Several studies have shown that MLPA were useful in detecting mutation quantitatively, not only for deletions but also duplications and female carriers in the DMD gene [20
]. We applied MLPA in a subset of DMD/BMD patients in northern China. Our results indicated that 66.25% (106/160) of our patients carry deletion and 6.25% (10/160) patients had a duplication, which brought the sensitivity of detecting DNA rearrangement up to 72.5%. In addition, MLPA may present higher sensitivity. In this study, three patients who had negative results in their previous DNA testing by multiplex PCR (data not shown) were identified. Furthermore, an advantage of employing MLPA in clinical genetic testing is that it is capable of identifying female carriers, as we have demonstrated among 14 out of 19 females who came to our prenatal clinics. Certainly, MLPA should be considered as the initial test for the clinically suspected DMD/BMD patients as well as for women who have a DMD/BMD family history, as suggested previously [29
], to provide both confirmation of genetic defects on DMD gene and better genetic counseling.
Exons 2–20 and exons 44–53 had been previously reported as the two-hotspot regions [8
]. In this study, we have analyzed the distribution of individual exons regarding their frequency of deletion or duplication (Fig. ). Our data indicates that the deletion occurs at exons 45 to 55 accounting for 47.67%, followed by exons 21–44 (26.67%) and exons 1–20 (23.66%). If both deletions and duplications are counted together, the most frequent mutant region occurs within exons 45–55 (41.06%) in the DMD gene, followed by exons 21–44 (30.43%), exons 1–20 (25.32%) and exons 56–79 (3.19%). We found the most common region in our study is the exons 45–55, and the least common region is the exons 56–79. These data are in agreement with other global populations [26
]. We have noticed that the frequency of duplication is more than 20% and it appears there is no model peak of deletion in Taiwan (Fig. ) [21
]. As well, the deletion rate in Hong Kong [31
] showed difference from the northern Chinese. We hypothesize that the frequency of deletion is different in variant regions of China, and we propose that further investigation with molecular screening of deletion (and duplication) should be pursued among newborns in China. The pattern of duplication did not show significant difference among the global variant populations (Fig. ).
Figure 4 Comparison of exon deletion (A) and duplication (B) among global populations. All these data showed that exons 45–55 are the most frequent deletion region of DMD gene. Deletion and duplication were not common in region of exons 56–79, (more ...)
The structure of introns of dystrophin gene determines the breakpoint that is responsible for the deletion [33
]. There were many repeated sequences in introns of dystrophin gene which resulted in many breakpoints. For example, in intron 44 there were 13 breakpoints resulted in the hot-spot deletion region [34
]. The repeated sequences included direct repeated sequence, inverted repeat sequence and homology sequence. It was reported that 22 repeated sequences had been found in intron 51 and intron 52, which was a possible reason for the high frequency of single-exon deletion at exon 51 [35
]. We believe this might be the same case for the most common deletion at exon 50.
Recently, White et al [25
] reported that as high as 87% of duplications can be detected among three subsets of selected DMD/BMD cases, among which no deletion or point mutation had been found. In this study, duplication was only detected in 18.51% of the cases where no deletion could be detected. This lower rate possibly resulted from either our small size of samples or a high rate of point mutation, which should be further investigated. To combine the rates of duplication with deletion, we estimate that a point mutation might account for 20–28% of our cases. In addition, the duplications in our cases mainly involved a larger fragment; however, among 118 cases of the reported duplications [25
], there were only 12 cases where the duplication was more than 20 exons.
In summary, we have applied MLPA to detect DNA rearrangements of deletions and duplications among DMD/BMD male patients and female potential carriers. We have determined that deletions and duplications were 72.5% of our samples. We observed that the majority of deletions was small in size that the number of deleted exons was less than 10 but duplications usually involved larger sizes. Finally, we determined that the most common region of DMD gene rearrangement is between exons 45 and 55 and the 5' hotspot region was not a hotspot region in our study.