Konkoľová et al. BMC Medical Genetics (2017) 18:59 DOI 10.1186/s12881-017-0421-8 CASE REPORT Open Access Severe child form of primary hyperoxaluria type 2 - a case report revealing consequence of GRHPR deficiency on metabolism Jana Konkoľová1,2*, Ján Chandoga1,2, Juraj Kováčik3, Marcel Repiský2, Veronika Kramarová2, Ivana Paučinová3 and Daniel Böhmer1,2 Abstract Background: Primary hyperoxaluria type 2 is a rare monogenic disorder inherited in an autosomal recessive pattern. It results from the absence of the enzyme glyoxylate reductase/hydroxypyruvate reductase (GRHPR). As a consequence of deficient enzyme activity, excessive amounts of oxalate and L-glycerate are excreted in the urine, and are a source for the formation of calcium oxalate stones that result in recurrent nephrolithiasis and less frequently nephrocalcinosis. Case presentation: We report a case of a 10-month-old patient diagnosed with urolithiasis. Screening of inborn errors of metabolism, including the performance of GC/MS urine organic acid profiling and HPLC amino acid profiling, showed abnormalities, which suggested deficiency of GRHPR enzyme. Additional metabolic disturbances observed in the patient led us to seek other genetic determinants and the elucidation of these findings. Besides the elevated excretion of 3-OH-butyrate, adipic acid, which are typical marks of ketosis, other metabolites such as 3- aminoisobutyric acid, 3-hydroxyisobutyric acid, 3-hydroxypropionic acid and 2-ethyl-3-hydroxypropionic acids were observed in increased amounts in the urine. Direct sequencing of the GRHPR gene revealed novel mutation, described for the first time in this article c.454dup (p.Thr152Asnfs*39) in homozygous form. The frequent nucleotide variants were found in AGXT2 gene. Conclusions: The study presents metabolomic and molecular-genetic findings in a patient with PH2. Mutation analysis broadens the allelic spectrum of the GRHPR gene to include a novel c.454dup mutation that causes the truncation of the GRHPR protein and loss of its two functional domains. We also evaluated whether nucleotide variants in the AGXT2 gene could influence the biochemical profile in PH2 and the overproduction of metabolites, especially in ketosis. We suppose that some metabolomic changes might be explained by the inhibition of the MMSADH enzyme by metabolites that increase as a consequence of GRHPR and AGXT2 enzyme deficiency. Several facts support an assumption that catabolic conditions in our patient could worsen the degree of hyperoxaluria and glyceric aciduria as a consequence of the elevated production of free amino acids and their intermediary products. Keywords: Primary hyperoxaluria type 2, GRHPR, Oxalate, Glyoxylate reductase, Hydroxypyruvate reductase * Correspondence: [email protected] 1Institute of Medical Biology, Genetics and Clinical Genetics, Comenius University, Faculty of Medicine & University Hospital Bratislava, Sasinkova 4, 811 08 Bratislava, Slovakia 2Department of Molecular and Biochemical Genetics – Centre of Rare Genetic Diseases, Faculty of Medicine & University Hospital Bratislava, Mickiewiczova 13, 813 69 Bratislava, Slovakia Full list of author information is available at the end of the article © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Konkoľová et al. BMC Medical Genetics (2017) 18:59 Page 2 of 11 Background [10] with consequences similar to PH2. Our study focuses Primary hyperoxalurias (PHs) are a group of rare auto- on the deficiency of the GRHPR enzyme that possesses somal recessive inherited diseases. PHs are characterized glyoxylate reductase (GR), hydroxypyruvate reductase by a defect in glyoxylate metabolism which results in en- (HPR), and D-glycerate dehydrogenase activities (DGDH) dogenous oxalate overproduction. The clinical conse- [11–14], which is causative of PH2. This homodimeric en- quences of excessive oxalate excretion are nephrolithiasis zyme consists of 328 amino acids per subunit and is and/or nephrocalcinosis, and/or the early-onset end-stage encoded by the GRHPR gene, located in the centromeric renal disease (ESRD) in childhood. Glyoxylate is a highly region of chromosome 9 and contains nine exons span- reactive metabolite that is normally effectively removed ning 9 kbp. Though GRHPR deficiency is very rare, the through its conversion to glycine and less effectively to current mutation database includes about 30 different glycolate. In humans, the glycine producing pathway is types of mutations in the human GRPHR gene [2, 3, catalysed by the liver-specific peroxisomal enzyme ala- 15–17]. We report the case of a 10 month-old female pa- nine/glyoxylate aminotransferase (AGXT1; EC 2.6.1.44) tient with a clinical finding of urolithiasis who a clinician also known as: serine-pyruvate aminotransferase (AGT1, suspected of having a genetic disorder. Results of specific AGXT, SPT, SPAT). The second metabolic pathway in biochemical analyses and genetic examination led to the which the glyoxylate is consumed by reduction to glyco- diagnosis of PH2 and the disclosure of a novel mutation late in the cytosol and mitochondria is catalysed by the in the GRHPR gene. Given the unexpected and unclear enzyme glyoxylate reductase-hydroxypyruvate reductase biochemical findings in relation to PH2, we subsequently (GRHPR; EC 1.1.1.79, alternative symbol: GLXR). In the sought genetic variants in the relevant gene - AGXT2,the case of the deficiency of one of the above-mentioned en- protein of which is functionally closely coupled to zymes, a compensative mechanism takes over – the oxida- GRHPR. Finally we evaluate all the important biochemical tion of glyoxylate to oxalate by cytosolic L-lactate changes and genetic data observed in the patient, and dis- dehydrogenase (LD). Also hydroxypyruvate that is nor- cuss the possible metabolic consequences of GRPHR mally reduced by GRHPR to D-glycerate is converted to deficiency. L-glycerate by the same LD in the deficiency of enzyme GRHPR [1–4]. Under normal conditions, the competition Case presentation between the reduction and oxidation of glyoxylate is bal- A 10-month-old girl with a history of tonsilopharyngi- anced and controlled mainly by the cytoplasmic pool of tis, bilateral inborn hip dysplasia with improvement, NADPH, which can be utilized by GRHPR but not by LD dispenzarised by an orthopaedist, was referred to hos- and favours the production of glycolate, whereas LD activ- pital because of a one-day febrilities and positive urin- ity favours the production of oxalate [5, 6]. Mutations in ary finding of ketone bodies, proteins, leukocyturia and either the AGXT1 or GRHPR gene that result in the syn- haematuria. She was born after a normal pregnancy at thesis of deficient proteins alter this equilibrium, and full term with an uncomplicated perinatal and neonatal cause the overproduction of the main metabolites respon- course. There was no history of gross haematuria or sible for PHs. To date, the three types of PH (PH1, PH2 colicky abdominal pain, and she had received no medi- and PH3) have been described [7, 8]. Primary hyperoxa- cation except for antipyretics. The family history was luria type 1 (PH1; OMIM #259900) is the most prevalent negative with respect to renal and metabolic disease, in- and most severe form of primary hyperoxaluria caused by cluding urolithiasis and nephrocalcinosis, however, gen- AGXT1 deficiency. Primary hyperoxaluria type 2 (PH2; etic counselling revealed that the parents have common OMIM #260000 also known as L-glyceric aciduria) is less ancestors in the fourth generation. common than PH1 (exact incidence is unknown), and is On physical examination the child appeared mildly characterized by a GRHPR enzyme defect. Recently, a dehydrated with increased body temperature of 37.6 ° third type of primary hyperoxaluria (PH3; OMIM C. A laboratory examination found moderate anaemia #613616) has been described that is caused by the defi- (haemoglobin 93.0 g/l), leucocytosis (15x109/l) and ciency of the mitochondrial enzyme 4-hydroxy-2-oxoglu- moderately elevated CRP (42.6 mg/l). Serum electro- tarate aldolase (HOGA), the apical enzyme in the lyte, urea and creatinine levels were normal. The cal- mitochondrial hydroxyproline catabolism. Under physio- cium/creatinine ratio and 24 h calcium excretion was logical conditions, the enzyme splits HOG into pyruvate within the reference range. Blood gas analysis was and glyoxylate, the latter being subsequently oxidized by normal with no evidence of metabolic acidosis. Ultra- the cytosolic LD to oxalate [8, 9]. HOGA enzyme defi- sonography of the abdomen raised suspicion of ureter- ciency results in HOG accumulation, however, the mech- olithiasis l.sin. Cystourethrography was indicated, anism by which this deficiency causes hyperoxaluria has showing concrement sized 2 cm x 0.8 cm in the distal not been elucidated in detail yet. The inhibitory effect of part of the left ureter with mild dilatation of the renal HOG on the GRHPR enzyme has been assumed by Riedel pelvis and ureter (Fig. 1). Konkoľová et al. BMC Medical Genetics (2017) 18:59 Page 3 of 11 Fig. 1 X-ray findings of urolithiasis in patient with PH2 At the age of 11 months, an ureterolithotomy was per- on 30 m DB5 column with 0.25 i.d. (Agilent) and constant formed at the University Children Hospital Bratislava. A helium column flow 1.2 ml/min during 28 min. Equal ali- chemical analysis of the extracted stone was performed, quot of the internal standard (0.61 mmol of 4- and the stone was characterised as a composite of virtu- phenylbutyric acid) was added to 1 ml - 2 ml of urine ally pure calcium oxalate. The following course was sample, and organic acids were extracted by ethylacetate.