GENETICA MOLECOLARE, EPIDEMIOLOGIA, PATOGENESI DEL RENE MIDOLLARE A SPUGNA

Medullary Sponge Kidney (MSK) was recognized for the first time in Padua, in 1939 by Lenarduzzi utilizing a new urography technique and subsequently described in 1949 by Cacchi and Ricci respectively urologist and pathologist at the University of Padua. Since its association with other congenital renal and extrarenal malformations, MSK may be considered among the congenital renal malformations also due to the presence of precalyceal ectasia of collecting ducts . The renal function and life span are normal in MSK patients, neverthless MSK is often complicated by the development of nephrocalcinosis and nephrolithiasis. The pathogenesis of MSK has not been totally elucidated but many authors agree that it is a congenital disease with delayed expression. A genetic cause for MSK could be suggested by the description of some familial cases, by its link to the group of congenital malformations and by its association with other inherited diseases. Recently, the discovery of the genes responsible for this disease has attracted the attention of our group work and in particular of my PhD work. The working hypothesis is that MSK is the consequence of a disturbance in the ureteric bud- metanephric blastema interface, which might be due either to disease- causing mutation/ specific polymorphism of RET, GDNF and other genes involved in the inter-facing, or to particular RET/GDNF genotype interaction. The results of direct sequencing and RFLP testing of GDNF on a population of 112 , unrelated of Venetian origin MSK patients selected on the basis of strict urographic criteria led to the identification of a rare variant of GDNF promoter region (27+18G>A) significantly associated with MSK (p=0.02). In addition, the possibility to extend GDNF screening to the family members of MSK patients carrying rare variants allowed us to discover that the variants were inherited and that they were associated with the MS K phenotype. In the second part of the study it has been placed attention to the familial cases of MSK. Sampling 50 cases from the cohort of 112 sporadic MSK cases, and analyzing family members of the 1° and 2° generations by ultrasonography and /or UroTAC, we found that in 27 families MSK was inherited as an autosomal dominant trait, with variable expressivity and incomplete penetrance. In 19 families the screening of GDNF gene was conducted and, in addition, the screening of Six1, Spry1 and PAX2 genes that are involved in nephrogenesis with an important role in the mechanisms of GDNF regulation was performed. Sequencing of exon and intron boundaries of GDNF gene on the 19 families did not reveal any nucleotide substitutions or rare variants. Similarly, no causative mutation was found for Six1, Spry1 and Pax2. For Spry1 and Pax2 polymorphisms have been found but without any significance in the allele frequency as compared to control population. Another objective of the study was to understand the functional significance od GDNF rare variants in the context of MSK phenotype. The removal of a renal cell carcinoma in a MSK patient with the GDNF intronic (-27+18 G / C )rare variant gave us the opportunity to study MSK papillary cells in culture. We observed an exceptional phenomenon, the appearance of an osteogenic-like phenotype with deposition of calcium phosphate. We wonder if the down-regulation of GDNF expression that was lower in MSK cells in respect to control cells were the culprit of the observed phenomenon. To investigate if GDNF down-regulation most likely due to the mutation in the promoter region, may have had a role in the process of spontaneous calcification, we have stably silenced GDNF gene in human renal epithelial cells (HK2), through the technique of RNA interference. Preliminary data from these experiments showed in the silenced HK2 cells cultured under osteogenic conditions the presence of aggregates that Won Kossa staining and SEM analysis confirmed to be of Ca2PO4. The presence of calcium phosphate deposits were not observed in control negative as well as in silenced clones cultured in normal conditions, while in control negative cells under osteogenic stimulation few deposits were seen, however in lower number in respect to silenced cells. Although preliminary, our results suggest that GDNF down regulation in HK2 cells may favor Ca2PO4 deposition throught a mechanism not yet identified. Our hypothesis is that apoptosis might be the key.IL Rene con Midollare a Spugna (MSK) riconosciuto per la prima volta a Padova, da Lenarduzzi nel 1939 grazie all’impiego dell’allora nuova tecnica dell’urografia e successivamente descritto nel 1949 da Cacchi e Ricci, rispettivamente urologo e patologo dell’Università patavina può essere annoverato tra le nefropatie congenite malformative per la presenza di ectasie precaliceali dei dotti collettori e per la sua frequente associazione con altre malformazioni congenite renali ed extrarenali. La funzione renale e la durata della vita nei pazienti MSK è normale; spesso si complica con lo sviluppo di nefrocalcinosi e nefrolitiasi. La patogenesi di MSK non è stata completamente chiarita ma la maggior parte degli autori concorda che si tratta di una patologia congenita con espressione ritardata. Che la causa di MSK potesse essere di natura genetica era suggerito dalla descrizione di alcuni casi familiari, dall’appartenenza di MSK al gruppo delle patologie malformative e dalla sua associazione con altre malattie ereditarie. La scoperta del gene/i responsabili della patologia è stato negli ultimi anni obiettivo del nostro gruppo di lavoro ed in particolare del mio lavoro di dottorato. L’ipotesi che ha dato inizio allo studio è che MSK sia la conseguenza di un disturbo durante lo sviluppo renale dell’interfaccia “gemma ureterale- mesenchima metanefrico” a causa di mutazioni/polimorfismi di RET, GDNF, o di altri geni coinvolti nell’embriogenesi renale, o in particolare a causa dell’interazione GDNF/RET. I risultati del sequenziamento diretto e test RFLP di GDNF su una casistica veneta di 112 pazienti, ben selezionata sulla base di stretti criteri urografici hanno permesso di identificare 1 variante rara della regione del promotore di GDNF (-27+18G>A) significativamente associata a MSK (p=0.02). Inoltre, la possibilità di estendere lo screening di GDNF su alcuni familiari di pazienti MSK ci ha permesso di scoprire che le varianti erano ereditate e che nelle famiglie erano associate al fenotipo MSK. Nella seconda parte del lavoro di dottorato è stata posta l’attenzione sui casi familiari di MSK. In collaborazione con la Clinica Nefrologica di Verona sono stati indagati 50 gruppi famigliari scelti random dalla coorte di 112 pazienti con MSK . Dall’’analisi mediante ecografia e/o Uro-TC estesa ai consanguinei della stessa generazione, e di 1, se possibile, 2 generazioni precedenti e/o successive è emerso che in 27 famiglie MSK segregava come carattere autosomico dominante, con espressività variabile e ridotta penetranza. Sui probandi di queste famiglie è stato fatto lo screening per il gene GDNF. Inoltre, in collaborazione con il laboratorio di Nefrologia Pediatrica dell’Università di Padova è stato fatto, lo screening di Six1, Spry1 e Pax2 che sono geni coinvolti nel processo di nefrogenesi renale e con un importante ruolo nei meccanismi di regolazione di GDNF, per un totale di 19 casi famigliari. Lo screening di GDNF mediante sequenziamento diretto delle regioni esoniche e delle giunzioni introne-esone non ha evidenziato nessuna variante rare né altre sostituzioni nucleotidiche. Allo stesso modo nessuna mutazione causativa è stata evidenziata per Six1, Spry1 e Pax2. Per Spry1 e Pax2 solo polimorfismi noti ma senza alcuna significatività statistica nelle frequenze alleliche confrontata con quella di popolazioni di controllo riportate in letteratura. Un altro obiettivo del mio lavoro di dottorato è stato quello di aver cercato di capire come varianti rare di GDNF possano essere associate al fenotipo MSK, in un sottogruppo di pazienti con nefrolitiasi, nefrocalcinosi ed MSK bilaterale. L’asportazione di un carcinoma renale in una paziente con MSK e mutazione di GDNF (-27+18G/C) ha dato l’opportunità di studiare per la mutazione di GDNF il suo significato funzionale e di verificare che cellule papillari renale prelevate da polo indenne al carcinoma renale e con bassi profili di espressione per GDNF, differenziavano spontaneamente verso un lineaggio osteogenico con la sintesi di proteine tipiche della matrice osteoide. Per cercare di approfondire se la down regolazione di GDNF, molto probabilmente dovuta alla mutazione che cade nella regione del promotore, possa avere avuto un coinvolgimento nel fenomeno di calcificazione osservato si è cercato di ottenere su cellule epiteliali renali (HK2), attraverso la tecnica di RNA interference, un silenziamento stabile di GDNF. I dati preliminari di questi ultimi esperimenti hanno mostrato che nelle cellule HK2 silenziate per GDNF vi era la presenza di depositi di Ca2PO4, confermati sia dalla colorazione von Kossa sia dall’analisi SEM. la presenza dei depositi di calcio fosfato non sono state osservate né nel controllo negativo né nei cloni silenziati in normali condizioni di coltura, mentre nel controllo negativo in condizione osteogenica i depositi erano presenti, seppur in minore quantità rispetto al clone silenziato. Sebbene preliminari, i nostri suggeriscono che la down regolazione di GDNF potrebbe favorire la deposizione di Ca2PO4 attraverso un meccanismo non ancora identificato. La nostra ipotesi è che l’apoptosi potrebbe essere la chiave

Identification of GDNF Gene Sequence Variations in Patients with Medullary Sponge Kidney Disease

Background and objectives: Medullary sponge kidney (MSK) is a rare nephropathy characterized by cystic anomalies of precalyceal ducts, nephrocalcinosis, renal stones, and tubule dysfunctions. Its association with various malformations and cases of familial aggregation supports the conviction that genetic factors are involved, but no genetic studies have been conducted to date. It is hypothesized that MSK is due to a disruption at the “ureteric bud/metanephric blastema” interface caused by critical developmental genes functioning abnormally

Identification of GDNF Gene Sequence Variations in Patients with Medullary Sponge Kidney Disease

Abstract BACKGROUND AND OBJECTIVES: Medullary sponge kidney (MSK) is a rare nephropathy characterized by cystic anomalies of precalyceal ducts, nephrocalcinosis, renal stones, and tubule dysfunctions. Its association with various malformations and cases of familial aggregation supports the conviction that genetic factors are involved, but no genetic studies have been conducted to date. It is hypothesized that MSK is due to a disruption at the "ureteric bud/metanephric blastema" interface caused by critical developmental genes functioning abnormally. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: Fifty-five apparently sporadic MSK patients were analyzed by direct DNA sequencing of all exons and exon-intron boundaries of glial cell-derived neurotrophic factor (GDNF) gene and rearranged during transfection (RET) gene, which have a leading role in renal development. RESULTS: Two novel variants were found in heterozygosity in the MSK case population: GDNF{ENST00000344622}:c.-45G>C and c.-27+18G>A in a putative binding domain for paired-box 2 transcription factor. As a whole, eight patients showed these variations: four patients carried the c.[-45G>C; -27+18G>A] complex allele, and the others had the c.-27+18G>A alone. A case-control study revealed that these two alleles were significantly associated with MSK. Five of the eight cases were found to be familial, and the allele variants cosegregated with the disease in a seemingly dominant pattern of inheritance. Patients revealed no mutations in the RET gene. CONCLUSIONS: This is the first report identifying GDNF gene sequence variations in patients with MSK and suggesting a role for this gene in the pathogenesis of some cases of the disease

An atypical Dent\u2019s disease phenotype caused by co-inheritance of mutations at CLCN5 and OCRL genes

Dent's disease is an X-linked renal tubulopathy caused by mutations mainly affecting the CLCN5 gene. Defects in the OCRL gene, which is usually mutated in patients with Lowe syndrome, have been shown to lead to a Dent-like phenotype called Dent disease 2. However, about 20% of patients with Dent's disease carry no CLCN5/OCRL mutations. The disease's genetic heterogeneity is accompanied by interfamilial and intrafamilial phenotypic heterogeneity. We report on a case of Dent's disease with a very unusual phenotype (dysmorphic features, ocular abnormalities, growth delay, rickets, mild mental retardation) in which a digenic inheritance was discovered. Two different, novel disease-causing mutations were detected, both inherited from the patient's healthy mother, that is a truncating mutation in the CLCN5 gene (A249fs*20) and a donor splice-site alteration in the OCRL gene (c.388+3A>G). The mRNA analysis of the patient's leukocytes revealed an aberrantly spliced OCRL mRNA caused by in-frame exon 6 skipping, leading to a shorter protein, but keeping intact the central inositol 5-phosphatase domain and the C-terminal side of the ASH-RhoGAP domain. Only wild-type mRNA was observed in the mother's leukocytes due to a completely skewed X inactivation. Our results are the first to reveal the effect of an epistatic second modifier in Dent's disease too, which can modulate its expressivity. We surmise that the severe Dent disease 2 phenotype of our patient might be due to an addictive interaction of the mutations at two different genes