Amplification-created Restriction Site Research Articles

BRCA mutations: screening for germ-line founder mutations among early-onset Syrian breast cancer patients

BackgroundBreast cancer (BC) is the most common female cancers in many countries including Syria. Familial breast cancer or previous family cancer history are considered significant risk factors. Therefore, detecting the prevalence and founder mutations in the population facilitates genetic counselling, risk assessment and the development of a cost-effective screening strategy. In this study, we investigated the three germ-line founder mutations in the BRCA1/2 genes: [NM_007294.4 (BRCA1):c.68_69del (p.Glu23fs), NM_007294.4 (BRCA1):c.5266dup (p.Gln1756fs) and NM_000059.4 (BRCA2):c.5946del (p.Ser1982fs)], to examine their incidence and frequency in early-onset breast cancer cases and determine if they are connected to familial breast cancer. One hundred early diagnosed BC females (≤ 40 years old) with no other type of cancer were recruited. Genomic DNA was isolated from peripheral blood samples, and mutations were investigated using the Amplification-Created Restriction Site (ACRS) method.ResultsThe family history of cancer was observed in 61% of the cases, of which 35% were breast cancer; however, none of the screened mutations were detected among BC patients.ConclusionsThe investigated germ-line mutations were not common among Syrian female patients with early-onset BC and were not associated with familial BC. Other mutations in the BRCA1/2 genes or other genes may have a contributing role. Future studies and the need to launch nationwide mutation screening tests for BRCA 1/BRCA2 in the Syrian population are recommended.

Analysis of the Effect of Polymorphisms within the CATHL7 Gene on Dairy Performance Parameters.

Antimicrobial peptides, including cathelicidins, play a significant role in farm animals, influencing animal welfare, immunity, and thus the quality of animal products. The study used amplification-created restriction site and PCR-restriction fragment length polymorphism to analyse single nucleotide polymorphisms of the CATHL7 gene encoding the BMAP-34 protein in cattle, at positions 2,383 G > C and 2,468 G > C. The material was collected from 279 Polish Black-and-White Holstein-Friesian dairy cows. There were statistically significant differences between milk performance parameters in cows with the CATHL7/HhaI and CATHL7/HinfI genotypes. In the case of the CATHL7/HhaI polymorphism, the highest milk yield and protein and lactose content and the lowest somatic cell count in milk were observed for the CC genotype, while the fat content was the highest in milk from cows with the GG genotype. In the case of the CATHL7/HinfI polymorphism, the highest protein and lactose content in milk was observed for the CC genotype. The results were statistically significant, which suggests that the search for relationships can be continued, and that the results can be used to improve selection programmes supporting dairy farming.

Modification of a previously patented method to unequivocally score A2-like and A1-like bovine β-casein variants

A growing interest in the production and commercialization of A2 cow's milk has been observed in many countries in the last few years due to the beneficial properties for human health attributed to A2 β-casein variant. Methods of varying complexity and different equipment requirements have been proposed for the determination of the β-casein genotype of individual cows. We proposed herein a modification of a previously patented method based on an amplification-created restriction site PCR followed by restriction fragment length polymorphism analysis. This method allows to identify and differentiate A2-like from A1-like β-casein variants, after differential endonuclease cleavage flanking the nucleotide that determines the amino acid at position 67 of β-casein. The advantages of this method are that it:• enables to unequivocally score A2-like as well as A1-like β-casein variants,• can be performed at low cost in simply equipped molecular biology laboratories, and• can be scaled up to analyze hundreds of samples per day.For these reasons, and based on the results obtained from the analysis carried out in this work, it showed to be a reliable method for the screening of herds to selective breeding of homozygous cows and bulls for A2 or A2-like alleles.

“A2 milk” authentication using isoelectric focusing and different PCR techniques

Genetic variants of milk proteins have attracted great interest for decades as they are related to important issues such as the composition and technological properties of milk. More recently, an “A1/A2 hypothesis” was developed saying that β-casein variant A1 may be a dietary risk factor for cardiovascular diseases, type 1 diabetes, sudden infant death syndrome and neurological disorders due to the release of β-casomorphin-7, whereas no evidence for such adverse effects was assumed for β-casein A2. Thus, the aim of this study was to adapt and establish analytical methods for the identification of genetic variants of β-casein using isoelectric focusing of milk proteins as well as appropriate PCR techniques. Allele-specific polymerase chain reaction (AS-PCR) proved to be a reliable method for differentiating most common β-casein variants (A1, A2, B, C), amplification-created restriction site (ACRS)-PCR using three different restriction enzymes allowed also the detection of variant A3, and the restriction fragment length polymorphism (RFLP)-PCR method enabled the reliable discrimination between A2 (homozygote/heterozygote) and non-A2 animals. Since traces of β-casein A1 were also found in commercial “A2 milk” in Austria, the authentication of such expensive dairy products is urgently recommended, both by genotyping of all dairy cows at farm level (to confirm that all cows are homozygous β-casein A2A2) and by screening commercial products on the market (to confirm the absence of β-casein variants A1, B, and C in dairy products labelled “A2 milk”) to protect consumers from this unexpected fraud.

Association of single nucleotide polymorphisms in genes coding insulin-like growth factor 1 system and milk production traits in Montbeliarde cows

The insulin-like growth factor 1 system plays a central role in the growth and development of the mammary gland. Insulin-like growth factor 1 (IGF1) and insulin-like growth factor 1 receptor (IGF1R) have been proposed as candidate genes for milk production traits. This study involved a population of 163 Montbeliarde cows. Five polymorphic sites were analysed using the polymerase chain reaction: restriction fragment length polymorphism (PCR-RFLP) (TaiI and MspI restriction enzymes) and amplification-created restriction site (ACRS) (SnaBI, TasI and TaqI restriction enzymes). The frequencies of the most common alleles were 0.67 for the T allele (IGF1/SnaBI), 0.85 for the A allele (IGF1/TasI), 0.95 for the C allele (IGF1R/TaiI), 0.84 for the G allele (IGF1R/MspI) and 0.69 for the G allele (IGF1R/TaqI). In the first lactation, IGF1/TasI polymorphisms and all single nucleotide polymorphisms in the IGF1R gene were associated with differences in milk, fat and protein yields, without further confirmation in the second lactation. No differences were found in milk production traits between IGF1/SnaBI genotypes and combined genotypes. To date, this is the first association study based on polymorphisms of the primary genes encoding the IGF-1 system in a small herd of Montbeliarde cows. If specific haplotypes could be determined in large-scale studies, based on Montbeliarde and other dairy breeds, it would provide a valuable genetic tool to identify causative mutations. ______________________________________________________________________________________ Keywords : IGF1, IGF1R, milk production traits, polymorphism

An improved polymerase chain reaction-restriction fragment length polymorphism assay for the detection of a PON2 gene polymorphism.

In recent research, it has been shown that there have been variants of rs12026 within the paraoxonase 2 (PON2) gene, which have been associated with cardiovascular disease, cerebrovascular disease, diabetes and other diseases. The isochizomers, such as the BsoFI enzyme, required for the detection of this polymorphism are expensive. Therefore, an improved and less expensive polymerase chain reaction (PCR)-restriction fragment length polymorphism method was established for the detection of the single-nucleotide polymorphism rs12026 in the exon 5 of chromosome 7 of the human PON2 gene using the method of amplification-created restriction site. Subsequent to assessing 302 individuals, the genotype frequencies were 68.9% for CC, 29.8% for CG and 1.3% for GG, and the allelic frequencies were 83.8% for C and 16.2% for G. The PCR results were confirmed by DNA sequencing. The χ2 test showed that the genotype and allele frequencies of PON2-148 do not deviate from Hardy-Weinberg equilibrium, and the sequences of amplified products were consistent with the sequence published in GenBank with the exception of a mismatched base.

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.

Mediterranean river buffalo CSN1S1 gene: search for polymorphisms and association studies

The aim of the present study was to investigate the variability at CSN1S1 locus of the Italian Mediterranean river buffalo and to study possible allele effects on milk yield and its composition. Effects of parity, calving season and month of production were also evaluated. Three single-nucleotide polymorphisms were detected. The first mutation, located at position 89 of the 17th exon (c.628C>T), is responsible for the amino acid change p.Ser178 (B allele)/Leu178 (A allele). The other two polymorphisms, detected at the positions 144 (c.882G>A) and 239 (c.977A>G) of 19th exon, respectively, are silent (3ʹ UTR, untranslated region). Associations between the CSN1S1 genotypes and milk production traits were investigated using 4122 test day records of 503 lactations from 175 buffalo cows. Milk yield, fat and protein percentages were analysed using a mixed linear model. A significant association between the c.628C>T SNP and the protein percentage was found. In particular, the CC genotype showed an average value ~0.04% higher than the CT and TT genotypes. The allele substitution effect of cytosine into thymine was –0.014, with a quite low (0.3%) protein percentage contribution to total phenotypic variance. A large dominance effect was detected. Characterisation of the CSN1S1 transcripts and a method based on MboI amplification created restriction site PCR for a rapid genotyping of c.628C>T are provided.

SNP identification in FBXO32 gene and their associations with growth traits in cattle

The F-box protein 32 (FBXO32), also known as Atrogin-1, is one of the four subunits of the ubiquitin protein ligase complex. FBXO32 has been previously shown to be involved in regulation of initiation and development of muscle mass. In the present study, we investigated the polymorphism of FBXO32 gene in 1313 cattle from seven bovine breeds using DNA sequencing, polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) and PCR-based amplification-created restriction site (PCR-ACRS) methods. Four novel single nucleotide polymorphisms (SNPs) were identified within bovine FBXO32, and were deposited in the GenBank database. The association studies between these four SNPs and growth traits were performed in NanYang cattle. Notably, the SNPs ss411628932 and ss411628936 were shown to be significantly associated with body length of 24-month-old NanYang cattle. Based on the above four SNPs, 16 haplotypes were identified. The main haplotype was AATA, which occurred at a frequency of more than 40%. Additionally, phylogenetic analysis showed that geographical distance was essential to gene flow among seven cattle breeds. Indigenous bovine breeds displayed genetic difference in comparison to hybrid bovine breeds that have foreign origins. We herein describe for the first time a comprehensive study on the variability of bovine FBXO32 gene that is predictive of genetic potential for body length phenotype.

Analysis of the CYP21A1P pseudogene: Indication of mutational diversity and CYP21A2-like and duplicated CYP21A2 genes

The CYP21A1P gene downstream of the XA gene, carrying 15 deteriorated mutations, is a nonfunctional pseudogene that shares 98% nucleotide sequence homology with CYP21A2 located on chromosome 6p21.3. However, these mutations in the CYP21A1P gene are not totally involved in each individual. From our analysis of 100 healthy ethnic Chinese (i.e., Taiwanese) ( n = 200 chromosomes) using the polymerase chain reaction (PCR) products combined with an amplification-created restriction site (ACRS) method and DNA sequencing, we found that approximately 10% of CYP21A1P alleles ( n = 195 chromosomes) presented the CYP21A2 sequence; frequencies of P30, V281, Q318, and R356 in that locus were approximately 24%, 21%, 11%, and 34%, respectively, and approximately 90% of the CYP21A1P alleles had 15 mutated loci. In addition, approximately 2.5% ( n = 5 chromosomes) showed four haplotypes of the 3.7-kb TaqI-produced fragment of the CYP21A2-like gene and one duplicated CYP21A2 gene. We conclude that the pseudogene of the CYP21A1P mutation presents diverse variants. Moreover, the existence of the CYP21A2-like gene is more abundant than that of the duplicated CYP21A2 gene downstream of the XA gene and could not be distinguished from the CYP21A2– TNXB gene; thus, it may be misdiagnosed by previously established methods for congenital adrenal hyperplasia caused by a 21-hydroxylase deficiency.

Polymorphism of the beta-casein gene and its associations with breeding value for production traits of Holstein–Friesian bulls

Beta-casein A1 protein variant might be one of the risk factors in the etiology of human disorders like diabetes and ischemic heart disease. However, from the practical perspective selecting for the A2 allele in dairy cattle requires knowledge of whether the A1/A2 polymorphism is associated with breeding values for production traits. A DNA fragment containing A1/A2 polymorphic region was amplified and genotyped using the PCR-ACRS (Amplification Created Restriction Site) technique in 478 Holstein–Friesian bulls yielding the allele frequencies of 0.35 and 0.65 for the A1 and the A2 variants, respectively. A linear regression model was used for testing the association between the polymorphism and breeding values for production traits and showed that the allele coding the A2 protein variant increases breeding values for milk and protein yield and decreases breeding values for fat percentage in milk. Genotyping A.I. bulls at beta-casein locus and preferring A2 allele may lead to two benefits: increasing breeding value for protein yield and decreasing frequency of A1 protein variant, being considered as risk factor for human health.

Amplification created restriction sites for genotyping SNPs in the bovine ABCG2 and its association with milk production traits (Brief Report)

Abstract. ABCG2 (ATP-binding cassette, subfamily G, member 2) belongs to the superfamily of ATPbinding cassette (ABC) transporters. In ATP-dependent processes, ABCG2 is responsible for transporting xenobiotics and cytostatic drugs across various cellular membranes. The ABCG2 gene is expressed in the apical membrane of alveolar mammary epithelial cells and is responsible for the active secretion of substrates into mouse milk. Other members of the ABC subfamily G are sterol transporters. It therefore appears that ABCG2 might transport cholesterol into milk (COHEN-ZINDER et al. 2005). In the study by COHEN-ZINDER et al. (2005), several SNPs were detected in the ABCG2 gene but only two were genotyped – in exon 14 and in intron 3. In the case of SNP A/C in exon 14 resulting in an amino acid change Y581S, it was demonstrated that this substitution affects milk yield and composition. To detect theses polymorphisms in our study we used a new PCR-RFLP method based on an amplification created restriction site (ACRS). This method has been frequently used by various researchers in recent times (e.g. ZYCH et al. 2007). The aim of this study was to estimate the frequencies of genotypes and alleles and to investigate possible associations between ABCG2 polymorphisms and milk production traits in Jersey cows.

Prevalence of the serpin peptidase inhibitor (alpha-1-antitrypsin) PI*S and PI*Z alleles in Brazilian children with liver disease

Serpin peptidase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), member 1 (SERPINA1) deficiency is one of the main genetic causes related to liver disease in children. In SERPINA1 deficiency the most frequent SERPINA1 alleles found are the PI*S and PI*Z alleles. We used the polymerase chain reaction and the amplification created restriction site (ACRS) technique to investigate the prevalence of the PI*S and PI*Z alleles in a group of Brazilian children (n = 200) with liver disease and established the general frequency of the PI*S allele in our population. We found a significant association of the PI*Z allele and liver disease, but no such relationship was found for the PI*S allele. Our results show that SERPINA1 deficiency due to the PI*Z allele, even when heterozygous, is a frequent cause of liver disease in our group of Brazilian children but that the PI*S allele does not confer an increased risk of hepatic disorders in our group of Brazilian children

Tests for the measurement of factor VII-activating protease (FSAP) activity and antigen levels in citrated plasma, their correlation to PCR testing, and utility for the detection of the Marburg I-polymorphism of FSAP

The single nucleotide Marburg I (MRI) polymorphism of the factor VII-activating protease (FSAP) gene, the prourokinase-activating activity of FSAP, and antigen levels of FSAP in plasma have been associated with incidence and progression of carotid stenosis and venous thromboembolism. However, more information on the extent of these associations, potential further ones, and respective clinical utilities remain to be determined. At present, testing is performed mainly by PCR assays based on probes or SYBR Green I. Some studies include testing for antigen levels of total FSAP and its ability to activate prourokinase. To test large cohorts, it is beneficial to rely on assays that are cost-effective, reliable, easy to use, rapid to perform, and that may eventually be automated. In addition, it appears advantageous to use functional tests or tests that determine antigen levels as they may relate more closely to the phenotype than the genotype does. Tests for the measurements of antigen levels of FSAP and its prourokinase-activating activity were improved and performance characteristics assessed. To determine the FSAP genotypes, an amplification created restriction site (ACRS) PCR test was developed. Key performance characteristics of the FSAP activity and antigen tests were as follows: measuring range: 350-1400 mPEU/mL and 1.8-120 ng/mL, total coefficients of variation (CV): 5%-20% and 5%-14%, within-run CV: 4%-11% and 2.3%-12%, and run-to-run CV: 2%-17% and 4.3%-8.3%, respectively. The ratio of the activity and antigen level of FSAP correctly identified the FSAP genotypes of 126 samples tested. The ACRS PCR test is useful for laboratories that do not have the equipment to perform probe or SYBR Green I based real-time PCR. Furthermore, the tests developed for the determination of FSAP activity and antigen levels are convenient for determining clinical correlations, even for large population studies. The ratio of activity and antigen level of FSAP appears to be a promising and efficient alternative to molecular diagnostic techniques to detect the MRI polymorphism of FSAP.

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.

Phenotype and genotype characteristics of Indonesian 21-hydroxylase deficient patients

Background Congenital adrenal hyperplasia (CAH) is the mostcommon cause of ambiguous genitalia in children and 90-95%cases show 21-hydroxylase deficiency. More than 100 mutationshave been described and of these, four mutations have beenfrequently reported in Asia. Those mutations are deletion/largegene conversion (LGC), intron2 splice mutation (I2 splice), pointmutations at codon 172 (I172N) and codon 356 (R356W).Genotyping is very valuable since close correlation observedbetween genotype and phenotype.Objective To identify phenotype and genotype characteristics ofCAH due to 21-hydroxylase deficiency (CAH-21OH) andcorrelation between them.Methods From June to November 2006 we analyzed 37 confirmedCAH-21OH patients treated at the Department of Child Health,Cipto Mangunkusumo Hospital during the period of 1990-2006.Polymerase chain reaction (PCR) followed by restriction fragmentlength polymorphism (RFLP) analysis or amplification-createdrestriction site (ACRS) were performed. We first identified deletion/LGC and I172N mutation that had been mostly reported in saltwasting (SW) and simple virilizing (SV) form patients respectively.Results There were 37 patients, consisted of 6 males and 31females with the ratio 1:5.2. Of those, 25, 10, 2 patients wereSW, SV and non-classic (NC) form, respectively. PCR-RFLP orACRS was performed to detect two mutations in 32 patients (64alleles). Deletion/LGC was found in 6 alleles while I172Nmutations in two. All deletion alleles showed SW phenotype butI172 mutated alleles showed SW and SV phenotype.Conclusion There is a consistent close association betweengenotype and phenotype in our CAH-21OH patients.

Serum calcium, urine calcium and polymorphisms of the calcium sensing receptor gene

Recent studies have suggested a correlation between the A986S polymorphism of the calcium sensing receptor (CASR), and serum total and ionized calcium. This study aimed to assess the prevalence of three CASR polymorphisms in a West of Scotland population and relate genotype to serum and urine calcium levels. Fasting blood and urine samples were obtained from 121 healthy male and female volunteers aged 20-60 years. Volunteers were genotyped for the A986S, Q1011E and R990G polymorphisms using allele-specific amplification and amplification-created restriction site techniques. Total calcium, ionized calcium and urine calcium excretion were measured using automated clinical chemistry analysers. Genotype frequencies for the A986S polymorphism were: AA, 74.4%; AS, 24.8%; SS, 0.8%. There was a small but statistically significant (P < 0.01) increase in ionized calcium concentration in AS individuals compared with the wild type (1.22 versus 1.20 mmol/L). No statistical difference was detected in serum total calcium or parameters of urine calcium excretion. Genotype frequencies for the remaining polymorphisms were: RR, 82.6%; RG, 16.5%; GG, 0.8% and QQ, 93.4%; QE, 6.6%; EE, 0%. Biochemical parameters in these individuals were not statistically different from the wild type. The increase in serum ionized calcium in the AS group was small and, therefore, unlikely to be of clinical significance.

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.