D-GLYCERIC ACIDURIA IS CAUSED BY GENETIC DEFICIENCY OF D-GLYCERATE KINASE Jörn Oliver Sass, Kathleen Fischer, Raymond Wang, Ernst Christensen, Sabine Scholl-Bürgi, Richard Chang, Klaus Kapelari, Melanie Walter
To cite this version:
Jörn Oliver Sass, Kathleen Fischer, Raymond Wang, Ernst Christensen, Sabine Scholl-Bürgi, et al.. D- GLYCERIC ACIDURIA IS CAUSED BY GENETIC DEFICIENCY OF D-GLYCERATE KINASE. Human Mutation, Wiley, 2010, 31 (12), pp.1280. 10.1002/humu.21375. hal-00591289
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D-GLYCERIC ACIDURIA IS CAUSED BY GENETIC DEFICIENCY OF D-GLYCERATE KINASE
For Peer Review
Journal: Human Mutation
Manuscript ID: humu-2010-0428.R1
Wiley - Manuscript type: Rapid Communication
Date Submitted by the 23-Sep-2010 Author:
Complete List of Authors: Sass, Jörn Oliver; Universitätsklinikum Freiburg, Zentrum für Kinder- und Jugendmedizin, Labor für Klinische Biochemie und Stoffwechsel Fischer, Kathleen; Universitätsklinikum Freiburg, Zentrum für Kinder- und Jugendmedizin, Labor für Klinische Biochemie und Stoffwechsel Wang, Raymond; Children's Hospital of Orange County, Division of Metabolic Disorders Christensen, Ernst; Rigshospitalet, Juliane Marie Centre, Dept. of Clinical Genetics Scholl-Bürgi, Sabine; Medizinische Universität Innsbruck, Department für Kinder- und Jugendheilkunde, Universitätsklinik für Pädiatrie IV Chang, Richard; Children's Hospital of Orange County, Division of Metabolic Disorders Kapelari, Klaus; Medizinische Universität Innsbruck, Department für Kinder- und Jugendheilkunde, Universitätsklinik für Pädiatrie I Walter, Melanie; Universitätsklinikum Freiburg, Zentrum für Kinder- und Jugendmedizin, Labor für Klinische Biochemie und Stoffwechsel
inborn error of metabolism , D-glycerate, serine, fructose, chirality, Key Words: enantiomer
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1 2 3 4 5 6 7 D-GLYCERIC ACIDURIA IS 8 9 CAUSED BY GENETIC DEFICIENCY OF D-GLYCERATE KINASE 10 11 12 13 14 15 Jörn Oliver Sass 1 , Kathleen Fischer 1 , Raymond Wang 2 , Ernst Christensen 3 , 16 17 4 2 5 1 18 Sabine Scholl-Bürgi , Richard Chang , Klaus Kapelari , Melanie Walter 19 20 For Peer Review 21 22 23 1Labor für Klinische Biochemie & Stoffwechsel, Zentrum für Kinder- und Jugendmedizin, 24 25 2 26 Universitätsklinikum Freiburg, Germany; Division of Metabolic Disorders, Children's 27 28 Hospital of Orange County, Orange, CA, USA; 3Department of Clinical Genetics, Juliane 29 30 4 31 Marie Centre, Rigshospitalet, Copenhagen, Denmark; Universitätsklinik für Pädiatrie IV, 32 33 Department für Kinder- und Jugendheilkunde, Medizinische Universität Innsbruck, Austria; 34 35 5Universitätsklinik für Pädiatrie I, Department für Kinder- und Jugendheilkunde, 36 37 38 Medizinische Universität Innsbruck, Austria 39 40 41 42 43 44 45 46 47 48 Address correspondence to: 49 50 51 Prof. Dr. Jörn Oliver Sass, Labor für Klinische Biochemie & Stoffwechsel, Zentrum für 52 53 Kinder- und Jugendmedizin, Universitätsklinikum Freiburg, Mathildenstr. 1, 54 55 D-79106 Freiburg, Germany 56 57 58 Phone: +49-761-270 4371; Fax: +49-761-270 4527; 59 60 e-mail: [email protected]
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1 2 3 Abstract 4 5 6 7 D-glyceric aciduria is a rare inborn error of serine and fructose metabolism that was first 8 9 described in 1974. Most affected individuals have presented with neurological symptoms. The 10 11 12 molecular basis of D-glyceric aciduria is largely unknown; possible causes that have been 13 14 discussed are deficiencies of D-glycerate dehydrogenase, triokinase, and D-glycerate kinase. 15 16 In 1989, van Schaftingen has reported decreased D-glycerate kinase activity in the liver of a 17 18 19 single patient with D-glyceric aciduria. However, this analysis has not been performed in 20 For Peer Review 21 other affected individuals, and the underlying defect has remained unknown on the gene level 22 23 until now. We report three patients with deficiency of D-glycerate kinase. They are of 24 25 26 Serbian, Mexican, and Turkish origin and include the patient initially reported in 1974. All 27 28 had homozygous mutations in exon 5 of the GLYCTK gene encoding D-glycerate kinase: 29 30 31 c.1448delT (p.Phe483SerfsX2), c.1478T>G (p.Phe493Cys) or c.1558delC 32 33 (p.Leu520CysfsX108). Transient overexpression of the variant GLYCTK genes in HEK293 34 35 cells clearly showed loss of enzyme activity and immunoreactivity when compared to the 36 37 38 reference enzyme. Our work has revealed mutations in the GLYCTK gene as the cause of D- 39 40 glycerate kinase deficiency and D-glyceric aciduria and provides a non-invasive approach for 41 42 further diagnostic work-up and research. 43 44 45 46 47 48 49 Key Words: 50 inborn error of metabolism, D-glycerate, serine, fructose, chirality, enantiomer 51 52 53 54 55 56 57 58 59 60
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1 2 3 Introduction 4 5 6 7 L-glyceric aciduria is a well known indicator for primary hyperoxaluria type II (MIM ID 8 9 260000), a disease which may lead to urolithiasis and nephrocalcinosis due the low solubility 10 11 12 of calcium oxalate. This is a clinically well defined and biochemically well understood inborn 13 14 error of metabolism, caused by mutations in the GRHPR gene, which result in deficiency of 15 16 glyoxylate reductase/hydroxypyruvate reductase (D-glyceric acid dehydrogenase; EC 17 18 19 1.1.1.26) (Cramer et al., 1999). 20 For Peer Review 21 22 23 In contrast, the clinical presentation of patients with D-glyceric aciduria (MIM ID 220120) is 24 25 26 rather heterogeneous. D-glyceric acid originates from serine catabolism and – to a minor 27 28 extent – from fructose metabolism. Up to now, only 10 children with D-glyceric aciduria have 29 30 31 been described in the literature (Table 1). Their clinical phenotypes range from an 32 33 encephalopathic presentation leading to death at 2.5 months of age, chronic metabolic 34 35 acidosis, seizures and severe mental retardation, microcephaly and speech delay to apparently 36 37 38 healthy siblings. Three more cases have been mentioned, but not been characterized except 39 40 for the report of D-glyceric acid identification in their urine (Rashed et al., 2002). Since 41 42 extraction of glyceric acid is often not effective in routine determinations of urinary organic 43 44 45 acids (Wadman et al., 1976, and own observations), it may well be that D-glyceric aciduria is 46 47 considerably underdiagnosed. 48 49 50 51 In the past, three different metabolic defects have been considered possible causes of D- 52 53 glyceric aciduria. Deficiency of D-glyceric acid dehydrogenase as has been suggested early 54 55 (Kølvraa et al. 1976). However, this hypothesis can be rejected today, not only, because this 56 57 58 enzyme deficiency is known to result in primary hyperoxaluria type 2 with accumulation of 59 60 the L-enantiomer of glyceric acid and not of the D-form, but also because the leukocyte
enzyme activities reported by Kølvraa et al. 1976 for a patient with D-glyceric aciduria are
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1 2 3 too similar to the control values to suggest deficiency of D-glyceric acid dehydrogenase if 4 5 6 compared to other reports (Williams & Smith, 1968; Chalmers et al., 1984). 7 8 9 In a patient with normal erythrocyte triokinase (EC 2.7.1.28) activity, hepatic triokinase 10 11 12 deficiency has been suggested as an underlying cause of D-glyceric aciduria, because oral 13 14 loading with fructose or dihydroxyacetone yielded a major increase in the excretion of D- 15 16 glycerate, while administration of 200mg L-serine/ kg body weight did not (Duran et al., 17 18 19 1987). However, van Schaftingen (1989) has noted on this case that triokinase deficiency 20 For Peer Review 21 would not explain the excretion of D-glycerate on a fructose-free regimen. Bonham et al. 22 23 (1990) explained that a higher dose of L-serine may have been necessary in the patient of 24 25 26 Duran et al. (1987) in order to get plasma L-serine levels high enough to raise excretion of D- 27 28 glycerate. This implies impaired catabolism of serine in the patient – as well as in other 29 30 31 individuals with D-glyceric aciduria – thus advocating against hepatic triokinase deficiency as 32 33 an underlying cause. 34 35 36 Since it is understood that in the human D-glycerate can only be metabolized to D-2- 37 38 39 phosphoglycerate (Snell 1986), it is reasonable to suspect D-glycerate kinase (EC 2.7.1.31) as 40 41 the deficient enzyme. While liver activities of D-glycerate dehydrogenase and triokinase were 42 43 44 normal, van Schaftingen (1989) demonstrated deficiency of hepatic D-glycerate kinase in a 45 46 patient with D-glyceric aciduria described by Fontaine et al., 1989, and Largillière et al., 47 48 1991. However, confirmation of that finding in other individuals with D-glyceric aciduria was 49 50 51 never reported. Possibly, this can be explained by the need for liver tissue for the enzyme 52 53 assay, and by the fact that the latter is complicated by the low stability of the hepatic enzyme. 54 55 56 57 Until now, the possibility of D-glycerate kinase deficiency has never been investigated on the 58 59 gene level ( GLYCTK gene, MIM ID 610516). This has prompted us to search for its 60
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1 2 3 molecular basis in the patient first described by Brandt et al. in 1974 and in two newly 4 5 6 diagnosed children with D-glyceric aciduria. 7 8 9 Patients, Materials and Methods 10 11 12 13 Patients 14 15 16 Patient A was the second son of Serbian parents. From the first days of life on he was 17 18 19 severely hypotonic and showed almost no spontaneous movements. Generalized seizures 20 For Peer Review 21 started when the boy was 8 weeks old. He was fed a normal diet, but showed no mental 22 23 development until he died from pneumonia at 3.5 years of age. This patient was the first 24 25 26 individual in whom D-glyceric acidemia was described (Brandt et al., 1974+1976) and was in 27 28 the focus of several reports which have been published until 1984 (Kølvraa 1979; Kølvraa et 29 30 31 al. 1976, 1980a+b, 1984). In this patient, clinical symptoms, high glycine levels in serum, 32 33 cerebrospinal fluid and urine and decreased activity of the glycine cleavage system in an 34 35 autopsy liver sample suggest that he may have been affected by nonketotic hyperglycinemia 36 37 38 in addition to D-glyceric aciduria. 39 40 41 42 Patient B was born prematurely at 27 weeks’ gestational age due to placental abruption, to a 43 44 45 16 year-old primigravida of Mexican origin. There was no known parental consanguinity or 46 47 family history of unexplained neonatal illnesses. Complications related to prematurity 48 49 50 included neonatal respiratory distress syndrome requiring mechanical ventilation for six 51 52 weeks, intraventricular hemorrhage, patent ductus arteriosus that was surgically ligated, and 53 54 gastroesophageal reflux. Ophthalmologic examination demonstrated bilateral optic nerve 55 56 57 hypoplasia. 58 59 60
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1 2 3 She demonstrated poor weight gain in the neonatal period that persisted beyond her discharge 4 5 6 from the neonatal intensive care unit at 5.5 months of age, necessitating placement of a 7 8 gastrostomy tube. The patient developed focal clonic seizures. At 11 months of age (8 months 9 10 adjusted), an inborn errors of metabolism evaluation was requested. She had severe failure to 11 12 13 thrive and microcephaly. 14 15 Physical examination was significant for microcephaly, roving eye movements with 16 17 18 intermittent fixation on faces, tongue thrusting, hypotonia, appendicular spasticity, and brisk 19 20 deep tendon reflexes. ForBrain MRI Peerdemonstrated Reviewdelayed myelination and mild cortical atrophy 21 22 without contrast enhancement, heterotopias, or lesions in the basal ganglia and brainstem. 23 24 25 Initial electroencephalogram showed status epilepticus. Qualitative urine organic acid analysis 26 27 revealed a large peak of glyceric acid without oxalic aciduria, and excretion of 3- 28 29 methylglutaconic acid. 30 31 32 33 34 Despite the provision of more than 120 kilocalories per kg body weight per day, at 31 months 35 36 of age she has continued to demonstrate poor weight gain and microcephaly. Severe global 37 38 39 developmental delay is evident, as she has no verbal milestones and no gross or fine motor 40 41 skills. Her seizures evolved into a mixture of generalized tonic-clonic and focal clonic types 42 43 44 that have been refractory to management despite multiple antiepileptic medications. 45 46 47 Patient C is the second child of healthy consanguineous Turkish parents. Pregnancy and 48 49 delivery in the 41 st gestational week were uneventful. Some dysmorphic features were noted, 50 51 52 i.e., sunken-in eyeballs and nasal ridge, transversal palmar crease, micropenis. At the age of 53 54 12 hours the boy showed a cyanotic attack following milk aspiration. 55 56 57 58 Slow capillary refill, muscular hypotonia, bradycardia, and an episode of profound 59 60 hypoglycemia (14 mg/dl) were observed as well. He was substituted with glucose 5 mg
kg -1 min -1. The patient has subsequently had recurrent episodes of hypoglycemia. Follow-up
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1 2 3 investigations showed pituitary insufficiency with complete absence of TSH, hGH, ACTH, 4 5 6 cortisol, sexual hormones and prolactin and agenesis of the hypophysis on MRI (empty sella), 7 8 which was explained by a homozygous mutation in the HESX1 gene (Prof. Pfäffle, Leipzig, 9 10 Germany). Both adrenal glands were not detected in ultrasound investigations. A basic 11 12 13 metabolic work-up yielded an elevated signal of glyceric acid in the analysis of urinary 14 15 organic acids, but no oxalic aciduria. 16 17 18 19 Substitution with L-thyroxine and hydrocortisone immediately, and intermittent growth 20 For Peer Review 21 hormone and testosterone corrected endocrine parameters and stabilized the patient. Full oral 22 23 feeds could only be introduced slowly due to gastroesophageal reflux and muscular 24 25 26 hypotonia. However, at the age of eight weeks the patient was dismissed in good condition, 27 28 adequately thriving and developing for age. 29 30 31 32 Glyceric acid determination 33 34 Glyceric acid was quantified by GC-MS analysis (using a CP-Wax 58 (FFAB) column; 35 36 Varian) following liquid extraction from random urine or plasma and derivatization with 37 38 39 diazomethane (Lehnert 1994). Isopropylmalonic acid served as internal standard. Prior to the 40 41 extraction, plasma protein was precipitated with 50 mg solid sulfosalicylic acid per 1 ml 42 43 44 plasma and removed by centrifugation. External calibration was performed using calibration 45 46 curves obtained with 0 – 100 mM D-glyceric acid calcium salt (Sigma-Aldrich) in water (for 47 48 the urine) or in serum (for serum samples). Enantiomers of methylated enantiomers of 49 50 51 glyceric acid were separated on a CP-Cyclodex B column (Varian). 52 53 54 55 Analysis of the GLYCTK gene 56 57 58 Genomic DNA was extracted from fibroblasts (patient A) or peripheral blood (patients B, C, 59 60 and their parents, if available; controls) by standard methods.
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1 2 3 All exons and exon-intron boundaries of the GLYCTK gene of the patients were amplified by 4 5 6 PCR and prepared for direct sequencing by a commercial provider (LGC Genomics, Berlin, 7 8 Germany). Primer sequences and PCR conditions are available upon request. DNA sequences 9 10 were analyzed using CodonCode Aligner software (CodonCode Coporation, Dedham, MA, 11 12 13 USA). Nucleotide numbering reflects cDNA numbering with +1 corresponding to 14 15 the A of the ATG translation initiation codon in the GenBank reference 16 17 18 sequence NM_145262.3, according to journal guidelines (www.hgvs.org/mutnomen). The 19 20 initiation codon is codonFor 1. The Peer corresponding Review reference intron sequence is available from 21 22 GenBank file NG_023246.1. 23 24 25 In order to exclude that the novel sequence variations, namely the missense mutation 26 27 p.Phe493Cys (patient B), represent polymorphisms, 107 samples (214 chromosomes) 28 29 (Collins & Schwartz, 2002), were analyzed by sequence analysis of the coding region of exon 30 31 32 5 of the GLYCTK gene, using primers 5’-TTGGAGCAGAGTTGAGAAGGTG-3’ (forward) 33 34 and 5’-TGTCCTCATCCTCAGGGGTAG-3’ (reverse). In view of the origin of patient B, 73 35 36 of the 107 control samples were anonymized samples from Hispanic women (kindly provided 37 38 39 by Prof. Jan Kraus, University of Colorado). 40 41 42 43 44 Cloning and site-directed Mutagenesis 45 46 A human cDNA clone with transcript variant 1 of GLYCTK (NCBI NM_145262.2) in the 47 48 vector pCMV6-AC was obtained from Origene (cat. no. SC319873) . It was used for 49 50 51 transformation of DH10B competent cells (Invitrogen). The Stratagene QuickChange site- 52 53 directed mutagenesis kit was applied to introduce alternatively the mutations c.1448delT, 54 55 c.1478 T>G and c.1558delC into the GLYCTK cDNA sequence carried by the vector pCMV6- 56 57 58 AC. Successful mutagenesis was confirmed by sequence analysis. Subsequently, the different 59 60 GLYCTK cDNA sequences were cloned into the mammalian expression vector pCDNA3.1(+)
(Invitrogen) via the restriction sites HindIII and XhoI.
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1 2 3 4 5 6 Cell culture and transfection of HEK293 cells 7 8 Expression studies for GLYCTK cDNA reference sequence, GLYCTK cDNA with any of the 9 10 three mutations or the empty vector (mock control) were performed in human embryonic 11 12 13 kidney cells of the cell line HEK293. HEK293 cells were purchased from DSMZ (Deutsche 14 15 Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany) and 16 17 18 cultivated in DMEM (Dulbecco's Modified Eagle's Medium) supplemented with 10 % heat- 19 20 inactivated fetal bovineFor serum Peer and antibiotic-antimycotic Review no. 15240 (all from Gibco). 21 22 Transfections of 10 µg plasmid DNA into HEK293 cells were performed using Xfect 23 24 25 transfection reagent (Clontech). After 68.5 hours, cell samples were harvested and frozen at - 26 27 80°C until analysis. The pEGFP-C1 plasmid (Clontech) was used to confirm successful 28 29 transfection by fluorescence microscopy. 30 31 32 33 34 SDS-PAGE and Western Blot analysis 35 36 GLYCTK expression was studied on the protein level by SDS-PAGE followed by Western 37 38 39 blot analysis. Homogenates of HEK293 cells transfected with a pcDNA3.1(+) vector carrying 40 41 the GLYCTK cDNA reference sequence or the GLYCTK cDNA with one of the mutations 42 43 identified in the patients A-C, or transfected with the empty vector, were used. Following 1 44 45 46 min centrifugation of the homogenates at 10,000 g and 4°C, protein assessment according to 47 48 Lowry et al., 1951, and denaturation, 30 µg of supernatant protein were loaded per lane. The 49 50 D-glycerate kinase protein was identified in the Western blot using a mouse polyclonal 51 52 53 antibody against full-length human GLYCTK protein (Abnova H00132158-B01). The 54 55 secondary antibody was a goat-anti mouse antibody (horseradish peroxidase conjugate, 56 57 58 Southern Biotech 1010-05). For comparison, signal intensities were compared after 59 60 reincubating the blot with a rabbit polyclonal antibody against the housekeeping enzyme
GAPDH (Gene Tex, GTX100118). Here, the secondary antibody was a goat-anti rabbit
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1 2 3 antibody (horseradish peroxidase conjugate, Southern Biotech 4010-05). Molecular weights 4 5 6 of proteins were estimated using the Biorad protein standard cat.no. 161-0374. 7 8 9 10 Assessment of D-glycerate kinase activity 11 12 13 Immediately after thawing on ice, transfected HEK293 cells were homogenized in the cold. 14 15 For stabilization of the D-glycerate kinase and in order to bind enzyme-inhibiting calcium 16 17 18 present in commercially available D-glycerate, 50 µM glycerate and 1 mM EGTA were 19 20 added to 10 mM potassiumFor buffer Peer (pH 7.1) used Review for homogenization (van Schaftingen, 1989). 21 22 Following 1 min of centrifugation at 10,000 g and 4°C the resulting supernatant was directly 23 24 25 submitted to enzyme activity testing. Only two samples were investigated at the time, in order 26 27 to analyze the samples within 5 min after the homogenization, as indicated in order to 28 29 minimize degradation of the enzyme (van Schaftingen 1989). Enzyme activity was 30 31 32 determined with a coupled assay (van Schaftingen, 1989), based on the description by 33 34 Lamprecht et al., 1959. Within this assay, 2-phosphoglycerate formed from D-glycerate is 35 36 converted to pyruvate by added enolase and pyruvate kinase. In order to increase sensitivity, 37 38 39 we monitored NADH-dependent reduction of pyruvate to lactate (catalyzed by lactate 40 41 dehydrogenase) not in a photometric assay, but by fluorescence analysis in a BioTek Synergy 42 43 44 HT microplate reader (excitation 360 nm, emission 460 nm). For determination of the K m 45 46 value, variable substrate concentrations of up to 1mM D-glycerate were applied, otherwise the 47 48 concentration of D-glycerate was 1 mM. The protein concentrations of the samples were 49 50 51 determined by the Lowry method using bovine serum albumin as the standard (Lowry et al., 52 53 1951). 54 55 56 57 58 59 60
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1 2 3 Results 4 5 6 D-glycerate in body fluids 7 8 The determination of urinary organic acids, a fundamental diagnostic test in the diagnostic 9 10 work-up of a suspected inborn error of metabolism, revealed an abnormal pattern for the 11 12 13 investigated patients. High concentrations of glyceric acid were detected, which is normally 14 15 undetectable. 16 17 18 For patient A, deceased more than 30 years ago, no body fluids were available anymore for 19 20 new analyses. However,For for this Peerpatient D-glyceric Review acid concentrations of 1.0 to 1.3 mM in 21 22 plasma (normal: not detectable) and 24-hour urinary excretion with ranges of 14 – 23, 46.1 – 23 24 25 62.2 and 25.0 – 85.0 mmol have been reported previously (Brandt et al., 1976; Kølvraa et al., 26 27 1984). We found elevated urinary glyceric concentrations of 9.32 mol/mol creatinine in a 28 29 sample of patient B, 11.3, 8.61 and 18.6 mol/mol creatinine in three samples of patient C 30 31 32 (normal: not detectable); all glyceric acid was in the D-form. For patient C, the glyceric acid 33 34 analysis in a serum sample yielded 1 mM glyceric acid. 35 36 37 38 39 Mutations in the GLYCTK gene 40 41 All three patients presented with homozygous mutations in exon 5 of the GLYCTK gene 42 43 44 encoding D-glycerate kinase, but no other mutations (Figure 1). 45 46 In patient A the mutation c.1448delT was identified, which predicts a frameshift resulting in 47 48 premature stop of translation (p.Phe483SerfsX2). Child B presented with a homozygous 49 50 51 missense mutation c. 1478T>G, resulting in an amino acid exchange p.Phe493Cys. In the 52 53 third patient (C) a frameshift mutation c.1558delC was revealed, which suggests a massively 54 55 altered, overlong product of protein synthesis (p.Leu520CysfsX108), which is probably prone 56 57 58 to degradation. Analyses of DNA of the mother of individual B and of both parents of patient 59 60 C revealed that the parents were heterozygous for the mutations. In contrast, none of 107
control samples (214 chromosomes) showed any of the three mutations.
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1 2 3 4 5 6 Expression analysis of the GLYCTK gene in HEK293 cells 7 8 Transfection of HEK293 cells with the vector carrying the cDNA reference sequence of 9 10 GLYCTK resulted in a major signal in the Western blot analysis, which was much stronger 11 12 13 than the faint band to be seen with the mock control (Figure 2; lanes 1,2,6). 14 15 Western blot data were confirmed by results of enzyme activity tests which raised enzyme 16 17 -1 -1 -1 -1 18 activity from trace amounts < 0.5 µmol min g protein to 30.7 µmol min g protein 19 20 (Figure 3). For the recombinantFor Peer human enzyme Review analyzed in HEK293 cell homogenate we 21 22 obtained a K m value of 0.1 mM (mean of two independent transfection series). In contrast, 23 24 25 expression of any of the three mutated forms of GLYCTK, c.1448delT, c. 1478T>G and 26 27 c.1558delC, yielded neither an enhanced signal for GLYCTK in Western blot analysis nor 28 29 activity in the enzyme activity test. 30 31 32 33 34 Discussion 35 36 Although several reports on D-glyceric aciduria have been published between 1974 and 2002, 37 38 39 the molecular basis of this inborn error of metabolism has remained obscure. D-glycerate 40 41 dehydrogenase deficiency was excluded as an underlying cause and convincing evidence is 42 43 44 lacking for ascribing D-glyceric aciduria to triokinase deficiency (see Introduction). In 45 46 contrast, activity testing of D-glycerate kinase which van Schaftingen has performed in the 47 48 liver of a single patient with D-glyceric aciduria (van Schaftingen, 1989), suggested that this 49 50 51 may be the key enzyme. However, for more than twenty years those investigations have not 52 53 been continued. The molecular basis of D-glyceric aciduria remained to be elucidated. We 54 55 have now expressed the GLYCTK enzyme in a human expression system. Protein size, 56 57 58 immunoreactivity and enzyme activity of cell homogenate confirmed that this approach has 59 60 been successful. The K m value for D-glycerate which we have obtained (0.1 mM) is
compatible with the wide range of values reported earlier for the liver enzyme of different
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1 2 3 species: 0.015 mM (van Schaftingen, 1989) and - applying acetone powder - 2.5 – 3.5 mM 4 5 6 (Thieden et al., 1972) for human liver, 0.03 mM (Thieden et al., 1972) and 0.3 mM (Holzer 7 8 & Holldorf, 1957) for rat liver, 2.4 mM for horse liver (Ichihara & Greenberg, 1957). Archaea 9 10 and bacteria which possess class II glycerate kinases (like animals do), have been reported 11 12 13 with K m values of several hundreds of µmol/l (Kehrer et al., 2007). As noted already by 14 15 Ichihara & Greenberg (1957) and by van Schaftingen (1989) for hepatic D-glycerate kinase, 16 17 18 the enzyme is quite labile. We can confirm this experience for the recombinant enzyme. 19 20 Rapid processing of theFor samples Peer proved to beReview important for reliable analyses, as they are 21 22 needed for the evaluation of the biological impact of sequence variations in the GLYCTK 23 24 25 gene. 26 27 28 29 Sequencing of the GLYCTK gene in three unrelated patients with D-glyceric aciduria revealed 30 31 32 mutations in exon 5 which will affect the N-terminus of the protein and have not been 33 34 reported before. For patient A, a mutation (c.1448delT) was identified, which predicts a 35 36 frameshift resulting in premature stop of translation (p.Phe483SerfsX2), which may yield a 37 38 39 translation product which is prone to immediate degradation. In agreement with such a 40 41 consequence, the protein was neither detected in Western blot analysis performed with an 42 43 44 antibody raised against the full-length protein, nor resulted its expression in HEK293 cells in 45 46 detectable GLYCTK enzyme activity. 47 48 49 50 51 Child B presented with a homozygous missense mutation c.1478T>G, resulting in an amino 52 53 acid exchange p.Phe493Cys. Neither this (nor any other of the patients’ mutations) has been 54 55 detected in 214 control chromosomes, most of them with a similar ethnic background than 56 57 58 patient B. This makes it unlikely that the missense mutation simply represents a 59 60 polymorphism. The high degree of conservation of the phenylalanine residue 493 next to a
second phenylalanine advocates for a functional role of this residue. All vertebrates studied
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1 2 3 showed this sequence (Figure 4). Even in fruit fly and nematode one phenylalanine residue is 4 5 6 still present and the other replaced only by another aromatic amino acid, tyrosine or 7 8 tryptophan. Like the frameshift mutations the amino acid exchange p.Phe493Cys yielded no 9 10 protein that was detectable in the immunoassay and completely abolished GLYTK activity. 11 12 13 14 15 An ENSEMBL search for domains of the GLYCTK protein indicated that its amino acids 401 16 17 18 – 514, i.e., the part directly affected by the mutations found in patients A and B, form a 19 20 MOFRL (“multi-organismFor fragment Peer with rich Review leucine”) domain. Since this is characteristic 21 22 for class II glycerate kinases, which comprise animal glycerate kinases, (Kehrer et al., 2007) it 23 24 25 may be well be that sequence variations in this part of the protein affect enzyme function and/ 26 27 or stability. 28 29 30 31 32 In the third patient (C) a frameshift mutation c.1558delC was identified which results not only 33 34 in the replacement of a evolutionary conserved branched-chain amino acid residue by 35 36 cysteine, but encodes elongation of the translation product by more than 100 amino acids, 37 38 39 which suggests that no stable protein will be formed. Indeed, GLYCTK protein was not 40 41 detected in the immunoblot or in the enzyme activity test. 42 43 44 45 46 14 tentatively protein coding transcripts of the GLYCTK gene are listed in NCBI and 47 48 EBSEMBL data bases. However, except for transcript variant 1, which we have now 49 50 51 expressed in HEK293 cells, no enzyme activity derived from another GLYCTK transcript has 52 53 been reported. Notably, any of the three mutations in exon 5 which we have identified, has 54 55 resulted in the loss of detectable protein and enzyme activity, including the missense mutation 56 57 58 p.Phe493Cys. Hence, it is not surprising if transcript variants presenting with sequence 59 60 variations or deletions affecting exon 5 will not result in functional protein. As summarized
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1 2 3 by Sorek et al. (2004), aberrant rather than regulated splicing can account for many of such 4 5 6 transcript variants. 7 8 Excretion of D-glyceric acid in the patients reported here resembled the range of values 9 10 previously reported for most other children with D-glyceric aciduria (Wadman et al., 1976; 11 12 13 Grandgeorge et al., 1980; Duran et al., 1987; Fontaine et al., 1989; Bonham et al., 1990), thus 14 15 rendering patients A, B and C quite typical representatives of children with D-glyceric 16 17 18 aciduria. Interestingly, mutations could be identified in all three individuals in whom we have 19 20 identified D-glyceric aciduria.For Thus, Peer we provide Review no evidence for genetic heterogeneity of D- 21 22 glyceric aciduria. Segregation analyses revealed that all parents studied were heterozygous 23 24 25 carriers of the mutations, which is compatible with an autosomal recessive trait of inheritance. 26 27 D-glyceric aciduria has been identified in patients of various ethnic origins. However, 28 29 knowledge about D-glyceric aciduria is very limited. This may not only reflect that standard 30 31 32 protocols used for the assessment of urinary organic acids may show rather low sensitivity for 33 34 glyceric acid and that this disorder therefore may be underdiagnosed. In addition, the limited 35 36 diagnostic options beyond invasive liver biopsy can have prevented detailed investigations. In 37 38 39 view of the heterogeneous clinical presentation of children with D-glyceric aciduria, further 40 41 work is needed to clarify whether D-glycerate kinase deficiency is a disease or a merely 42 43 44 biochemical variant. It is possible that D-glycerate kinase deficiency has consequences later in 45 46 life, perhaps as a predisposing factor. Long-term follow-up studies with a greater number of 47 48 individuals may be required to detect such effects; observations on the basis of three pediatric 49 50 51 patients alone may not be sufficient. Our finding that homozygous mutations in the GLYCTK 52 53 gene cause D-glycerate kinase deficiency and D-glyceric aciduria, opens the opportunity to 54 55 learn more about this inborn error of metabolism. Mutation analysis in the GLYCTK gene 56 57 58 provides a non-invasive tool for confirmatory tests as well as for epidemiological studies. 59 60
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1 2 3 4 5 6 Acknowledgements 7 8 The authors are grateful to Prof. Daniela Karall (Medizinische Universität Innsbruck, 9 10 Innsbruck, Austria) for her help regarding patient C and thank Sidney Behringer, Peter 11 12 13 Pfeiffer and Frauke Beermann for excellent technical assistance. We give thanks to Prof. Jan 14 15 Kraus (University of Colorado, CO, USA) for kindly providing control DNA samples and 16 17 18 acknowledge financial support by the Jürgen Manchot Stiftung (Düsseldorf, Germany). 19 20 For Peer Review 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
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1 2 3 References 4 5 6 7 Bonham JR, Stephenson TJ, Carpenter KH, Rattenbury JM, Cromby CH, Pollitt RJ, 8 9 Hull D. 1990. D(+)-glyceric aciduria: etiology and clinical consequences. Pediatr Res 28: 38- 10 11 12 41. 13 14 Brandt NJ, Brandt S, Rasmussen K, Schønheyder F. 1974. Hyperglycericacidaemia 15 16 with hyperglycinaemia: A new inborn error of metabolism. Br Med J 4: 344. 17 18 19 Brandt NJ, Rasmussen K, Brandt S, Kølvraa S, Schønheyder F. 1976. D-glyceric- 20 For Peer Review 21 acidaemia and non-ketotic hyperglycinaemia. Clinical and laboratory findings in a new 22 23 syndrome. Acta Paediatr Scand 65: 17-22. 24 25 26 Chalmers RA, Tracey BM, Mistry J, Griffiths KD, Green A and Winterborn MH. 1984. 27 28 L-Glyceric aciduria (primary hyperoxaluria type 2) in siblings in two unrelated families. J 29 30 31 Inherit Metab Dis 7 (suppl.2):133-134. 32 33 Collins JS, Schwartz CE. 2002. Detecting polymorphisms and mutations in candidate 34 35 genes. Am J Hum Genet 71:1251-1252. 36 37 38 Cramer SD, Ferree PM, Lin K, Milliner DS, Holmes RP. 1999. 39 40 The gene encoding hydroxypyruvate reductase (G RHPR ) is mutated in patients with primary 41 42 hyperoxaluria type II. Hum Mol Genet 8:2063-2069. 43 44 45 Duran M, Beemer FA, Bruinvis L, Ketting D and Wadman SK. 1987. D-glyceric 46 47 acidemia: an inborn error associated with fructose metabolism. Pediatr Res 21: 502-506. 48 49 Fontaine M, Porchet N, Largilliere C, Marrakchi S, Lhermitte M, Aubert JP and 50 51 52 Degand P. 1989. Biochemical contribution to diagnosis and study of a new case of D-glyceric 53 54 acidemia/aciduria. Clin Chem 35: 2148-2151. 55 56 57 Grandgeorge D, Favier A, Bost M, Frappat P, Boujet C, Garrel S, Stoebner P. 1980. 58 59 L’acidémie D-glycérique: a propos d’une nouvelle observation anatomo-clinique. Arch Fr 60 Pediatr 37 : 577-584
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1 2 3 Holzer H, Holldorf A. 1957. Anreicherung, Charakterisierung und biologische 4 5 6 Bedeutung einer D-Glyceratkinase aus Rattenleber. Biochem Z 329:283-291. 7 8 9 Ichihara A, Greenberg DM.1957. Studies on the purification and properties of the D- 10 11 12 glyceric acid kinase of liver. J Biol Chem 225:949-958. 13 14 Kehrer D, Ahmed H, Brinkmann H, Siebers B. 2007. Glycerate kinase of the 15 16 hyperthermophilic archaeon Thermoproteus tenax: new insights into the phylogenetic 17 18 19 distribution and physiological role of members of the three different glycerate kinase classes. 20 For Peer Review 21 BMC Genomics 8:301 doi:10.1186/1471-2164-8-301. 22 23 Kølvraa S. 1979. Inhibition of the glycine cleavage system by branched-chain amino 24 25 26 acid metabolites. Pediatr Res 13:889-893. 27 28 Kølvraa S, Christensen E, Brandt NJ. 1980a. Studies on the glycine metabolism in a 29 30 31 patient with D-glyceric acidemia and hyperglycinemia. Pediatr Res 14:1029-1034. 32 33 Kølvraa S, Gregersen N, Brandt NJ. 1980b. Excretion of short-chain N-acylgylcines in 34 35 the urine of a patient with D-glyceric acidemia. Clin Chim Acta 106:215-221. 36 37 38 Kølvraa S, Gregersen N, Christensen E. 1984. In vivo studies on the metabolic 39 40 derangement in a patient with D-glyceric acidemia and hyperglycinemia. J Inherit Metab Dis 41 42 7:49-52. 43 44 45 Kølvraa S, Rasmussen K, Brandt NJ. 1976. D-glyceric acidemia: biochemical studies 46 47 of a new syndrome. Pediatr Res 10:825-830. 48 49 Largillière C, van Schaftingen E, Fontaine M, Farriaux JP. 1991. D-glyceric acidaemia: 50 51 52 clinical report and biochemical studies in a patient. J Inherit Metab Dis 14:263-264. 53 54 Lehnert W. 1994. Long-term results of selective screening for inborn errors of metabolism. 55 56 57 Eur J Pediatr 153: S9-S13. 58 59 60 Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. 1951. Protein measurement with the
folin phenol reagent. J Biol Chem 193:265–275.
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1 2 3 Rashed MS, Aboul-Enein HY, AlAmoudi M, Jakob M, Al-Ahaideb LY, Abbad A, 4 5 6 Shabib S, Al-Jishi E. 2002. Chiral liquid chromatography tandem mass spectrometry in the 7 8 determination of the configuration of glyceric acid in urine of patients with D-glyceric and 9 10 L-glyceric acidurias. Biomed Chromatogr 16:191-198. 11 12 13 14 Snell K. 1986. The duality of pathways for serine biosynthesis is a fallacy. Trends 15 16 Biochem Sci 11:241-243. 17 18 Sorek R, Shamir R, Ast G. 2004. How prevalent is functional alternative splicing in the 19 20 For Peer Review 21 human genome? Trends Genet 20:68-71. 22 23 Thieden HID, Grunnet N, Damgaard SE, Sestoft L. 1972. Effect of fructose and 24 25 gkyceraldehyde in ethanol metabolism in human liver and rat liver. Eur J Biochem 30:250-61. 26 27 28 Topcu M, Saatci I, Haliloglu G, Kesimer M, Coskun T. 2002. D-glyceric aciduria in a 29 30 six-month-old boy presenting with West syndrome and autistic behaviour. Neuropediatrics 31 32 33:47-50. 33 34 35 van Schaftingen E. 1989. D-Glycerate kinase deficiency as a cause of D-glyceric 36 37 aciduria. FEBS Lett 1989; 243:127-131. 38 39 40 Wadman SK, Duran M, Ketting D, Bruinvis L, De Bree PK, Kamerling JP, Gerwig GJ, 41 42 Vliegenthart JFG, Przyrembel H, Becker K, Bremer HJ. 1976. D-Glyceric acidemia in a 43 44 patient with chronic metabolic acidosis. Clin Chim Acta 71:477-484. 45 46 47 Williams HE, Smith LH Jr. 1968. L-Glyceric aciduria: a new genetic variant of primary 48 49 hyperoxaluria. N Engl J Med 278:233-239. 50 51 52 53 54 55 56 57 58 59 60
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1 2 3 Figure legends 4 5 6 7 8 Figure 1: DNA sequencing results indicate that patients were homozygous for the mutations 9 10 c.1448delT (p.Phe483SerfsX2; patient A), c. 1478T>G (p.Phe493Cys; patient B) and 11 12 13 c.1558delC (p.Leu520CysfsX108; patient C) in the GLYCTK gene. Nucleotide numbering 14 15 reflects cDNA numbering with +1 corresponding to 16 17 18 the A of the ATG translation initiation codon in the reference 19 20 sequence. The initiationFor codon is Peercodon 1. Review 21 22 23 24 25 26 27 Figure 2: Expression of GLYCTK protein in HEK293 cells. Centrifugated homogenates of 28 29 HEK293 cells transfected with a pcDNA3.1(+) vector carrying the GLYCTK cDNA reference 30 31 32 sequence (corresponding to GLYTK transcript variant 1; lanes 1 and 6), the empty vector 33 34 (mock control, lane 2), or the GLYCTK cDNA with the mutations identified in patient C 35 36 (c.1558delC, p.Leu520CysfsX108, lane 3), patient B (c. 1478T>G, p.Phe493Cys, lane4), or 37 38 39 patient A (c.1448delT, p.Phe483SerfsX2, lane 5) were analyzed by SDS page analysis and 40 41 subsequent Western blotting using a mouse polyclonal antibody against full-length human 42 43 44 GLYCTK protein (molecular weight 55 kDa) as primary antibody. 30 µg of homogenate 45 46 protein (assessed by the Lowry method) were loaded per lane. For comparison, signal 47 48 intensities were compared after reincubating the blot with a rabbit polyclonal antibody against 49 50 51 the housekeeping enzyme GAPDH as primary antibody. The molecular weight of GAPDH is 52 53 36 kDa. Molecular weights of proteins were estimated using the Biorad protein standard 54 55 cat.no. 161-0374. 56 57 58 59 60 Figure 3: HEK293 cells transfected with a pcDNA3.1(+) vector without an insert (mock
control), cells carrying the GLYCTK cDNA reference sequence (corresponding to GLYCTK
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1 2 3 transcript variant 1), or the GLYCTK cDNA with the mutations identified in patients A 4 5 6 (c.1448delT, p.Phe483SerfsX2), B (c.1478T>G, p.Phe493Cys) and C (c.1558delC, 7 8 p.Leu520CysfsX108) were homogenized, centrifugated, and immediately submitted to the 9 10 fluorometric enzyme activity test described above. Mean values for duplicate analyses are 11 12 13 given, except for the single value for the mock control. 14 15 16 17 N 18 Figure 4: Sequence alignment of the -terminus of human D-glycerate kinase (GLYCTK) 19 20 protein and correspondingFor orthologs Peer from various Review species underlines strong conservation of 21 22 GLYCTK during evolution. The first amino acids which are changed in the patients A 23 24 25 (Phe483), B (Phe493) and Leu520) are printed in bold. The multiple-protein alignment was 26 27 constructed using the NCBI HomoloGene search. There is a high degree of conservation 28 29 between the orthologous proteins, advocating functional consequences of the missense 30 31 32 mutation resulting in an amino acid exchange in the GLYCTK gene of patient B 33 34 (p.Phe493Cys). The corresponding accession numbers of the protein sequences are: for 35 36 Homo sapiens NP_660305.2, for Pan troglodytes XP_001171685.1, for Canis lupus familiaris 37 38 39 XP_541854.1, for Bos taurus NP_001039452.1, for Mus musculus NP_777271.3, for Gallus 40 41 gallus XP_425150.2, for Danio rerio NP_001070856.1, for Drosophila melanogaster 42 43 44 NP_608684.1, and for Caenorhabditis elegans NP_498462.3. 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
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1 2 3 Table 1: Previously reported patients with D-glyceric aciduria 4 5 6 Key features Reference 7 8 Serbian boy, see description of patient A in this report, death at the age of 3.5 years Brandt et al., 1974+1976 9 10 Afghan boy with chronic metabolic acidosis from birth and failure to thrive Wadman et al. 1976 11 12 13 Boy of Swedish and Madagascan origin,For with severe neurological Peer impairment/ Review encephalopathy, Grandgeorge et al., 1980 14 15 rapid clinical deterioration, death at the age of 2.5 months 16 Girl examined in the Netherlands, mentally retarded, epileptic seizures Duran et al., 1987 17 18 19 Turkish girl, hypotonia and feeding difficulties noted at 2 months, first generalized seizures at 1 year of age; at 3 Fontaine et al., 1989; 20 21 years severe spastic tetraplegia, profound encephalopathy and hypotrophy Largillière et al., 1991 22 13-year-old Asian boy with mild speech delay and microcephaly, normal school Bonham et al., 1990 23 24 25 5-year-old Asian boy with mild speech delay and microcephaly, normal school Bonham et al., 1990 26 27 9-year-old Asian girl, considered developmentally normal, normal school Bonham et al., 1990 28 29 7-year-old Asian girl, considered developmentally normal, normal school Bonham et al., 1990 30 31 32 Boy from Turkey, infantile spasms, autistic behaviour and white matter lesions with cerebral atrophy Topcu et al., 2002 33 34 35 36 37 38 39 40 41 42 43 44 45 John Wiley & Sons, Inc. 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 23 of 26 Human Mutation
1 2 3 4 5 A 6 7 A C C T C C T A G c.1448delT 8 Thr Ser 9 p.Phe483SerfsX2 10 11 Reference A C C T T C C T A [40 tripletts] T G A 12 Thr Phe Leu 13 For Peer Review 14 15 16 17 18 19 B 20 T T C T G C T G C c.1478T>G 21 Phe Cys Cys 22 p.Phe493Cys 23 24 Reference T T C T T C T G C 25 Phe Phe Cys 26 27 28 29 30 c.1558delC 31 p.Leu520CysfsX108 32 C 33 T T C T G C G G C C T C G G T G A T [102 tripletts] T G A 34 Phe Cys Gly Leu Gly Asp 35 36 37 38 Reference T T C C T G C G G C C T C G G T G A 39 40 Phe Leu Arg Pro Arg 41 42 43 44 45 46 John Wiley & Sons, Inc. 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Human Mutation Page 24 of 26
1 2 3 4 5 6 1 2 3 4 5 6 7 [kDa] 8 9 10 11 75 __ 12 13 For Peer Review 14 15 50 __ 16 17 18 37 __ 19 20 21 22 25 __ 23 24 25 26 27 __ 28 29 30 31 32 37 33 __
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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 For Peer Review 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 Figure 3 419x593mm (150 x 150 DPI) 48 49 50 51 52 53 54 55 56 57 58 59 60 John Wiley & Sons, Inc. Human Mutation Page 26 of 26
1 2 3 4 5 6 7 8 9 10 11 12 13 For Peer Review 14 15 16 17 18 19 20 p.Phe483SerfsX2 p.Phe493Cys p.Leu520CysfsX108 21 • • • 22 Homo sapiens 465 VTPELASQAAAEGL--DIAT FLAHNDSHTF FCCLQGGAHLLHTGMTGTNVMDTHLLF LRPR------523 23 Pan troglodytes 465 VTPELASQAAAEGL--DIAT FLAHNDSHTF FCRLQGGAHLLHTGMTGTNVMDTHLLF LRPR------523 24 Canis lupus familiaris 465 VVPELASQAATEGL--DVAT FLSHNDSHTF FRCFRGGAHLLHTGLTGTNVMDVHFLF LRLR------523 25 Bos taurus 465 VRPELTSQAAAEGL--DVAT FLAHNDSHTF FCRFQGGAHLLHTGLTGTNVTDAHFLF LHPQ------523 26 S F L 27 Mus musculus 465 VMSDLISQASAESL--DIAT LANNDSYTF CRFRGGTHLLHTGLTGTNVMDVHLLI HPQ------523 28 Gallus gallus 444 CSPELVAEALQEGL--DAET FLSNNDSYTF FSQFQRGRHLLVTGLTGTNVMDIQLIL IRATDRS--- 505 29 Danio rerio 438 ADGELKEEAASQGL--DTDS FLANNDSFTF FSKLSEGRRLLNPGLTGTNVMDVHVML LPPSPQKDLQ 502 30 Drosophila melanogaster 429 GDSSVVESYLGDHTLDELAE TLRNCDSYNF YKNLAQGEHHVLTGHTGTNVMDLHFLV VP------487 31 Caenorhabditis elegans 402 ISNE--DLPLNSLL--NSSE FLQNSDSYNF WRQFKGGANHILTGPSGTNVMDIQILL LDQL------458 32 33 34 35 36 37 38 39 40 41 42 43 44 45 John Wiley & Sons, Inc. 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60