Analisi mutazionale di geni coinvolti nella nefrogenesi e ricerca di variazioni di sequenza nelle loro regioni target per mirna

Congenital anomalies of kidney and urinary tract (CAKUT) are prominent diseases in the pediatric population in terms of prevalence and morbidity (3-6/1000 live births) and responsible of 50% chronic renal failure in pediatric populations. CAKUT encompass a wide phenotypic variability whose clinical relevance varies from less severe forms with mild renal functional impairment, to serious forms such as agenesis and renal hypodysplasia. Medical literature suggests that diseases could result from aberration in spatio-temporal regulation of nephrogenetic program, which involves a complex gene network. Mutations in several kidney developmental genes, have been associated with some of these anomalies, but much remains to be clarified on the etiopathogenesis of most CAKUT. It has been recently confirmed the role of short non-coding RNA named microRNA (miRNA) as gene regulators during the mammalian nephrourogenesis. Studies of conditional loss of kidney miRNAs during embryonic development in mice, suggest that their deregulation may underlie CAKUT. Mutational analysis was performed in 53 CAKUT patients for SIX1, PAX2, GATA3 and SPRY1 genes, which are involved in nephrogenesis. We used an integrated bioinformatics resource for animal miRNA-target interactions, to define which miRNA may be involved in the regulation of our selected kidney developmental genes. Analysis of miRNA target regions of these genes was performed in order to identify sequence variations that can modify homology/complementarity features to the targeted microRNAs and their translational mechanism. Sequence variation were checked also in miRNA gene identified by predictive bioinformatic tools Five causative mutations have been identified in six patients with syndromic or isolated renal hypodysplasia: 4 mutations in the PAX2 gene and 1 mutation in GATA3 gene. No mutations were observed in SIX1 and SPRY1 genes, and also in 3'UTR targets and in related MIRNA genes analyzed. In one patient with syndromic hypodysplasia CGH array analysis showed a proximal microdeletion of about 1.4Mb in 17q12. We hypothesized that the absence of sequence’s variations, both in genomic regions regulated by miRNA and in genes codifying for miRNA, observed in our CAKUT patients, may be due to a very low incidence of these mutations. It must be considered that molecular interactions predicted by in silico studies for miRNA should be validated by functional studies to confirm their involvement during the nephrourogenesis. Identification of causing-disease mutation in PAX2 and GATA3 genes confirmed their role in CAKUT phenotype. Molecular screening of PAX2 and GATA3 genes is mainly performed only in patients with complete phenotype of papillorenal syndrome and hypoparathyroidism, sensorineural deafness, and renal disease syndrome. The identification of these mutations in not syndromic pediatric subjects confirms the necessity to extend the analysis to a higher number of patients. The increased understanding of the post-transcriptional molecular mechanisms responsible of renal and uretheral anomalies, will contribute to the identification of a “targeted gene panel” that could be used in an early and accurate diagnosis of these pathologies

QueryOR: a comprehensive web platform for genetic variant analysis and prioritization

Background: Whole genome and exome sequencing are contributing to the extraordinary progress in the study of human genetic variants. In this fast developing field, appropriate and easily accessible tools are required to facilitate data analysis. Results: Here we describe QueryOR, a web platform suitable for searching among known candidate genes as well as for finding novel gene-disease associations. QueryOR combines several innovative features that make it comprehensive, flexible and easy to use. Instead of being designed on specific datasets, it works on a general XML schema specifying formats and criteria of each data source. Thanks to this flexibility, new criteria can be easily added for future expansion. Currently, up to 70 user-selectable criteria are available, including a wide range of gene and variant features. Moreover, rather than progressively discarding variants taking one criterion at a time, the prioritization is achieved by a global positive selection process that considers all transcript isoforms, thus producing reliable results. QueryOR is easy to use and its intuitive interface allows to handle different kinds of inheritance as well as features related to sharing variants in different patients. QueryOR is suitable for investigating single patients, families or cohorts. Conclusions: QueryOR is a comprehensive and flexible web platform eligible for an easy user-driven variant prioritization. It is freely available for academic institutions at http://queryor.cribi.unipd.it/

Deciphering Variability of PKD1 and PKD2 in an Italian Cohort of 643 Patients with Autosomal Dominant Polycystic Kidney Disease (ADPKD)

Autosomal Dominant Polycystic Kidney Disease (ADPKD) is the most common hereditary kidney disease. We analysed PKD1 and PKD2, in a large cohort of 440 unrelated Italian patients with ADPKD and 203 relatives by direct sequencing and MLPA. Molecular and detailed phenotypic data have been collected and submitted to the PKD1/PKD2 LOVD database. This is the first large retrospective study in Italian patients, describing 701 variants, 249 (35.5%) already associated with ADPKD and 452 (64.5%) novel. According to the criteria adopted, the overall detection rate was 80% (352/440). Novel variants with uncertain significance were found in 14% of patients. Among patients with pathogenic variants, in 301 (85.5%) the disease is associated with PKD1, 196 (55.7%) truncating, 81 (23%) non truncating, 24 (6.8%) IF indels, and in 51 (14.5%) with PKD2. Our results outline the high allelic heterogeneity of variants, complicated by the presence of variants of uncertain significance as well as of multiple variants in the same subject. Classification of novel variants may be particularly cumbersome having an important impact on the genetic counselling. Our study confirms the importance to improve the assessment of variant pathogenicity for ADPKD; to this point databasing of both clinical and molecular data is crucial

Analisi mutazionale di geni coinvolti nella nefrogenesi e ricerca di variazioni di sequenza nelle loro regioni target per mirna

Congenital anomalies of kidney and urinary tract (CAKUT) are prominent diseases in the pediatric population in terms of prevalence and morbidity (3-6/1000 live births) and responsible of 50% chronic renal failure in pediatric populations. CAKUT encompass a wide phenotypic variability whose clinical relevance varies from less severe forms with mild renal functional impairment, to serious forms such as agenesis and renal hypodysplasia. Medical literature suggests that diseases could result from aberration in spatio-temporal regulation of nephrogenetic program, which involves a complex gene network. Mutations in several kidney developmental genes, have been associated with some of these anomalies, but much remains to be clarified on the etiopathogenesis of most CAKUT. It has been recently confirmed the role of short non-coding RNA named microRNA (miRNA) as gene regulators during the mammalian nephrourogenesis. Studies of conditional loss of kidney miRNAs during embryonic development in mice, suggest that their deregulation may underlie CAKUT. Mutational analysis was performed in 53 CAKUT patients for SIX1, PAX2, GATA3 and SPRY1 genes, which are involved in nephrogenesis. We used an integrated bioinformatics resource for animal miRNA-target interactions, to define which miRNA may be involved in the regulation of our selected kidney developmental genes. Analysis of miRNA target regions of these genes was performed in order to identify sequence variations that can modify homology/complementarity features to the targeted microRNAs and their translational mechanism. Sequence variation were checked also in miRNA gene identified by predictive bioinformatic tools Five causative mutations have been identified in six patients with syndromic or isolated renal hypodysplasia: 4 mutations in the PAX2 gene and 1 mutation in GATA3 gene. No mutations were observed in SIX1 and SPRY1 genes, and also in 3'UTR targets and in related MIRNA genes analyzed. In one patient with syndromic hypodysplasia CGH array analysis showed a proximal microdeletion of about 1.4Mb in 17q12. We hypothesized that the absence of sequence’s variations, both in genomic regions regulated by miRNA and in genes codifying for miRNA, observed in our CAKUT patients, may be due to a very low incidence of these mutations. It must be considered that molecular interactions predicted by in silico studies for miRNA should be validated by functional studies to confirm their involvement during the nephrourogenesis. Identification of causing-disease mutation in PAX2 and GATA3 genes confirmed their role in CAKUT phenotype. Molecular screening of PAX2 and GATA3 genes is mainly performed only in patients with complete phenotype of papillorenal syndrome and hypoparathyroidism, sensorineural deafness, and renal disease syndrome. The identification of these mutations in not syndromic pediatric subjects confirms the necessity to extend the analysis to a higher number of patients. The increased understanding of the post-transcriptional molecular mechanisms responsible of renal and uretheral anomalies, will contribute to the identification of a “targeted gene panel” that could be used in an early and accurate diagnosis of these pathologies.Le anomalie congenite del rene e delle vie urinarie (CAKUT) rappresentano circa il 30% di tutte le anomalie di sviluppo embrionale (prevalenza e morbilità nella popolazione pediatrica 3-6/1000 nati vivi in Europa). Sono malformazioni caratterizzate da un’ampia variabilità fenotipica la cui rilevanza clinica varia da forme meno severe con lievi alterazioni della funzionalità renale, a forme gravi quali l’agenesia e l’ipodisplasia renale. Circa il 50% dei bambini in dialisi o portatori di trapianto renale sono affetti da CAKUT. I dati della letteratura suggeriscono che l’origine di queste malattie sia una disregolazione del complesso programma nefrogenetico. La presenza di mutazioni in geni fortemente coinvolti nello sviluppo del rene è stata associata ad alcune di queste malattie, ma ancora molto resta da chiarire sull’eziopatogenesi della maggior parte delle CAKUT. Recentemente è stato confermato il ruolo rilevante di corte sequenze di RNA non-codificante (microRNA), in qualità di regolatori genici post-trascrizionali, durante la nefrourogenesi dei mammiferi. Studi di perdita condizionale dell’espressione dei microRNA durante lo sviluppo embrionale del topo, suggeriscono che la loro disregolazione possa portare alle CAKUT. Pochi sono a tutt’oggi i dati relativi al loro coinvolgimento nella nefrourogenesi umana. È stata condotta l’analisi mutazionale dei geni SIX1, PAX2, GATA3 e SPRY1, fortemente coinvolti nello sviluppo del rene e delle vie urinarie, su una popolazione di 53 pazienti con CAKUT. In tutti i soggetti che non presentavano mutazioni nei geni dello sviluppo analizzati, sono state ricercate variazioni di sequenza nelle regioni 3’UTR target per miRNA, identificate attraverso uno studio bioinformatico predittivo. La ricerca di variazioni di sequenza è stata quindi estesa anche ai geni codificanti per i MIRNA, che la predizione in silico aveva identificato come ipotetici regolatori post-trascrizionali dei geni SIX1, PAX2, GATA3 e SPRY1. Sono state identificate 5 mutazioni causative di cui 4 a carico del gene PAX2, 1 a carico del gene GATA3, in sei pazienti con ipodisplasia renale in forma sindromica o isolata. Non è stata osservata alcuna mutazione a carico dei geni SIX1 e SPRY1, né a carico dei target 3’UTR e dei relativi MIR analizzati. E’ stata inoltre osservata una microdelezione di 1.4Mb (CNV) nel braccio lungo del cromosoma 17 in una paziente con una forma sindromica di ipodisplasia renale. E’ ipotizzabile che l’assenza di variazioni di sequenza nelle regioni geniche regolate dai microRNA e nei geni codificanti per i MIRNA osservata nella nostra coorte di pazienti con CAKUT, possa essere imputabile a frequenze di mutazione molto basse. Bisogna tuttavia considerare che l’interazione molecolare predetta per i microRNA dallo studio in silico, dovrebbe essere validata mediante studi funzionali per confermare il loro coinvolgimento durante il processo della nefrourogenesi. L’identificazione di mutazioni causative di geni coinvolti nello sviluppo renale, ci permette di confermare il ruolo dei geni PAX2 e GATA3 nella determinazione delle CAKUT. L’analisi molecolare del gene PAX2 e GATA3 viene prevalentemente eseguita in pazienti con fenotipo completo delle sindromi associate (papillo-renale e iperparatiroidismo, sordità e rene). L’aver identificato mutazioni di questi geni anche in soggetti pediatrici non sindromici, è un’ulteriore conferma della necessità di estendere l’analisi anche ad una più ampia categoria di pazienti. I risultati di questa ricerca, aumentando le conoscenze relative ai meccanismi molecolari anche post-trascrizionali coinvolti nella determinazione di anomalie renali e ureterali, possono inoltre contribuire a meglio definire un “targeted gene panel” da analizzare per una più precisa e precoce diagnosi di queste patologi

PAX2 and CAKUT Phenotypes: Report on Two New Variants and a Review of Mutations from the Leiden Open Variation Database

PAX2 is a transcription factor expressed during embryogenesis in the eye, ear, CNS, and genitourinary tract, and is one of the major regulators of kidney development. Mutations in this gene are associated with papillorenal syndrome (PAPRS), a genetic condition characterized by optic nerve dysplasia and renal hypo/dysplasia. In the last 28 years, many cohort studies and case reports highlighted PAX2’s involvement in a large spectrum of kidney malformations and diseases, with or without eye abnormalities, defining the phenotypes associated with PAX2 variants as “PAX2-related disorders”. Here, we reported two new sequence variations and reviewed PAX2 mutations annotated on the Leiden Open Variation Database 3.0. DNA was extracted from the peripheral blood of 53 pediatric patients with congenital abnormalities of the kidney and urinary tract (CAKUT). PAX2 gene-coding exonic and flanking intronic regions were sequenced with Sanger technology. Two unrelated patients and two twins carrying one known and two unknown PAX2 variations were observed. The frequency of PAX2-related disorders in this cohort was 5.8%, considering all CAKUT phenotypes (16.7% in the PAPRS phenotype and 2.5% in non-syndromic CAKUT). Although PAX2 mutations have a higher frequency in patients with PAPRS or non-syndromic renal hypoplasia, from the review of variants reported to date in LOVD3, PAX2-related disorders are detected in pediatric patients with other CAKUT phenotypes. In our study, only one patient had a CAKUT without an ocular phenotype, but his twin had both renal and ocular involvement, confirming the extreme inter- and intrafamilial phenotypic variability

PAX2 gene mutations in pediatric and young adult transplant recipients: kidney and urinary tract malformations without ocular anomalies.

Heterozygous humans for PAX2 mutations show autosomal dominant papillorenal syndrome (PRS), consisting of ocular colobomas, renal hypo/dysplasia and progressive renal failure in childhood. PAX2 mutations have also been identified in patients with isolated renal hypo/dysplasia. Twenty unrelated children and young adults with kidney and urinary tract malformations and no ocular abnormalities were retrospectively recruited for PAX2 mutational analysis. All patients had undergone renal transplantation after end-stage renal disease. We identified two new sequence variations: (i) a deletion causing a frameshift (c.69delC) and (ii) a nucleotide substitution determining a splice site mutation (c.410+5 G/A) by predictive analysis. Therefore, we suggest PAX2 molecular analysis to be extended to all patients with congenital malformations of kidney and urinary tract (CAKUT)