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Letters to the Editor |
1 Service de Biochimie Pédiatrique and
5 Service de Neuropédiatrie Hôpital Debrousse Lyon, France
2 VU University Medical Center Amsterdam, The Netherlands
3 Unité de Biologie Foetomaternelle Centre de Biologie Nord Hôpital de la Croix-Rousse Lyon, France
4 Centre Pluridisciplinaire de Diagnostic Prénatal Hôpital E Herriot Lyon, France
aAddress correspondence to this author at: Service de Biochimie Pédiatrique, Hôpital Debrousse, 29 rue S
ur Bouvier, 69322, Lyon cedex 05, France. Fax 33-4-72-38-58-84; e-mail david.cheillan{at}chu-lyon.fr.
To the Editor:
Guanidinoacetate methyltransferase (GAMT; EC 2.1.1.2) deficiency (OMIM 601240) is an autosomal recessive disorder of creatine biosynthesis, characterized clinically by mental retardation, language delay, extrapyramidal movements, epilepsy, and autistic behavior (1). Biochemically, GAMT deficiency is characterized by depletion of creatine and accumulation of guanidinoacetate (GAA) in the brain and body fluids (2). Treatment by creatine supplementation (combined with arginine restriction and ornithine supplementation) partially restores (
70%) cerebral creatine, reduces seizures, and improves behavior, but it does not reverse the mental retardation (3). We have described a method to measure GAA and creatine in plasma and urine by liquid chromatography–tandem mass spectrometry (LC-MS/MS) (4). In the present study, we validated this method for measurement of GAA and creatine in amniotic fluid, and we report the first GAMT prenatal diagnosis based on a combination of molecular and biochemical investigations.
We adapted a previously reported method to measure GAA and creatine in plasma (4) for amniotic fluid. Briefly, 50 µL of supernatant of amniotic fluid or aqueous calibrators were mixed with internal standards {d3-creatine (CDN isotopes) and [13C2]GAA (Dr. Ten Brink, VU University Medical Center, Amsterdam, The Netherlands)}. Samples were extracted and derivatized as butyl esters. LC-MS/MS analysis was performed in the multiple-reaction monitoring mode (API 2000; Sciex Applied Biosystems). Calibration curves were constructed by linear regression analysis of the ratios of the GAA and creatine calibrators to their respective internal standards.
The intraday imprecision (CV) of the global procedure (extraction and quantification) was estimated on a pool of amniotic fluids extracted 10 times and injected; the CVs were 9% for GAA and 5% for creatine. Interday imprecision was assessed in the same pool, extracted and injected on 5 different days; the CVs were 7% for GAA and 5% for creatine. For recovery, we added 2 concentrations of GAA and creatine (n = 5 for each concentration) to pooled amniotic fluid. The recoveries of 10 and 20 µmol/L GAA were 94% (CV = 4%) and 106% (CV = 3%), respectively, and the recoveries of 100 and 200 µmol/L creatine were 88% (CV = 3%) and 98% (CV = 2%), respectively.
We established control values by measuring GAA and creatine in 82 amniotic fluid samples from women at risk for carrying a fetus with trisomy 21; samples were obtained by amniocentesis between 15 and 20 weeks of amenorrhea. In all cases, the karyotype was normal. The results are given in Table 1
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We used this method to determine a prenatal diagnosis in a family with a 10-year-old child affected with GAMT deficiency. The diagnosis of GAMT deficiency was based on increased urinary concentrations of GAA and was confirmed by mutational analysis of the GAMT gene. A novel homozygous missense mutation was detected in exon 1 (c.148A>C; p.Met50Leu), which affected a highly conserved amino acid. The parents were heterozygous for this mutation. At the time of diagnosis of the index patient, the mother was pregnant, and amniocentesis was performed at 15 weeks of amenorrhea for prenatal diagnosis of GAMT deficiency by screening for the familial mutation in amniocytes and GAA measurement in amniotic fluid.
Assay results (Table 1
) indicated increased GAA (11.4 µmol/L) and normal creatine (48 µmol/L). The fetus was homozygous for the familial mutation, confirming the biochemical diagnosis of GAMT deficiency. The parents decided—partly because of the lack of experience with treatment at early age—to terminate the pregnancy.
We have validated a rapid method to measure GAA and creatine in amniotic fluid and have determined expected values at 15–20 weeks of amenorrhea. The feasibility of GAA measurement in amniotic fluid by gas chromatography–mass spectrometry has been reported (5). Here we describe the first prenatal diagnosis confirming that increased GAA in amniotic fluid appears to be pathognomonic for GAMT deficiency. Measurement of GAA in amniotic fluid may therefore be useful in the prenatal diagnosis of GAMT deficiency.
Acknowledgments
We thank Brigitte Soubeyrand and Luc Anselmini for excellent technical support. This work was supported by a grant from the French Ministry of Health–PHRC regional 2003; Pr des Portes.
References
The following articles in journals at HighWire Press have cited this article:
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  R S Carling, S L Hogg, T C Wood, and J Calvin Simultaneous determination of guanidinoacetate, creatine and creatinine in urine and plasma by un-derivatized liquid chromatography-tandem mass spectrometry Ann Clin Biochem, November 1, 2008; 45(6): 575 - 584. [Abstract] [Full Text] [PDF]
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