The role of RET proto-oncogene methylation in the pathogenesis of Hirschsprung's disease / 天津医药

Objective To investigate the relationship between the methylation of RET (proto-oncogene, RET) and Hirschsprung disease (HD), and understand its significance in the development of intestinal wall ganglion cells. Methods Twenty-one surgical removal specimens, which were all dilation segment of HD in Tianjin Children’s Hospital, were used as experimental group, and 5 samples of non-HD normal colon tissues were used as control group. The bisulfite sequencing (BSP)-direct detecting method was used to detect RET CpG island methylation status. The expression of RET protein was detected by immunohistochemistry in experimental group and control group. Results In the experimental group 12 cases (57.14%) were found methylation, but no methylation was found in control group. The average optical density of methylated RET protein was 0.201±0.015 in 12 cases. The average optical density of un-methylated RET protein was 0.364±0.023 in 9 cases (P<0.05). Conclusion RET CpG island methylation reduced protein expression levels of RET. The corollary RET gene methylation may influence the expression levels of RET protein, thereby affecting the ganglion cell development, and thus participating in the occurrence of HD.

Clinical Significance of GDNF/GFRα1/RET Expression in Distal Rectum with Congenital Anorectal Malformation / 天津医药

Objective To evaluate the significance and expression of the GDNF/GFRα1/RET genes in distal rectum with Congenital Anorectal Malformations(ARMs) Methods Specimens were collected from resected colon which is 3 cm away to the anus and the distal of rectum in 12 ARMs patients. Haematoxylin and Eosin (HE) staining, immunohistochemical (IHC) staining and reverse transcription PCR (RT-PCR) were used to test RET, GDNF and GFRα1 expression in the differ-ent site of samples with ARMs. Results Expression of ganglia and RET, GDNF and GFRα1 were all negative in distal rec-tum in 12 ARMs patients. Ganglionic cells were found in tissues 3 cm away from the anus where RET, GDNF and GFRα1 expression were also positive in those ARMs patients. Expression of RET, GDNF and GFRα1 were strong positive in middle and low imperforate anus indicated by brown color and week positive in high imperforate anus indicated by yellow color. One case of faeces contamination and one case of loose motion without anal incontinence were found in post-op follow up of high ARMs patients. By contrast, one case of rectal mucosa prolapse and one case of occasional faeces contamination which recov-ered with hip bath were found in post op follow up in middle or low ARMs. Conclusion The unsatisfactory anorectal func-tion is possibly related to the decrease or lost of neurotrophic factors (GDNF/GFRα1/RET) and ganglia in the myenteric plex-uses post plastic surgery in ARMs patients.

The 5-hydroxytryptamine 4 Receptor Agonist-induced Actions and Enteric Neurogenesis in the Gut

We explored a novel effect of 5-hydroxytryptamine 4 receptor (5-HT4R) agonists in vivo to reconstruct the enteric neural circuitry that mediates a fundamental distal gut reflex. The neural circuit insult was performed in guinea pigs and rats by rectal transection and anastomosis. A 5-HT4R-agonist, mosapride citrate (MOS) applied orally and locally at the anastomotic site for 2 weeks promoted the regeneration of the impaired neural circuit or the recovery of the distal gut reflex. MOS generated neurofilament-, 5-HT4R- and 5-bromo-2'-deoxyuridine-positive cells and formed neural network in the granulation tissue at the anastomosis. Possible neural stem cell markers increased during the same time period. These novel actions by MOS were inhibited by specific 5-HT4R-antagonist such as GR113808 (GR) or SB-207266. The activation of enteric neural 5-HT4R promotes reconstruction of an enteric neural circuit that involves possibly neural stem cells. We also succeeded in forming dense enteric neural networks by MOS in a gut differentiated from mouse embryonic stem cells. GR abolished the formation of enteric neural networks. MOS up-regulated the expression of mRNA of 5-HT4R, and GR abolished this upregulation, suggesting MOS differentiated enteric neural networks, mediated via activation of 5-HT4R. In the small intestine in H-line: Thy1 promoter green fluorescent protein (GFP) mice, we obtained clear 3-dimensional imaging of enteric neurons that were newly generated by oral application of MOS after gut transection and anastomosis. All findings indicate that treatment with 5-HT4R-agonists could be a novel therapy for generating new enteric neurons to rescue aganglionic disorders in the whole gut.

Análise do gene CDKN1B/p27kip1 em pacientes com neoplasia endócrina múltipla tipo 2 / CDKN1B/p27kip1 gene analysis in patients with multiple endolcrine neopasia type 2 (MEN2)

INTRODUÇÃO: Na Neoplasia Endócrina Múltipla tipo 2 (NEM2), o desenvolvimento do Carcinoma Medular de Tireoide (CMT), Feocromocitoma (FEO) e Hiperparatireoidismo primário (HPT) está associado à mutações germinativas ativadoras no proto-oncogene RET. Casos de CMT esporádico podem apresentar mutações somáticas no RET (~40%). A variabilidade fenotípica observada em casos de CMT e FEO familiais associados à NEM2 indica o envolvimento de eventos genéticos adicionais que seriam responsáveis pelas diferenças clínicas observadas nos indivíduos afetados (idade de desenvolvimento, progressão e agressividade do tumor). Outras alterações genéticas no RET como duplas mutações, SNPs e haplótipos específicos podem influenciar na susceptibilidade, agressividade e modulação do fenótipo NEM2. Entretanto, os estudos de outros genes envolvidos no processo da tumorigênese NEM2 ainda estão em andamento. Recentemente foi mostrado que RET ativado controla a expressão de proteínas inibidoras do ciclo celular (p18 e p27). Mutações germinativas no gene p27 foram recentemente associadas à susceptibilidade de tumores neuroendócrinos e estão associadas à síndrome NEM4 (Neoplasia endócrina múltipla tipo 4). Mutações somáticas, inativadoras de p27, são raramente encontradas em vários tipos de tumores. Entretanto, diversos estudos documentaram que a redução na expressão e a sublocalização citoplamática de p27 são controladas por alterações pós-transducionais e/ou epigenéticas. OBJETIVOS: o estudo teve como objetivos avaliar a participação de genes, recentemente associados ao RET ativado, em tumores de pacientes com NEM2 e também verificar se polimorfismos no gene p27 estariam atuando como moduladores de fenótipo em uma grande família com NEM2. CASUÍTICA: foram analisadas 66 amostras tumorais advindas de 36 pacientes com diagnóstico clínico e genético de NEM2 e 28 indivíduos pertencentes a uma grande família com NEM2A-CMTF e mutação C620R no gene RET. MÉTODOS:...

INTRODUCTION: In Multiple Endocrine Neoplasia type 2 (MEN2) the development of medullary thyroid carcinoma (MTC), pheochromocytoma (PHEO) and primary hyperparathyroidism (HPT) are associated with activating germline mutations in RET proto-oncogene. Cases of sporadic MTC may have somatic RET mutations (~ 40%). The phenotypic variability observed in cases with familial MTC/MEN2 and PHEO/MEN2 indicates the probable involvement of additional genetic events that could be responsible for the clinical differences observed in the affected individuals (age development, progression and aggressiveness of the tumor). Other genetic alterations such as RET double mutations, SNPs and specific haplotypes may influence susceptibility, aggressiveness and MEN2 phenotype modulation. However, studies of other genes involved in the tumorigenesis of MEN2 are still in progress. Recently, it was shown that the activated RET controls the expression of cell cycle inhibitory proteins (p18 and p27). Germline mutations in the p27 gene have recently been associated with the susceptibility to neuroendocrine tumors and are associated with the MEN4 syndrome (Multiple endocrine neoplasia type 4). Somatic inactivating mutations p27 are rarely found in many types of tumors. However, several studies have documented that reduced expression and subcellular location of p27 is controlled by post-transductional changes and/or epigenetic factors. OBJECTIVES: This study aimed to evaluate the role of genes recently associated with RET activated in tumors from MEN2 patients and also check whether polymorphisms in the p27 gene would be acting as modulators of phenotype in a large MEN2 family. PATIENTS: We analyzed 66 tumor samples from 36 patients with clinical and genetic diagnosis of MEN2 and from 28 individuals belonging to a large family with FMTC/MEN2A and RET C620R mutation. METHODS: The analyses of somatic p27, p15, p18 and RET...

Two Cases of Multiple Endocrine Neoplasia Type 2B, Early Diagnosis by Genetic Analysis and Prophylactic Total Thyroidectomy / 대한소아내분비학회지

Multiple endocrine neoplasia (MEN) 2B is charaterized by tumors of endocrine glands, consisting of medullary thyroid carcinoma (MTC), pheochromocytoma and mucosal neuromas of the tongue, lips and other sites. Especially, MTC is the main cause of death in patients who have not received early prophylactic treatment, and MTC in MEN 2B represents more aggressive progress than that of MEN 2A. We encountered two cases of multiple endocrine neoplasia type 2B. One was a 13 month old boy who had familial history of MEN 2B without any symptoms, and the other was a 6-year old boy who manifested multiple mucosal neuromas of the tongue which had been aggravated in four months. Their genetic analysis revealed a point mutation 918th cordon in the RET proto-oncogene. Both of them underwent an operation for prophylactic total thyroidectomy and the 6 year old boy's specimen turned out to be thyroid medullary carcinoma. We encountered two cases of MEN 2B with prophylactic thyroidectomy by early diagnosis of RET proto-oncogene, and report the cases with review of literature.