Novel germline mutation in the transmembrane region of RET gene close to Cys634Ser mutation associated with MEN 2A syndrome

Two mutations on the same allele of RET gene were revealed in a family with predisposition to multiple endocrine neoplasia (MEN) type 2A. The first mutation changes codon 634 from cysteine to serine. The second, a novel mutation in codon 641, changes alanine to serine in the transmembrane domain of the RET protein. Two mutations were present in close proximity in both the patients germline and tumor DNA and were absent in DNA isolated from healthy family members and control blood donors. All MEN 2A affected family members suffered from medullary thyroid carcinoma and two of ten patients for pheochromocytoma. No parathyroid gland alterations were observed in patients with two RET gene mutations. Analysis of four genetic polymorphisms in the RET gene showed higher incidence of polymorphisms of exons 11 and 15. The observed allelic imbalance in favor of mutated allele in pheochromocytoma corresponded to higher expression of the RET gene. These observations confirm the multifactorial process leading to development of MEN 2A syndrome.     

Effect of pentoxifylline on tumor suppressor and proto-oncogene apoptosis in sperm

Purpose : Pentoxifylline (PTX), a methylxanthine phosphodiesterase inhibitor reduces superoxide anions responsible for DNA apoptosis. The null hypothesis was that PTX was equally effective in reducing damage to specific cell genes. The objective was to determine the DNA integrity of the BRCA1 tumor suppressor gene and the c-myc proto-oncogene after PTX. Methods : Sperm (64 samples, 4 patients) were preincubated in either 0 (control) or 3.6 mM PTX (30 min), washed and incubated for 4 h at either 37 or 40°C heat shock activation. Single primer polymerase chain reactions (PCR) were carried out on lysed sperm targeting either BRCA1 exon 11 or c-myc exon 1. Control single-stranded DNA (ssDNA) were stained with 9 M Hoechst 33342 (blue) while PTX-treated ssDNA were stained with SYBR Gold (green). Nytran membrane discs with control ssDNA were hybridized to PTX-derived ssDNA. Fluorescent images stored in a microarray design were analyzed using ANOVA and Students' t-test for (P < 0.05) significance. Results : BRCA1 integrity was higher with PTX pretreatment (93.3 + 10.4 vs. control 50.5 + 9.2; mean + SEM). In contrast, there was no difference in c-myc integrity (56.8 + 9.0 vs. 41.7 + 6.4). Sense or antisense primers gave similar DNA fragmentation results. Conclusions : The data showed PTX pretreatment protected BRCA1 but not c-myc suggesting that PTX did not equally protect different cell genes. A possible explanation was that proto-oncogenes had more fragile sites. The study involved the DNA disc chip assay to assess separate PCR-amplified sense and antisense strands. The results suggested that both strands were equally affected by PTX pretreatment.     

干细胞因子受体基因在再生障碍性贫血患儿中的表达

目的探讨再生障碍性贫血（再障）患儿造血干细胞因子受体（C-KIT）基因Exon9、11、13的表达和序列构象及与发病机制的关系。方法应用逆转录-聚合酶链反应（RT-PCR）和聚合酶链反应单链构象多态分析（PCR-SSCP）和序列测定技术,测定再障与正常对照儿童各15例C-KIT基因Exon9、11、13的表达水平和结构是否存在变异。结果1.再障组C-KIT基因Exon9、11、13阳性表达率（40.0%）与正常对照组（46.67%）比较无显著性差异（P〉0.05）;2.经PCR-SSCP分析和基因序列测定C-KIT基因Exon9、11、13均未见碱基序列改变或碱基数目增多、缺失。结论C-KIT基因Exon9、11、13表达与儿童再障发病关系不大,提示儿童再障的发病可能不是由于Exon9、11、13突变引起。     

C-kit原癌基因在白血病细胞上的表达分析

目的　研究C kit(CD117)原癌基因在白血病细胞上的表达规律和意义。方法　采用CD4 5 SSC双参数 (对数 )散点图设门法进行三色流式细胞术分析。结果　急性髓细胞白血病 (AML)患者CD117表达率为 5 9 6 % ;慢性髓细胞白血病 (CML)CD117表达率为 16 7% ,且都为急变期 ;急性淋巴细胞性白血病 (ALL)极少表达 ,慢性淋巴细胞性白血病 (CLL)不表达 ;干细胞性白血病 (AUL)、双表型白血病 (BAL)高表达。结论　CD117有助于鉴别诊断髓系白血病 ,并能作为慢粒病程监测的指标     

c-cbl反义RNA抑制K562细胞的增殖

为探讨原癌基因c-cbl对CML细胞增殖的影响,我们用RT-PCR克隆了包括5′端非编码区的人类c-cbl基因部分序列,将之反向插入pcDNA3载体,并转染K562及NB4细胞。经MTT实验、半固体集落培养和流式细胞术观察细胞增殖能力的改变。结果表明,限制性酶切分析及序列测定证明所克隆的基因核苷酸序列正确,MTT实验显示反义载体转染K562细胞生长明显受抑,24、48及72小时增殖抑制率分别为30.07％,42.53％和55.75％(P值均<0.001),FCM-BrdU法测定的细胞倍增时间之比为1∶1.87(P<0.02),细胞集落形成抑制率为33.01％,反义转染基因NB4细胞增殖率无改变。上述结果直接地证明了c-cbl在CML细胞生长中的重要性。     

内脏不适与甜味刺激对大鼠孤束核神经元激活的相互作用

目的观察内脏不适刺激和甜味觉刺激单独给予和先后联合给予对脑干孤束核(nucleustractussolitarius,NTS)中相关神经元的激活作用,为内脏感觉和味觉在NTS中的信息处理提供形态学方面的资料。方法SD雄性大鼠16只,以抽签法随机分为4组,分别给予4种不同方式的刺激,即处理组T1:胃内灌注LiCl+口内灌注糖精(I/GLiCl+I/OSac);T2:胃内灌注LiCl+口内灌注蒸馏水(I/GLiCl+I/OWater);T3:胃内灌注蒸馏水+口内灌注糖精(I/GWater+I/OSac);对照组C4:胃内灌注蒸馏水+口内灌注蒸馏水(I/GWater+I/OWater)。应用免疫组织化学方法观察刺激后大鼠孤束核吻尾方向上不同区域的FOS蛋白表达情况。结果与对照组C4相比,T1组大鼠在孤束核吻尾方向上5个区域的FOS蛋白表达增加(P1=0.001,P2=0.000,P3=0.000,P4=0.002,P5=0.002);T2组(P1=0.049,P2=0.001,P3=0.021;P4=0.002;P5=0.001)和T3组(P1=0.005,P2=0.000,P3=0.031,P4=0.002,P5=0.000)在这些区域的FOS蛋白表达也增加。两者联合刺激对孤束核尾侧最后区周围的内脏亚核(N2)和吻侧的味觉区(N4,N5)的FOS蛋白表达有交互作用(分别为F2=11.87,P2<0.01;F4=6.83,P4<0.05;F5=12.81,P5<0.01)。结论不适内脏刺激和甜味觉刺激对孤束核相关神经元的激活具有交互抑制作用。     

多发性内分泌腺瘤病2A型家系临床特点分析及RET基因突变研究

目的检测互不相关的3个多发性内分泌腺瘤病2A型（MEN2A）家系中RET原癌基因突变情况，以探寻其发病的分子机制，同时总结其临床特点。方法收集3个MEN2A家系，共有8例MEN2A患者，3个家系有28位同意进行基因检测，提取28位外周血基因组DNA，对RET原癌基因21个外显子进行聚合酶链反应（PCR），PCR产物进行直接测序，对发现新的突变点进一步进行克隆测序。结果家系1RET原癌基因存在外显子11的C634R突变，家系2为C634Y突变，家系3的4例MEN2A患者均存在D631密码子（GAC）的杂合缺失，碱基序列由TGC∧GACGAGCTG变为TGCGAGCTG，导致代表天冬氨酸的D631的缺失，即del D631。8例MEN2A患者中7例有MTC（87．5％），8例有PCC（100％），未发现有HPT的发生，其中6例（75％）患者是以PCC起病，而且PCC中7例（87．5％）为双侧。结论本研究结果提示中国大陆MEN2A家系存在C634Y突变，也有exon11的D631杂合缺失突变，其中RET基因第11号外显子的D631缺失突变（delD631）是首例报道。D631 del临床特点为发病年龄较迟，肾上腺嗜铬细胞瘤可先于甲状腺髓样癌的发生。     

雌激素对福尔马林致痛后小鼠脊髓背角c-Fos和P物质的影响

目的观察雌激素对福尔马林致痛后小鼠痛觉评分及脊髓背角c-Fos和P物质(SP)表达的影响。方法选用雌性C57/BL6小鼠15只,分成对照组(control)、去卵巢手术组(OVX+V)、去卵巢手术加雌激素组(OVX+E),每组5只。采用痛觉评分分析雌激素对小鼠福尔马林足底注射后痛行为的影响,结合c-Fos及SP免疫组化,观察雌激素对脊髓L3～L5节段背角浅层神经元激活及痛觉传入的影响。结果小鼠福尔马林致痛后control组、OVX+V组和OVX+E组在第一时相的痛觉评分组间无统计学差异(P>0.05);间歇期及第二时相早期的痛觉评分OVX+V组明显高于control组和OVX+E组(P<0.05)。卵巢摘除增加福尔马林致痛后引起的脊髓背角FLIN数(P<0.01)和SP免疫阳性反应物,而雌激素能有效抑制福尔马林引起的注射侧背角FLIN数的升高(P<0.01)以及SP免疫阳性反应物显著降低。结论雌激素通过调节脊髓背角浅层感觉神经元的活动及痛觉纤维的传入参与对小鼠福尔马林致痛的镇痛作用。     

Familial medullary carcinoma prevention, risk evaluation, and RET in children of families with MEN2

The ability to predict the risk of MEN2 and medullary thyroid carcinoma (MTC) by genetic RET proto-oncogene analysis has provided an essential tool in identifying patients in whom thyroid cancer can be prevented by prophylactic thyroidectomy but emphasizes the need for clear policy guidelines. Children of families with RET cysteine mutations (exons 10, 11, 13, and 16) may develop early metastatic tumours and require prophylactic thyroidectomy. The 918 mutation associated with MEN2B is associated with early aggressive behaviour and distant metastatic spread. This has led to active screening of affected families underlining the need for specific intervention strategies.

Aim

To evaluate the risk to children of families with MEN2 and to assess the risk and determine the treatment.

Methods

Twenty-five patients from 10 families with MEN2 phenotypes were screened for RET mutations. Polymerase chain reaction amplification was performed on all 21 exons of the RET proto-oncogene, followed by heteroduplex single-strand conformation polymorphism (HEX-SSCP) analysis. Polymerase chain reaction products demonstrating variation in the HEX-SSCP gels were subjected to automated DNA sequencing analysis.

Results

Eleven significant RET mutations were detected in affected families. Eight index cases received initial thyroidectomy for established MTC (plus 2 advised). In the family members screened, 3 prophylactic thyroidectomies (2 with early MTC) were performed and a further 2 recommended. An exon 10 C620W missense mutation (the “Janus” gene) was detected in a patient with Hirschsprung's disease plus 1 family member.

Conclusion

RET analysis of MEN has revolutionized the management of children of families with MEN2 and allowed surgical prediction and prophylaxis to take place. The presence of an exon 10 C620W mutation in association with Hirschsprung's disease was difficult to assess. We suggest possible guidelines for management of families with MTC and the role of genetic testing in their evaluation.