OBJECTIVE: To determine the incidence and genotypes of glucose-6-phosphate dehydrogenase (G6PD) deficiency in Dongguan region of Guangdong Province and assess the efficacy and feasibility of flow-through hybridization. METHODS: Peripheral blood samples were randomly selected and detected by modified G6PD/6PGD ratio method. Flow-through hybridization was used to detect 14 G6PD mutations among all samples. RESULTS: In total 1005 samples were collected, the detection rate for modified G6PD/6PGD ratio method and flow-through hybridization were 2.79% and 20.90%, respectively. The consistency of the two methods was poor(Kappa=0.187). When c.1311C>T mutation is excluded, the consistency of the two methods was good for males (Kappa=0.952) but still poor for females (Kappa=0.194). The most common mutations were c.1376G>T, c.1388G>A and c.95A>G. No G6PD deficiency was found among those only carrying the c.1311C>T mutation. CONCLUSION Flow-through hybridization can simultaneously detect 14 loci, covering over 90% of common mutations in Chinese population, and can be easily expanded. The routine method may miss many females carrying homozygous, compound heterozygous and heterozygous mutations, but the detection rate for male hemizygous mutation was much higher.
China ; DNA Mutational Analysis ; Female ; Genetic Testing ; Genotype ; Glucosephosphate Dehydrogenase ; genetics ; Glucosephosphate Dehydrogenase Deficiency ; diagnosis ; Humans ; Male ; Mutation

OBJECTIVESince glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common hereditary hemolytic erythrocyte enzyme deficiency, most cases have single nucleotide mutations in the coding region, and current test methods for gene mutation have some missed detections, this study aimed to investigate the feasibility of RT-PCR sequencing in the detection of gene mutation in G6PD deficiency.

METHODSAccording to the G6PD/6GPD ratio, 195 children with anemia of unknown cause or who underwent physical examination between August 2013 and July 2014 were classified into G6PD-deficiency group with 130 children (G6PD/6GPD ratio <1.00) and control group with 65 children (G6PD/6GPD ratio≥1.00). The primer design and PCR amplification conditions were optimized, and RT-PCR sequencing was used to analyze the complete coding sequence and verify the genomic DNA sequence in the two groups.

RESULTSIn the G6PD-deficiency group, the detection rate of gene mutation was 100% and 13 missense mutations were detected, including one new mutation. In the control group, no missense mutation was detected in 28 boys; 13 heterozygous missense mutations, 1 homozygous same-sense mutation (C1191T) which had not been reported in China and abroad, and 14 single nucleotide polymorphisms of C1311T were detected in 37 girls. The control group showed a high rate of missed detection of G6PD deficiency (carriers) in the specimens from girls (35%, 13/37).

CONCLUSIONSRT-PCR sequencing has a high detection rate of G6PD gene mutation and a certain value in clinical diagnosis of G6PD deficiency.

Adolescent ; Child ; Child, Preschool ; Female ; Glucosephosphate Dehydrogenase ; genetics ; Glucosephosphate Dehydrogenase Deficiency ; diagnosis ; genetics ; Humans ; Infant ; Male ; Mutation ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; Sequence Analysis, DNA

OBJECTIVETo study the feasibility of genetic diagnosis for female carriers of human glucose-6-phosphate dehydrogenase (G6PD) deficiency by reverse transcriptase-PCR-denaturing gradient gel electrophoresis (RT-PCR-DGGE).

METHODSBlood samples were collected from suspected 54 female carriers of G6PD deficiency. Total RNAs of peripheral blood were prepared and reverse-transcripted into cDNA. Design of 6 primer pairs for DGGE was based on 17 mutation sites of G6PD cDNA described in the Chinese population. Mutations in the coding region of G6PD gene were screened and genotyped by combination of PCR-DGGE and DNA sequencing.

RESULTSOne case of 1024C/T, 20 cases of 1376G/T and 12 cases of 1388G/A were detected in the 54 samples. The total detection rate was 66.1% (33/54).

CONCLUSIONSHeterozygous mutation rate in female carriers of G6PD deficiency detected by RT-PCR-DGGE is high. RT-PCR-DGGE is value of clinical diagnosis for G6PD-deficiency female carriers.

Adolescent ; Adult ; Child ; Child, Preschool ; Electrophoresis, Polyacrylamide Gel ; Female ; Glucosephosphate Dehydrogenase Deficiency ; diagnosis ; genetics ; Heterozygote ; Humans ; Infant ; Reverse Transcriptase Polymerase Chain Reaction ; methods

OBJECTIVETo detect gene mutations of children with glucose-6-phosphorate dehydrogenase (G-6-PD) deficiency and of carriers of G-6-PD deficiency gene with the technique of polymerase chain reaction and denatured gradient gel electrophoresis (PCR-DGGE), and to explore the value of the technique in the diagnosis of G-6-PD deficiency and G-6-PD deficiency gene carrying.

METHODScDNAs were harvested by reverse transcription method after RNAs had been extracted from peripheral blood of 43 children with G-6-PD deficiency and of their family members (36 lineages). Electrophoresis behaviors of the fragment from exons 11-12 of G-6-PD cDNA were detected with the technique of PCR-DGGE. Gene sequencing was then performed for the abnormal electrophoresis bands.

RESULTSAbnormal electrophoresis bands were found in the 1304-1520 fragment of G-6-PD cDNA in 33 out of 36 family lineages. The G-6-PD/6-PGD ratio was below 1.00 in 9 mothers of patients. Three of them had the G-6-PD/6-PGD ratio lower than 0.50. The PCR-DGGE bands were the same in the 3 mothers. Gene sequencing showed double heterozygote in the 3 mothers, but the maternal carriers of G-6-PD deficiency gene who had normal G-6-PD/6-PGD ratio showed mono-heterozygote in gene sequencing. Three mutational sites were found in the 1304-1520 fragment, i.e., C1311TG1376T and G1388A. The electrophoresis behaviors were different among the 3 gene mutational sites.

CONCLUSIONSPCR-DGGE is a sensitive and reliable technique in the screening of gene mutations. It is useful in the diagnosis of G-6-PD deficiency, especially in the diagnosis of female G-6-PD deficiency gene carrying.

Base Sequence ; Electrophoresis, Polyacrylamide Gel ; Female ; Glucosephosphate Dehydrogenase Deficiency ; diagnosis ; genetics ; Humans ; Male ; Molecular Sequence Data ; Mutation ; Polymerase Chain Reaction ; methods ; Sequence Analysis, DNA

Although glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common enzyme disorder worldwide, it has rarely been reported among Korean. The patient was previously healthy 39 yr old male who showed severe hemolytic anemia and acute renal failure accompanied by hyperbilirubinemia after hepatitis A infection. The additional studies for differential diagnosis of hemolytic anemia showed a moderate deficiency of G6PD enzyme. Because hepatitis A infection in patient with G6PD deficiency present much more severe clinical symptoms, G6PD enzyme should be examined in patients with triggering factors of hemolysis such as hepatitis A infection.
Adult ; Diagnosis, Differential ; Glucosephosphate Dehydrogenase/genetics ; Glucosephosphate Dehydrogenase Deficiency/*complications/diagnosis ; *Hemolysis ; Hepatitis A/*complications/diagnosis ; Hepatitis A Virus, Human/isolation & purification ; Humans ; Hyperbilirubinemia/etiology ; Kidney Failure, Acute/*diagnosis/etiology ; Male

OBJECTIVEOf denaturing high performance liquid chromatography (DHPLC), a technique platform was developed for screening G6PD deficient variants.

METHODSWhen applied to screen and identify the G6PD deficient variants from 124 patients who come from 11 nations in China, the DHPLC was compared with amplification refractory mutation system (ARMS) or DNA sequence technique and assessed carefully in its accuracy, sensitivity, efficiency and the cost of experiment.

RESULTSThe G6PD-deficient variants, such as 1388 G-->A (36/124 cases), 1376 G-->T(35), 95 A-->G (14), 1024 C-->T (3), 392 G-->T (4), 871 G-->A /1311 C-->T /IVS XI +93 t-->c (9), 871 G-->A (1), 1311 C-->T/IVS XI +93 t-->c (4), 1376 G-->T /1388 G-->A (1) and so on, were characterized as sharp peaks by DHPLC and verified by DNA sequence. Further, the standard chromatograms were put into database for 8 kinds of common G6PD deficient variants in Chinese populations. And also DHPLC found 3 G6PD variants (1388 G-->A) from 103 negative controls. With taking 8.8 minutes and costing 1 dollar for each sample, DHPLC successfully detected and identified 34 heterozygous females from patients with G6PD deficiency. However, ARMS checked 83 positive controls but got 12 false G6PD mutants, of which 5 were false positive, 7 false negative. Above results show that DHPLC sounds like to be more convenience, sensitive and accurate than ARMS and DNA sequence techniques for checking G6PD mutants.

CONCLUSIONDHPLC is of great advantage to screen the G6PD deficient variants with accuracy, convenience, automation and less cost, and significantly to identify the female heterozygote and clinical type IV individuals with G6PD deficiency.

Chromatography, High Pressure Liquid ; methods ; DNA Mutational Analysis ; Female ; Glucosephosphate Dehydrogenase ; genetics ; Glucosephosphate Dehydrogenase Deficiency ; diagnosis ; genetics ; Humans ; Male ; Mutation ; Reproducibility of Results

OBJECTIVETo investigate the relationship between complex 1311 mutation of C-->T in exon 11 and 93 T-->C in intron 11 of G6PD gene and the G6PD deficiency.

METHODSUsing NBT paper strip method to screen and quantitative NBT method to confirm G6PD deficiency. PCR-SSCP technique was used to find the abnormal exon 11 and the amplification refractory mutation system (ARMS) to identify 1311 mutation, and DNA sequencing to identify the complex mutation at 1311 in exon 11 and 93 in intron 11.

RESULTSAbnormal band in exon 11 was found in 12 cases. DNA sequencing showed that they were 1311 mutation together with 93 mutation.

CONCLUSIONThis complex mutation may be the cause of reduced activity of G6PD enzyme.

Base Sequence ; DNA Mutational Analysis ; Exons ; genetics ; Genetic Testing ; Glucosephosphate Dehydrogenase ; genetics ; Glucosephosphate Dehydrogenase Deficiency ; diagnosis ; enzymology ; genetics ; Humans ; Introns ; genetics ; Molecular Sequence Data ; Point Mutation ; Polymerase Chain Reaction ; Polymorphism, Single Nucleotide ; Polymorphism, Single-Stranded Conformational

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common hereditary enzyme disorder and more than 200 million people have a deficiency in this enzyme. It is a globally important cause of neonatal jaundice and causes life-threatening hemolytic crisis in childhood. At later ages, certain drugs such as antimalarials, and fava beans cause hemolysis among G6PD deficiency patients. The frequency and severity is influenced by genetic and cultural factors. It is common in Mediterranean, African, and some East Asian populations but rare in Korea. Four cases of G6PD deficiency which were first noticed in Korea are investigated with their clinical features.
Child ; Child, Preschool ; *Glucosephosphate Dehydrogenase Deficiency/diagnosis/genetics ; Humans ; Male ; Pedigree