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Technical Briefs |
1 Department of Pediatrics, University Hospital and General Hospital (AKH), Währingerstrasse 18-20, A-1090 Vienna, Austria
2 Department of Pediatrics, University Hospital, D-69120 Heidelberg, Germany
3 Institute of Child Health, University College, WC1N 2AP London, United Kingdom
4 Dipartimento di Scienze Neurologiche e Psichiatriche dell'Età Evolutiva, Università "La Sapienza", 00185 Rome, Italy
5 VU University Medical Center, De Boelelaan 1117, 1007 Amsterdam, The Netherlands
aauthor for correspondence: fax 43-1-4063484, e-mail stoeckler{at}metabolic-screening.at
Guanidinoacetate methyltransferase [(GAMT); EC 2.1.1.2] deficiency is the first recognized inborn error of creatine biosynthesis, manifesting in infancy with severe neurologic symptoms such as epilepsy, mental retardation, muscular hypotonia, and progressive extrapyramidal movement disorder (1). Patients with GAMT deficiency have shown favorable responses to oral supplementation of creatine-monohydrate, but complete reversal of symptoms has not been observed (2). Biochemical findings include high urinary excretion of guanidinoacetate (the immediate precursor of creatine and substrate of GAMT), low urinary excretion of creatinine [conversion product of intracellular creatine; see Ref. (3)], and depletion of creatine in brain and muscle (4). After assessing two index patients (5)(6), we aimed to establish methods for the noninvasive molecular diagnosis of GAMT deficiency. We recently developed a radiochemical method for the determination of GAMT activity in cultured skin fibroblasts and in virus-transformed lymphoblasts (7). Here we report a denaturing gradient gel electrophoresis (DGGE) technique for the screening of mutations in the GAMT gene in DNA extracted from dried-blood spot filter-paper samples and from fibroblasts and virus-transformed lymphoblasts.
Three index patients with mutations confirmed by techniques other than DGGE [P1, P2, and P6; see Refs. (5), (8)] and three new patients (P3, P4, and P5) with undetectable GAMT activity but unknown GAMT deficiency mutations (9)(10) were investigated. Details of the patient studies are summarized in Table 1
, and the locations and frequencies of the mutations identified in these patients are shown in Fig. 1
.
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We extracted DNA from dried-blood spot, filter-paper samples and from fibroblasts and lymphoblasts using Chelex-100 particles [Promega; see Ref. (11)] and the Nucleospin C+T reagent set (Machery), respectively. Primer pairs for nested PCR are available as a data supplement at Clinical Chemistry Online (Table 2; http://www.clinchem.org/content/vol48/issue5). Seven DNA fragments were amplified, including exon 1/promoter, the 3'-untranslated region (UTR), exons 2–6, and adjacent intron boundaries. The first round of PCR was carried out in a mixture containing 500 ng of DNA extracted from dried-blood spots, 800 nM each outside primer, 10 mM Tris-HCl (pH 8.3), 1.5 mM KCl, 2 mM MgCl2, 200 µM each deoxyribonucleotide, and 2.5 U of Ampli-Taq Gold DNA polymerase in a total reaction volume of 50 µL. Alternatively, 100 ng of DNA extracted from fibroblasts and lymphoblasts and 400 nM each of the outside primers were used. PCR conditions for a Perkin-Elmer-Cetus DNA Thermal Cycler were as follows: denaturation at 95 °C for 5 min followed by 40 cycles at 95 °C for 1 min, 65 °C for 1 min, 72 °C for 1 min, and a final elongation at 72 °C for 10 min. In a second PCR round, thermal cycling was carried out for 35 cycles as above with 0.5 µL of PCR product from the first round of amplification with 400 nM each of the nested primers.
DGGE was carried out essentially as described previously (12) with 20 µL of sample loaded onto a 10% polyacrylamide gel containing a 40–70% denaturing gradient (exon 2b); 8% polyacrylamide gel containing a 25–65% or a 40–70% denaturing gradient (exons 3, 4, 5, and 6); 12% polyacrylamide gradient gel containing a 40–80% denaturing gradient (exon 2a, 3'-UTR). A 100% denaturing gradient gel contains 7 mol/L urea and 400 mL/L (by volume) deionized formamide.
A one-step reversed transcription-PCR reaction with the Qiagen reagent set was used.
GAMT activity was measured in concentrated and dialyzed homogenates of cultured skin fibroblasts and/or Epstein-Barr virus-transfected lymphoblasts as described (7).
Using a combination of DGGE analysis of exons 2–6 and the 3'-UTR, and direct sequencing of the exon 1/promoter regions, we evaluated the established sensitivity of the DGGE assay with DNA from two index patients (P1 and P2) already known to be either compound heterozygous for c.327G-A/c.309ins13 or homozygous for c.327G-A in exon 2 of the GAMT gene (7) and a recently characterized patient (P6) with a c.491insG mutation in exon 5 and a IVS5–3C-G mutation in intron 5 (11). The zygosity of the samples investigated by DGGE was confirmed before sequencing by mixing the respective samples with the wild type (Fig. 2 in the online data supplement at Clinical Chemistry Online). Mutation screening in three new patients with enzymatically confirmed GAMT deficiency revealed homozygosity for the known c.327G-A mutation in two patients (P3 and P4). The third new patient (P5) was homozygous for a novel 151-bp deletion (g.1637–1787del) spanning 86 bp of exon 2 and 65 bp of intron 2 (Fig. 3A; online data supplement at Clinical Chemistry Online). The deletion predicted a frameshift with the first affected amino acid at position 81. The new reading frame terminated at a stop codon at position 97 (A81X97) in exon 3, with the addition of 16 novel amino acids. Sequencing of the mutant reverse transcription-PCR product from P5 revealed an in-frame skipping of exons 2 and 3 of the cDNA (Fig. 3B; online data supplement at Clinical Chemistry Online). A possible explanation of the in-frame skipping of exon 2 and of the neighboring exon 3 is the complete removal of the consensus sequence of the 5' splice site of exon 2/intron 2 (13) and splicing alterations circumventing the nonsense codon in exon 3 of the cDNA so that translation terminates normally (14). In P1, a novel A
G transition at nucleotide position 2366 of the genomic DNA in exon 4 was found in addition to the known c.327G-A/c.309ins13 mutations on exon 2. This 438GA sequence change is a silent mutation because both the wild-type ACA and the mutated ACG triplet encode for threonine at residue 146 of the GAMT peptide (T146T). P1 maternally coinherited in cis the c.438A-G mutation in exon 4 and the c.309ins13 mutation in exon 2 (data not shown). To date, distribution of the c.438A-G mutation as a possible polymorphism has not been examined in the general population.
The parents of P1, P2, P4, and P5, the mother of P3, and the brother of P1 were investigated. Heteroduplex bands on DGGE indicated heterozygosity for the wild-type and mutated alleles in all probands. Sequencing of the fragments carrying the mutant alleles revealed the sequence changes as expected from the findings in the affected patients. The brother of P1 also maternally coinherited in cis the c.438A-G mutation in exon 4 and the c.309ins13 mutation in exon 2 (data not shown).
The clinical phenotype of GAMT deficiency is highly variable, irrespective of residual enzyme activity and of the underlying gene mutations. Whereas P1, P2, P3, and P6 have a severe clinical phenotype with mental retardation, epilepsy, and progressive pyramidal and extrapyramidal motor handicaps, P4 and P5 have only moderate, nonprogressive mental retardation and behavioral problems. Ten of the 12 alleles investigated (83%) carried a mutation on exon 2. Among these mutations, the c.327G-A point mutation was the most frequent, as indicated by its presence in eight alleles. We did not perform haplotype analysis, but a common founder effect is unlikely because all patients carrying the c.327G-A point mutation are from different ethnic origins.
In conclusion, the DGGE method as described here is a valid tool for mutation screening in patients with clinical and biochemical features of GAMT deficiency, allowing the identification of point mutations and insertions and deletions on various sites of the GAMT gene. Newborn screening for GAMT deficiency based on the detection of characteristic metabolites (e.g., guanidinoacetate) by tandem mass spectrometry (15) can now be complemented by a genetic screening method applicable in dried blood spots.
References
The following articles in journals at HighWire Press have cited this article:
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  S. Mercimek-Mahmutoglu, S. Stoeckler-Ipsiroglu, A. Adami, R. Appleton, H. C. Araujo, M. Duran, R. Ensenauer, E. Fernandez-Alvarez, P. Garcia, C. Grolik, et al. GAMT deficiency: Features, treatment, and outcome in an inborn error of creatine synthesis Neurology, August 8, 2006; 67(3): 480 - 484. [Abstract] [Full Text] [PDF]
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