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NTN or SOX10 gene mutations and long-term results after surgery for total colonic aganglionosis with small bowel involvement
Relationship Between the Type of RET/GDNF/NTN or SOX10 Gene Mutations and Long-Term Results After Surgery for Total Colonic Aganglionosis With Small Bowel Involvement By Hideki Tomiyama, Takashi Shimotake, Shigeru Ono, Osamu Kimura, Kazuaki Tokiwa, and Naomi Iwai Kyoto, Japan
Background/Purpose: Germline mutations of the RET-mediated or SOX10-mediated signaling pathway genes have been reported in total colonic aganglionosis (TCA). The authors investigated the possible relationship between the type of such genomic abnormalities and surgical outcomes.
total intestinal aganglionosis, only 2 survived beyond 2 years of age, both of whom underwent Ziegler’s myectomy-myotomy. A SOX10 mutation was identified in an infant with Shah-Waardenburg’s syndrome, and he showed persistent bowel malfunction.
Methods: Sixteen patients with TCA with extensive small bowel involvement were studied. DNA sequences of all the RET/GDNF/NTN and SOX10 coding regions were determined by the direct DyeDeoxy Terminator Cycle method. Data on the patients’ clinical courses were obtained retrospectively from their medical charts and surgical records.
Conclusion: The existence or type of RET mutation usually did not affect surgical results in this series of TCA patients, whereas the mutational analysis suggested 2 disease categories of TCA showing different postoperative courses, which may reflect the disparate pathogenesis in the enteric nervous system development induced by impaired RET or SOX10 signaling pathway. J Pediatr Surg 36:1685-1688. Copyright © 2001 by W.B. Saunders Company.
Results: RET or SOX10 germline mutations were identified in 11 of the 16 patients (68.8%). In children with aganglionosis up to the jejunum or ileum, most grew up within normal ranges, and the frequency of bowel movements decreased to 2 to 4 times per day within 5 years. However, in 5 infants with
H
IRSCHSPRUNG’S DISEASE is characterized by the absence of intramural ganglion cells in the distal gut, resulting in bowel obstruction in infancy and childhood.1 The majority of enteric ganglion cells are derived from the vagal neural crest and migrate into the intestinal wall in a caudal direction between the 5th and 12th gestational weeks.2 Total colonic aganglionosis (TCA) is a rare condition in which aganglionosis involves the entire colon and, in other rare cases, the more proximal bowel.3,4 Recently, germline mutations of RET, its natural ligand of glial cell– derived neurotrophic factor (GDNF), neurturin (NTN), and the SOX10 gene have been reported in patients with TCA.5-7 However, the frequency, location, and nature of possible gene mutations in TCA remain unclear, and the question of whether the existence or type of such gene mutations can predict surgical outcomes of TCA children remains unanswered. In this study, we investigated the possible relationship between the type of genomic abnormalities and surgical outcomes in 16 children with TCA with small bowel involvement. MATERIALS AND METHODS From 1982 to 2000, we treated 16 children with total colonic aganglionosis with extensive small bowel involvement. The diagnosis
INDEX WORDS: Hirschsprung’s disease, RET, SOX10, longterm results.
of intestinal aganglionosis was made based on histopathology, and the levels of involvement of aganglionic bowel segments were determined by intraoperative leveling biopsies from the stomach, duodenum, jejunum, ileum, and colon. Surgery was performed at the age of 7 months to 2 years using a modified Martin’s procedure (using GIA stapler) for TCA patients with mild small bowel involvement,3 whereas proximal jejunostomy with myectomy-myotomy following Ziegler’s method was used for children with extensive bowel involvement up to the duodenum or stomach.8
RET/GDNF/NTN and SOX10 Gene Analysis Genomic DNAs were extracted from peripheral blood lymphocytes from the patients and their parents after informed consent was obtained. Polymerase chain reaction (PCR) of exons 1 to 20 of the RET
From the Division of Surgery, Children’s Research Hospital, Kyoto Prefectural University of Medicine, Kyoto, Japan. Presented at the 34th Annual Meeting of the Pacific Association of Pediatric Surgeons, Kyoto, Japan, April 4-8, 2001. This work was supported by grants from the Scientific Research Fund of the Ministry of Education, Science, and Culture of Japan (Nos. 13671870 & 13557146). Address reprint requests to Hideki Tomiyama, MD, Division of Surgery, Children’s Research Hospital, Kyoto Prefectural University of Medicine, 465 Kawaramachi-Hirokoji, Kamigyo-ku, Kyoto 6020841, Japan. Copyright © 2001 by W.B. Saunders Company 0022-3468/01/3611-0020$35.00/0 doi:10.1053/jpsu.2001.27960
Journal of Pediatric Surgery, Vol 36, No 11 (November), 2001: pp 1685-1688
1685
1686
(10q11.2), exons 1 and 2 of the GDNF (5p13.1-p12), exons 1 and 2 of the NTN gene (19p13.3), and exons 3 to 5 of the SOX10 gene (22q13) was done using oligonucleotide primers as previously described.7,9-10 Amplification was achieved with a reaction mixture (40 L) containing 100 ng of lymphocyte DNA, 2.5 mol/L of each set of primers, 200 mol/L dNTP, 10 mmol/L Tris HCl, pH 8.3, 1.5 mmol/L MgCl2, 50 mmol/L KCl, and 1.0U Taq DNA polymerase. Thermocycling conditions comprised denaturation at 95°C for 9 minutes, 45 cycles of annealing at 60°C for 2 minutes, and extension at 95°C for 30 seconds, followed by a denaturing step at 60°C for 3 minutes in an automatic thermocycler (GeneAmp PCR System 2400; Perkin Elmer, Norwalk, CT). DNA sequencing was carried out using the direct DyeDeoxy Terminator Cycle method with a fluorescent automatic DNA sequencer (Model 373A DNA Sequencing systems; Applied Biosystems, Foster City, CA). In cases in which an abnormal DNA direct sequence pattern was exhibited, amplicon cloning was performed to confirm the presence of gene mutations. The PCR products were ligated with a pMosBlue TA-cloning plasmid vector (Amersham Pharmacia Biotech, Buckinghamshire, England) and subcloned in a routine way. DNA from the plasmid vector was extracted and sequenced using an automated DNA sequencer.
Assessment of TCA Patients’ Clinical Courses Postoperative follow-up for children with TCA ranged from 3 years to 18 years. As nutritional and growth indicators, height and body weight were recorded in each case. For clinical assesment of bowel function, the number of bowel movements per day and consistency of
TOMIYAMA ET AL
stools were consecutively recorded at 6 months, 1, 2, 3, 5, 7, 10, 13, 15, and 17 years after definitive surgery for intestinal aganglionosis.
RESULTS
In the DNA analysis, 8 different types of RET germline mutations were found in 10 of the 16 children (Fig 1). Of the 10 patients, 3 (1 boy and 2 girls) were siblings from the same kindred. A SOX10 mutation was found in a male infant with TCA associated with congenital deafness, skin hypopigmentation, and iris heterochromia. ShahWaardenburg’s syndrome was diagnosed. No GDNF or NTN gene mutations were identified. In the postoperative assessments, most TCA patients with limited small bowel involvement showed satisfactory growth, and height and body weight remained within normal ranges (Fig 2). In these children, the frequency of bowel movements decreased gradually to 2 to 5 times per day, and the stools became loose or formed patterns within 5 years of surgery. However, in one patient, the number of stools rapidly increased up to 8 to 9 times per day at the age of 5 years (Fig 3). Further examination verified an internal fistula between the jejunum and the residual colonic pouch. After resection of
Fig 1. Results of RET and SOX10 mutational analysis in 16 patients with total colonic aganglionosis. RET mutations were found in 10 of the 16 patients and appeared to be accumulated in the tyrosine kinase domain. The male:female ratio of the 10 patients carrying RET mutations was 5:5. On the other hand, 5 patients without RET or SOX10 mutations were all boys. Asterisks indicate syndromic Hirschsprung’s disease. Type of mutation indicates RET codon number, normal and altered amino acid codes. Del, deletion; Cd, cadherinlike homology; TM, transmembrane domain.
GENE MUTATIONS IN TCA
Fig 2. Height and body weight of the patients with total colonic aganglionosis with reference to possible RET or SOX10 gene mutations. Most patients with mild small bowel involvement showed satisfactory growth and height and body weight remained within ⴞ 2 SD values irrespective of the type of RET mutation.
the fistula she had 2 to 3 bowel movements per day, and the stools returned to a formed pattern. However, in 5 infants who had total intestinal aganglionosis, the stools remained watery. Of these 5, only 2 survived beyond 2 years of age. The remaining 3 patients could not lead normal lives because of enteritis, sepsis, or TPN-associated liver dysfunction.11,12 A mutated SOX10 gene carrier presenting with Shah-Waardenburg’s syndrome showed persistent bowel malfunction. His stools remained watery for long periods, and he suffered from repeated enteritis. DISCUSSION
In recent years, the pathogenesis of intestinal aganglionosis has been disclosed by varied basic and clinical studies. Genetic analyses have suggested that Hirschsprung’s disease is a multigenic disorder caused by dysfunction of the Ret-GDNF, the EDN3-EDNRB receptor, or the SOX10-mediated signaling pathway or genetic transcription factor.7,13,14 Histologic studies in murine models derived from natural mutants (sl/sl, ls/ls, Dom) or gene targeting technology (such as ret, gdnf, edn3,
1687
ednrb, and sox10 knockouts) clearly have shown the absence of enteric ganglia in the distal gut.15,16 In addition, microenvironmental factors causing the migration arrest or the deficient innervation of neural crest– derived cells may play significant roles in the pathogenesis of Hirschsprung’s disease.17,18 In patients with TCA, aganglionosis involves the entire colon, leading to severe disruption of enteric neuroblast development. Previous reports frequently have described RET and SOX10 mutations in children with extensive forms of intestinal aganglionosis.5,7,19 In this study, we found RET and SOX10 gene mutations in 10 and 1 of the 16 TCA children, respectively. Compared with the results of mutational analysis in typical Hirschsprung’s disease (short segment aganglionosis), this patient group appeared to exhibit a higher percentage of RET mutations and accumulation of mutations in the tyrosine kinase domain (Fig 1).20 The male to female ratio of the 10 TCA patients carrying RET mutations was 5:5. However, 5 patients without RET or SOX10 mutations were all boys. These results suggest that RET mutations causing severe disruption of the RET signal transduction may originate in severe types of intestinal aganglionosis, regardless of gender. In clinical analysis, we studied the patients’ body weight, height, frequency of bowel movements, and consistency of stools to examine whether the type of RET or SOX10 mutation can predict surgical outcomes of TCA children. In RET mutational carriers, the type of genomic alteration did not significantly affect the longterm results after surgery for TCA. Instead, it appeared to depend primarily on the length of intestinal aganglionosis. In this series of patients, only 2 of 5 infants with total intestinal aganglionosis survived beyond the age of 2 years. These 2 patients can feed enterally, but parenteral nutritional support has not been discontinued completely in either case. Considering that the outlook for gut absorption and adaptation is hardly optimistic in patients with total intestinal aganglionosis, this disease entity remains a candidate for intestinal transplantation, irrespective of the type of gene mutation.12,21,22 A male infant carrying a SOX10 mutation showed persistent bowel malfunction despite limited small bowel involvement by aganglionosis. His stools remained watery and he showed repeated enteritis. The gut malabsorption and susceptibility to enteritis in this case were barely improved compared with those in mutated RET gene carriers presenting with TCA. These results may classify TCA patients into 2 disease categories of TCA showing different postoperative courses. These differences between mutated RET and SOX10 carriers may reflect the disparate pathogenesis in the enteric nervous system development induced by impaired RET-mediated or SOX10-mediated signaling pathway.
1688
TOMIYAMA ET AL
Fig 3. Frequency of bowel movements in the 16 patients after surgery for total colonic aganglionosis. In most patients with mild small bowel aganglionosis involvement, the number of bowel movements gradually decreased to 2 to 5 times per day, and the stools changed to a formed pattern within 5 years of surgery. However, in an infant with Shah-Waardenburg’s syndrome, the stools were persistently loose and enteritis frequently occurred.
REFERENCES 1. Swenson O, Bill AH: Resection of rectum and rectosigmoid with preservation of the sphincter for benign spastic lesion producing megacolon. Surgery 24:212-220, 1948 2. Okamoto E, Ueda T: Embryogenesis of intramural ganglia of the gut and its relation to Hirschsprung’s disease. J Pediatr Surg 2:437-243, 1967 3. Martin LW: Surgical management of Hirschsprung’s disease involving the small intestine. Arch Surg 97:183-189, 1968 4. Caniano DA, Ormsbee HS III, Polito W, et al: Total intestinal aganglionosis. J Pediatr Surg 20:456-460, 1985 5. Edery P, Lyonnet S, Mulligan LM, et al: Mutations of the RET proto-oncogene in Hirschsprung’s disease. Nature 367:378-380, 1994 6. Ivanchuk SM, Myers SM, Eng C, et al: De novo mutation of GDNF, ligand for the RET/GDNFR-alpha receptor complex, in Hirschsprung disease. Hum Mol Genet 5:2023-2026, 1996 7. Pingault V, Bondurand N, Kuhlbrodt K, et al: SOX10 mutations in patients with Waardenburg-Hirschsprung disease. Nat Genet 18:171173, 1998 8. Ziegler MM, Ross AJ, Bishop HC: Total intestinal aganglionosis: A new technique for prolonged survival. J Pediatr Surg 22:82-83, 1987 9. Ceccherini I, Hofstra RM, Luo Y, et al: DNA polymorphisms and conditions for SSCP analysis of the 20 exons of the ret proto-oncogene. Oncogene 9:3025-3029, 1994 10. Doray B, Salomon R, Amiel J, et al: Mutations of the RET ligand, neurturin, supports multigenic inheritance in Hirschsprung’s disease. Hum Mol Genet 7:1449-1452, 1998 11. Moss RL, Das JB, Raffensperger JG: Total pareteral nutritionassociated cholestasis: Clinical and histopathologic correlation. J Pediatr Surg 28:1270-1275, 1993 12. Zerella JT, Ingebo KR: A 3-year survivor of near-total intestinal aganglionosis. J Pediatr Surg 28:1589-1591, 1993
13. Puffenberger EG, Hosoda K, Washington SS, et al: A missense mutation of the endothelin-B receptor gene in multigenic Hirschsprung’s disease. Cell 79:1257-1266, 1994 14. Pasini B, Borrello MG, Greco A, et al: Loss of function effect of RET mutations causing Hirschsprung disease. Nat Genet 10:35-40, 1995 15. Schuchardt A, D’Agati V, Larsson-Blomberg L, et al: Defects in the kidney and enteric nervous system of mice lacking the tyrosine kinase receptor Ret. Nature 367:380-383, 1994 16. Baynash AG, Hosoda K, Giaid A, et al: Interaction of endothelin-3 with endothelin-B receptor is essential for development of epidermal melanocytes and enteric neurons. Cell 30:1277-1285, 1994 17. Shimotake T, Iwai N, Yanagihara J, et al: Impaired proliferative activity of mesenchymal cells affects the migratory pathway for neural crest cells in the developing gut of mutant murine embryos. J Pediatr Surg 30:445-447, 1995 18. Jacob-Cohen RJ, Payette RF, Gershon MD, et al: Inability of neural crest cells to colonize the presumptive aganglionic bowel of ls/ls mutant mice: Requirement for a permissive microenvironment. J Comp Neurol 255:425-438, 1987 19. Attie T, Pelet A, Edery P, et al: Diversity of RET protooncogene mutations in familial and sporadic Hirschsprung’s disease. Hum Mol Genet 4:1381-1386, 1995 20. Inoue K, Shimotake T, Iwai N: Mutational analysis of RET/ GDNF/NTN genes in children with total colonic aganglionosis with small bowel involvement. Am J Med Genet 93:278-284, 2000 21. MacKinnon AE, Cohen SJ: Total intestinal aganglionosis, an autosomal recessive condition? Arch Dis Child 52:898-899, 1977 22. Di Lorenzo M, Yazbeck S, Brochu P: Aganglionosis of the entire bowel: Four new cases and review of the literature. Br J Surg 72:657-658, 1985
Background/Purpose: Germline mutations of the RET-mediated or SOX10-mediated signaling pathway genes have been reported in total colonic aganglionosis (TCA). The authors investigated the possible relationship between the type of such genomic abnormalities and surgical outcomes.
total intestinal aganglionosis, only 2 survived beyond 2 years of age, both of whom underwent Ziegler’s myectomy-myotomy. A SOX10 mutation was identified in an infant with Shah-Waardenburg’s syndrome, and he showed persistent bowel malfunction.
Methods: Sixteen patients with TCA with extensive small bowel involvement were studied. DNA sequences of all the RET/GDNF/NTN and SOX10 coding regions were determined by the direct DyeDeoxy Terminator Cycle method. Data on the patients’ clinical courses were obtained retrospectively from their medical charts and surgical records.
Conclusion: The existence or type of RET mutation usually did not affect surgical results in this series of TCA patients, whereas the mutational analysis suggested 2 disease categories of TCA showing different postoperative courses, which may reflect the disparate pathogenesis in the enteric nervous system development induced by impaired RET or SOX10 signaling pathway. J Pediatr Surg 36:1685-1688. Copyright © 2001 by W.B. Saunders Company.
Results: RET or SOX10 germline mutations were identified in 11 of the 16 patients (68.8%). In children with aganglionosis up to the jejunum or ileum, most grew up within normal ranges, and the frequency of bowel movements decreased to 2 to 4 times per day within 5 years. However, in 5 infants with
H
IRSCHSPRUNG’S DISEASE is characterized by the absence of intramural ganglion cells in the distal gut, resulting in bowel obstruction in infancy and childhood.1 The majority of enteric ganglion cells are derived from the vagal neural crest and migrate into the intestinal wall in a caudal direction between the 5th and 12th gestational weeks.2 Total colonic aganglionosis (TCA) is a rare condition in which aganglionosis involves the entire colon and, in other rare cases, the more proximal bowel.3,4 Recently, germline mutations of RET, its natural ligand of glial cell– derived neurotrophic factor (GDNF), neurturin (NTN), and the SOX10 gene have been reported in patients with TCA.5-7 However, the frequency, location, and nature of possible gene mutations in TCA remain unclear, and the question of whether the existence or type of such gene mutations can predict surgical outcomes of TCA children remains unanswered. In this study, we investigated the possible relationship between the type of genomic abnormalities and surgical outcomes in 16 children with TCA with small bowel involvement. MATERIALS AND METHODS From 1982 to 2000, we treated 16 children with total colonic aganglionosis with extensive small bowel involvement. The diagnosis
INDEX WORDS: Hirschsprung’s disease, RET, SOX10, longterm results.
of intestinal aganglionosis was made based on histopathology, and the levels of involvement of aganglionic bowel segments were determined by intraoperative leveling biopsies from the stomach, duodenum, jejunum, ileum, and colon. Surgery was performed at the age of 7 months to 2 years using a modified Martin’s procedure (using GIA stapler) for TCA patients with mild small bowel involvement,3 whereas proximal jejunostomy with myectomy-myotomy following Ziegler’s method was used for children with extensive bowel involvement up to the duodenum or stomach.8
RET/GDNF/NTN and SOX10 Gene Analysis Genomic DNAs were extracted from peripheral blood lymphocytes from the patients and their parents after informed consent was obtained. Polymerase chain reaction (PCR) of exons 1 to 20 of the RET
From the Division of Surgery, Children’s Research Hospital, Kyoto Prefectural University of Medicine, Kyoto, Japan. Presented at the 34th Annual Meeting of the Pacific Association of Pediatric Surgeons, Kyoto, Japan, April 4-8, 2001. This work was supported by grants from the Scientific Research Fund of the Ministry of Education, Science, and Culture of Japan (Nos. 13671870 & 13557146). Address reprint requests to Hideki Tomiyama, MD, Division of Surgery, Children’s Research Hospital, Kyoto Prefectural University of Medicine, 465 Kawaramachi-Hirokoji, Kamigyo-ku, Kyoto 6020841, Japan. Copyright © 2001 by W.B. Saunders Company 0022-3468/01/3611-0020$35.00/0 doi:10.1053/jpsu.2001.27960
Journal of Pediatric Surgery, Vol 36, No 11 (November), 2001: pp 1685-1688
1685
1686
(10q11.2), exons 1 and 2 of the GDNF (5p13.1-p12), exons 1 and 2 of the NTN gene (19p13.3), and exons 3 to 5 of the SOX10 gene (22q13) was done using oligonucleotide primers as previously described.7,9-10 Amplification was achieved with a reaction mixture (40 L) containing 100 ng of lymphocyte DNA, 2.5 mol/L of each set of primers, 200 mol/L dNTP, 10 mmol/L Tris HCl, pH 8.3, 1.5 mmol/L MgCl2, 50 mmol/L KCl, and 1.0U Taq DNA polymerase. Thermocycling conditions comprised denaturation at 95°C for 9 minutes, 45 cycles of annealing at 60°C for 2 minutes, and extension at 95°C for 30 seconds, followed by a denaturing step at 60°C for 3 minutes in an automatic thermocycler (GeneAmp PCR System 2400; Perkin Elmer, Norwalk, CT). DNA sequencing was carried out using the direct DyeDeoxy Terminator Cycle method with a fluorescent automatic DNA sequencer (Model 373A DNA Sequencing systems; Applied Biosystems, Foster City, CA). In cases in which an abnormal DNA direct sequence pattern was exhibited, amplicon cloning was performed to confirm the presence of gene mutations. The PCR products were ligated with a pMosBlue TA-cloning plasmid vector (Amersham Pharmacia Biotech, Buckinghamshire, England) and subcloned in a routine way. DNA from the plasmid vector was extracted and sequenced using an automated DNA sequencer.
Assessment of TCA Patients’ Clinical Courses Postoperative follow-up for children with TCA ranged from 3 years to 18 years. As nutritional and growth indicators, height and body weight were recorded in each case. For clinical assesment of bowel function, the number of bowel movements per day and consistency of
TOMIYAMA ET AL
stools were consecutively recorded at 6 months, 1, 2, 3, 5, 7, 10, 13, 15, and 17 years after definitive surgery for intestinal aganglionosis.
RESULTS
In the DNA analysis, 8 different types of RET germline mutations were found in 10 of the 16 children (Fig 1). Of the 10 patients, 3 (1 boy and 2 girls) were siblings from the same kindred. A SOX10 mutation was found in a male infant with TCA associated with congenital deafness, skin hypopigmentation, and iris heterochromia. ShahWaardenburg’s syndrome was diagnosed. No GDNF or NTN gene mutations were identified. In the postoperative assessments, most TCA patients with limited small bowel involvement showed satisfactory growth, and height and body weight remained within normal ranges (Fig 2). In these children, the frequency of bowel movements decreased gradually to 2 to 5 times per day, and the stools became loose or formed patterns within 5 years of surgery. However, in one patient, the number of stools rapidly increased up to 8 to 9 times per day at the age of 5 years (Fig 3). Further examination verified an internal fistula between the jejunum and the residual colonic pouch. After resection of
Fig 1. Results of RET and SOX10 mutational analysis in 16 patients with total colonic aganglionosis. RET mutations were found in 10 of the 16 patients and appeared to be accumulated in the tyrosine kinase domain. The male:female ratio of the 10 patients carrying RET mutations was 5:5. On the other hand, 5 patients without RET or SOX10 mutations were all boys. Asterisks indicate syndromic Hirschsprung’s disease. Type of mutation indicates RET codon number, normal and altered amino acid codes. Del, deletion; Cd, cadherinlike homology; TM, transmembrane domain.
GENE MUTATIONS IN TCA
Fig 2. Height and body weight of the patients with total colonic aganglionosis with reference to possible RET or SOX10 gene mutations. Most patients with mild small bowel involvement showed satisfactory growth and height and body weight remained within ⴞ 2 SD values irrespective of the type of RET mutation.
the fistula she had 2 to 3 bowel movements per day, and the stools returned to a formed pattern. However, in 5 infants who had total intestinal aganglionosis, the stools remained watery. Of these 5, only 2 survived beyond 2 years of age. The remaining 3 patients could not lead normal lives because of enteritis, sepsis, or TPN-associated liver dysfunction.11,12 A mutated SOX10 gene carrier presenting with Shah-Waardenburg’s syndrome showed persistent bowel malfunction. His stools remained watery for long periods, and he suffered from repeated enteritis. DISCUSSION
In recent years, the pathogenesis of intestinal aganglionosis has been disclosed by varied basic and clinical studies. Genetic analyses have suggested that Hirschsprung’s disease is a multigenic disorder caused by dysfunction of the Ret-GDNF, the EDN3-EDNRB receptor, or the SOX10-mediated signaling pathway or genetic transcription factor.7,13,14 Histologic studies in murine models derived from natural mutants (sl/sl, ls/ls, Dom) or gene targeting technology (such as ret, gdnf, edn3,
1687
ednrb, and sox10 knockouts) clearly have shown the absence of enteric ganglia in the distal gut.15,16 In addition, microenvironmental factors causing the migration arrest or the deficient innervation of neural crest– derived cells may play significant roles in the pathogenesis of Hirschsprung’s disease.17,18 In patients with TCA, aganglionosis involves the entire colon, leading to severe disruption of enteric neuroblast development. Previous reports frequently have described RET and SOX10 mutations in children with extensive forms of intestinal aganglionosis.5,7,19 In this study, we found RET and SOX10 gene mutations in 10 and 1 of the 16 TCA children, respectively. Compared with the results of mutational analysis in typical Hirschsprung’s disease (short segment aganglionosis), this patient group appeared to exhibit a higher percentage of RET mutations and accumulation of mutations in the tyrosine kinase domain (Fig 1).20 The male to female ratio of the 10 TCA patients carrying RET mutations was 5:5. However, 5 patients without RET or SOX10 mutations were all boys. These results suggest that RET mutations causing severe disruption of the RET signal transduction may originate in severe types of intestinal aganglionosis, regardless of gender. In clinical analysis, we studied the patients’ body weight, height, frequency of bowel movements, and consistency of stools to examine whether the type of RET or SOX10 mutation can predict surgical outcomes of TCA children. In RET mutational carriers, the type of genomic alteration did not significantly affect the longterm results after surgery for TCA. Instead, it appeared to depend primarily on the length of intestinal aganglionosis. In this series of patients, only 2 of 5 infants with total intestinal aganglionosis survived beyond the age of 2 years. These 2 patients can feed enterally, but parenteral nutritional support has not been discontinued completely in either case. Considering that the outlook for gut absorption and adaptation is hardly optimistic in patients with total intestinal aganglionosis, this disease entity remains a candidate for intestinal transplantation, irrespective of the type of gene mutation.12,21,22 A male infant carrying a SOX10 mutation showed persistent bowel malfunction despite limited small bowel involvement by aganglionosis. His stools remained watery and he showed repeated enteritis. The gut malabsorption and susceptibility to enteritis in this case were barely improved compared with those in mutated RET gene carriers presenting with TCA. These results may classify TCA patients into 2 disease categories of TCA showing different postoperative courses. These differences between mutated RET and SOX10 carriers may reflect the disparate pathogenesis in the enteric nervous system development induced by impaired RET-mediated or SOX10-mediated signaling pathway.
1688
TOMIYAMA ET AL
Fig 3. Frequency of bowel movements in the 16 patients after surgery for total colonic aganglionosis. In most patients with mild small bowel aganglionosis involvement, the number of bowel movements gradually decreased to 2 to 5 times per day, and the stools changed to a formed pattern within 5 years of surgery. However, in an infant with Shah-Waardenburg’s syndrome, the stools were persistently loose and enteritis frequently occurred.
REFERENCES 1. Swenson O, Bill AH: Resection of rectum and rectosigmoid with preservation of the sphincter for benign spastic lesion producing megacolon. Surgery 24:212-220, 1948 2. Okamoto E, Ueda T: Embryogenesis of intramural ganglia of the gut and its relation to Hirschsprung’s disease. J Pediatr Surg 2:437-243, 1967 3. Martin LW: Surgical management of Hirschsprung’s disease involving the small intestine. Arch Surg 97:183-189, 1968 4. Caniano DA, Ormsbee HS III, Polito W, et al: Total intestinal aganglionosis. J Pediatr Surg 20:456-460, 1985 5. Edery P, Lyonnet S, Mulligan LM, et al: Mutations of the RET proto-oncogene in Hirschsprung’s disease. Nature 367:378-380, 1994 6. Ivanchuk SM, Myers SM, Eng C, et al: De novo mutation of GDNF, ligand for the RET/GDNFR-alpha receptor complex, in Hirschsprung disease. Hum Mol Genet 5:2023-2026, 1996 7. Pingault V, Bondurand N, Kuhlbrodt K, et al: SOX10 mutations in patients with Waardenburg-Hirschsprung disease. Nat Genet 18:171173, 1998 8. Ziegler MM, Ross AJ, Bishop HC: Total intestinal aganglionosis: A new technique for prolonged survival. J Pediatr Surg 22:82-83, 1987 9. Ceccherini I, Hofstra RM, Luo Y, et al: DNA polymorphisms and conditions for SSCP analysis of the 20 exons of the ret proto-oncogene. Oncogene 9:3025-3029, 1994 10. Doray B, Salomon R, Amiel J, et al: Mutations of the RET ligand, neurturin, supports multigenic inheritance in Hirschsprung’s disease. Hum Mol Genet 7:1449-1452, 1998 11. Moss RL, Das JB, Raffensperger JG: Total pareteral nutritionassociated cholestasis: Clinical and histopathologic correlation. J Pediatr Surg 28:1270-1275, 1993 12. Zerella JT, Ingebo KR: A 3-year survivor of near-total intestinal aganglionosis. J Pediatr Surg 28:1589-1591, 1993
13. Puffenberger EG, Hosoda K, Washington SS, et al: A missense mutation of the endothelin-B receptor gene in multigenic Hirschsprung’s disease. Cell 79:1257-1266, 1994 14. Pasini B, Borrello MG, Greco A, et al: Loss of function effect of RET mutations causing Hirschsprung disease. Nat Genet 10:35-40, 1995 15. Schuchardt A, D’Agati V, Larsson-Blomberg L, et al: Defects in the kidney and enteric nervous system of mice lacking the tyrosine kinase receptor Ret. Nature 367:380-383, 1994 16. Baynash AG, Hosoda K, Giaid A, et al: Interaction of endothelin-3 with endothelin-B receptor is essential for development of epidermal melanocytes and enteric neurons. Cell 30:1277-1285, 1994 17. Shimotake T, Iwai N, Yanagihara J, et al: Impaired proliferative activity of mesenchymal cells affects the migratory pathway for neural crest cells in the developing gut of mutant murine embryos. J Pediatr Surg 30:445-447, 1995 18. Jacob-Cohen RJ, Payette RF, Gershon MD, et al: Inability of neural crest cells to colonize the presumptive aganglionic bowel of ls/ls mutant mice: Requirement for a permissive microenvironment. J Comp Neurol 255:425-438, 1987 19. Attie T, Pelet A, Edery P, et al: Diversity of RET protooncogene mutations in familial and sporadic Hirschsprung’s disease. Hum Mol Genet 4:1381-1386, 1995 20. Inoue K, Shimotake T, Iwai N: Mutational analysis of RET/ GDNF/NTN genes in children with total colonic aganglionosis with small bowel involvement. Am J Med Genet 93:278-284, 2000 21. MacKinnon AE, Cohen SJ: Total intestinal aganglionosis, an autosomal recessive condition? Arch Dis Child 52:898-899, 1977 22. Di Lorenzo M, Yazbeck S, Brochu P: Aganglionosis of the entire bowel: Four new cases and review of the literature. Br J Surg 72:657-658, 1985