Mutation spectrum of probands in the DMD families
For the 303 probands, the detection rate of DMD mutation is 97.7%. The families we included in our hospital were mainly DMD deletions and duplications. The DMD gene deletions were detected in 248/303 (81.8%) probands and DMD gene duplications were detected in 36/303 (11.9%) probands. In addition, four cases (1.3%) with both deletion and duplication were also detected. And the remaining 8/303(2.6%) were small mutations (Figure 1).
Deletion, duplication and small mutation patterns
To further analyze the mutation patterns in 303 probands, each exon deletion times were accounted. We found 97 different deletion patterns. Interestingly, exon deletion hotspot was between exon 43-55 at the 3` end of DMD gene (Figure 2A). In addition, the deletion of exon 50 was detected 102 times, which appeared to be the most detected exon deletion in the probands (Figure 2A). And the most frequent large deletion pattern was deletion of exon 48 to exon 50, which was detected 14 times (Figure 2B). Besides, the largest deletion was from exon 2 to exon 79. In 97 different deletion patterns, a single exon deletion pattern was also accounted for lots of proportion (21.6%).
Furthermore, 30 duplication patterns were also detected in the probands. Likewise, two hotspots of duplication were also detected, exon 8-11 at the 5` of the DMD gene and exon 51 at 3` end (Figure 3A). In addition, we also found that the DMD gene exon 8-11 and exon 51-71 as the most common duplication patterns which appeared 4 times (Figure 3B). And the largest duplication extended 38 exons which is from exon 3 to exon 40. Complex rearrangements were also identified in four cases, three of which were deletion of exon 22 combined with duplication of exons 8 to 11.
However, a small part of the DMD families included in our hospital was small mutations including nonsense mutation, frameshift mutation and splice site mutation. A total of 8 (2.7%) different small mutations were identified in our population including 5 nonsense mutations, 2 frameshift mutations and 1 splice site mutation (Table 1). All in all, our findings indicate that many mutation patterns exist in the DMD gene.
Carrier testing results for prenatal mothers
To identify the carrier status of DMD gene mutation in pregnant mothers, 204 pregnant women who decided to do the carrier testing were screened according to the mutation types of the proband. Of the 204 cases, 108 (52.9%) cases were carriers and 96 (47.1%) showed no pathogenic mutations in the blood. A single exon deletion rate (20.5%) was much lower compared with multi exon deletions (79.5%) among the total deletion carriers (Table 2). And the rate of multi exon duplications showed 42.8% more inheritance than a single exon duplication among the total duplication carriers (Table 2). Even complex mutations that included deletions combined with duplications were found in two mothers. For the small mutations, the carrier rate is about 66.7% (Table 2).
Prenatal diagnosis and cytogenetic analysis
Among the 305 pregnancies, there were 173 (56.7%) male fetuses and 132 (43.3%) female fetuses. The mean age of mothers in pregnancies was 33.0 ±4.1 years. In order to test the heredity of the DMD gene mutation, we analyzed the mutation type of 305 pregnancies. The results showed that a total of 55 male fetuses (18%) prenatally diagnosed as disease-positive, 40 female fetuses (13.1%) carrying DMD gene mutations and the remaining fetuses (68.9%) were healthy. Among 224 collected chorion villus samples, there were 37 disease positive male fetuses (16.5%), 29 female carriers (13.0%) and 158 healthy fetuses (70.5%) (Table 3). In addition, we also collected 81 amniotic fluid samples and found 18 disease positive male fetuses (22.2%), 11 female carriers (13.6%) and 52 healthy fetuses (64.2%) (Table 3).
Surprisingly, we also found 3 fetuses with trisomy 21 (Down syndrome) by karyotype analysis and two of three fetuses were not affected by DMD. Moreover, we firstly discovered a male fetus with DMD gene mutation of exon 48 deletion and trisomy 21 (Figure 4). Thus, our analysis showed that prenatal diagnosis is crucial in DMD families to prevent the birth of DMD children. Prenatal diagnosis was suggested to mothers with a proband whether they carried the causative mutation or not.