Rapid diagnosis of hemoglobin constant spring and hemoglobin E by amplified created restriction sites.

To the Editor: The diagnoses of these cases were based on cellulose acetate electrophoresis, isoelectric focusing, or direct sequencing of the entire globin genes. For the detection of Hb CS, two pairs of primers were used by alteration of the structure of one of the globin chains of the hefor PCR. The first pair of primers (upstream primer, 5’moglobin (Hb) molecule.’ The diagnosis of these disorders depends CGTGCTGACCTCCAGACACCGT-3’; downstream primer, 5’on Hb electrophoresis, isoelectric focusing, high performance liquid GTCTGAGACAGGTAAACACCTCCAT-3’ ) was used to recognize chromatography, and amino acid sequencing.’ Another approach is the base “C” of the codon “CAA,” which is the mutant codon genetic analysis. Many hemoglobinopathies are caused by point muat the position of the normal ($ termination codon. This primer pair tation of globin genes.’ Traditionally, detection of known point mucan also amplify part of the 3’ end of the pseudo al gene, but not tations or of small deletions is dependent on allele-specific hybridthe nl gene. The second pair of primers (upstream primer, ization, direct sequencing of polymerase chain reaction (PCR) 5’-AGCCACTGCCTGCTGGTGAC-3’; downstream primer, 5’products,’ ligase-mediated allele detection, or cleavage mismatch deGAACGGCTACCGAGGCTCAAGC-T ) was used to rec-nize the tection. If the mutations create or abolish a restriction site, it is usually bases “AA” of the codon “CAA” of Hb CS. The PCR product of the easy to detect the mutations after digestion of the PCR product by ne’ gene with the first primer pair will create a restriction site for using specific restriction enzymes. Unfortunately, not all of the muTthl I1 I and with the second primer pair will create a restriction site tations will create or abolish a restriction site, and some enzymes are for lfindlll. Figure IA shows the results ofdigestion of PCR products very expensive or difficult to handle. To solve these problems, we from individuals with and without Hb CS. For a”, a 186-bp fragment have devised a nonradioactive method by using sitedirected mutawas formed aRer digestion with Tthl I1 I and a 122-bp fragment was genesis to create specific restriction sites to diagnose two common noted after digestion with Hindlll. For normal and other termination hemoglobinopathies in Chinese. codon mutations, only one of the digested fragments could be noted. In thisstudy, weobtained DNA from40individualswith HbConCase I is an Hb H patient with Hb CS with a genotype of a a”/-. stant Spring (Hb CS) and from four individuals with Hb E disease. An almost completely digested band ( I 86 bp) was formed after TIhlll Hemoglobinopathies are a group of genetic disorders charawnzed