A novel frameshift mutation (+G) at codons 15/16 in a beta0 thalassaemia gene results in a significant reduction of beta globin mRNA values.

AIMS: To identify a novel beta globin gene mutation found in a Chinese family, and also to assess its functional consequences. METHODS: Haematological analysis was performed on all family members. The 23 common mutations of beta thalassaemia found in Chinese populations were detected by means of a reverse dot blot method. Direct DNA sequencing of polymerase chain reaction (PCR) amplified complete beta globin gene was carried out to identify the novel mutation. A real time, one step reverse transcription PCR assay was used to measure beta globin mRNA in the reticulocytes of heterozygous patients. RESULTS: A novel frameshift mutation-an insertion of G between codons 15 and 16 in a homonucleotide run of four guanines-was determined, which generates a new premature chain terminator at the 22nd codon. Relative quantitative analysis of the beta globin mRNA in heterozygous subjects demonstrated a 39.83% reduction compared normal controls. CONCLUSIONS: The significantly lower amounts of beta globin mRNA found in mutation carriers is probably caused by the rapid nonsense mediated degradation of the mutant mRNA. These data, combined with haematological analysis, suggest that this novel mutation of CDs 15/16 (+G) results in a beta(0) thalassaemia phenotype.

Optimization and head-to-head comparison of MISSR-PCR, ERIC-PCR, RAPD and 16S rRNA evolutionary clock for the genotyping of Vibrio cholerae isolated in China.

PURPOSE: To establish a new genotyping method for Vibrio cholerae and compare it with other methods. MATERIALS AND METHODS: In the current study, a modified inter simple sequence repeat-polymerase chain reaction (MISSR-PCR) system was developed via several rounds of optimisation. Comparison study was then conducted between MISSR-PCR and three other methods, including enterobacterial repetitive intergenic consensus sequences-based PCR (ERIC-PCR), randomly amplified polymorphic DNA (RAPD) and 16S rRNA evolutionary clock, for the detection and genetic tracing of Vibrio cholerae isolated from seafood in China. RESULT: The results indicated that the MISSR-PCR system could generate the highest polymorphic fingerprinting map in a single round PCR and showed the best discriminatory ability for Vibrio cholerae genotyping by clearly separating toxigenic/nontoxigenic strains, local/foreign strains, and O1/O139/non-O1/non-O139 serogroup strains, comparing to ERIC-PCR, RAPD and 16S rRNA evolutionary clock. Moreover, the MISSR-PCR is superior to previously described traditional simple sequence repeat based PCR method on genotyping by more clearly separating different clusters. CONCLUSION: To the best of our knowledge, this is the first head-to-head comparison of four detection and genotyping methods for Vibrio cholerae The MISSR-PCR system established here could serve as a simple, quick, reliable and cost-effective tool for the genotyping and epidemiological study.

Rapid, accurate genotyping of the common -alpha(4.2) thalassaemia deletion based on the use of denaturing HPLC.

AIMS: To develop an alternative assay for specific genotyping of the -alpha(4.2) thalassaemia deletion based on the DNA sequence features surrounding the breakpoint. METHODS: The 5' and 3' ends of the breakpoint regions of the -alpha(4.2) allele and the normal homologous segments were sequenced in Chinese individuals. A sequence haplotype composed of four single nucleotide variations within the X2/X1 box of the -alpha(4.2) breakpoint region was found in all of the 10 Chinese -alpha(4.2) thalassaemia alleles studied. Based on these findings, a novel polymerase chain reaction (PCR)/denaturing high performance liquid chromatography (DHPLC) assay was developed for rapid genotyping of the -alpha(4.2) allele instead of traditional Southern blotting or Gap-PCR. This method involves amplification of the alpha globin target sequence encompassing these four polymorphic sites, followed by a partially denaturing HPLC analysis using the transgenomic WAVE DNA fragment analysis system. RESULTS: The three major genotypes (-alpha4.2/alphaalpha, -alpha(4.2)/--SEA, and alphaalpha/alphaalpha) could be distinguished through the characteristic chromatograms generated by the WAVE system. The accuracy of this technique was evaluated blindly, and the results were 100% (40 of 40) concordant with the genotypes previously characterised by Southern blotting or Gap-PCR. CONCLUSIONS: This study validates the PCR/DHPLC approach as a simple, rapid, highly accurate, and cost effective method, potentially adaptable for use in epidemiological surveys, genetic screening, and diagnosis of silent alpha+ thalassaemia and Hb H disease.

Alpha0 thalassaemia as a result of a novel 11.1 kb deletion eliminating both of the duplicated alpha globin genes.

AIMS: To characterise a novel 11.1 kb deletion that eliminated both of the duplicated alpha globin genes, giving rise to a typical alpha0 thalassaemia phenotype in four carriers from a Chinese family. METHODS: Haematological investigations were carried out on all family members. The seven common forms of alpha thalassaemia were screened for by the polymerase chain reaction (PCR) and Southern blotting was used to analyse the alpha globin gene cluster. DNA sequence analysis of the entire alpha1 and alpha1 globin gene region was carried out and reverse transcription (RT)-PCR was used to investigate the transcription levels of the alpha and beta globin genes. RESULTS: The breakpoints were found to lie between coordinates 31695-31724 and 42846-42867 of the alpha globin gene cluster (NG_000006), with a total of about 11,135 nucleotides deleted. These sequences are involved in (CA)n repeats, suggesting a homologous recombination event. RT-PCR analysis gave a transcription level of the alpha globin gene in heterozygotes comparable with that of SEA deletion heterozygotes, confirming no output of alpha globin from the linked pair of alpha globin genes. The heterozygosity for this novel deletion was confirmed by PCR diagnosis in all four carriers from this family. CONCLUSIONS: This rare mutation constitutes an additional heterogeneous defect causing alpha thalassaemia in the Chinese population.

The prevalence and spectrum of alpha and beta thalassaemia in Guangdong Province: implications for the future health burden and population screening.

AIM: Thalassaemia is a good candidate disease for control by preventive genetic programmes in developing countries. Accurate population frequency data are needed for planning the control of thalassaemia in the high risk Guangdong Province of southern China. METHODS: In total, 13397 consecutive samples from five geographical areas of Guangdong Province were analysed for both haematological and molecular parameters. RESULTS: There was a high prevalence of carriers of alpha thalassaemia (8.53%), beta thalassaemia (2.54%), and both alpha and beta thalassaemia (0.26%). Overall, 11.07% of the population in this area were heterozygous carriers of alpha and beta thalassaemia. The mutation spectrum of alpha and beta thalassaemia and its constitution were fully described in this area. This study reports the true prevalence of silent alpha thalassaemia in the southern China population for the first time. In addition, two novel mutations that give rise to alpha thalassaemia, one deletion resulting in beta thalassaemia, and a rare deletion (--(THAI) allele) previously unreported in mainland China were detected. The frequency of the most common mutation, the Southeast Asian type of deletion (--(SEA), accounting for 48.54% of all alpha thalassaemias) was similar to the total of two alpha(+) thalassaemia deletions (-alpha(3.7) and -alpha(4.2), accounting for 47.49% of alpha thalassaemia). CONCLUSION: Both alpha and beta thalassaemia are widely distributed in Guangdong Province of China. The knowledge gained in this study will enable the projected number of pregnancies at risk to be estimated and a screening strategy for control of thalassaemia to be designed in this area.

Molecular characterization and PCR detection of a deletional HPFH: application to rapid prenatal diagnosis for compound heterozygotes of this defect with beta-thalassemia in a Chinese family.

Hereditary persistence of fetal hemoglobin (HPFH) is one of the hemoglobinopathies in which the fetal gamma-globin genes remain active in adult life. Most HPFHs are caused by a large deletion involving a variable extent of DNA segment on the beta-globin gene cluster. We report the molecular defects associated with a deletional HPFH, which has previously been described in Cambodians and Vietnamese, in two unrelated Chinese individuals. To define the sequence around the breakpoints of the deletion, both the deletion junction fragment and the normal DNA across the breakpoints were cloned by PCR and sequenced. We found that the 5' breakpoint is located between nucleotides 986 and 987 upstream from the startpoint of the beta-globin gene, which further confirmed the Southeast Asian (SEA) HPFH deletion previously determined, whereas the 3' breakpoint, which is clarified for the first time by us, lies approximately 2.3 kb downstream from the 3' HS1 site of the beta-globin gene. It is suggested that deletions were the result of a non-homologous recombination event. Based on our novel sequence data, we designed a PCR amplification method with three primers bridging the 3' breakpoint. With this method and reverse dot blot (RDB) for detecting beta-thalassemia mutations, a Chinese family that had a 6-year-old propositus with severe thalassemia intermediate and that had requested prenatal diagnosis for the second pregnancy was found to be compound heterozygotes of HPFH defects with beta-thalassemia. The fetal genomic DNA diagnosis showed the same results as those in propositus, i.e., both of them inherited the deletion from their mother and inherited a codons 14-15 (+G) frameshift mutation causing beta-thalassemia from their father.