Mutations in the RARE and MARE regulatory sequences of HNF1β are not a frequent cause of kidney/urinary tract malformation

Mutations in the RARE and MARE regulatory sequences of HNF1B are not a frequent cause of kidney/urinary tract malformation Sir, Mutations in several genes have been identified in congenital abnormalities of kidneys and urinary tract (CAKUT): heterozygous mutations in HNF1β account for 8%, while other genes (PAX2, EYA1, SALL1, SIX1) have been identified as causing isolated renal hypo/dysplasia to a lesser extent in humans [1]. Despite extensive HNF1β molecular analysis, including direct sequencing of the minimal promoter and of the nine exons associated with screening for exonic rearrangement, HNF1β genetic testing failed to identify mutations in a majority of patients with CAKUT, even in individuals with very evocative renal phenotype [2,3]. Recently, specifically conserved cis-acting RARE (retinoic acid responsive element) and T-MARE (MafB responsive element) regulatory sequences have been identified [4]. RA signalling cascade and MafB bind RARE and T-MARE regulatory DNA sequences , respectively, and promote the expression of Hnf1β in both the caudal hindbrain (r4/r5 boundary) and pancreas. These sequences are both located in intron 4 of HNF1β and are not actually studied for the purpose of diagnosis. Finally , the roles of RA and MAFB in kidney development suggest that the RA-MAFB-HNF1β pathway may also be functional in human kidney. In this study, we hypothesized that mutations in the highly conserved (see Figure 1) RARE and T-MARE regulatory sequences could disturb the early embryologic process of intermediate mesoderm, from which kidneys and urinary tract both derived, and consequently lead to renal phenotype mimicking HNF1β-related nephropathy. Among the 63 individuals we tested for HNF1β, a mutation was identified in 12 (19%). We therefore performed molecular analysis of these regulatory sequences in the 51 remaining patients with isolated or syndromic CAKUT of unknown origin for whom previous molecular analysis of HNF1β (QMPSF and direct sequencing) had failed to identify any causative mutation. All patients gave informed consent according to French law. Renal and extra-renal features of the 51 tested patients [31 males, 20 females , median age 9 years (0–53)] are summarized in Tables 1 and 2, respectively. RARE and T-MARE regulatory sequences were amplified by PCR using a set of primers deduced from the genomic sequence (forward: TC CCCAGAACCCTCTTCCTA, reverse: TGGTTCAAAGC CCCAACTTA), corresponding to amplification of a 344 bp fragment (G1266–17138 to G1266–16794). Among the 51 tested patients, no sequence variation of the RARE