Detection of the most common G6PD gene mutations in Chinese using amplification refractory mutation system.

Glucose-6-phosphate dehydrogenase (G6PD) is the most common human enzymopathy. To date more than 122 mutations in the G6PD gene have been discovered, among which 12 point mutations are found in the Chinese. The 2 most common mutations, G1388A and G1376T, account for more than 50% of mutations representing various regions and ethnic groups in China. Setting up a simple and accurate method for detecting these mutations is not only useful for studying the frequency of the G6PD genotypes, but also for finding new mutations. The purpose of this study was to find a simple, inexpensive and accurate method for detecting these common mutations. The amplification refractory mutation system (ARMS) method was used in this study. Samples from 28 G6PD-deficient males were investigated. The natural and mismatched amplification and restriction enzyme digestion method was used as a standard method to evaluate the nature of the point mutations. Sixteen cases were found carrying the G1388A mutation and 12 the G1376T mutation. Fourteen cases of G1388A and 10 cases of G1376T were confirmed by ARMS. Four cases were not in concordance with the results obtained by the mismatched amplification-restriction enzyme digestion. These 4 cases were then judged by direct PCR sequencing at exon 12. The DNA sequencing data supported the results obtained by ARMS. Thus we concluded that the ARMS is a rapid, simple, inexpensive and accurate method for detecting the most common G6PD gene mutations among the Chinese.

[Shear bond test of HF acid etching porcelain bonded to enamel with different concentration and disposing time]

OBJECTIVE:Understand the effect of shear bond test of HF acid etching porcelain bonded to enamel with different concentration and disposing time. METHODS:After HF acid etching under 30 groups composed of 5 different HF concentrations and 6 exposing times,shear bonding strength of porcelain to enamel was tested under imitating occlusion and debonding types of the composite were examined by microscope.RESULTS:The favorable concentration-time groups for clinics were 2.5%-5min;5.0%-2.5min;10%-30s;7.5%,15%-5min.The debonding type of porcelain resin enamel composite body was mixed one.CONCLUSION:The results showed HF acid etching technique had a positive influence on the bonding strength of porcelain to enamel.

[Expression and biochemical characterization of human G6PD gene 1376 and 1388 mutation in G6PD-deficient Escherichia coli].

Nine types of human G6PD gene mutated at the positions of nt 1376 and nt 1388 by site-directed mutagenesis were transformed into the strain of G6PD dificent E. coli HB 351(DE3). The mutated gene was expressed successfully and the enzyme kinetic studies undertaken according to WHO standardization. The results showed that the arginine residues at the positions of 459 and 463 of G6PD gene play an important role in maintaining activity of the enzyme. The amino acid structure, polarity, and electronic property may be responsible for it. The arginine residues at the positions of 459 and 463 are also important for the enzyme-NADP+ binding, but it was not interfered by the lysine-arginine substitution. By inducing a non-sense mutation, it was further demonstrated that the amino acids residueds behind the position of 459 were extremely significant for G6PD activity.

[Study on the compatibility of slip casting aluminous ceramic crowns]

One of the key factors for a good slip casting aluminous ceramic crown is good compatibility between its core material and the veneering porcelain.The chemical and thermal compatibility of two slip casting aluminous ceramic crown systems(In-Ceram and GI-I) were investigated by means of SEM and EDAX,thermal shock tests were also performed to evaluate the crazing resistance.The results showed: the crazing resistance of In-Ceram was 158 degrees centigrade,and that of GI-I was degrees centigrade;there existed tightly bonded interfaces between the slip casting aluminous ceramic cores and the veneering porcelains in both of the two systems,where ion transferences were found.The results also suggested good compatibility of the two slip casting aluminous ceramic crown systems.

[The effect of different size of aluminoxide for sandblasting on bonding strength of porcelain to metal].

In this article, 10um, 50um, 110um, 200um and 280um aluminoxide particles were used separatively for producing various roughness metal surfaces. The effects of different roughness of metal surface on porcelain--metal bonding strength were studied by porcelain--metal bonding strength measurement, scanning electron microscope (SEM) observation and energy-dispersive analysis of x-rays (EDAX). The result shows: the size of aluminoxide particles should be over 50 microns for non-precious Ni-Cr allay Surface blasting. In certain range, the roughness of metal surface can increase bonding strength significantly.

[Restriction fragment length polymorphism of Bam HI and Rsa I loci in the retinoblastoma gene].

The retinoblastoma (Rb) gene probe p123 M1.8 and p68 Rs2.0 were used to study the frequencies of the Bam HI and Rsa I restriction fragment length polymorphisms (RFLPs) in the RB gene among the population of Han nationality in Guangdong province. The result showed that the heterozygote rate of the Rsa I locus was only 55.0%, remarkably different from reports in the literature. Linkage study of the Bam HI and the Rsa I RFLPs demonstrated that 10.5% of the Rsa I polymorphic loci were heterozygous for the Bam HI RFLP. Thus the combined use of the two RFLPs can give information for about 65.5% of the population.

Molecular characterization of glucose-6-phosphate dehydrogenase (G6PD) deficiency in patients of Chinese descent and identification of new base substitutions in the human G6PD gene.

The underlying DNA changes associated with glucose-6-phosphate dehydrogenase (G6PD)-deficient Asians have not been extensively investigated. To fill this gap, we sequenced the G6PD gene of 43 G6PD-deficient Chinese whose G6PD was well characterized biochemically. DNA samples were obtained from peripheral blood of these individuals for sequencing using a direct polymerase chain reaction (PCR) sequencing procedure. From these 43 samples, we have identified five different types of nucleotide substitutions in the G6PD gene: at cDNA 1388 from G to A (Arg to His); at cDNA 1376 from G to T (Arg to Leu); at cDNA 1024 from C to T (Leu to Phe); at cDNA 392 from G to T (Gly to Val); at cDNA 95 from A to G (His to Arg). These five nucleotide substitutions account for over 83% of our 43 G6PD-deficient samples and these substitutions have not been reported in non-Asians. The substitutions found at cDNA 392 and cDNA 1024 are new findings. The substitutions at cDNA 1376 and 1388 account for over 50% of the 43 samples examined indicating a high prevalence of these two alleles among G6PD-deficient Chinese. Our findings add support to the notion that diverse point mutations may account largely for much of the phenotypic heterogeneity of G6PD deficiency.

[Clinical study on the prevention of kernicterus caused by hereditary glucose-6-phosphate dehydrogenase deficiency].

A combined preventive scheme was conducted in four hospitals in Guangzhou to lower the rate of kernicterus and mental retardation caused by related neonatal jaundice due to G6PD deficiency. Observation was focused on 330 G6PD deficient infants, and the effects were measured according to the incidence of hyperbilirubinemia and kernicterus. The results, as compared to those of a retrospective study, showed that the incidence of hyperbilirubinemia was significantly decreased (51.4% to 21.2%), and neither kernicterus nor mental retardation infant was found in this series (12.5% in the control group). The authors conclude that this combined scheme is extremely effective and can be used in any large population area in which there is a high gene frequency of G6PD deficiency.

A new mutation responsible for severe G6PD deficiency in two ethnic Chinese with different clinical presentations: determination by a direct PCR sequencing technique.

To test the hypothesis that clinical manifestation in G6PD deficiency correlates with a molecular lesion, we investigated the G6PD gene of two Chinese Americans both of whom had G6PD deficiency, but who manifested different clinical presentations. In this study, we have developed a direct PCR sequencing protocol to examine the human G6PD gene. By using optimized PCR conditions with internal primers, we were able to amplify a 4.2 kb DNA fragment (covering exon 3 through 13 of the G6PD gene) of consistently high quality. From this we were then able to generate high quality single-stranded DNA templates by asymmetric PCR for subsequent sequencing. We also overcame the crossband problem by using internal primers, high temperature reaction with Taq I DNA polymerase, and/or sequencing with gene 32 protein. We could consistently amplify exons 1 and 2 despite their high G/C content by substituting 75% of dGTP with deoxy-7-deaza-guanosine triphosphate. By using this novel approach, we have identified a new mutation at cDNA position 1376 from G to T, which causes substitution of Leu for Arg at amino acid position 459. This mutation has not been reported in other ethnic groups. It is the only genetic defect in the coding regions of the G6PD gene of these two G6PD deficient individuals. We speculate that in addition to a defect in the G6PD gene, other factors also play a role in the clinical manifestation of G6PD deficiency.

Two commonly occurring nucleotide base substitutions in Chinese G6PD variants.

Using a direct PCR sequencing technique, we have identified two DNA base substitutions in 8 different biochemical G6PD variants of Chinese origin. Neither one of these abnormalities has been reported in other ethnic groups. An abnormality (C1) of G to T substitution at cDNA 1376 causing an amino acid change from Arg to Leu has been found in 3 variants. Another abnormality (C2) of G to A substitution at cDNA 1388 causing an amino acid change from Arg to His has been found in 5 variants. Both C1 and C2 are located in exon 12 of the G6PD gene and are only 12 base pairs apart. However, C1 is associated with a significant increase in the deamino-NADP utilization rate, whereas C2 is not. Taken together, our data suggest that C1 and C2 are very common among Chinese with a G6PD deficiency and exon 12 may define an important functional domain of the human G6PD.

[Restriction fragment length polymorphisms in FVIII:C gene and their application in the linkage analysis of hemophilia A].

The Han people in Guangong province were studied to determine the frequencies of BclI, XbaI, and BglI restriction fragment length polymorphisms (RFLPs) in FVIII:C gene. The incidences for positive BclI, XbaI, and BglI sites were 63.5%, 43.5%, and 100%, respectively. Linkage of BclI and XbaI RFLPs were studied and the result showed that 19.5 percent of positive BclI polymorphic site homozygotes were heterozygous for the XbaI RFLP. Hence combining these two RFLPs was informative for 65.9 percent of females. We implemented carrier detection in two hemophilia A families and prenatal diagnosis in another hemophilia A family with these RFLPs.

Glucose-6-phosphate dehydrogenase variants and their frequency in Guangdong, China.

Erythrocyte glucose-6-phosphate dehydrogenase (G6PD) was characterized in blood samples obtained from 97 randomly selected males with enzyme deficiency from various regions of Guangdong Province, China. Nine new variants (Gd Kaiping, Gd Boluo, Gd Huiyang, Gd Gaomin, Gd Qing-Baijiang, Gd Gaozhou, Gd Huazhou, Gd Nanhai, and Gd Guangzhou) were identified. Of the 31 variants found in this province, Gd Kaiping, Gd Taiwan-Hakka, Gd Haad Yai, Gd Haad Yai-like and Gd Huiyang occurred most frequently. The frequency of each variant was calculated. The results demonstrated that the genetic heterogeneity of G6PD deficiency was high in this area.

Glucose-6-phosphate dehydrogenase deficiency among three national minorities in Hainan Island, China.

A total of 296 healthy males belonging to three national minority groups, Li, Miao and Hui, in Hainan Island, China, were screened for G6PD deficiency. 9 out of 139 Li (6.47%), 8 out of 48 Miao (16.67%), and 2 out of 109 Hui (1.83%) individuals were found to be G6PD deficient. Of these 19 variant samples, three were subjected to G6PD characterization and found to be different from each other. They were considered to be G6PD Miaozu-Baisha, G6PD Qingyuan, and a new variant tentatively named G6PD Huizu-Sanya.