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Letters to the Editor |
Clinical Biochemistry, UCL Hospitals, London, United Kingdom
aAddress correspondence to this author at: Clinical Biochemistry, UCL Hospitals, 60 Whitfield St., London W1T 4EU, United Kingdom. Fax 020-7380-9584; e-mail gill.rumsby{at}uclh.nhs.uk.
To the Editor:
Primary hyperoxaluria type 2 (PH2; OMIM 260000) is an inherited disease of endogenous oxalate overproduction arising from mutations in the GRHPR gene encoding glyoxylate reductase. The disease typically presents with urolithiasis or recurrent urinary tract infections and increased urinary oxalate (1). The diagnosis may be supported by L-glyceraciduria, although this does not occur in all cases (2). Definitive diagnosis is currently based on demonstration of diminished glyoxylate reductase activity in a liver biopsy (3), although DNA analysis offers a noninvasive method.
The GRHPR gene maps to the centromeric region of chromosome 9 (4) and, from Northern blot analysis(5), is ubiquitously expressed, although the bulk of enzyme activity is found in the liver (3)(5). Among the described mutations and polymorphisms (5)(6), c.103delG accounts for 37% of mutant alleles, allowing diagnosis of PH2 to be made by genetic testing (5). One of the polymorphisms, c.579G>A, occurs in exon 6, and the G allele has been shown to have a frequency of 0.68 in genomic DNA from PH2 patients (5).
While evaluating leukocyte cDNA for identifying mutations and demonstrating potential splice defects in this gene, we found a lack of expression of mutations in leukocyte cDNA, in contrast to liver cDNA and genomic DNA from the same individual.
We studied liver and blood samples from a patient who presented clinically with features of PH and who subsequently was found, on liver enzyme analysis, to have PH2 (2). Genomic DNA was prepared from EDTA–whole blood with the QIAamp DNA Blood Mini Kit (Qiagen). RNA was isolated from liver by homogenization in RNA isolator (Sigma Genosys) and from blood with the QIAamp Blood Mini Kit (Qiagen), according to the manufacturer’s instructions. Reverse transcription-PCR was carried out using a Sensiscript® Reverse Transcriptase Kit (Qiagen). Genomic DNA was amplified with intronic primers flanking exons 6 and 7, and cDNA was amplified with primers designed to amplify across intron–exon boundaries (5). Sequencing of PCR products was performed on an ABI 3100 (Applied Biosystems) sequencer either directly after purification using the QIAquick PCR Purification Kit (Qiagen) or after cloning into TA-vector (Invitrogen).
Analysis of GRHPR genomic DNA showed a homozygous 2-bp deletion (c.608_609delCT) mutation and a G allele at position c.579 of cDNA (Fig. 1A
). The same genotype was observed in liver cDNA (Fig. 1B
). Leukocyte cDNA, however, showed a heterozygous pattern for the mutation as well as the c.579G/A polymorphism. After cloning, 2 transcripts were identified. One of these had the CT deletion mutation and c.579G (Fig. 1C
); the other transcript had no mutation and contained the A allele at c.579 (Fig. 1D
). The discrepancy was verified by repeat analysis and by analysis of other family members. All controls, including reverse transcriptase-negative control and DNA-free PCR reactions, were negative.
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The most likely explanation for these findings, which affect expression of mutations and a polymorphic site, is the presence of 2 highly homologous genes with different expression profiles. A BLAST search (http://www.ncbi.nlm.nih.gov/BLAST/) of the human genome using GRHPR cDNA picked up only 1 hit at the expected location at the centromeric region of chromosome 9. This finding in itself does not exclude the possibility that a duplicated gene might be present as there is known to be a large amount of duplication in this particular chromosomal region (7). Duplication can lead to genes with a high degree of homology in the flanking and coding regions, as in glutathione S-transferase (8), making it difficult to recognize them as separate entities.
We have now extended our studies to an additional 6 patients with PH2, with a variety of genotypes, and we have found the same mismatch between leukocyte cDNA and genomic DNA. Further work, including sequence analysis of the 5'- and 3'-untranslated regions of liver and leukocyte GRHPR cDNA and of cosmids containing GRHPR genomic DNA, is in progress. However, until a satisfactory explanation is found, it would be ill advised to use GRHPR enzyme or cDNA derived from tissues other than liver for the diagnosis of PH2.
References
class glutathione S-transferase genes. Biochem Biophys Res Commun 1994;200:1665-1671.[CrossRef][Web of Science][Medline]
[Order article via Infotrieve]The following articles in journals at HighWire Press have cited this article:
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  J. Knight, R. P. Holmes, D. S. Milliner, C. G. Monico, and S. D. Cramer Glyoxylate reductase activity in blood mononuclear cells and the diagnosis of primary hyperoxaluria type 2 Nephrol. Dial. Transplant., August 1, 2006; 21(8): 2292 - 2295. [Abstract] [Full Text] [PDF]
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G. Rumsby Is liver analysis still required for the diagnosis of primary hyperoxaluria type 2? Nephrol. Dial. Transplant., August 1, 2006; 21(8): 2063 - 2064. [Full Text] [PDF]
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