Research Paper Volume 11, Issue 4 pp 1252—1261
Pleiotropic effect of common PHOX2B variants in Hirschsprung disease and neuroblastoma
- 1 Department of Pediatric Surgery, Guangzhou Institute of Pediatrics, Guangzhou Women and Children’s Medical Center, Guangzhou Medical University, Guangzho, Guangdong 510623, China
- 2 Department of Pediatric Surgery, The First Affiliated Hospital of Zhengzhou University, Zhengzho, Henan 450052, China
- 3 Department of Pediatric Surgery, the Second Affiliated Hospital of Xi'an Jiaotong University, Xi'a, Shaanxi 710004, China
received: December 28, 2018 ; accepted: February 17, 2019 ; published: February 22, 2019 ;https://doi.org/10.18632/aging.101834
How to Cite
Copyright: Zhao et al. This is an open‐access article distributed under the terms of the Creative Commons Attribution License (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Hirschsprung disease (HSCR) is a heterogeneous congenital disorder that affects the enteric nervous system, while neuroblastoma is an embryonal tumor of the sympathetic nervous system. Familial cases of both HSCR and neuroblastoma appear to be functionally linked to PHOX2B, which plays a key role in the development of neural crest derivatives. However, the association between common PHOX2B variants and disease risk is contested. Additionally, large-scale examination for pleiotropy or shared genetic susceptibility in sporadic HSCR and neuroblastoma cases lacks theoretical support. Here, we report the first examination of PHOX2B in 1470 HSCR and 469 neuroblastoma patients with matched healthy controls. The PHOX2B rs28647582 polymorphism was found to be associated with HSCR (P = 2.21E-03, OR = 1.26), and each subtype of the ailment (3.22E-03 ≤ P ≤ 0.43, 1.11 ≤ OR ≤ 2.32). The association between rs28647582 and NB risk was consistent with HSCR in a recessive model, though the P value was marginal (P = 0.06). These new genetic findings indicate the potential pleiotropic effects of PHOX2B in both HSCR and neuroblastoma, which could guide the development of therapeutic targets for the treatment of related neurodevelopmental disorders.
Hirschsprung’s disease (HSCR) is a congenital disorder characterized by a partial or complete absence of ganglion cells in the nerve plexuses of the lower digestive tract. The incidence is about 1/5,000 live births in Caucasians and about 1.4/5,000 of live births in Asians, and males are 3.5-4.0 times more likely to be affected than females . HSCR is divided into three subtypes – short-segment HSCR (S-HSCR), long-segment HSCR (L-HSCR) and total colonic aganglionosis (TCA) – based on the length of the intestinal segment lacking nerve cells [2,3]. As a result of extensive mutation screening in familial and sporadic patients, several genes are now known to be associated with HSCR, including RET, EDNRB, SOX10 and PHOX2B, among others [4,5]. Most of those genes have been implicated in the migration, differentiation, and maturation of enteric neural crest cells, and HSCR reflects the failure of enteric neural crest-derived cells to complete their anteroposterior migration to the end of the bowel .
Neuroblastoma (NB) is an embryonal tumor of the sympathetic nervous system and is generally considered to originate from neural crest cells in the paravertebral sympathetic ganglia and the adrenal medulla . It is the most commonly occurring extracranial cancer of early childhood, with an estimated incidence of 1/8,000-10,000 births . Most NB patients are sporadic, but about 1-2% are familial cases . For instance, paired mesoderm homeobox 2b gene (PHOX2B) and anaplastic lymphoma kinase gene (ALK) predict the main etiology of NB [10–12]. A subset of patients with Congenital Central Hypoventilation Syndrome (CCHS) present both HSCR and NB [12–14].
PHOX2B is located on chromosome 4p12 and encodes a transcription factor involved in the development of enteric neuron populations. In mice, its expression coincides with neural crest cell invasion of the foregut mesenchyme and continues throughout the process of cellular differentiation into enteric neurons . In mice and zebrafish, PHOX2B deficiency impairs neuronal sympathetic differentiation, and results in a failure of enteric neural crest-derived cells to normally colonize the gut [15–17]. Mutant mice harboring a non-polyalanine repeat expansion mutation of PHOX2B exhibit the CCHS-HSCR-NB phenotype . The predisposition of CCHS patients to HSCR and developmental and functional intestinal defects reflect PHOX2B haploinsufficiency in these patients .
PHOX2B mutations in HSCR-NB cases suggest the gene has pleiotropic effects that influence sporadic cases of both HSCR and NB [20–22]. The association between a common PHOX2B variant (rs28647582) and HSCR risk has been investigated in case-control studies, but the results are inconsistent [23–26]. On the other hand, PHOX2B mutations have been detected in a familial case of NB (mutation R100L) and in an isolated case of NB associated with HSCR (mutation R141G) . Thus, much about the role of PHOX2B in sporadic HSCR and NB cases remains unclear. We therefore conducted a replication study of HSCR and NB cases and controls to evaluate the independent contribution of PHOX2B to the two ailments. Our findings offer additional insight into their etiology and provide a better understanding of the role of PHOX2B in sporadic HSCR and NB.
Characteristics of the study participants
Supplementary Table 1 summarizes the subclinical information for a South Chinese population that included 1470 HSCR patients and 1473 healthy controls. The age and gender distributions were similar between the cases and controls (P > 0.05). Clinical subtype manifestation and total intestine involved are listed in the table. The Supplementary Table 2 summarizes the subclinical information for a total of 469 NB patients and 998 healthy controls. Also detailed are the distribution of clinical stages and sites of tumor origin.
PHOX2B and HSCR susceptibility
We initially detected a significant association between PHOX2B SNP rs28647582 and HSCR (P = 2.21E-03, OR = 1.26) (Table 1). To further specify the association between rs28647582 and HSCR, we classified the samples using genotypic, additive, dominant, and recessive models (7.35E-04 ≤ P ≤ 0.02; 1.21 ≤ OR ≤ 2.37) (Table 2). We observed the most significant association with disease in the recessive model (7.35E-04 ≤ P ≤ 0.02; 1.21 ≤ OR ≤ 2.37). Somewhat surprisingly, PHOX2B rs28647582 showed a strong association with S-HSCR, L-HSCR and TCA (3.22E-03 ≤ P ≤ 0.43; 1.11 ≤ OR ≤ 2.32), which highlights the crucial role played by PHOX2B rs28647582 in the disease (Table 3). By contrast, most research to date reports an impact of common variants on only one or two of the subtypes. This inconsistency may be attributable to environmental and genetic diversity among different ethnicities [4,5,28].
Table 1. Replication results of PHOX2B SNP (rs28647582) in a South Chinese population using 1470 cases and 1473 controls.
|SNP, single Nucleotide Polymorphism; CHR, chromosome; BP, base pair where the SNP is located; A1/A2, risk allele and protective allele to disease; F_A/F_U, risk allele frequency of the SNP in cases or controls; OR, odds ratio; CI, confidence interval. The P value indicates the significance based on allelic association tests. Calculation of the OR was also based on the risk allele of each SNP.|
Table 2. Detailed replication results with different genetic models.
|HWE, Hardy-Weinberg equilibrium; A1/A2, risk allele and protective allele to disease; AFF/UNAFF, cases and controls; DF, degree of freedom; OR, odds ratio; CI, confidence interval. The P value indicates the significance based on allelic association tests. Calculation of OR was also based on the risk allele of each SNP.|
Table 3. The association results of PHOX2B gene SNP (rs28647582) to different subclinical features classified by aganglionosis length.
|CHR||SNP||Length of aganglionic segment||A1/A2||F_A||F_U||P||OR|
|SNP, single nucleotide polymorphism; CHR, chromosome; S-HSCR, short-segment HSCR; L-HSCR, long-segment HSCR; TCA, total colonic aganglionosis; A1/A2, risk allele and protective allele to disease; F_A/F_U, risk allele frequency of the SNP in cases or controls; OR, odds ratio; CI, confidence interval. The P value indicates the significance based on allelic association tests. Calculation of OR was also based on the risk allele of each SNP.|
PHOX2B and NB susceptibility
Genotype and allele frequencies of PHOX2B rs28647582 and associations with NB risk are summarized in Table 4. In both the combined and subgroup analyses, the genotype distribution of PHOX2B rs28647582 in the controls was consistent with Hardy-Weinberg equilibrium (P = 0.538). Although the TT and TC distributions did not significantly differ between the patients and controls, we observed a marginal difference between the CC distributions (P = 0.058). Consistent with that finding, we found a similar marginal association between the CC genotype and NB risk in the recessive model (P = 0.060). These findings suggest similar patterns of association between PHOX2B rs28647582 and HSCR and NB risk, suggesting a potential effect of PHOX2B pleiotropy in the diseases.
Table 4. Association of PHOX2B rs28647582T>C polymorphism with neuroblastoma risk.
|P a||Crude OR|
(95% CI) b
|TT||320 (68.23)||662 (66.33)||1.00||1.00|
|TC||140 (29.85)||298 (29.86)||0.97 (0.76-1.24)||0.817||0.97 (0.76-1.23)||0.794|
|CC||9 (1.92)||38 (3.81)||0.49 (0.23-1.03)||0.058||0.49 (0.23-1.02)||0.058|
|Additive||0.155||0.88 (0.72-1.08)||0.214||0.88 (0.71-1.08)||0.204|
|Dominant||149 (31.77)||336 (33.67)||0.471||0.92 (0.73-1.16)||0.471||0.91 (0.72-1.16)||0.454|
|Recessive||460 (98.08)||960 (96.19)||0.055||0.49 (0.24-1.03)||0.060||0.49 (0.24-1.03)||0.060|
|OR, odds ratio; CI, confidence interval; HWE, Hardy-Weinberg equilibrium.|
|a χ2 test for genotype distributions between neuroblastoma patients and controls.|
|b Adjusted for age and gender.|
Stratification analysis of PHOX2B rs28647582 and NB risk
To further evaluate the contributions of PHOX2B rs28647582 T>C polymorphisms to the risk of NB, stratification analysis based on age, gender, tumor sites of origin, and clinical stage was performed (Table 5). Although we failed to detect significant association between PHOX2B rs28647582 T>C polymorphism and NB risk in any of the evaluated subgroups, we observed a marginal association in patients older than 18 years of age.
Table 5. Stratification analysis of the association between PHOX2B rs28647582 T>C polymorphism and neuroblastoma risk.
|Crude OR||P||Adjusted OR a||P a|
|TT/TC||CC||(95% CI)||(95% CI)|
|≤18||165/378||4/12||0.76 (0.24-2.40)||0.645||0.79 (0.25-2.49)||0.685|
|>18||295/582||5/26||0.38 (0.14-1.00)||0.050||0.38 (0.15-1.00)||0.051|
|Females||192/397||4/17||0.49 (0.16-1.47)||0.200||0.49 (0.16-1.47)||0.202|
|Males||268/563||5/21||0.50 (0.19-1.34)||0.169||0.49 (0.18-1.33)||0.162|
|Sites of origin|
|Adrenal gland||157/960||5/38||0.81 (0.31-2.08)||0.653||0.80 (0.31-2.06)||0.641|
|Retroperitoneal||135/960||3/38||0.56 (0.17-1.84)||0.341||0.56 (0.17-1.85)||0.342|
|Mediastinum||120/960||1/38||0.21 (0.03-1.55)||0.126||0.21 (0.03-1.52)||0.122|
|I+II+4s||229/960||4/38||0.44 (0.16-1.25)||0.123||0.44 (0.15-1.24)||0.119|
|III+IV||211/960||5/38||0.60 (0.23-1.54)||0.287||0.60 (0.23-1.53)||0.283|
|OR, odds ratio; CI, confidence interval.|
|aAdjusted for age and gender, omitting the corresponding stratification factor.|
HSCR and NB are neurocristopathies of the sympathetic and enteric nervous systems. HSCR is an aganglionosis with a strong genetic component affecting intestinal segments of various length. A number of genes are associated with HSCR, including PHOX2B, haploinsufficiency of which predisposes people to defects in intestinal development and function [19,29]. Since PHOX2B was first found to be associated with HSCR in 2003, eight PHOX2B SNPs have been reported [24,30,31]. However, not all patient populations exhibited a significant association between PHOX2B and HSCR [23–26]. In the present study, we confirmed the significant association between PHOX2B rs28647582 and HSCR in a Southern Chinese population of 1470 cases, which is a much larger sample than in earlier studies. Moreover, our findings predict an increased risk for every subtype of HSCR.
PHOX2B encodes a highly conserved protein of about 314 amino acids . The rs28647582 polymorphism is a variant within the intron of the gene. It is unclear whether rs28647582 directly affects PHOX2B expression. Our findings indicate that rs28647582 is associated with HSCR in our study population. This association may indicate a direct contribution of rs28647582 to the HSCR phenotype or that rs28647582 is a linkage disequilibrium with another susceptibility locus. Direct involvement of rs28647582 in HSCR would be through alteration of intronic sequences crucial for splicing and/or regulation of PHOX2B expression. In addition to SNPs, the coexistence of RET mutations with mutations or SNPs in EDNRB and GDNF have also been described in HSCR patients [33,34]. We therefore hypothesize that rs28647582 or other PHOX2B variants must occur together or act in concert with mutations in the RET and/or EDNRB signaling pathways to produce the HSCR phenotype. Remarkably, this study is the first to propose a significant link between PHOX2B rs28647582 and the risk of all HSCR subtypes. Relationships between HSCR subtypes and PHOX2B have not been reported previously, though several common genetic variants in RET, EDNRB, and SOX10 reportedly associate with one or two of the subtypes [4,5,28]. The different HSCR subtypes reflect the severity of the intestinal neuronal precursor cell migration defect during embryonal development . Our findings thus highlight the idea that dysfunction related to PHOX2B variants directly affect the extent of aganglionosis that arises during enteric nervous system development, ultimately leading to any of the three HSCR subtypes.
In the present study, PHOX2B mutations were most frequent in familial HSCR and NB cases or HSCR-NB syndrome cases [8,17]. PHOX2B variants had previously been identified as the major disease-causing gene in sporadic cases and in CCHS [8,11,36]. Several SNP studies examining PHOX2B’s association with NB in sporadic cases have been previously reported [20,37,38]; however, to our knowledge, this is the first exploration of the relationship between the PHOX2B 12345678 T>C polymorphism and NB risk in sporadic cases. We did not detect an association between the PHOX2B rs28647582 T>C polymorphism and NB risk in sporadic cases, but the trend is consistent with the findings for HSCR risk. There are several possible reasons for this negative result. First, NB mutations tend to be missense alterations in highly conserved regions or nonsense mutations that lead to a truncated protein lacking a second polyalanine motif [11,39–41]. The common genetic variants would not necessarily exert a marked influence. Second, NB is a multifactorial disease resulting from multiplicative interactions among genetic backgrounds and environmental factors. Our study lacked several valuable parameters that likely exert effects, including parental exposures, dietary intake, and living environment . Third, our study includes only the rs28647582 T>C polymorphism. It is nevertheless clear that only a small fraction of SNPs influence cancer susceptibility, while most do not. Finally, there is likely some selection bias. Our study was hospital-based with subjects recruited from the provinces of Guangdong, Henan and Shaanxi. This population may not be representative of the general Chinese population.
In summary, this was the first exploration of the pleiotropic effects of common PHOX2B variants in HSCR and NB. Our results show that PHOX2B rs28647582 is independently associated with HSCR risk, but that the PHOX2B rs28647582 T>C polymorphism has no effect on NB risk. Nonetheless, this study has compelling biological applicability as the largest population-based study of the potential pleiotropic effects of PHOX2B genetic polymorphisms in HSCR and NB. Although the mechanisms remain largely undetermined, our findings provide proof-of-principle that certain genetic susceptibility loci could potentially be used to stratify individuals with respect to HSCR risk. Based on our findings, we suggest future multicenter and multiracial studies of the relation between PHOX2B SNPs and HSCR are warranted.
The study included 1470 HSCR cases (age range 8.37 ± 20.50 months; 83.67% males) and 1473 controls. The patients were diagnosed with HSCR by the Pediatric Clinic of Guangzhou Women and Children’s Medical Center in Guangzhou, China between 2000 and 2015. These diagnoses were based on histologic examination of biopsy or surgical resection samples revealing the absence of enteric nerve plexuses. After dividing the cases into three subgroups based on the segment lengths exhibiting aganglionosis, there were 1033 patients with S-HSCR, 294 with L-HSCR and 82 with TCA (Supplementary Table 1). In addition, a total of 469 NB cases and 998 healthy controls were also included in this study . Of those, 275 NB cases and 531 controls were from Guangzhou Women and Children’s Medical Center [44–46], 118 cases and 281 controls were from The First Affiliated Hospital of Zhengzhou University [47–49], and 76 NB cases and 186 controls were from the Second Affiliated Hospital of Xi'an Jiaotong University (Supplementary Table 2). The cases were patients diagnosed with NB, and the controls were recruited from the same hospitals. Ethical approved was obtained from the Institutional Review Board of each hospital and written informed consent was obtained from each subject.
SNP genotyping and quality control
A common PHOX2B variant (rs28647582) was selected using the GTEx portal website (http://www.gtexportal.org/home/) to predict potential associations between the SNP and PHOX2B expression levels . Past studies covering 372 cases and 511 controls have suggested that the PHOX2B rs28647582 genotype is not associated with HSCR [24,25,51], but we chose to re-assess PHOX2B rs28647582 using a much larger sample. For the 1470 HSCR cases and 1473 controls, SNPs were genotyped using a MassARRAY iPLEX Gold system (Sequenom). Hardy-Weinberg equilibrium tests were performed.
For all NB cases and controls, rs28647582 was genotyped using a TaqMan real-time PCR system on a 7900 Sequence Detection System (Applied Biosystems, Foster City, CA), as described previously [52–54]. The call rate for the SNPs was 99%, which met the pre-set criterion. For quality control, eight duplicate positive and eight negative controls without DNA were used in each 384-well plate.
The χ2 test was used to evaluate differences in the frequency distributions of the demographics and genotypes between the cases and controls. A genotype test of 3 × 2 contingency tables and Cochran-Armitage trend test were used to test genotypic, additive, dominant and recessive models. These tests were carried out using PLINK software Version 1.9 . SNPs were tested for associations with disease using a case comparison approach, with and without indicating the disease subtype. Odds ratios (ORs) and 95% confidence intervals (CIs) calculated using the Woolf approximation method were used to assess the correlation between the PHOX2B rs28647582 and HSCR and NB risk. The Hardy-Weinberg equilibrium was assessed using the goodness-of-χ2 test. Crude and age- and gender-adjusted ORs were assessed using the unconditional logistic regression method. SAS software (Version 9.4; SAS Institute, Cary, NC, USA) was used for these analyses. All statistical tests were two-sided. Values of P < 0.05 were considered significant.
We thank Yanlu Tong and Hezhen Wang for their assistance in DNA extraction and the collection of medical histories. The authors have no conflicts of interest to declare. All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional review board of Guangzhou Women and Children’s medical center (No. 2016042036) and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.
Conflicts of Interest
The authors declare no competing financial interests.
Huimin Xia thanks the supporting of National Natural Science Foundation of China (Grant No. 81771629). Ruizhong Zhang thanks the National Natural Science Foundation of China (Grant No. 81600399), Science and Technology Planning Project of Guangdong Province (No. 2017A020214017). Yan Zhang thanks the supporting by the grant of National Natural Science Foundation of China (Grant No. 81601423) and the Science and Technology Project of Guangzhou (No.201707010014). Lihua Huang thanks the grant of National Natural Science Foundation of China (No 81600400), the Natural Science Foundation of Guangdong Province (No.2016A030310250) and the China Postdoctoral Science Foundation (No.2017T100621).
- 1. Zhao J, Xie X, Yao Y, He Q, Zhang R, Xia H, Zhang Y. Association of VAMP5 and MCC genetic polymorphisms with increased risk of Hirschsprung disease susceptibility in Southern Chinese children. Aging (Albany NY). 2018; 10:689–700. https://doi.org/10.18632/aging.101423 [PubMed]
- 2. Ruttenstock E, Puri P. A meta-analysis of clinical outcome in patients with total intestinal aganglionosis. Pediatr Surg Int. 2009; 25:833–39. https://doi.org/10.1007/s00383-009-2439-2 [PubMed]
- 3. Moore SW. Total colonic aganglionosis in Hirschsprung disease. Semin Pediatr Surg. 2012; 21:302–09. https://doi.org/10.1053/j.sempedsurg.2012.07.004 [PubMed]
- 4. Zhang Y, He Q, Zhang R, Zhang H, Zhong W, Xia H. Large-scale replication study identified multiple independent SNPs in RET synergistically associated with Hirschsprung disease in Southern Chinese population. Aging (Albany NY). 2017; 9:1996–2009. https://doi.org/10.18632/aging.101294 [PubMed]
- 5. Amiel J, Sproat-Emison E, Garcia-Barcelo M, Lantieri F, Burzynski G, Borrego S, Pelet A, Arnold S, Miao X, Griseri P, Brooks AS, Antinolo G, de Pontual L, et al, and Hirschsprung Disease Consortium. Hirschsprung disease, associated syndromes and genetics: a review. J Med Genet. 2008; 45:1–14. https://doi.org/10.1136/jmg.2007.053959 [PubMed]
- 6. Torroglosa A, Alves MM, Fernández RM, Antiñolo G, Hofstra RM, Borrego S. Epigenetics in ENS development and Hirschsprung disease. Dev Biol. 2016; 417:209–16. https://doi.org/10.1016/j.ydbio.2016.06.017 [PubMed]
- 7. Ke XX, Zhang D, Zhao H, Hu R, Dong Z, Yang R, Zhu S, Xia Q, Ding HF, Cui H. Phox2B correlates with MYCN and is a prognostic marker for neuroblastoma development. Oncol Lett. 2015; 9:2507–14. https://doi.org/10.3892/ol.2015.3088 [PubMed]
- 8. Schleiermacher G, Janoueix-Lerosey I, Delattre O. Recent insights into the biology of neuroblastoma. Int J Cancer. 2014; 135:2249–61. https://doi.org/10.1002/ijc.29077 [PubMed]
- 9. Wu Q, Zhuo ZJ, Zeng J, Zhang J, Zhu J, Zou Y, Zhang R, Yang T, Zhu D, He J, Xia H. Association between NEFL Gene Polymorphisms and Neuroblastoma Risk in Chinese Children: A Two-Center Case-Control Study. J Cancer. 2018; 9:535–39. https://doi.org/10.7150/jca.22681 [PubMed]
- 10. Trochet D, O’Brien LM, Gozal D, Trang H, Nordenskjöld A, Laudier B, Svensson PJ, Uhrig S, Cole T, Niemann S, Munnich A, Gaultier C, Lyonnet S, Amiel J. PHOX2B genotype allows for prediction of tumor risk in congenital central hypoventilation syndrome. Am J Hum Genet. 2005; 76:421–26. https://doi.org/10.1086/428366 [PubMed]
- 11. McConville C, Reid S, Baskcomb L, Douglas J, Rahman N. PHOX2B analysis in non-syndromic neuroblastoma cases shows novel mutations and genotype-phenotype associations. Am J Med Genet A. 2006; 140:1297–301. https://doi.org/10.1002/ajmg.a.31278 [PubMed]
- 12. Mossé YP, Laudenslager M, Longo L, Cole KA, Wood A, Attiyeh EF, Laquaglia MJ, Sennett R, Lynch JE, Perri P, Laureys G, Speleman F, Kim C, et al. Identification of ALK as a major familial neuroblastoma predisposition gene. Nature. 2008; 455:930–35. https://doi.org/10.1038/nature07261 [PubMed]
- 13. Chen Y, Takita J, Choi YL, Kato M, Ohira M, Sanada M, Wang L, Soda M, Kikuchi A, Igarashi T, Nakagawara A, Hayashi Y, Mano H, Ogawa S. Oncogenic mutations of ALK kinase in neuroblastoma. Nature. 2008; 455:971–74. https://doi.org/10.1038/nature07399 [PubMed]
- 14. Trochet D, Bourdeaut F, Janoueix-Lerosey I, Deville A, de Pontual L, Schleiermacher G, Coze C, Philip N, Frébourg T, Munnich A, Lyonnet S, Delattre O, Amiel J. Germline mutations of the paired-like homeobox 2B (PHOX2B) gene in neuroblastoma. Am J Hum Genet. 2004; 74:761–64. https://doi.org/10.1086/383253 [PubMed]
- 15. Pattyn A, Morin X, Cremer H, Goridis C, Brunet JF. The homeobox gene Phox2b is essential for the development of autonomic neural crest derivatives. Nature. 1999; 399:366–70. https://doi.org/10.1038/20700 [PubMed]
- 16. Elworthy S, Pinto JP, Pettifer A, Cancela ML, Kelsh RN. Phox2b function in the enteric nervous system is conserved in zebrafish and is sox10-dependent. Mech Dev. 2005; 122:659–69. https://doi.org/10.1016/j.mod.2004.12.008 [PubMed]
- 17. Pei D, Luther W, Wang W, Paw BH, Stewart RA, George RE. Distinct neuroblastoma-associated alterations of PHOX2B impair sympathetic neuronal differentiation in zebrafish models. PLoS Genet. 2013; 9:e1003533. https://doi.org/10.1371/journal.pgen.1003533 [PubMed]
- 18. Nagashimada M, Ohta H, Li C, Nakao K, Uesaka T, Brunet JF, Amiel J, Trochet D, Wakayama T, Enomoto H. Autonomic neurocristopathy-associated mutations in PHOX2B dysregulate Sox10 expression. J Clin Invest. 2012; 122:3145–58. https://doi.org/10.1172/JCI63401 [PubMed]
- 19. Fernández RM, Mathieu Y, Luzón-Toro B, Núñez-Torres R, González-Meneses A, Antiñolo G, Amiel J, Borrego S. Contributions of PHOX2B in the pathogenesis of Hirschsprung disease. PLoS One. 2013; 8:e54043. https://doi.org/10.1371/journal.pone.0054043 [PubMed]
- 20. van Limpt V, Schramm A, van Lakeman A, Sluis P, Chan A, van Noesel M, Baas F, Caron H, Eggert A, Versteeg R. The Phox2B homeobox gene is mutated in sporadic neuroblastomas. Oncogene. 2004; 23:9280–88. https://doi.org/10.1038/sj.onc.1208157 [PubMed]
- 21. Perri P, Bachetti T, Longo L, Matera I, Seri M, Tonini GP, Ceccherini I. PHOX2B mutations and genetic predisposition to neuroblastoma. Oncogene. 2005; 24:3050–53. https://doi.org/10.1038/sj.onc.1208532 [PubMed]
- 22. Szymońska I, Borgenvik TL, Karlsvik TM, Halsen A, Malecki BK, Saetre SE, Jagła M, Kruczek P, Talowska AM, Drabik G, Zasada M, Malecki M. Novel mutation-deletion in the PHOX2B gene of the patient diagnosed with Neuroblastoma, Hirschsprung’s Disease, and Congenital Central Hypoventilation Syndrome (NB-HSCR-CCHS) Cluster. J Genet Syndr Gene Ther. 2015; 6:269. https://doi.org/10.4172/2157-7412.1000269 [PubMed]
- 23. Liang CM, Ji DM, Yuan X, Ren LL, Shen J, Zhang HY. RET and PHOX2B genetic polymorphisms and Hirschsprung’s disease susceptibility: a meta-analysis. PLoS One. 2014; 9:e90091. https://doi.org/10.1371/journal.pone.0090091 [PubMed]
- 24. Garcia-Barceló M, Sham MH, Lui VC, Chen BL, Ott J, Tam PK. Association study of PHOX2B as a candidate gene for Hirschsprung’s disease. Gut. 2003; 52:563–67. https://doi.org/10.1136/gut.52.4.563 [PubMed]
- 25. Liu CP, Li XG, Lou JT, Xue Y, Luo CF, Zhou XW, Chen F, Li X, Li M, Li JC. Association analysis of the PHOX2B gene with Hirschsprung disease in the Han Chinese population of Southeastern China. J Pediatr Surg. 2009; 44:1805–11. https://doi.org/10.1016/j.jpedsurg.2008.12.009 [PubMed]
- 26. Xiao D, Liu L, Miao XP, Mao JX. Association between genetic polymorphisms of PHOX2B and susceptibility of Hirschsprung’s disease in Shenzhen region. J Shanxi Med Univ. 2009.
- 27. van Limpt V, Schramm A, van Lakeman A, Sluis P, Chan A, van Noesel M, Baas F, Caron H, Eggert A, Versteeg R. The Phox2B homeobox gene is mutated in sporadic neuroblastomas. Oncogene. 2004; 23:9280–88. https://doi.org/10.1038/sj.onc.1208157 [PubMed]
- 28. Zhang Y, Xie X, Zeng J, Wu Q, Zhang R, Zhu D, Xia H. Association of NRG1 and AUTS2 genetic polymorphisms with Hirschsprung disease in a South Chinese population. J Cell Mol Med. 2018; 22:2190–99. https://doi.org/10.1111/jcmm.13498 [PubMed]
- 29. Jennings LJ, Yu M, Rand CM, Kravis N, Berry-Kravis EM, Patwari PP, Weese-Mayer DE. Variable human phenotype associated with novel deletions of the PHOX2B gene. Pediatr Pulmonol. 2012; 47:153–61. https://doi.org/10.1002/ppul.21527 [PubMed]
- 30. Liu CP, Li XJ. Association analysis of the PHOX2B gene with Hirschsprung disease in the Han Chinese population of Southeastern China. J Pediatr Surg. 2010; 48:496–503. [PubMed]
- 31. Lauriola M, Ugolini G, Rivetti S, Nanì S, Rosati G, Zanotti S, Montroni I, Manaresi A, Zattoni D, Belluzzi A, Castellani L, D’Uva G, Mattei G, et al. IL23R, NOD2/CARD15, ATG16L1 and PHOX2B polymorphisms in a group of patients with Crohn’s disease and correlation with sub-phenotypes. Int J Mol Med. 2011; 27:469–77. https://doi.org/10.3892/ijmm.2010.591 [PubMed]
- 32. Di Lascio S, Benfante R, Di Zanni E, Cardani S, Adamo A, Fornasari D, Ceccherini I, Bachetti T. Structural and functional differences in PHOX2B frameshift mutations underlie isolated or syndromic congenital central hypoventilation syndrome. Hum Mutat. 2018; 39:219–36. https://doi.org/10.1002/humu.23365 [PubMed]
- 33. Salomon R, Attié T, Pelet A, Bidaud C, Eng C, Amiel J, Sarnacki S, Goulet O, Ricour C, Nihoul-Fékété C, Munnich A, Lyonnet S. Germline mutations of the RET ligand GDNF are not sufficient to cause Hirschsprung disease. Nat Genet. 1996; 14:345–47. https://doi.org/10.1038/ng1196-345 [PubMed]
- 34. Hofstra RM, Osinga J, Buys CH. Mutations in Hirschsprung disease: when does a mutation contribute to the phenotype. Eur J Hum Genet. 1997; 5:180–85. [PubMed]
- 35. Kim JH, Cheong HS, Sul JH, Seo JM, Kim DY, Oh JT, Park KW, Kim HY, Jung SM, Jung K, Cho MJ, Bae JS, Shin HD. A genome-wide association study identifies potential susceptibility loci for Hirschsprung disease. PLoS One. 2014; 9:e110292. https://doi.org/10.1371/journal.pone.0110292 [PubMed]
- 36. Mossé YP, Laudenslager M, Longo L, Cole KA, Wood A, Attiyeh EF, Laquaglia MJ, Sennett R, Lynch JE, Perri P, Laureys G, Speleman F, Kim C, et al, and YP M. Identification of ALK as a major familial neuroblastoma predisposition gene. Nature. 2008; 455:930–35. https://doi.org/10.1038/nature07261 [PubMed]
- 37. Trochet D, Bourdeaut F, Janoueix-Lerosey I, Deville A, de Pontual L, Schleiermacher G, Coze C, Philip N, Frébourg T, Munnich A, Lyonnet S, Delattre O, Amiel J. Germline mutations of the paired-like homeobox 2B (PHOX2B) gene in neuroblastoma. Am J Hum Genet. 2004; 74:761–64. https://doi.org/10.1086/383253 [PubMed]
- 38. Caglar HO, Tumer S, Olgun HN, Altungoz O. Analysis of sporadic neuroblastic tumors reveals a novel PHOX2B mutation in neuroblastoma. Gene Rep. 2018; 11:239–43.
- 39. Amiel J, Laudier B, Attié-Bitach T, Trang H, de Pontual L, Gener B, Trochet D, Etchevers H, Ray P, Simonneau M, Vekemans M, Munnich A, Gaultier C, Lyonnet S. Polyalanine expansion and frameshift mutations of the paired-like homeobox gene PHOX2B in congenital central hypoventilation syndrome. Nat Genet. 2003; 33:459–61. https://doi.org/10.1038/ng1130 [PubMed]
- 40. Berry-Kravis EM, Zhou L, Rand CM, Weesemayer DE. Congenital central hypoventilation syndrome: PHOX2B mutations and phenotype. Am J Respir Crit Care Med. 2006; 74:1139–44. https://doi.org/10.1164/rccm.200602-305OC [PubMed]
- 41. Mosse YP, Laudenslager M, Khazi D, Carlisle AJ, Winter CL, Rappaport E, Maris JM. Germline PHOX2B mutation in hereditary neuroblastoma. Am J Hum Genet. 2004; 75:727–30. https://doi.org/10.1086/424530 [PubMed]
- 42. Stoll C, Baretton G, Löhrs U. The influence of p53 and associated factors on the outcome of patients with oral squamous cell carcinoma. Virchows Arch. 1998; 433:427–33. https://doi.org/10.1007/s004280050270 [PubMed]
- 43. Cheng J, Zhuo Z, Xin Y, Zhao P, Yang W, Zhou H, Zhang J, Gao Y, He J, Li P. Relevance of XPD polymorphisms to neuroblastoma risk in Chinese children: a four-center case-control study. Aging (Albany NY). 2018; 10:1989–2000. https://doi.org/10.18632/aging.101522 [PubMed]
- 44. He J, Zou Y, Liu X, Zhu J, Zhang J, Zhang R, Yang T, Xia H. Association of Common Genetic Variants in Pre-microRNAs and Neuroblastoma Susceptibility: A Two-Center Study in Chinese Children. Mol Ther Nucleic Acids. 2018; 11:1–8. https://doi.org/10.1016/j.omtn.2018.01.003 [PubMed]
- 45. Zhuo ZJ, Liu W, Zhang J, Zhu J, Zhang R, Tang J, Yang T, Zou Y, He J, Xia H. Functional Polymorphisms at ERCC1/XPF Genes Confer Neuroblastoma Risk in Chinese Children. EBioMedicine. 2018; 30:113–19. https://doi.org/10.1016/j.ebiom.2018.03.003 [PubMed]
- 46. Yang X, He J, Chang Y, Luo A, Luo A, Zhang J, Zhang R, Xia H, Xu L. HOTAIR gene polymorphisms contribute to increased neuroblastoma susceptibility in Chinese children. Cancer. 2018; 124:2599–606. https://doi.org/10.1002/cncr.31353 [PubMed]
- 47. Zhang J, Lin H, Wang J, He J, Zhang D, Qin P, Yang L, Yan L. LMO1 polymorphisms reduce neuroblastoma risk in Chinese children: a two-center case-control study. Oncotarget. 2017; 8:65620–26. https://doi.org/10.18632/oncotarget.20018 [PubMed]
- 48. Zhang J, Zhuo ZJ, Wang J, He J, Yang L, Zhang D, Qin P, Yan L. CASC15 gene polymorphisms reduce neuroblastoma risk in Chinese children. Oncotarget. 2017; 8:91343–49. https://doi.org/10.18632/oncotarget.20514 [PubMed]
- 49. Zhang J, Zhuo Z, Li W, Zhu J, He J, Su J. XRCC1 gene polymorphisms and risk of neuroblastoma in Chinese children. Aging (Albany NY). 2018; 10:2944–53. https://doi.org/10.18632/aging.101601 [PubMed]
- 50. Kellis M, and GTEx Consortium. The Genotype-Tissue Expression (GTEx) project. Nat Genet. 2013; 45:580–85. https://doi.org/10.1038/ng.2653 [PubMed]
- 51. Dong X, Liu L, Miao XP, Mao JX. Association between genetic polymorphisms of PHOX2B and susceptibility of Hirschsprung’s disease in Shenzhen region. J Shanxi Med Univ. 2009.
- 52. He J, Qiu LX, Wang MY, Hua RX, Zhang RX, Yu HP, Wang YN, Sun MH, Zhou XY, Yang YJ, Wang JC, Jin L, Wei QY, Li J. Polymorphisms in the XPG gene and risk of gastric cancer in Chinese populations. Hum Genet. 2012; 131:1235–44. https://doi.org/10.1007/s00439-012-1152-8 [PubMed]
- 53. He J, Wang F, Zhu J, Zhang R, Yang T, Zou Y, Xia H. Association of potentially functional variants in the XPG gene with neuroblastoma risk in a Chinese population. J Cell Mol Med. 2016; 20:1481–90. https://doi.org/10.1111/jcmm.12836 [PubMed]
- 54. He J, Yang T, Zhang R, Zhu J, Wang F, Zou Y, Xia H. Potentially functional polymorphisms in the LIN28B gene contribute to neuroblastoma susceptibility in Chinese children. J Cell Mol Med. 2016; 20:1534–41. https://doi.org/10.1111/jcmm.12846 [PubMed]
- 55. Gama-Castro S, Salgado H, Santos-Zavaleta A, Ledezma-Tejeida D, Muñiz-Rascado L, García-Sotelo JS, Alquicira-Hernández K, Martínez-Flores I, Pannier L, Castro-Mondragón JA, Medina-Rivera A, Solano-Lira H, Bonavides-Martínez C, et al. RegulonDB version 9.0: high-level integration of gene regulation, coexpression, motif clustering and beyond. Nucleic Acids Res. 2016; 44:D133–43. https://doi.org/10.1093/nar/gkv1156 [PubMed]