Frequency of glucose-6-phosphate dehydrogenase (G6PD) mutations in Chinese, Filipinos, and Laotians from Hawaii.

In a Hawaii Hereditary Anemia Screening Project, 4,984 participants were tested for glucose-6-phosphate dehydrogenase (G6PD) deficiency by a filter paper blood spot fluorescence test. Abnormal samples and suspected heterozygotes were checked by quantitative G6PD assay (normal 4.5 to 14 units/g Hb). G6PD was deficient (< 1.5 units/g Hb) in 188 of 2,155 males; 7 other males had low activity (1.5 to 2.8 units/g Hb). The gene frequency, estimated from males after excluding referred and related cases, was 0.037 for Chinese, 0.134 for Filipinos, and 0.203 for Laotians. Among 2,829 females tested, family data showed 111 females were obliged to be at least heterozygous, regardless of G6PD activity, and 43 others had low G6PD activity. Most heterozygotes probably remained undetected by G6PD screening. In 28 females, activity was under 10%; in another 9 females, activity was < 1.5 units/g Hb. Since only 25 homozygotes would be predicted, this apparent excess of females with deficient activity could be due to unequal X-inactivation in some heterozygotes. DNA analysis by polymerase chain reaction amplification and special analytic procedures revealed 10 different missense mutations in 75 males. The nucleotide 835 A-->T and 1360 C-->T transitions were first detected in this Hawaiian Project; we found that the nucleotide 1360 mutation was the most common cause of G6PD deficiency in Filipinos. This is the first report of G6PD screening and analysis of molecular G6PD mutations in Filipino and Laotian populations.

A PCR procedure to determine the sequence of large polypeptides by rapid walking through a cDNA library.

A procedure that uses the PCR to make rapid successive steps through a random-primed cDNA library has been developed to provide a method for sequencing very long genes that are difficult to obtain as a single clone. In each successive step, the portions of partial clones that extend out from the region of known DNA sequence are amplified by two stages of PCR with nested, outward-directed primers designed approximately 50 bases in from the end of the known sequence, together with a general primer based on the sequence of the vector. This procedure has been used to determine the coding sequence of the cDNA for the beta heavy chain of axonemal dynein from embryos of the sea urchin Tripneustes gratilla. By starting from a single parent clone, whose translated amino acid sequence overlapped the microsequence of a tryptic peptide of the beta heavy chain, and making 3 such walk steps downstream and 14 walk steps upstream, we obtained a sequence of 13,799 base pairs that had an open reading frame of 13,398 base pairs. This sequence encodes a polypeptide with 4466 residues of Mr 511,804 that is believed to correspond to the complete beta heavy chain of ciliary outer arm dynein.

Molecular screening for haemoglobin constant spring.

Haemoglobin H/Constant Spring is an important cause of severe haemoglobin H disease, but the Constant Spring protein is difficult to detect by electrophoresis. A technique for allele specific polymerase chain amplification of the 3'-end of the alpha 2 globin gene improved detection of the alpha cs alpha haemoglobin variant in DNA samples by slot-blot hybridisation. The alpha cs alpha mutation was confirmed in subjects that had been previously diagnosed by haemoglobin electrophoresis, and it was also detected in patients who were negative by protein electrophoresis. 10 of 103 unrelated Laotians with HbE were alpha cs alpha heterozygotes. Of these, 3 were negative to the normal probe because they had -alpha 3.7/alpha cs alpha with a single alpha globin deletion. 5 samples did not amplify or hybridise to either probe because they had deletions of both alpha 2 globin regions. The gene frequency for alpha cs alpha is about 0.05 for Laotians. This technique, which is highly specific and sensitive for rapid detection of the alpha cs alpha mutation, is suitable for clinical diagnoses and population studies. The true incidence of alpha cs alpha may prove to be greater than previously suspected from protein electrophoresis.