Hirschsprung Disease and Congenital Anomalies of the Kidney ... : Medicine (original) (raw)

INTRODUCTION

Hirschsprung disease (HSCR) is a congenital anomaly of the enteric nervous system characterized by the absence of intestinal intramural ganglia (myenteric and submucosal plexuses) with variable distal bowel involvement. The disease develops as a consequence of abnormal migration/differentiation of neural crest derived neuroblasts into the developing gut.5 In 1994, mutations affecting the RET proto-oncogene were identified in HSCR patients,13,29 the same as were also involved in different diseases such as multiple endocrine neoplasia type 2A and 2B and sporadic and familial medullary thyroid carcinoma.18,21 Since then, loss-of-function of RET tyrosine-kinase has been demonstrated in approximately 50% of familial and 10%-15% of sporadic HSCR cases.18 Moreover, a common RET haplotype comprising single nucleotide polymorphisms (SNPs) from the promoter region down to intron 5 has been found to be strongly overrepresented among HSCR patients compared to controls. A variant in linkage disequilibrium with such a haplotype has been identified in a RET transcriptional enhancer of intron 1 and was found to be associated with reduced expression of the tyrosine-kinase receptor mRNA.1 Other HSCR susceptibility genes that belong to the RET signaling pathway, including glial cell line-derived neurotrophic factor (GDNF) and GDNF family receptor α1 gene (GFRα1), have been identified to date.

RET, GDNF, and GFRα1 also play an important role in the development of the human urinary tract by regulating the interaction of ureteric bud and metanephric blastema. The fusion of these embryologic structures is mandatory for the correct development of the kidney and urinary tract.7,31,35 The existence of different phenotypes, incomplete penetrance, variable aggregation, and clinical presentation makes uniform data collection difficult and introduces selection bias. A clinically useful diagnostic scheme is to classify malformations depending on whether the kidney, the collecting system, or both are affected. Uncertainty in the clinical classification resulted in the aggregation of different entities under the popular single-label acronym "CAKUT": Congenital Anomalies of the Kidney and Urinary Tract.27 There is also genetic support for the use of CAKUT, since mutations in a single gene can have pleiotropic effects on the development of the urogenital tract. For example, mutations in the PAX2 gene are associated with the renal-coloboma syndrome, a rare autosomal dominant disorder in which different individuals in the same family can present with various anomalies, including renal agenesis/hypodysplasia, vesicoureteral reflux, and/or secondary obstruction.12 Another example is the gene for autosomal dominant renal hypodysplasia (Online Mendelian Inheritance in Man [OMIM] %610805) localized on chromosome 1p32-33, in which family members of index cases with hypodysplastic kidneys showed ureteric anomalies.32

Murine animal models knocked-out for RET, GDNF, and GFRα1 can develop HSCR and associated renal abnormalities.14,23,36 Furthermore, patients with HSCR and associated CAKUT28,33,37,40,41 or with medullary thyroid carcinoma and renal agenesis21 have already been reported, and the incidence of urogenital anomalies in HSCR has been found to be a nonrandom association.2,17,22 In a review of more than 4800 HSCR patients from 18 separate series, Moore22 described an overall incidence of 6.05% of genitourinary anomalies, including conditions such as kidney malformations, ureteric duplications, and hydronephroses compatible with the CAKUT definition, which were found in roughly 3% of patients. In the same paper, the author also described hypospadias, undescended testes, and disorders of bladder function, which do not belong to the CAKUT definition. In a series of 302 patients with HSCR, Sarioglu and coworkers34 reported an incidence of roughly 7.3% for genitourinary anomalies, alone or in conjunction with other abnormalities. Finally, Amiel and coworkers1,2 reported a 4.4% incidence of renal agenesis or dysplasia plus 2%-3% incidence of genital abnormalities, suggesting a measure of underreporting.

Based on the common genetic background of the enteric nervous system and kidney development and on the above epidemiologic findings, we designed a prospective study to determine the prevalence of CAKUT in patients with HSCR and to identify RET, GDNF, and GFRα1 mutations or haplotypes possibly associated with this subset of HSCR patients.

PATIENTS AND METHODS

Patients

HSCR Patients

Eighty-four patients with HSCR were admitted to the Giannina Gaslini Research Institute in the 12 months between July 2006 and July 2007 and were screened for the presence of CAKUT. To avoid population stratification bias, we enrolled in this study only patients of Italian origin who had received the diagnosis of aganglionosis in our institution with a proper diagnostic evaluation including histochemistry on adequate rectal suction biopsies.

Following approval of the Institutional Ethical Committee, all patients were asked to sign an informed consent form and in case of acceptance, patients or parents (when appropriate) were interviewed to collect clinical and demographic data, personal and familial history, and any information regarding urogenital anomalies detected previously but not yet acknowledged to the medical staff of our department. Only some of the patients who gave informed consent also agreed to have a molecular study for HSCR susceptibility genes.

The following data were collected for each patient and stored in a digital database according to the Personal Data Protection Act:

After enrollment, each patient underwent the following:

Isolated CAKUT Patients

We used a subset of 27 patients with isolated CAKUT as a control group for the molecular genetics study. All patients were children of Italian origin and were being followed in the nephrology department of our institution. None of these children had extrarenal involvement, and all had a normal karyotype. Mean age was 8 years (range, 2-13 yr). All children had renal hypoplasia occurring either as an isolated defect (n = 14) or together with other CAKUT (6 hydronephrosis, 6 vesicoureteral reflux, 1 horseshoe kidney).

CAKUT Screening and Definition

The current study focused on the following abnormalities27: renal agenesis, renal dysplasia-hypoplasia, hydronephrosis, vesicoureteral reflux, duplex collecting system, and horseshoe kidney. We used the following criteria to define CAKUT variants:

Renal agenesis: Renal agenesis was defined as the absence of a kidney on renal ultrasonography confirmed by static scintigraphic study.

Renal dysplasia-hypoplasia: These were considered a single entity due to the impossibility of reliable distinction, and defined as reduction of renal size (longitudinal diameter) by 2 standard deviations from mean size for age and height. The diagnosis was confirmed by static scintigraphy.

Hydronephrosis: An anteroposterior pelvic diameter greater than 7 mm was considered suggestive for hydronephrosis, which was investigated by repeated ultrasound scan or dynamic scintigraphy based on the degree of pelvic dilatation.

Vesicoureteral reflux: Vesicoureteral reflux was diagnosed with retrograde voiding cystourethrogram followed by static scintigraphy to detect possible renal scarring.

Duplex collecting system and horseshoe kidney: The diagnosis was obtained with a combined ultrasound-scintigraphy study describing position, shape, and size of the kidneys.

Imaging Methods for CAKUT Diagnostic Workup

We used the following methods for CAKUT diagnosis:

Renal ultrasound scan: We used an ATL 3000 plus real-time scanner (ATL Ultrasound, Bothell, WA) equipped with convex 2- or 4- to 7-MHz multiple frequency electronic transducers.

Static renal scintigraphy: Nephrograms were recorded 3-4 hours after injection of a weight-scaled dose of technetium-99m DMSA to obtain views in the posterior and both posterior-oblique projections for 300 kilocounts or more. Renal scarring was defined as decreased uptake with distortion of renal contours or as cortical thinning with loss of parenchymal volume.

Mag3 scintigraphy: Nephrograms were recorded at various intervals up to 25 minutes after injection of technetium-99m Mag3. A diuretic test was performed by intravenous injection of 1 mg/kg of furosemide to enhance urine output and discriminate the cause of hydronephrosis. Two nuclear physicians, blinded to the test results, interpreted the scans independently and resolved discrepancies by discussion.

Voiding cystourethrography and cystosonography: These studies were performed as previously described by Piaggio et al25 in 2003.

Molecular Analysis of the RET, GDNF, and GFRα-1 Genes

Patients with HSCR and CAKUT (isolated or in association) were analyzed for mutations and for susceptibility haplotypes of the whole RET gene. A screening study was also performed on selected GDNF and GFRα-1 exons (see below). Genomic DNA was extracted from peripheral blood using Puregene DNA Isolation Kit (Gentra Systems, Inc, Minneapolis, MN). PCR primers and annealing temperature conditions for each considered amplicon are reported in Table 1. PCR reactions were performed on a total volume of 25 μL of 1X solution buffer containing 100 ng of genomic DNA, 0.1 μM of each respective primer, 100 μM of dNTPs, 5% DMSO, 1.5 mM of MgCl[inf]2[r], and 1 unit of Taq Gold (PE Applied Biosystems, Foster City, CA). Reactions were performed in a PE Applied Biosystems 2400 thermal cycler. Amplifications were carried out with an initial denaturation for 4 minutes at 94°C, followed by 36 cycles of 94°C for 20 seconds, T annealing for 30 seconds, and 72°C for 30 seconds, and concluding with a final denaturation at 95°C for 5 minutes followed by slow cooling to room temperature. Polymerase chain reaction products were analyzed either by denaturing high-performance liquid chromatography (DHPLC) (Wave optimizer, Transgenomic Inc, Omaha, NE) or, following Exo-Sap treatment, by automatic sequencing using the Applied Biosystems Dye-Terminator Kit on a ABI Prism 377 DNA sequencer.

T1-2

TABLE 1:

Primer Sequences and Amplification Conditions of Specific Exons at 3 Loci, GDNF, GFRα1, and RET

Mutational analysis of the whole RET proto-oncogene had already been carried out in all patients with HSCR referred to the Department of Pediatric Surgery and to the Molecular Genetics Laboratory of our institution, regardless of the associated CAKUT. The results are reported here for the first time for most of the patients included in this study.

Statistical Analysis

Descriptive statistics were reported as percentage for categorical variables and as mean and standard deviation for continuous data. Differences in the frequencies of each categorical variable were evaluated by the chi-square test, or by the Fisher exact test when appropriate. Comparison of continuous data was performed using the 2-tailed unpaired t test or ANOVA, when appropriate. A p value < 0.05 was considered statistically significant. Analyses were performed using Stata for Windows statistical package (release 9.0, Stata Corporation, College Station, TX).

RESULTS

Demographics and Epidemiology (Overall Series of HSCR Patients)

Eighty-four consecutive patients with HSCR were enrolled and underwent interviews and screening for CAKUT. Mean age, weight, and height were 6.9 ± 5.5 yr, 27 ± 16 kg, and 123 ± 28 cm, respectively. Male to female ratio was 2.8:1. Nineteen of these patients had total colonic aganglionosis, 6 had L-HSCR, and 59 had S-HSCR (Table 2).

T2-2

TABLE 2:

Demographic Data of Current Series of Patients

HSCR Associated With CAKUT (HSCR + CAKUT)

Twenty-one of 84 patients (25%) with HSCR had associated CAKUT involving 29 renal units (Table 3), with a slight right-side preponderance (16 right kidneys and 11 left kidneys for a 1.45:1 ratio). The male to female ratio of HSCR + CAKUT patients was 3.2:1, with the majority of patients having classic aganglionosis confined to the splenic flexure (5 total colonic aganglionosis, 3 L-HSCR, and 13 S-HSCR). Three patients showed a clear familial association of CAKUT (see Table 3) with an autosomal dominant pattern of inheritance (in 2 families the father had renal agenesis and in another family the mother had horseshoe kidney like her son). We observed the following CAKUT phenotypes: 7 hydronephrosis, 7 renal hypoplasia, 3 duplex collecting system, 3 vesicoureteral reflux, and 1 horseshoe kidney. In 9 patients (9/84 = 10.7%) the CAKUT proved to be symptomatic with past urinary tract infections requiring antibiotic therapy. Twelve patients (12/84 = 14.3%) were asymptomatic and had the CAKUT diagnosed during the present screening. Specific medical treatment for recurrent urinary tract infection was required in 3 children with vesicoureteral reflux who were effectively managed with conservative antibiotic prophylaxis for 12 months. One patient with severe hydronephrosis due to pyeloureteral junction obstruction required surgical treatment (dismembered pyeloplasty according to Anderson-Hynes).

T3-2

TABLE 3:

Patients With HSCR + CAKUT, Current Series*

Other associated anomalies were identified in this group of patients. Three children had Down syndrome, 3 had major congenital heart anomalies, 2 had sacral dysmorphism or cerebral palsy. Finally, 2 patients had abnormal skin pigmentation that was interpreted as associated neurocristopathy. Informed consent for DNA sampling was given by 12 of these 21 HSCR + CAKUT patients (57%) who therefore underwent molecular genetic analysis.

HSCR Without CAKUT

The remaining 63 patients of our cohort had no associated CAKUT. The extent of aganglionosis (14 total colonic aganglionosis, 3 L-HSCR, 46 S-HSCR) and the male to female ratio (2.7:1) were not significantly different from those of the HSCR + CAKUT group (see Table 2). Two patients had a familial history of CAKUT (brother with renal agenesis and grandfather with renal hypoplasia). Associated anomalies among these patients were 5 central nervous system anomalies, 3 heart anomalies, 1 Down syndrome, 1 congenital central hypoventilation syndrome ("Ondine's curse"), and 8 others-namely, thalassemic trait, celiac disease, Gilbert syndrome, sucrase-isomaltase deficiency, polycystic ovary, strabismus, juvenile rectal polyp, and bleeding duodenal ulcers.

Additional urogenital findings not belonging to the CAKUT definition were detected in 6 other patients (urinary stones in 3, unspecified glomerular anomalies in 2, and Barter syndrome in 1). However, to avoid selection bias and for homogeneity purposes, these patients were excluded from association studies and molecular genetic analysis.

Molecular Analysis of RET, GDNF, and GFRα1

We screened for RET mutations and analyzed the HSCR-predisposing RET haplotype in a total of 34 patients with HSCR, either associated (12 patients) or not associated (22 patients) with CAKUT, in addition to a group of 27 patients presenting with CAKUT as an isolated malformation. All these patients also underwent partial GDNF and GFRα1 mutation screening. The frequency of the HSCR-predisposing RET haplotype has been estimated by a tag variant lying in a functional noncoding region of the RET intron 1 (rs2435357) (Table 4). The T allele of this SNP was found in 63.6% of the HSCR without CAKUT chromosomes, 50% of patients with HSCR + CAKUT, and 24.1% in the isolated CAKUT group. This latter proportion is similar to the 22.4% previously observed in a normal Italian control population.16 Consistent with what has been already reported,19 such a gradient of frequencies may be accounted for by an excess of the TT genotype in patients with HSCR without CAKUT and an excess of the CC genotype in patients with isolated CAKUT. However, differences in genotype distribution among the individual subgroups also may be due to a deficit of heterozygotes that seems to be present not only in isolated CAKUT but also in HSCR + CAKUT patients, both deviating from the Hardy-Weinberg equilibrium. Conversely, no differences were observed regarding the proportion of alleles at an SNP already known in exon 11 among these 3 groups of patients, all showing a frequency similar to that reported in a normal control group16 (see Table 4). Mutation screening of the entire RET coding region, carried out in all patients with HSCR without CAKUT and HSCR + CAKUT, detected 4 mutations in the HSCR without CAKUT group (Y791F, R912Q, F893C, and c.2829insGGAG) and 1 nonsense mutation E921X in the HSCR + CAKUT group. We did not observe any nucleotide changes in RET exons 10, 11, and 13 of patients affected with isolated CAKUT.

T4-2

TABLE 4:

Frequency of RET SNP Alleles and RET Mutations Detected in the 3 Patient Groups

The results of GDNF and GFRα1 analyses are reported in Table 5. A significant underrepresentation of the G allele of an exon 1 SNP of the GDNF gene (rs2973033), lying in the 5′UTR region of the gene, was found in HSCR (18.2%) and in HSCR + CAKUT (8.4%) patients, whereas in isolated CAKUT patients the G allele had a frequency of 29.6%, similar to that observed in 87 control individuals of European origin reported in HapMap (35.1%) (International HapMap Consortium). The GDNF mutation p.W93R (rs2973033) was found in 2 HSCR without CAKUT patients and in 1 isolated CAKUT patient, whereas no mutations were detected in HSCR + CAKUT patients. A new mutation in exon 3 of the GDNF gene causing the glycine to arginine substitution at the amino acid 105 (p.G105R) was detected in an isolated CAKUT patient presenting with horseshoe kidney.

T5-2

TABLE 5:

Frequency of 1 GDNF SNP alleles and GDNF and GFRα1 Mutations Detected in the 3 Patient Groups

Finally, as reported in Table 5, no significant variations in exons 2 and 9 of the GFRα1 gene were found in any of the 3 patient groups.

DISCUSSION

Our data confirm that CAKUT can be observed in patients with HSCR with a frequency higher than expected in the normal population, thus supporting the concept of a novel syndromic association between the 2 conditions.2,15,25,30,36,37 This hypothesis is not new, since it was already suggested by various authors, especially Amiel and coworkers,1,2 Sarioglu et al34 and Moore.22 These authors reported an overall incidence of genitourinary anomalies ranging between 6% and 7% in HSCR patients with a preponderance of renal agenesis, suggesting a measure of underestimation. Indeed, we observed a 4- to 6-fold higher prevalence of CAKUT in our cohort of HSCR patients compared with that previously reported, and also confirmed that renal hypoplasia/dysplasia represents the most frequent anomaly observed in these patients (9% of cases in the current series).

It seems reasonable that the underestimation of CAKUT in the cohorts of patients mentioned above occurred because certain CAKUT variants remain asymptomatic for years, being discovered as unexpected incidental findings or, alternatively, because of heterogeneous inclusion and diagnostic criteria of CAKUT. If we consider that in the general population vesicoureteral reflux has an incidence of about 10/1000, hydronephrosis of 1/100015, and other CAKUT variants of 1.5/1000 live births (0.3/1000 for renal agenesis and 1.2/1000 for renal hypoplasia),24 we can conclude that HSCR patients have a 3- to 18-fold higher risk of developing CAKUT than the normal population.

It is noteworthy that the male to female ratio in the overall HSCR patient population as well as in patients with HSCR with or without CAKUT was lower than expected in a normal population (approximately 4:1). This is likely due to the high incidence of total colonic aganglionosis (22.6% of cases in the current series), which is known to have a slight female preponderance. The higher incidence of total colonic aganglionosis in our series compared to a normal control population depends on the centralization of care in Italy. Nonetheless, this bias should be irrelevant if we consider that length of aganglionosis and sex did not statistically interfere with the incidence of CAKUT in our cohort of HSCR patients (see Table 2).

The prevalence of HSCR in patients with CAKUT is relatively low and ranges from 1% to 4% in previous reports.3,6,10,11,30 On the other hand, the incidence of HSCR increases with great variability (ranging from 5% to 80%) in patients who have other syndromes such as Down, congenital central hypoventilation, Bardet-Biedl, cartilage-hair-hypoplasia, Smith-Lemli-Opitz, Goldberg-Sprintzsen, congenital X-linked hydrocephalus, Waardenburg-Shah, and Mowat-Wilson.8,9,22

Based on these considerations, the high prevalence of CAKUT in HSCR patients suggests the possibility of a syndromic association, whereas the relatively low prevalence of HSCR in CAKUT series is consistent with a low penetrance of the HSCR phenotype.

RET plays a pivotal role in both isolated and syndromic HSCR. Various authors have described epistatic interactions with the common HSCR-predisposing RET allele in Down syndrome, congenital central hypoventilation syndrome, and Bardet-Biedl syndrome, but not in Mowat-Wilson syndrome and Waardenburg-Shah syndrome type 4, in which HSCR occurrence does not seem to depend on the RET genotype but rather on 1 or more other genes.8,9

A common genetic background involving the RET gene has been suggested for both HSCR and renal hypoplasia or renal agenesis based on several pieces of evidence: 1) animal models knocked-out for RET, GDNF, and GFRα1 develop both kidney agenesis and intestinal aganglionosis14,23,36; 2) the RET mutation p.Cys620Arg, which predisposes to HSCR and thyroid cancer in humans, has been demonstrated to induce HSCR and renal agenesis in knock-in mouse models4,43; 3) the RET mutation p.Cys620Ser has been reported in a 32-year-old woman with familial medullary thyroid carcinoma whose son also had unilateral renal agenesis.20 Finally, it was recently shown that both activating (gain-of-function) and inactivating (loss-of-function) RET and GDNF mutations can significantly contribute to abnormal kidney development in stillborn fetuses with renal agenesis or severe renal dysgenesis who underwent postmortem assessment for diagnostic reasons.38

Another instructive aspect of the current HSCR series is the relatively high percentage of patients with vesicoureteral reflux (4%). This is not surprising based on the suggested association with RET anomalies. In fact, Yu and coworkers44 demonstrated that transgenic HoxB7/RET mice with a constitutive RET overexpression (driven by the HoxB7 promoter) develop primary vesicoureteral reflux. In addition, 70% of patients with primary vesicoureteral reflux belonging to the French-Canadian population in Quebec carry the rare A allele of the c.2071G>A SNP that results in a p.Gly691Ser missense substitution.42

It is noteworthy that our molecular genetic workup showed that the incidence of the predisposing RET T allele in HSCR + CAKUT patients (50%) is lower compared to HSCR without CAKUT patients (64%) and higher compared to isolated CAKUT patients (24%). Despite the fact that the overall genotype frequencies are significantly different between isolated CAKUT patients and a healthy Italian control population, the RET T allele frequencies are similar (24% vs 22.4%). Indeed, deviations observed from the Hardy-Weinberg equilibrium in the RET genotypes of the HSCR + CAKUT and isolated CAKUT groups of patients may be explained either by effective involvement of the T containing HSCR-predisposing haplotype in inducing not only intestinal aganglionosis but also renal developmental abnormalities, or by a potential overdominant genetic effect. A larger sample size is required to distinguish between these 2 possibilities.

These results are consistent with the inverse logarithmic relationship described by de Pontual et al8 between the incidence of the predisposing RET haplotype and the penetrance of the HSCR phenotype in a certain patient population. In this view, HSCR + CAKUT can be regarded as a novel syndromic association, with the frequent, low penetrant, predisposing allele of the RET gene acting as a risk factor for the HSCR phenotype. The explanation for the significantly lower frequency of the G allele of an exon 1 SNP of the GDNF gene in HSCR + CAKUT patients is still unclear, but this event might further contribute to increasing the penetrance of the HSCR phenotype.

Although we did not identify any clear functional variants in the RET, GDNF, and GFRα1 gene portions that underwent mutational analysis, these results do not allow us to fully exclude these genes as causative of this novel syndromic association. Presumably, other genes are responsible for the development of urinary tract abnormalities. We can speculate that RET acts to modify the effects of 1 or more other genes. Conversely, 1 or more genes could modify RET effects. At this point no clear conclusions can be drawn about the molecular details of the HSCR + CAKUT association.

In conclusion, the current study supports the existence of a novel syndromic association (HSCR + CAKUT) with low penetrance of the intestinal phenotype. Based on our data, one can expect that 1 in 3 patients with HSCR also presents with CAKUT. This aspect is important in terms of clinical management and outcome. Based on these results, we strongly suggest including ultrasound screening of the urinary tract in the diagnostic workup of any patient who presents with HSCR.

REFERENCES

1. Amiel J, Sproat-Emison E, Garcia-Barcelo M, Lantieri F, Burzynski G, Borrego S, Pelet A, Arnold S, Miao X, Griseri P, Brooks AS, Antinolo G, de Pontual L, Clement-Ziza M, Munnich A, Kashuk C, West K, Wong KK, Lyonnet S, Chakravarti A, Tam PK, Ceccherini I, Hofstra RM, Fernandez R; Hirschsprung Disease Consortium. Hirschsprung disease, associated syndromes and genetics: a review. J Med Genet. 2008;45:1-14.

2. Amiel J, Lyonnet S. Hirschsprung disease, associated syndromes, and genetics: a review. J Med Genet. 2001;38:729-739.

3. Barakat A, Drougas JC, Barakat R. Association of congenital abnormalities of the kidney and urinary tract with those of other organ system in 13775 autopsies. Child Nephrol Urol. 1988;9:269-272.

4. Carniti C, Belluco S, Riccardi E, Cranston AN, Mondellini P, Ponder BA, Scanziani E, Pierotti MA, Bongarzone I. The Ret(C620R) mutation affects renal and enteric development in a mouse model of Hirschsprung's disease. Am J Pathol. 2006;168:1262-1275.

5. Chakravarti A, Lyonnet S. Hirschsprung disease. In: Scriver CR, Beaudet AL, Valle D, Sly WS, Childs B, Kinzler KW, Vogelstein B, eds. The Metabolic and Molecular Bases of Inherited Disease, International Edition. 8th ed, vol 4. New York: McGraw-Hill; 2001: 6231-6255.

6. Cocchi G, Magnani C, Morini MS, Garani GP, Milan M, Calzolari E. Urinary tract abnormalities (UTA) and associated malformations: data of the Emilia-Romagna Registry. IMER Group. Emilia-Romagna Registry on Congenital Malformations. Eur J Epidemiol. 1996;12:493-497.

7. Costantini F, Shakya R. GDNF/Ret signaling and the development of the kidney. Bioessays. 2006;28:117-127.

8. de Pontual L, Pelet A, Clement-Ziza M, Trochet D, Antonarakis SE, Attie-Bitach T, Beales PL, Blouin JL, Dastot-Le Moal F, Dollfus H, Goossens M, Katsanis N, Touraine R, Feingold J, Munnich A, Lyonnet S, Amiel J. Epistatic interactions with a common hypomorphic RET allele in syndromic Hirschsprung disease. Hum Mutat. 2007;28:790-796.

9. de Pontual L, Pelet A, Trochet D, Jaubert F, Espinosa-Parrilla Y, Munnich A, Brunet JF, Goridis C, Feingold J, Lyonnet S, Amiel J. Mutations of the RET gene in isolated and syndromic Hirschsprung's disease in human disclose major and modifier alleles at a single locus. J Med Genet. 2006;43:419-423.

10. Domini R, Ruggeri G, Appignani A, De Castro R. Idronefrosi primitiva. In: Domini R, De Castro R, eds. Chirurgia delle Malformazioni Urinarie e Genitali. Padova, Italy: Piccin; 1998: 145-174.

11. Domini R, De Castro R, Mordenti M, Perrotta ML. Reflusso vescicoureterale in congenite. In: Domini R, De Castro R, eds. Chirurgia delle Malformazioni Urinarie e Genitali. Padova, Italy: Piccin; 1998:273-312.

12. Eccles MR, Schimmenti LA. Renal-coloboma syndrome: a multi-system developmental disorder caused by PAX2 mutations. Clin Genet. 1999;56:1-9.

13. Edery P, Lyonnet S, Mulligan LM, Pelet A, Dow E, Abel L, Holder S, Nihoul-Fekete C, Ponder BAJ, Munnich A. Mutations of the RET proto-oncogene in Hirschsprung's disease. Nature. 1994;367:378-379.

14. Enomoto H, Araki T, Jackman A, Heuckeroth RO, Snider WD, Johnson EM Jr, et al. GFR alpha1-deficient mice have deficits in the enteric nervous system and kidneys. Neuron. 1998;21:317-324.

15. Flashner SC, King LR. Ureteropelvic junction. In: Kelalis PP, King LR, Belman BA, eds. Clinical Pediatric Urology. 3rd ed. Philadelphia: WB Saunders; 1992:693.

16. Griseri P, Bachetti T, Puppo F, Lantieri F, Ravazzolo R, Devoto M, Ceccherini I. A common haplotype at the 5′ end of the RET proto-oncogene, overrepresented in Hirschsprung patients, is associated with reduced gene expression. Hum Mutat. 2005;25:189-195.

17. Kaiser G, Bettex M. Disorder and congenital malformations associated to Hirschsprung's disease. In: Holschneider AM, ed. Hirschsprung Disease. Stuttgart: Hypokrates-Verlag; 1982:49-53.

18. Lantieri F, Griseri P, Amiel J, Martucciello G, Ceccherini I, Romeo G, Lyonnet S. The molecular genetics of Hirschsprung's disease. In: Holschneider AM, Puri P, eds. Hirschsprung's Disease and Allied Disorders. 3rd ed. Berlin: Springer-Verlag; 2008:63-78.

19. Lantieri F, Griseri P, Puppo F, Campus R, Martucciello G, Ravazzolo R, Devoto M, Ceccherini I. Haplotypes of the human RET proto-oncogene associated with Hirschsprung disease in the Italian population derive from a single ancestral combination of alleles. Ann Hum Genet. 2006;70(Pt 1):12-26.

20. Lore F, Talidis F, Di Cairano G, Renieri A. Multiple endocrine neoplasia type 2 syndromes may be associated with renal malformations. Intern Med. 2001;250:37-42.

21. Lore F, Di Cairano G, Talidis F. Unilateral renal agenesis in a family with medullary thyroid carcinoma. N Engl J Med. 2000;20(342):1218-1219.

22. Moore SW. The contribution of associated congenital anomalies in understanding Hirschsprung's disease. Pediatr Surg Int. 2006;22:305-315.

23. Moore MW, Klein RD, Farinas I, Sauer H, Armanini M, Philips H, et al. Renal and neuronal abnormalities in mice lacking GDNF. Nature. 1996;382:76-79.

24. Nazer J, Fernandez P, Silva C. Urinary tract malformations in newborns at the Clinical Maternity Hospital of the University of Chile. Rev Med Chil. 1998;126:1472-1477.

25. Piaggio G, Degl'Innocenti ML, Toma P, Calevo MG, Perfumo F. Cystosonography and voiding cystourethrography in the diagnosis of vesicoureteral reflux. Pediatr Nephrol. 2003;18:18-22.

26. Pini Prato A, Gentilino V, Giunta C, Avanzini S, Parodi S, Mattioli G, Martucciello G, Jasonni V. Hirschsprung's disease: 13 years' experience in 112 patients from a single institution. Ped Surg Int. 2008;24:175-182.

27. Pope JC IV, Brock JW III, Adams MC, Stephens FD, Ichikawa I. How they begin and how they end: classic and new theories for the development and deterioration of congenital anomalies of the kidney and urinary tract, CAKUT. J Am Soc Nephrol. 1999;10:2018-2028.

28. Reish O, Gorlin RJ, Hordinsky M, Rest EB, Burke B, Berry SA. Brain anomalies, retardation of mentality and growth, ectodermal dysplasia, skeletal malformations, Hirschsprung disease, ear deformity and deafness, eye hypoplasia, cleft palate, cryptorchidism, and kidney dysplasia/hypoplasia (BRESEK/BRESHECK): new X-linked syndrome? Am J Med Genet. 1997;11:386-390.

29. Romeo G, Ronchetto P, Luo Y, Barone V, Seri M, Ceccherini I, Pasini B, Bocciardi R, Lerone M, Kaariainen H, et al. Point mutations affecting the tyrosine kinase domain of the RET proto-oncogene in Hirschsprung's disease. Nature. 1994;367:377-378.

30. Ruggeri G, Domini M, Tizzone V. Incidenza delle uropatie congenite in portatori di altre malformazioni congenite. In: Domini R, De Castro R, eds. Chirurgia delle Malformazioni Urinarie e Genitali. Padova, Italy: Piccin; 1998:47-58.

31. Sadler TW. Urogenital system. In: Sadler TW, ed. Medical Embryology. Baltimore: Lippincott Williams & Williams; 2006:229-236.

32. Sanna-Cherchi S, Caridi G, Weng PL, Dagnino M, Seri M, Konka A, Somenzi D, Carrea A, Izzi C, Casu D, Allegri L, Schmidt-Ott KM, Barasch J, Scolari F, Ravazzolo R, Ghiggeri GM, Gharavi AG. Localization of a gene for nonsyndromic renal hypodysplasia to chromosome 1p32-33. Am J Hum Genet. 2007;80:539-549.

33. Santos H, Mateus J, Leal MJ. Hirschsprung disease associated with polydactyly, unilateral renal agenesis, hypertelorism, and congenital deafness: a new autosomal recessive syndrome. J Med Genet. 1988;25:204-205.

34. Sarioglu A, Tanyel FC, Buyukpamukcu N, Hisconmez A. Hirschsprung-associated congenital anomalies. Eur J Pediatr Surg. 1997;7:331-337.

35. Sariola H, Sainio K. The tip-top branching ureter. Curr Opin Cell Biol. 1997;9:877-884.

36. Schuchardt A, D'Agayi V, Larsson-Blomberg L, Costantini F, Pachnis V. Defects in the kidney and enteric nervous system of mice lacking the tyrosine kinase receptor Ret. Nature. 1994;367:380-383.

37. Sinnassamy P, Yazbeck S, Brochu P, O'Regan S. Renal anomalies and agenesis associated with total intestinal aganglionosis. Int J Pediatr Nephrol. 1986;7:1-2.

38. Skinner MA, Safford SD, Reeves JG, Jackson ME, Freemerman AJ. Renal aplasia in humans is associated with RET mutations. Am J Hum Genet. 2008;82:344-351.

39. Sweetser DA, Froelick GJ, Matsumoto AM, Kafer KE, Marck B, Palmiter RD, Kapur RP. Ganglioneuromas and renal anomalies are induced by activated RET(MEN2B) in transgenic mice. Oncogene. 1999;18:877-886.

40. Van Nesselrooij BP, Spliet W, Beemer FA. Unusual association of congenital malformations: craniosynostosis, heart defect, abnormal intestinal innervation and urogenital abnormalities. Clin Dysmorphol. 1998;7:51-53.

41. Virdi VS, Cheema AS. Neonatal Hirschsprung disease with multicystic dysplastic kidneys presenting as multiple gastrointestinal perforations. Trop Gastroenterol. 2003;24:99-101.

42. Yang Y, Houle AM, Letendre J, Richter A. RET Gly691Ser mutation is associated with primary vesicoureteral reflux in the French-Canadian population from Quebec. Hum Mutat. 2008;29:695-702.

43. Yin L, Puliti A, Bonora E, Evangelisti C, Conti V, Tong WM, Medard JJ, Lavoue MF, Forey N, Wang LC, Manie S, Morel G, Raccurt M, Wang ZQ, Romeo G. C620R mutation of the murine ret proto-oncogene: loss of function effect in homozygotes and possible gain of function effect in heterozygotes. Int J Cancer. 2007;121:292-300.

44. Yu OH, Murawski IJ, Myburgh DB, Gupta IR. Overexpression of RET leads to vesicoureteric reflux in mice. Am J Physiol Renal Physiol. 2004;287:F1123-F113.

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