A 17-year-old boy was referred to the department of neurology with recurrent myalgia following exercise. He had suffered an easy fatigability and myalgia several minutes after exercise such as running or tracking the mountain every time since childhood. These symptoms had recovered spontaneously within a day. The second wind phenomenon was not seen. Incidentally, he was found to have an elevated aspartate aminotransferase (73 U/L, normal range < 40 U/L, AST) and alanine aminotransferase (65 U/L, normal range < 41 U/L) at age thirteen years old. Spontaneous recover had left him undiagnosed. A year later, he experienced a dark urine for a day after 500-meter running. In seventeen years old, he presented a myalgia on bilateral thigh with a dark urine after 10-minute walking, which led to visit our hospital. The laboratory performed on a same day in referring hospital showed an elevated level of serum creatine kinase (73,529 U/L, normal range 39-308 U/L, CK), AST (88 U/L), myoglobin (>1,000 ng/mL, normal range 0-110 ng/mL), and myoglobinuria. These laboratory disarrangements brought the patient to our hospital.
In neurological examination at our hospital, the muscle strengths of bilateral upper and lower extremities were normal. The deep tendon reflexes were mildly reduced. The atrophy or arthrogryposis was not identified.
The routine laboratory showed an elevated level of serum CK (1,484 U/L), AST (45 U/L), myoglobin (430 ng/mL), and aldolase (185 U/L, normal range < 7.6 U/L). Complete blood count, thyroid stimulating hormone, alanine aminotransferase, and urine myoglobin were normal. The ischemic forearm exercise test was normal, showing an elevated serum level of lactic acid and ammonia after exercise (Fig. 1A).
The electrocardiogram showed a normal sinus rhythm. The findings of nerve conduction studies were normal. The needle electromyography demonstrated positive sharp waves with small amplitude and short duration motor unit action potentials on right first dorsal interosseous, biceps, and tibialis anterior muscles, indicating active myopathic changes.
Left biceps brachii biopsy was performed. Routine stain including hematoxylin and eosin, nicotinamide adenine dinucleotide tetrazolium reductase, and modified Gomori trichrome could not be evaluated due to the cryostat artifact. However, the immunohistochemistry (IHC) of muscle biopsy in paraffin block revealed an absent expression of dystrophin using the antibody against the C-terminal region (Thermofisher scientific, PA5-16734, USA, 1:200 dilution, Fig 1B) and has been repeated to confirm.
Whole exome sequencing revealed a novel hemizygotic missense mutation c.119T>A (p.Leu40His, exon 3, NM_004006.2, NG_012232.1, Fig. 1C) in DMD by investigating variants which affect protein function, show a depth of more than 30, and filtered by allele frequency of PopFreqMax less than 0.0001 consisting of the Genome Aggregation Database (gnomAD), the Exome Aggregation Consortium (ExAC), and 1000 Genome (1000genome). This variant is not present in Human Gene Mutation Database (HGMD) or Leiden Open Variation Database (https://databases.lovd.nl/shared/genes/DMD) and predicted to be pathogenic by using SIFT/PROVEAN and Mutation taster system. Structure of mutated dystrophin protein was predicted to be destabilizing using SDM web server (ΔΔG = 0.88 kcal/mol) and FoldX (ΔΔG = 19.5414 kcal/mol) [3, 4]. Both wild type (Leu40) and mutated (His40) residues were expected not to be part of aggregation-prone regions by an Aggrescan3D server [5], although the mutated residue (His40) can become solvent exposed by JPred4 server [6].