A De Novo Mutation in the AGXT Gene Causing Primary Hyperoxaluria Type 1 Emma L. Williams, PhD, Markus J. Kemper, MD, and Gill Rumsby, PhD, FRCPath ● Primary hyperoxaluria type 1 is caused by mutations in the alanine-glyoxylate aminotransferase (AGXT) gene. In cases in which no mutation was identified, linkage analysis can be used to confirm or exclude the diagnosis in other siblings. We present a family in which a sibling of the index case predicted to have primary hyperoxaluria type 1 by means of linkage analysis failed to show hyperoxaluria during the following 7 years, putting the diagnosis into question. Whole-gene sequence analysis identified 2 causative mutations in the index case, of which only 1, c.646A (Gly216Arg), was inherited. The other sequence change, c.33_34insC, was a de novo mutation occurring on the paternal allele. This particular mutation is a relatively common cause of primary hyperoxaluria type 1. It occurs in a run of 8 cytosines and therefore potentially is susceptible to polymerase slippage. This case illustrates 2 important points. First, biochemical confirmation of a genetic diagnosis should always be made in siblings diagnosed by using genetic tests. Second, de novo mutations should be considered as a potential, albeit rare, cause of primary hyperoxaluria type 1. Am J Kidney Dis 48:481-483. © 2006 by the National Kidney Foundation, Inc. INDEX WORDS: Hyperoxaluria; primary hyperoxaluria type 1; genotype; phenotype; de novo mutation; oxalate; AGXT.
RIMARY HYPEROXALURIA type 1 is caused by a deficiency of hepatic alanineglyoxylate aminotransferase and typically presents in childhood with nonspecific symptoms of urinary tract infection or hematuria, which may be related to underlying renal stones or nephrocalcinosis. The disease has a relatively severe course, with up to 28% of patients in end-stage renal failure by the age of 15 years.1 Since the alanine-glyoxylate aminotransferase (AGXT) gene was cloned, a number of mutations have been identified (reviewed recently in2), although there are still a number of patients for whom the pathological mutation has not been described. In individuals for whom no mutation has been identified, linkage analysis using either intragenic or extragenic markers has proved useful for prenatal diagnosis and the identification of other affected family members.3,4 In some affected siblings, a number of years may pass before abnormal oxalate excretion is documented.5 This finding possibly may reflect difficulties assessing the oxalate-creatinine ratio in random urine samples with their age-related reference range,6,7 rather than a full 24-hour urine collection corrected for body surface area. Random samples also are susceptible to hydration status and diet.8 We present a case in which a sibling of a child with primary hyperoxaluria type 1 was predicted by means of intragenic linkage analysis to be affected, but who continued to show normal oxalate
excretion over several years. The diagnosis was reviewed after identification of the pathological mutations in the index case, and it was found that one of the mutations was de novo. To the best of our knowledge, this is the first reported case of a de novo mutation in the AGXT gene. CASE REPORT A 4.5-year-old boy was referred from the Urology Department for further diagnostic workup and treatment of urolithiasis. He presented at the age of 3.8 years with colicky abdominal pain caused by left-sided urolithiasis that was diagnosed by using ultrasound and radiology. This was treated successfully by means of extracorporeal shock wave lithotripsy. Stone analysis showed calcium oxalate monohydrate. The boy was otherwise healthy with no other symptoms. Family history for renal or metabolic disorders and urolithiasis was negative. On physical examination, he was well, and body weight and height were within normal percentiles. Blood pressure was 110/60 mm Hg. His abdomen was soft and nontender, with no flank pain. Funduscopy was normal.
From Clinical Biochemistry, UCL Hospitals, London, UK; and University Children’s Hospital, Hamburg, Germany. Received March 31, 2006; accepted in revised form May 23, 2006. Originally published online as doi:10.1053/j.ajkd.2006.05.022 on July 14, 2006. Support: None. Potential conflicts of interest: None. Address reprint requests to Gill Rumsby, PhD, FRCPath, Clinical Biochemistry UCL Hospitals, 60 Whitfield St, London W1T 4EU, UK. E-mail: [email protected]
© 2006 by the National Kidney Foundation, Inc. 0272-6386/06/4803-0015$32.00/0 doi:10.1053/j.ajkd.2006.05.022
American Journal of Kidney Diseases, Vol 48, No 3 (September), 2006: pp 481-483
Laboratory analysis showed normal hematologic workup and venous blood gas analysis results. Serum electrolyte levels were normal, and serum creatinine level was 0.6 mg/dL (53 mol/L). Urinalysis showed pH of 6.5, no proteinuria, but microscopic hematuria. Urinary calcium excretion was 0.02 mmol/kg/d (normal), and oxalate excretion was 248 mg/1.73 m2/d (normal, ⬍40.5 mg/1.73 m2/d [2.76 mmoL/1.73 m2/d; normal, ⬍0.45 mmoL/1.73 m2/d]), the latter suggestive of primary hyperoxaluria. Urinary glycolate excretion was elevated at 186 g/mg creatinine (275 mol/mmol; normal, ⬍37 g/mg), and plasma oxalate level was slightly elevated at 11 mol/L (normal, ⬍10 mol/L nonfasting). Renal ultrasound and plain abdominal X-rays at the time of first outpatient presentation were normal, with no evidence of nephrocalcinosis. Initial studies of the family showed they were negative for the common c.508A and c.731C mutations; therefore, linkage analysis using intragenic and extragenic polymorphisms was used to examine the status of 2 other siblings. The family was fully informative for these markers, and results were consistent with a diagnosis of primary hyperoxaluria type 1 in individual II3, a 2-year-old girl, and her 5-year-old brother (II2) being a carrier for the paternal disease allele (Fig 1). Treatment of the index case with pyridoxine and citrate was started, including high fluid intake. Urinary oxalate
WILLIAMS, KEMPER, AND RUMSBY
excretion did not diminish with pyridoxine. During a 12-year follow-up, 6 episodes of urolithiasis occurred, requiring 5 additional extracorporeal shock wave lithotripsy sessions. This was attributable in part to noncompliance, especially with citrate medication. Presently, at 16 years of age, the patient is obese (body mass index, 29.4 kg/m2), still has no nephrocalcinosis, and has a serum creatinine level of 1.0 mg/dL (88 mol/L) and glomerular filtration rate of 98 mL/min/1.73 m2 (1.63 mL/s). The apparently affected sibling had oxalate excretion within the normal range on repeated occasions during the next 7 years. Full gene sequencing subsequently showed that the index case was a compound heterozygote for the c.33_34insC (numbering based on complementary DNA sequence NM_000030 [AGXT], with nucleotide 1 denoting the first coding base) and c.646A (Gly216Arg) mutations. Additional studies of other family members showed that the mother and sister (II3) were heterozygous for the c.646A allele, but neither the father (I1), brother (II2), nor sister (II3) had the c.33_34insC mutation although the 2 children shared the same paternal polymorphic haplotype as the affected child. The most likely explanation for the discrepancy (nonpaternity was excluded by means of microsatellite analysis) is that a de novo mutation occurred in the index case.
Fig 1. Family tree showing results of linkage analysis with 2 polymorphic markers, Int 4 (a variable number of tandem repeats in exon 4 of the AGXT gene) and D2S140 (a microsatellite close to the AGXT gene) with the predicted disease alleles shown in boxes. Subsequent mutation analysis showed that II:1 had a de novo mutation on the paternal allele that changed the predicted genotype of II:2 from carrier to unaffected and that of II:3 from affected to carrier.
DE NOVO MUTATION IN THE AGXT GENE
More than 50 mutations have been described in the AGXT gene, ranging from nonsense and missense mutations to major and minor deletions and insertions.2 The c.33_34insC mutation occurs in a run of 8 cytosine residues in exon 1 in a region encoding the so-called “major allele,” with a proline encoded by codon 11.9 Both insertions and deletions have been described in this region10-12 and are likely to be caused by slipped mispairing.13 The frequency of the c.33_34insC insertion mutation was shown to be 12%,14 and to date, it is always associated with the major allele, suggesting that the C¡T change in the “minor allele” may alter susceptibility to slippage by reducing the number of consecutive C nucleotides from 8 to 6. Unfortunately, we are unable to determine the incidence of de novo mutations at this site in our 43 patients homozygous or heterozygous for the change because most have been studied in isolation, ie, without family studies. However, finding this de novo germline mutation reinforces the importance of tracking identified mutations within families because the risk for disease recurrence in that family will be decreased significantly if a de novo change is present. The case also illustrates the importance of biochemical confirmation of genotype in family members predicted to be affected, although this may take some time to be shown reliably, particularly in childhood.5 REFERENCES 1. Cochat P, Deloraine A, Rotily M, Liponski I, Deries N: Epidemiology of primary hyperoxaluria type 1. Nephrol Dial Transplant 10:3-7, 1995 2. Coulter-Mackie MB, Rumsby G: Genetic heterogeneity in primary hyperoxaluria type 1: Impact on diagnosis. Mol Genet Metab 83:38-46, 2004
3. von Schnakenburg C, Weir T, Rumsby G: Linkage of microsatellites to the AGXT gene on chromosome 2q37.3 and their role in prenatal diagnosis of primary hyperoxaluria type 1. Ann Hum Genet 61:365-368, 1997 4. Rumsby G, Mandel H, Avey C, Geraerts A: Polymorphisms in the alanine:glyoxylate aminotransferase gene and their application to the prenatal diagnosis of primary hyperoxaluria type 1. Nephrol Dial Transplant 8:30-32, 1995 5. Hoppe B, Danpure CJ, Rumsby G, et al: A vertical (pseudodominant) pattern of inheritance in the autosomal recessive disease primary hyperoxaluria type 1: Lack of relationship between genotype, enzymic phenotype, and disease severity. Am J Kidney Dis 29:36-44, 1997 6. Barratt TM, Kasidas GP, Murdoch I, Rose GA: Urinary oxalate and glycolate excretion and plasma oxalate concentration. Arch Dis Child 66:501-503, 1991 7. von Schnakenburg C, Byrd DJ, Latta K, Reusz GS, Graf D, Brodehl J: Determination of oxalate excretion in spot urines of healthy children by ion chromatography. Eur J Clin Chem Clin Biochem 32:27-29, 1994 8. Milliner DS: The primary hyperoxalurias: An algorithm for diagnosis. Am J Nephrol 25:154-160, 2005 9. Purdue PE, Takada Y, Danpure CJ: Identification of mutations associated with peroxisome-to-mitochondrion mistargeting of alanine/glyoxylate aminotransferase in primary hyperoxaluria type 1. J Cell Biol 111:2341-2351, 1990 10. Pirulli D, Puzzer D, Ferri L, et al: Molecular analysis of hyperoxaluria type 1 in Italian patients reveals eight new mutations in the alanine:glyoxylate aminotransferase gene. Hum Genet 104:523-525, 1999 11. Milosevic D, Rinat C, Batinic D, Frishberg Y: Genetic analysis—A diagnostic tool for primary hyperoxaluria type 1. Pediatr Nephrol 17:896-898, 2002 12. Coulter-Mackie MB, Applegarth D, Toone JR, Henderson H: The major allele of the alanine:glyoxylate aminotransferase gene: Seven novel mutations causing primary hyperoxaluria type 1. Mol Genet Metab 82:64-68, 2004 13. Antonarkis SE, Krawczak M, Cooper DN: Diseasecausing mutations in the human genome. Eur J Pediatr 159:S173-S178, 2000 (suppl 3) 14. Rumsby G, Williams E, Coulter-Mackie MB: Evaluation of mutation screening as a first line test for the diagnosis of the primary hyperoxalurias. Kidney Int 66: 959-963, 2004