves as a modulator to enhance elimination of precore stop mutant by IFN, which advocates the benefit of corticosteroid pretreatment in an area where the precore mutants are prevalent.

Identification of four novel mutations in the CYP21 gene in congenital adrenal hyperplasia in the Chinese.

Congenital adrenal hyperplasia (CAH) is a common autosomal recessive disorder mainly caused by defects in the steroid 21-hydroxylase (CYP21) gene. We have analyzed CYP21 gene sequences in 65 CAH families in Taiwan. All ten exons of the CYP21 gene were analyzed by differential polymerase chain reaction followed by single-strand conformation polymorphism electrophoresis and the amplification-created restriction site method. About 95% (123 chromosomes) contain mutations due to conversion of DNA sequences into its neighboring homologous pseudogene, CYP21P. Four novel mutations representing 5% of the total chromosomes have also been identified. The mutations were confirmed by sequencing an aberrant DNA fragment. These four mutations included a base change of the splicing donor site at intron 2 from GT to AT, a base substitution of C to T at codon 316, deletion of ten bases (TCCAGCTCCC) at codons 330-333 of exon 8, and duplication of 16 bases (CCTGGATGACACGGTC) at codons 393-397 of exon 9. The loss of the splicing donor site at intron 2 and the premature stop at codon 316 may result in aberrant splicing to reduce enzyme activity and a truncated protein with no enzyme activity, respectively. Likewise, both the duplication and the deletion forms create a frameshift and premature stop during translation. The resulting proteins lack the heme-binding domain and hence are expected to lose enzymatic activity. Since these mutations are not found in the neighboring CYP21P pseudogene, gene conversion should not be the cause of these novel mutations.

Prenatal and molecular diagnosis of hemophilia B

Prenatal diagnosis was carried out on a woman who had previously given birth to a son with a spontaneous mutation of C → T transition at nt 31133 of the factor IX (F.IX) gene. The diagnosis was performed on chorionic villi sampling by the method of amplification-created restriction site (ACRS). It revealed a female fetus with a normal F.IX gene, as confirmed by DNA sequencing after delivery. Meanwhile, a survey using the ACRS method to evaluate the inheritance of 63 individuals from 8 hemophilia B families was done. A different single-point mutation in each family was proved by DNA sequencing. One individual had a mutation with a naturally-created restriction site. In each of the remaining patients, we were able to show an enzyme-cutting site in their DNA amplification product for ACRS with the designed mutagenesis primers. All patients and carriers could be diagnosed accurately by comparing ACRS results with clinical and laboratory findings. There were new novel mutations among the patients. © 1996 Wiley-Liss, Inc.

Precore mutant of hepatitis B virus prevails in acute and chronic infections in an area in which hepatitis B is endemic.

By using an amplification-created restriction site method, the precore TAG mutant of hepatitis B virus was detected in 6 (75%) of 8 acute fulminant hepatitis B patients, 7 (58%) of 12 acute self-limiting hepatitis B patients, 35 (81%) of 43 hepatitis B virus surface antigen carriers with fulminant hepatitis, and 42 (70%) of 60 hepatitis B virus surface antigen carriers with chronic hepatitis. The precore TAG mutant prevails in acute and chronic hepatitis B of various severity in this area where hepatitis B is endemic.