Introduction 1 2 3 4 5 6 8 9 10 11 12 13 14 16 17 20 21 22 23 24 25 26 USF2 27 28 PAX2 6 7 19 20 29 ROBO2 18 Here, we describe the results of the second genome-wide scan for primary VUR. Differently from previous studies and aiming to collect a homogeneous sample set, our patients were ascertained in a single geographic region. Our results suggest the presence of several novel loci for primary VUR, giving further evidence for the genetic heterogeneity of this disorder. Methods Patients and families 1 30 31 Fig. 1 VUR  32 33 1 Informed consent from patients and family members (parents for their children) and approval from the Ethic Committee at Second University of Naples were obtained previously. Laboratory analysis Genomic DNA was isolated from peripheral blood leukocytes by standard techniques and was sent from the Paediatrics Department of Second University of Naples to the Department of Clinical Genetics, Erasmus Medical Centre in Rotterdam. A systematic genome scan was performed using the ABI Prism MD-10 set (Applied Biosystems) consisting of 382 short-tandem-repeat polymorphisms markers (STRPs), average spaced 10 cM. Additional markers for further characterization of candidate regions were selected from the gender-average Marshfield genetic map. Information about marker order and distances were obtained from the National Center for Biotechnology Information (NCBI) physical map and Marshfield integrated genetic map. Polymerase chain reaction (PCR) products were resolved on an ABI3100 automated sequencer, and genotypes were analyzed using the GeneMapper software v.2.0 (Applied Biosystems). Linkage analysis 34 35 36 A parametric analysis was performed as well. An autosomal dominant mode of inheritance with reduced penetrance, as described above (power calculations), was used. Marker allele frequencies were calculated using all spouses (unrelated individuals) coming from the same geographic area. Due to the genetic heterogeneity of the disease, the genome scan data was analyzed, maximizing the heterogeneity LOD score (HLOD) with respect to the proportion of linked families (α). Nonparametric (NPL) and parametric (LOD) scores were calculated for each of the families, and then total NPL and HLOD scores were obtained. Results Patients 1 A total of 78 primary VUR patients belonging to 25 pedigrees were identified (33 based on a positive VCUG/RNC, 18 based on detection of RN, 21 diagnosed as having both reflux and RN, and six with ESRF). During the course of the study, a new patient affected with RN was detected in family 1 and included in the second stage. Twenty-eight patients were males and 51 were females (ratio M/F = 0.57). The median age at diagnosis was 3.5 years (range 1 month–68 years). Among the 79 patients, 19 were treated surgically, and four out of six who developed ESRF underwent renal transplantation. 2 2 Fig. 2 Squares circles grey white asterisk number 3 a b VUR RN ESRF UTIs  Genome search 18 p 28 p p 1 p D1S468-D1S255, D1S213-D1S2785 D3S3681 D3S1569 D4S402-D4S1597 D22S539-D22S280 p p ROBO2 29 ROBO2 Table 1 p Pedigrees D1S468-D1S255 D1S213-D1S2785 D3S3681-D3S1569 D4S402-D4S1597 1p36.32–1p34.3 1q41–1q43 3p12.3–3q24 4q26–4q32.3 4–65 cM 242–266 cM 109–158 cM 117–169 cM 1 −0.80 (−1.09) 0.59 (0.19) 2.48 (0.87) −0.37 (0.07) 2 1.34 (0.27) −0.42 (−0.76) 1.34 (0.28) 0.44 (0.27) 4 1.14 (0.72) 0.26 (−0.36) 1.04 (−0.31) 3.05 (1.3) 5 1.9 (0.55) 1.78 (0.55) 1.89 (0.58) 1.69 (0.57) 7 1.59 (0.54) 1.07 (0.42) 1.75 (0.58) −0.33 (−1.37) 11 1.34 (0.27) 1.34 (0.27) 1.34 (0.28) 0.44 (0.28) 12 1.47 (0.19) 1.58 (0.35) 0.65 (0.10) −0.04 (−1.66) 13 1.25 (0.54) 1.97 (0.54) 1.25 (0.57) 1.25 (0.57) p p p p p Total HLOD (α) 1.13 (0.62) 0.61 (0.57) 1.52 (0.76) 1.31 (0.51) p NPL HLOD α p p D2S165-D2S337 p 2 p D1S2667 D1S255 Refinement of the chromosome 3 locus D3S3681 D3S1569 p p 2 p D3S3641 D3S1764 3 Fig. 3 grey white blocks  Table 2 p Pedigrees D1S468-D1S255 D3S3681-D3S1569 1p36.32–1p34.3 3p12.3–3q24 4–65 cM 109–158 cM 1 −0.79 (−2.19) 1.43 (0.74) 2 1.34 (0.28) 1.34 (0.28) 4 1.59 (1.17) 0.46 (−0.43) 5 1.89 (0.56) 1.79 (0.58) 7 1.6 (0.57) 1.75 (0.57) 11 1.34 (0.28) 1.34 (0.28) 12 1.51 (0.05) 0.81(−0.73) 13 1.26 (0.57) 1.98 (0.57) 14 1.41 (0.27) 0.004 (0.001) 15 −0.24 (−0.07) 1.41 (0.28) 16 −0.07 (−0.02) 0.0004 (−0.008) 17 1.41 (0.28) 1.41 (0.28) 18 −0.37 (−0.32) −0.57 (−0.48) 19 0.9 (0.28) −0.81 (−0.9) 20 −0.37 (−0.29) 1.73 (0.48) 21 −0.48 (−0.45) −0.58(−0.48) 22 1.37 (0.27) −0.57 (−0.48) 23 2.43 (0.86) −1.41 (−0.96) 25 0.22 (0.28) 4.63 (1.45) p p p Total HLOD (α) 1.65 (0.55) 1.24 (0.36) p NPL HLOD α Refinement of chromosome 1 locus D1S468 D1S255 2 p D1S228 D1S255 Candidate genes PPP2R3A 37 FNDC6 38 39 RBP1 RBP2 40 AGTR1 41 Discussion This study reports the second genome-wide search for primary VUR. Our results suggest the existence of several loci mapping to chromosomes 1, 3, 4, and 22, further supporting the hypothesis that primary VUR is genetically heterogeneous. We, as others, encountered several pitfalls when studying VUR. We observed clinical variability among and within families and the presence of obligate carriers (individuals who carry and transmit the disease allele but do not manifest any disease sign or symptom). Although the appropriate clinical investigations were performed in several apparently healthy individuals (carriers), no evidence of disease was found, indicating a reduced or age-dependent penetrance. As most of these individuals were recruited during their adulthood, we could not exclude an earlier disease condition that evolved to a spontaneous resolution. To overcome these problems, we first performed a careful clinical evaluation of patients and available relatives. All individuals older than 5 years of age with insufficient or no evidence of VUR were classified as diagnosis unknown, despite the consequent loss of power for the statistical analysis. 18 GATA128C02 D3S1763 p 42 The complex disease etiology of primary VUR has shown to be difficult to disentangle. Genetic heterogeneity and lack of knowledge of the true genetic model for VUR are probably the main difficulties in the identification of the genetic etiology of VUR. Although genetic studies in VUR are still in an early phase, we can presume that primary VUR is likely a complex disorder, with a number of not fully penetrant genes causing most of the familiar cases. Finally, primary VUR could be caused by simultaneous gene-environment interactions. In conclusion, our results show further evidence for the genetic heterogeneity in primary VUR. We will next focus on the refinement of the identified genomic regions and the sequence analysis of the candidate genes according to their tissutal expression and biological function. Replication of the results in additional families will be essential, first to confirm and eventually to evaluate the contribution of these loci to the pathogenesis of primary, nonsyndromic VUR.