Use Urease Pretreatment-gas Gas Chromatography-Mass Spectrometry And Gene Analysis To The Diagnosis Of Methylmalonic Aciduria

Objective To investigate the value of urease pretreatment-gaschromatography-mass spectrometry (UP-GC-MS) in the diagnosis ofmethylmalnic aciduria (MMA). To analyze the mutation types of methylmalonicaciduria patients by gene sequencing.Methods 157 urine samples were collected from the populations withhigh risk of Inherited metabolic disorders. Urea was decomposed with urease,heptadecanoic acid was added as an internal standard, protein wasdenaturated with ethanol and precipitate was removed by centrifugation,evaporation was performed to dryness, the residue was trimethylsilyllyderivatized with BSTFA/TMCS, and then analyzed with GC-MS. The exons ofMMACHC gene were amplified from the 2 MMA families with the patients andtheir parents by PCR, The PCR products were analyzed by sequencing.Results Large quantity of methylmalnic acid and methylcitrate weredetected in 8 urine samples. In TIC profile, their retention time (relativeretention time) were 6.78min, 0.396min, 8.52min, and 0.24min, respectively,with characteristic ions of MMA in mass spectrums(m/z TMS) were 73, 147,247. The mutations were found in all the cases, including 146₁54delCCTTCCTGG in exon 2 and 609 G/A instead in exon 4 of MMACHC gene.Conclusions Analysis of urinary metabolites by UP-GC-MS is animportant and reliable method for the diagnosis of MMA. The Chinese patientsand their parents found the new mutation of 146₁54delCCTTCCTGG inMMACHC gene.