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[Application of PCR-DGGE technique in G-6-PD deficiency]
Chang-Gan Li1, Xiao-Wen Chen, Yun-Sheng Chen
1Children's Hospital of Shenzhen, Shenzhen, Guangdong, China. licg6336@sina.com
Insights
Polymerase chain reaction and denatured gradient gel electrophoresis (PCR-DGGE) effectively detects glucose-6-phosphorate dehydrogenase (G-6-PD) gene mutations. This method is valuable for diagnosing G-6-PD deficiency and identifying carriers, particularly females.
Area of Science:
- Genetics
- Molecular Biology
- Biochemistry
Context:
- Glucose-6-phosphorate dehydrogenase (G-6-PD) deficiency is a common genetic disorder.
- Accurate diagnosis of G-6-PD deficiency and carrier status is crucial for genetic counseling and clinical management.
- Traditional diagnostic methods may have limitations in detecting all genotypes, especially in female carriers.
Purpose:
- To evaluate the utility of polymerase chain reaction and denatured gradient gel electrophoresis (PCR-DGGE) for detecting gene mutations in individuals with G-6-PD deficiency and their family members.
- To assess the diagnostic value of PCR-DGGE for identifying G-6-PD deficiency and carrier states.
- To identify specific mutation sites within the G-6-PD gene.
Summary:
- RNA was extracted from peripheral blood of 43 children with G-6-PD deficiency and 36 family lineages.
- PCR-DGGE was employed to analyze the G-6-PD cDNA fragment from exons 11-12, followed by gene sequencing of abnormal bands.
- Three novel mutation sites (C1311T, G1376T, and G1388A) were identified, with distinct electrophoresis patterns for each mutation.
Impact:
- PCR-DGGE demonstrates high sensitivity and reliability in screening for G-6-PD gene mutations.
- The technique proves beneficial for diagnosing G-6-PD deficiency, offering improved accuracy in identifying female carriers.
- This advancement aids in precise genetic diagnosis and family screening for G-6-PD-related disorders.
Objective:
To 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.
Methods:
cDNAs 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.
Results:
Abnormal 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.
Conclusions:
PCR-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.
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