Direct DNA Sequencing Of Exons Research Articles

Amelogenin gene influence on enamel defects of cleft lip and palate patients.

The aim of this study was to investigate the occurrence of mutations in the amelogenin gene (AMELX) in patients with cleft lip and palate (CLP) and enamel defects (ED). A total of 165 patients were divided into four groups: with CLP and ED (n=46), with CLP and without ED (n = 34), without CLP and with ED (n = 34), and without CLP or ED (n = 51). Genomic DNA was extracted from saliva followed by conducting a Polymerase Chain Reaction and direct DNA sequencing of exons 2 through 7 of AMELX. Mutations were found in 30% (n = 14), 35% (n = 12), 11% (n = 4) and 13% (n = 7) of the subjects from groups 1, 2, 3 and 4, respectively. Thirty seven mutations were detected and distributed throughout exons 2 (1 mutation - 2.7%), 6 (30 mutations - 81.08%) and 7 (6 mutations - 16.22%) of AMELX. No mutations were found in exons 3, 4 or 5. Of the 30 mutations found in exon 6, 43.34% (n = 13), 23.33% (n = 7), 13.33% (n = 4) and 20% (n = 6) were found in groups 1, 2, 3 and 4, respectively. c.261 C > T (rs2106416), a silent mutation, was detected in 26 subjects, and found more significantly (p = 0.003) in patients with CLP (groups 1 and 2 - 23.75%), compared with those without CLP (groups 3 and 4 - 8.23%). In the groups without ED, this silent mutation was also found more significantly (p = 0.032) among subjects with CLP (17.65% in group 2), compared with those without CLP (7.8% in group 4). In conclusion, this study suggested that AMELX may be a candidate gene for cleft lip and palate.

Oncogenic PIK3CA gene mutations and HER2/neu gene amplifications determine the sensitivity of uterine serous carcinoma cell lines to GDC-0980, a selective inhibitor of Class I PI3 kinase and mTOR kinase (TORC1/2)

To evaluate PIK3CA mutational status and c-erbB2 gene amplification in a series of primary uterine serous carcinomas (USC) cell lines. To assess the efficacy of GDC-0980, a potent inhibitor of Class I PI3 kinase and mTOR kinase (TORC1/2), against primary USC harboring HER2/neu gene amplification and/or PIK3CA mutations. Twenty-two primary USC cell lines were evaluated for c-erbB2 oncogene amplification by fluorescence in situ hybridization (FISH) assays and for PIK3CA gene mutations by direct DNA sequencing of exons 9 and 20. In vitro sensitivity to GDC-0980 was evaluated by flow-cytometry-based viability and proliferation assays. Downstream cellular responses to GDC-0980 were assessed by measuring phosphorylation of the 4-EBP1 protein by flow-cytometry. Five of 22 (22.7%) USC cell lines contained oncogenic PIK3CA mutations although 9 (40.9%) harbored c-erbB2 gene amplification by FISH. GDC-0980 caused a strong differential growth inhibition in FISH+ USC when compared with FISH- (GDC-0980 IC50 mean ± SEM = 0.29 ± 0.05 μM in FISH+ vs 1.09 ± 0.20 μM in FISH- tumors, P = .02). FISH+ USC harboring PIK3CA mutations were significantly more sensitive to GDC-0980 exposure when compared with USC cell lines harboring wild-type PIK3CA (P = .03). GDC-0980 growth-inhibition was associated with a significant and dose-dependent decline in phosphorylated 4-EBP1 levels. Oncogenic PIK3CA mutations and c-erbB2 gene amplification may represent biomarkers to identify patients harboring USC who may benefit most from the use of GDC-0980.

A case of clear cell variant of solid-pseudopapillary tumor of the pancreas in an adult male patient

A clear cell variant of solid-pseudopapillary tumor (SPT) of the pancreas was initially reported in 2006 as a tumor that arose in the pancreatic body and tail in young adults; to date, only 4 cases of this entity have been reported. Here, we present the case of a 58-year-old man with clear cell variant of SPT with distinctive clinicopathologic features. The tumor was well demarcated, was 2.6 cm in size and mostly composed of multivacuolated clear cells with solid growth, and exhibited the characteristic immunohistochemical positivity of β-catenin in the cytoplasm and nuclei of the neoplastic cells. In contrast to classical SPT with nuclear positivity, this case was negative for E-cadherin. Direct DNA sequencing of exon 3 of β-catenin gene demonstrated a single amino acid substitution (serine to phenylalanine) in codon 37, which is the phosphorylation site by GSKβ and frequently found in classical SPT. Electron microscopy demonstrated enlarged mitochondria and endoplasmic reticulum. Despite the fact that previous cases of clear cell variant of SPT arose mainly in the pancreatic body and tail in female young adults (age, 26-32 years), this case suggested that it is possible for a clear cell variant of SPT to arise in the pancreatic head in a middle-aged man. Because the recognition of the clear cell variant of SPT is important for the appropriate diagnosis of primary pancreatic tumor, the present case with its distinctive characteristics may provide new information for a more profound understanding of the pancreatic SPT.

Identification of VEGFR3 gene mutation in a Chinese family with autosomal dominant primary congenital lymphoedema

To identify the disease-causing gene in a four-generation Chinese family with 9 members affected with primary congenital lymphoedema (PCL, also known as Milroy disease). Linkage analysis was performed with a few microsatellite markers flanking the candidate genetic loci for PCL, including 3 known genes associated with autosomal dominant PCL. For mutation analysis, VEGFR3 gene was sequenced with DNA from the proband. Direct DNA sequencing of exon 25 of the VEGFR3 gene was performed in all family members. The disease gene in the family was mapped to chromosome 5q35.3 with a maximum Lod score of 2.07. Direct DNA sequencing of VEGFR3 gene revealed a heterozygous C to T transition at nucleotide 3341, resulting in p.Pro1114Leu mutation. The p.Pro1114Leu mutation co-segregated with all affected individuals in the family. This study identified a C3341T (p.Pro1114Leu) mutation in the VEGFR3 gene in a Chinese family with PCL, provided evidence that VEGFR3 mutation can cause PCL in Chinese.

A 14-Year-Old Female Patient With FIGO Stage IB Endometrial Carcinoma

This is the second report on the conservative treatment of endometrial carcinoma in a female patient younger than fifteen years. Case A, a 14-year-old teenager, presented with menorrhagia. An endometrial biopsy revealed grade 2 endometrioid adenocarcinoma. The estrogen receptor and progesterone receptor were strongly positive in 60% and 90% of the tumor, respectively. Although she was administered medroxyprogesterone acetate for a month, it was not effective. She underwent standard surgery including a hysterectomy. She was thereafter free of disease 1 year after surgery. No estrogen receptor staining of the surgical specimen was observed, and 30% of the tumor was strongly positive for progesterone receptor. Direct DNA sequencing of exons 7 and 8 of PTEN and the K-ras codon 12 demonstrated the presence of no mutation. In addition, no dominant-negative p53 mutation was found by a yeast functional assay. A uterine malignancy should therefore be included in the differential diagnosis in a young female patient complaining of abnormal genital bleeding.

Comparison of genotyping with direct sequencing as a method to detect EGFR mutations in NSCLC

22013 Background: EGFR mutations are detected in 10–15% of patients with non-small cell lung cancer (NSCLC). The most common clinically used method, and used at our institution, of EGFR mutation detection is based on direct DNA sequencing of exons 18–21. Most of the clinically significant EGFR mutations have been characterized and hence an alternative mutation detection method could be appropriate; we explored a genotyping method using scorpion probes (DXs; Manchester, UK). Methods: We compared genotyping (EGFR 29 mutation kit; Dxs) in a blinded fashion using 147 NSCLC specimens that had been previously sequenced in a CLIA (Clinical Laboratory Improvement Amendments) certified sequencing laboratory at Harvard. DNA specimens were from adenocarcinomas and typically had ≥ 50% tumor content. Results: In the 147 specimens, direct sequencing identified 42 (29%) EGFR mutations (Exons 18/19/20/21: 1/22/4/15). Of these, genotyping using scorpion probes (DxS) identified 39 (93%; sensitivity 93%). This method did not detect 3 mutations that were detected by sequencing including 2 exon 19 deletions and 1 exon 20 insertion; 1 of the exon 19 deletions is not assayed for in the DxS 29 mutation EGFR genotyping kit. Genotyping detected 5 additional EGFR mutations (Exon 19 deletion:1; EGFR T790M: 2 and L858R: 2) not detected by direct sequencing (specificity 96%). Four of the 5 (80%) were confirmed using a second methodology (WAVE/Surveyor). One of the 5 patients (L858R) has been treated with erlotinib and her tumor has regressed with treatment. No treatment information is available on the other 4 pts. Conclusions: Genotyping (sensitivity 93%; specificity 96%) provides an alternative method to direct sequencing for EGFR mutation detection. The biological and clinical significance of EGFR mutations detected by genotyping but not detected by direct sequencing is not fully defined. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Expert Testimony Other Remuneration AstraZeneca, Boehringer Ingelheim, Roche Pfizer

Molecular analysis of MEFV gene mutations among Palestinian patients with Behçet's disease

Objectives: The aim of our study was to determine the prevalence of Mediterranean fever gene (MEFV) mutations among Palestinian patients with Behçet's disease (BD).Methods: We screened 42 BD patients from the West Bank and Jerusalem for most of the MEFV mutations known to date. Patients diagnosed clinically according to the International Study Group (ISG) criteria were recruited from Makassed Islamic Charitable Hospital and private clinics. We performed the DNA testing using direct DNA sequencing of exon 10 of the MEFV gene and using the amplification refractory mutation system (ARMS) technique for mutations located in other exons.Results: We found that 40.5% of the samples had nine different MEFV mutations and one polymorphism. E148Q was the most prevalent mutation, found in 38.1% of the mutated alleles. M694V, V726A, M694I, A744S, P369S, R408Q, and F479L were each detected in 4.8% of the mutated alleles studied. The polymorphism P706 was detected in 9.5% of the mutated alleles. The mutations A744S, P369S, R408Q, and F479L were reported for the first time in BD patients. V722M, a novel MEFV mutation that has not been reported before in either FMF or BD patients, was identified in this study.Conclusion: This study is the first genetic analysis of MEFV mutations among Palestinian BD patients. It reflects their mutations profile, providing further data that MEFV mutations are an additional genetic susceptibility factor in BD.

Human Bone Marrow-Derived Mesenchymal Stem Cells Do Not Undergo Transformation after Long-Term In Vitro Culture and Do Not Exhibit Telomere Maintenance Mechanisms.

Mesenchymal sem cells (MSCs) are multipotent progenitors with the ability to differentiate along multiple cell lineages and are today considered a useful tool for cell therapy and tissue engineering approaches. Concerns that adult human MSCs may undergo malignant transformation have been recently raised. In fact, human adipose tissue-derived MSCs have been shown to spontaneously transform after long-term in vitro culture. Aim of this study was to investigate the susceptibility to transformation of human bone marrow (BM)-derived MSCs at different time points of in vitro culture. MSCs were isolated from BM of 10 healthy donors and propagated continuously in vitro until reaching a senescence phase or passage (P) 25. MSCs in the senescence phase were closely monitored for 8–12 weeks, to look for the appearance of a crisis phase. MSCs were characterized by morphology, differentiation capacity and immunophenotype at different time-points in culture. The genetic characterization of MSCs was investigated through array comparative genomic hybridization (array-CGH), classical cytogenetics and subtelomeric FISH analysis both before and after prolonged in vitro culture. MSCs were tested for the expression of telomerase activity (TA), hTERT transcripts and alternative mechanisms of telomere lengthening (ALT) at different time-points in culture. MSCs from 5 donors were also analyzed for telomere length at P3 and P15. p53 gene status was also analyzed in MSCs from donors #1 and #2 that rapidly reached senescence in culture. A huge variability between donors was noted in terms of proliferative capacity and in vitro life-span of MSCs. All MSCs maintained the typical spindle-shaped morphology, phenotype and ability to differentiate into osteoblasts and adipocytes throughout the culture period. All MSCs displayed a progressive decrease in the proliferative capacity until reaching senescence, not followed by any further growth. Array-CGH, karyotype and subtelomeric FISH analyses demonstrated that MSCs expanded in vitro did not show chromosomal rearrangements, also after long term culture. TA was not evidenced in the samples tested, including a post-senescence culture. hTERT transcripts (full-length and the additional splice variants a−, b−, a−b−) were found not to be expressed in any of the examined cultures. Telomeres shortened during the culture period. ALT were not evidenced in the MSCs tested, as indicated by the lack of ALT-associated promyelocytic leukemia bodies. Direct DNA sequencing of exons 2–11 in pre-senescence cultures from donors #1 and #2 did not evidence the presence of mutation in the p53 gene. Human BM-derived MSCs do not display an aptitude for spontaneous transformation and can be safely expanded in vitro without any sign of immortalization or development of chromosomal abnormalities. Our results provide support to the concept that the biological properties of human BM-derived MSCs after ex vivo expansion remain suitable for use in cell-therapy approaches; however, it is strongly recommended that phenotype, functional and genetic characteristics of MSCs after in vitro culture and before in vivo infusion are tested, to further guarantee safety for the patient.

P3-141: Assessments of the epidermal growth factor receptor mutations and amplification and their clinical implication in patients with lung adeno-carcinoma

Although the epidermal growth factor receptor (EGFR) has emerged as a promising target for patients with EGFR overexpressing non-small cell lung cancer, the impetus to standardize for their prognostic and predictive value is still much needed. In this study, we performed a study to establish a relationship among EGFR mutation, EGFR gene copy, and responses for gefitinib therapy in patients with lung adenocarcinoma. The EGFR status was evaluated by direct DNA sequencing of exons 18, 19, and 21 and fluorescence in situ hybridization (FISH) in 284 patients, and the clinical data including gefitinib treatment were gathered. Among 284 biopsy tumor samples, which were more than 0.1 cm, EGFR mutation with lung adenocarcinoma, mutation analysis was possi-ble in 263 cases (92.4%) and gene amplification analysis was possible in 270 cases (95.1%). Male:female ratio was 44.4%:55.6% and median age was 58 (range, 21-84). One hundred and eighteen patients (44.9%) had EGFR mutations and 30 patients (11.1%) had EGFR gene amplifications. The mutations included deletion or mutation of exon 19 in 65 patients (55.1%), mutation of exon 21 in 43 patients (36.4%) and mutation of exon 18 in 10 patients (8.5%). Of 172 non-smoking patients, 92 patients (53.5%) had EGFR mutations in contrast to 26 of 91 (28.6%) in smoking patients. Of 177 non-smoking patients, 24 patients (13.6%) had EGFR gene amplification in contrast to 6 of 93 (6.5%) in smoking patients. Of 30 patients with EGFR gene amplification, 27 patients (90%) harbored EGFR mutation simultaneously. One hundred and twenty-two patients were treated with gefitinib and 84 patients had measurable lesion. In the patients with measurable lesion, response rate in patients with EGFR mutation was 69.7% (23 of 33 patients), in contrast to 24.4% (10 of 41 patients) in patients without mutation. The response rate in the patients with EGFR gene amplification was 83.3% (5 of 6 patients). Of the patients treated with gefitinib, fifty eight patients with EGFR mutation had significantly prolonged time to progression (TTP) than 50 patients without mutation (median survival, 13.1 vs. 2.6 months; p = .0003). Of 58 patients with EGFR mutation, 13 patients with simultaneous EGFR gene amplification showed much longer TTP (median survival time not reached). EGFR mutation and EGFR gene amplification could be tested with small sized biopsy sample. We showed its clinical importance with re-gard to gefitinib. Lung adenocarcinoma that harbored EGFR mutation and gene amplification simultaneously showed better tumor response and TTP with gefitinib.

Mutation identification and characterization of a Taiwanese patient with fucosidosis

Fucosidosis is a rare lysosomal storage disease caused by a defect of the alpha-L: -fucosidase (FUCA1) gene. Worldwide 26 mutations underlying the disease have been reported. By direct DNA sequencing of exons and flanking introns, homozygous Y126X mutation and Q281R polymorphism were found in a Taiwanese patient with fucosidosis. Upon expressing in COS-7 cells, 97.4% of alpha-L: -fucosidase activity compared with that of the wild-type construct was observed in the cDNA containing Q281R polymorphism. Western blot analysis revealed a 58-kDa precursor and 56-kDa mature forms for cells transfected with wild-type and Q281R enzymes. Using the fluorogenic substrate, the Michaelis constants and maximal velocities of both enzymes were very similar. While no appreciable enzyme activity (0.0%) was observed with Y126X mutation, no apparent decrease in FUCA1 mRNA level was seen with Y126X mutation. The expressed truncated Y126X protein was unstable and largely degraded. The delineation of the molecular defect could serve to complement future prenatal diagnosis for this family when necessary.

PIK3CA and PTEN Mutations in Uterine Endometrioid Carcinoma and Complex Atypical Hyperplasia

The tumor suppressor PTEN gene and the PIK3CA oncogene are frequently mutated in uterine endometrioid carcinoma (UEC). PTEN mutations are also common in complex atypical hyperplasia (CAH), the precursor lesion of UEC. The status of PIK3CA has not yet been explored in CAH. In this study, we evaluated both CAH and UEC for PTEN and PIK3CA mutations. Neoplastic tissue was microdissected, and DNA was extracted from 29 cases of CAH. DNA was available from 44 UEC cases previously characterized for PTEN mutations. Direct DNA sequencing of exons 9 and 20 of the PIK3CA gene was done on all 73 cases. In addition, CAH cases were analyzed for PTEN mutations. Statistical analyses were done using the Fisher's exact test. Two (7%) of 29 CAH and 17 (39%) of 44 UEC cases contained a PIK3CA mutation (P = 0.003). Fourteen (48%) of 29 CAH cases had a PTEN mutation, but none contained both a PTEN and PIK3CA mutation. Twenty-five (57%) of 44 UEC cases had a PTEN mutation, and 12 (48%) of these 25 cases also contained a PIK3CA mutation. Coexistent PIK3CA and PTEN mutations were significantly correlated with UEC compared with CAH (P = 0.002), but the association in UEC did not reach statistical significance (P = 0.21). PIK3CA is the most commonly mutated oncogene in UEC; however, mutations are uncommon in CAH. Thus, mutations in PIK3CA, unlike PTEN mutations, are associated with invasion. These findings suggest that mutations in PIK3CA may serve as a marker of invasion with potential clinical use. Furthermore, PIK3CA and PTEN mutations may play distinct roles in endometrial tumorigenesis.

Description of the First Two Seemingly Unrelated Greek Cypriot Families with a Common C618R RET Proto-Oncogene Mutation

Germ-line mutations of the RET proto-oncogene cause three different cancer syndromes: multiple endocrine neoplasia type 2A (MEN2A), multiple endocrine neoplasia type 2B, and familial medullary thyroid carcinoma (FMTC). The objective of the present study was the clinical and molecular characterization of the first two Greek Cypriot families diagnosed with MEN2A and FMTC. The clinical diagnosis of the probands was based on clinical presentation and supported with laboratory findings (calcitonin and carcinoembryonic antigen tumor marker levels). We screened the RET gene by direct DNA sequencing of exons 10, 11, and 16 using genomic DNA as templates. After identification of the mutation, we also developed the amplification refractory mutation system (ARMS) as an alternative method to direct sequencing for genetic diagnosis of 22 additional individuals from both families. We identified the germ-line missense mutation T → C of codon 618 of exon 10 (C618R) in the probands of both families. By using ARMS, two members of the MEN2A family and five members of the FMTC family were also found positive for the C618R mutation. These are the first seemingly unrelated families in Cyprus investigated clinically and molecularly in detail and shown to transmit this common RET proto-oncogene mutation.

Description of the first two seemingly unrelated Greek Cypriot families with a common C618R RET proto-oncogene mutation.

Germ-line mutations of the RET proto-oncogene cause three different cancer syndromes: multiple endocrine neoplasia type 2A (MEN2A), multiple endocrine neoplasia type 2B, and familial medullary thyroid carcinoma (FMTC). The objective of the present study was the clinical and molecular characterization of the first two Greek Cypriot families diagnosed with MEN2A and FMTC. The clinical diagnosis of the probands was based on clinical presentation and supported with laboratory findings (calcitonin and carcinoembryonic antigen tumor marker levels). We screened the RET gene by direct DNA sequencing of exons 10, 11, and 16 using genomic DNA as templates. After identification of the mutation, we also developed the amplification refractory mutation system (ARMS) as an alternative method to direct sequencing for genetic diagnosis of 22 additional individuals from both families. We identified the germ-line missense mutation T --> C of codon 618 of exon 10 (C618R) in the probands of both families. By using ARMS, two members of the MEN2A family and five members of the FMTC family were also found positive for the C618R mutation. These are the first seemingly unrelated families in Cyprus investigated clinically and molecularly in detail and shown to transmit this common RET proto-oncogene mutation.

Orthostatic Intolerance Is Not Necessarily Related to a Specific Mutation (Ala457Pro) in the Human Norepinephrine Transporter Gene

Orthostatic intolerance (OI) is a syndrome characterized by lightheadedness, palpitations, fatigue, blurred vision, dizziness, chest discomfort, cognitive impairment, and occasionally syncope. These symptoms usually occur after upright posture and are associated with tachycardia and high plasma concentrations of norepinephrine. It has been proposed that a mutation in exon 9 of the norepinephrine transporter gene (Ala457Pro), resulting in more than 98% loss of function compared with the wild type, might provide a pathogenetic mechanism to explain the clinical symptoms of patients with OI. We studied 46 young men from military service who had sought medical advice because of dizziness while standing. Every patient underwent a tilt-table test, with monitoring of blood pressure, heart rate, and plasma catecholamines in supine position and during 30 minutes of standing. Fourteen patients showing the full-blown OI syndrome (30 bpm increase in heart rate and 600 pg/mL plasma norepinephrine levels while standing) underwent direct DNA sequencing of exon 9 of the norepinephrine-transporter gene. The specific mutation (Ala457Pro) was not detected in any of the 14 OI patients. Based on these findings, we doubt that this specific genetic transport defect is a frequent cause of the impaired uptake of norepinephrine in OI patients. Its routine determination will therefore not be helpful to establish the clinical diagnosis of OI.

Sequence-Based analysis of the HLA-DRB1 polymorphism in Metalsa Berber and Chaouya Arabic-speaking groups from Morocco

To examine the genetic diversity in Morocco, the polymorphism at the HLA-DRB1 locus was investigated in two populations: the Metalsa group consisting of Berbers from north Morocco (who speak the Tarifit language and live in the Nador area), and the Chaouya group who are Arabic-speaking people from west Morocco (Atlantic coast) living in the Settat area. The DRB1 alleles of 197 healthy unrelated individuals were identified by direct DNA sequencing of exon 2 using fluorescently-labeled primers. A total of 28 and 29 alleles at DRB1 locus were identified in the Metalsa and Chaouya groups, respectively. The most frequent alleles in the Metalsa group are DRB1∗03011 (20.2%), DRB1∗0701 (12.12%), and DRB1∗1302 (11.11%). In the Chaouya group, DRB1∗0701 (16.33%), DRB1∗15011 (12.76%), and DRB1∗03011 (11.73%) are most common. Each population exhibits some specific variants and some uncommon alleles. The frequency of the DRB1∗03011 allele differs significantly between the two populations ( p = 0.0311). The DRB1 frequency distributions in the two groups suggest the effects of balancing selection. The interpopulation analysis highlighted a strong relatedness, based on genetic distances, between the two Moroccan groups and the other north Africans (the Moroccans from El Jadida area, Moroccan Souss Berbers, Algerians, and Tunisians), and to a lesser extent with the Iberians, French, and Ethiopians.

Analysis of p53 mutations and Helicobacter pylori infection in human and animal models.

p53 gene mutations are believed to play a critical role in the development of gastric carcinoma. We examined the relation between Helicobacter pylori infection and p53 gene mutations of the gastric mucosa in human and animal models. To detect the original p53 DNA sequences of the Japanese monkey and Mongolian gerbil, the p53 genes of these animals were amplified using the nested polymerase chain reaction method with primers for the human p53 gene. Direct DNA sequencing of exons 5, 6, 7, and 8 of the p53 genes was performed by the dideoxy terminator method for gastric mucosa of humans, the Japanese monkey, and the Mongolian gerbil. The expression of p53 was examined immunohistochemically in a Japanese monkey model. Mutations of the p53 gene were identified in 52.4% of human H. pylori-positive mucosa and in 100% of monkey H. pylori-positive mucosa. However, no mutations of the p53 gene were found in the H. pylori-positive gastric mucosa of Mongolian gerbils. There were no mutations in H. pylori-negative gastritis mucosa of humans, monkeys, or Mongolian gerbils. Nuclear staining of p53 was seen in the glandular cells of the H. pylori-infected mucosa of Japanese monkeys, especially in the neck region of the glands. These findings demonstrate that the H. pylori infection can induce p53 point mutations in humans and the Japanese monkey and appear to be involved in the pathway leading to dysplasia or carcinoma. However, our direct DNA sequencing method showed no p53 mutations in the Mongolian gerbil model at present. Further studies with this model are needed.

Distinctive Molecular Genetic Alterations in Sporadic and Familial Adenomatous Polyposis-Associated Pancreatoblastomas: Frequent Alterations in the APC/β-Catenin Pathway and Chromosome 11p

Pancreatoblastomas are unusual malignant neoplasms of the pediatric pancreas that may also rarely affect adults. The molecular pathogenesis of pancreatoblastomas is unknown. They are clinicopathologically distinct from adult pancreatic ductal adenocarcinomas, but their occasional occurrence in patients with Beckwith-Wiedemann syndrome and the case presented here of a pancreatoblastoma in an adult patient with familial adenomatous polyposis (FAP) suggests that they might bear a genetic similarity to other infantile embryonal tumors such as hepatoblastomas. We analyzed a series of nine pancreatoblastomas for mutations common to other embryonal malignancies including somatic alterations in the adenomatous polyposis coli (APC)/beta-catenin pathway and chromosome 11p, using immunohistochemistry for beta-catenin, 5q and 11p allelic loss assays, and direct DNA sequencing of exon 3 of the beta-catenin gene and the mutation cluster region of the APC gene. In addition, we analyzed the pancreatoblastomas for alterations found in adult-type pancreatic ductal adenocarcinomas including mutations in the K-ras oncogene and the p53 and DPC4 tumor suppressor genes, using direct DNA sequencing of exon 1 of K-ras and immunohistochemistry for p53 and Dpc4. Allelic loss on chromosome 11p was the most common genetic alteration in pancreatoblastomas, present in 86% (six of seven informative cases). Molecular alterations in the APC/beta-catenin pathway were detected in 67% (six of nine), including five neoplasms with activating mutations of the beta-catenin oncogene and the one FAP-associated tumor with biallelic APC inactivation (germline truncating mutation combined with loss of the wild-type allele); seven neoplasms showed abnormal nuclear accumulation of beta-catenin protein. In contrast, loss of Dpc4 protein expression was present in only two cases (one diffuse and one focal), and no alterations in the K-ras gene or p53 expression were detected. Our findings indicate that pancreatoblastomas are genetically distinct from the more common pancreatic ductal adenocarcinomas, but bear a close molecular pathogenesis to hepatoblastomas. In addition, pancreatoblastoma may represent an extracolonic manifestation of FAP.

Mutation analysis of the p73 gene in nonastrocytic brain tumours.

Loss of heterozygosity (LOH) involving the distal chromosome 1p36region occurs frequently in nonastrocytic brain tumours, but the tumour suppressor gene targeted by this deletion is unknown. p73is a novel gene that has high sequence homology and similar gene structure to thep53 gene; it has been mapped to 1p36, and may thus represent a candidate for this tumour suppressor gene. To determine whether p73is involved in nonastrocytic brain tumour development, we analysed 65 tumour samples including 26 oligodendrogliomas, 4 ependymomas, 5 medulloblastomas, 10 meningiomas, 2 meningeal haemangiopericytomas, 2 neurofibrosarcomas, 3 primary lymphomas, 8 schwannomas and 5 metastatic tumours to the brain, for p73 alterations. Characterization of allelic loss at 1p36–p35 showed LOH in about 50% of cases, primarily involving oligodendroglial tumours (22 of 26 cases analysed; 85%) and meningiomas (4 of 10; 40%). PCR-SSCP and direct DNA sequencing of exons 2 to 14 of p73 revealed a missense mutation in one primary lymphoma: a G-to-A transition, with Glu291Lys change. 8 additional cases displayed no tumour-specific alterations, as 3 distinct polymorphic changes were identified: a double polymorphic change of exon 5 was found in one ependymoma and both samples derived from an oligodendroglioma, as follows: a G-to-A transition with no change in Pro 146, and a C-to-T variation with no change in Asn 204: a delG at exon 3/+12 position was identified in 4 samples corresponding to 2 oligodendrogliomas, 1 ependymoma and 1 meningioma, and a C-to-T change at exon 2/+10 position was present in a metastatic tumour. Although both LOH at 1p36 and p73 sequence changes were evidenced in 4 cases, it is difficult to establish a causal role of the p73 variations and nonastrocytic brain tumours development. © 2001 Cancer Research Campaign http://www.bjcancer.com

Analysis of p53 mutations and Helicobacter pylori infection in human and animal models

Background. p53 gene mutations are believed to play a critical role in the development of gastric carcinoma. We examined the relation betweenHelicobacter pylori infection andp53 gene mutations of the gastric mucosa in human and animal models.Methods. To detect the originalp53 DNA sequences of the Japanese monkey and Mongolian gerbil, thep53 genes of these animals were amplified using the nested polymerase chain reaction method with primers for the humanp53 gene. Direct DNA sequencing of exons 5, 6, 7, and 8 of thep53 genes was performed by the dyedeoxy terminator method for gastric mucosa of humans, the Japanese monkey, and the Mongolian gerbil. The expression ofp53 was examined immunohistochemically in a Japanese monkey model.Results. Mutations of thep53 gene were identified in 52.4% of humanH. pylori-positive mucosa and in 100% of monkeyH. pylori-positive mucosa. However, no mutations of thep53 gene were found in theH. pylori-positive gastric mucosa of Mongolian gerbils. There were no mutations inH. pylori-negative gastritis mucosa of humans, monkeys, or Mongolian gerbils. Nuclear staining of p53 was seen in the glandular cells of theH. pylori-infected mucosa of Japanese monkeys, especially in the neck region of the glands.Conclusions. These findings demonstrate that theH. pylori infection can inducep53 point mutations in humans and the Japanese monkey and appear to be involved in the pathway leading to dysplasia or carcinoma. However, our direct DNA sequencing method showed nop53 mutations in the Mongolian gerbil model at present. Further studies with this model are needed.

Sporadic Fundic Gland Polyps: Common Gastric Polyps Arising Through Activating Mutations in the β-Catenin Gene

Fundic gland polyps (FGPs) are the most common gastric polyps. FGPs traditionally have been regarded as nondysplastic hamartomatous or hyperplastic lesions, but their pathogenesis remains unclear. We have recently shown that somatic adenomatous polyposis coli (APC) gene alterations are frequently present in FGPs associated with familial adenomatous polyposis (FAP), raising the possibility that mutations of the beta-catenin gene affecting the APC/beta-catenin pathway might be involved in the pathogenesis of sporadic FGPs. We analyzed somatic beta-catenin gene mutations in 57 sporadic FGPs from 40 patients without FAP and in 19 FGPs from 13 FAP patients. Direct DNA sequencing of exon 3 encompassing the glycogen synthase kinase-3beta phosphorylation region for beta-catenin was used with confirmation by HIN:fI restriction endonuclease digestion. The foveolar epithelium and dilated fundic glands of the polyps were separately microdissected and analyzed in 22 of 57 sporadic FGPs. Activating beta-catenin gene mutations were present in 91% (52 of 57) of sporadic FGPs. Both the foveolar epithelium and the dilated fundic gland epithelium comprising the polyps were shown to have the same somatic beta-catenin mutation in 21 of 22 (95%) sporadic FGPs. In contrast, beta-catenin gene mutations were not present in any of the 19 FAP-associated FGPs (P: < 0.000001). The high frequency of beta-catenin mutations in sporadic FGPs indicates that these lesions arise through activating mutations of the beta-catenin gene. Beta-catenin mutations in gastrointestinal tract polyps have previously only been demonstrated in a subset of adenomatous (dysplastic) or neoplastic polyps. Sporadic FGPs are therefore the only lesions of the gastrointestinal tract to demonstrate beta-catenin mutations while lacking dysplastic morphology.