NDT Advance Access originally published online on May 17, 2007
Nephrology Dialysis Transplantation 2007 22(8):2371-2374; doi:10.1093/ndt/gfm271
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A novel mutation in the GRHPR gene in a Japanese patient with primary hyperoxaluria type 2
1Department of Cancer Biology, Wake Forest University School of Medicine, Medical Center Blvd, Winston-Salem, NC 27157, USA 2Department of Urology, Hamamatsu University School of Medicine, 1-20-1 Handayama, Hamamatsu, Shizuoka 431-3192, Japan and 3Department of Renal & Genitourinary Surgery, Graduate School of Medicine, Hokkaido University, North-15 West-7, Kitaku, Sapporo, Hokkaido 060-8638, Japan
Correspondence and offprint requests to: Dr Tatsuya Takayama, Department of Cancer Biology, Wake Forest University School of Medicine, Medical Center Blvd, Winston-Salem, NC 27157, USA. Email: ttakayam{at}hama-med.ac.jp
Keywords: genotype; glyoxylate reductase/hydroxypyruvate reductase (GRHPR); hyperoxaluria; phenotype; primary hyperoxaluria type2
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Introduction |
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 Top Introduction Case reportDNA extraction and amplification...Site-directed mutagenesis...Transient transfection and...Results and DiscussionAcknowledgementsReferences |
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Primary hyperoxaluria type 2 (PH2) is a rare monogenic genetic disorder with an autosomal recessive pattern of inheritance. The disease is caused by mutations in the GRHPR gene encoding the glyoxylate/hydroxypyruvate reductase enzyme [2,12]. The high urinary excretion of oxalate and L-glycerate is a characteristic biochemical feature of PH2. Pathologically, the increased plasma and urinary oxalate leads to calcium oxalate supersaturation in the collecting ducts, which causes progressive renal deposition of calcium oxalate in the kidney, in the form of urolithiasis and/or nephrocalcinosis. In severe cases, this occasionally leads to renal failure and/or systemic oxalosis.
The diagnostic tools of PH2 include the measurement of urinary oxalate and L-glycerate, genotyping for mutations of the GRHPR gene, the measurement of GRHPR enzymatic activity in the blood cells [5,13] and measurement of enzymatic activity in liver tissue. None of these assays are ideal. Measurement of enzymatic activity in liver biopsy is complicated by comorbidity, measurement in blood cells requires sensitive assays not generally available. Measurement of urinary metabolites can occasionally lead to misdiagnosis. While genetic screening offers potential solutions to problems created by other methods, there are currently too few mutations described and evaluated for functional consequences to use this method for efficient diagnosis.
The GRHPR gene is located in the pericentromeric region of chromosome 9 [2]. The gene has 9 exons and encodes a 328 amino acid, 36 kDa protein [2,8]. To date, 13 mutations have been identified [2–4,6,11,12]. All mutations reported lead to a loss of enzyme expression or function. The most common of all the mutations is 103delG in exon 2, which results in a frameshift and induces a premature stop at codon 45 [2]. The prevalence of this mutation in PH2 is around 40% [3,11] of all reported mutations and appears to have originated in a founder of Northern European origin [11]. However, it has not been found in any patient of Asian origin. The 103delG mutation is a candidate mutation for DNA screening especially in patients of Caucasian origin. The sensitivity of diagnosis using 103delG mutation in DNA samples from the individuals referred for PH2 was 33% [9].
We describe a Japanese patient with PH2 who suffers from recurrent urolithiasis without nephrocalcinosis. This 19 year old patient was diagnosed through biochemical urine analysis by gas chromatography-mass spectrometry at 10 months of age [14]. In the current report, we provide the molecular basis of PH2 in this patient.
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Case report |
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TopIntroductionCase reportDNA extraction and amplification...Site-directed mutagenesis...Transient transfection and...Results and DiscussionAcknowledgementsReferences |
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The patient was a 19-year-old man. He had haematuria and bilateral renal and ureteral stones at 7 months of age. Urinary excretion of organic acids was measured by gas chromatography-mass spectrometry and they showed markedly elevated glyceric acid (3032 ± 1276 mg/day/1.73 m2) in the presence of increased oxalate (227 ± 120 mg/day/1.73 m2) and normal glycolate concentrations. He was, therefore, diagnosed with primary hyperoxaluria type 2 in 1989 [14]. There was no family history of urinary stones and his parents and brother showed normal urinary organic acids. In recent clinical data using capillary electrophoresis, serum oxalate concentration (16.9 µM; reference interval, 0.5–3 µM) and urinary oxalate excretion (270 mg/day/1.73 m2) were significantly elevated. In spite of the elevated serum oxalate, the serum creatinine level (0.74 mg/dl; reference interval, 0.70–1.30 mg/dl) and creatinine clearance for 24 h (231 l/day; reference interval, 93–238 l/day) was normal. The patient had some renal stones that comprised of calcium oxalate in the absence of nephrocalcinosis.
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DNA extraction and amplification and sequence analysis |
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TopIntroductionCase reportDNA extraction and amplification...Site-directed mutagenesis...Transient transfection and...Results and DiscussionAcknowledgementsReferences |
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Genomic DNA of the patient was extracted from peripheral blood samples by the QIAamp blood kit (Qiagen, Hilden, Germany) according to the manufacturer's instruction in Japan and shipped on ice to Wake Forest University. All protocols were performed by the Institutional Review Board of Wake Forest University School of Medicine. Informed consent was obtained from the patient in accordance with the Institutional Review Board of Hamamatsu University School of Medicine. PCR was applied to all nine exons with all splice acceptor and donor sites of the patient, by using methods previously described [2,12] and the nucleotide sequences were deduced. For Exon 8 we also first subcloned the PCR product and sequenced individual clones.
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Site-directed mutagenesis-construction of a missense mutant |
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TopIntroductionCase reportDNA extraction and amplification...Site-directed mutagenesis...Transient transfection and...Results and DiscussionAcknowledgementsReferences |
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Site-directed mutagenesis was used to construct mutant clones to represent the missense mutation of the patient, using the QuickChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA) and standard molecular methods. Detailed methods are available upon request.
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Transient transfection and enzymatic activities |
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TopIntroductionCase reportDNA extraction and amplification...Site-directed mutagenesis...Transient transfection and...Results and DiscussionAcknowledgementsReferences |
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Transfection experiments were performed by using Lipofectamine (Gibco BRL, Grand Island, NY, USA) as previously described by Cramer et al. [2]. Each co-transfection was performed in triplicate. Twenty-four hours after transfection, the cells were scraped off the plates, spun briefly and lysed by lysis buffer (25 mM Hepes, pH 7.1, 0.1% Triton-X) with 9-times V/W of the cell pellet. Cell lysates were stored at –70°C until required.
DGDH activities were determined as previously described [2] and expressed as V (pmoles hydroxyupyrvate phenyhydrazone/min/mg protein/luciferase unit).
Student's t-test was applied (P < 0.05) to the raw data from the enzyme assay in order to determine whether the mean enzyme activities of each clone were significantly different.
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Results and Discussion |
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TopIntroductionCase reportDNA extraction and amplification...Site-directed mutagenesis...Transient transfection and...Results and DiscussionAcknowledgementsReferences |
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Prior to this report, no data existed on mutations in GRHPR from Japanese PH2 patients. DNA was isolated from a previously reported Japanese PH2 patient and shipped to Wake Forest University School of Medicine for genetic analysis. Nucleotide sequence analysis identified the patient as a compound heterozygote at the GRHPR locus. The DNA was heterozygous for a single nucleotide substitution, an adenosine for a guanine at nucleotide 337 (relative to the first codon nucleotide) in codon 113, exon 4, i.e. Glu113Lys (Figure 1A) and for a two nucleotide deletion in exon 8 at codon 288, i.e. 864_865delTG (Figure 1B). No other exons contained mutations (data not shown).
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The 864_865delTG mutation occurs at the interface between exon 8 and intron H [2] and is predicted to disrupt splicing or lead to a truncated protein. This mutation has been described previously in a Chinese patient with PH2, who was homozygous for the mutation. This patient developed nephrocalcinosis and end-stage renal failure requiring renal transplantation. In addition, one of the patient's sisters was heterozygous for the mutation and was asymptomatic [6]. The previous finding of this mutation in a Chinese PH2 patient and our finding in a Japanese patient suggests that this mutation is a candidate, which needs to be incorporated into strategies that use genetics to detect/diagnose PH2 in east-Asian patients.
The missense Glu113Lys mutation has not been previously described and its functional consequences on enzymatic activity are not known. Therefore, we tested the effect of the Glu113Lys mutation on enzyme activity. Site-directed mutagenesis was used to introduce the G337A mutation into the wild-type GRHPR cDNA. The fidelity of mutagenesis was confirmed by DNA sequencing in both directions (data not shown). Cos-1 cells were transfected with control (empty vector, pcDNA3.1), the wild-type (pGRHPR3.1), and the mutant clone (mpcGRHPR Ex4 MG) and enzymatic activity was measured in total cell lysates. Figure 2 demonstrates that DGDH activity of the mutant clone is significantly less than that of the wild type and not significantly different from the control. These data confirm that this mutation results in the loss of GRHPR enzymatic activity. A recent publication reported the crystal structure of human GRHPR [1]. The human GRHPR protein is composed of a substrate binding domain (residues 5–106 and 299–328), coenzyme-binding domain (residues 107–298), and dimer-forming loop (residues 123–149) and forms a homodimer in solution. The Glu113Lys mutation is located at the dimerization interface. When compared with the GRHPR protein sequence from various organisms, residue 113 is either a glutamate or an aspartate [2]. The substitution reported here of a lysine, which represents a charge change, may destabilize the structure and interfere with dimerization of the GRHPR protein, triggering proteolytic degradation. The prevalence of the G337A mutation in the Japanese population or other populations is unknown. However, the presence of this mutation in a Japanese PH2 patient and our demonstration of the loss of enzyme function leads us to propose its inclusion in screening strategies of patients originating from this region of the world.
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Milliner recently proposed an algorithm for the differential diagnosis of primary hyperoxaluria [7]. This algorithm relies on a combination of biochemical urinary analyte measurements, enzymatic assays in tissue homogenates and genetic screening. We have proposed that genetic screening, with its lower morbidity and minimal invasiveness, should take a more prominent role in PH diagnostics [10]. However, a lack of sufficient reports on mutations and their validation through functional enzyme assays will continue to hamper this approach. Additionally, little data are available on population specific mutations in the GRHPR gene. Our report, that confirms the 864_865delTG mutation and describes a novel G337A mutation, specifically in a confirmed PH2 patient of Japanese descent, will further progress towards the goal of efficient genetic screening for PH2.
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Acknowledgements |
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TopIntroductionCase reportDNA extraction and amplification...Site-directed mutagenesis...Transient transfection and...Results and DiscussionAcknowledgementsReferences |
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We are especially grateful to Katie Ruff, Lina Romero and Jessica Kaplan for their technical assistance. This work is supported by a grant to S.D.C. from the National Institute of Health (DK069331).
Conflict of interest statement. None declared.
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References |
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TopIntroductionCase reportDNA extraction and amplification...Site-directed mutagenesis...Transient transfection and...Results and DiscussionAcknowledgementsReferences |
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- Booth MP, Conners R, Rumsby G, et al. Structural basis of substrate specificity in human glyoxylate reductase/hydroxypyruvate reductase. J Mol Biol (2006) 360:178–189.[CrossRef][Web of Science][Medline]
- Cramer SD, Ferree PM, Lin K, et al. The gene encoding hydroxypyruvate reductase (GRHPR) is mutated in patients with primary hyperoxaluria type II. Hum Mol Genet (1999) 8:2063–2069.
[Abstract/Free Full Text] - Cregeen DP, Williams EL, Hulton S, et al. Molecular analysis of the glyoxylate reductase (GRHPR) gene and description of mutations underlying primary hyperoxaluria type 2. Hum Mutat (2003) 22:497.[Medline]
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Accepted in revised form: 6. 4.07
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