High promoter hypermethylation frequency of p14/ARF in supratentorial PNET but not in medulloblastoma

AIMS: Medulloblastoma (MB) is the most common primitive neuroectodermal tumour (PNET) of the central nervous system. Although supratentorial PNET (sPNET) and MB are histologically similar, their clinical behaviour differs, sPNET being more aggressive than MB. The aim of this study was to determine whether sPNET and MB are genetically different entities. METHODS AND RESULTS: We investigated 32 PNET primary tumour samples (23 MB and nine sPNET) and four PNET cell lines, for the presence of CDKN2A homozygous deletions at exon 1-alpha of p16/INK4 and exon 1-beta of p14/ARF, and promoter hypermethylation of both genes. No homozygous deletion of either p16/INK4 or p14/ARF was demonstrated in any of the PNET primary tumour samples. Methylation of p16/INK4 was found in one of six sPNET and in one of 23 MB, while p14/ARF methylation was observed in three of six sPNET and in three of 21 MB. No methylation of p16/INK4 or p14/ARF was found in any of the PNET cell lines analysed. The three MB cell lines did not show p16/INK4 expression, and only the MB Daoy cell line (homozygously deleted at CDKN2A) presented loss of p14/ARF expression. CONCLUSIONS: Our results in this limited series of central PNET show that p14/ARF is frequently involved in PNET carcinogenesis, with a higher frequency, but not statistically significant, for sPNET than for MB.     

Haplotype analysis of two recurrent CDKN2A mutations in 10 melanoma families: Evidence for common founders and independent mutations

Germ-line mutations in CDKN2A have been shown to predispose to cutaneous malignant melanoma. We have identified 2 new melanoma kindreds which carry a duplication of a 24bp repeat present in the 5′ region of CDKN2A previously identified in melanoma families from Australia and the United States. This mutation has now been reported in 5 melanoma families from 3 continents: Europe, North America, and Australasia. The M53I mutation in exon 2 of CDKN2A has also been documented in 5 melanoma families from Australia and North America. The aim of this study was to determine whether the occurrence of the mutations in these families from geographically diverse populations represented mutation hotspots within CDKN2A or were due to common ancestors. Haplotypes of 11 microsatellite markers flanking CDKN2A were constructed in 5 families carrying the M53I mutation and 5 families carrying the 24bp duplication. There were some differences in the segregating haplotypes due primarily to recombinations and mutations within the short tandem-repeat markers; however, the data provide evidence to indicate that there were at least 3 independent 24bp duplication events and possibly only 1 original M53I mutation. This is the first study to date which indicates common founders in melanoma families from different continents. Hum Mutat 11:424–431, 1998. © 1998 Wiley-Liss, Inc.     

Expression and localization of mutant p16 proteins in melanocytic lesions from familial melanoma patients

Little is known about the correlation between the loss of p16 expression and tumor progression in familial melanoma; no systematic study has been conducted on p16 expression in melanocytic tumors from patients carrying germline CDKN2A mutations. We analyzed 98 early primary lesions from familial patients, previously tested for germline CDKN2A status, by quantitative immunohistochemistry using 3 p16 antibodies. We found that p16 expression was inversely correlated with tumor progression and was significantly lower in melanomas, including in situ lesions, than in nevi. Of other features analyzed, tumor thickness showed the most significant correlation with p16 levels. Lesions from mutation-negative patients displayed combined nuclear and cytoplasmic staining. However, some mutation-positive lesions (ie, G101W, 113insR, M53I, R24P, and 33ins24), including benign nevi, showed nuclear mislocalization, confirming previous studies suggesting that subcellular distribution indicates functional impairment of p16.     

CDKN2A (p16INK4A) somatic and germline mutations

The cell cycle is composed of a series of steps that can be negatively or positively regulated by various factors. A group of low-molecular-weight proteins have recently been identified that specifically inhibit the function of cyclin-dependent kinases in mammalian cells. Inactivation of the CDKN2A gene (also known as p16^INK4A and MTS1) attracted considerable interest after it was mapped to 9p21, a locus for familial melanoma. In an effort to standardize the information regarding human CDKN2A mutations detected in cancers, a database with information of 146 point mutations has been created. Cancer type, origin of cells, specific mutation, amino acid change, literature citation, and other data are provided for each mutation entry. Studies of biochemical and biological functions of both wild-type and mutant proteins are central to our understanding of the role of p16^INK4a mutations in tumorigenesis, a summary of these studies is also included in the present update. © 1996 Wiley-Liss, Inc.     

High prevalence of the G101W germline mutation in the CDKN2A (P16ink4a) gene in 62 Italian malignant melanoma families

CDKN2A germline mutation frequency estimates are commonly based on families with several melanoma cases. When we started counseling in a research setting on gene susceptibility analysis in northern and central Italy, however, we mostly found small families with few cases. Here we briefly characterize those kindred, estimate CDKN2A/CDK4 mutation test yields, and provide indications on the possibility of implementing formal DNA testing for melanoma‐prone families in Italy. In September 1995 we started genetic counseling in a research setting at our Medical Genetics Center. Screening for CDKN2A/CDK4 mutations was performed on families with two melanoma patients, one of whom was younger than 50 years at onset, the other complying with one of the following: 1) being a first‐degree relative, 2) having an additional relative with pancreatic cancer, or 3) having multiple primary melanomas. Sixty‐two of 67 (80%) melanoma cases met our criteria. Four previously described CDKN2A mutations (G101W, R24P, V126D, and N71S) were found in 21 of the 62 families (34%) with a high prevalence of G101W (18/21). The percentage of families with two melanoma cases/family harboring a mutation was low (7%, 2/27), but rose to 45% (9/20) if one of the melanoma patients carried multiple melanomas or if pancreatic cancer was present in that family. In the 15 families with three melanoma cases the presence of a mutation was higher (67%, 10/15) and reached 100% in the 4 families with four or more melanoma cases. Our results suggest that CDKN2A/CDK4 counseling‐based mutational analysis may be reasonably efficient also for families with two melanoma cases, if one patient carries multiple melanomas or if pancreatic cancer is present in the family. © 2002 Wiley‐Liss, Inc.     

大肠癌组织p14ARF与p53基因变异研究

目的：观察大肠癌组织p14ARF基因遗 传和表观遗传变化及p53基因突变状况，探讨两者改变的关系及p14ARF-p53通路功能 破坏在大肠癌发生中的作用。方法：应用PCR、直接测序、甲基化特异PC R和RT-PCR分别检测56例原发性大肠癌及相应癌旁正常组织p14ARF基因纯合性缺失、 突变、5′CpG岛甲基化、mRNA表达及p53基因突变状况。结果：①大肠 癌组织p14ARF总变异率为27% (15/56)，其中1例纯合性缺失，14例5′CpG岛甲基化,未见突变发生。②15例p14ARF基因变异的大肠癌组织其相应mRNA表达阴性（13例）或 低水平表达（2例），41例未发生基因变异的大肠癌组织和所有癌旁正常组织p14ARF mRNA均显示明显表达。③大肠癌组织p53突变率为48% (27/56)。④56例大肠癌组织中，12例 仅发生p14ARF变异，24例仅显示p53突变，3例同时有p14ARF 5′CpG岛甲基化 和p53突变，17例p14ARF和p53均无变异，p14ARF-p53通路总变异率为70%（39/ 56），p14ARF 5′CpG岛高甲基化与野生型p53状态有关（P＜0.05）。⑤低分化 腺癌组p14ARF变异率（44%，7/16）明显高于高中分化腺癌组（20%，8/40）（P＜ 0.05），但两组间p53突变率无显著差异（P＞0.05）。结论:①大肠癌p14ARF基因5′ CpG岛高甲基化是其表达失活的主要机制；②大肠癌p14 ARF基因高甲基化与p53基因突变可能是相互独立的事件，两者的失活可能各自出现于不同 的大肠癌亚组中；③p14ARF高甲基化或p53突变引起的p14ARF-p53通路功能 破坏在大肠癌的发生中具有重要作用。     

单启动子p53/p14ARF非融合表达载体的构建及其在MG-63中的表达鉴定

目的构建单启动子非融合表达载体pIRES-p53-p14~(ARF)并观察其对骨肉瘤细胞增殖生长的影响及其功能初探。方法将p53与p14~(ARF)全长cDNA编码区依次亚克隆至pIRES载体中,并通过PCR、酶切鉴定重组质粒。用Lipofectamin 2000将野生型pIRES-p53-p14~(ARF)真核表达载体转染MG-63细胞,48h后采用axiovent200型倒置显微镜、荧光免疫细胞化学,观察其在细胞内的表达并初步探究其抑瘤功能。结果成功构建出含p53与p14~(ARF)全长基因片段的单启动子非融合表达载体pIRES-p53-p14~(ARF)。RT-PCR,酶切鉴定质粒大小、位点均符合实验设计。荧光免疫细胞化学检测显示p53阳性细胞同时也表现为p14~(ARF)免疫反应阳性,转染后瘤细胞可见散在凋亡现象。结论pIRES-p53-p14~(ARF)非融合表达载体的构建与真核细胞导入、目的基因表达成功。     

CDKN2A mutations in Spanish cutaneous malignant melanoma families and patients with multiple melanomas and other neoplasia

The CDKN2A gene has been implicated in cutaneous malignant melanoma (CMM) in about 40% of families with linkage to chromosome 9p21, while a small proportion of families have mutations in the CDK4 gene. In order to estimate the importance of these genes in the predisposition to CMM in Spanish families and patients we have analysed, by SSCA, a total of 56 subjects belonging to 34 CMM families, and nine patients with multiple CMM and other neoplasia. We have detected germline CDKN2A mutations in six out of the 34 families (17%). A frameshift mutation (358delG) and four missense mutations (G59V, G101W (two cases), D84Y, and R87W) were identified. Five CMM patients from different families (14%) carried the A148T variant, which is known not to affect p16 activity. No mutations were detected in the patients with multiple CMM or other neoplasms. We have not found mutations either in exon 1 beta of the CDKN2A gene or in exon 2A of CDK4. Linkage analysis of the 9p21 region showed exclusion for one of the families for CMM and for four families for CMM/dysplastic naevi. This study indicates a small role for CDKN2A in Spanish CMM families and suggests that other genes are also responsible for CMM predisposition.     

Immunohistochemical inactivation of p14ARF concomitant with MDM2 overexpression inversely correlates with p53 overexpression in oral squamous cell carcinoma

The CDKN2 gene encodes two structurally different proteins: a cyclin-dependent kinase inhibitor called p16, which regulates retinoblastoma protein (pRb)-dependent G1 arrest, and a cell cycle inhibitor designated p14ARF, which arrests cell growth in G1-S and also in G2-M. Whereas inactivation of p16 has been described as a frequent event in various cancers, including oral cancer, the current function of p14ARF is still poorly understood. A physical association between p14ARF and MDM2 blocks MDM2-induced p53 degradation, resulting in increased levels of p53, which in turn leads to cell cycle arrest. The present study immunohistochemically examined the expression of p16 and p14ARF together with pRb, MDM2 and p53 status in a series of oral cancers. The results showed that p14ARF was frequently absent in the oral cancers (15/37, 41%) as was p16 immunostaining. Concomitant immunopositivity for p14ARF and MDM2 overexpression was frequently observed in a subset of the cancers, whereas an inverse correlation between p14ARF and MDM2 expression and the diffuse staining of p53 was clearly detected. Moreover, the results showed that in most cases of oral cancer (35/37, 95%) at least one protein was altered, and lymph node metastasis was more frequent in the tumors with alterations in both the p16/pRb and p14ARF/p53 pathway (8/16, 50%) than in the tumors with one or no alteration of these two major pathways.     

Analysis of germline CDKN1C (p57KIP2) mutations in familial and sporadic Beckwith-Wiedemann syndrome (BWS) provides a novel genotype-phenotype correlation.

Beckwith-Wiedemann syndrome (BWS) is a human imprinting disorder with a variable phenotype. The major features are anterior abdominal wall defects including exomphalos (omphalocele), pre- and postnatal overgrowth, and macroglossia. Additional less frequent complications include specific developmental defects and a predisposition to embryonal tumours. BWS is genetically heterogeneous and epigenetic changes in the IGF2/H19 genes resulting in overexpression of IGF2 have been implicated in many cases. Recently germline mutations in the cyclin dependent kinase inhibitor gene CDKN1C (p57KIP2) have been reported in a variable minority of BWS patients. We have investigated a large series of familial and sporadic BWS patients for evidence of CDKN1C mutations by direct gene sequencing. A total of 70 patients with classical BWS were investigated; 54 were sporadic with no evidence of UPD and 16 were familial from seven kindreds. Novel germline CDKN1C mutations were identified in five probands, 3/7 (43%) familial cases and 2/54 (4%) sporadic cases. There was no association between germline CDKN1C mutations and IGF2 or H19 epigenotype abnormalities. The clinical phenotype of 13 BWS patients with germline CDKN1C mutations was compared to that of BWS patients with other defined types of molecular pathology. This showed a significantly higher frequency of exomphalos in the CDKN1C mutation cases (11/13) than in patients with an imprinting centre defect (associated with biallelic IGF2 expression and H19 silencing) (0/5, p<0.005) or patients with uniparental disomy (0/9, p<0.005). However, there was no association between germline CDKN1C mutations and risk of embryonal tumours. No CDKN1C mutations were identified in six non-BWS patients with overgrowth and Wilms tumour. These findings (1) show that germline CDKN1C mutations are a frequent cause of familial but not sporadic BWS, (2) suggest that CDKN1C mutations probably cause BWS independently of changes in IGF2/H19 imprinting, (3) provide evidence that aspects of the BWS phenotype may be correlated with the involvement of specific imprinted genes, and (4) link genotype-phenotype relationships in BWS and the results of murine experimental models of BWS.     

Clinical,In Silico,and Experimental Evidence for Pathogenicity of Two Novel Splice Site Mutations in the SH3TC2 Gene

Abstract: Charcot-Marie-Tooth (CMT) neuropathy is the most common inherited neuromuscular disorder. CMT is genetically very heterogeneous. Mutations in the SH3TC2 gene cause Charcot-Marie-Tooth neuropathy type 4C (CMT4C), a demyelinating form with autosomal recessive inheritance. In this study, two novel splice site mutations in the SH3TC2 gene have been studied (c.279G → A, c.3676–8G → A). Mutation c.279G → A was detected on one allele in two unrelated families with CMT4C in combination with a known pathogenic mutation (c.2860 C →T in one family, c.505T → C in the other) on the second allele of SH3TC2 gene. Variant c.3676–8G → A was detected in two patients from unrelated families on one allele of the SH3TC2 gene in combination with c.2860C →T mutation on the other allele. Several in silico tests were performed and exon trap experiments were undertaken in order to prove the effect of both mutations on proper splicing of SH3TC2. Fragments of SH3TC2 were subcloned into pET01 exon trap vector (Mobitec) and transfected into COS-7 cells. Aberrant splicing was predicted in silico for both mutations, which was confirmed by exon trap analysis. For c.279G → A mutation, 19 bases from intron 3 are retained in cDNA. The mutation c.3676–8G→ A produces a novel splice acceptor site for exon 17 and complex changes in splicing were observed. We present evidence that mutations c.279G → A and c.3676–8G →A in the SH3TC2 gene cause aberrant splicing and are therefore pathogenic and causal for CMT4C.     

Overexpression of p16 and p14ARF is associated with human papillomavirus infection in cervical squamous cell carcinoma and dysplasia

The CDKN2 gene encodes two structurally different proteins: a cyclin-dependent kinase inhibitor, p16, which regulates retinoblastoma protein (pRb)-dependent G1 arrest, and a cell cycle inhibitor, p14ARF, which blocks MDM2-induced p53 degradation resulting in an increase in p53 levels that leads to cell cycle arrest. Recent studies have revealed that expression of p16 and p14ARF is influenced markedly by the status of pRb and p53, and p16 overexpression has been demonstrated in cervical neoplasia because of functional inactivation of pRb by the human papillomavirus (HPV) E7 protein. To clarify the p14ARF status and the relationship between p16/p14ARF and other cell cycle molecules in cervical carcinogenesis, immunohistochemical analysis of p16, p14ARF, p53 and MDM2 was performed on 65 samples of cervical and genital condylomatous and neoplastic lesions, including nine HPV-negative tumors. In most cervical cancers and preneoplastic lesions with HPV infection of high and intermediate risk, a marked overexpression of p14ARF as well as the p16 protein (i.e. dotted nuclear immunostaining) was observed. All condyloma acuminata except one and low-grade dysplasia with HPV infection of low risk, such as HPV 6, immunohistochemically showed completely negative staining for p14ARF, also seen in non-neoplastic and mesenchymal cells. Our results clearly show that the mode of p14ARF overexpression in cervical neoplastic cells with HPV association differs from that in cancers of other organs without HPV association, and the p14ARF overexpression may be attributable to a negative feedback result in the functional inactivation of the pRb and p53 proteins by HPV oncoproteins.     

HCV肝炎肝硬化中核心蛋白、p14ARF和p21WAF1的表达

目的检测HCV肝炎、肝硬化组织中的核心蛋白、p14ARF、p21WAF1的表达及其相关性,探讨HCV核心蛋白对p14ARF、p21WAF1的表达作用.方法收集HCV表达阳性的肝炎、肝硬化组织23例,采用免疫组织化学EnVision两步法检测HCV感染的肝炎、肝硬化组织的核心蛋白、p14ARF、p21WAF1的表达,并分析三者间的关系.结果核心蛋白、p14ARF、p21WAF1的阳性表达主要定位于细胞核. 在核心蛋白均阳性的组织中,p14ARF的阳性表达率为69.6%,p21WAF1表达率为13%;三组间的阳性强度比较,差异有显著性(P=0.01),HCV核心蛋白与p14ARF、p21WAF1各组间的P值均<0.01;HCV核心蛋白与p14ARF、p21WAF1阳性强度两者间相关性检验,相关系数rs分别为-0.053、0.45(P值分别为0.72、0.20),表明无相关性.结论 HCV核心蛋白对p14ARF、p21WAF1的表达有影响可能间接促进p14ARF的表达,但抑制p21WAF1的表达.