The two male patients with clinical diagnosis of DMD were incidentally found to have marked elevations of serum CK levels (more than 50 times higher than control) in early childhood, despite a negative family history for muscular dystrophy. To confirm the clinical diagnosis of DMD, dystrophin gene mutations were extensively searched for using not only genomic DNA but also mRNA. However, no mutations could be identified, even when we included a deep intron mutation [15
To clarify pathological changes in these two patients, muscle biopsies were performed. In patient 1 (KUCG 527), H-E staining revealed marked replacement of muscle by adipose tissue along with increases in endomysial connective tissue. We also found a few muscle fibers that were remarkably different in size (data not shown). In spite of the clinical diagnosis of DMD, the immunostaining pattern for both dystrophin and merosin staining was completely normal. Unexpectedly, there was no staining for γ-sarcoglycan, while there was only a mild reduction for α-sarcoglycan and almost normal results for β- and δ-sarcoglycan (Figure ). In order to confirm the γ-sarcoglycan deficiency, we looked for mutations in the SGCG gene. When PCR was used to amplify the eight exons that encompassed the regions of the SGCG gene, all regions other than exon 6 could be obtained. This suggests a homozygous deletion of exon 6 (Figure ). To confirm this, we used RT-PCR to analyze the SGCG mRNA from the patient's muscle. Amplification of the fragment encompassing exons 5 to 8 resulted in a small-sized product. Subsequent sequencing of this product revealed a complete absence of the exon 6 sequence (data not shown). Therefore, we concluded that this patient had a homozygous deletion of exon 6 in the SGCG gene. In addition, both the patient's mother and father were found to carry this deletion in one allele (data not shown). Because the exon 6 deletion removes 73 bp (nt 506 - 578) from the mRNA, it was expected that a stop codon would appear in exon 7, thereby leading to γ-sarcoglycan deficiency. It became clear that this patient had LGMD2C rather than DMD.
Figure 1 Immunostaining of skeletal muscle. Results of immunohistochemical examination using dystrophin, α-, β-, γ-, and δ-sarcoglycans and dystrophin antibodies are shown. There was clear staining of every protein along the plasma (more ...)
Figure 2 Mutation analysis of the SGCG gene in patient 1. Amplification products of exons 5, 6, and 7 are shown (c; control, p: patient) in the upper part of the figure. Although PCR amplification of eight exons of the SGCG gene was conducted, no products were (more ...)
In patient 2 (KUCG 280), H-E staining indicated excess variability of muscle fiber size, clusters of regenerating fibers, degenerating fibers, some acutely necrotic fibers, and scattered inflammatory cells. Immunohistochemistry revealed the absence of γ-sarcoglycan and a patchy reduction in α- and β-sarcoglycan. The staining patterns for dystrophin, δ-sarcoglycan, and merosin all appeared normal (Figure ). Since the findings suggested γ-sarcoglycan deficiency, PCR amplification of the patient's genomic DNA was performed for all eight exons in the SGCG gene. Amplification of all fragments resulted in products that were of the expected size, and thus, subsequently could be used for direct sequencing. With the exception of exon 7, sequencing of the amplified products demonstrated a completely normal sequence. In exon 7, subcloning of an ambiguous sequence of the amplified product resulted in one clone with a completely normal sequence and a second clone that contained a novel single T nucleotide insertion between nt 602 and 603 within exon 7 (c.602_603insT). This mutation created a stop codon in exon 7. The patient's mother was heterozygous for this mutation, whereas the father had a normal exon 7 sequence (data not shown). Since patient 1 had a homologous deletion of exon 6 of the SGCG gene, we supposed that patient 2 carried this deletion in one allele. When semi-quantitative PCR amplification of exon 6 of the patient's SGCG gene was performed, the results showed nearly half the genomic dose for the exon 6 encompassing region, indicating a heterozygous deletion of exon 6 (Figure ). Further analysis of the SGCG mRNA from the patient's muscle revealed two kinds of mRNA: one exhibited an exon 6 deletion and the other demonstrated the above-mentioned novel single nucleotide insertion within exon 7 (Figure ). Therefore, the γ-sarcoglycan deficiency in this patient was caused by a hemizygous c.602-603insT in exon 7 along with the deletion of exon 6 on the other allele.
Figure 3 Mutation analysis of the SGCG gene in patient 2. Capillary electrophoretic patterns of the PCR products are shown in the upper portion of the figure. Five genomic regions were co-amplified in one PCR reaction, with the products separated using capillary (more ...)
To date, our analysis of patients suspected to have DMD has revealed that two of the entire cohort examined can be regarded as having LGMD2C. Thus, the relative incidence of LGMD2C among Japanese patients suspected to have DMD can be calculated as 1 in 161 (2 of 324 patients = 0.6%). When the DMD incidence is taken into consideration for the overall population (1/3,500 males), the incidence of LGMD2C can be estimated as 1 per 560,000 or 1.8 per million.