Many patients with a family history of DMD have visited our centre. If a proband was confirmed in a family, they would be worried about their next generation and strongly wish to determine the genetic causes to avoid the same disease. In the present study, data from patients with DMD were retrieved and analysed.
Methods of DMD gene detection
MLPA is one of the most widely used methods [10], but it cannot be applied to the detection of very small mutations, while Sanger sequencing is expensive and time consuming in the detection of DMD gene point mutations. Compared with Sanger sequencing technology, NGS has the advantages of large flux, a short time, high accuracy and abundant information, which significantly shortens the detection cycle and reduces the detection cost [11, 12].
The methods of DMD prenatal diagnosis
DMD/BMD prenatal diagnosis can be classified into direct and indirect gene diagnoses. Methods of direct diagnosis, such as MLPA and Sanger sequencing, are the gold standard, but mutations in probands need to be defined to detect DMD gene mutations in the foetus [13]. NGS improves the efficiency of proband mutation detection [14, 15]. Indirect prenatal diagnosis predicts prenatal risks using STR linkage analysis, with the advantages of simplicity and quickness [16]. However, the risk of gene combinations cannot be ignored [17]. The different results between direct and indirect prenatal diagnoses in one foetus (DMD147) confirmed that separate STR linkage analysis had the risk of misdiagnosis in the DMD/BMD prenatal diagnosis. Therefore, the combination of direct diagnosis and indirect diagnosis is the current routine prenatal diagnostic strategy (Fig. 3).
In this research, MLPA was used to exclude de novo large deletions/duplications in the DMD gene for all foetuses. Based on the consideration of the high cost of NGS and the low percentage of small mutations among all de novo mutations, we did not use NGS to screen small de novo mutations for all DMD/BMD prenatal diagnoses. However, NGS was required by the families that had probands with small mutations.
DMD gene mutation and genetic analyses
Among all 931 foetuses (twins in one pregnancy were counted twice), 20.73% (193/931) were males expected to develop dystrophinopathies, 16.33% (152/931) were female carriers, 37.59% (350/931) were males without DMD mutations and 25.35% (236/931) were females without DMD mutations. Moreover, 13 pregnant women (4.48%, 13/290) without DMD mutations at risk of having a foetus who was a carrier gave birth again. Thus, pregnant women without the DMD mutation have an extremely low chance of having another child at risk again. However, it is suggested that women who have given birth to a child with DMD need a prenatal diagnosis when she becomes pregnant again.
In our research, five mothers without DMD gene mutations in peripheral blood had a foetus with DMD, but their foetuses had the same DMD mutations as the proband. This result might be caused by gonadal mosaicism, but the possibility of de novo mutations cannot be ruled out. In addition, we cannot exclude the possibility of somatic mosaicism in these five mothers. In theory, MLPA and NGS can be used to exclude mosaicism. MLPA cannot distinguish the low percentage of mosaicism. However, no research has confirmed that NGS can be used to test somatic mosaicism. Thus, it was unfortunate that we could not exclude the possibility of somatic mosaicism in these five mothers. In summary, without considering the possibility of de novo mutations and somatic mosaicism, 1.78% of proband mothers without mutations in peripheral blood had gonadal mosaicism in our study. Our data were similar to another study that indicated a recurrence risk of 8.6% for non-carrier females due to germline mosaicism [18]. Thus, with the high de novo mutation rate, we suggest that a prenatal diagnosis is still necessary for families.
Our data indicated that 32.91% of DMD/BMD probands had de novo mutations, which was similar to previous reports [19, 20]. Among these de novo mutations, large deletions/duplications accounted for approximately 70%. These data and the discovery of a foetus with a de novo mutation (deletion of exons 45–50) (DMD188) that was different from the mutation in the probands and mother carriers (deletion of exons 3–29) illustrate the high de novo mutation rate of the DMD gene. Hence, de novo mutations in foetuses should be considered in prenatal diagnosis.
An atypical family (DMD303) with a DMD female proband (II2) was detected (Fig. 4). The 17-year-old female proband, her mother and her elder sister all carried the heterozygous deletion of exons 8–21. The female proband was clinically diagnosed with DMD, but her mother and elder sister had no symptoms. The prenatal diagnosis result indicated that the female foetus of her sister (III1) carried the deletion of the DMD gene exons 8–21. In genetic counselling, we told the above information and the low percentage of DMD female patients in the population, and only 3 female probands in our data chose to continue pregnancy and had a female infant after term delivery. The post-natal conformation of the female foetus was normal (creatine kinase level was 186 U/L). At present, the pathogenesis of symptomatic female DMD carriers is unclear, but some research has reported that approximately 20% of female DMD mutation carriers have varying degrees of clinical symptoms [21–23]. It is worth noting that prenatal diagnosis for families with DMD/BMD female patients is necessary and provides genetic counselling for such families.
The expectation of a DMD prenatal diagnosis
In conclusion, the basic strategy in our study was to first use MLPA and NGS to identify DMD mutations in probands and then use MLPA and Sanger sequencing combined with STR linkage analysis to detect definite mutations for a DMD/BMD prenatal diagnosis. In this protocol, gene recombination, gonadal mosaicism and de novo mutations of the DMD gene were also taken into consideration. In general, prenatal diagnosis is currently limited to hereditary diseases. However, it is not practical for all pregnant women to have a prenatal diagnosis via amniotic fluid puncture. Although there are several treatments for DMD, the likelihood of a cure is extremely low.
Owing to the high de novo mutation rate of the DMD gene, the following questions are worth discussion: Should a prenatal diagnosis of DMD/BMD be limited to the defined DMD mutation locus in a family, or should it be extended to the full length of the DMD gene? Moreover, issues should be discussed regarding how to implement prenatal diagnosis and genetic counselling for DMD/BMD families with missense mutations and what principles should be met in the DMD/BMD prenatal diagnosis for families with female DMD/BMD patients.
In addition, the functional verification of missense mutations was lacking, and prenatal diagnosis was the only option for these families. However, since the pathogenicity of the missense mutation of DMD cannot be determined at present, the proband diagnosed with a missense mutation was not included in the research scope.
The treatment expectation of DMD
In our study, 193 foetuses were diagnosed with DMD. In China, pregnant women have the option of having a voluntary termination of pregnancy. However, not all countries allow a voluntary termination of pregnancy. Therefore, many paediatric patients are still born ceaselessly. Hence, not only is a prenatal diagnosis crucial, but the treatment of patients should also be valued.
At present, there is no cure for DMD patients, and gene therapy is the only hope, which has attracted much attention worldwide. According to the structure of the DMD genome, DMD can be treated by splicing exons to restore the loss of the open reading frame (ORF), including the complete reading of nonsense mutations [24–27] (Ataluren (PTC124)) and skipping of exons [28–33] (Eteplirsen (Exondys 51) and Golodirsen (Vyondys 53). Ultimately, the DMD phenotype is converted to mild BMD. In addition, the percentages of patients treated by jumping exon 51 or the complete reading of nonsense mutations were 14.51% and 11.67%, respectively, which is almost consistent with the percentage of 15% [34] in Scoto M’s study and 16–17% in McDonald C M’s study [35].