Deletion detection in the dystrophin gene by multiplex gap ligase chain reaction and immunochromatographic strip technology.

The purpose of this study is to demonstrate the value of a multiplex amplification and readout system. The validation was done using as a model system the detection of deletions in nine possible dystrophin exons: 4, 8, 12, 17, 19, 44, 45, 48, and 51. The amplification system was gap ligase chain reaction, adapted to amplify selected regions of multiple exons simultaneously. The amplified products were read out with an immunochromatographic methodology, adapted from that used in the Abbott product line commercialized under the name Test Pack Plus. In each amplification, the beta-globin gene was incorporated and served as a procedural control. The complete process takes < 3 hr from DNA sample to result. The procedure is therefore rapid and simple, as well as being potentially very cost effective. The combination of these two technologies is shown to be a useful tool for the determination of deletions in the nine exons of the dystrophin gene. The results of a 100-patient sample study showed concordance with cDNA and PCR in current use. Equivalent performance at two sites was shown.

Simultaneous analysis of mutant and normal alleles for multiple cystic fibrosis mutations by the ligase chain reaction.

The ligase chain reaction (LCR) involves repetitive cycles of ligation of two adjacent pairs of oligonucleotides to form longer ligated products in a template-dependent manner. This study demonstrates the application of LCR for analysis of multiple small mutations. We adapted the technology for the simultaneous determination of the normal and mutant alleles in a competition format, as well as multiple mutations in a multiplex format. For these purposes, we used mutations causing cystic fibrosis, namely the delta F508, W1282X, and G551D mutations. Blunt ligation was compared to a strategy with a single base gap on one or both strands to be filled by thermostable polymerase prior to ligation. Blunt or gap strategies worked well for detection of the delta F508 mutation. Detection of the W1282X mutation worked well with a blunt strategy when high K+ concentration (180-220 mM) was used to reduce template-independent ligation. For reliable detection of the G551D mutation, we used mismatches in the oligonucleotides 2-5 bp away from the ligation site and hot start of the reaction to achieve allele specificity. Excellent discrimination of mutations was achieved using competitive LCR with six oligonucleotides (two common on one side of the mutation plus two wild type and two mutant on the opposite side with the mutation site at the end adjacent to the common oligonucleotides) and with multiplex-competitive LCR using 12 oligonucleotides to detect both alleles for two mutations in a single tube.(ABSTRACT TRUNCATED AT 250 WORDS)

Glucose absorption and weight change in 18 months of continuous ambulatory peritoneal dialysis.

Previous studies have suggested that weight gain is a potential problem for patients using continuous ambulatory peritoneal dialysis (CAPD) due to the influx of glucose from the dialysate. To document the relationship between weight change and glucose absorption, the authors reviewed the medical charts of eight patients from the University of Michigan Hospitals Home Training Dialysis Unit who have used CAPD for at least 18 months. The average daily glucose absorption for each patient was determined by using the equation of Grodstein et al. (y = 11.3(x) -10.9, r = .96, p less than or equal to .001). The mean weight change after 18 months of CAPD was +4.0 +/- 8.9 kg (range = -2.5 to +24.1 kg). Two patients became obese (greater than 120% IBW); one remained obese; four patients maintained desirable weight for height; and one patient became nutritionally depleted (less than 80% IBW). Differences between weights before and after 18 months of CAPD were not significant (p greater than or equal to .05); however, mean weight change was significantly correlated (r = .62, p less than or equal to .05) with average daily amounts of glucose absorbed from the dialysate. This study indicates that nutritional outcomes can be optimized if total energy intake and the passive uptake of significant amounts of glucose across the peritoneal membrane are monitored along with body weight in patients using long-term CAPD.

A mechanism of erythrocyte lysis by lysophosphatidylcholine.

Lysophosphatidylcholine micelles liberate several cell surface polypeptides from erythrocyte membranes, inducing a sodium-selective permeability defect which leads to colloid osmotic lysis. Evidence is presented to support the hypothesis that at the lowest lytic lysophospholipid concentrations, selective disruption of membrane protein function, rather than gross structural reorganization of the membrane, is the primary lytic mechanism.

Selective extraction of membrane-bound proteins by phospholipid vesicles.

Extraction of membrane proteins from erythrocytes into sonicated phosphatidylcholine vesicles is described. In a process involving phospholipid and neutral lipid exchange, cell membrane proteins associate with the vesicles and can be separated from the cells by centrifugation. The protein transfer appears to be reversible; phospholipid vesicles mediate the delivery of small amounts of previously extracted protein into cell membranes. Prior to extraction, all but one of the proteins are accessible to lactoperoxidase iodination, and lipid analysis indicates that primarily the outer monolayer of the cell is involved in phospholipid exchange. Among the extracted proteins is acetylcholinesterase which is removed much more efficiently by this procedure than by concentrated salt solutions. The most abundant proteins of the erythrocyte membrane are not represented in the vesicle extract.