An Analysis On Mutation Spectrum And Promoter Methylation In G6PD Gene

Objectives:To investigate mutations in whole coding sequencing and promoter region and promoter methylation in G6PD gene so as to improve molecular diagnosis in patients with G6PD deficiency.Methods:Sequencing followed RT-PCR was performed to screen mutations in the coding region of G6PD. Real-time quantitative polymerase chain reaction (Real-time PCR) was used to detect mRNA expression. DNA amplification and sequencing were carried to screen mutations in the promoter region and identify heterozygous or homozygous mutations in females, as well as novel mutations. Both biulfite sequencing PCR and MS-High resolution melting curve analysis were combined to detect the methylation of G6PD promoter. Results:One hundred and ninety five children were included in the study(145 males and 50 females). All the children were divided into two groups according to G6PD/6PGD quantitative ratio,130 of the children with the ratio below 1.00 were considered as G6PD deficiency and the other 65 children with the ratio?1.00 as control. Eighteen mutations were identified in the coding sequencing of the 195 samples, among which G1376T, G1388A and A95G were the most common mutations, including a novel missense mutation (A1088T) and one synonymous mutation (C1191T). The RT-PCR sequencing detected mutations in the coding area of the all samples in all the samples with G6PD deficiency, which was consistent with G6PD/6PGD ratio method. For the controls, G6PD/6PGD method failed to detect 28 female samples with missense or synonymous mutations which were identified by RT-PCR sequencing. Except for one missense mutation and one synonymous mutation, all female mutants were verified as heterozygotes by using DNA amplification and sequencing. Twenty two samples with reduced mRNA expression(Ct ratio<0.5) were investigated by mutation scanning and methylation analysis in the promoter region, no missense or synonymous mutation was shown; 92 percent of females (46/50) had the methylation, while all males present non-methylation.Conclusions:Single base mutations in the coding sequence are the most common in G6PD deficiency and two new mutations have been revealed. G6PD/6PGD method is sufficient to diagnose G6PD deficiency in male children (100%), but RT-PCR combined with sequencing is necessary to improve detection rate of G6PD deficiency for female cases. DNA amplification and sequencing are needed to identify heteozygotes. No mutations shown in the promoter region suggests that G6PD deficiency may not be associated with the promoter mutations of G6PD gene. Only female children have methylation in the promoter region which is not related to the reduced enzyme activity of G6PD. The methylation may be associated with X chromosome inactivation. Non-methylation observed in 4 females could be related to G6PD gene that escape X inactivation, which is reported in Chinese pupolation for the first time.