Study On Gene Mutation Of Glucose-6-phosphate Dehydrogenase Deficiency In Heze Newborns

Objective:Glucose-6-phosphate dehydrogenase（G-6-PD）deficiency is a common X-linked incomplete dominant neonatal genetic disorder caused by G-6-PD gene mutation,resulting in erythrocyte enzyme deficiency and hemolytic anemia.This study explored the prevalence of G-6-PD deficiency and the characteristics of gene mutations in newborns in Heze area,aiming to provide clinical reference data for screening and treatment of G-6-PD deficiency.Methods:From January 2021 to June 2022,GSP^?automatic fluorescence immunoanalyzer was used to detect G-6-PD activity in 114285 neonates born in Heze area,and genomic DNA was extracted from 80 confirmed cases of dry blood spots.9 pairs of primers containing all exons of G-6-PD gene were designed and Sanger sequencing technology was used to study the gene mutation types and characteristics of neonatal G-6-PD deficiency.The prevalence of G-6-PD deficiency was calculated,and the data of gender,gestatory age,maternal age,birth weight,glucose-6-phosphate dehydrogenase activity and mother’s native place of newborn infants screened in Heze region were analyzed.According to the ACMG guidelines,Poly Phen＿2,PROVEAN and Mutation Taster were used to predict the risk of new mutations of G-6-PD gene,and multi-species protein conserved analysis and three-dimensional structure analysis of G-6-PD mutant protein were also performed.In this study,Chi-square test was used for counting data and T-test was used for measurement data,and P<0.05 was statistically significant.Results:1.A total of 80 children with G-6-PD deficiency were diagnosed in the screening of114285 neonates with G-6-PD activity in Heze,and the prevalence was 0.07%（80/114285）.There were 75 males and 5 females,with the prevalence rate of 93.75%（75/80）in males significantly higher than 6.25%（5/80）in females（²=51.913,P<0.001）.The prevalence rate was 0.32%（17/5271）in children aged 34 to 36⁺⁶ weeks,which was higher than that in other groups（²=66.864,P<0.001）.The incidence rate of macrosomia with birth weight>4000g was 0.15%（7/4825）,which was significantly higher than that of other weight groups（²=6.431,P<0.001）.The prevalence of maternal age≥33 years was 0.10%（24/23330）higher than that of other age groups（²=5.023,P<0.001）.2.In the analysis and study of all G-6-PD gene exon mutations,a total of 17 mutation types were detected,among which 13 were reported G-6-PD mutation types in Chinese population:c.1388G>A、c.487G>A、c.1376G>T、c.95A>G、c.1024C>T、c.871G>A、c.392G>T、c.1192G>A、c.486-34del T、c.1360C>T、c.592C>T、c.1387C>T、c.196T>A;Among them,male hemizygous type 16,female heterozygous type 4 and female homozygous type 1.81.25%（65/80）of G-6-PD mutations were located in exons 12,6,9 and 2,of which 75.00%were c.1388G>A、c.487G>A、c.1376G>T、c.1024C>T and c.95A>G.The mutation hotspots in Heze region were c.1388G>A 27.50%（22/80）,c.487G>A 20.00%（16/80）,c.1376G>T 13.75%（11/80）.The activity value of G-6-PD in group c.1388G>A was（8.51±2.97）U/g Hb,and that in group c.487G>A was（13.29±5.50）U/g Hb,and the difference was significant（t=3.889,P<0.05）.The activity value of G-6-PD in male semzygous group（10.57±5.72）U/g Hb was significantly different from that in female heterozygous group（22.51±4.17）U/g Hb（t=3.453,P<0.05）.3.Through the retrieval of Master Mind,NCBI Clinvar and the Chinese National Genome Database,four new G-6-PD gene mutations were found in the Chinese population for the first time in this study,among which three exon missense mutations had not been reported in the Chinese population:c.682G>A、c.479G>A、c.404A>T;One intron variation was not reported in the Chinese population:c.486-7C>G.Bioinformatics analysis software predicted the harminess of three newly discovered missense mutations:c.682G>A/p.D228N,c.479G>A/p.S160N and c.404A>T/p.N135I;The protein sequences of G-6-PD gene in six mammal species were compared,and D228,S160 and N135 were all located in highly conserved sequences.3D protein model prediction results showed that the three newly discovered G-6-PD gene missense mutations were more likely to affect protein structure and function.4.The four common genotypes of G-6-PD deficiency children of Heze mothers were c.1388G>A 27.59%（8/29）,c.1024C>T 13.79%（4/29）,c.487G>A 13.79%（4/29）and c.95A>G 13.79%（4/29）,The four common genotypes of G-6-PD deficiency in non-Heze mothers were c.1388G>A 27.45%（14/51）,23.53%（12/51）for c.487G>A,15.69%（8/51）for c.1376G>T,5.88%（3/51）for c.871G>A,In addition,the most common mutation type of G-6-PD deficiency in both Heze native and non-Heze native mothers was c.1388G>A.Conclusions:1.The prevalence of G-6-PD deficiency in neonates in Heze region was low,and the mutation hotspots of neonates in Heze region were as follows:c.1388G>A 27.50%,c.487G>A 20.00%,c.1376G>T 13.75%,indicating that the prevalence of G-6-PD deficiency and the type of gene mutation of neonates in Heze region have regional characteristics.2.In this study,four new G-6-PD gene mutations were found in the Chinese population for the first time（three exon missense mutations and one intron mutation were not reported in the Chinese population）,which enriched the database of G-6-PD gene mutations and provided rich reference materials for the screening and treatment of this disease.