4-hydroxy-2-oxoglutarate metabolism in a mouse model of Primary Hyperoxaluria Type 3.

Primary Hyperoxaluria Type 3 (PH3) results from 4-hydroxy-2-oxoglutarate (HOG) aldolase (HOGA) deficiency, which causes an increase in endogenous oxalate synthesis leading to calcium oxalate kidney stone disease. The mechanisms underlying HOG metabolism and increased oxalate synthesis in PH3 are not well understood. We used a Hoga1 knock-out mouse model of PH3 to investigate two aspects of HOG metabolism: reduction to dihydroxyglutarate (DHG), a pathway that may limit oxalate synthesis in PH3, and metabolism to glyoxylate, which is a direct precursor to oxalate. The metabolism of HOG to DHG was highest in liver and kidney cortical tissue, enhanced in the cytosolic compartment of the liver, and preferred NADPH as a cofactor. In the absence of HOGA, HOG to glyoxylate aldolase activity was highest in liver mitoplasts, with no activity present in brain tissue lysates. These findings will assist in the identification of enzymes responsible for the metabolism of HOG to DHG and glyoxylate, which may lead to novel therapeutic approaches to limit oxalate synthesis in those afflicted with PH3.

Characterization of Stone Events in Patients With Type 3 Primary Hyperoxaluria.

PURPOSE: Hallmarks of primary hyperoxaluria type 3 are nephrolithiasis and hyperoxaluria. However, little is known about factors influencing stone formation in this disease. We characterized stone events and examined associations with urine parameters and kidney function in a primary hyperoxaluria type 3 population. MATERIALS AND METHODS: We retrospectively analyzed clinical, and laboratory data of 70 primary hyperoxaluria type 3 patients enrolled in the Rare Kidney Stone Consortium Primary Hyperoxaluria Registry. RESULTS: Kidney stones occurred in 65/70 primary hyperoxaluria type 3 patients (93%). Among the 49 patients with imaging available, the median (IQR) number of stones was 4 (2, 5), with largest stone 7 mm (4, 10) at first imaging. Clinical stone events occurred in 62/70 (89%) with median number of events per patient 3 (2, 6; range 1-49). Age at first stone event was 3 years (0.99, 8.7). Lifetime stone event rate was 0.19 events/year (0.12, 0.38) during follow-up of 10.7 (4.2, 26.3) years. Among 326 total clinical stone events, 139 (42.6%) required surgical intervention. High stone event rates persisted for most patients through the sixth decade of life. Analysis was available for 55 stones: pure calcium oxalate accounted for 69%, with mixed calcium oxalate and phosphate in 22%. Higher calcium oxalate supersaturation was associated with increased lifetime stone event rate after adjusting for age at first event (IRR [95%CI] 1.23 [1.16, 1.32]; P < .001). By the fourth decade, estimated glomerular filtration rate was lower in primary hyperoxaluria type 3 patients than the general population. CONCLUSIONS: Stones impose a lifelong burden on primary hyperoxaluria type 3 patients. Reducing urinary calcium oxalate supersaturation may reduce event frequency and surgical intervention.

Primary Hyperoxaluria Type 3 Can Also Result in Kidney Failure: A Case Report.

Primary hyperoxaluria (PH) is a group of genetic disorders that result in an increased hepatic production of oxalate. PH type 3 (PH3) is the most recently identified subtype and results from mutations in the mitochondrial 4-hydroxy-2-oxoglutarate aldolase gene (HOGA1). To date, there have been 2 cases of kidney failure reported in PH3 patients. We present a case of a young man with a history of recurrent urinary tract infections and voiding dysfunction who developed kidney failure at 33 years of age. He developed a bladder stone and bilateral staghorn calculi at 12 years of age. Initial metabolic evaluation revealed hyperoxaluria with very low urinary citrate excretion on multiple measurements for which he was placed on oral citrate supplements. Further investigation of the hyperoxaluria was not completed as the patient was lost to follow-up observation until he presented at 29 years of age with chronic kidney disease stage 4 (estimated glomerular filtration rate 24mL/min/1.73m2). Hemodialysis 3 times a week was started at 33 years of age, and subsequent genetic testing revealed a homozygous HOGA1 mutation (C.973G>A p.Gly325Ser) diagnostic of PH3. The patient is currently being evaluated for all treatment options including possible liver/kidney transplantation. All cases of a childhood history of recurrent urinary stone disease with marked hyperoxaluria should prompt genetic testing for the 3 known PH types. Hyperhydration and crystallization inhibitors (citrate) are standard of care, but the role of RNA interference agents for all 3 forms of PH is also under active study.

Rapid liquid chromatography tandem mass-spectrometry screening method for urinary metabolites of primary hyperoxaluria.

BACKGROUND: The primary hyperoxalurias are inherited disorders of glyoxylate metabolism that lead to overproduction of oxalate, urolithiasis and renal failure. Delays in diagnosis can be costly in terms of preserving renal function. Here we present a rapid liquid chromatography tandem mass-spectrometry screening method for the analysis of metabolites (primary hyperoxaluria metabolites) produced in excess by primary hyperoxaluria patients that include glycolate, glycerate and 2,4-dihydroxyglutarate. METHODS: Assay performance was compared to our existing gas chromatography-mass spectrometry method and clinical utility established by analysis of urine samples from patients with confirmed primary hyperoxalurias (11 PH1, 12 PH2 and 8 PH3) and controls ( n = 12). An additional 67 urine samples from patients with PH3 were used postvalidation to confirm the derived 2,4-dihydroxyglutarate cut-off. RESULTS: Glycolate, glycerate and 2,4-dihydroxyglutarate showed a mean bias of 3.3, -22.8 and 5.7%, respectively, compared to our previously published gas chromatography-mass spectrometry method. The mean total imprecision for glycolate, glycerate and 2,4-dihydroxyglutarate was shown to be 6.4, 10 and 11%, respectively. Clinical assessment confirmed that mean urinary glycolate, glycerate and 2,4-dihydroxyglutarate excretion were significantly elevated in patients with PH1, PH2 and PH3, respectively. The greatest sensitivity and specificity for PH1, PH2 and PH3 was achieved at cut-offs of 193, 100 and 4.9 µmol/mmol for glycolate, glycerate and 2,4-dihydroxyglutarate, respectively. CONCLUSIONS: A rapid screening method for the identification and differentiation of patients with suspected PH1, PH2 and PH3 is presented that allows focussing of genetic testing, saving time, money and, with earlier treatment, potential preservation of renal function for these patients.

Combined liver-kidney transplantation for primary hyperoxaluria type I in children: Single Center Experience.

Primary hyperoxalurias are rare inborn errors of metabolism with deficiency of hepatic enzymes that lead to excessive urinary oxalate excretion and overproduction of oxalate which is deposited in various organs. Hyperoxaluria results in serious morbid-ity, end stage kidney disease (ESKD), and mortality if left untreated. Combined liver kidney transplantation (CLKT) is recognized as a management of ESKD for children with hyperoxaluria type 1 (PH1). This study aimed to report outcome of CLKT in a pediatric cohort of PH1 patients, through retrospective analysis of data of 8 children (2 girls and 6 boys) who presented by PH1 to Wadi El Nil Pediatric Living Related Liver Transplant Unit during 2001-2017. Mean age at transplant was 8.2 ± 4 years. Only three of the children underwent confirmatory genotyping. Three patients died prior to surgery on waiting list. The first attempt at CLKT was consecutive, and despite initial successful liver transplant, the girl died of biliary peritonitis prior to scheduled renal transplant. Of the four who underwent simultaneous CLKT, only two survived and are well, one with insignificant complications, and other suffered from abdominal Burkitt lymphoma managed by excision and resection anastomosis, four cycles of rituximab, cyclophosphamide, vincristine, and prednisone. The other two died, one due to uncontrollable bleeding within 36 hours of procedure, while the other died awaiting renal transplant after loss of renal graft to recurrent renal oxalosis 6 months post-transplant. PH1 with ESKD is a rare disease; simultaneous CLKT offers good quality of life for afflicted children. Graft shortage and renal graft loss to oxalosis challenge the outcome.

Extended substrate range of thiamine diphosphate-dependent MenD enzyme by coupling of two C-C-bonding reactions.

Carboligations catalyzed by aldolases or thiamine diphosphate (ThDP)-dependent enzymes are well-known in biocatalysis to deliver enantioselective chain elongation reactions. A pyruvate-dependent aldolase (2-oxo-3-deoxy-6-phosphogluconate aldolase [EDA]) introduces a chiral center when reacting with the electrophile, glyoxylic acid, delivering the (S)-enantiomer of (4S)-4-hydroxy-2-oxoglutarate [(S)-HOG]. The ThDP-dependent enzyme MenD (2-succinyl-5-enol-pyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate synthase (SEPHCHC synthase)) enables access to highly functionalized substances by forming intermolecular C-C bonds with Michael acceptor compounds by a Stetter-like 1,4- or a benzoin-condensation 1,2-addition of activated succinyl semialdehyde (ThDP adduct formed by decarboxylation of 2-oxoglutarate). MenD-catalyzed reactions are characterized by high chemo- and regioselectivity. Here, we report (S)-HOG, in situ formed by EDA, to serve as new donor substrate for MenD in 1,4-addition reactions with 2,3-trans-CHD (2,3-trans-dihydroxy-cyclohexadiene carboxylate) and acrylic acid. Likewise, (S)-HOG serves as donor in 1,2-additions with aromatic (benzaldehyde) and aliphatic (hexanal) aldehydes. This enzyme cascade of two subsequent C-C bond formations (EDA aldolase and a ThDP-dependent carboligase, MenD) generates two new stereocenters.

Folding Defects Leading to Primary Hyperoxaluria.

Protein misfolding is becoming one of the main mechanisms underlying inherited enzymatic deficits. This review is focused on primary hyperoxalurias, a group of disorders of glyoxylate detoxification associated with massive calcium oxalate deposition mainly in the kidneys. The most common and severe form, primary hyperoxaluria Type I, is due to the deficit of liver peroxisomal alanine/glyoxylate aminotransferase (AGT). Various studies performed in the last decade clearly evidence that many pathogenic missense mutations prevent the AGT correct folding, leading to various downstream effects including aggregation, increased degradation or mistargeting to mitochondria. Primary hyperoxaluria Type II and primary hyperoxaluria Type III are due to the deficit of glyoxylate reductase/hydroxypyruvate reductase (GRHPR) and 4-hydroxy-2-oxoglutarate aldolase (HOGA1), respectively. Although the molecular features of pathogenic variants of GRHPR and HOGA1 have not been investigated in detail, the data available suggest that some of them display folding defects. Thus, primary hyperoxalurias can be ranked among protein misfolding disorders, because in most cases the enzymatic deficit is due to the inability of each enzyme to reach its native and functional conformation. It follows that molecules able to improve the folding yield of the enzymes involved in each disease form could represent new therapeutic strategies.

Dihydrodipicolinate Synthase: Structure, Dynamics, Function, and Evolution.

Enzymes are usually comprised of multiple subunits and more often than not they are made up of identical subunits. In this review we examine lysine biosynthesis and focus on the enzyme dihydrodipicolinate synthase in terms of its structure, function and the evolution of its varied number of subunits (quaternary structure). Dihydrodipicolinate synthase is the first committed step in the biosynthesis of lysine, which occurs naturally in plants, bacteria, archaea and fungi, but is not synthesized in mammals. In bacteria, there have been four separate pathways identified from tetrahydrodipicolinate to meso-diaminopimelate, which is the immediate precursor to lysine. Dihydrodipicolinate synthases from many bacterial and plant species have been structurally characterised and the results show considerable variability with respect to their quaternary structure, hinting at their evolution. The oligomeric state of the enzyme plays a key role, both in catalysis and in the allosteric regulation of the enzyme by lysine. While most bacteria and plants have tetrameric enzymes, where the structure of the dimeric building blocks is conserved, the arrangement of the dimers differs. We also review a key development in the field, namely the discovery of a human dihydrodipicolinate synthase-like enzyme, now known as 4-hydroxy-2-oxoglutarate aldolase . This discovery complicates the rationale underpinning drug development against bacterial dihydrodipicolinate synthases, since genetic errors in 4-hydroxy-2-oxoglutarate aldolase cause the disease Primary Hyperoxaluria Type 3 and therefore compounds that are geared towards the inhibition of bacterial dihydrodipicolinate synthase may be toxic to mammalian cells.

HOGA1 Gene Mutations of Primary Hyperoxaluria Type 3 in Tunisian Patients.

BACKGROUND: Primary hyperoxaluria type 3 (PH3) is due to mutations in the recently identified 4-hydroxy-2-oxoglutarate aldolase (HOGA1) gene. PH3 might be the least severe form with a milder phenotype with good preservation of kidney function in most patients. The aim of this study was to report three PH3 cases carrying mutations in HOGA1. MATERIALS AND METHODS: Genetic analysis of HOGA1 was performed in patients with a high clinical suspicion of PH after sequencing of AGXT and GRHPR genes, which was negative. Also, a complete AGXT/GRHPR MLPA was performed in these patients in order to detect large deletions/insertions. RESULTS AND DISCUSSION: Two different HOGA1 gene mutations were identified: the p.Pro190Leu in a homozygous state and the p.Gly287Val in two patients in homozygous and heterozygous carriers. The median age at onset of clinical symptoms was 3.93 years. Most of the patients had a positive family history for recurrent urolithiasis. The p.Pro190Leu mutation was reported with impaired renal function at follow-up; however, the p.Gly287Val was presented with normal renal function. All patients were presented with urolithiasis, but only one had a nephrocalcinosis. CONCLUSION: This study expanded the number of PH3 patients from 63 to 66 cases. The p.Pro190Leu and the p.Gly287Val mutations found in this study can provide a first-line investigation in Tunisian PH1 patients.

Use of a novel microtitration protocol to obtain diffraction-quality crystals of 4-hydroxy-2-oxoglutarate aldolase from Bos taurus.

The enzyme 4-hydroxy-2-oxoglutarate aldolase (HOGA) catalyses the retro-aldol degradation of 4-hydroxy-2-oxoglutarate to pyruvate and glyoxylate as part of the hydroxyproline catabolic pathway in mammals. Mutations in the coding region of the human HOGA gene are associated with primary hyperoxaluria type 3, a disease characterized by excessive oxalate production and ultimately stone deposition. Native HOGA was purified from bovine kidney using an improved and streamlined purification protocol from which two crystal forms were obtained using two different approaches. Vapour diffusion using PEG 3350 as a precipitant produced monoclinic crystals that belonged to space group C2 and diffracted to 3.5 Šresolution. By comparison, orthorhombic crystals belonging to space group I222 or I212121 and diffracting to beyond 2.25 Šresolution were obtained using a novel microtitration protocol with ammonium sulfate. The latter crystal form displayed superior diffraction quality and was suitable for structural determination by X-ray crystallography.