Carrier detection in Duchenne muscular dystrophy

Abstract

The identification of apparently healthy carriers of the lethal, X- linked gene for Duchenne muscular dystrophy (DMD) is of importance for genetic counselling purposes. At present the accepted most reliable test for determining DMD carrier status is the estimation of serum creatine kinase activity. However, approximately one third of genetic carriers remain undetected by this method. This study was designed to evaluate other possible methods of DMD heterozygote recognition with a view to improving the carrier detection rate.

There is considerable evidence to suggest a generalised membrane defect is responsible for the degenerative muscle fibre alterations which occur in DMD. Increased intracellular calcium has been implicated as an early biochemical change which would account for many of the membrane abnormalities reported. Several changes exhibited by DMD erythrocytes could be attributed to an increased intraerythrocytic calcium content. If this increased calcium level is related to the primary defect then, according to the Lyon hypothesis, a proportion of erythrocytes from a genetic carrier of DMD should manifest this abnormality. The relative intraerythrocytic calcium content of individual erythrocytes within DMD patient, DMD carrier and control erythrocyte populations were examined. Quantitative measurement of the elemental content of single erythrocytes was carried out using electron probe X -ray microanalysis (EPXMA). However the extremely low level of intraerythrocytic calcium and the wide variation in relative calcium concentrations of each erythrocyte population render EPXMA insufficiently sensitive to be capable of distinguishing two erythrocyte populations within a DMD carrier.

An alternate approach to the demonstration of cellular mosaicism is the additional measurement of secondary biochemical parameters which, if non -correlated, could improve the carrier detection rate over that obtained using serum creatine kinase estimation alone. Serum levels of haemopexin and myoglobin were found to be raised significantly in a series of 10 DMD patients compared to 10 age and sex matched, normal, healthy controls. Measurements of serum pyruvate kinase, haemopexin and myoglobin levels were then carried out in a series of 15 DF'T carriers and 15 age matched, normal, healthy women, in conjunction with serum creatine kinase estimation. The mean serum pyruvate kinase activity of the DMD carrier group was significantly higher than that of the control group but this test selected only 4/14 (28.5%) carriers and failed to identify 3 carriers whose serum creatine kinase levels were within normal limits. The mean serum haemopexin level of the carrier group was not significantly different from that of the control group. The mean serum myoglobin level of the carrier group was significantly higher than that of the control group. Nine of 15 (60%) carriers had myoglobin levels outside the 95% confidence limits. However serum myoglobin was within normal limits in all 3 carriers with normal serum creatine kinase levels. Discriminant analysis suggested that combining serum myoglobin and creatine kinase measurements might improve the detection rate. Serum levels of myoglobin and creatine kinase were measured in a series of 20 DMD carriers (all with serum creatine kinase activities within normal limits) and 20 age matched, normal, healthy women. Three of 20 (15 %) carriers had myoglobin levels outside the calculated 95% confidence limits but all 3 had serum creatine kinase activities close to the 95th percentile of the control series. The positive correlation between serum myoglobin and creatine kinase levels implies the combined use of these tests may be of value only in cases where the subjects' creatine kinase level is borderline normal. Otherwise the results of this study suggest the additional measurement of either serum pyruvate kinase of haemopexin in combination with serum creatine kinase estimation will have little value in DMD carrier detection.