ras Mutations in Endocrine Tumors : Mutation Detection by Polymerase Chain Reaction‐Single Strand Conformation Polymorphism

To elucidate the molecular basis for endocrine tumorigenesis, ras mutations in human endocrine tumors were analyzed using polymerase chain reaction‐single strand conformation polymorphism (PCR‐SSCP) analysis. Mutations of the H‐, K‐, N‐ras genes were examined in genomic DNAs from 169 successfully amplified primary endocrine tumors out of 189 samples. Four out of 24 thyroid follicular adenomas analyzed contained mutated N‐ras codon 61, and one contained the mutated H‐ras codon 61. One of the 19 pheochromocytomas revealed mutation of the H‐ras codon 13. No mutations of the ras gene were detected in pituitary adenomas, parathyroid tumors, thyroid cancers, endocrine pancreatic tumors, and adrenocortical tumors. Based on these findings we conclude that activation of the ras gene may play a role in the tumorigenesis of a limited number of thyroid follicular adenomas and pheochromocytomas, and that mutation of the ras gene is not frequent in other human endocrine tumors

Synthetic Receptors for the Differentiation of Phosphorylated Peptides with Nanomolar Affinities

Artificial ditopic receptors for the differentiation of phosphorylated peptides varying in i+3 amino acid side chains were synthesized, and their binding affinities and selectivities were determined. The synthetic receptors show the highest binding affinities to phosphorylated peptides under physiological conditions (HEPES, pH 7.5, 154 mM NaCl) reported thus far for artificial systems. The tight and selective binding was achieved by high cooperativity of the two binding moieties in the receptor molecules. All receptors interact with phosphorylated serine by bis(ZnII-cyclen) complex coordination and a second binding site recognizing a carboxylate or imidazole amino acid side chain functionality

Telomerase activity, P53 mutation and Ki-ras codon 12 point mutation of the peripheral blood in patients with hepato pancreato biliary diseases

BackgroundWith progress in molecular biology, the presence of telomerase activity, P53 mutation and Ki‐ras codon 12 point mutation has been reported in malignant tumours of the liver, pancreas and biliary tree. The purpose of this paper is to clarify the clinical implications of finding these three biomarkers in the peripheral blood of affected patients.MethodsTelomerase activity, P53 mutation, and Ki‐ras codon 12 point mutation in the peripheral blood were examined among 86 patients with hepato pancreato biliary disease, both benign and malignant, and the results were compared with clinical findings.ResultsOf 20 patients with benign conditions, only one patient with intraductal papillary adenoma showing severe dysplasia exhibited a biomarker (telomerase activity) in the peripheral blood. In total, there were 66 patients with various HPB carcinomas. Of 56 cancer patients studied preoperatively, 16 were positive for more than one biomarker, 13 were positive for telomerase activity, 4 for P53 mutation (three at exon 7 and another at exon 8), and 2 for Ki‐ ras codon 12 point mutation (both in the second letter). Twelve of the 16 biomarker‐postiive patients had stage IV disease as opposed to 23 of 40 biomarker‐negative patients. The resectability rate of the cancer was 38% in positive patients and 50% in negative patients. The one‐year survival rate after resection was zero in positive patients and 15% in negative patients, but the difference was not significant (P = 0.65). Of 32 patients with liver metastasis at the time of the molecular examination, eight were positive and 24 negative. Of 34 patients without liver metastasis, nine were positive and 25 negative. The development of subsequent liver metastases in those without them at the start was not significantly different in those with and without biomarkers (56 vs 36%: P = 0.31).ConclusionsThe three novel biomarkers of the peripheral blood seemed to be of little value for screening of early malignant HPB neoplasms but may help to predict liver metastasis

Technical Evaluation: Identification of Pathogenic Mutations in <i>PKD1</i> and <i>PKD2</i> in Patients with Autosomal Dominant Polycystic Kidney Disease by Next-Generation Sequencing and Use of a Comprehensive New Classification System

<div><p>Genetic testing of <i>PKD1</i> and <i>PKD2</i> is expected to play an increasingly important role in determining allelic influences in autosomal dominant polycystic kidney disease (ADPKD) in the near future. However, to date, genetic testing is not commonly employed because it is expensive, complicated because of genetic heterogeneity, and does not easily identify pathogenic variants. In this study, we developed a genetic testing system based on next-generation sequencing (NGS), long-range polymerase chain reaction, and a new software package. The new software package integrated seven databases and provided access to five cloud-based computing systems. The database integrated 241 polymorphic nonpathogenic variants detected in 140 healthy Japanese volunteers aged >35 years, who were confirmed by ultrasonography as having no cysts in either kidney. Using this system, we identified 60 novel and 30 known pathogenic mutations in 101 Japanese patients with ADPKD, with an overall detection rate of 89.1% (90/101) [95% confidence interval (CI), 83.0%–95.2%]. The sensitivity of the system increased to 93.1% (94/101) (95% CI, 88.1%–98.0%) when combined with multiplex ligation-dependent probe amplification analysis, making it sufficient for use in a clinical setting. In 82 (87.2%) of the patients, pathogenic mutations were detected in <i>PKD1</i> (95% CI, 79.0%–92.5%), whereas in 12 (12.8%) patients pathogenic mutations were detected in <i>PKD2</i> (95% CI, 7.5%–21.0%); this is consistent with previously reported findings. In addition, we were able to reconfirm our pathogenic mutation identification results using Sanger sequencing. In conclusion, we developed a high-sensitivity NGS-based system and successfully employed it to identify pathogenic mutations in <i>PKD1</i> and <i>PKD2</i> in Japanese patients with ADPKD.</p></div

Summary of the pathogenic mutations in Japanese patients with ADPKD.

<p>Summary of the pathogenic mutations in Japanese patients with ADPKD.</p