Introduction

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 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; Garcia-Barcelo 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

Patients and control samples

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. 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-μl 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 37°C 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 (χ2) analysis was used to determine the significance of the association of allele frequencies between patients and normal control groups.

Results

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 Δ1059 and L1061P indicates that Y1062 is a critical multifunctional intracytoplasmic 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).

Fig. 1
figure 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

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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; 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). 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. (Garcia-Barcelo 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 nucleotide 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 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.

Table 1 Allele frequencies of RET and EDNRB polymorphisms in Hirschsprung disease (HSCR )patients and controls
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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
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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 LSA/TCA patients were homozygous for allele A of c135G>A and allele G of c2307T>G (82%).

Table 3 Frequencies of RET genotypes in Hirschsprung disease (HSCR) patients and controls in Taiwan
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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.

Table 4 Allele frequencies of RET polymorphisms in Hirschsprung disease (HSCR) patients and parents
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Table 5 Transmission disequilibrium test (TDT) by individual polymorphic locus in RET
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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 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 disease-causing 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 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.