A Sensitive Assay of Tumor Necrosis Factor α in Sera from Duchenne Muscular Dystrophy Patients

Duchennne muscular dystrophy (DMD) is a genetically determined disorder resulting from absence of the protein encoded by the dystrophin gene, which is located on chromosome Xq21 (1)(2). Almost all patients develop progressive muscular weakness and atrophy that begin at a young age in association with myofiber degeneration and necrosis (3)(4)(5). The pathophysiologic significance of serum cytokines in DMD is unclear. Lundberg et al. (6) have reported recently that tumor necrosis factor α (TNFα) mRNA is expressed in muscle from DMD patients. Other investigators have demonstrated TNFα protein expression in muscle from DMD patients, using immunohistochemical staining (7). However, correlation between TNFα in serum and mechanisms of progression of DMD has been difficult to study because serum concentrations of TNFα often are too low to measure by conventional methods (8)(9). We recently established a highly sensitive method for measurement of TNFα in serum by an immuno-PCR assay (10), which has a sensitivity 5 × 104 times greater than that of a conventional ELISA. The new method can determine serum TNFα concentrations in both DMD patients and healthy subjects. In the present study, we measured serum TNFα concentrations in 65 DMD patients by immuno-PCR and investigated relationships between TNFα, patient age, and biochemical markers, including muscle-related enzymes. To investigate whether TNFα could be important in the pathogenesis of DMD, we first measured serum TNFα concentrations in 65 patients with DMD (mean age, 20.7 years; range, 7–46 years) and 54 healthy subjects (mean age, 38.6 years; range, 21–61 years), using immuno-PCR. The mean value in DMD patients (27.8 ng/L; range, 0.009–619.3 ng/L) was ∼1000 times higher than that in healthy subjects (0.027 ng/L; range, 0.004–0.120 (Fig. 1 ). Fifty-seven of the DMD patients (87.7%) had a TNFα concentration below the detection limit of a conventional ELISA. To confirm progression of muscle destruction in young patients with DMD, we investigated the relationship between age and serum biochemical markers such as creatine kinase (CK) and myoglobin (Mb) concentrations. A negative correlation was found in DMD patients between age and CK as well as age and Mb (age vs CK: r = −0.70; age vs Mb: r = −0.69). Patients younger than 20 years of age showed a much higher mean CK concentration (2838.0 U/L) than patients >20 years of age (351.2 U/L). Mb concentration in patients <20 years of age (405.6 μg/L) also was higher than that in the cases >20 years of age (66.1 μg/L). Defining TNFα positivity as a value exceeding the mean + 2 SD for control subjects, we next investigated positivity rates by immuno-PCR and the mean concentration of TNFα in DMD patients younger and older than 20 years (Table 1 ). No difference in positivity rate was seen between patients <20 years of age (86.2%) and those >20 years of age (75.0%). In contrast, the mean TNFα concentration in the patients <20 years of age (60.7 ng/L) was approximately five times higher than that in patients >20 years of age (12.9 ng/L). In addition, the mean TNFα concentration in the patients <20 years of age was significantly higher than that in normal healthy subjects (P < 0.05 by the Student t-test after ANOVA. Consequently, an increase of serum TNFα is particularly marked in the early progressive stage of the disease. Whether an increase of serum TNFα concentration in DMD patients simply reflects muscle destruction has been an unresolved issue. Lundberg et al. (6) examined mRNA expression of several cytokines in muscle from DMD patients by reverse transcription-PCR, detecting TNFα mRNA expression in muscle samples from three of five cases. Tews and Goebel (7) recently performed immunohistochemistry for several cytokine proteins in inflammatory cells and muscle specimens from patients with inflammatory myopathy and DMD. These investigators found that TNFα protein was expressed in the muscle fibers of five of eight DMD patients examined, whereas inflammatory cells did not stain for TNFα. TNFα immunoreactivity was present in myofibers of only 2 of 21 inflammatory myopathy patients who were examined. These previous findings suggested that serum TNFα concentrations could be increased as a result of muscle fiber destruction. Therefore, to investigate whether an increase of serum TNFα concentration could simply reflect the muscle destruction in DMD patients, we examined the relationship between TNFα and various biochemical markers, including CK and Mb, using the samples identical to those that were used for the measurement of TNFα. However, we found no significant correlation between the serum concentrations of TNFα and those of CK or Mb in the present study, arguing against such an explanation. Considering findings from various studies, TNFα is likely to play an important but presently undetermined pathophysiologic role in DMD. Previous reports have indicated that sudden death from thrombotic disease can occur in DMD patients, in addition to the usual deaths from respiratory and heart failure (11)(12)(13)(14). TNFα is well known to activate the coagulation system (15)(16)(17). It was speculated that TNFα increases in lower concentrations that could not be detected by conventional ELISA may predict not only steady progression but also the progressive and sudden change of disease state such as a hypercoagulable state in DMD patients. In addition, in eight of the present cases, the serum TNFα concentration was high enough to be measured by a conventional ELISA. Among these patients, four who were <20 years of age showed particularly high TNFα concentrations and had progressive difficulty in walking in the weeks after examination. These observations suggested that TNFα may participate in unusual but acute progression of DMD. Comparison of TNFα concentrations between sera from 54 healthy subjects and 65 DMD patients by immuno-PCR. The shaded area shows the range in which TNFα could also be detected by ELISA. Serum TNFα concentrations in patients with DMD as measured by immuno-PCR. Serum samples in which the concentration exceeded the mean ± 2 SD of measurements in control subjects. Serum TNFα concentrations in patients with DMD as measured by immuno-PCR. Serum samples in which the concentration exceeded the mean ± 2 SD of measurements in control subjects.