Identification of RFLP G6PD mutations by using microcapillary electrophoretic chips (Experion)

Area of Science:

• Human Genetics
• Molecular Biology
• Clinical Diagnostics

Background:

• Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a prevalent human genetic disorder affecting over 400 million people worldwide.
• Currently, no commercial kits exist for widespread mutation screening of G6PD deficiency.

Purpose of the Study:

• To evaluate and compare the efficacy of two techniques, agarose gel electrophoresis and the Experion system, for identifying G6PD gene mutations.
• To screen G6PD-deficient Italian individuals for common mutations and identify novel variants.

Main Methods:

• Allele-specific PCR followed by restriction fragment length analysis was employed.
• Comparison of agarose gel electrophoresis with the Experion system for restriction pattern identification.
• Direct sequencing of the G6PD gene using ABI 3100 Avant Instrumentation for negative screening results.

Main Results:

• The Experion system demonstrated more accurate size band determination than agarose gel electrophoresis.
• Identified common G6PD variants: Mediterranean (75.7%), Seattle (7.1%), A(-) 202 + 376 (7.1%), and Cassano (2.8%).
• Discovered two novel mutations: Buenos Aires and Rignano.

Conclusions:

• The Experion system is a valid, user-friendly, and reproducible diagnostic method for G6PD mutation screening.
• This study advances G6PD deficiency diagnostics by comparing advanced techniques and identifying new mutations.