The Study Of The Polymorphisms Of RET Gene And PHOX2B Gene On Hirschsprung's Disease & The Study Of Pathological And Enterocolitis Features Of Intestinal Neuronal Dysplasia

Part Ⅰ The Study of the Polymorphisms of RET gene and PHOX2B gene on Hirschsprung's Disease IntroductionHirschsprung's disease (HD), or congenital aganglionosis, is a neurocristopathy characterized by the absence of ganglion cells in submucosal and myenteric plexuses along a variable length of the gastrointestinal tract. The incidence varies among different ethnic groups, with 1.5,2.1, and 2.8 per 10,000 live births in Caucasians, Africans, and Asians, respectively. HD appeared to be a multifactorial malformation, several genes involved in HD genetic aetiology including RET, GDNF, GFRα-1, EDNRB, EDN3, ECE-1, SOX10 and PHOX2B. The RET gene plays a crucial role in neural crest cell development in the bowel wall and has been identified as the most important one of HD associated genes.Linkage analyses of multiplex HD families revealed that the RET gene locus at 10q11.2 is the major gene locus for Hirschsprung's disease, however, RET mutations in codon region were detected in only 20% of sporadic and 50% of familial cases of HD, with low, sex dependent penetrance and lack of genotype-phenotype correlation. This support that the existence of moreRET variants or other modified genes in HD.To date, several RET polymorphisms were found associated with the HD in Caucasians, but no study of RET promoter has been published on the genetics of HD in the Chinese mainland population and no study of functional analysis of RET promoter's haplotypes polymorphism.PHOX2B is a transcription factor involved in the development of the noradrenergic nervous system, so it may associated with malformations of neural-crest origin such CCHS and HD. Only one study showed that the SNP of PHOX2B 1364 A>G( intron 2) was associated with increased risk of HD but no more studies about the relationship between PHOX2B and HD.Materials and MethodsPatients and controlsThis study consisted of 123 HD patients of Zhejiang Han ethnic Chinese. All patients were histologically confirmed with the criteria of the fourth International Symposium on Hirschsprung's disease and related neurocristopathies. Matched control were unselected, unrelated of Zhejiang Han ethnic Chinese without HD or other congenital malformation.Polymorphism AnalysisGenomic DNA was extracted from blood samples, genotypes were analyzed using PCR and direct sequencing,Immunohistochemical stainningAccording to the genotypes of RET promoter, forty tissue of normal ganglion segment of HD cases embedded in paraffin were cut with 5-μm-thick sections and immunohistochemical stained with C-Ret. The number of neuronal cells were examined.Statistical AnalysisChi square tests were performed to determine whether each SNP was in Hardy-Weinberg equilibrium within each group.The associations between SNPs and risk of HD were estimated by ORs and their 95% CIs, tested by x~2 tests. Data were considered significant at a level of P<0.05.Mann-Whitney Test was used for analysis the data of immunohistochemical staining.Haplotypes and their frequencies were estimated by means of the PHASE software.ResultsAssociation between genotypes and the risk of HDIncreased risk of HD was observed in homozygous genotypes of the RET allele -5AA, -1CC, cl35AA or c2307GG when compared with the other genotypes(P<0.05). No increased risk of HD was found in PHOX2B 1364A>G.Association between frequency of allele and the risk of HDIncreased risk of HD was observed in allele of RET -5A, -1C, cl35A, c2307G when compared with the other allele(P<0.05). In PHOX2B allele 1364A>G, no increased risk of HD was found.Association between frequency of allele and the types of HD.No associated risk was found between frequency of allele and the types of HD.Association between haplotypes frequencies and the risk of HDThe haplotype -5A/-1C of RET promoter was the most frequently one in HD (82.5%). The difference in promoter haplotype frequencies was highly significant between the HD and controls(P<0.001). The haplotype ACAG of four RET SNPs account for 75.2% of the HD, and the difference between the two distribution was highly significant(P<0.001).Frequency of allele in different ethnic peopleThe frequencies of HD-associated allele of RET -5A, c135A, c2307G in Chinese was higher than in the Caucasians or Africans significantly(.P<0.05).Functional analysis of RET promoter SNPsThe decreased express of RET was observed in promoter phenotype -5AA/-1CC or in homozygous of haplotype -5A/-1C than in others(P<0.05).ConclusionsThe RET SNPs -5G>A, -1A>C, c135G>A, c2307T>G is associated with HD and the haplotype ACAG is the core one in Zhejiang Han ethnic Chinese. It is no relationshipsbetween frequency of allele and the types of HD.The frequencies allele of RET -5A, cl35A, c2307G in controls is higher than in Caucasians or Africans.The different RET promoter haplotypes or genotypes associated with express of RETIt is no relationships between SNPs PHOX2B1364A>G and HD in Zhejiang Han ethnic Chinese.Part Ⅱ The Study of Pathological and Enterocolitis Features of Intestinal Neuronal DysplasiaIntroductionIn past few years, a group of disease named Allied Disorders of Hirschsprung's Disease (ADHD) were found, which show the clinical features similar to Hirschsprung's disease (HD). ADHD consist of Intestinal Neuronal Dysplasia (IND), Hypoganglionosis and Immaturity of ganglion cells. IND is the most frequently kind of all ADHD. The pathological features include presence of giant ganglia of the submucosal plexuses and myenteric plexuses, giant ganglia containing more than seven nerve cells, increase of Ache activity in nerve fibres of the lamina propria, ectopic ganglion cells in the colonic circular muscle and lamina propria, deficiency of sympathetic innervation in the muscle. Depending on the length of the neuronal dysplasia intestine, IND B may have a localized or disseminated form, it may occur in an isolated pure form or in combination with HD.IND as a cause for severe chronic constipation remains controversial. The congenital origin of IND B is supposed; nevertheless, IND were found secondary to intestinal obstruction orinflammatory disease. The precise etiology remains unknown, and, to date, no specific diagnostic test exists other than morphology. The criteria for diagnosis is still uncertain, the most approbatory technique is laparoscopic full-thickness intestinal biopsy or resected surgical specimens with HE stain or Ache stain, additional immunohistochemical staining is used to identify the dysplasia nerve cells.IND has no unified concept of treatment. There is an opinion that majority of patients with IND can be treated successfully with conservative treatment such as diet, laxatives and prokinetic drugs, however, in some severe cases a transient enterostomy or a segmental resection is unavoidable.The postoperative bowel function is well in most of megacolon, but part of them is complicated with recurrent postoperative enterocolitis (EC). But now there are still few studies about the relationship between morphological findings and postoperative EC. The features of IND under optic microscopy is well known, but there is still no picture of dysplasia neurons under electron microscopy.Materials and MethodsClinical features of INDThe data of clinical features of 293 cases with HD, IND and HD/IND were collect. The postoperative bowel function were followed. The histopathologic features of all cases were rechecked according to Holschneider's criteria.Immunohistochemical stainningTwenty cases of IND tissue embedded in paraffin and 7 with normal neurons were cut with 5-μm-thick sections and immunohistochemical stained with NSE,Cathepsin-D and S-100. The morphology of plexuses, neurons and neuroglia cells were examined.Transmission electron microscopy examiningFresh tissue of IND and normal colon were shaped and followed a series of process. The morphological of dysplasia and normal neurons were examined by TEM.Statistical analysis.All data were analyzed by x~2 test. Data were considered significant at a level of P<0.05.ResultsNeurons distribution and EC features of INDIn the distension segment of colon, the frequencies of dysganglion is 11.9%, 75.6% and 81.6% in HD, HD/IND and IND respectively (P<0.01).Incidences of EC in cases with the residual IND margins and with the normal margins were 38.2% and 8.7% respectively (P<0.01).Eight cases underwent another procedure because of severe persistent constipation or EC after operation, including 5 cases with IND of the proximal segment (4 HD/IND, 1 IND), 3 cases with the proximal segment aganglion.Histopathologic features of IND with immunohistochemical stainingWith immunohistochemical stained with NSE, the whole plexuses are positive. Giant ganglia with a amount of cells in different size (dysplasia neurons and neuroglia cells) were showed in IND sections.With immunohistochemical stained with Cathepsin-D, only the cytoplasm of neurons is positive. In IND sections, the picture of dysplasia neurons can be distinguished from the normal easily with a smaller cell and cytoblast diameter, a distinct increase of number, less and stain thickened cytoplasm, nucleolus exit or not.With immunohistochemical stained with S-100, the nerve fibres and neuroglia cells are positive. In IND sections, we can see the giant ganglia present with a number of stain-deficiency area in different size.Morphological features of IND under TEMIn normal neurons, the membrane of cells and nucleus is intact, the cell organs such as mitochondria and endoplasmic reticulum and ribosome are clear, the nucleolus are distinct, the chromatin is loosen, the cytoplasm is filled with neuroendocrine vesicle. In dysplasia neurons, whereas, neuroendocrine vesicle in cytoplasm is deficient, chromatin is collected near the nuclearmembrane, cell shape shrunk, smaller cytoblast and less cytoplasm.ConclusionsNeurons distribution of IND is inconsistent with macropathology. Recurrent postoperative EC is more likely occur in patients with IND, especially in margins with residual IND. However, the extension of excision about IND is uncertain and need to be further studied.IND can be diagnosed easily with immunohistochemical stained with NSE, Cathepsin-D and S-100 showing different part of plexuses.Dysplasia neurons is different from the normal. Neuroendocrine vesicle in cytoplasm is deficient, chromatin is collected near the nuclear membrane.