Studio delle basi genetiche del rene con midollare a spugna, una rara nefropatia malformativa

Medullary sponge kidney (MSK) is a rare renal disorder characterized by cystic anomalies of precalyceal ducts, nephrocalcinosis, renal stones and nephronic tubule dysfunctions. Its pathogenesis has yet to be elucidated, but its association with different renal and extra-renal malformative conditions supports the idea of a developmental disorder and its involvement in familial cases shows the role of genetic factors. To date, no genetic study has been conducted. The working hypothesis is that genes regulating renal embryogenesis are good candidates to play a role in the pathogenesis of the disease. Particularly, GDNF and RET genes are necessary for the developing nephro-urological system and their expression is finely regulated during embryogenesis, so mutations of these genes may be important. This study investigated whether they may be really considered disease-causing genes or disease-susceptibility genes, which should modify the expression of key genes of the nephrogenetic process, in MSK patients. A well characterized population of 50 Venetian nephrolitiasic unrelated patients, all of them affected with MSK diagnosed by intravenous urography, was collected. All cases were sporadic. A mutation analysis of all exons and exon-intron boundaries of both GDNF and RET genes was performed using direct DNA sequencing and RFLP approaches. Association and expression studies were also conducted to define the role of the identified mutations. Seven patients had GDNF variants in heterozygosity: a complex allele, constituted by the novel -45G>C and IVS3+18G>A nucleotide substitutions in the 5' UTR, an allele with the intronic mutation IVS3+18G>A alone and the R93W mutation in the coding region, previously described as a disease-susceptibility allele in Hirschsprung disease, congenital central hypoventilation syndrome and phaeocromocytoma and often identified in association with mutations in other genes, including RET. Both the first two alleles showed a frequency below 1% in a Venetian general population and they were never found in a specific control population constituted by individuals who had recurrent calcium nephrolithiasis but not MSK. Investigating the family history, three of these cases were discovered to be indeed familial cases of MSK and the sequence variants were found to co-segregate with the disease in the pedigrees, except for the R93W mutation. An involvement of the RET gene also was hypothesized to be responsible for MSK phenotype in the last case. GDNF gene expression was evaluated in the renal tissue of one MSK patient with the intronic variant resulting, however, not significantly different from control samples. Therefore, it was impossible to characterize further the effects of the identified mutation. No mutations but several single nucleotide polymorphisms (SNPs), already described, were found in the RET gene in MSK patients. Functionally important polymorphisms of the promoter as well as the coding region were focused. The G allele of SNP c2307 T>G in exon 13 showed a frequency different from the control sample (p=0.006). The particular haplotype AA at the -5 and -1 RET promoter SNP loci, never found in controls, also was observed in one patient. The analysis of the genotype frequencies of the promoter revealed that the A/A genotype of SNP-5 was represented in controls but not in MSK patients, while the G/A genotype showed a statistically significant overrepresentation in MSK patients (p=0.040). No AC/AC genotype at the RET promoter SNP loci was identified in MSK patients. Moreover, the AG haplotype composed by alleles at two SNPs in the coding region (c2071 G>A in exon 11 and c2712 C>G in exon 15) was found in homozygosity in three patients and only in heterozygosity in controls (p=0.024). These SNPs were observed to co-segregate as AG and GC haplotypes, but the anomalous GG haplotype was identified in homozygosity in two patients. Reconstructing RET haplotypes using the most significant SNPs in the pedigrees of the probands with GDNF mutations, one was identified recurrent and often associated with MSK and GDNF mutations. This also could explain the familial case without R93W/MSK co-segregation. These data suggest that GDNF gene variants and specific RET haplotypes may be highly associated with MSK phenotype and may have, alone or in association, a potential pathogenicity at least in a subset of MSK patients, demonstrating for the first time that genetic determinants are involved in the pathogenesis of the disease.