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We developed a rapid and simple method to diagnose the molecular defects of beta-thalassemia in Chinese patients. This method involves the selective amplification of a DNA fragment from human beta globin gene with specific oligonucleotide primers, followed by digestion with restriction enzymes that recognize artificially created or naturally occurring restriction sites. To detect the 4-nucleotide deletion of codon 41–42, we introduced a single mismatch nucleotide into the 3′ end of the upstream primer to create an artificial Taq I restriction site. With a similar approach, an artificial Rsa I site was generated to detect the nucleotide 654 mutation (C-->T) of IVS-2, and Alu I restriction site was created to detect the codon 17 mutation (A-->T), and EcoRI restriction site was created for the -28 mutation (A-->G), a Rsa I restriction site was created for the nucleotide 5 mutation (G-- >C) of IVS-1, and a Spe I restriction site was created to distinguish the codon 71 (+T) and codon 71/72 (+A) mutations from a normal sequence. The other eight rare mutations that occur in the genes of the Chinese people naturally create or abolish restriction sites. Using this kind of approach, we are able to provide a simple, rapid, accurate, and nonradioactive method to detect the genetic defects of beta-thalassemia in the Chinese population. It should be used not only for routine screening but also for prenatal diagnosis.

We have developed a rapid and simple method to diagnose the molecular defects of glucose-6-phosphate dehydrogenase (G6PD) deficiency in Chinese in Taiwan. This method involves the selective amplification of a DNA fragment from human G6PD gene with specific oligonucleotide primers followed by digestion with restriction enzymes that recognize artificially created or naturally occurring restriction sites. Ninety- four Chinese males with G6PD deficiency were studied. The results show that 50% (47 of 94) were G to T mutation at nucleotide (nt) 1376, 21.3% (20 of 94) were G to A mutation at nt 1388, 7.4% (7 of 94) were A to G mutation at nt 493, 7.4% (7 of 94) were A to G mutation at nt 95, 4.2% (4 of 94) were C to T mutation at nt 1024, 1.1% (1 of 94) was G to T mutation at nt 392, and 1.1% (1 of 94) was G to A mutation at nt 487. These results show that the former five mutations account for more than 90% of G6PD deficiency cases in Taiwan. Aside from showing that G to T change at nt 1376 is the most common mutation, our research indicates that nt 493 mutation is a frequent mutation among Chinese in Taiwan. We compared G6PD activity among different mutations, without discovering significant differences between them.

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We have developed a rapid and simple method to detect the relation between HLA-DQ beta 57 Asp and Chinese IDDM patients. The method involved the selective amplification of a DNA fragment from the HLA-DQ B1 gene by using the mutagenic primers. After PCR, if the HLA-DQ beta 57 was Asp, then there was an artificially created restriction enzyme cutting site. We then can accurately obtain the results by enzyme digestion and electrophoresis. Sixty-nine IDDM patients and 30 nondiabetic control subjects were analyzed using this method. Twenty-two (42%) IDDM patients had non-Asp 57 homozygous, 31/45%) were Asp/non-Asp 57 heterozygous, and 9 (13%) had Asp-57 homozygous. Of the 30 control subjects, the number of cases for these three types were 6 (20%), 18 (60%), and 6 (20%), respectively. The relative risk of homozygous DQ beta 57 non-Asp in our group was 2.9 and the p value was greater than 0.05. Using this kind of approach, we were able to provide a simple, rapid, and non-radioactive method to detect whether the HLA DQ beta 57 was Asp or not.