Argininosuccinate synthetase gene is silenced by CpG methylation in children with phenylketonuria

• PKU is an autosomal recessive hereditary disease with amino acid metabolism disorder.
• Blood arginine level and the expression of ASS in PKU are not clear.
• 25 PKU patients and 65 normal healthy persons were investigated.
• Blood arginine level decreased significantly in PKU patients.
• The promoter of ASS was methylated which silence the transcription of ASS in PKU.

ObjectivesThe concentration of tyrosine and the ratio of branch-amino acid to the aromatic amino acid in phenylketonuria (PKU) patients are much lower than that of normal people, which reveal that PKU patients have amino acid metabolism disorder. The aim of the present study was to investigate the arginine level in blood, the expression of argininosuccinate synthetase (ASS), the rate-limiting enzyme in arginine synthesis pathway, and the methylation of ASS in patients with PKU.Design and MethodsTwenty-five children with PKU and 65 healthy controls were investigated in this study. Blood concentration of arginine was analyzed by automatic amino acid analyzer. The methylation of ASS gene promoter was evaluated by using methylation-specific polymerase chain reaction (MSP) and bisulfite sequencing PCR (BSP) methods, and the mRNA level of ASS was evaluated by semi-quantitative RT-PCR.ResultsBlood concentration of arginine in PKU patients without dietary control was 0.017 ± 0.009 mmol/L while in normal persons was 0.129 ± 0.007 mmol/L, which is statistically significant (P < 0.001). The promoter of ASS was methylated in PKU (15/15, 100%) but not in normal persons (0/15). The mRNA level of ASS in PKU patients was lower than that of normal people, which was well correlated with its methylation status.ConclusionsThe silencing of ASS due to aberrant promoter CpG methylation may be an important mechanism for arginine biosynthesis disorders in PKU. High levels of phenylalanine and low levels of arginine are common characteristics in PKU patients. These findings would extend the current understanding of arginine, ASS in the development of PKU disease.

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