K. Tang

Neonatal jaundice and molecular mutations in glucose‐6‐phosphate dehydrogenase deficient newborn infants

Molecular mutations of the glucose‐6‐phosphate dehydrogenase (G6PD) gene and clinical manifestations of neonatal jaundice in 112 male and 50 female Chinese neonates with G6PD deficiency were studied. In the 112 males, the nucleotide (nt) 1376 (G→T) mutation was the dominant type (50.0%), followed by nt 1388 (G→A) (16.1%), nt 493 (A→G) (8.0%), nt 1024 (C→T) (6.2%), nt 95 (A→G) (5.4%), nt 392 (G→T) (1.8%), nt 487 (G→A) (1.8%), nt 871 (G→A) (0.9%), and nt 1360 (C→T) (0.9%). The nt 871 variant has not been reported in Taiwan before. The occurrence rates for nt 1376, nt 1388, nt 493, nt 95, and nt 1024 mutations in the 50 females were 44.0%, 18.0%, 12.0%, 6.0%, and 6.0%, respectively. The type of G6PD mutation in 10 male and 7 female neonates has not been identified yet. Although G6PD deficient neonates had higher frequency of phototherapy than G6PD normal neonates in both sexes, a significant difference in the prevalence of hyperbilirubinemia (peak bilirubin ≥ 15.0 mg/dl) between G6PD deficient and normal neonates was found only in males. Further analysis showed that duration of phototherapy was longer in G6PD deficient male neonates than in the control group, while the outcome of phototherapy was better in subjects with non‐nt 1376 mutations than subjects with the nt 1376 mutation. Most (78.3%) of the 23 G6PD deficient neonates who subsequently suffered from neonatal hyperbilirubinemia carried the nt 1376 mutation. The results of this study indicate that the nucleotide substitution at 1376 is the most common and important mutation for G6PD deficiency in Chinese neonates in Taiwan.

G6PD NanKang (517 T→C; 173 Phe→Leu): A New Chinese G6PD Variant Associated with Neonatal Jaundice

Using a non-radioactive PCR-SSCP technique, we identified a novel glucose-6-phosphate dehydrogenase (G6PD) mutation in a Chinese newborn with neonatal jaundice. This new variant (G6PD NanKang) causes a T to C change at nucleotide position 517, producing a Phe173Leu substitution in the human G6PD protein. Since the 517 mutation does not create or remove any known restriction site, we introduced an artificially created site by adding a primer containing a mismatched base to the PCR reaction mixture. The mismatched base accompanying the nearby 517 T-->C mutation generates an XhoI site which is suitable for distinguishing normal from mutant alleles. Using this approach, the 517 mutation can be diagnosed quickly at the DNA level.

Content of Reduced Glutathione and Consequences in Recipients of Glucose-6-Phosphate Dehydrogenase Deficient Red Blood Cells

The red blood cell glucose‐6‐phosphate dehydrogenase (G6PD) activity of every donor was examined with automatic enzyme‐coupled method. The technique of molecular biology was applied to determine the DNA mutations for the 97 donors with undetectable G6PD activity. The concentration of reduced glutathione (GSH) in the stored RBC of the 97 G6PD‐deficient donors and 124 normal donors was determined with the technique of high performance liquid chromatography. Routine blood counts, bilirubin and haptoglobin levels were used to evaluate posttransfusional hemolysis for the 48 adult patients transfused with 1 U G6PD deficient and 1 U normal RBC. Most (88, 90.7%) of the 97 donors were confirmed to be G6PD deficient at the DNA level. At each age interval of storage, the GSH concentration of G6PD‐deficient RBC was significantly different from that of normal RBC. The total average value of GSH (μmol/gHb) was 2.52 ± 0.95 (mean ± 1 standard deviation) vs. 3.74 ± 1.43 (P < 0.001). Hemoglobin, hematocrit, bilirubin, and haptoglobin levels in the patients receiving G6PD‐deficient RBC were not statistically different from those in the recipients of normal RBC; even though the age of stored blood was 26–35 days. Within the same group of patients, the results of bilirubin and haptoglobin were not significantly changed before and after transfusion. The results of this study show that the GSH concentration in the stored blood of G6PD deficient donors was 67% of that in the normal donors. However, hemolysis does not occur in adult patients transfused with 1 U G6PD‐deficient RBC. It seems unnecessary to screen G6PD activity for donors of adult recipients in Taiwan. Am. J. Hematol. 57:187–192, 1998.

Molecular Diagnosis of Glucose-6-Phosphate Dehydrogenase (G6PD) Mutations

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is one of the most common human inherited diseases affecting people worldwide. We and others have recently identified that several different types of mutations are respon-sible for G6PD polymorphism in Chinese. In this communication, we described the PCR-mediated Restriction Site Modification (PRSM) method for quick di-agnosis of the 95A-＞G mutation, the 1376G-＞T mutation, and a compound heterozygosity (487G-＞A/1388G-＞A) in three affected Chinese families with G6PD deficiency. Since no isotopic labeling and no genomic DNA preextrac-tion are required for DNA diagnosis, the PRSM method will greatly benefit the detection of known G6PD mutations at the DNA level. In this report, we also summarize all naturally or artificially created or deleted restriction sties gen-erated by all reported G6PD mutations. This information will be very helpful for designing suitable primers to detect all known G6PD mutations.

Diagnosis of Glucose-6-Phosphate Dehydrogenase (G6PD) Mutations by DNA Amplification and Allele-Specific Oligonucleotide Probes

We have recently identified that at least four types of mutation are responsible for the glucose-6-phosphate dehydrogenase (G6PD) polymorphism in the Chinese of Taiwan. Two mutations (487 G-->A and 493 A-->G) occurring at nucleotide position 487 and 493, respectively, create Alu I and Ava II recognition sites which enabled us to directly examine these two mutations by PCR/restriction enzyme (RE) digestion. However, the other two mutations (1376 G-->T and 1388 G-->A), which do not generate any recognizable restriction sites, were detected by DNA sequencing method which is not suitable for rapid diagnosis. Using the PCR technique, we have successfully developed a simple and rapid method for the detection of 1376 and 1388 mutations. This method involves the selective amplification of a DNA fragment from human G6PD gene with specific oligonucleotide primers, followed by hybridization with allele-specific oligonucleotide (ASO) probes. Using the PCR/ASO and PCR/RE methods, we have successfully examined two families and 20 unrelated subjects with G6PD deficiency. Our results indicate that the PCR/ASO method is suitable for the rapid determination of 1376 and 1388 mutations. The combined use of PCR/ASO and PCR/RE methods will be suitable for rapid diagnosis of four known G6PD mutations in Chinese.