A Preliminary Application Study Based On MALDI TOF MS Of G6PD Hot Spot Mutations

BackgroundGlucose-6-phosphate dehydrogenase deficiency remains the most common inherited enzyme disorder in human beings.Several southern cities of China are listed as the regions with a high G6PDd allele frequency.Enzyme deficiency can cause acute hemolytic anemia,chronic non-spherical cell hemolytic anemia and neonatal hyperbilirubinemia that gives rise to adverse outcome and even neonatal death.Currently,two probing strategies are generally used,dot-blot hybridization and fluorescent in situ hybridization.However,their diagnostic efficiency of female heterozygotes is limited.As for genetic testing,Sanger sequencing and the next generation sequencing are most common,but they are rather inefficient and expensive.ObjectivesIn order to meet the needs of a fast,efficient and accurate detection of G6PD dehydrogenase deficiency,this dissertation uses a method based on multiplex polymerase chain reaction(MPCR)technology and a matrix-assisted laser desorption ionization time of flight mass spectrometry(MALDI-TOF MS)technology,in the hope of providing the basic research and verification of a new rapid detection method.It is of great significance to improve the level of genetic diagnosis of G6PD in China.MethodsBased on current epidemiological studies,this dissertation selected 16 common glucose-6-phosphate dehydrogenase deficiency disease-causing mutation sites in the Chinese population.A total of 411 patients with clinical diagnosis of "glucose-6-phosphate dehydrogenase deficiency" were tested with peripheral blood samples.DNA were extracted,the corresponding primers of 16 sites were designed,multiplexed PCR and single base extension were performed,and finally the samples were detected by time-of-flight mass spectrometry,and mutations were interpreted.For those cannot be tested in the list of 16 sites,we performed Sanger sequencing to confirm.And finally we did Sanger sequencing to the blood samples.ResultsThe mutation sites detected in this paper mainly include a total of 279 homozygous mutations.Therein,the most common are c.1388 G>A(37.28%),c.1376 G>T(34.41%),c.95 A>G(11.47%).A total of 86 heterozygous mutations include c.1376 G>T(46.51%),c.1388 G>A(31.40%),c.95 A>G(11.63%).Besides,we detected 19 compound heterozygous mutations such as c.1388 and c.1376(52.63%).These results basically in line with China’s epidemiological investigation.There are 27 samples cannot be tested in the designed 16 sites,and were confirmed in the sanger sequencing.We also did Sanger sequencing in blood samples,and it turns out that the results are accord with mass spectrometry’s.ConclusionsThe hot spot mutation detection method based on multiplex PCR and time-of-flight mass spectrometry used in this dissertation has the characteristics of high throughput,fast and low cost.It can improve the efficiency of clinical diagnosis at the genetic level and avoid missed diagnosis.Furthermore,the combination of multiplex PCR and time-of-flight mass spectrometry can provide new detection ideas for other genetic diseases.