Efficient 12-mutation testing in the CFTR gene: a general model for complex mutation analysis

The identification of the cystic fibrosis transmembrane conductanceregulator (CFTR) gene has led to the identification of morethan 225 presumed disease-causing mutations at the locus. Thediagnosis of cystic fibrosis or the carrier state by directDNA analysis is hindered by this large number. A practical assaymust be able to detect enough mutations to achieve clinicallysignificant sensitivity. The use of allele-specifk oligonucleotideprobes is the most promising of the available methods. However,to date this has generally involved tedious probe-by-probe hybridizations,due to variations in the oligonucleotides' denaturation temperaturescaused by differences in their G-C base-pair content. We havedeveloped a rapid, cost-effective assay that simultaneouslydetects 12 CFTR mutations after multiplex polymerase-chain-reactionamplification of genomic DNA. The test may be readily extendedto detect additional mutations at minimal increase in the costper test or the turnaround time. We improve specificity andavoid the need for individual hybridizations by the use of tetramethylammoniumchloride to virtually eliminate the effects of G-C differences.Coupled with non-invasive sample-collection methods, this isan immediately practical assay for cystic fibrosis. More generally,it will serve as a model for the development of diagnostic testsin other genetic disorders involving complex mutation analysis.     

Localised endosteal bone lysis in relation to the femoral components of cemented total hip arthroplasties

Four cases are described of localised endosteal bone lysis in the femur occurring in association with cemented femoral components that were not obviously 'loose' radiologically. In each, the area of lysis was shown at operation to be related directly to a region in which there was a local defect in the cement mantle surrounding the stem. Via the space between the stem and cement, such defects provide a route through which the contents of the joint cavity may reach the endosteal surface of the femur, subsequently leading to localised bone lysis, and later to frank loosening.     

A Chinese adolescent girl with Fechtner-like syndrome

We describe a 15-y-old girl with Fechtner-like syndrome, who is the first Chinese reported to have this rare syndrome. She presented with left homonymous hemianopia and neuroimaging revealed haemorrhage in both parietal and occipital lobes. Peripheral blood smear showed macrothrombocytopenia and intracytoplasmic inclusion bodies inside leucocytes. Thrombocytopenia and proteinuria responded to intravenous immunoglobulin and pulsed methylprednisolone. This case illustrates that life-threatening haemorrhage can occur in patients with Fechtner syndrome. Although there was no effective treatment reported in the literature, high dose steroid and immunoglobulin seemed to be useful in our patient. Our patient also had nephritic-nephrotic syndrome with renal insufficiency, which is unusual in adolescent female patients.     

Serum and Cerebrospinal Fluid Pharmacokinetics of Intravenous and Oral Lamivudine in Human Immunodeficiency Virus-Infected Children

We studied the pharmacokinetics of intravenously and orally administered lamivudine at six dose levels ranging from 0.5 to 10 mg/kg of body weight in 52 children with human immunodeficiency virus infection. A two-compartment model with first-order elimination from the central compartment was simultaneously fitted to the serum drug concentration-time data obtained after intravenous and oral administration. The maximal concentration at the end of the 1-h intravenous infusion and the area under the concentration-time curve after oral and intravenous administration increased proportionally with the dose. The mean clearance of lamivudine (± standard deviation) in the children was 0.53 ± 0.19 liter/kg/h (229 ± 77 ml/min/m² of body surface area), and the mean half-lives at the distribution and elimination phases were 0.23 ± 0.18 and 2.2 ± 2.1 h, respectively. Clearance was age dependent when normalized to body weight but age independent when normalized to body surface area. Lamivudine was rapidly absorbed after oral administration, and 66% ± 25% of the oral dose was absorbed. Serum lamivudine concentrations were maintained above 1 μM for ≥8 h of 24 h on the twice daily oral dosing schedule with doses of ≥2 mg/kg. The cerebrospinal fluid drug concentration measured 2 to 4 h after the dose was 12% (range, 0 to 46%) of the simultaneously measured serum drug concentration. A limited-sampling strategy was developed to estimate the area under the concentration-time curve for concentrations in serum at 2 and 6 h.     

A guar-enriched wholemeal bread reduces postprandial glucose and insulin responses

We have developed a guar wholemeal bread and tested its physiological efficacy and sensory qualities. The objective of the study was to estimate the optimum concentration of guar in bread required to reduce postprandial glycaemia and insulinaemia without adversely affecting the quality of the bread. Following overnight fasts, 15 normal weight, non-diabetic subjects consumed meals of wholemeal bread alone (controls) and supplemented with guar at three different concentrations (5, 10 and 15% replacement of wheat flour). Blood samples for glucose and insulin analysis were taken preprandially, and at 30 and 60 min after the start of each meal. The acceptability of the wholemeal control and guar breads was assessed using a hedonic scaling method. A significantly lower blood glucose rise at 30 min was found after the 15% guar bread meal compared to the control. Plasma insulin responses at 30 and 60 min were significantly lower after the 10 and 15% guar bread meals compared to the controls. Hedonic scores indicated that the subjects found the 5 and 10% guar breads acceptable and in the case of the 5% variety the mean score was identical to the control. We suggest that a guar concentration of between 5 and 10% replacement of wheat flour (wholemeal or white) should be used for the baking of guar wholemeal bread but further work is needed to define the precise quantity of guar required.     

The interaction of cocaine and alcohol on schedule-controlled responding

 Within a number of physiological preparations, the effects of alcohol and cocaine in combination are reported to be greater than the effects of either drug given alone. Little has been reported, however, on the behavioral effects of the interaction. The present study investigated this issue by assessing the effects of cocaine and alcohol (alone and in combination) on schedule-controlled responding. Specifically, rats were trained to respond on an FR20 schedule for a water reinforcer. They were then administered cumulative doses of cocaine or alcohol. Following this, subjects were administered ineffective doses of alcohol prior to further dose-response assessments with cocaine and with ineffective doses of cocaine prior to further dose-response assessments with alcohol. Cocaine and alcohol alone produced dose-related decreases in responding. Furthermore, the dose-response function for cocaine was shifted to the left by alcohol and the dose-response function for alcohol was shifted to the left by cocaine. An isobolographic analysis revealed that the interaction between cocaine and alcohol was additive in nature. The possible bases for the interaction (e.g., changes in cocaine pharmacokinetics by alcohol and the formation of cocaethylene following co-administration of cocaine and alcohol) were discussed. Received: 22 February 1996 / Final version: 23 August 1996     

Carbon Monoxide Mass Exposure in a Pediatric Population

Abstract. Objectives:To describe the outcomes of a mass carbon monoxide (CO) intoxication, and to calculate the CO half-life in a pediatric school-aged population.
Methods:A retrospective chart review was performed based on Regional Poison Center database information, hospital laboratory data, and medical records of the pediatric patients who sought care at one of 3 St. Louis area hospitals, after exposure to high levels of CO. Exposures occurred on January 5, 1996, after evidence of a CO leak was discovered at an area elementary school. Charts were reviewed for major demographics, symptoms reported, carboxyhemoglobin (COHb) levels and times, and level of effect.
Results:Information about 177 (35%) of the 504 children in attendance at school that day was available. Mean age was 8.7 ± 1.8 years (range 4–12 years). Symptoms were present in 155 (88%) of the 177 children for whom data were available. Initial COHb levels were obtained for 147 (83.1%) of the 177 children. First mean COHb level was 7.0% (95% CI = 6.6–7.5%). Second COHb level was obtained for 26 children with a mean of 2.7% (95% CI = 2.2–3.2%). Calculated half-life of COHb, on 100% 0₂ at 1 atm, was 44.0 minutes (95% CI = 39.6–48.2 minutes).
Conclusion:Some children had symptoms at COHb levels that traditionally have been considered nontoxic. The elimination of COHb was found to be more rapid in this population of children than reported in other studies.     

Clonidine Use and Abuse Among Methadone Program Applicants and Patients

Forty-eight consecutive applicants and 30 known clonidine-abusing methadone patients at three methadone treatment programs were surveyed regarding their use of clonidine. Two distinct patterns of clonidine use emerged. Of 22 applicants who took clonidine illicitly, 15 used it primarily to decrease opioid withdrawal, as well as for its sedating effect. Applicants mostly obtained it from physicians, used an average dose of 0.37 mg at a time, and about one third believed clonidine to be addictive. In contrast, clonidine-using patients took clonidine primarily for its psychoactive effects, including the interaction with methadone, in addition to decreasing opioid withdrawal. Patients obtained clonidine frequently on the street and from family or friends, but less from physicians. The average reported dose for patients was 0.6 mg. The vast majority of these patients felt clonidine was addictive. Our findings, when coupled with the risk inherent in clonidine overdose, suggest that further research into the identification and treatment of clonidine abuse among methadone patients is warranted.     

Phase II evaluation of merbarone in renal cell carcinoma

Summary The Southwest Oncology Group (SWOG) studied the response rate and toxicity of merbarone (1,000 mg/m² IV continuous infusion days 1–5, q 21 days) in patients with advanced metastatic renal cell carcinoma. Among 36 eligible patients, there was one partial response for a response rate of 3% (95% C.I. 0.1–15%). There were no mixed responses. There were no treatment related deaths or adverse drug reactions. Significant anemia, diarrhea, and hypercalcemia were observed. Mild to moderate degrees of malaise/fatigue/lethargy, dizziness/vertigo, hyperglycemia, creatinine increase, nausea, vomiting, weight loss, pedal edema, dyspnea, and granulocytopenia were noted. Merbarone does not have significant activity as a single agent in advanced renal cell carcinoma.