Molecular and clinical characteristics in 32 families affected with familial adenomatous polyposis

Germ‐line mutations in the 5′ half of the Adenomatous Polyposis Coli (APC) gene are found in about 80% of the patients affected with familial adenomatous polyposis (FAP). The vast majority of these are nonsense or frameshift mutations which result in the loss of the carboxyl terminus of the APC protein. Using an in vivo assay in yeast, we have identified pathogenic germ‐line mutations in 26 of 32 (81%) unrelated Swiss families affected with FAP. Nine mutations were novel and eight families were shown to harbor two recurrent mutations. Correlations were attempted between the location of APC germ‐line mutations and clinical manifestations of the disease. © 2001 Wiley‐Liss, Inc.     

APC germline mutations identified in Czech patients with familial adenomatous polyposis

Familial adenomatous polyposis (FAP) is an autosomal dominantly inherited predisposition to colorectal cancer, which is caused by germline mutations in the adenomatous polyposis coli (APC) gene. The APC mutations have been investigated in 46 Czech unrelated FAP families and 9 suspected FAP families using DGGE analysis and direct DNA sequencing. We found 25 germline APC mutations and identified 11 which were not previously reported. Of the identified mutations, 10 were 1 to 5 bp deletions, four were 1 bp insertions and six were nonsense, all leading to the formation of premature stop codon. In addition, we detected two mutations in the splice-donor region of APC intron 11, one missense and two samesense mutations. Phenotypes of patients with known and novel types of mutations are presented and discussed.     

Novel germline mutations in the APC gene of Cypriot patients with familial and sporadic adenomatous polyposis

Familial adenomatous polyposis (FAP) is one of the two commonest familial syndromes that predispose to colorectal cancer. FAP is caused by mutations in the adenomatous polyposis coli (APC) tumour suppressor gene that has a high penetrance. The disease is characterized by the occurrence of hundreds to thousands of colorectal polyps, which if left untreated give rise to colorectal cancer. In Cyprus, there are no molecular data available as yet on families with FAP. This work presents the results of APC analysis in our population for the first time. The APC gene was analyzed in 33 DNA samples from 20 individuals belonging to four FAP families and 13 patients with sporadic polyposis. We identified three truncating mutations, four missense mutations and 11 polymorphisms. It is of interest that two of the three truncating mutations, 2307delA and Q1242X, are novel, which supports the existence of a unique genetic pool in the Cypriot population. This ethnic molecular study in addition to highlighting population heterogeneity also contributes to phenotype-genotype associations that are essential for the clinical management of FAP families in Cyprus.     

Mutation analysis of the adenomatous polyposis coli (APC) gene in northwest Spanish patients with familial adenomatous polyposis (FAP) and sporadic colorectal cancer

Germline mutations in the tumor‐suppresor APC gene are associated with hereditary familial adenomatous polyposis (FAP) and somatic mutations are common in sporadic colorectal cancer. In this study, we report the identification of three novel germline mutations: 1682‐1683insA, 3252‐3253insAT, 3544A>T and a new somatic mutation 4130‐4131delTT, all giving rise to truncated APC proteins. The majority of the mutations we found originate a truncated APC protein and cause the FAP phenotype. However, special attention must be given to the missense mutations Asp1822Val and Ser2621Cys since their segregation with the FAP phenotype is questionable. In our FAP families we did not find any genetical alterations at codon 1309, being this mutation the most frequent reported in APC. Differences in the recurrence of pathological mutations in APC could exist among populations. However, epidemiological studies must be performed to confirm this hypothesis. Hum Mutat 18:355, 2001. © 2001 Wiley‐Liss, Inc.     

中国人家族性腺瘤性患肉病患者结肠腺瘤性患肉病基因的胚系突变

目的 探讨中国人家族性腺瘤性息肉病(familial adenomatous polyposis,FAr)患者的结肠腺瘤性息肉病(adenomatous polyposis coli,APC)基因的胚系突变类型.方法 对9个FAP家系18名成员进行多重连接依赖性探针扩增(multiplex ligation-dependent probe amplification,MLPA)检测APC基因有无大片段缺失.再应用PCR扩增APC基因的15个外显子区域,经变性高效液相色谱(denaturing high performance liquid chromatography,DHPLC)对每个扩增片段进行筛查,流出峰异常的片段,经DNA测序验证小片段的改变.结果 9个家系中有3个家系发现有APC基因的胚系突变:家系2为c.3184-3187 del CAhA,家系4为c.5432C＞T,家系9为c.3925-3929 del AAAAG.3种突变中c.5432C＞T在数据库中未见报道.结论 中国人不同的APC基因的胚系突变可引起FAP;无APC胚系突变的FAP患者的发病可能存在其他的机制.     

Germline mutations in APC and MUTYH are responsible for the majority of families with attenuated familial adenomatous polyposis

A small fraction of families with familial adenomatous polyposis (FAP) display an attenuated form of FAP (AFAP). We aimed to assess the presence of germline mutations in the MUTYH and adenomatous polyposis coli (APC) genes in AFAP families and to compare the clinical features between the two causative genes. Families with clinical AFAP were selected from the Dutch Polyposis Registry according to the following criteria: (a) at least two patients with 10-99 adenomas diagnosed at age >30 years or (b) one patient with 10-99 adenomas at age >30 years and a first-degree relative with colorectal cancer (CRC) with a few adenomas, and, applying for both criteria, no family members with more than 100 polyps before the age of 30 years. All probands were screened for germline mutations in the APC and MUTYH genes. Twenty-five of 315 Dutch families with FAP (8%) met our criteria for AFAP. These families included 146 patients with adenomas and/or CRC. Germline APC mutations were identified in nine families and biallelic MUTYH mutations in another nine families. CRC was identified at a mean age of 54 years (range 24-83 years) in families with APC and at 50 years (range 39-70 years) in families with MUTYH (p = 0.29). APC and biallelic MUTYH mutations are responsible for the majority of AFAP families. Based on our results and those reported in the literature, we recommend colonoscopy once every 2 years in AFAP families, starting surveillance from the late teens in APC mutation carriers and from age 20-25 years in biallelic MUTYH mutation carriers.     

Size-dependent expression of cyclooxygenase-2 in sporadic colorectal adenomas relative to adenomas in patients with familial adenomatous polyposis

Several studies have indicated that administration of non-steroidal anti-inflammatory drugs (NSAID) to patients with familial adenomatous polyposis (FAP) results in a regression of colorectal adenomas through inhibition of cyclooxygenase-2 (COX-2). It is thought that sporadic colorectal adenomas might also be useful targets for the chemoprevention of colorectal cancer, but a marked effect of NSAID on the regression of sporadic adenomas has not been observed. We investigated the immunohistochemical expression of COX-2 in sporadic tubular adenomas (n = 100) from 63 patients and in tubular adenomas (n = 121) from 12 patients with FAP, in order to determine if chemoprevention might be more successful in sporadic adenomas once they have reached a certain size. COX-2 scores were significantly lower (P < 0.0001) in small (< 5 mm in diameter) adenomas than in large (> or = 5 mm) adenomas. This was observed in both sporadic cases and in cases involving patients with FAP. With regard to small (< 5 mm) adenomas, significantly higher (P = 0.02) COX-2 scores were obtained in adenomas resulting from FAP than sporadic adenomas. The variation in COX-2 expression observed among sporadic adenomas of different sizes should be taken into account when making decisions regarding attempts at chemoprevention using NSAID. Sporadic adenomas 5 mm or larger with upregulated COX-2 expression are potentially useful targets for the antiproliferative effects of NSAID.     

Molecular analysis of the APC gene in 105 Dutch kindreds with familial adenomatous polyposis: 67 germline mutations identified by DGGE,PTT, and southern analysis

Germline mutations of the adenomatous polyposis coli (APC) gene are responsible for familial adenomatous polyposis (FAP), an autosomal dominant predisposition to colorectal cancer. We screened the entire coding region of the APC gene for mutations in an unselected series of 105 Dutch FAP kindreds. For the analysis of exons 1–14, we employed the GC-clamped denaturing gradient gel electrophoresis (DGGE), while the large exon 15 was examined using the protein truncation test. Using this approach, we identified 65 pathogenic mutations in the above 105 apparently unrelated FAP families. The mutations were predominantly either frameshifts (39/65) or single base substitutions (18/65), resulting in premature stop codons. Mutations that would predict abnormal RNA splicing were identified in seven cases. In one of the families, a nonconservative amino acid change was found to segregate with the disease. In spite of the large number of APC mutations reported to date, we identified 27 novel germline mutations in our patients, which reiterates the great heterogeneity of the mutation spectrum in FAP. In addition to the point mutations identified in our patients, structural rearrangements of APC were found in two pedigrees, by Southern blot analysis. The present study indicates that the combined use of DGGE, protein truncation test, and Southern blot analysis offers an efficient strategy for the presymptomatic diagnosis of FAP by direct mutation detection. We found that the combined use of the currently available molecular approaches still fails to identify the underlying genetic defect in a significant subset of the FAP families. The possible causes for this limitation are discussed. © 1997 Wiley-Liss, Inc.     

Intracellular distribution of beta-catenin in human medulloblastoma cell lines with different degree of neuronal differentiation

Gene mutations impairing the functions of the WNT signaling transduction pathway have been found in approximately 15% of human sporadic medulloblastomas. To understand the functional role of the WNT pathway in medulloblastoma, we have investigated the intracellular distribution of β-catenin in a series of 17 human medulloblastomas to correlate such expression with neuronal differentiation and in cultured cell models following functional silencing of the APC gene by small-interference RNA (siRNA). Transient siRNA transfection resulted in a 50% reduction of the APC gene product levels in both DAOY and D283MED cell lines. In the former, less-differentiated cell line, β-catenin levels remained unchanged or were slightly reduced, but β-catenin translocated in the nucleus following APC gene siRNA silencing. In contrast, in the more differentiated D283MED cells, β-catenin levels increased about twofold while mainly maintaining the cytoplasmic and cell membrane localization. Cytoplasmic/nuclear localization of β-catenin was present in 12 of 17 cases of medulloblastoma with a prevalent distribution in the classic, 6/7 cases, and large cell/anaplastic variant, 4/4 cases. The nodular/desmoplastic lesions showed strongly positivity in the cell membrane mainly of intranodular cells with advanced neuronal differentiation. These observations support an important functional role of WNT/β-catenin pathway in neuronal differentiation in medulloblastoma.     

Effects of Plasmodium berghei on thymus: high levels of apoptosis and premature egress of CD4(+)CD8(+) thymocytes in experimentally infected mice

We have previously showed alterations in the thymus during experimental infection with Plasmodium berghei, the causative agent of Malaria. Such alterations comprised histological changes with loss of delimitation between cortical and medullar regions, a profound atrophy with depletion of CD4⁺CD8⁺ double-positive (DP) thymocytes, and severe changes in the expression of cell migration-related molecules, belonging to the extracellular matrix and chemokine protein families. Taken together, these considerations prompted us to evaluate if the acute thymic atrophy observed during Plasmodium infection was correlated with increased apoptotic levels of thymocytes or with their premature emigration to the periphery. Our results confirmed that the marked reduction of the thymus weight in infected animals was accompanied by histological alterations, which included a very large number of cells showing nuclear condensation and karyorrhectic changes surrounded by histiocytes suggesting increased levels of apoptosis. This was confirmed by immunohistochemistry and flow cytometry techniques. In order to verify if an accelerated emigration of thymic cells to the peripheral lymphoid organs was also occurring we analyzed the spleen and mesenteric lymph nodes from control and infected mice. No significant differences were found in the spleen, but were seen after 14 days of infection between control and infected mice in the mesenteric lymph nodes. The main alteration was the presence of double negative (CD4⁻CD8⁻) and double positive (CD4⁺CD8⁺) cells. We concluded that both apoptosis of thymocytes and premature egress of immature cells take place during infection. Additional studies will be necessary to verify how such alterations might influence the systemic immune response to the parasite.     

Abnormal beta-catenin expression in oral cancer with no gene mutation: correlation with expression of cyclin D1 and epidermal growth factor receptor, Ki-67 labeling index, and clinicopathological features

Beta-Catenin not only acts as a regulator of E-cadherin-mediated cell-cell adhesion but also plays an important role in Wnt signaling. To assess the prevalence of Wnt signaling, we examined beta-catenin mutation and its immunohistochemical protein expression in oral cancers. The results were linked with expression of cyclin D1, one of the target genes of Wnt signaling, expression of epidermal growth factor receptor (EGFR) relevant to beta-catenin tyrosine phosphorylation, Ki-67 labeling index, clinicopathological features, and survival. In the analysis based on membranous expression of beta-catenin, 75 (68.2%) of 110 cases showed a reduced membranous pattern, and the remaining 35 (31.8%) had a preserved membranous pattern similar to that in oral epithelium. In the analysis of another category of beta-catenin expression, a cytoplasmic/nuclear pattern was observed in 21 (19.1%) of the 110 tumors. Most (19/21, 90.5%) of these tumors had a concomitant reduction of membranous expression of beta-catenin. The reduced membranous or cytoplasmic/nuclear pattern of beta-catenin was significantly associated with an invasive growth pattern, EGFR expression, an increased Ki-67 labeling index, and shorter survival but not with cyclin D1 expression. Mutational analyses of beta-catenin were performed for 39 cases, including the 21 tumors with a cytoplasmic/nuclear pattern, but no mutations in the beta-catenin gene exon 3 were detected in these samples. Our data indicate that altered expression of beta-catenin may play an important role in tumor progression through increased proliferation and invasiveness under EGFR activation. However, mutations of beta-catenin do not appear to be responsible for tumor development and abnormal expression of beta-catenin in oral cancers.     

Regulation of T lymphocyte apoptotic markers is associated to cell activation during the acute phase of dengue

Dengue fever, a public health problem in Brazil, may present severe clinical manifestations as result of an increased vascular permeability and coagulation disorders. T cell activation is a critical event for an effective immune response against infection, including the production of cytokines. We aim to reveal mechanisms that modulate the virus-cell interaction, with an emphasis on cell death. Apoptosis is involved in lymphocyte homeostasis, contributes to the clearance of virus-infected cells but also may play a role in the pathogenesis. Phosphatidylserine exposure on CD8T lymphocytes from dengue patients support early apoptotic processes and loss of genomic integrity, observed by DNA fragmentation in T lymphocytes and indicating late apoptosis. These T cells express activation and cytotoxic phenotypes as revealed by CD29 and CD107a upregulation. Higher frequencies of CD95 were detected in T lymphocytes mainly in those with the cytotoxic profile (CD107a⁺) and lower levels of anti-apoptotic molecule Bcl-2, suggesting that both CD4⁺ and CD8⁺ T cell subsets are more susceptible to apoptosis during acute dengue. The analysis of apoptosis-related protein expression profile showed that not only molecules with pro- but also those with anti-apoptotic functions are overexpressed, indicating that survival mechanisms could be possibly protecting cells against apoptosis caused by viral, immune, oxidative and/or genotoxic stresses. These observations led us to propose that in dengue patients there is an association between T cell susceptibility to apoptosis and the activation state. The mechanisms for understanding the immunopathogenesis during dengue infection are discussed.     

The role of intracellular amyloid beta in Alzheimer's disease

Extracellular amyloid beta (Abeta) that confers neurotoxicity and modulates synaptic plasticity and memory function has been central to the amyloid hypothesis of Alzheimer's disease (AD) pathology. Like many other misfolded proteins identified in neurodegenerative disorders, Abeta also accumulates inside the AD neurons. This intracellular Abeta affects a variety of cellular physiology from protein degradation, axonal transport, autophagy to apoptosis, further documenting the role of Abeta in AD. Therapeutics targeting intracellular Abeta could be effective treatment for AD.