Highsmith, W. Edward

AbstractConversion Technology (CT) is a streamlined version of somatic cell hybrid preparation that was developed to be amenable for use as a mutation detection method and as a diagnostic tool for individual patients. It is also a powerful research tool. There are two broad categories of potential applications for CT: research applications in gene mapping and identification, and clinical applications in the detection of disease‐causing mutations in individual patients. CT may emerge as the gold standard for both mutation detection and haplotyping. It will likely prove valuable in mutation detection due to its ability to detect mutations that are not amenable to detection by sequencing (e.g., deep intron or promoter mutations). It will likely prove valuable in gene identification projects because genotyping of haploid chromosomes yields unequivocal haplotypes. Curr. Protoc. Hum. Genet. 55:3.6.1‐3.6.38. © 2007 by John Wiley & Sons, Inc.

AbstractBackground: Laboratory testing in suspected α-1-antitrypsin (A1AT) deficiency involves analysis of A1AT concentrations and identification of specific alleles by genotyping or phenotyping. The purpose of this study was to define and evaluate a strategy that provides reliable laboratory evaluation of A1AT deficiency.Methods: Samples from 512 individuals referred for A1AT phenotype analysis were analyzed by quantification, phenotype, and genotype. A1AT concentrations were measured by nephelometry. Phenotype analysis was performed by isoelectric focusing electrophoresis. The genotype assay detected the S and Z deficiency alleles by a melting curve analysis.Results: Of the 512 samples analyzed, 2% of the phenotype and genotype results were discordant. Among these 10 discordant results, 7 were attributed to phenotyping errors. On the basis of these data we formulated an algorithm, according to which we analyzed samples by genotyping and quantification assays, with a reflex to phenotyping when the genotype and quantification results were not concordant. Retrospective analyses demonstrated that 4% of samples submitted for genotype and quantitative analysis were reflexed to phenotyping. Of the reflexed samples, phenotyping confirmed the genotype result in 85% of cases. In the remaining 15%, phenotyping provided further information, including identifying rare deficiency alleles and suggesting the presence of a null allele, and allowed for a more definitive interpretation of the genotype result.Conclusions: The combination of genotyping and quantification, with a reflex to phenotyping, is the optimal strategy for the laboratory evaluation of A1AT deficiency.

Abstract Background: Single-base pair missense mutations in exons 10, 11, 13, 14, 15, and 16 of the RET protooncogene are associated with the autosomal dominant multiple endocrine neoplasia type 2 (MEN2) syndromes: MEN2A, MEN2B, and familial medullary thyroid carcinoma. The current widely used approach for RET mutation detection is sequencing of the exons. Methods: Because RET mutations are rare and the majority are heterozygous mutations, we investigated RET mutation detection by high-resolution amplicon melting analysis. This mutation scanning technique uses a saturating double-stranded nucleic acid binding dye, LCGreen®, and the high-resolution melter, HR-1™, to detect heterozygous and homozygous sequence variations. Mutant genotypes are distinguished from the wild-type genotype by an altered amplicon melting curve shape or position. Results: Samples of 26 unique RET mutations, 4 nonpathogenic polymorphisms, or the wild-type genotype were available for this study. The developed RET mutation-scanning assay differentiated RET sequence variations from the wild-type genotype by altered derivative melting curve shape or position. A blinded study of 80 samples (derived from the 35 mutant, polymorphism, or wild-type samples) demonstrated that 100% of RET sequence variations were differentiated from wild-type samples. For exons 11 and 13, the nonpathogenic polymorphisms could be distinguished from the pathogenic RET mutations. Some RET mutations could be directly genotyped by the mutation scanning assay because of unique derivative melting curve shapes. Conclusion: RET high-resolution amplicon melting analysis is a sensitive, closed-tube assay that can detect RET protooncogene sequence variations.

R115777 is a nonpeptidomimetic enzyme-specific inhibitor of farnesyl protein transferase (FT) that was developed as a potential inhibitor of Ras protein signaling, with antitumor activity in preclinical models. This study was a phase 1 trial of orally administered R115777 in 35 adults with poor-risk acute leukemias. Cohorts of patients received R115777 at doses ranging from 100 mg twice daily (bid) to 1200 mg bid for up to 21 days. Dose-limiting toxicity occurred at 1200 mg bid, with central neurotoxicity evidenced by ataxia, confusion, and dysarthria. Non–dose-limiting toxicities included reversible nausea, renal insufficiency, polydipsia, paresthesias, and myelosuppression. R115777 inhibited FT activity at 300 mg bid and farnesylation of FT substrates lamin A and HDJ-2 at 600 mg bid. Extracellular signal-regulated kinase (ERK), an effector enzyme of Ras-mediated signaling, was detected in its phosphorylated (activated) form in 8 (36.4%) of 22 pretreatment marrows and became undetectable in 4 of those 8 after one cycle of treatment. Pharmacokinetics revealed a linear relationship between dose and maximum plasma concentration or area under the curve over 12 hours at all dose levels. Weekly marrow samples demonstrated that R115777 accumulated in bone marrow in a dose-dependent fashion, with large increases in marrow drug levels beginning at 600 mg bid and with sustained levels throughout drug administration. Clinical responses occurred in 10 (29%) of the 34 evaluable patients, including 2 complete remissions. Genomic analyses failed to detect N-ras gene mutations in any of the 35 leukemias. The results of this first clinical trial of a signal transduction inhibitor in patients with acute leukemias suggest that inhibitors of FT may have important clinical antileukemic activity.