Primary, Nonsyndromic Vesicoureteric Reflux and Nephropathy in Sibling Pairs: a United Kingdom Cohort for a DNA Bank

CJASN ePress. Published on March 24, 2011 as doi: 10.2215/CJN.04580510 Article

Primary, Nonsyndromic Vesicoureteric Reflux and Nephropathy in Sibling Pairs: A United Kingdom Cohort for a DNA Bank

Heather J. Lambert,*† Aisling Stewart,* Ambrose M. Gullett,‡ Heather J. Cordell,* Sue Malcolm,‡ Sally A. Feather,§ Judith A. Goodship,* Timothy H. J. Goodship,* and Adrian S. Woolf, on behalf of the UK VUR Study Group

*Newcastle University, Summary Newcastle upon Tyne, Background and objectives Primary vesicoureteric reflux (VUR) can coexist with reflux nephropathy (RN) United Kingdom; †The and impaired renal function. VUR appears to be an inherited condition and is reported in approximately Great North Children’s one third of siblings of index cases. The objective was to establish a DNA collection and clinical database Hospital, Newcastle upon Tyne, United from U.K. families containing affected sibling pairs for future VUR genetics studies. The cohort’s clinical Kingdom; ‡UCL characteristics have been described. Institute of Child Health, London, United § Design, setting, participants, & measurements Most patients were identified from tertiary pediatric nephrol- Kingdom; St James’ University Hospital, ogy centers; each family had an index case with cystography-proven primary, nonsyndromic VUR. Affected Leeds, United Kingdom; siblings had radiologically proven VUR and/or radiographically proven RN. and ʈUniversity of Manchester and Results One hundred eighty-nine index cases identified families with an additional 218 affected siblings. Manchester Children’s Ͻ Hospital, Manchester, More than 90% were 20 years at the study’s end. Blood was collected and leukocyte DNA extracted from United Kingdom all 407 patients and from 189 mothers and 183 fathers. Clinical presentation was established in 122; 92 had urinary tract infections and 16 had abnormal antenatal renal scans. RN was radiologically proven in 223 Correspondence: Dr. patients. Four patients had been transplanted; none were on dialysis. In 174 others aged Ͼ1 year, estimated Heather J. Lambert, GFR (eGFR) was calculated. Five had eGFR 15 to 59 and 48 had eGFR 60 to 89 ml/min per 1.73 m2. Values Department of Paediatric were lower in bilateral RN patients than in those with either unilateral or absent RN. Nephrology, Royal Victoria Infirmary, Newcastle NE1 4LP, Conclusions The large DNA collection from families with VUR and associated RN constitutes a resource for UK. Phone: 44-0- researchers exploring the most likely complex, genetic components predisposing to VUR and RN. 1912336161; Fax: Clin J Am Soc Nephrol 6: 760–766, 2011. doi: 10.2215/CJN.04580510 44-0-1912820077 E-mail heather.lambert@ ncl.ac.uk

Introduction receiving kidney transplants (6). These may be under- Vesicoureteric reflux (VUR) describes urine flowing estimations because some RN patients are probably into the upper urinary tract. It can be secondary to hidden in diagnostic categories including “tubulo- bladder outflow obstruction (1) and can occur in sev- interstitial disease” and “unknown” (7). RN occurs in eral multiorgan congenital disorders (2). Usually, 5% of children investigated after the first UTI, allow- VUR is primary and nonsyndromic, this article’s ing estimation that around 0.5% of girls aged 0 to 16 topic. VUR occurs in 25 to 40% of children presenting years have RN (8). As addressed below, hypertension with urinary tract infection (UTI) and in 3 to 19% of and proteinuria sometimes accompany RN. infants with hydronephrosis on antenatal ultrasound Perceptions that UTI with VUR leads to RN via scan (USS) screening (3). VUR’s true prevalence re- ascending infection stimulated an era of “active-treat- mains uncertain because early diagnosis requires in- ment” of VUR (9). Certainly, RN can follow UTI, vasive radiology and most VUR spontaneously re- especially without prompt antibiotic treatment solves. It may be as low as 1 to 2% but may be higher (10,11). Prospective studies comparing antireflux sur- (3). Although VUR can exist in isolation, Hodson and gery with antibiotic prophylaxis fail to show signifi- Edwards (4) associated VUR with renal segmental cant differences in acquisition of kidney defects (12– parenchymal thinning and calyceal clubbing. This 14) or progression to ESRF (15). However, not all “chronic pyelonephritis” was subsequently called chronic radiologic kidney defects after UTIs are asso- “reflux nephropathy” (RN). In the United Kingdom ciated with VUR (16) and not all RN is pyelonephritic (U.K.), RN respectively accounts for 8 and 12% of because some individuals born with VUR have mal- end-stage renal failure (ESRF) in children and adults formed kidneys (17,18). Accordingly, the rationale for (5), and RN exists in 5% of North American children active treatment of VUR has been questioned (9) and

prospective trials have been initiated in children with VUR and/or an abnormal split kidney function over 60/40%. A and UTI, including a “watchful waiting” arm (19–22). normal DMSA scan excluded RN but a normal USS was Brandstrom et al. (20,21) reported that either prophylaxis or inconclusive because this modality is less sensitive at de- endoscopic VUR correction reduced risks of febrile UTIs tecting RN (36). Each family also contained one or more and acquisition of renal defects and Craig et al. (22) pro- “affected siblings” with VUR and/or RN. A minority of vided evidence for efficacy of antibiotic prophylaxis in affected siblings had RN but not VUR. These tended to be reducing UTI. older siblings and we believe that they would have had Primary VUR can occur in the renal coloboma syn- been shown to have VUR if investigated when younger drome, caused by mutations of PAX2 (23) encoding a because VUR usually spontaneously resolves by the sec- transcription factor expressed in developing renal tracts ond decade (37). For each index case and affected siblings, (24). Although PAX2 mutations have not been found in verification of VUR and RN status was essential and es- nonsyndromic VUR (25), defects in other genes might be tablished from clinical records and review of original im- relevant. Indeed, numerous genes control ureteric morpho- aging pictures and reports. genesis and functional maturation of the bladder and ure- For the index cases and affected siblings, medical re- ter (2,3,26). There is a high concordance of VUR in identical cords were interrogated for information about BP, plasma twins (27) and kindreds with multiple affected members creatinine, and proteinuria. “Hypertension” was present with apparent dominant (28,29) or recessive (30) inheri- either when an individual was on hypotensive medication tance exist. When first-degree relatives of index cases are and/or had a raised BP; when height data were unavail- screened by cystography, around one third of siblings (31) able, age-related BP percentiles for children of 50th percen- and up to two thirds of offspring (32,33) have VUR. Thus, tile height were used (38). For some patients, plasma cre- although yet-to-be defined environmental factors might atinine data existed, allowing calculation of estimated GFR predispose to primary VUR, genetics must be important. (eGFRs). The most recently available creatinine result was The definition of genetic factors that contribute to VUR and used (providing there was no contemporaneous UTI) in RN will illuminate mechanisms of normal human renal the Schwartz formula (39) for those over 1 but under 18 tract development. Information from such studies may also years old and the Modification of Diet in Renal Disease have clinical utility by giving families reasons why a mal- (MDRD) formula (40) for the 13 older patients. We defined formation has occurred and may also allow the prediction “proteinuria” as Նϩϩ on dipstick testing. GFRs were not of VUR status in relatives of index cases. As a step toward calculated in patients under 1 year old because of the these ends, we established a DNA collection and clinical known maturation of filtration rate in infancy, which can database from U.K. families containing affected sibling occur even with structurally abnormal kidneys (41) and the pairs. This bank, which we have called “The UK VUR limitations of available formulae. DNA collection” (34), could then be used as a resource Normality of the data for age and eGFR was tested using with which researchers can begin to unravel the perhaps the Kolmogorov-Smirnov test. Comparisons between complex genetics underlying VUR. We here present the groups for age were undertaken using Kruskal-Wallis and detailed clinical description of the patient cohort. Mann-Whitney tests. Comparisons between groups for eGFR were undertaken using ANOVA and pairwise com- parison. Materials and Methods Clinical details of parents and other family members After approval by the South West Research Ethics were sought but only 21 mothers and 10 fathers had un- Committee, informed consent/assent was obtained. All dergone traceable renal investigations. U.K. tertiary pediatric nephrology centers were ap- proached initially through the British Association for Pediatric Nephrology, and subsequently by individual Results contact, letters, and visits from the researchers to those Overview and some regional pediatric centers. Information sheets Index cases were indentified from 189 overtly unrelated and a Web site link (http://www.vur.org.uk/) were publi- families and there were 218 affected siblings, giving a total cized to encourage family involvement and inform of 407 patients (223 female and 184 male), of which 96% Study Group members. Families were recruited from were white. Over 90% of them were under 20 years old at January 2003 to July 2006. Databases in nephrology and the end of the study period (Figure 1). VUR was proven in radiology departments were searched for validating re- 363 (89%) of the 407 patients, the others (i.e., 44 of the ports. Secondary or syndromic VUR was excluded. For affected siblings) having radiologic evidence of RN only. In this collection, no new patients were identified nor were 161 families there was 1 affected sibling, 25 families had 2 additional tests undertaken, other than collection of affected siblings, and 2 families had 3 affected siblings. blood for leukocyte DNA (35). Thus, most families contained one affected sibling pair. In Each family comprised an “index case” with VUR of any total, there were 250 affected sibling pairs: 55 male/male grade diagnosed by micturating cystourethrography or pairs; 111 male/female pairs; 84 female/female pairs. Leu- radionucleotide cystography. Some index cases also had kocyte DNA samples were collected from all 407 patients RN. RN was defined as an abnormality on most recent and from 189 mothers and 183 fathers. Clinical presenta- imaging, predominantly using renogaphy with 99mTc-di- tion was established in 122 (65%) of the 189 families; 92 mercaptosuccinic acid (DMSA). In a minority, RN was (75%) had UTI and 16 (13%) had abnormal antenatal renal diagnosed using USS or intravenous urography. A diag- scans. The other 12% comprised a variety of presentations nosis of RN was accepted by finding focal or global defects including renal impairment and hypertension. Duplex kid- 762 Clinical Journal of the American Society of Nephrology

BP and Proteinuria

Systolic BP data were available in 351 of 407 patients. Four had undergone renal transplantation, and each re-

ceived antihypertensives. Excluding these, 4 of the 351 had

bilateral RN and been treated for hypertension, one by unilateral nephrectomy and the others with hypotensive

medications only. An additional 10 had multiple BP read- Ͼ ings of 95th percentile and, of these, 4 had bilateral RN, 2 had unilateral RN, and 4 had normal DMSA scans. Four

others had a single BP recording of Ͼ95th percentile, two of them having unilateral RN and two with normal renal scans. Eight other patients had BP recordings including one Ն95th percentile but others in the normal range. Ex- Figure 1. | Age (in years) distribution of 407 individuals. cluding the four post-transplant patients, there was information on urine analysis on 302 individuals. Eight had ney was noted in three index cases (bilateral in one) and in proteinuria of ϩϩ or more on dipstick testing. Six of these six affected siblings (bilateral in two). Other anomalies had RN, one had a normal DMSA scan, and one lacked noted were one case each of vesicoureteric junction ob- appropriate imaging to assess RN status. struction, megaureter, and pelvic position of kidney.

Nephropathy Renal Excretory Function Of the 407 patients, radiologically proven RN was pres- eGFR was calculated in 174 patients over a year old and ent in 223 (55%), being bilateral in 61 (15% of the 407). In these, together with the four transplanted cases, are shown 122 (30%) of the 407 patients, appropriate imaging had in Table 2. eGFR was normally distributed and data were been undertaken, permitting the conclusion that they did expressed as mean Ϯ SD. eGFR was normal in 121 patients. not have RN, whereas for the other 62 (15% of the 407) Even after excluding the four patients with ESRF who had patients, definitive imaging to assign a RN status did not been transplanted (who inevitably would have had bilat- exist. Of the 189 index cases (all, by definition, with proven eral RN in their native kidneys), there was a significant VUR), 120 (63%) had RN, 60 (32%) did not have RN, and in difference in eGFR (ANOVA; P Ͻ 0.001) between the three 9 the RN status could not be established. Of the 218 af- subsets: with no RN, with unilateral RN, and with bilateral fected siblings, 174 had evidence of VUR; 74 of these also RN (Figure 2). Pairwise analysis using Fisher 95% confi- had RN, 62 had no RN (normal DMSA), and 38 had insuf- dence intervals showed that eGFR in the bilateral RN ficient information to assign RN status. The remaining 44 group (n ϭ 48: mean Ϯ SD, 92.2 Ϯ 27.8 ml/min per 1.73 siblings only had evidence of RN; 8 had a negative mic- m2) was significantly lower than those in unilateral RN turating cysto-urethrogram (MCUG) and 36 had not had a (n ϭ 88: 104.8 Ϯ 22.1) and the group without RN (n ϭ 38: test for VUR (Table 1). 112.8 Ϯ 21.5). There was no significant difference, how- In 40% of all (250) sibling pairs in the cohort, both ever, between the last two groups. Notably, there was also individuals had RN. After a UTI, some DMSA defects a significant difference (Kruskal-Wallis test; P Ͻ 0.001) represent acute pyelonephritis and these may resolve over between the ages of the three groups (Figure 3). Age was 6 months (42). Accordingly, we noted that 165 patients not normally distributed and was therefore expressed as with RN assigned on the basis of an abnormal first DMSA median (range). Mann-Whitney test showed that the bilat- scan had a subsequent scan Ͼ6 months later. Overwhelm- eral RN group (median 12.3 years, range 1 to 33) was ingly, in 92%, renography was unchanged (136 patients) or significantly older than the unilateral RN (median 6.4 had deteriorated (16 patients). In an additional five pa- years, range 1 to 23) and the group without RN (median tients, there was both improvement and deterioration in 3.7, range 1 to 20 years) (P Ͻ 0.001 for both comparisons). DMSA appearance and in an additional eight patients There was also a significant difference (P Ͻ 0.05) in age there was improvement, with complete resolution in three. between the last two groups.

Table 1. Radiologically proven VUR and RN status of the 407 individuals who together comprise summary of cases and phenotypes “index cases” and “affected siblings”

Vesicoureteric Reflux and Reflux Nephropathy Status of Index Cases and Affected Siblings

VUR, VUR, RN, VUR, RN, VUR, RN, VUR, RN, VUR, RN, ϩ ϩ ϩ ϩ Ϫ ϩ ? Ϫ ϩ ? ϩ

Index cases 189 189 120 60900 Affected siblings 218 174 74 62 38 8 36 Individuals 407

Key: ϩ, present; Ϫ, absent; ?, uncertain. Clin J Am Soc Nephrol 6: 760–766, April, 2011 The UK VUR DNA Bank, Lambert et al. 763

Table 2. Chronic kidney disease status of patients over 1 year of age

Bilateral RN Unilateral RN No RN (n ϭ 52) (n ϭ 88) (n ϭ 38)

GFR Ն90 ml/min per 1.73 m2 (stage 1 CKD) 23 65 33 GFR 60–89 ml/min per 1.73 m2 (stage 2 CKD) 21 22 5 GFR 15–59 ml/min per 1.73 m2 (stage 3/4 CKD) 41 ESRF (transplanted; stage 5 CKD) 4

CKD, chronic kidney disease.

affected individuals (35,44). Studies using genetic association (35,45–47) and candidate gene sequencing (45,48) strategies are also being undertaken. The DNA Bank from the current cohort represents one of the largest such collections, nearly rivaled by Irish (44) and Slovenian (35) collections. Cordell et al. (35) undertook an initial genetic study using both the U.K. and Slovenian collections. In studies of white subsets, to control for potentially con- founding racial variations in genetic background, modest evidence of linkage was found to several loci but there was no clear overlap with previous linkage studies (29,30,43,44). Indeed, major loci may not exist for this common renal tract malformation within some European pop- ulations. Furthermore, Cordell et al. (35) found no association with polymorphisms in candidate genes AGTR2, HNF1B, PAX2, RET, ROBO2, and UPK3A. Of the 407 patients in the U.K. cohort, 96% were white. In the 2001 census (http://www.statistics.gov.uk/focuson/ -Figure 2. | Mean (؎95% confidence intervals) eGFR in VUR patients ethnicity/), 8% (i.e., 4.6 of 57.5 million) of the U.K. popu with bilateral, unilateral, and no reflux nephropathy (RN). lation were non-white, of which approximately 1 million were black and 2 million were from either India, Pakistan, or Bangladesh. Thus, non-whites are apparently under- represented (␹2 test, P ϭ 0.002) in the UK VUR DNA collection versus the whole population. Perhaps this represents a putative inequality of health care access but could also be explained by a different incidence of VUR between racial groups. Indeed, when Chand et al. (49) analyzed 15,504 UTI patients, black children were less likely as white children (P Ͻ 0.0001) to have VUR. Duplex kidney was noted in nine patients in the U.K. cohort. The incidence of duplication in the general population is approximately 2% (50) and VUR and duplex kidney can coexist in an individual (51). Families have also been described in which some members can have either VUR or duplex kidney (51). The incidence of RN in the U.K. cohort is similar to some other reports (14) whereas in some series of children with VUR the incidence of RN was lower (52). Williams et al. (3) estimated that for each 6000 individuals who are born with VUR, 1600 (27%) will have their clinical course compli- Figure 3. | Box plot of age (in years) in VUR patients with bilateral, cated by UTI and 2000 (33%) will have RN, with just one unilateral, and no RN. The boundary of the box closest to zero indicates the 25th percentile, the line within the box shows the median, and the (0.02%) having ESRF. Compared with this, the current boundary of the box farthest from zero indicates the 75th percentile. patient cohort therefore appears enriched for RN, which Whiskers (error bars) above and below the box indicate the 90th and was present in 55% of all patients; furthermore, four (1%) 10th percentiles. Outlying points are shown. had ESRF. The cohort may not be representative of all cases of VUR as most referrals for entry into the collection were Discussion from specialist pediatric nephrology centers likely to have Researchers have begun to undertake linkage analyses seen the more severe end of the VUR/RN spectrum of using both families with numerous members affected by disease. Centers differ in their clinical policies on use of VUR and/or RN (29,30,43) and kindreds with two to three MCUG for early diagnosis of VUR after UTI and in sib- 764 Clinical Journal of the American Society of Nephrology

lings. Furthermore, those families with severely affected geted sequencing of the several tens of candidate VUR members may have been more likely to agree to be in- genes which already exist; seeking copy number variants cluded in the collection. (e.g., duplications and deletions) by comparative genomic Of interest, the fact that each member of 40% of all hybridization by microarray. Potential investigators are sibling pairs had RN raises the possibility of there being a now directed to “The United Kingdom Vesicoureteric Re- genetic influence on development of nephropathy as well flux (VUR) DNA Collection” Web site (http://www.vu- as VUR itself. We are unable to unravel what proportion of r.org.uk) in which instructions on how to access, and also RN in the current U.K. cohort represents congenital anom- terms and conditions of using, the collection are detailed alies versus defects acquired from pyelonephritis; indeed, it (see also Supplemental Data). is debated what (nonhistologic) criteria can be used to dis- tinguish these possibilities (10). There may be roles played by Acknowledgments environmental factors such as variable access to health care Physicians who referred families who were included in the VUR and/or delay in treatment of UTIs common to members of DNA Bank were designated as being members of the UK VUR the same family and which would result in RN. Study Group. This study was supported by grants from the Well- Our data confirm that bilateral RN is a risk factor for come Trust (066647) and the Medical Research Council decreased eGFR. However, the bilateral RN group was (G0600040). A.S.W. is supported by the Manchester NIHR Bio- older than those with either unilateral or no RN. GFR medical Research Centre. We thank the following for their general normally begins to decline after 20 years of age (53), so we support of the project: the British Association for Pediatric Ne- cannot exclude that the tendency to lower eGFR is related phrology, the UK VUR DNA Development Group, and Kidney to aging. Having said this, most patients with bilateral RN Research UK. reliminary data were presented in abstract form and and eGFR Ͻ90 ml/min per 1.73 m2 were Ͻ20 years old. and as a poster at the May 14–16, 2008 Renal Association meeting Previous studies found that children with RN entering Glasgow. studies with a GFR of Ն70 ml/min per 1.73 m2 show barely Hospitals involved in, and individual members of, the UK VUR any risk of functional deterioration up to 10 years (14,15), Study Group: Addenbrookes Hospital, Cambridge: R. Sandford; although one would remain cautious about the long-term Birmingham Children’s Hospital: S. Hulton, D. Milford, S. Ste- prognosis of these individuals (54). The number of phens, C.M. Taylor; Bristol Royal Hospital for Sick Children, younger patients with VUR but no RN in this cohort may Bristol: J. Dudley, C. Inward, M. McGraw, J. Tizard; Burnley have been influenced by changes in clinical practice such General Hospital, Burnley: J. Iqbal; Cumberland Infirmary, Carl- as the recognition of the importance of prompt treatment isle: J. Storr; Derriford Hospital, Plymouth: R. Jones; Evelina Chil- of UTI in babies and increased testing for VUR in newborn dren’s Hospital, London: G. Haycock, C. Reid, S. Rigdon; Notting- siblings of index cases. ham City Hospital: A. Watson; Gloucester Royal Hospital, In this U.K. cohort, predominantly under 20 years old, Gloucester: L. Jadresic; Great Ormond Street Hospital, London: P. the incidence of hypertension (excluding four patients who Cuckow, S. Marks, L. Rees, R. Trompeter, K. Tullus, W.Van’t Hoff, had received a renal transplant) was only 4%. Accelerated D. Wilcox, A. Woolf; James Cook University Hospital, Middles- arterial hypertension may be a presenting feature when borough: S. Sinha; Royal Victoria Infirmary, Newcastle upon RN coexists with impaired renal function (55), and pro- Tyne: M. Coulthard, H. Lambert, E. Hunter, M. Kier, N. Moghal, spective studies (13,56) show that some individuals with M. Ognanovic, S. Vernon; Leeds Teaching Hospitals NHS Trust: RN develop hypertension (56–58). Wennerstrom et al. (59), M. Fitzpatrick, S. Feather; Leicester Royal Infirmary, Leicester: P. however, reported that individuals with RN not in renal Houtman; Northampton General Hospital, Northampton: N. Grif- failure had no increased risk of hypertension versus con- fin; Queen Elizabeth the Queen Mother Hospital, Margate: E. trols without RN. Indeed, the fact that, in this study, sev- Rfidah; Royal Belfast Hospital for Sick Children: M. Savage, M. eral of the patients with VUR but without radiologic evi- O’Connor, M. Convery; Royal Hospital for Sick Children, Edin- dence of RN had hypertension shows that hypertension is burgh: S. Taheri; Royal Hospital for Sick Children, Glasgow: H. multifactorial in this reflux population. Perhaps longer Maxwell, J. Beattie; Royal Liverpool Children’s Hospital: D. term follow-up will show an increased incidence of hyper- Hughes, C. Jones, B. Judd; Royal Manchester’s Children’s Hospi- tension in this cohort and indeed several had borderline tal: M. Lewis, N. Webb, M. Bradbury, N. Plant, R. Postlethwaite; high BPs. Proteinuria is common when RN and renal in- Queen Elizabeth Hospital, Kings Lynn: J. Dossetor, A. Hughes; Salford Royal Hospital, Salford; D.J. O’Donoghue; Street Mary’s sufficiency coexist (60) and, in a retrospective adult study, Hospital, Portsmouth: J. Scanlan; University College Hospital, Lon- progressive loss of kidney excretory function positively don: D. Hodes, A. Kilby; University Hospital of North Tees, Cleve- correlated with proteinuria (54). In our cohort, only 8 of land: I. Verber; University Hospital of Wales, Cardiff: K. Verrier- 302 individuals had proteinuria of Նϩϩ; of these, 6 had Jones; Walsgrave Hospital, Coventry: N. Coad; West Cumberland bilateral RN. Quantification of urine albumin/creatinine Infirmary, Carlisle: J. Jackson; Whittington Hospital, London: M. ratios may have revealed a higher incidence of abnormal Jaswon. proteinuria. In conclusion, the current patient cohort represents a resource which investigators can use to unravel the genetic Disclosures bases of VUR and/or RN. Studies for which the UK VUR None. 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