Glutamine synthetase plays a major role in ammonia detoxification, interorgan nitrogen flux, acid–base homeostasis, and cell signaling. We report on two unrelated newborns who had congenital human glutamine synthetase deficiency with severe brain malformations resulting in multiorgan failure and neonatal death. Glutamine was largely absent from their serum, urine, and cerebrospinal fluid. Each infant had a homozygous mutation in the glutamine synthetase gene (R324C and R341C). Studies that used immortalized lymphocytes expressing R324C glutamine synthetase (R324C-GS) and COS7 cells expressing R341C-GS suggest that these mutations are associated with reduced glutamine synthetase activity.
Diminished glutamine synthetase expression or a secondary deficiency of glutamine synthetase has been described but is rare.10,11,12,13 We describe two unrelated patients with congenital systemic glutamine synthetase deficiency due to mutations in the glutamine synthetase gene.
Methods
Patient 1
Patient 1, a boy, was the second child of consanguineous Turkish parents. He was resuscitated at birth and was found to be neurologically compromised, with marked flaccidity, and also to have cardiac insufficiency. He died at two days of life (Table 1).
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The infant's skin fibroblasts failed to grow, but peripheral blood lymphocytes from the patient and his parents were immortalized successfully by transfection of the Epstein–Barr virus. Two years before his birth, his mother had had a pregnancy terminated at 34 weeks because ultrasonography of the fetal brain revealed abnormal gyrations with mild dilatation of the ventricles suggesting lissencephaly. Micromelia was also evident. However, fluorescence in situ hybridization (FISH) analysis of the region 17p13.3 on amniotic cells ruled out a deletion at the LIS1 locus.
Patient 2
Patient 2, a girl, was the third child of consanguineous Turkish parents. On the first day of life, convulsions and respiratory failure required intubation and ventilation. During the first weeks of life, the infant had voluminous yellowish stools and progressive weight loss, despite enteral feeding. After two weeks, a generalized blistering erythematous rash developed that on histologic examination supported a diagnosis of epidermal necrolysis. Brain MRI showed markedly attenuated gyri and subependymal cysts. She died during the fourth week of life from multiorgan failure. The only material available from the patient post mortem was her DNA, since her skin fibroblasts failed to grow. However, paternal skin fibroblasts were cultured successfully.
Glutamine was largely absent in the serum from both patients, whereas the glutamate levels were normal. The levels of both glutamine and glutamate were normal in the parents, for whom serum samples, urine samples, or both, were investigated after written informed consent was obtained (Table 1).
Elucidation of the Structure of the Glutamine Synthetase Gene
A search was performed with the glutamine synthetase transcript sequence (GenBank accession number NM_002065 [GenBank] ) and data derived from the Human Genome Project with the use of the Basic Local Alignment Search Tool (BLAST) algorithm (www.ncbi.nlm.nih.gov). Sequence alignments permitted the elucidation of the genomic structure of glutamine synthetase, which was confirmed by the sequencing of human liver–derived complementary DNA (cDNA) (Marathon Ready, Clontech) and DNA obtained from healthy control subjects. A GenBank search with the elucidated entire glutamine synthetase messenger RNA (mRNA) sequence was performed with the use of the nucleotide–nucleotide BLAST as well as the single-nucleotide polymorphism BLAST.
For clarification of the 5' region, 5' rapid amplification of cDNA ends (5' RACE) experiments were performed with the use of glutamine synthetase–specific primers according to the manufacturer's instructions (Boehringer).
Mutation Analysis
DNA was isolated from peripheral blood lymphocytes by standard methods. Polymerase-chain-reaction (PCR) amplification of the glutamine synthetase gene was performed exon by exon with the use of oligonucleotides designed for the flanking intronic regions of all coding exons. Direct sequencing of PCR products was performed with the use of a DNA sequencer (ABI Prism 3700, Applied Biosystems).
To rule out the possibility that the findings represented a common polymorphism, the frequency of the sequence alterations detected was investigated in 50 healthy white control subjects, most of whom were German but some of whom were from other countries in Europe (100 alleles), and in 34 healthy white Turkish controls (68 alleles). Race was determined by the investigators. All subjects provided written informed consent. For the mutation R324C, the enzyme BfrBI (New England Biolabs) was used to digest DNA at the site of the mutation. For the mutation R341C, allele-specific amplification with a primer carrying parts of the restriction site for ClaI (New England Biolabs) permitted the detection of mutated alleles.
Investigations of material obtained from the patients and their parents were performed after written informed consent was obtained from the parents. Healthy controls provided written informed consent for the use of DNA samples rendered anonymous for the analysis of polymorphisms. The study was approved by the institutional review board of the University of Münster.
Alignment of Mutations
Sequence alignment with eukaryotic and prokaryotic species was performed with the use of the GenBank BLAST algorithm in order to estimate the degree of evolutionary conservation. For estimation of the functional relevance of the mutations found, a model of the human enzyme was constructed on the basis of the x-ray structure of glutamine synthetase from Salmonella typhimurium (protein data bank code 1FPY [PDB] ).14
Cloning and Transfection of Wild-Type and Mutated Glutamine Synthetase Plasmids
As a template for PCR amplification of the entire glutamine synthetase cDNA (1122 bp), cDNA derived from human liver (Marathon-Ready, Clontech) was used. PCR amplification was performed with glutamine synthetase–specific primers with the use of plaque-forming unit–DNA–polymerase (Clontech) at an annealing temperature of 67°C. The product was purified (PCR Purification Kit, Qiagen) and directly ligated into the previously BamHI- and XbaI-digested eukaryotic expression vector pcDNA3.1HisA (Invitrogen). The ligation product was transformed into a Top10F' Escherichia coli strain. Correct plasmids were selected by sequencing and were named pcDNA3.1GS-WT.
Site-directed in vitro mutagenesis with the use of the wild-type plasmid as a target was performed for introduction of the alteration c.1021C>T into the expression plasmid, thereafter coding for R341C glutamine synthetase (R341C-GS). Methylation and mutagenesis reactions were performed with the use of the GeneTailor Site-Directed Mutagenesis System (Invitrogen). In brief, methylated plasmids (20 ng) were used for the mutagenesis reaction catalyzed by Platinum Taq DNA Polymerase High Fidelity. DH5
T1 E. coli were transformed with the mutagenesis product, and the resultant plasmid was control sequenced and was named pcDNA3.1GS-Mut.
COS7 cells (i.e., a line of African green-monkey kidney cells transformed by simian virus [SV] 40 were transfected with the plasmids pcDNA3. 1GS-WT and pcDNA3.1GS-Mut with the use of cationic lipids (in a molar lipid-to-DNA ratio of 2:1) (TransFast Transfection Reagent, Promega). Successfully transfected cells were selected by adding G418 (Geneticin, GIBCO) to the culture medium (200 µg per milliliter). GS-WT, GS-Mut, and a nontransfected cell line serving as a control were grown for 14 to 21 days at 37°C in Dulbecco's modified Eagle's medium with 10 percent fetal-calf serum (Invitrogen) in a humidified atmosphere with 5 percent carbon dioxide in six 75-cm2 flasks (Falcon).
Analysis of Glutamine Synthetase Expression
Cells were harvested in buffer containing 10 mM TRIS hydrochloride (pH 7.4), 1 percent Triton X-100, 150 mM sodium chloride, 1 mM EDTA, 1 mM ethylene glycol-bis(
-aminoethyl ether)-N,N-tetraacetic acid, 1 mM sodium vanadate oxide, 20 mM -glycerophosphate, 20 mM sodium fluoride, 10 mM sodium diphosphate, and a protease-inhibitor cocktail (1 tablet per 50 ml) (Boehringer). The protein content was estimated15 with the use of a reagent from BioRad. Proteins were analyzed by Western blotting with the use of 10 percent sodium dodecyl sulfate–polyacrylamide gels and a semi-dry transfer apparatus (Amersham Pharmacia). Blots were probed with antisera against glutamine synthetase (1:5000 solution) (polyclonal antibody, Santa Cruz Biotechnology; monoclonal antibodies, Transduction Laboratories and Chemicon) and against His-Tag (1:5000 solution) (Invitrogen) for two hours. After washing and incubation with horseradish-peroxidase–coupled antimouse-IgG or antigoat-IgG antibody diluted in a 1:10,000 solution at 4°C for two hours, the blots were developed with the use of Western-Lightning Chemiluminescence Reagent Plus (PerkinElmer).
Analysis of Glutamine Synthetase Activity
The glutamine synthetase–catalyzed formation of 
-glutamylhydroxamate from glutamine and hydroxylamine was measured.16 Aliquots of cell lysates containing equal amounts of protein were incubated with 60 mM of L-glutamine, 15 mM of hydroxylamine-hydrochloride, 20 mM sodium arsenite, 0.4 mM adenine diphosphate, 3 mM magnesium chloride, and 60 mM imidazole–hydrochloride buffer (pH 6.8) in a final volume of 1 ml at 37°C. The reaction was terminated by the addition of 1 ml of stop solution containing 0.2 M of trichloroacetic acid, 0.67 M hydrochloride, and 0.37 M iron chloride. The solution was cleared of protein by centrifugation (14,000xg) at 4°C and glutamylhydroxamate measured in the supernatant at 500 nm.
Statistical Analysis
Results (±SE) were compared with the use of Student's t-test. A difference was considered statistically significant if the P value was less than 0.05.
Results
Gene Structure
The GenBank-based search revealed the putative genomic structure of glutamine synthetase. After sequencing, the confirmed sequences of glutamine synthetase mRNA and DNA were deposited (GenBank accession numbers AY486122 [GenBank] and AY486123 [GenBank] , respectively). The glutamine synthetase mRNA consists of 1122 bp comprising 6 exons encoding a 374-amino-acid polypeptide with an estimated molecular mass of 42 kD.
Mutation Analysis
Sequencing of PCR fragments in both directions and restriction digestion showed different mutations in each of the two families: c.970C>T (R324C) in the family of Patient 1 and c.1021C>T (R341C) in the family of Patient 2 in exon 6 of the glutamine synthetase gene (Figure 1). The mutations were ruled out as common polymorphisms, since none of the 160 control alleles (including 60 alleles from Turkish persons) showed these sequence alterations.
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Sequence alignment showed R324 (human) to be strictly conserved in prokaryotic and eukaryotic glutamine synthetase and, likewise, that R341 was highly conserved. According to a model of human glutamine synthetase based on the x-ray structure of glutamine synthetase of S. typhimurium, R324 is part of the glutamate binding pocket, which stabilizes the -phosphate 1 moiety of ATP and is part of a sequence motif (ASIRIP). The arginine residue at codon R324 appears to be strictly conserved in prokaryotic and eukaryotic species. The arginine residue at codon R341 is in close proximity to highly conserved residues that are part of the active site of glutamine synthetase, forming a pocket for ATP and glutamate.
Analysis of Glutamine Synthetase Expression and Activity
In immortalized lymphocytes from Patient 1, glutamine synthetase activity was reduced to about 12 percent of that found in immortalized lymphocytes from wild-type controls. In the cells of the parents of Patient 1, glutamine synthetase activity was reduced to about 71 percent (in the father) and 49 percent (in the mother) (Figure 2A, gray bars). Increased glutamine synthetase expression was observed in the lymphocytes from Patient 1 and his mother. The normalization of glutamine synthetase activity to the level of glutamine synthetase expression found in the Western blotting test (Figure 2A, black bars) indicates that increased glutamine synthetase expression may, in part, compensate for the loss of specific glutamine synthetase activity.
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Discussion
Congenital systemic glutamine deficiency was the main biochemical finding in two neurologically devastated neonates with multisystem disease. The most striking features were marked brain malformations with abnormal gyration and marked white-matter lesions. Substitutions of arginine for cysteine at codons 324 and 341 were detected in Patient 1 and Patient 2, respectively. Parental heterozygosity suggested an autosomal recessive mode of inheritance and confirmed segregation of the mutations in the two families.
Despite an increase by about a factor of six in R324C-GS protein expression, immortalized lymphocytes from Patient 1 exhibited a considerable loss of glutamine synthetase activity as compared with lymphocytes from his parents and from the controls. As previously suggested,17 low levels of intracellular glutamine may up-regulate glutamine synthetase expression within a feedback regulatory loop. In the case of heterozygous carriers, this up-regulated expression may help to achieve glutamine synthetase activity sufficient to permit normal cell function. Transfection studies delineated the inactivation of glutamine synthetase as a result of the R341C exchange, supporting the disease-causing role of the mutation in Patient 2.
Glutamine is a major source of energy and is involved in cell proliferation, apoptosis inhibition, and cell signaling.18,19,20 Fetal glutamine requirements are very high and depend largely on active glutamine synthesis and the release of glutamine into the fetal circulation by the placenta.21 Although the levels of serum glutamine in heterozygous carriers were normal, maternal glutamine synthesis may be insufficient to guarantee an adequate supply of glutamine to the fetus. In addition, a loss of specific glutamine synthetase activity in the placenta derived from the fetus may further limit the glutamine fetal supply. In samples obtained from both Patient 1 and Patient 2 and in a fetal sample from a terminated pregnancy of the mother of Patient 1, fibroblast cultures failed to grow in spite of the presence of extracellular glutamine, pointing to a need for glutamine synthesis in cultures of fibroblasts from such patients. Interestingly, the concentration of glutamate (i.e., the substrate proximal to the metabolic block) was not elevated in either of the two patients, but this lack of elevation might be due to the various other pathways of glutamate catabolism.
In summary, we describe a congenital syndrome that results from systemic glutamine deficiency caused by a defect of the glutamine synthetase gene. Brain malformation was the principal manifestation in these two patients. This report might prompt others to consider this disorder in neonates with similar brain malformations. Obtaining serum glutamine levels in such neonates would be useful in screening for the disorder.
Supported by the German Research Foundation through the Collaborative Research Center SFB575 (Experimental Hepatology), Düsseldorf, Germany; and by a grant from Innovative Medizinische Forschung, Münster (to Dr. Häberle).
We are indebted to I. DuChesne, M. Grüneberg, M. Herting, V. Sablitzky, and L. Vogelpohl for excellent technical assistance and to C.O. Kalayci for supplying Turkish DNA samples.
Source Information
From the Universitätsklinikum Münster, Klinik und Poliklinik für Kinderheilkunde und Jugendmedizin, Münster, Germany (J.H., F.R., E.S., H.G.K.); Heinrich-Heine-University Düsseldorf, Clinic for Gastroenterology, Hepatology and Infectiology, Düsseldorf, Germany (B.G., F.S., D.H.); the Service de Génétique, Centre Hospitalier Universitaire Bretonneau (A.T.), and the Département de Pédiatrie, Centre Hospitalier Universitaire Clocheville (A.-L.S.) — both in Tours, France; the Hôpital Robert Debré, Service de Biochimie-Hormonologie (J.-F.B.), and the Unité de Neuropathologie, Hôpital Armand Trousseau (A.G.) — both in Paris; and the Charité-Universitätsmedizin Berlin, Institut für Biochemie, Berlin (W.H.).
References
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Related Letters:
Congenital Glutamine Deficiency with Glutamine Synthetase Mutations
Kölker S., Hoffmann G. F., Okun J. G., Rose C., Jalan R., Häberle J., Schliess F., Häussinger D.
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N Engl J Med 2006;
354:1093-1094, Mar 9, 2006.
Correspondence
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