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Technical Briefs |
in Sera from Duchenne Muscular Dystrophy Patients
1
Department of Laboratory Diagnosis and
2
Clinical Laboratory Medicine, Sapporo Medical University, School of Medicine, Sapporo 060-0061, Japan
3
National Yakumo Hospital, Department of Pediatrics, Yakumo, Japan
a address correspondence to this author at: Department of Laboratory Diagnosis, Sapporo Medical University, School of Medicine, South-1, West-16, Chuo-ku, Sapporo 060-0061, Japan; fax 81-11-622-7502, e-mail watanabn{at}sapmed.ac.jp
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 
(TNF) mRNA is expressed in muscle from DMD
patients. Other investigators have demonstrated TNF protein
expression in muscle from DMD patients, using immunohistochemical
staining (7). However, correlation between TNF in serum
and mechanisms of progression of DMD has been difficult to study
because serum concentrations of TNF often are too low to measure by
conventional methods (8)(9). We recently
established a highly sensitive method for measurement of TNF in
serum by an immuno-PCR assay (10), which has a sensitivity
5 x 104 times greater than that of a
conventional ELISA. The new method can determine serum TNF
concentrations in both DMD patients and healthy subjects. In the
present study, we measured serum TNF concentrations in 65 DMD
patients by immuno-PCR and investigated relationships between TNF,
patient age, and biochemical markers, including muscle-related enzymes.
To investigate whether TNF could be important in the pathogenesis of
DMD, we first measured serum TNF 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 TNF
concentration below the detection limit of a conventional ELISA.
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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 TNF 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 TNF 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 TNF 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 TNF
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 TNF is particularly marked in the early
progressive stage of the disease.
|
Whether an increase of serum TNF 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 TNF 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 TNF protein was expressed in the
muscle fibers of five of eight DMD patients examined, whereas
inflammatory cells did not stain for TNF. TNF immunoreactivity
was present in myofibers of only 2 of 21 inflammatory myopathy patients
who were examined. These previous findings suggested that serum TNF
concentrations could be increased as a result of muscle fiber
destruction. Therefore, to investigate whether an increase of serum
TNF concentration could simply reflect the muscle destruction in DMD
patients, we examined the relationship between TNF and various
biochemical markers, including CK and Mb, using the samples identical
to those that were used for the measurement of TNF. However, we
found no significant correlation between the serum concentrations of
TNF and those of CK or Mb in the present study, arguing against such
an explanation.
Considering findings from various studies, TNF 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). TNF is well known
to activate the coagulation system (15)(16)(17). It was
speculated that TNF 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 TNF concentration was high enough to
be measured by a conventional ELISA. Among these patients, four who
were <20 years of age showed particularly high TNF concentrations
and had progressive difficulty in walking in the weeks after
examination. These observations suggested that TNF may participate
in unusual but acute progression of DMD.
References
in healthy donors, using a highly sensitive immuno-PCR assay. Clin Chem 1999;45:665-669. and its soluble TNF receptor I. Angiology 1999;50:703-706.
The following articles in journals at HighWire Press have cited this article:
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  J. D. Porter, A. P. Merriam, P. Leahy, B. Gong, and S. Khanna Dissection of temporal gene expression signatures of affected and spared muscle groups in dystrophin-deficient (mdx) mice Hum. Mol. Genet., August 1, 2003; 12(15): 1813 - 1821. [Abstract] [Full Text] [PDF]
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 A. Cittadini, L. Ines Comi, S. Longobardi, V. Rocco Petretta, C. Casaburi, L. Passamano, B. Merola, E. Durante-Mangoni, L. Sacca, and L. Politano A preliminary randomized study of growth hormone administration in Becker and Duchenne muscular dystrophies Eur. Heart J., April 1, 2003; 24(7): 664 - 672. [Abstract] [Full Text] [PDF]
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