were prepared for luciferase assay as previously described [Sinner et al., 2007]. Quantitative RT-PCR Analysis For quantitative RT-PCR, total RNA was extracted from cells using a NucleoSpin RNA II Kit (Macherey and Nagel, Germany) and treated with RNase-free DNase (Roche, Indianapolis, IN). cDNA was synthesized with random hexamer primers. Real-time RT-PCR was performed using an Opticon machine (MJ Research-Bio Rad, Hercules, CA). The primers used in this study were: hSox17 forward 50 GAC GAC CAG AGC CAG ACC 30 and hSox17 reverse 50 CGC CTC GCC CTT CAC C 30; hb-tubulin forward 50 GAT ACC TCA CCG TGG CTG CT 30 and hb-tubulin reverse 50 AGA GGA AAG GGG CAG TTG AGT 30. SYBR green dye (Qiagen, Chatsworth, CA) was included in the PCR mix. The amount of product in each sample was estimated at the log-linear amplification phase, and these values were normalized to the expression level of tubulin and the data presented as a ratio of tubulin expression. Results Our study began with the observation of a young girl with congenital defects of the urinary tract, chronic constipation, and mild mental retardation, who carried a de novo pseudodicentric duplicated chromosome 8 (Family 1; Fig. 1A). The de novo duplication dup(8)(q11.1q12.1) of 13.1 Mb on chromosome 8 was demonstrated by CGH-array (Agilent 44B) and confirmed by FISH analysis using BAC clones known to map to the duplicated region (Fig. 2). A direct duplication was demonstrated by dual-color FISH with BAC RP11-53M11 and CEP8. A pseudodicentric chromo- some 8 with a inactive centromere was evidenced by FISH with CEP8 and by immunostaining with CENP-C antibody (Fig. 2D). These results allow us to define the karyotype of our proposita as: 46,XX,psudicdup(8)(q11.1q12.1) (chr8:46,958,053..60,381,578). Among the 38 genes located in the duplicated region of chromosome 8, SOX17 seemed a good candidate to study because of its expression throughout development of the urogenital tract in the mouse and its role in regulating b-catenin signaling. We identified a point mutation at nucleotide 775 with respect to the translation start site (c.775T4A) in the SOX17 gene. This mutation results in a protein change from tyrosine at position 259 to an asparagine residue (p.Y259N). The mutation is localized between the HMG-box and the glycine–proline-rich segment at the C-terminal of the protein (Fig. 1B). The same mutation was found in another family with VUR (out of 10 families for an overall of 58 affected) and in several sporadic subjects presenting VUR and renal scars. Segregation analysis of the first identified patient showed the maternal origin of the mutation. In fact, we analyzed 56 somatic cell hybrid clones, using chromosome 8q11 markers and selected three clones containing only the duplicated chromosome 8 and three containing only the normal chromosome 8. Typing with a panel of polymorphic markers demonstrated that the duplication was of maternal origin and intrachromatidic (Supp. Table S1). Amplification and sequencing of exon 2 of the SOX17 gene from the proposita, her parents, and selected hybrid clones demon- strated that in both the proposita and her mother, chromosome 8 carried the p.Y259N mutation, while the paternal chromosome 8 had a normal SOX17 allele. The screening for SOX17 mutations was extended to 178 individuals with sporadic VUR and in 305 controls consisting of 135 children with nephrotic syndrome, 88 normal subjects without a history of urinary infection who were not further characterized, and 82 cord blood samples (610 chromosomes). Children with nephrotic syndrome were chosen because all had been followed for years with frequent urine analysis and echosonograpic evaluation. The same p.Y259N mutation was found in two patients of the sporadic VUR cohort, whereas two additional children carried different SOX17 mutations (Table 1) (chi-squuare, Po0.01): one was a point mutation (p.G178C) producing a glycine to cysteine change at position 178, and the other (p.17Q_18SinsTQ) was characterized by an in-frame insertion of threonine–glutamine at position 17 (Fig. 1B and Table 1). Finally, p.Y259N was found in one normal blood donor DNA who was recruited from our Transfusion Unit without having any further clinical studies, including renal sonography (Table 1). Two (p.G178C and p.Y259N) of the three mutations described here are localized between the HMG-box and the glycine–proline- rich segment at the C-terminal of the protein; the other is at the N-terminus near the HMG-box (Fig. 1B). Comparison of the predicted amino acid sequences indicates that human and mouse Sox17 share about 85% identity over their entire sequence, whereas zebrafish (Dr) Sox17 shows nearly perfect conservation only in the HMG box and the N- and C-terminal portions (Supp. Fig. S1). The Glycine 178 residue is conserved across all three species, while Tyrosine 259 is conserved between human and mouse (and the frog Xenopus laevis, data not shown); the 17Q_18S residues are located in the conserved N-terminal portion of the protein, but are not themselves conserved (Supp. Fig. S1). Analysis of SOX17 p.Y259N Protein Activity We investigated the impact of the p.Y259N mutation on SOX17 protein levels, SOX17 mRNA levels, transcriptional activity, and Wnt-inhibitory activity in the kidney cell line HEK293T. Western blot analysis of total cell extracts demonstrated that, at equal amounts of transfected DNA, the mutant SOX17-p.Y259N protein accumulated to much higher levels than SOX17-WT protein (Fig. 3A). In numerous experiments we observed between 6- and 20-fold increase in protein levels as measured by Western blot and densitometry analysis. These differences were not due differences in mRNA levels from the transfected plasmids, because quanti- tative RT-PCR on total RNA isolated from transfected cells revealed that mRNA levels of wild-type and SOX17– p.Y259N- transfected cells were largely comparable (Fig. 3B). In the example shown, there was a slight increase (1.4-fold) in levels of SOX17– p.Y259N mRNA relative to wild type, but this is minimal compared to the 6- to 20-fold increase in protein levels of SOX17–p.Y259N. We analyzed SOX17 transcriptional and Wnt- b-catenin repressing activity by cotransfection of SOX17 with a SOX17 reporter plasmid [Sinner et al., 2007] (Fig. 3C) and a Wnt reporter plasmid (Fig. 3D). In all cases, the p.Y259N mutant protein levels were significantly higher and this correlated with higher transcriptional activity as measured by the SOX17 reporter plasmid. Moreover, SOX17–p.Y259N suppressed Wnt-b-catenin signaling activity two- to threefold better than wild-type SOX17 (Fig. 3D). However, when luciferase activity was approximately normalized to SOX17 protein levels, the p.Y259N mutation did not appear to alter the inherent transcriptional or Wnt/b-catenin inhibitory activity of SOX17 [Sinner et al., 2007; Wodarz and Nusse, 1998; Zorn et al., 1999]. Because we have previously demonstrated that SOX17 can interact with b-catenin and TCF/ LEF proteins and target them for degradation, one likely interpretation for these data is that elevated p.Y259N mutant HUMAN MUTATION, Vol. 31, No. 12, 1352–1359, 2010 1355