J Hum Genet (2005) 50:168–174 DOI 10.1007/s10038-005-0236-x
O R I GI N A L A R T IC L E
Trang-Tiau Wu Æ Tsui-Wei Tsai Æ Chao-Ta Chu Zen-Fung Lee Æ Chuan-Mao Hung Æ Ching-Chyuan Su Shuan-Yow Li Æ Mingli Hsieh Æ Chuan Li
Low RET mutation frequency and polymorphism analysis of the RET and EDNRB genes in patients with Hirschsprung disease in Taiwan Received: 9 December 2004 / Accepted: 20 January 2005 / Published online: 15 April 2005 The Japan Society of Human Genetics and Springer-Verlag 2005
Abstract Hirschsprung disease (HSCR), or congenital intestinal aganglionosis, is a relatively common disorder characterized by the absence of ganglion cells in the nerve plexuses of the lower digestive tract, resulting in intestinal obstruction in neonates. Mutations in genes of the RET receptor tyrosine kinase and endothelin receptor B (EDNRB) signaling pathways have been shown to be associated in HSCR patients. In this study, we collected genomic DNA samples from 55 HSCR patients in central Taiwan and analyzed the coding regions of the RET and EDNRB genes by PCR amplification and DNA sequencing. In the 55 patients, an A to G transition was detected in two (identical twin brothers). The mutation was at the end of RET exon 19 at codon 1062 (Y1062C), a reported critical site for the signaling pathways. Single nucleotide polymorphisms (SNP) in exons 2, 7, 11, 13, and 15 of RET and exon 4 of
EDNRB in the HSCR patients or controls were detected. The differences between patients and controls in allele distribution of the five RET polymorphic sites were statistically significant. The most frequent genotype encompassing exons 2 and 13 SNPs (the polymorphic sites with the highest percentage of heterozygotes) was AA/GG in patients, which was different from the AG/ GT in the normal controls. Transmission disequilibrium was observed in exons 2, 7, and 13, indicating nonrandom association of the susceptibility alleles with the disease in the patients. This study represents the first comprehensive genetic analysis of HSCR disease in Taiwan.
T.-T. Wu Department of Pediatric Surgery, Chung Shan Medical University Hospital, Taichung, Taiwan, Republic of China
Introduction
T.-T. Wu Æ C.-M. Hung Æ C.-C. Su Institute of Medicine, Chung-Shan Medical University, Taichung, Taiwan, Republic of China T.-W. Tsai Æ C.-T. Chu Æ C.-M. Hung Æ S.-Y. Li Æ M. Hsieh Æ C. Li (&) Department of Life Sciences, Chung Shan Medical University, 110 Sec. 1, Chein-Kuo North Road, Taichung, Taiwan, Republic of China E-mail:
[email protected] Tel.: +886-4-2473-0022 ext 11807 Fax: +886-4-2475-7412 Z.-F. Lee Division of Pediatric Surgery, God’s Heart Hospital, Chiayi, Taiwan, Republic of China C.-C. Su Tian-Sheng Memorial Hospital, Tong Kang, Pin-Tong, Taiwan, Republic of China M. Hsieh Department of Biology, Tunghai University, Taichung, Taiwan, Republic of China
Keywords Hirschsprung disease (HSCR) Æ Polymorphism Æ RET Æ EDNRB
Hirschsprung disease (HSCR) is characterized by the absence of intramural ganaglion cells in the nerve plexuses of the distal gut (Badner et al. 1990; Reyna 1993).Typically, HSCR are present in neonates or early childhood, with symptoms ranging from chronic constipation to acute ileus, but late manifestation in adults has occasionally been described (Lesser et al. 1979). It is the most common cause of neonatal intestinal obstruction, affecting one in 5,000 live newborns with a male predominance (3:1–5:1) (Angrist et al. 1993; Badner et al. 1990; Lyonnet et al. 1993; Reyna 1993). The phenotype in HSCR can be classified into two groups: short-segment aganglionosis (SSA), which includes patients with aganglionosis as far as the rectosigmoid junction, and long-segment aganglionosis (LSA), which includes patients with aganglionosis beyond the rectosigmoid junction and total colon or universal intestinal aganglionosis (TCA) (Reyna 1993). Most HSCR cases occur sporadically, but familial ones have been reported to be 3.6–7.8% (Kusafuka and Puri 1998). Affected
169
infants may have other pathologies and malformations (Badner et al. 1990; Frost 1992). The disease has a complex genetic etiology and is likely to be of multifactorial inheritance with a couple of susceptibility genes, including members of the RET (Angrist et al. 1996; Doray et al. 1998; Edery et al. 1994; Romeo et al. 1994) or the endothelin (EDNRB)-regulated signaling pathways (Hofstra et al. 1996; Puffenberger et al. 1994) and the SOX-10-mediated transcriptional regulation (Pingault et al. 1998). The RET proto-oncogene is a protein tyrosine kinase gene expressed in cells derived from the neural crest and may play a critical part in embryogenesis of the mammalian enteric nervous system (Takahashi et al. 1988). The RET gene has been described to be the most frequently mutated gene in HSCR patients with dominant mutations identified in its 20 different exons. RET mutations were associated with about 50% of familial and 7–35% of sporadic HSCR patients (Angrist et al. 1995; Edery et al. 1994; GarciaBarcelo et al. 2004; Romeo et al. 1994; Sakai et al. 2000; Seri et al. 1997; Svensson et al. 1998). The activation of the EDNRB gene is also involved in the development and migration of human enteric ganglion cells. Recessive mutations of the EDNRB gene have also been found in a small portion of HSCR patients (Puffenberger et al. 1994). Mutations in other susceptibility genes such as the RET ligand GDNF (glial-derived neurotrophic factors; Angrist et al. 1996), NTN (neurturin; Doray et al. 1998), the EDNRB ligand EDN3 (endothelin-3; Hofstra et al. 1996), ECE1 (endothelin-converting enzyme 1; Hofstra et al. 1999), SOX 10 (Pingault et al. 1998), and SIP1 (Samd interacting protein 1; Wakamatsu et al. 2001) are uncommon, and none are fully penetrant. Thus, in this study we examined the most disease-relevant RET and EDNRB gene in HSCR patients in Taiwan. This is the first comprehensive genetic study of the RET and EDNRB genes in HSCR patients in Taiwan.
Materials and methods
Genomic DNA from 52 parents of 26 patients was available. Control DNA samples were obtained from individuals in central or southern Taiwan normal in HSCR. The study was approved by the local Institutional Research Board. Polymerase chain reaction and DNA sequence analysis Genomic DNA was extracted from blood samples by a Puregene Genomic DNA Purification Kit (Gentra systems, MN, USA) following the protocols of the manufacturer. PCR amplifications of all exons of the RET and EDNRB genes were performed using oligonucleotide primers, as previously described (Sakai et al. 2000). Basically, for each PCR reaction, 150 ng of genomic DNA was amplified in a 50-ll reaction containing 20 pmol of each primer and 2 U of Taq DNA polymerase. The PCR products were purified by a PCR-M Clean Up System (VIOGENE, Taiwan) and sequenced using an ABI PRISM Big Dye assay (Applied Biosystems, Foster City, CA, USA) and ABI automated sequencers. Restriction cleavages Polymorphisms in exons 2, 13, and 15 of the RET gene were further examined by sequencing or restriction enzyme cleavage. The PCR products of exon 2 or 15 were digested with the EagI or RsaI enzyme at 37C for 2– 4 h, respectively. The PCR products of exon 13 were cleaved with the TaqI enzyme at 65 C for 1.5 h. Statistical analysis Chi-square (v2) analysis was used to determine the significance of the association of allele frequencies between patients and normal control groups.
Patients and control samples
Results Fifty-five peripheral blood samples were obtained from patients diagnosed as HSCR at infancy who received a definite pull-through operation at Chung-Shan Medical College Hospital and Affiliate Hospitals from 1976 to 2004. Patients with Down syndrome or anorectal malformation were excluded from this study. Most of these 55 cases showed SSA and a sporadic occurrence of the disease (six familiar patients with relationships as identical twin brothers, cousins, and uncle/nephew; the remaining 49 were sporadic). There were 44 patients (M:F 37:7) with SSA and 11 (M:F 7:4) with LSA, including two (M:F1:1) with TCA. For all patients, the diagnosis of HSCR was confirmed histologically by the absence of ganglionic cells in rectal biopsy and resected bowel specimens. All patients had been treated by surgical resection material for absence of enteric plexuses.
Sequence alteration in RET and low RET mutation frequency in HSCR patients We analyzed the coding regions, including the exon/intron junctions, of the RET and EDNRB genes in 55 HSCR patients with typical phenotypes. An A to G transition at the end of exon 19 at codon 1062 (Y1062C) of the RET gene was detected in two of the 55 patients. The two patients were identical twin brothers with SSA. This alteration was not observed in 20 functional gastrointestinal disorder patients and 50 normal controls. Even though no Y1062 mutation has been identified in HSCR patients previously, investigation of nearby mutations such as D1059 and L1061P indicates that Y1062 is a critical multifunctional intracytoplasmic
170 Fig. 1 DNA sequence analysis of exon 19 of the RET gene. a Normal control. b The Hirschsprung disease (HSCR) patient demonstrating a heterozygous 1062 A to G mutation supposedly resulting in a mutation of Tyr to Cys. c The mother of the HSCR patient in b
docking site for downstream signaling pathways such as Shc (Geneste et al. 1999). This Y1062C mutation is present in a heterozygous state in the twin patients and is in their asymptomatic mother but not the father (Fig 1). To our surprise, we were unable to detect any other mutations in the RET gene in the rest of the 53 HSCR patients. RET mutations have been previously reported in HSCR patients of various ethic groups, including Chinese, with mutation frequencies ranging from 7% to 50% (Angrist et al. 1995; Edery et al. 1994; GarciaBarcelo et al. 2004; Garcia-Barcelo et al. 2003; Romeo et al. 1994; Sakai et al. 2000; Seri et al. 1997; Svensson et al. 1998). In the present study, RET mutations were only detected in two (identical twins) of 55 typical HSCR patients (3.6%). This frequency is much lower than the 19% frequency of the Chinese HSCR patients in Hong Kong studied by Garcia-Barcelo et al. (GarciaBarcelo et al. 2004). RET gene polymorphism Furthermore, apart from the low RET mutation rate, less polymorphic sites in the coding regions of the RET gene were identified in our 55 patients than those described previously in the Chinese population in Hong Kong (Garcia-Barcelo et al. 2003). Four single nucleo-
tide polymorphisms (SNPs) in exons 2, 7, 11, and 13 of the RET gene were detected in the HSCR patients (Table 1). The polymorphisms are a G to A transition (c135 G>A) at codon 45 (A45A) in exon 2, a G to A transition (c1296 G>A) at codon 432 (A432A) in exon 7, a G to A transition (c2071 G>A) at codon 691 (G691S) of exon 11, and a T to G transversion (c2307 T>G) at codon 769 (L769L) of exon 13. These polymorphisms have been described previously in various populations (Borrego et al. 1999; Fitze et al. 1999; Gath et al. 2001). We did not detect any sequence alteration in our patients for other polymorphic sites in the RET coding regions reported by Garcia-Barcelo et al. in the Chinese HSCR patients in Hong Kong, including a c2712C>G site in exon 15 (S904S) that accounts for 4.5% of their patients (Garcia-Barcelo et al. 2003). However, the frequencies of the alleles of the SNPs in exons 2, 7, 11, 13, and 15 were determined in the control population. Statistically, the difference of the allele frequencies of all five polymorphic sites between patients and controls were significant (P<0.05) (Table 1). The allele frequencies were very close to those reported in Hong Kong (Garcia-Barcelo et al. 2003). The allele frequencies in exon 2, 13, and 15 in the normal population were also close to those reported by Chattopadhyay et al. (Chattopadhyay et al. 2003). The allele frequencies of allele A of c135 G>A (exon2), allele G of
Table 1 Allele frequencies of RET and EDNRB polymorphisms in Hirschsprung disease (HSCR) patients and controls RET Exon
Codon
Nucleotide change
2
A45A
c135G>A
7
A432A
c1296G>A
11
G691S
c2071G>A
13
L769L
c2307T>G
15
S904S
c2712C>G
EDNRB Exon 4 L277L
c831A>G
Allele frequency Patients (55)
Controls (50)
G: 0.32 A: 0.68 G: 0.98 A: 0.02 G: 0.98 A: 0.02 T: 0.28 G: 0.72 C: 1.0 G: 0
0.46 0.54 0.77 0.23 0.91 0.09 0.47 0.53 0.92 0.08
A:0.7 G:0.3
0.65 0.35
Statistic
Allele frequency Chinese (Hong Kong)
2
P
Patients
Controls
v2
4.41
0.035
<0.0001
<0.0001
5.3
0.02
5.44
0.02
4.53
0.03
7.95
0.005
28.05
<0.0001
9.14
0.002
0.59 0.41 0.79 0.21 0.895 0.105 0.51 0.49 0.895 0.105
38.62
22.42
0.27 0.73 0.88 0.12 0.955 0.045 0.248 0.752 0.955 0.045
4.53
0.03
0.60
0.44
v
P
171 Table 2 Allele frequencies of RET polymorphisms in SSA and LSA patients and controls. SSA short segment aganglionosis, LSA long segment aganglionosis, C control samples RET Exon
Codon
Nucleotide change
2
A45A
c135G>A
7
A432A
c1296G>A
11
G691S
c2071G>A
13
L769L
c2307T>G
Allele frequency
Statistic
SSA (44)
LSA (11)
C (50)
v2 (SSA vs LSA)
P
G:0.36 A:0.64 G:0.98 A:0.02 G:0.98 A:0.02 T:0.34 G:0.66
0.14 0.86 1.00 0 1 0 0.05 0.95
0.46 0.54 0.77 0.23 0.91 0.09 0.47 0.53
4.19
0.04
0.51
Table 3 Frequencies of RET genotypes in Hirschsprung disease (HSCR) patients and controls in Taiwan
v2 (LSA vs C)
P
v2 (SSA vs C)
P
7.86
0.005
1.79
0.18
0.48
6.24
0.013
17.4
<0.0001
0.51
0.48
2.14
0.144
3.8
0.05
7.59
0.006
13.62
<0.0001
3.22
0.07
LSA/TCA patients were homozygous for allele A of c135G>A and allele G of c2307T>G (82%).
Genotypes A45A
L769L
Patients (n=55)
controls (n=50)
AA AG AA AG GG GG GG AG
GG GT GT GG TT GG GT TT
0.36 0.20 0.16 0.11 0.07 0.05 0.02 0.02
0.14 0.36 0.14 0.08 0.10 0.02 0.08 0.08
c1296 G>A (exon 7), allele G of c2071 G>A (exon 11), allele G of c2307 T>G (exon 13), and allele C of c2712C>G (exon 15) were higher in our HSCR patients than in controls. Interestingly, even though the sample size of our LSA patients was small, the differences of allele frequencies of the polymorphic sites in exon 2 and exon 13 between the LSA and SSA patients were statistically significant (P<0.05 and P<0.01, respectively; Table 2). However, the difference between the SSA patients and controls were not significant in both polymorphic sites of exon 2 and 13. RET genotypes Since the polymorphisms in exons 7, 11, and 15 were either not detected or of low frequencies (0.02 for the rare allele of exons 7 and 11) in our HSCR patients, we only combined exon 2 and exon 13 SNPs for the genotype analyses (Table 3). It is apparent that the genotype distribution differs significantly between patients and controls. Homozygotes of both the A allele of c135 G>A (A45A) and G allele of c2307T>G (L769L) was the most frequent genotype in HSCR patients (36%) while heterozygotes of both polymorphic sites were most common in controls (36%). The results were similar with the Hong-Kong studies (Garcia-Barcelo et al. 2004) but different with the Caucasian populations (Borrego et al. 1999; Fitze et al. 1999). Furthermore, all but two of the
Transmission disequilibrium of the A allele of exon 2 and the G allele of exon 13 SNP The nonrandom association of the A allele of exon 2, the G allele of exon 7, and the G allele of exon 13 SNP in our HSCR patients was further indicated by transmission analysis. Twenty–six HSCR trios were available for the analysis. The allele frequencies of the three polymorphic sites for parents and patients with parents are indicated in Table 4. Basically, the allele frequencies for patients with parents were similar to those of total patients. The frequencies of the parents were not statistically different from the control population, either. However, the difference for the allele frequencies of the c1296 G>A SNP in exon 7 between the patients and their parents was significant (P=0.003). Due to limited sample numbers in patients with parents, the difference for exons 2 and 13 SNP was not significant. Nevertheless, when the allele frequencies of the parent and of the total patient were compared, the differences of the SNP in exons 2 and 13 were significant (P<0.05). We further analyzed the association of specific alleles by the transmission disequilibrium test (TDT), as shown in Table 5. Surprisingly, in transmissions of the A432A SNP (c1296 G>A) from 16 heterozygous parents, only transmission of the G allele was observed. The A allele of exon 2 (c135G>A) was preferentially transmitted to the proband from their A/G heterozygous parents (A:G 24:11). For the transmission of the G/T SNP in exon 13 (c2307 T>G), the G allele was preferentially transmitted from the G/T heterozygous parents (G:T 23:10). The results indicate nonrandom association of transmitting specific alleles of the three polymorphic sites to the HSCR patients. Furthermore, for the inheritance of alleles of exon 7 SNP, we observed that one male G/G homozygous proband with a heterozygous father (G/A) and a G/G homozygous mother received the G allele from his father. His twin sister and another older sister, who
172 Table 4 Allele frequencies of RET polymorphisms in Hirschsprung disease (HSCR) patients and parents RET Exon
Codon
Nucleotide change
Allele frequency a
b
Pp (26)
Pt (55) 2
A45A
c135G>A
7
A432A
c1296G>A
13
L769L
c2307T>G
a
G:0.32 A:0.68 G:0.98 A:0.02 T:0.28 G:0.72
Statistic Parents (52)
0.38 0.62 1 0 0.29 0.71
C (50)
0.47 0.53 0.85 0.15 0.44 0.56
0.46 0.54 0.77 0.23 0.47 0.53
Pt vs Pp value
Pp vs parents
Pt vs parents
C vs parents
v2
P
v2
v2
P
v2
P
value
1.146
0.284
1.05
0.31
6.59
0.01
0.189
0.664
0.994
0.319
8.91
0.003
12.77
<0.0001
1.912
0.167
0.008
0.93
3.38
0.065
5.84
0.016
0.169
0.681
P
value
value
Pt: total patients Pp: patients with parents available included in this analysis
b
Table 5 Transmission disequilibrium test (TDT) by individual polymorphic locus in RET Exon
2 7 13
SNP (allele)
c135G>A(A) c1296G>A(G) c2307T>G(G)
TDT TR
NT
24 16 23
11 0 10
v2
P value
4.83 16 5.12
<0.03 <0.001 <0.03
received the A allele from their father, were normal. Although the data are limited, it is consistent with the association of the G allele with the disease or the exclusion of the A allele in the HSCR patients.
EDNRB polymorphisms In the EDNRB gene, we did not observe any sequence alteration, except one reported polymorphism at codon L277L (A to G) in exon 4. No statistically significant differences were observed for the frequency of the polymorphic alleles in our HSCR patients and controls (Table 1).
Discussion This study represents the first comprehensive genetic analysis of HSCR disease in Taiwan. After thorough analysis of the coding regions of both the RET and EDNRB genes, however, only one mutation (Y1062C) in exon 19 of the RET gene was identified in two twin brothers of the 55 HSCR patients in our study. Even though we did not provide direct evidences for the causative effect the Y1062C mutation on HSCR, other studies have already indicated the importance of this site in signaling pathways (Geneste et al. 1999; Lorenzo et al. 1997). Ret protein with point mutation of Y1062F apparently decreases its association with Shc in vitro and in vivo (Geneste et al. 1999). Thus, the Y1062C missense mutation identified in this study is
consistent with the loss of function of germline mutation in RET and is likely to be a novel diseasecausing mutation. Interestingly, even though it is likely to be a severe mutation, the mother of the probands who contained this allele was asymptomatic. Whether there are other genetic factors or gender-specific factors involved in the development of HSCR is an interesting question. However, it is consistent with the low penetrance of many RET mutations. The RET mutation rate in our HSCR patients seems to be low compared with those reported in other populations. In general, studies on sporadic HSCR yield lower frequencies of RET mutation studies than the familial cases (7–20% compared with 50%; Angrist et al. 1995; Edery et al. 1994; Garcia-Barcelo et al. 2004; Garcia-Barcelo et al. 2003; Romeo et al. 1994; Sakai et al. 2000; Seri et al. 1997; Svensson et al. 1998). However, germline RET mutation is associated with only 3% of a population-based series of isolated HSCR (Svensson et al. 1998). As our HSCR patients were mostly sporadic without familial history, this may partially explain the low RET mutation frequency. Nevertheless, from polymorphism analyses, we found the association of specific alleles in some SNP sites in the RET coding region in our patients. Generally, RET is the major susceptibility gene of HSCR, with incomplete penetrance. Beside the mutations in the RET coding regions, specific polymorphisms or haplotypes of RET (Borrego et al. 2000; Borrego et al. 2003; Burzynski et al. 2004; Fitze et al. 1999; Fitze et al. 2003; Garcia-Barcelo et al. 2003; Griseri et al. 2002) and variations at the RET locus, together with variations at unknown loci on 3p21 and 19q12 (Gabriel et al. 2002), have all been proposed to account for the genetic susceptibility to HSCR. In this study, significant deviation of random transmission of the A allele of c135G>A, the G allele of c1296G>A, and the G allele of c2307T>G, indicates that these alleles are in strong linkage disequilibrium with the disease-causing alleles. In the 55 patients, the gender ratio was 4 (44 males and 11 females). The ratios were apparently higher in the SSA (5.29) than in the LSA and TCA (1.75). The ratios were very close to another HSCR study in the Chinese
173
population (Garcia-Barcelo et al. 2003). The decrease in gender bias with the increase in the length of aganglionosis indicates that certain gender-specific modifying loci must be present, and the effects decrease in cases with severe genetic defects (more frequent in LSA) (Attie et al. 1995). In our analyses, the probability of the presence of allele A of c135G>A and allele G of c2307T>G were significantly higher in LSA than in SSA patients. The allele frequency of the c135G>A polymorphism was not different significantly in the LSA and SSA Chinese patient in Hong Kong (Garcia-Barcelo et al. 2003) but has been suggested to be associated with the disease phenotype in Germans (Fitze et al. 2002). However, in Germans, A/A homozygosity accounts for 63% of SSA and 18% of LSA patients while A/A homozygosity comprised 82% of our LSA patients and only 43% of our SSA patients. The significant difference of the allelic distribution of exon 7 SNP between LSA and SSA in Hong Kong patients (Garcia-Barcelo et al. 2003) was not detected in our patients. The results implicate that even if the SNP might be associated with genetic factors involved in development of LSA and SSA, they might be different in different populations. Noticeably, in our study, we excluded clinically atypical patients with Down syndrome or imperforate anus and included only HSCR patients with radical surgical resection of aganglionic segments. Interestingly, two male SSA patients, both with AG/GG genotype, were born by ‘‘foreign-bride’’ mothers—one from Vietnam and another from Indonesia. There were two male LSA patients with typical AA/GG genotype with both parents being Aboriginals (from the Bunun tribe) of Taiwan. Another SSA male patient had an Aboriginal (from the Ami tribe) father. The ratios of the HSCR patients born by the foreign-bride mothers or Aboriginals are consistent with the diverse ethic conditions of the general new-born babies in Taiwan. The RET genotypes and SNP allele distributions in these HSCR patients were not different from other HSCR patients and thus were not excluded. However, the two Bunun LSA patients account for 2/7 of our male LSA patients. The Bunun tribe resides mostly in the central mountain range of Taiwan locally, close to our hospital, while other Aboriginal tribes reside in other parts of Taiwan and are less likely to be collected in this study. Whether there are specific genetic factors involved in the severe HSCR phenotype in the Bunun population requires further investigation. In summery, the results of our study further support that the susceptibility RET alleles in ethnic Chinese patients are likely to be different from those in the Caucasian populations. We demonstrated that five SNP sites in the RET coding region might be associated with HSCR in Taiwanese. Among these, transmission disequilibrium was observed in exons 2, 7, and 13 SNPs, and the transmission of the G allele of c1296G>A in exon 7 was most strongly suggested by TDT test. The c135G>A (exon 2) and c2307T>G (exon 13) might be associated with the phenotypic HSCR severity, as their
allele frequencies were statistically different in LSA and SSA. Other genetic factors or environmental factors involved in our patients require further investigation. Acknowledgements This study was supported in part by National Science Council (NSC 89-2314-B040-036, 89-2745-P040-002, 902745-P040-002) and Chung Shan Medical University (CSMC 89OM-A038). We thank the patients and their parents involved in this study.
References Angrist M, Kauffman E, Slaugenhaupt SA, Matise TC, Puffenberger EG, Washington SS, Lipson A, Cass DT, Reyna T, Weeks DE (1993) A gene for Hirschsprung disease (megacolon) in the pericentromeric region of human chromosome. Nat Genet 4:351–356 Angrist M, Bolk S, Thiel B, Puffenberger EG, Hofstra RM, Buys CH, Cass DT, Chakravarti A (1995) Mutation analysis of the RET receptor tyrosine kinase in Hirschsprung disease. Hum Mol Genet 4:821–830 Angrist M, Bolk S, Halushka M, Lapchak PA, Chakravarti A (1996) Germline mutations in glial cell line-derived neurotrophic factor (GDNF) and RET in a Hirschsprung disease patient. Nat Genet 14:341–344 Attie T, Pelet A, Edery P, Eng C, Mulligan LM, Amiel J, Boutrand L, Beldjord C, Nihoul-Fekete C, Munnich A (1995) Diversity of RET proto-oncogene mutations in familial and sporadic Hirschsprung disease. Hum Mol Genet 4:1381–1386 Badner JA, Sieber WK, Garver KL, Chakravarti A (1990) Specific polymorphisms in the RET proto-oncogene are over-represented in patients with Hirschsprung disease and may represent loci modifying phenotypic expression. Am J Hum Genet 46:568–580 Borrego S, Saez ME, Ruiz A, Gimm O, Lopez-Alonso M, Antinolo G, Eng C (1999) Specific polymorphisms in the RET protooncogene are over-represented in patients with Hirschsprung disease and may represent loci modifying phenotypic expression. J Med Genet 36: 771–774 Borrego S, Ruiz A, Saez ME, Gimm O, Gao X, Lopez-Alonso M, Hernandez A, Wright FA, Antinolo G, Eng C (2000) RET genotypes comprising specific haplotypes of polymorphic variants predispose to isolated Hirschsprung disease. J Med Genet 37:572–578 Borrego S, Wright FA, Fernandez RM, Williams N, Lopez-Alonso M, Davuluri R, Antinolo G, Eng C (2003) A founding locus within the RET proto-oncogene may account for a large proportion of apparently sporadic Hirschsprung disease and a subset of cases of sporadic medullary thyroid carcinoma. Am J Hum Genet 72:88–100 Burzynski GM, Nolte IM, Osinga J, Ceccherini I, Twigt B, Maas S, Brooks A, Verheij J, Plaza Menacho I, Buys CH, Hofstra RM (2004) Localizing a putative mutation as the major contributor to the development of sporadic Hirschsprung disease to the RET genomic sequence between the promoter region and exon 2. Eur J Hum Genet 12: 604–612 Chattopadhyay P, Pakstis AJ, Mukherjee N, Iyengar S, Odunsi A, Okonofua F, Bonne-Tamir B, Speed W, Kidd JR, Kidd KK(2003) Global survey of haplotype frequencies and linkage disequilibrium at the RET locus. Eur J Hum Genet 11(10):760– 769 Doray B, Salomon R, Amiel J, Pelet A, Touraine R, Billaud M, Attie T, Bachy B, Munnich A, Lyonnet S (1998) Mutation of the RET ligand, neurturin, supports multigenic inheritance in Hirschsprung disease. Hum Mol Genet 7:1449–1452 Edery P, Lyonnet S, Mulligan LM, Pelet A, Dow E, Abel L, Holder S, Nihoul-Fekete C, Ponder BA, Munnich, A (1994) Mutations of the RET proto-oncogene in Hirschsprung’s disease. Nature 367:378–380
174 Fitze G, Schreiber M, Kuhlisch E, Schackert HK, Roesner D (1999) Association of RET protooncogene codon 45 polymorphism with Hirschsprung disease. Am J Hum Genet 65: 1469– 1473 Fitze G, Cramer J, Ziegler A, Schierz M, Schreiber M, Kuhlisch E, Roesner D, Schackert HK (2002) Association between c135G/A genotype and RET proto-oncogene germline mutations and phenotype of Hirschsprung’s disease. Lancet 359:1200–1205 Fitze G, Appelt H, Konig IR, Gorgens H, Stein U, Walther W, Gossen M, Schreiber M, Ziegler A, Roesner D, Schackert HK (2003) Functional haplotypes of the RET proto-oncogene promoter are associated with Hirschsprung disease (HSCR). Hum Mol Genet 12:3207–3214 Frost G (1992) Hirschsprung disease in infants and children. Gastroenterol Nurs 15:45–48 Gabriel SB, Salomon R, Pelet A, Angrist M, Amiel J, Fornage M, Attie-Bitach T, Olson, JM, Hofstra R, Buys C, Steffann J, Munnich A, Lyonnet S, Chakravarti A (2002) Segregation at three loci explains familial and population risk in Hirschsprung disease. Nat Genet 31:89–93 Garcia-Barcelo MM, Sham MH, Lui VC, Chen BL, Song YQ, Lee WS, Yung SK, Romeo G, Tam PK (2003) Chinese patients with sporadic Hirschsprung’s disease are predominantly represented by a single RET haplotype. J Med Genet 40:e122 Garcia-Barcelo M, Sham MH, Lee WS, Lui VC, Chen BL, Wong KK, Wong JS, Tam PK (2004) Highly recurrent RET mutations and novel mutations in genes of the receptor tyrosine kinase and endothelin receptor B pathways in Chinese patients with sporadic Hirschsprung disease. Clin Chem 50:93–100 Gath R, Goessling A, Keller KM, Koletzko S, Coerdt W, Muntefering H, Wirth S, Hofstra RM, Mulligan L, Eng C, von Deimling A (2001) Analysis of the RET, GDNF, EDN3, and EDNRB genes in patients with intestinal neuronal dysplasia and Hirschsprung disease. Gut 48:671–675 Geneste O, Bidaud C, De Vita G, Hofstra RM, Tartare-Deckert S, Buys CH, Lenoir GM, Santoro M, Billaud, M (1999) Two distinct mutations of the RET receptor causing Hirschsprung’s disease impair the binding of signalling effectors to a multifunctional docking site. Hum Mol Genet 8: 1989–1999 Hofstra RM, Osinga J, Tan-Sindhunata G, Wu Y, Kamsteeg EJ, Stulp RP, van Ravenswaaij-Arts C, Majoor-Krakauer D, Angrist M, Chakravarti A, Meijers C, Buys CH (1996) A homozygous mutation in the endothelin-3 gene associated with a combined Waardenburg type 2 and Hirschsprung phenotype (Shah-Waardenburg syndrome). Nat Genet 12:445–447 Hofstra RM, Valdenaire O, Arch E, Osinga J, Kroes H, Loffler BM, Hamosh A, Meijers C, Buys CH (1999) A loss-of-function mutation in the endothelin-converting enzyme 1 (ECE-1) associated with Hirschsprung disease, cardiac defects, and autonomic dysfunction. Am J Hum Genet 64:304–308 Kusafuka T, Puri P (1998) Genetic aspects of Hirschsprung’s disease. Semin Pediatr Surg 7:148–155
Lesser PB, El-Nahas AM, Lukl P, Andrews P, Schuler JG, Filtzer HS (1979) Adult-onset Hirschsprung’s disease. Jama 242:747– 748 Lorenzo MJ, Gish GD, Houghton C, Stonehouse TJ, Pawson T, Ponder BA, Smith DP (1997) RET alternate splicing influences the interaction of activated RET with the SH2 and PTB domains of Shc, and the SH2 domain of Grb2.Oncogene 14:763– 771 Lyonnet S, Bolino A, Pelet A, Abel L, Nihoul-Fekete C, Briard M., Mok-Siu V, Kaariainen H, Martucciello G, Lerone M (1993) A gene for Hirschsprung disease maps to the proximal long arm of chromosome 10. Nat Genet 4:346–350 Pingault V, Bondurand N, Kuhlbrodt K, Goerich DE, Prehu MO, Puliti A, Herbarth B, Hermans-Borgmeyer I, Legius E, Matthijs G, Amiel J, Lyonnet S, Ceccherini I, Romeo G, Smith JC, Read AP, Wegner M, Goossens M (1998) SOX10 mutations in patients with Waardenburg-Hirschsprung disease. Nat Genet 18:171–173 Puffenberger EG, Hosoda K, Washington SS, Nakao K, deWit D, Yanagisawa M, Chakravart A (1994) A missense mutation of the endothelin-B receptor gene in multigenic Hirschsprung’s disease. Cell 79:1257–1266 Reyna TM (1993) Hirschsprung’s disease. J Pediatr Surg 28:1522– 1523 Romeo G, Ronchetto P, Luo Y, Barone V, Seri M, Ceccherini I, Pasini B, Bocciardi R, Lerone M, Kaariainen H (1994) Point mutations affecting the tyrosine kinase domain of the RET proto-oncogene in Hirschsprung’s disease. Nature 367:377–378 Sakai T, Nirasawa Y, Itoh Y, Wakizaka A (2000) Japanese patients with sporadic Hirschsprung: mutation analysis of the receptor tyrosine kinase proto-oncogene, endothelin-B receptor, endothelin-3, glial cell line-derived neurotrophic factor and neurturin genes: a comparison with similar studies. Eur J Pediatr 159:160–167 Seri M, Yin L, Barone V, Bolino A, Celli I, Bocciardi R, Pasini B, Ceccherini I, Lerone M, Kristoffersson U, Larsson LT, Casasa JM, Cass DT, Abramowicz MJ, Vanderwinden JM, Kravcenkiene I, Baric I, Silengo M, Martucciello G, Romeo G (1997) Frequency of RET mutations in long- and short-segment Hirschsprung disease.Hum Mutat 9:243–9 Svensson PJ, Molander ML, Eng C, Anvret M, Nordenskjold A (1998) Low frequency of RET mutations in Hirschsprung disease in Sweden. Clin Genet 54:39–44 Takahashi M, Buma Y, Iwamoto T, Inaguma Y, Ikeda H, Hiai H (1988) Cloning and expression of the ret proto-oncogene encoding a tyrosine kinase with two potential transmembrane domains.Oncogene 3:571–578 Wakamatsu N, Yamada Y, Yamada K, Ono T, Nomura N, Taniguchi H, Kitoh H, Mutoh N, Yamanaka T, Mushiake K, Kato K, Sonta S, Nagaya M (2001) Mutations in SIP1, encoding Smad interacting protein-1, cause a form of Hirschsprung disease. Nat Genet 27:369–370