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NTF-3, a gene involved in the enteric nervous system development, as a candidate gene for Hirschsprung disease
Journal of Pediatric Surgery (2008) 43, 1308–1311
www.elsevier.com/locate/jpedsurg
NTF-3, a gene involved in the enteric nervous system development, as a candidate gene for Hirschsprung disease Macarena Ruiz-Ferrer a,b , Raquel M. Fernandez a,b , Guillermo Antiñolo a,b , Manuel Lopez-Alonso b,c , Salud Borrego a,b,⁎ a
Unidad Clínica de Genética y Reproducción, Hospitales Universitarios Virgen del Rocío, 41013 Seville, Spain Centre for Biomedical Research on Rare Diseases (CIBERER), 41013 Seville, Spain c Servicio de Cirugía Infantil, Hospitales Universitarios Virgen del Rocío, 41013 Seville, Spain b
Key words: Hirschsprung; Haplotype; Neurotrophins; Germline mutation; dHPLC
Abstract Hirschsprung disease (HSCR) is a congenital disorder caused by a failure of neural crest cells to migrate, proliferate, and/or differentiate during the enteric nervous system (ENS) development. The requirement of the NTF-3/TrkC signaling for the proper development of the ENS, together with the evidences presented by animal models, led us to investigate the involvement of NTF-3 gene in HSCR. We performed both a mutational screening of NTF-3 and a complete evaluation of 3 polymorphisms as genetic susceptibility factors for HSCR. We identified a novel sequence variant, G76R, present in 2 different patients and absent in controls. We postulate that this variation could generate a lack of mature functional NTF-3 proteins in neural crest cell precursors; thus, altering the NTF-3/TrkC signaling pathway and influencing in the adequate ENS development. Although these results do not provide complete assurance of the involvement of this gene in HSCR, given the polygenic nature of the disease and its etiology, investigation of the genes encoding protein members of the signaling pathways governing the ENS development could provide new key findings in the elucidation of this complex disease. © 2008 Elsevier Inc. All rights reserved.
Hirschsprung disease (HSCR, OMIM 142623) is a developmental disorder of the enteric nervous system (ENS) characterized by the absence of intramural ganglion cells in the myenteric and submucosal plexuses along a variable portion of the distal gastrointestinal tract. Its incidence is approximately 1 in 5000 live births and it most commonly presents in sporadic cases, although it can be familial and may be inherited as autosomal dominant or autosomal recessive, with reduced penetrance and male predominance [1,2]. ⁎ Corresponding author. Unidad Clínica de Genética y Reproducción, Hospitales Universitarios Virgen del Rocío, Avda. Manuel Siurot s/n, 41013 Seville, Spain. Tel.: +34 955 012780; fax: +34 955 013473. E-mail address: [email protected] (S. Borrego). 0022-3468/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.jpedsurg.2008.02.076
Hirschsprung disease has a complex genetic etiology with many studies indicating the RET protooncogene (OMIM 164761) as its major susceptibility gene. Loss-of-function mutations within the coding region of RET account for up to 50% of familial cases and 7% to 35% of sporadic cases [2,3]. Moreover, various studies have shown the existence of a predisposing RET haplotype linked to the sporadic forms of HSCR [4-8]. Thus, in addition to traditional mutations, these results support growing evidence that common low-penetrance variants can also underlie HSCR. Apart from RET, mutations in other genes implicated in intestinal neurodevelopment have been identified in patients with HSCR [2]. Mutations in these genes account for only 7% of the cases. In this way, several studies on mice have
NTF-3 gene and HSCR
1309
provided evidence that neurotrophin 3 (NTF-3, OMIM 162660) is secreted by the non–crest-derived enteric mesenchyme and promotes the development and/or survival of neurons and glia from enteric crest-derived cells expressing the receptor TrkC [9]. Moreover, analysis of the localization of neurotrophins and their receptors in developing and postnatal human intestine, both in normal individuals and in patients with HSCR, revealed that NTF-3 was absent in the aganglionic colon and reduced in transitional intestine [10]. Therefore, the requirement of the NTF-3/TrkC signaling for the proper development of the ENS, together with the evidences presented by the murine models, has prompted us to investigate the possible involvement of the human NTF-3 in Hirschsprung disease.
Polymorphisms) were constructed. On the other hand, a dinucleotide repeat (CA)n polymorphism in the promoter region of the NTF-3 gene was genotyped in an ABI Prism®3130xl Genetic Analyzer (Applied Biosystems). Among all sporadic HSCR cases, allelic and haplotypic frequencies were compared between different features such as sex, length of aganglionosis and the presence of a RET traditional coding mutation, or risk haplotype. All statistical comparisons were performed using χ2 analysis with Yate correction, and statistical significance was considered when P b .05.
1. Methods
Mutational screening and subsequent sequence analysis revealed a novel variant in NTF-3 gene consisting in a nonconservative change of glycine to arginine in codon 76 (G76R, c.226GNA). This mutation was detected in 2 independent patients, and in both cases it had been inherited from one of their unaffected parents but did not appear in other healthy family members, or in any of 200 healthy controls tested. One of the patients was affected with S-HSCR, whereas the other was catalogued as having L-HSCR. To examine the potential pathogenicity of this novel variant, we used PolyPhen (http://genetics.bwh.harvard.edu/pph/), a tool that predicts possible impact of an amino acid substitution on the structure and function of a human protein. However, the substitution G76R was predicted to be benign and the alignment of the NTF-3 protein sequences corresponding to different species showed that the homology degree of the region is quite high among the species but such particular position was not conserved. Moreover, the same codon is affected by a polymorphism described in humans, G76E (rs18005149). Nevertheless, although the novel variant affects the same amino acid position that the previously described polymorphism, neither the nucleotide substitution is the same (c.226GNA vs c.227GNA) nor it results in the same amino acid variation (Gly76Arg vs Gly76Glu). On the other hand, we analyzed and compared the allelic and genotypic distribution of 2 cSNPs, P84P and P185P, in sporadic HSCR and controls, and statistical analysis showed no significant differences (Table 1). Generation of the haplotypes comprising combinations of the 2 SNPs was possible for 149 patients (95%) and 88 controls (89%). We found 4 different haplotypes, but no differences were either detected in their distribution in the 2 groups (χ2 = 0.64, P = .89). In addition, the dinucleotide repeat (CA)n in the promoter region of the NTF-3 revealed a similar distribution in both groups (χ2 = 8.75, P = .27). With the aim to analyze a possible genotype-phenotype or haplotype-phenotype correlation, the distribution of each SNP and haplotype was compared in the group of patients against the length of aganglionosis (TCA/L-HSCR vs SHSCR) and sex (males vs females), but no particular
1.1. Patients and control subjects We have included a total of 182 patients with HSCR from Spain (20% female, 80% male). Of these patients, 157 have sporadic cases, whereas 25 have familial cases belonging to 15 different families. Regarding the phenotype, 104 cases were catalogued as S-HSCR (80%), 16 cases as L-HSCR (12.3%), and 10 patients presented with a total colonic aganglionosis (7.7%). The length of the aganglionic segment was not available for the remaining 52 cases. We have also included a group of 200 normal controls comprising unselected, unrelated race-, age-, and sexmatched individuals. An informed consent was obtained from all the participants for clinical and molecular genetic studies. The study conformed to the tenets of the declaration of Helsinki.
1.2. Mutational screening of NTF-3 Genomic DNA was extracted according to standard protocols [11]. Mutational screening of NTF-3 coding sequence was undertaken by denaturing high-performance liquid chromatography. Those samples with aberrant wave profiles were subjected to sequence analysis in an ABI Prism3130xl Genetic Analyzer (Applied Biosystems, Foster City, CA). Thus, when sequence analysis revealed a novel NTF-3 variant, a group of 200 normal controls were also sequenced to determine if the variant identified was a mutation or just a common polymorphism.
1.3. Analysis of NTF-3 polymorphisms We analyzed the variants P84P (c.252GNA, rs6332) and P185P (c.555CNT, rs10849277) in the 157 sporadic patients and 100 controls by Taqman technology, using a 7500 Fast Real-Time PCR System (Applied Biosystems) and haplotypes comprising both cSNP (Coding Single Nucleotide
2. Results
1310 Table 1
M. Ruiz-Ferrer et al. Distribution of NTF-3 cSNPs in patients with HSCR and controls
Genotypic distribution P84P (c.252GNA) GG GA AA P185P (c.555CNT) CC CT TT
HSCR 27 (17.20%) 91 (57.96%) 39 (24.84%) χ2 = 4.18, P = .12 HSCR 114 (72.61%) 39 (24.84%) 4 (2.55%) χ2=2.18, P = .34
Allelic distribution Controls 25 (25.25%) 45 (45.46%) 29 (29.29%)
P84P (c.252GNA) G allele A allele
Controls 64 (64.65%) 33 (33.33%) 2 (2.02%)
P185P (c.555CNT) C allele T allele
association was detected (P ~ 1). In addition, with the available data from previous studies [3], we sought to determine if there existed any relationship between the distribution of NTF-3 SNPs/haplotypes and the presence of RET mutations or risk haplotype, although no significant results were obtained (P ~ 1).
3. Discussion Several lines of evidence support that neurotrophic influences are involved in ENS development and survival, with potential importance in functional differentiation disorders such as Hirschsprung disease. The effects of neurotrophins have been analyzed on separated populations of crest-derived cells obtained from the fetal rat gut, and it has been observed that the crest-derived cells colonizing the fetal bowel respond specifically to NTF-3 [9]. Furthermore, mice lacking NTF-3 or its receptor TrkC have reduced numbers of both myenteric and submucosal neurons, and mice overexpressing NTF-3 have increased numbers of myenteric neurons. On the other hand, it was demonstrated that there was no expression of NTF-3 and its functional receptor TrkC in the aganglionic colon of patients with HSCR [10]. All these findings taken together led us to consider NTF-3 an excellent candidate gene for HSCR. The lack of genotype-phenotype correlation in HSCR suggests that most cases arise from the effects of both traditional mutations and polymorphisms in multiple susceptibility genes, acting either alone or in combination. Therefore, in the present study, we performed both a mutational screening of NTF-3 and a complete evaluation of 2 SNPs (P84P and P185P). If the results derived from our statistical analyses showed no particular association of any of the polymorphisms or haplotypes to the manifestation of the disease, our most relevant finding was the identification of a novel sequence variant present in 2 different patients. This new change, G76R, is located in the amino-terminal propeptide of the initially synthesized precursor protein, which is proteolytically cleavaged from
HSCR 145 (46.18%) 169 (53.82%)
Controls 95 (47.98%) 103 (52.02%)
χ2 = 0.09, P = .76 HSCR 267 (85.03%) 47 (14.97%)
Controls 161 (81.31%) 37 (18.69%)
χ2=0.97, P = .33
the mature protein [12]. Suter et al [13] showed that the propeptide in the NGF precursor was obligatory for the secretion of correctly processed and biologically active protein. Because strong conserved amino acid segments are present in neurotrophin propeptides, this finding suggests that the propeptides of NGF and NTF-3 share important functional aspects. Therefore, we postulate that the change of an uncharged amino acid to a positively charged in the NTF-3 propeptide could alter severely the production of the properly folded protein, hampering the action of proteases for efficiently cleavage and activation. In this sense, the lack of mature NTF-3 proteins in neural crest-cell precursors might be causing a failure in the ENS development. This proposal of functional mechanism for the variant, together with its absence in 200 healthy controls, provide arguments to consider that G76R may contribute to the pathogenesis of HSCR in the 2 patients where it has been found. The fact that in both patients, the mutation was inherited from one of their unaffected parents would confirm again the complex nature of this disease, in which a unique mutational event may be necessary but not sufficient to produce the phenotype. On the other hand, in silico analyses predict no significant effect of G76R. However, 30% of false-negative error rates have been reported for the PolyPhen and, hence, the possibility of a false negative exists [14]. In summary, this is the first report evaluating NTF-3 as a candidate gene for HSCR. Although the results of the present study do not provide complete assurance of the involvement of this gene in HSCR, we cannot either exclude it. Additional analyses on this and other candidate genes are warranted to dig in the complex mechanisms underlying Hirschsprung disease.
Acknowledgments This study was funded by Fondo de Investigación Sanitaria, Spain (PI040266) y Consejería de Salud de la Junta de Andalucía (CAA 138/06). We would like to thank the families who participated in the study.
NTF-3 gene and HSCR
References [1] Badner JA, Sieber WK, Garver KL, et al. A genetic study of Hirschsprung disease. Am J Hum Genet 1990;46:568-80. [2] Chakravarti A, Lyonnet S. Hirschsprung disease. In: Scriver CS, et al, editors. The metabolic and molecular bases of inherited disease. New York: McGraw-Hill; 2002. p. 6231-55. [3] Ruiz-Ferrer M, Fernandez RM, Antiñolo G, et al. A complex additive model of inheritance for HSCR is supported by both RET mutations and predisposing RET haplotypes. Genet Med 2006;8:704-10. [4] Fernandez RM, Boru G, Pecina A, et al. Ancestral RET haplotype associated with Hirschsprung's disease shows linkage disequilibrium breakpoint at -1249. J Med Genet 2005;42:322-7. [5] Griseri P, Bachetti T, Puppo F, et al. 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: 189-95. [6] Garcia-Barcelo M, Ganster RW, Lui VC, et al. TTF-1 and RET promoter SNPs: regulation of RET transcription in Hirschsprung's disease. Hum Mol Genet 2005;14:191-204.
1311 [7] Burzynski GM, Nolte IM, Bronda A, et al. Identifying candidate Hirschsprung disease–associated RET variants. Am J Hum Genet 2005;76:850-8. [8] Pelet A, de Pontual L, Clement-Ziza M, et al. Homozygosity for a frequent and weakly penetrant predisposing allele at the RET locus in sporadic Hirschsprung disease. J Med Genet 2005;42:e18. [9] Chalazonitis A. Neurotrophin-3 in the development of enteric nervous system. Prog Brain Res 2004;146:246-63. [10] Hoehner JC, Wester T, Pahlman S, et al. Alterations in neurotrophin and neurotrophin-receptor localization in Hirschsprung's disease. J Pediatr Surg 1996;31:1524-9. [11] Dracapoli NH, Haines JL, Korf BR, editors. Current protocols in human genetics. New York: John Wiley & Sons; 1994. [12] Edwards RH, Selby MJ, Mobley WC, et al. Processing and secretion of nerve growth factor: expression in mammalian cells with a vaccinia virus vector. Mol Cell Biol 1998;8:2456-64. [13] Suter U, Heymach Jr JV, Shooter EM. Two conserved domains in the NGF propeptide are necessary and sufficient for the biosynthesis of correctly processed and biologically active NGF. EMBO J 1991;10:2395-400. [14] Ng PC, Henikoff S. Predicting the effects of amino acid substitutions on protein function. Annu Rev Genomics Hum Genet 2006;7:61-80.
www.elsevier.com/locate/jpedsurg
NTF-3, a gene involved in the enteric nervous system development, as a candidate gene for Hirschsprung disease Macarena Ruiz-Ferrer a,b , Raquel M. Fernandez a,b , Guillermo Antiñolo a,b , Manuel Lopez-Alonso b,c , Salud Borrego a,b,⁎ a
Unidad Clínica de Genética y Reproducción, Hospitales Universitarios Virgen del Rocío, 41013 Seville, Spain Centre for Biomedical Research on Rare Diseases (CIBERER), 41013 Seville, Spain c Servicio de Cirugía Infantil, Hospitales Universitarios Virgen del Rocío, 41013 Seville, Spain b
Key words: Hirschsprung; Haplotype; Neurotrophins; Germline mutation; dHPLC
Abstract Hirschsprung disease (HSCR) is a congenital disorder caused by a failure of neural crest cells to migrate, proliferate, and/or differentiate during the enteric nervous system (ENS) development. The requirement of the NTF-3/TrkC signaling for the proper development of the ENS, together with the evidences presented by animal models, led us to investigate the involvement of NTF-3 gene in HSCR. We performed both a mutational screening of NTF-3 and a complete evaluation of 3 polymorphisms as genetic susceptibility factors for HSCR. We identified a novel sequence variant, G76R, present in 2 different patients and absent in controls. We postulate that this variation could generate a lack of mature functional NTF-3 proteins in neural crest cell precursors; thus, altering the NTF-3/TrkC signaling pathway and influencing in the adequate ENS development. Although these results do not provide complete assurance of the involvement of this gene in HSCR, given the polygenic nature of the disease and its etiology, investigation of the genes encoding protein members of the signaling pathways governing the ENS development could provide new key findings in the elucidation of this complex disease. © 2008 Elsevier Inc. All rights reserved.
Hirschsprung disease (HSCR, OMIM 142623) is a developmental disorder of the enteric nervous system (ENS) characterized by the absence of intramural ganglion cells in the myenteric and submucosal plexuses along a variable portion of the distal gastrointestinal tract. Its incidence is approximately 1 in 5000 live births and it most commonly presents in sporadic cases, although it can be familial and may be inherited as autosomal dominant or autosomal recessive, with reduced penetrance and male predominance [1,2]. ⁎ Corresponding author. Unidad Clínica de Genética y Reproducción, Hospitales Universitarios Virgen del Rocío, Avda. Manuel Siurot s/n, 41013 Seville, Spain. Tel.: +34 955 012780; fax: +34 955 013473. E-mail address: [email protected] (S. Borrego). 0022-3468/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.jpedsurg.2008.02.076
Hirschsprung disease has a complex genetic etiology with many studies indicating the RET protooncogene (OMIM 164761) as its major susceptibility gene. Loss-of-function mutations within the coding region of RET account for up to 50% of familial cases and 7% to 35% of sporadic cases [2,3]. Moreover, various studies have shown the existence of a predisposing RET haplotype linked to the sporadic forms of HSCR [4-8]. Thus, in addition to traditional mutations, these results support growing evidence that common low-penetrance variants can also underlie HSCR. Apart from RET, mutations in other genes implicated in intestinal neurodevelopment have been identified in patients with HSCR [2]. Mutations in these genes account for only 7% of the cases. In this way, several studies on mice have
NTF-3 gene and HSCR
1309
provided evidence that neurotrophin 3 (NTF-3, OMIM 162660) is secreted by the non–crest-derived enteric mesenchyme and promotes the development and/or survival of neurons and glia from enteric crest-derived cells expressing the receptor TrkC [9]. Moreover, analysis of the localization of neurotrophins and their receptors in developing and postnatal human intestine, both in normal individuals and in patients with HSCR, revealed that NTF-3 was absent in the aganglionic colon and reduced in transitional intestine [10]. Therefore, the requirement of the NTF-3/TrkC signaling for the proper development of the ENS, together with the evidences presented by the murine models, has prompted us to investigate the possible involvement of the human NTF-3 in Hirschsprung disease.
Polymorphisms) were constructed. On the other hand, a dinucleotide repeat (CA)n polymorphism in the promoter region of the NTF-3 gene was genotyped in an ABI Prism®3130xl Genetic Analyzer (Applied Biosystems). Among all sporadic HSCR cases, allelic and haplotypic frequencies were compared between different features such as sex, length of aganglionosis and the presence of a RET traditional coding mutation, or risk haplotype. All statistical comparisons were performed using χ2 analysis with Yate correction, and statistical significance was considered when P b .05.
1. Methods
Mutational screening and subsequent sequence analysis revealed a novel variant in NTF-3 gene consisting in a nonconservative change of glycine to arginine in codon 76 (G76R, c.226GNA). This mutation was detected in 2 independent patients, and in both cases it had been inherited from one of their unaffected parents but did not appear in other healthy family members, or in any of 200 healthy controls tested. One of the patients was affected with S-HSCR, whereas the other was catalogued as having L-HSCR. To examine the potential pathogenicity of this novel variant, we used PolyPhen (http://genetics.bwh.harvard.edu/pph/), a tool that predicts possible impact of an amino acid substitution on the structure and function of a human protein. However, the substitution G76R was predicted to be benign and the alignment of the NTF-3 protein sequences corresponding to different species showed that the homology degree of the region is quite high among the species but such particular position was not conserved. Moreover, the same codon is affected by a polymorphism described in humans, G76E (rs18005149). Nevertheless, although the novel variant affects the same amino acid position that the previously described polymorphism, neither the nucleotide substitution is the same (c.226GNA vs c.227GNA) nor it results in the same amino acid variation (Gly76Arg vs Gly76Glu). On the other hand, we analyzed and compared the allelic and genotypic distribution of 2 cSNPs, P84P and P185P, in sporadic HSCR and controls, and statistical analysis showed no significant differences (Table 1). Generation of the haplotypes comprising combinations of the 2 SNPs was possible for 149 patients (95%) and 88 controls (89%). We found 4 different haplotypes, but no differences were either detected in their distribution in the 2 groups (χ2 = 0.64, P = .89). In addition, the dinucleotide repeat (CA)n in the promoter region of the NTF-3 revealed a similar distribution in both groups (χ2 = 8.75, P = .27). With the aim to analyze a possible genotype-phenotype or haplotype-phenotype correlation, the distribution of each SNP and haplotype was compared in the group of patients against the length of aganglionosis (TCA/L-HSCR vs SHSCR) and sex (males vs females), but no particular
1.1. Patients and control subjects We have included a total of 182 patients with HSCR from Spain (20% female, 80% male). Of these patients, 157 have sporadic cases, whereas 25 have familial cases belonging to 15 different families. Regarding the phenotype, 104 cases were catalogued as S-HSCR (80%), 16 cases as L-HSCR (12.3%), and 10 patients presented with a total colonic aganglionosis (7.7%). The length of the aganglionic segment was not available for the remaining 52 cases. We have also included a group of 200 normal controls comprising unselected, unrelated race-, age-, and sexmatched individuals. An informed consent was obtained from all the participants for clinical and molecular genetic studies. The study conformed to the tenets of the declaration of Helsinki.
1.2. Mutational screening of NTF-3 Genomic DNA was extracted according to standard protocols [11]. Mutational screening of NTF-3 coding sequence was undertaken by denaturing high-performance liquid chromatography. Those samples with aberrant wave profiles were subjected to sequence analysis in an ABI Prism3130xl Genetic Analyzer (Applied Biosystems, Foster City, CA). Thus, when sequence analysis revealed a novel NTF-3 variant, a group of 200 normal controls were also sequenced to determine if the variant identified was a mutation or just a common polymorphism.
1.3. Analysis of NTF-3 polymorphisms We analyzed the variants P84P (c.252GNA, rs6332) and P185P (c.555CNT, rs10849277) in the 157 sporadic patients and 100 controls by Taqman technology, using a 7500 Fast Real-Time PCR System (Applied Biosystems) and haplotypes comprising both cSNP (Coding Single Nucleotide
2. Results
1310 Table 1
M. Ruiz-Ferrer et al. Distribution of NTF-3 cSNPs in patients with HSCR and controls
Genotypic distribution P84P (c.252GNA) GG GA AA P185P (c.555CNT) CC CT TT
HSCR 27 (17.20%) 91 (57.96%) 39 (24.84%) χ2 = 4.18, P = .12 HSCR 114 (72.61%) 39 (24.84%) 4 (2.55%) χ2=2.18, P = .34
Allelic distribution Controls 25 (25.25%) 45 (45.46%) 29 (29.29%)
P84P (c.252GNA) G allele A allele
Controls 64 (64.65%) 33 (33.33%) 2 (2.02%)
P185P (c.555CNT) C allele T allele
association was detected (P ~ 1). In addition, with the available data from previous studies [3], we sought to determine if there existed any relationship between the distribution of NTF-3 SNPs/haplotypes and the presence of RET mutations or risk haplotype, although no significant results were obtained (P ~ 1).
3. Discussion Several lines of evidence support that neurotrophic influences are involved in ENS development and survival, with potential importance in functional differentiation disorders such as Hirschsprung disease. The effects of neurotrophins have been analyzed on separated populations of crest-derived cells obtained from the fetal rat gut, and it has been observed that the crest-derived cells colonizing the fetal bowel respond specifically to NTF-3 [9]. Furthermore, mice lacking NTF-3 or its receptor TrkC have reduced numbers of both myenteric and submucosal neurons, and mice overexpressing NTF-3 have increased numbers of myenteric neurons. On the other hand, it was demonstrated that there was no expression of NTF-3 and its functional receptor TrkC in the aganglionic colon of patients with HSCR [10]. All these findings taken together led us to consider NTF-3 an excellent candidate gene for HSCR. The lack of genotype-phenotype correlation in HSCR suggests that most cases arise from the effects of both traditional mutations and polymorphisms in multiple susceptibility genes, acting either alone or in combination. Therefore, in the present study, we performed both a mutational screening of NTF-3 and a complete evaluation of 2 SNPs (P84P and P185P). If the results derived from our statistical analyses showed no particular association of any of the polymorphisms or haplotypes to the manifestation of the disease, our most relevant finding was the identification of a novel sequence variant present in 2 different patients. This new change, G76R, is located in the amino-terminal propeptide of the initially synthesized precursor protein, which is proteolytically cleavaged from
HSCR 145 (46.18%) 169 (53.82%)
Controls 95 (47.98%) 103 (52.02%)
χ2 = 0.09, P = .76 HSCR 267 (85.03%) 47 (14.97%)
Controls 161 (81.31%) 37 (18.69%)
χ2=0.97, P = .33
the mature protein [12]. Suter et al [13] showed that the propeptide in the NGF precursor was obligatory for the secretion of correctly processed and biologically active protein. Because strong conserved amino acid segments are present in neurotrophin propeptides, this finding suggests that the propeptides of NGF and NTF-3 share important functional aspects. Therefore, we postulate that the change of an uncharged amino acid to a positively charged in the NTF-3 propeptide could alter severely the production of the properly folded protein, hampering the action of proteases for efficiently cleavage and activation. In this sense, the lack of mature NTF-3 proteins in neural crest-cell precursors might be causing a failure in the ENS development. This proposal of functional mechanism for the variant, together with its absence in 200 healthy controls, provide arguments to consider that G76R may contribute to the pathogenesis of HSCR in the 2 patients where it has been found. The fact that in both patients, the mutation was inherited from one of their unaffected parents would confirm again the complex nature of this disease, in which a unique mutational event may be necessary but not sufficient to produce the phenotype. On the other hand, in silico analyses predict no significant effect of G76R. However, 30% of false-negative error rates have been reported for the PolyPhen and, hence, the possibility of a false negative exists [14]. In summary, this is the first report evaluating NTF-3 as a candidate gene for HSCR. Although the results of the present study do not provide complete assurance of the involvement of this gene in HSCR, we cannot either exclude it. Additional analyses on this and other candidate genes are warranted to dig in the complex mechanisms underlying Hirschsprung disease.
Acknowledgments This study was funded by Fondo de Investigación Sanitaria, Spain (PI040266) y Consejería de Salud de la Junta de Andalucía (CAA 138/06). We would like to thank the families who participated in the study.
NTF-3 gene and HSCR
References [1] Badner JA, Sieber WK, Garver KL, et al. A genetic study of Hirschsprung disease. Am J Hum Genet 1990;46:568-80. [2] Chakravarti A, Lyonnet S. Hirschsprung disease. In: Scriver CS, et al, editors. The metabolic and molecular bases of inherited disease. New York: McGraw-Hill; 2002. p. 6231-55. [3] Ruiz-Ferrer M, Fernandez RM, Antiñolo G, et al. A complex additive model of inheritance for HSCR is supported by both RET mutations and predisposing RET haplotypes. Genet Med 2006;8:704-10. [4] Fernandez RM, Boru G, Pecina A, et al. Ancestral RET haplotype associated with Hirschsprung's disease shows linkage disequilibrium breakpoint at -1249. J Med Genet 2005;42:322-7. [5] Griseri P, Bachetti T, Puppo F, et al. 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: 189-95. [6] Garcia-Barcelo M, Ganster RW, Lui VC, et al. TTF-1 and RET promoter SNPs: regulation of RET transcription in Hirschsprung's disease. Hum Mol Genet 2005;14:191-204.
1311 [7] Burzynski GM, Nolte IM, Bronda A, et al. Identifying candidate Hirschsprung disease–associated RET variants. Am J Hum Genet 2005;76:850-8. [8] Pelet A, de Pontual L, Clement-Ziza M, et al. Homozygosity for a frequent and weakly penetrant predisposing allele at the RET locus in sporadic Hirschsprung disease. J Med Genet 2005;42:e18. [9] Chalazonitis A. Neurotrophin-3 in the development of enteric nervous system. Prog Brain Res 2004;146:246-63. [10] Hoehner JC, Wester T, Pahlman S, et al. Alterations in neurotrophin and neurotrophin-receptor localization in Hirschsprung's disease. J Pediatr Surg 1996;31:1524-9. [11] Dracapoli NH, Haines JL, Korf BR, editors. Current protocols in human genetics. New York: John Wiley & Sons; 1994. [12] Edwards RH, Selby MJ, Mobley WC, et al. Processing and secretion of nerve growth factor: expression in mammalian cells with a vaccinia virus vector. Mol Cell Biol 1998;8:2456-64. [13] Suter U, Heymach Jr JV, Shooter EM. Two conserved domains in the NGF propeptide are necessary and sufficient for the biosynthesis of correctly processed and biologically active NGF. EMBO J 1991;10:2395-400. [14] Ng PC, Henikoff S. Predicting the effects of amino acid substitutions on protein function. Annu Rev Genomics Hum Genet 2006;7:61-80.