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1 A Common Variant of the PAX2 Gene Is Associated with Reduced Newborn Kidney Size Jacklyn Quinlan,* Mathieu Lemire, Thomas Hudson, Huiqi Qu,* Alice Benjamin, Anne Roy, Elena Pascuet,* Meigan Goodyer,* Chandhana Raju,* Zhao Zhang,* Fiona Houghton,* and Paul Goodyer* *Montreal Children s Hospital Research Institute and Departments of Obstetrics and Gynecology and Radiology, McGill University, and McGill University and Genome Quebec Innovation Centre, Montreal, Quebec, Canada Congenital nephron number ranges widely in the human population. Suboptimal nephron number may be associated with increased risk for essential hypertension and susceptibility to renal injury, but the factors that set nephron number during kidney development are unknown. In renal-coloboma syndrome, renal hypoplasia and reduced nephron number are due to heterozygous mutations of the PAX2 gene. This study tested for an association between a common haplotype of the PAX2 gene and subtle renal hypoplasia in normal newborns. A PAX2 haplotype was identified to occur in 18.5% of the newborn cohort, which was significantly associated with a 10% reduction in newborn kidney volume adjusted for body surface area. This haplotype was also associated with reduced allele-specific PAX2 mrna level in a human renal cell carcinoma cell line. Subtle renal hypoplasia in normal newborns may be partially due to a common variant of the PAX2 gene that reduces mrna expression during kidney development. J Am Soc Nephrol 18: , doi: /ASN Between 20 and 30% of North Americans develop hypertension during the course of their life, without any obvious cause (1). After the age of 18 yr, the risk for cardiovascular disease rises in parallel with BP 115/75 mmhg (2). Numerous theories have been advanced to explain the pathogenesis of essential hypertension, but for more than two decades, Brenner et al. (3) have argued that it may be the consequence of suboptimal nephron endowment. Indeed, normal human nephron number ranges widely from 300,000 to 1 million per kidney (4). Although this was once dismissed as a benign reflection of human diversity, Keller et al. (5) reported that patients with essential hypertension have 46% fewer nephrons than normotensive age-matched control subjects. Therefore, individuals who are born into the lower quartile of the nephron distribution may constitute a large subgroup that is destined to develop essential hypertension later in life (5). Little is known about how final nephron number is set. Individual nephrons are produced during fetal development through reciprocal interactions between the arborizing ureteric bud and the metanephric mesenchyme. Mature nephrons are connected, like apples, to the branches of a tree-like collecting system. Because nephrogenesis ends by 36 wk of gestation, the number of nephrons formed by that time will constitute the individual s nephron endowment for life (6). Environmental factors, such as retinol, can affect branching morphogenesis in Received October 11, Accepted March 26, Published online ahead of print. Publication date available at Address correspondence to: Dr. Paul Goodyer, 4060 Saint Catherine West, PT- 413, Montreal, Quebec, Canada H3Z 2Z3. Phone: ; Fax: ; experimental animals (7), but maternal vitamin A deficiency is uncommon in Western countries, where genetic factors are presumably the primary determinants of congenital nephron number (8). In 1995, Sanyanusin et al. reported that the renal-coloboma syndrome, an autosomal dominant form of renal hypoplasia, is caused by heterozygous mutations of the gene that encodes a developmental transcription factor, PAX2 (9 11). In this syndrome, nephron structure is relatively intact (except for compensatory glomerular hypertrophy), but absolute nephron number is strikingly reduced and patients were given the diagnosis oligomeganephronia in cases in which the ocular defect was not obvious (12). Studies of the 1Neu mouse strain, which bears a spontaneous Pax2 gene mutation, have shown that the nephron deficit is caused by a loss of the antiapoptotic effects of Pax2 during kidney development; heightened levels of programmed cell death in the ureteric bud compromise its arborization (10,13 16). A congenital nephron deficit has also been reported in heterozygous Gdnf mutant mice; these mice have a 30% decrease in nephron number and develop hypertension in adulthood (17). It is interesting that the Gdnf gene is regulated by Pax2 (18). Because Pax2 haploinsufficiency causes a severe congenital nephron deficit, we hypothesized that mild, common polymorphisms in the PAX2 gene might account for subtler reductions in nephron number that are seen among normal individuals. We used the HapMap project data (19) to identify common haplotype-tagging single-nucleotide polymorphisms (htsnp) in the PAX2 gene and examined their association with congenital kidney volume in a cohort of healthy white newborns in Montreal. Copyright 2007 by the American Society of Nephrology ISSN: /

2 1916 Journal of the American Society of Nephrology J Am Soc Nephrol 18: , 2007 Materials and Methods Study Subjects Healthy white infants who were born to women with uncomplicated pregnancies were recruited with informed parental consent at the final prenatal clinical visit (n 168) to the Royal Victoria Hospital (Montreal, QU, Canada). The study (PED ) was approved by the Montreal Children s Hospital Research Ethics Board. Mothers with twins, diabetes, intrauterine growth restriction, genetic abnormalities, renal malformations or hydronephrosis, or delivery 36 wk and who had newborns with low serum albumin were excluded. In a separate obstetric cohort from the same hospital, we previously found that vitamin A deficiency occurs in 5% of Montreal pregnancies (unpublished data). Newborns with low birth weight ( 2500 g) or combined kidney volume 2.5th percentile for published norms in healthy newborns (20) were excluded. Cord Blood Analyses At birth, cord blood was obtained for isolation of leukocyte DNA (in EDTA) and serum cystatin C determination. Genomic DNA was isolated with the FlexiGene DNA kit (Qiagen, Mississauga, ON, Canada) according to the manufacturer s protocol. DNA was quantified using the Quant-iT PicoGreen dsdna Assay Kit *2000 (Invitrogen, Carlsbad, CA). Cystatin C was measured by nephelometry (21) in serum that was separated from 5 ml of cord blood and stored at 70 C before assay. Renal Volume Left and right kidney volumes were measured by ultrasonography in newborns during the first 3 d of life with the following equation: [volume 4/3 ï (length/2)(height/2)(width/2)] (22). Body surface area (BSA) was estimated from newborn length and weight according to Mosteller (23). Choice of htsnp in the PAX2 Gene Region htsnp were chosen from a region that spanned the PAX2 gene (80 kb) plus 10 kb at both the 5 and 3 flanking segments (total chromosome location Chr10:102,160, ,260,517), using the HapMap human genome database ( NCBI_35, dbsnp_b135). All known SNP (n 248) from the Centre d Etude du Polymorphisme Humain (CEPH) population (white individuals from Western Europe who settled in Utah) for the PAX2 gene region were downloaded into Haploview (version 3.32) (24), and a plot of linkage disequilibrium (LD) between SNP was obtained. Using Haploview s pairwise Tagger program (25), we chose 23 htsnp (r 2 0.8) that occurred in at least 5% of the population, which spanned the entire gene plus 10 kb of the flanking DNA to either side. PAX2 htsnp Genotypes For each subject, 15 ng of genomic DNA was used for multiplex genotyping, using Sequenom iplex PCR technology (Sequenom, San Diego CA). This system involves extension of the PCR amplicon with modified nucleotides to distinguish SNP alleles by matrix-assisted laser desorption ionization time of flight technology. Primers for SNP detection were designed using MassARRAY AssayDesign software (Sequenom). Multiplex PCR conditions are outlined in Supplement 1. Statistical Analyses Data were analyzed with SPSS for Windows (version 11.0; SPSS, Chicago, IL). The distribution of each clinical end point was tested for skewness. Genotype frequencies for each htsnp were examined for divergence from Hardy-Weinberg equilibrium. A total of 168 subjects were genotyped for 23 htsnp. In preliminary analyses, the relationship between mean total renal volume and BSA or cord cystatin C was examined by linear regression. Association between htsnp genotype and each outcome variable was assessed by one-way ANOVA with post hoc t test (independent samples). Among the 168 subjects, it was possible to assign PAX2 haplotypes for a subset of 161, using the three tightly linked htsnp. Association between PAX2 haplotypes and total renal volume normalized for BSA was assessed by ANOVA followed by post hoc t test as described. Allele-Specific PAX2 Expression Assay From a panel of human renal cell carcinoma cell lines, we selected the A498 line that was heterozygous for a PAX2 exonic SNP (rs ) that was tightly linked (D 0.827, r ) to the htsnp (rs , rs , and rs ) and used to establish the PAX2 haplotypes. The cells were grown in minimal essential medium supplemented with calf serum (5%)/FBS (5%) and penicillin/streptomycin (1%) at 37 C in humidified 5% CO 2. Cells were trypsinized, pelleted, and washed for genomic DNA isolation using the Wizard Genomic DNA Purification Kit (Promega, Madison, WI). DNA was amplified by PCR using specific primers that flanked the rs SNP that spanned exons 8 and 9 as in Supplement 2. Total cellular RNA was isolated using Qiagen RNeasy Mini Column extraction kit (Qiagen), reverse-transcribed, and amplified in a one-step reverse transcription PCR (RT-PCR) reaction using the Qiagen OneStep RT-PCR kit as in Supplement 2. Relative levels of mrna expression from each allele were estimated by quantitative HPLC sequencing (Genome Quebec, Montreal, QC, Canada) of the RT-PCR amplicon as described by Pastinen et al. (26). PCR amplicons (six replicates) and RT-PCR amplicons (five replicates) were sequenced in duplicate. Results Characteristics of Study Subjects Characteristics of the newborn cohort (n 168) are shown in Table 1. Mean gestational age was 39.6 wk (37.1 to 42.2 wk), and approximately half of the study subjects were female (49.1%). Mean ( SD) newborn weight, height, and BSA were kg, cm, and m 2, respectively. Unadjusted left, right, and total kidney volumes were , , and ml, respectively. These values are similar to those of a large cohort of Danish newborns who were studied within the first5doflife (20). Total (left right) kidney volume corrected for BSA was ml/m 2. The mean level of cystatin C in newborn cord serum was mg/l. The distribution of these characteristics in our cohort approached normality (skewness 2 for all parameters). As seen in Figure 1A, total renal volume was correlated with BSA (r , P 0.001). Kidney volume was inversely correlated with cord cystatin C (r , P 0.001; Figure 1B). Frequencies of PAX2 Variants in Study Subjects and LD between htsnp To characterize the common variants of the human PAX2 gene, we chose 23 htsnp that captured 93% of alleles in the CEPH population and spanned the PAX2 gene plus 10 kb of DNA on either side. All subjects were genotyped at these loci; SNP frequencies were similar to those of the CEPH population, indicating no appreciable genetic drift in our cohort. Genotype distributions for each htsnp locus conformed to the expected Hardy-Weinberg

3 J Am Soc Nephrol 18: , 2007 Variant in PAX2 Gene and Reduced Newborn Kidney Size 1917 Table 1. Characteristics of study subjects a Characteristic n Value Minimum Maximum Gender (% female) Gestational age (wk) Weight (kg) Length (cm) BSA (m 2 ) Left kidney volume (ml) Right kidney volume (ml) Total kidney volume (ml) Total kidney volume/bsa (ml/m 2 ) Cord cystatin C (mg/l) a Data are means SD except where indicated. The gender, mean gestational age, weight, length, body surface area (BSA), and left and right kidney volume measurements for the cohort of white newborns (n 168) are presented. Total kidney volume (left right), total kidney volume normalized for BSA, and mean serum cystatin C measurements are also shown. Total kidney volume/bsa in our newborn cohort ranged approximately three-fold from 77.6 to ml/m 2. equilibrium. The degree of LD between htsnp in our cohort (Haploview version 3.32) is shown in Figure 2. Association between htsnp and Renal Volume/BSA Five htsnp variants were individually associated (P 0.05) with low total kidney volume/bsa. Four of these were clustered in a region that spanned intron 3 to intron 5 of the PAX2 gene. Three of the four (rs , rs , and rs ) were relatively common (major allele frequency approximately 0.70) and in tight LD with each other (D 0.911, r ) both in our cohort and in the CEPH population. These htsnp were used to construct PAX2 haplotypes for 161 of 168 subjects (Figure 3A); in seven subjects, haplotypes could not be assigned confidently. A total of 28% of the 161 subjects were heterozygous for the AAA haplotype; an additional 5% of infants were homozygous for this allele (AAA/ AAA). Total renal volume/bsa ( ml/m 2 ) among infants with at least one PAX2 AAA haplotype (32.5% of the cohort) was 10% lower than that ( ml/m 2 ) of subjects who were homozygous for the more common GGG/GGG haplotype (63% of the cohort; P 0.004). Figure 1. Correlation between total kidney volume and body surface are (BSA) or cord cystatin C. (A) Newborn total kidney volume is significantly correlated with BSA (n 168; Pearson r , P 0.001). (B) Newborn total kidney volume is inversely correlated with cord serum cystatin C (n 168; Pearson r , P 0.001). PAX2 Allele Expression Imbalance To determine whether PAX2 haplotype influences the level of mrna expression, we screened human renal cell carcinoma cell lines for heterozygosity at an exon 8 SNP (rs ) in tight LD (D 0.827, r ) with all three htsnp used to construct the PAX2 haplotype (HapMap). One cell line (A498) was shown to be heterozygous for both the exonic SNP and the three htsnp that were used to construct the PAX2 haplotype. Allele-specific PAX2 expression was examined in this cell line by amplifying an approximately 250-bp fragment of genomic DNA or the corresponding reverse-transcribed cdna that contained the rs locus (Figure 4). The estimated level of transcript that was derived from the more common rs (a) allele (70% of the CEPH population) was 65.3% of the total, whereas the transcript from the less common

4 1918 Journal of the American Society of Nephrology J Am Soc Nephrol 18: , 2007 Figure 2. Linkage disequilibrium (LD) between PAX2 haplotype-tagging single-nucleotide polymorphisms (htsnp) in the study cohort. The PAX2 gene region on chromosome 10 (Chr10:102,160, ,260,517) is shown. Twenty-three htsnp were chosen to span the region. The exon 8 SNP (rs ) that was used for allelic imbalance studies is also indicated (in green). The extent (D ) of LD between htsnp in our cohort is indicated inside each square as the value of pairwise D measure (D 1 when unspecified), according to HapMap convention. Five htsnp were associated with total kidney volume/bsa and are highlighted in gray. Minor allele frequencies for three of the five htsnp were relatively common (18.8 to 19.5% of the population) and were in very tight LD ( 95%); these were used to assign haplotypes for each subject. rs (c) allele (30% of the CEPH population) contributed only 34.5% of the total (P 0.001). Discussion Nephron number seems to correlate fairly well with renal size at birth, because placental clearance of fetal solutes buffers suboptimal renal function in the antenatal period. In a limited study of spontaneously aborted normal human fetuses, Hinchcliffe et al. (6) demonstrated a tight correlation between renal volume and glomerular number up to 40 wk of gestation Other small studies of humans who were autopsied in the perinatal period consistently showed an association between renal size and glomerular number (27 29). Similarly, in heterozygous Pax2 1Neu mice, kidney mass and nephron number were reduced in parallel (60 and 65%, respectively, in comparison with wildtype littermates) when measurements were made at 1 wk of age (15). We recently showed that newborn kidney volume correlates with serum creatinine at 1 mo of age (30), but long-term prospective studies of congenital nephron deficit have been difficult to perform because compensatory hypertrophy continues throughout the first few months of life, erasing the initial correlation between renal size and nephron number. Keller et al. (5) documented a 46% reduction in nephron number among subjects with essential hypertension by extensive glomerular counts in autopsy specimens, but glomerular volume was double that of age-matched control subjects and renal mass was equivalent in the two groups. Similarly, adult heterozygous Pax2 1Neu mice show glomerular hypertrophy, resulting in kidney mass equivalent to wild-type littermates by 1 yr of age (15). In this study, we used total kidney volume at birth (measured by ultrasonography) as a surrogate for congenital nephron number. Total newborn kidney volume (left right kidney volume) correlated inversely with umbilical cord cystatin C level, a low molecular weight protein that is used as a

5 J Am Soc Nephrol 18: , 2007 Variant in PAX2 Gene and Reduced Newborn Kidney Size 1919 Figure 3. Effect of PAX2 haplotype on newborn renal volume. (A) PAX2 haplotypes were constructed from three htsnp for each subject (n 168). A total of 63% of newborns were homozygous for the GGG haplotype. A total of 28% of newborns were heterozygous (GGG/AAA), and 5% were homozygous for the less common AAA haplotype. In seven (4%) individuals, a haplotype could not be confidently assigned. Newborns with one or more AAA alleles had lower total kidney volume/bsa ( ml/m 2 ) compared with GGG/GGG homozygotes ( ml/m 2 ;*P 0.004). Newborns with an AAA allele had slightly higher cord cystatin C (1.94 mg/l) compared with the GGG homozygotes (1.90 mg/l), but this difference was NS (P 0.05). (B) The effect of PAX2 haplotype on kidney volume/bsa percentile in our newborn cohort is compared with the effect of BP status on nephron number percentile in the normal adult population. Data from Keller et al. (5) on nephron number in adults with essential hypertension (EHT) versus normotensive control subjects (NT) are plotted according to the nephron number distribution reported by Nyengaard et al. (4). Nephron number percentile in relationship to the overall population mean ( ) were calculated from Keller s data, assuming that 25% of the normal population has essential hypertension (1). Accordingly, the presence of a PAX2 AAA haplotype decreases total kidney volume/bsa by 10% (versus GGG homozygotes), whereas the presence of EHT decreases nephron number by 46% (versus normotensive control subjects). marker of GFR in infants (31) and that does not seem to cross the placental barrier (32). To eliminate the confounding effect of body size (BSA) on newborn kidney volume, we tested for htsnp association with total kidney volume/bsa. Like other complex traits (33,34), congenital nephron number is normally distributed in human populations (4) and is likely to have multiple genetic determinants. We focused on the PAX2 gene as a candidate determinant of human nephron number because, in the autosomal dominant renal-coloboma syndrome, there is no compensation for the loss of one PAX2 allele (35) and PAX2 protein levels are reduced to approximately 50% of normal, in accordance with gene dosage. We identified a cluster of htsnp that defined a relatively common Figure 4. Allele-specific expression of PAX2. Allele-specific PAX2 expression in the human A498 renal cell carcinoma cell line was quantified by sequencing an approximately 250-bp amplicon that contained the exon 8 rs1800 (898) SNP from genomic DNA or the corresponding reverse-transcribed cdna according to the method of Pastinen et al. (26). The transcript level of the more common rs (a) allele was 65.3% of total PAX2 mrna, whereas the transcript from the less common rs (c) allele contributed only 34.5% of total PAX2 mrna (P 0.001). (18.5% of the population) PAX2 haplotype (AAA) associated with a 10% reduction in mean newborn total kidney volume/ BSA compared with the most common haplotype (GGG/GGG). The AAA haplotype was widely distributed in our population; combined renal volume in infants with an AAA haplotype ranged from to versus to ml in infants who were homozygous for the more common GGG haplotype. Therefore, the association between AAA haplotype and renal volume cannot be attributed to a few infants with very small kidneys. The observed decrease in renal volume associated with the PAX2 AAA haplotype is approximately one fifth of the 46% decrease in nephron number that was observed in individuals with essential hypertension (5) (Figure 3B). Therefore, the PAX2 AAA haplotype is presumably only one of several variables in a multifactorial model that accounts for low nephron number and seems to shift mean renal volume downward across the whole population. No common polymorphisms affect the amino acid sequence of human PAX2. However, our allele-specific expression studies in a human renal cell carcinoma cell line showed that an allele that bears a PAX2 exonic SNP that is tightly linked to the AAA haplotype generates only one third of the total PAX2 transcript (versus two thirds from the more common allele). Because this method compares expression of one allele versus the other within the same cell, the allelic expression ratio is independent of any unique features of the cell line. In a recent study, similar analyses of allele-specific expression showed that the majority of genes from the CEPH population exhibit allelic balance (26); individual measurements of allele-specific transcripts varied by no more than 10%, 95% of the time. Using independent replicate measurements (n 5), our finding of

6 1920 Journal of the American Society of Nephrology J Am Soc Nephrol 18: , 2007 allelic imbalance (65% GGG versus 35% AAA) cannot be attributed to variability in the assay. We hypothesize that the AAA haplotype identifies a fairly common ancestral allele that bears an intronic mutation that reduces the level of PAX2 expression during fetal kidney development. Reduced PAX2 expression from the AAA allele presumably then compromises branching of the ureteric bud and reduces congenital nephron number by 10%. Our study provides the first evidence of a common gene variant that contributes to subtle renal hypoplasia in normal newborns and, possibly, to the risk for essential hypertension or susceptibility to renal injury later in life. Acknowledgments This study was supported by an operating grant from the Canadian Institutes of Health Research (MOP 12954) and by a Pilot Project Grant from the McGill University Health Centre Research Institute. P.G. is the recipient of a James McGill (Canadian Institutes of Health Research) Research Chair. Disclosures None. References 1. Fields LE, Burt VL, Cutler JA, Hughes J, Roccella EJ, Sorlie P: The burden of adult hypertension in the United States 1999 to 2000: A rising tide. Hypertension 44: , Lewington S, Clarke R, Qizilbash N, Peto R, Collins R: Age-specific relevance of usual blood pressure to vascular mortality: A meta-analysis of individual data for one million adults in 61 prospective studies. Lancet 360: , Brenner BM, Garcia DL, Anderson S: Glomeruli and blood pressure. Less of one, more the other? Am J Hypertens 1: , Nyengaard JR, Bendtsen TF: Glomerular number and size in relation to age, kidney weight, and body surface in normal man. Anat Rec 232: , Keller G, Zimmer G, Mall G, Ritz E, Amann K: Nephron number in patients with primary hypertension. 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Hum Mol Genet 4: , Porteous S, Torban E, Cho NP, Cunliffe H, Chua L, McNoe L, Ward T, Souza C, Gus P, Giugliani R, Sato T, Yun K, Favor J, Sicotte M, Goodyer P, Eccles M: Primary renal hypoplasia in humans and mice with PAX2 mutations: Evidence of increased apoptosis in fetal kidneys of Pax2(1Neu) /- mutant mice. Hum Mol Genet 9: 1 11, Eccles MR, Schimmenti LA: Renal-coloboma syndrome: A multi-system developmental disorder caused by PAX2 mutations. Clin Genet 56: 1 9, Salomon R, Tellier AL, Attie-Bitach T, Amiel J, Vekemans M, Lyonnet S, Dureau P, Niaudet P, Gubler MC, Broyer M: PAX2 mutations in oligomeganephronia. Kidney Int 59: , Torban E, Eccles MR, Favor J, Goodyer PR: PAX2 suppresses apoptosis in renal collecting duct cells. Am J Pathol 157: , Dziarmaga A, Quinlan J, Goodyer P: Renal hypoplasia: Lessons from Pax2. 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7 J Am Soc Nephrol 18: , 2007 Variant in PAX2 Gene and Reduced Newborn Kidney Size 1921 neys: The relationship to birth weight. Kidney Int 63: , Manalich R, Reyes L, Herrera M, Melendi C, Fundora I: Relationship between weight at birth and the number and size of renal glomeruli in humans: A histomorphometric study. Kidney Int 58: , Goodyer P, Kurpad A, Rekha S, Muthayya S, Dwarkanath P, Iyengar A, Philip B, Mhaskar A, Benjamin A, Maharaj S, Laforte D, Raju C, Phadke K: Effects of maternal vitamin A status on kidney development: A pilot study. Pediatr Nephrol 22: , Filler G, Bokenkamp A, Hofmann W, Le Bricon T, Martinez-Bru C, Grubb A: Cystatin C as a marker of GFR: History, indications, and future research. Clin Biochem 38: 1 8, Cataldi L, Mussap M, Bertelli L, Ruzzante N, Fanos V, Plebani M: Cystatin C in healthy women at term pregnancy and in their infant newborns: Relationship between maternal and neonatal serum levels and reference values. Am J Perinatol 16: , Austin MA, Talmud PJ, Farin FM, Nickerson DA, Edwards KL, Leonetti D, McNeely MJ, Viernes HM, Humphries SE, Fujimoto WY: Association of apolipoprotein A5 variants with LDL particle size and triglyceride in Japanese Americans. Biochim Biophys Acta 1688: 1 9, Kitamura Y, Okumura K, Imamura A, Mizuno T, Tsuzuki M, Numaguchi Y, Matsui H, Murohara T: Association of plasminogen activator inhibitor-1 4G/5G gene polymorphism with variations in the LDL particle size in healthy Japanese men. Clin Chim Acta 347: , Eccles MR, Wallis LJ, Fidler AE, Spurr NK, Goodfellow PJ, Reeve AE: Expression of the PAX2 gene in human fetal kidney and Wilms tumor. Cell Growth Differ 3: , 1992 Supplemental information for this article is available online at