Familial form of Hirschsprung disease: Nucleotide sequence studies reveal point mutations in the RET proto‐oncogene in two of six families but not in other candidate genes

Hirschsprung disease (HSCR; McKusick 142623) or aganglionic megacolon is a frequent (1 in 5,000 live births) heritable disorder of the enteric nervous system. By haplotyping with a variety of microsatellite markers, by amplifying all 20 exons of the RET proto‐oncogene and by applying a direct DNA sequencing protocol, we have analyzed the DNA from HSCR patients in 6 different families. In one family with a joint occurrence of HSCR and FMTC (follicular medullary thyroid carcinoma), we have identified a mutation in codon 609 in one out of 6 cysteine residues encoded in exon 10 of the RET gene. This C609R point mutation has not previously been reported to cause HSCR. In 2 of the HSCR patients described here from different families, we have found a mutation in exon 2 (R77C) and a silent mutation in exon 3 (Y204Y), respectively, in the extracellular part of the RET proto‐oncogene. In introns 2 and 17 of the RET proto‐oncogene in 2 families, we have detected single nucleotide exchanges that are probably polymorphisms with unknown, if any, relations to HSCR. The DNA sequences of 5 further genes (GDNF, GDNFRα, EDN3, EDNRB, and NTN), that may contribute to the development of HSCR, have not shown mutations in the patients analyzed so far. In 2 of the reported families with several affected children and one grandchild, sequence analyses revealed no mutations in the coding regions of any of the candidate genes analyzed. Am. J. Med. Genet. 94:19–27, 2000. © 2000 Wiley‐Liss, Inc.     

KIF1A and EDNRB are differentially methylated in primary HNSCC and salivary rinses

Silencing of tumor suppressor genes plays a vital role in head and neck carcinogenesis. In this study, we aimed to evaluate to the utility of aberrant promoter hypermethylation for detection in a panel of 10 genes (KIF1A, EDNRB, CDH4, TERT, CD44, NISCH, PAK3, VGF, MAL and FKBP4) in head and neck squamous cell carcinoma (HNSCC) via a candidate gene approach. We investigated methylation of the gene promoters by bisulfite modification and quantitative methylation‐specific PCR (Q‐MSP) in a preliminary study of a limited cohort of salivary rinses from healthy subjects (n = 61) and patients with HNSCC (n = 33). The methylation status of 2 selected genes (EDNRB and KIF1A) were then analyzed in 15 normal mucosa samples from a healthy population, 101 HNSCC tumors and the corresponding salivary rinses from 71 out of the 101 HNSCC patients were collected before treatment. The promoter regions of CDH4, TERT, VGF, MAL, FKBP4, NISCH and PAK3 were methylated in normal salivary rinses while no methylation of CD44 was observed in either normal salivary rinses or tumor samples. However, KIF1A and EDNRB were methylated in 98 and 97% of primary HNSCC tissues respectively and were only methylated in 2 and 6.6% of normal salivary rinses. In addition, KIF1A and EDNRB were methylated in 38 and 67.6% of salivary rinses from HNSCC patients, respectively. Promoter hypermethylation of KIF1A and EDNRB is a frequent event in primary HNSCC, and these genes are preferentially methylated in salivary rinses from HNSCC patients. KIF1A and EDNRB are potential biomarkers for HNSCC detection.     

子宫内膜癌患者FHIT、SLIT2、EDNRB基因杂合性丢失

目的：探讨子宫内膜癌患者FHIT、SLIT2、EDNRB基因3个微卫星位点D3S1287、 D4S1593、D13S160杂合性丢失（loss of heterozygosity,LOH）,以确定侯选的抑癌基因。方法：应用PCR-变性 PAGE-银染方法分别对35例子宫内膜癌患者癌组织及相对应的正常子宫内膜组织在FHIT、SLIT2、EDNRB基因3个微卫星位点D3S1287、D4S1593、D13S160 行LOH检测。结果：LOH总检出率为54.3%,D3S1287、D4S1593、D13S160位点分别为34.5%、20.5%、19.3%。FHIT、SLIT2、EDNRB基因的3个微卫星位点发生LOH率与子宫内膜癌手术-病理分期无明显相关性。结论：子宫内膜癌患者肿瘤组织在FHIT、SLIT2、及EDNRB基因的微卫星位点D3S1287、D4S1593、D13S160 均有LOH。FHIT、SLIT2及EDNRB基因为抑癌基因,其失活可能与子宫内膜癌的发生有关。     

先天性巨结肠患者RET和血管内皮素-β受体的表达及其临床意义

目的 探讨RET抗体、血管内皮素-β受体（endothelin-β receptor，EDNRB）抗体在先天性巨结肠（Hirschsprung’s disease，HD）患者体内的分布情况及其意义。方法 结合光镜下肠壁的形态特征，并应用免疫组化技术对33例临床病理确诊为先天性巨结肠的病变肠段及10例正常结肠行RET抗体、EDNRB抗体免疫组化染色，比较RET和EDNRB的染色结果和分布特点。结果 RET在肠段神经纤维上表达，HD狭窄段中表达减少（P〈0．01）；EDNRB在肠段血管上表达，长、短型HD中表达减少（P〈0．01）。结论 RET、EDNRB基因与HD的发生及类型相关。可为HD的诊断与治疗提供新的研究方向。     

克隆大鼠内皮素B受体基因重组腺病毒穿梭质粒的构建及鉴定

目的克隆大鼠内皮素B受体（EDNRB）基因，构建含有EDNRB基因的重组腺病毒载体，为转染神经干细胞和基因治疗先天性巨结肠（HD）奠定基础。方法从大鼠心脏组织中提取总RNA，用RT—PCR方法扩增出约1580bp的片段，并将该片段克隆到载体pAdtrack—CMV中，进行酶切鉴定和序列分析。结果证实成功构建了含有EDNPd3基因的重组腺病毒质粒。结论克隆到正确的大鼠EDNPd3基因，并构建出重组质粒pAd—EDNRB，为下一步重组腺病毒颗粒和基因治疗打下基础。     

Genetics of human enteric neuropathies

Knowledge of molecular mechanisms that underlie development of the enteric nervous system has greatly expanded in recent decades. Enteric neuropathies related to aberrant genetic development are thus becoming increasingly recognized. There has been no recent review of these often highly morbid disorders. This review highlights advances in knowledge of the molecular pathogenesis of these disorders from a clinical perspective. It includes diseases characterized by an infantile aganglionic Hirschsprung phenotype and those in which structural abnormalities are less pronounced. The implications for diagnosis, screening and possible reparative approaches are presented.