Two novel missense mutations in the myostatin gene identified in Japanese patients with Duchenne muscular dystrophy

Abstract

Background: Myostatin is a negative regulator of skeletal muscle growth. Truncating mutations in the myostatin gene have been reported to result in gross muscle hypertrophy. Duchenne muscular dystrophy (DMD), the most common lethal muscle wasting disease, is a result of an absence of muscle dystrophin. Although this disorder causes a rather uniform pattern of muscle wasting, afflicted patients display phenotypic variability. We hypothesized that genetic variation in myostatin is a modifier of the DMD phenotype.

Methods: We analyzed 102 Japanese DMD patients for mutations in the myostatin gene.

Results: Two polymorphisms that are commonly observed in Western countries, p.55A>T and p.153K>R, were not observed in these Japanese patients. An uncommon polymorphism of p.164E>K was uncovered in four cases; each patient was found to be heterozygous for this polymorphism, which had the highest frequency of the polymorphism observed in the Japanese patients. Remarkably, two patients were found to be heterozygous for one of two novel missense mutations (p.95D>H and p.156L>I). One DMD patient carrying a novel missense mutation of p.95D>H was not phenotypically different from the non-carriers. The other DMD patient was found to carry both a novel mutation (p.156L>I) and a known polymorphism (p.164E>K) in one allele, although his phenotype was not significantly modified. Any nucleotide change creating a target site for micro RNAs was not disclosed in the 3' untranslated region.

Conclusion: Our results indicate that heterozygous missense mutations including two novel mutations did not produce an apparent increase in muscle strength in Japanese DMD cases, even in a patient carrying two missense mutations.

Figure 1

Myostatin and the human myostatin gene. The structure of myostatin, including the signal sequence (SS), and the regions of the propeptide and mature protein, is schematically described (Top). The numbers above the boxes indicate the amino-acid residue position. The vertical arrows indicate the locations of the two novel missense mutations and two nucleotides deletion identified in this study. The structure of the myostatin gene and the analyzed regions are schematically described (Bottom). Three coding regions (Exons 1, 2 and 3) and a part of the 3' untranslated region (3'UTR) of the myostatin gene were PCR amplified (boxes). The shaded and open boxes indicate the coding region and the sequenced region in the 3' untranslated region, respectively. Bold and thin horizontal lines indicate exons and introns, respectively. Horizontal arrows indicate the locations and directions of the primers used to amplify the regions. Numbers above the boxes indicate the nucleotide position according to the cDNA reference sequence in GenBank (accession no.: NM_005259), in which the "A" in the start codon is nucleotide #1. Numbers in the bottom indicate the size of each segment.

Figure 2

A novel mutation in exon 1 of the myostatin gene. A part of the sequencing results for exon 1 of the myostatin gene from a DMD patient (case 712) is shown (patient). Overlapping G and C peaks are present at the 283^rd nucleotide of the myostatin cDNA (c.283G>G/C) (Top). The single nucleotide change from G to C at the 283^rd nucleotide of the myostatin cDNA (c.283G>C) changed a GAT codon to a CAT codon at the position corresponding to the 95^th amino-acid residue of myostatin (p.95D>H)(boxes). The wild type sequence is shown below (control).

Figure 3

A novel mutation in exon 2 of the myostatin gene. A part of the sequencing results for exon 2 of the myostatin gene of one DMD case (case 549) is shown. Overlapping peaks were observed at two locations corresponding to c.466C>C/A and c.490G>G/A in one DNA sample (Top). Subcloning sequencing disclosed two different sequences: one had a completely normal sequence (Bottom 2), whereas the other one had two nucleotide changes (Bottom 1). The G to A change at the 490^th nucleotide of the myostatin cDNA (c. 490G>A) matched with the previously described p.164E>K mutation (box). The other nucleotide change from C to A at the 466^th nucleotide of the myostatin cDNA (c.466C>A) changes a CTA codon for Lys to a ATA codon for Ile at the position corresponding to the 156^th amino-acid residue of myostatin (p.156L>I) (box).

Figure 4

A novel deletion mutation in the 3' untranslated region of the myostatin gene. A part of the sequencing result for the 3' untranslated region of the myostatin gene is shown. In the control all of the sequences matched with that of the wild-type sequence (Genbank: AC073120), including the repeat of AT dinucleotides (boxes) (control). In the sequence of one DMD case (case 100) the repeat of AT dinucleotides was not present, removing AT dinucleotides at 2264 and 2265^th nucleotide (c.2264-2265delAT) (patient).