Evaluation of the human fragile X mental retardation 1 polymerase chain reaction reagents to amplify the FMR1 gene: testing in a clinical diagnostic laboratory.

Fragile X syndrome (FXS) is caused by the absence of a functional fragile X mental retardation protein (FMRP). In most cases, the molecular mutation is an expansion and consequent methylation of the CGG trinucleotide repeat in the 5' end of the FMR1 gene. Polymerase chain reaction (PCR)-based assays that overcome the limitations of amplifying >100-150 CGG repeats have been designed. One such product, Human FMR1 PCR Reagents, can detect expanded mutation alleles without determining methylation status. We used this assay to amplify 70 clinical samples previously tested in three clinical laboratories, including 28 full mutation alleles, 17 premutation alleles, 6 gray zone alleles, and 21 normal samples (51 normal alleles including 5 homozygous females). The results were concordant with previously reported results. All full and premutation alleles were identifiable: repeat sizes are not assigned when the CGG repeat number is >200 and all full and premutation alleles were scored in the same category using this assay. All normal and gray zone alleles were within 0-1 repeat of their previously reported allele sizes. This method identified a mosaic premutation/full mutation pattern in 12/21 samples previously identified as full mutation only and in 5/7 samples previously reported as mosaic premutation/full mutation. These results demonstrate that this assay provides comparable results to the combination of PCR/Southern blot methodologies. Additional issues such as technologist time, reagent costs, turnaround times, and sample requirements are comparable to the PCR/Southern blotting assays currently utilized; however, methylation status cannot be determined using this assay. It is likely that PCR-only based assays will eventually replace previous methods for FXS and that Southern blotting or another methylation assay will only be utilized when determination of methylation status is necessary. This type of assay may also be utilized for other nucleotide expansion disorders.

Phase II trial to evaluate gemcitabine and etoposide for locally advanced or metastatic pancreatic cancer.

Prior studies suggest that tumor cell lines harboring RAS mutations display remarkable sensitivity to gemcitabine and etoposide. In a phase II clinical trial of patients with locally advanced or metastatic pancreatic cancer, we evaluated the response rate to a combination of these drugs. Forty chemo-naïve patients with nonresectable and histologically confirmed pancreatic cancer were accrued. Patients received gemcitabine 1,000 mg/m(2) (days 1 and 8) and etoposide 80 mg/m(2) (days 8, 9, and 10; 21-day cycle). The primary end point was radiological response rate. Secondary objectives were determination of overall survival, response duration (time to progression), quality of life, toxicity, and CA 19-9 biomarker response. In 35 evaluable patients, 10 exhibited a radiological partial response and 12 had stable disease in response to treatment. Twenty patients exhibited a >20% decrease in CA 19-9 biomarker levels. Median overall survival was 6.7 months for all patients (40) and 7.2 months for evaluable patients (35). Notably, four patients survived for longer than 1 year, with two patients surviving for more than 2 years. Median time to progression for evaluable patients was 3.1 months. The median overall survival for locally advanced patients was 8.8 months and 6.75 months for metastatic patients. One-year survival was 10% for all patients and 11.4% for evaluable patients. Quality of life improved in 12 patients and remained stable in 3 of the evaluable patients. The primary dose-limiting toxicities were hematologic toxicity and fatigue. These results show that the gemcitabine and etoposide combination is generally well-tolerated and exhibits a response rate similar to other published studies.

Carnitine deficiency and supplementation do not affect the gene expression of carnitine biosynthetic enzymes in rats.

Starved male weanling rats supplemented with 20 mmol/L pivalate in their drinking water exhibit significantly depressed concentrations of carnitine in tissues and plasma. In addition, pivalate supplementation has been linked with increased renal and hepatic trimethyllysine hydroxylase (TMLH) activity, whereas carnitine supplementation has been associated with significantly decreased hepatic gamma-butyrobetaine hydroxylase (BBH) activity. The purpose of this study was to determine whether pivalate or carnitine supplementation affects the activity and genetic expression of 2 enzymes of carnitine (Cn) biosynthesis, TMLH and BBH, expressed as mRNA abundance, relative to the abundance of beta-actin mRNA. Male weanling rats were administered the control treatment (C; n = 6), the pivalate treatment (P; n = 7), or the pivalate treatment plus supplemental dietary carnitine (P+Cn; n = 7). Rats in group P had elevated renal TMLH activity, relative to the other groups (P < 0.05). The groups did not differ in the abundance of renal or hepatic TMLH or BBH mRNA. A previously unreported finding was the quantifiable level of renal BBH mRNA, which was verified by direct sequencing of the BBH cDNA product amplified from kidney RNA. The groups did not differ in renal BBH mRNA abundance and renal BBH enzyme activity was not detected. Thus, the alterations in enzyme activities in the pivalate-treated rats are not regulated at the transcriptional level, and are apparently related to post-transcriptional effects on the enzymes themselves.