Primary hyperoxalurias in Italy and Europe

Primary Hyperoxalurias (PH) are a group of rare disease caused by mutations in genes encoding hepatic enzymes of the glyoxalate detoxification pathway, causing the formation and deposition of calcium oxalate crystals at first in the urinary tract (nephrolithiasis and nephrocalcinosis) and, after end stage renal failure, in the whole body. At present, 3 PH types are known, with autosomal recessive inheritance: PH1, due to lack of function of the hepatic alanine-glioxylate aminotransferase (AGT) enzyme; PH2, due to lack of function of the glioxylate-reductase/hydroxypiruvate-reductase (GRHPR) enzyme; PH3, associated with HOGA1 gene mutations. HOGA1 encodes the 4hydroxy2oxoglutarate aldolase, a mitochondrial enzyme responsible for the final step of the mitochondrial hydroxyproline metabolism. The only two treatments available for PH are pyridoxine therapy, which is effective in a minority of patients, and liver transplantation, which is a very invasive and problematic procedure. Thus, approaches aimed at a deeper knowledge of genotype/phenotype correlations as well as at the development of new treatment strategies, appear to be desirable. The San Luigi Hospital is the only Italian Centre offering the genetic testing for primary Hyperoxaluria in Italy;, our Centre is part of the OxalEurope group, the European Hyperoxaluria Consortium. Aim of the PhD project was to refine the current knowledge on genotype/phenotype correlations, to better define the therapeutic strategy in patients and to extend the molecular analysis to HOGA1 gene. At the end of 2014 the Italian database includes 80 PH1 patients (28 females and 52 males) and 2 PH2 patient. Median age of the cohort is 22 years old (I-III quartile: 11-35,3). Symptoms at onset are mainly nephrolithiasis (52,5%) and nephrocalcinosis (35,2%), as generally reported in PH patients. Three patients were diagnosed at family screening before symptoms development. Median age at onset is age 4 years (0.5-11,5), median age at diagnosis is 11 years old (4-30). 55 patients have had ESRF. Median age at ESRD is 14 years of age (4-31). Ten patients had a kidney-only transplant and, among them, 2 had a second kidney-only transplant. In other 6 cases the second kidney transplant was combined with liver transplant. Some recurrent mutations are present; as reported in literature, also in the Italian cohort the most common mutation is p.Gly170Arg (28% of the alleles), in 14 patients in homozygosis, in 11 in heterozygosis with a missense mutation and in 4 with a null mutation. We set up the analysis of the AGXT promoter, of GRHPR and HOGA1 genes. In 15 patients with clinical diagnosis of PH, who resulted negative for pathogenic variants in the entire coding sequence and exon-intron boundaries of AGXT gene, we extended the analysis to GRHPR and HOGA1 genes. One patient was homozygous for a novel HOGA1 variant of undefined pathogenicity in intron 2 (c.341-81delT), that in silico tools predicted to affect splicing. However, a minigene splicing assay did not identify any abnormal splicing product. In two patients we detected heterozygosity for novel variants in the AGXT promoter (c.-647C>T, c.-424C>T), whose pathogenicity however remained uncertain, since they lay outside of known transcription regulatory sites. A genotype-phenotype study was conducted in the OxalEurope Consortium database. This retrospective report is based on 526 patients (410 with AGXT genotype). We grouped mutations by the predicted effect as null (N), missense leading to mistargeting (G170R) and other missense (M) and analyzed their phenotypic correlations. Median age of End-stage Renal Disease increased from 9.9 to 11.5, 16.9, 25.1, 31.2 and 33.9 years for the NN (n=88), MN (n=42), MM(n=116), G170RN(n=61), G170R/M(n=32) and G170R/G170R(n=71) respectively. The outcome of some recurrent missense mutations (p.I244T, p.F152I, p.M195R, p.D201E, p.S81L, p.R36C) and an unprecedented number of G170R homozygotes is described in detail. In collaboration with Prof. Borri-Voltattorni’s group (University of Verona) we developed a Telethon project (GGP10092), aiming to improve the clinical management of patients affected by PH1 by exploiting the knowledge on the genotype-enzymatic phenotype-clinical phenotype relationships to develop new specific and non-invasive treatment strategies. We focused our attention on the molecular defects underlying AGT deficiency in compound heterozygous patients. Until now, only “single protein” studies have been undertaken to investigate the molecular pathogenesis of PH1, and the possible interplay between two different pathogenic mutations at clinical and enzyme level has never been analysed. We started from the clinical data on two PH1 patients, one hemizygous for the S81L mutation associated with the major allele, and the other compound heterozygous for the S81L mutation on the major allele and the most common mutation in Caucasian patients, i.e. the G170R associated with the minor allele. With in vitro studies we demonstrated a phenomenon of positive interallelic complementation between the S81L and G170R monomers, mimicking heterozygous and heterozigous compound patients. In an attempt to identify molecules acting as pharmacological chaperones for AGT variants showing folding defects, the Telethon project was focused on aminoxyacetic acid (AOA) a known competitive inhibitor of AGT. The interaction of AOA with AGT and the pathogenic variants G41R, G170R and I244T were studied in CHO-GO cells stably expressing AGT, demonstrating that AOA behaves as a competitive inhibitor and is able to act as a pharmacological chaperone for the pathogenic variants because it decreases the aggregation propensity of G41R and promote the peroxisomal localization of G170R and I244T. These results provide the proof-of-principle that an enzyme enhancement therapy for PH1 could be possible and constitute the base for the identification of molecules more specific for AGT. In this regard, large scale in silico screening of libraries of commercially available compounds are ongoing. In conclusion, in this PhD thesis some contributions were made to the general understanding of this disease, both as regards the genotype-phenotype correlations and as functional characterization of AGXT mutations. A grey zone of cases in which suspicions based on clinical and biochemical findings are not confirmed by genetic testing is recognized in the diagnostic practice of most rare genetic diseases and in PH1 as well. To clarify the inconclusive diagnoses in our historical series of cases referred for genetic diagnosis of PH we selected 15 patients with primary oxaluria, AGXT mutationnegative, to address to the analysis of the AGXT-promoter and the additional known genes GRHPR and HOGA1. Our analyses did not provide evidence of pathogenic mutations of these genes in any of the 15 patients. Since oxalate metabolism is complex and not fully described, it is possible that other genes are presently uncovered and even a multiallelic PH inheritance has been suggested. In the future, next generation sequencing could be a useful tool to both discover novel genes involved in glyoxalate metabolism and PH phenotype modifiers. Thanks to the collaboration with Prof. Borri-Voltattorni's group, we were able, for the first time, to understand the effects of the S81L mutation on the major allele and the G170R on the minor allele under homozygous and heterozygous conditions. This work constitutes a valid example of an approach useful to understand the enzymatic phenotype in compound heterozygous PH1 patients, with potential applications to the study of the interaction between other pathogenic mutations. Besides providing a better knowledge of the disease pathogenesis, the results obtained could allow to develop proper therapeutic treatments, as the hybrid protein could have defects different from those of the parental enzymes. The genotype-phenotype studies were made possible by the collaboration with several European Centres, all being part of the OxalEurope group. This study of an unprecedented number of PH1 patients showed geno-phenotype associations that have not been previously described. These findings may have important implications for the management of PH1 patients and underline the necessity of genotyping PH patients. We also identified considerable diagnostic delay in this disease, which may partly explain the overall adverse outcome in some patients.