Table 1. SOX17 Mutation Analysis of Subjects with Vesicoureteral Reflux (VUR), DMSA-Positive (DMSA1UTI), and Renal Scars Associated with Recurrent Urinary Tract Infections Number (n) Sex (M/F) SOX17 p.Y259N n SOX17 p.G178C n SOX17 p.Q17_S18insTQ n Diagnosis (grade, position) VUR (III grade, R&L) double pelvis VUR (IV grade, R) VUR (III grade, L) VUR (III grade, L) ureter dilatation L VUR (III grade, L) VUR (I grade, R&L) VUR (III grade, R&L) n.a Tc99m DMSA Renal scars Normal Normal Renal scars Renal scars Renal scars Renal scars Renal scars n.a CAKUT Familial cases Sporadic cases Controls (CT) Cord blood Nephrotic syndrome 58 (10 fam.) Fam.1, II.1 Fam.1, I.2 Fam.2, II.1 Fam. 2, I.2 178 Pt 180 Pt 240 Pt 253 pt 36 88 ct 1 82 135 26/32 F F M F 57/121 F F M M 40/48 M 40/42 60/75 4 het het het het 2 het het 1 het 0 0 1 het 1 het Three groups of unselected control subjects were also analyzed. One of this included children with nephrotic syndrome who were excluded for VUR. VUR 5 vesicoureteral reflux; UTI, urinary tract infections; n, number; M, male; F, female; het, heterozygote; L, left; R, right; Tc99m DMSA, Technetium99-dimercaptosuccinic acid; n.a, not available. with BAC clones spanning the chromosomal 8q11.1q12.1 regions selected according to the University of California Santa Cruz (UCSC) Human Genome Assembly (March 2006 assembly). To investigate the activation status of the two centromeres present on the duplicated chromosome 8, immunofluorescence analysis was performed by CENP-C antibody, as described [Gimelli et al., 2000]. SOX17 Mutation Analysis DNA was extracted from lymphoblasts or whole blood sample with the High Pure PCR Template Preparation Kit (Roche Diagnostics, Italy). We also obtained genomic DNA from cultured skin biopsy fibroblasts and from the proband’s ureteric tissue. Amplification and sequencing of the SOX17 (NM_022454.2) gene and its promoter region were performed by PCR amplification using the following primer pairs: SOX17 Ex1a forward: 50-ggccacatctgtgcagaaaa-30 SOX17 Ex1a reverse: 50-CTCTGGGTCTGGCTCTGGT-30 SOX17 Ex1b forward: 50-GCATCTCAGTGCCTCACTCC-30 SOX17 Ex1b reverse: 50-cgtcaggctcgcaaagaa-30 SOX17 Ex2a forward: 50-tgcgcaattcaaagtctgag-30 SOX17 Ex2a reverse: 50-CGCCGTAGTACACGTGAAGG-30 SOX17 Ex2b forward: 50-CCGGCACCTACAGCTACG-30 SOX17 Ex2b reverse: 50-cacccttttcgaggatgaga-30 All amplification reactions were performed with standard PCR conditions in a GeneAmp 9700 PCR System (Applied Biosystems, Foster City, CA). Unincorporated dNTPs and primers were removed from 5 ml of PCR product by digestion with 2 ml of ExoSAP-IT (USB, Europe GmbH). Automated sequence analysis was performed by dye-terminator reactions on an ABI3130xl (Applied Biosystems) and electropherogram analysis by Sequencer Software 4.6 (GeneCodes Corporation, Ann Arbor, MI). Segregation Analysis and Somatic Cell Hybrids Parental origin of the duplication was studied by generating somatic cell hybrid clones to isolate the duplicated chromosome 8 from its normal homolog [Giorda et al., 2004]. Genomic DNA was extracted from the proposita’s EBV cell line and her parents’ 1354 HUMAN MUTATION, Vol. 31, No. 12, 1352–1359, 2010 lymphocytes using standard protocols; genomic DNA from hybrid clones was extracted using DNAzol (MRC Inc., Cincinnati, OH). Genotyping of polymorphic loci was performed by amplification with primers labeled with fluorescent probes (ABI 5-Fam, Hex and Tet) followed by analysis on a ABI 310 Genetic Analyzer (Applied Biosystems). Amplifications were performed with Taq Gold (Applied Biosystems) using standard protocols. Sequencing reactions were performed with a Big Dye Terminator Cycle Sequencing kit (Applied Biosystems) and run on an ABI Prism 3100 AV Genetic Analyzer. Western Blot Analysis and Luciferase Assays HEK293T cells were obtained from ATCC and cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS) (Hyclone, Logan, UT). Cells were plated into 24-well plates 24 hr prior to transfection. For Western blotting, cells were transfected with the Sox17 plasmids (100–300 ng), and a GFP plasmid (100 ng) as a transfection control, using Lipofectamine 2000 (Invitrogen, Carlabad, CA) according to the manufacturer’s protocol. Total amount of DNA was kept constant by adding empty vector DNA. Total cell lysates were prepared 48 hr posttransfection using RIPA buffer and analyzed by Western immunoblotting. Antibodies used were goat anti-Sox17 (1:5000, R&D Systems, Indianapolis, IN) and mouse antitubulin (1:5,000; Sigma, St. Louis, MO); the secondary antibodies were rabbit antigoat HRP (1:10,000; Vector Laboratories, Burlingame, CA), and goat antimouse HRP (1:10,000, Jackson ImmunoResearch, West Grove, PA). Each experiment was repeated at least three times and a representative sample is shown. For luciferase assays, cells were transfected with Sox17 plasmids (3–50 ng), either the TOPflash Wnt reporter plasmid or a SOX17 luciferase reporter plasmid (50 ng) [Sinner et al., 2007], a renilla plasmid (50 ng) as a transfection control, and a constitutively active S37A b-catenin plasmid (50 ng) for the TOPflash assay. The Sox17 reporter plasmid contained eight copies of a Sox17 binding site and the TOPflash reporter contains TCF/LEF binding sites and were previously described [Sinner et al., 2007; Zorn et al., 1999]. Total amount of DNA was kept constant by adding empty vector DNA, and cell lysates