Multiplex polymerase chain reaction.

The polymerase chain reaction (PCR) is a widely utilized assay for specifically amplifying small fragments of DNA. Multiplex PCR is the amplification of more than one DNA fragment per reaction and has many potential uses. When more than one primer set per reaction tube is utilized, the total number of tubes in any one experiment may be reduced, conserving expensive reagents and decreasing possible contamination. Multiplex PCR allows for an assay of the gene of interest and assures that the amplification process proceeds as expected with the use of a companion control genome primer set. Multiplex PCR is useful in assaying DNA extracted from samples of immunocompromised patients in which more than one infectious agent may be suspected such as simultaneous EBV and CMV detection. Multiplex PCR offers many advantages over single reaction PCR and has been found to be an useful adjunct in our laboratory.     

Partial pyruvate decarboxylase deficiency with profound lactic acidosis and hyperammonemia: responses to dichloroacetate and benzoate

We describe the successful use of sodium benzoate in a neonate with hyperammonemia associated with congenital lactic acidosis caused by a partial deficiency of the E1 component of pyruvate dehydrogenase (PDH); of note, this biochemical disturbance has not been previously described in PDH deficiency. The pyruvate dehydrogenase complex in skin fibroblasts had 48% of normal activity with a deficiency of the E1 component. The infant presented with rapid onset of a severe metabolic lactic acidosis, hyperventilation, hyperammonemia, and coma. At 30 hours of age continuous peritoneal dialysis was started; however, plasma NH3 concentrations remained in the 300-400 micrograms/dl range over the next 12 hours. Sodium benzoate, 250 mg/kg, was infused intravenously with a decrease in plasma ammonia of 25 micrograms/dl/hr. Hippurate was documented in the urine and peritoneal fluid after benzoate therapy. At 10.5 months of age, 50 mg/kg dichloroacetate was administered orally under fasting conditions, which resulted in a 56 and 62% reduction in the serum lactate and pyruvate levels, respectively; after 2 weeks on dichloroacetate his fasting levels were significantly decreased. Fibroblast PDH activity responded similarly to this drug. In our patient sodium benzoate was rapidly effective in producing a decline in plasma ammonia that was associated with clinical improvement. We feel that its use in organic acidemias deserves further evaluation and, furthermore, that any child with suspected PDH deficiency requires a clinical trial of dichloroacetate.     

Cell culture from rat renal glomeruli

The primary culture of rat renal glomeruli was found to result in the ready outgrowth of two cells types. One type designated c-cells were cytokeratin positive and exhibited microvilli and cilia. The second type designated f-cells were vimentin positive and showed rugose surfaces. C-cells were polygonal in culture on plastic surfaces and were derived from cells of parietal epithelial origin. F-cells assumed a more extended form on plastic and were judged to be a sub-set of parietal epithelial cells. Neither cell type was derived from the visceral epithelium which was found to have been destroyed during isolation of the glomeruli. When cultured on isolated glomerular basement membrane both the c-cells and f-cells assumed a polygonal morphology but when grown on Matrigel the cells assumed the form of long strands interconnecting the outgrowths between the glomeruli. The appearance of the cells in the strands, judged from scanning electron microscopy, suggested that these were formed from f-cells but other cell types were entrained in the structures. Glomeruli subjected to vigorous proteinase digestion of the basement membrane allowed culture of a wider variety of cells. These included endothelial cells, judged by OX-43 antibody and anti-von Willebrand Factor staining, and mesangial cells. In cultures from glomeruli polygonal cells are often assumed to be visceral epithelial cells, the results from this study indicate that this assumption is unsound. The very different behaviour of cells grown on isolated basement membrane as compared with cells grown on Matrigel suggests that Matrigel may not faithfully mimic basement membrane with respect to cell response in culture.     

Mutations associated with microsatellite unstable colorectal carcinomas exhibit widespread intratumoral heterogeneity

Although microsatellite instability (MSI) has been shown to be present in 15% of sporadic colorectal carcinomas, the genetic events underlying the development of these tumors have not been well described. By investigating intratumoral heterogeneity, this study attempts to elucidate whether MSI-positive colorectal carcinomas develop as the result of a random accumulation of mutations or as an ordered, stepwise sequence of genetic alterations. Eighty-six regions from 16 MSI-positive sporadic colorectal carcinomas were examined for mutations in repeat nucleotide sequences of the tumour suppressor genes transforming growth factor beta type II receptor (TGFBRII), insulin-like growth factor II receptor (IGFIIR), and BAX, and the mismatch repair genes MSH3 and MSH6. At least 2 and up to 5 of these genes were mutated in each tumour, and widespread intratumoral heterogeneity was observed for each gene. Regions of tumour with TGFBRII mutations were correlated with a poorly differentiated histology. Unlike the situation in microsatellite stable colorectal carcinomas, the findings of the present study did not suggest that a particular sequence of tumour suppressor and mismatch repair genes are mutated during colorectal tumorigenesis. It seems likely that a random accumulation of mutations, as a result of a defect in the mismatch repair pathway, drives tumour progression in this type of colorectal carcinoma.