Background Duchenne Muscular Dystrophy (DMD) is the most common muscle disease in children, and there are no effective therapies for DMD or Becker Muscular Dystrophy (BMD). Currently, targeted gene therapy treatments have emerged. As a result, genetic diagnosis is the basis of treatment. In addition, genetic and prenatal diagnosis significantly reduces their incidence rates. This study combines the application of multiplex ligation-dependent probe amplification technology (MLPA) and “next-generation” sequencing technology (NGS) as the most economical and efficient method of diagnosis. Therefore, in the diagnosis of DMD/BMD, patients’ MLPA data are first used to detect DMD gene deletions or duplications, and NGS and Sanger sequencing are then applied to exclude MLPA-negative samples. Meanwhile, polymerase chain reaction (PCR) is used to detect single exon deletions to exclude false-positives in MLPA caused by point mutations. Methods In this study, we recruited 1051 proband families of DMD from 2016 to 2018 and had access to information that could identify individual participants during or after data collection. Patients who were diagnosed with DMD were first tested by MLPA. MLPA results with single exon deletions were validated with PCR amplification and Sanger sequencing. The negative results of MLPA were further analysed with NGS and validated by Sanger sequencing. For novel missense mutations, phenotype-genotype correlations were analysed using PolyPhen2 and mutation taster. All methods were performed in accordance with the relevant guidelines and regulations. Results DMD mutations were identified in 1029 families (97.91%, 1029/1051). Large deletions, duplications, and small mutations accounted for 70.41% (740/1051), 8.28% (87/1051), and 19.12% (201/1051) of all cases, respectively. There were 205 small mutation types, 53 of which were novel. The rate of de novo mutations was 39.45% (187/474) and was higher in large duplications (49.53%, 157/317). Among 68 asymptomatic patients (< 3 years old) with unexplained persistent hyperCKaemia upon conventional physical examination, 63 were diagnosed as DMD/BMD according to genetic diagnosis. Conclusion Our results expand the spectrum of DMD mutations, which could contribute to the treatment of DMD/BMD and provide an effective diagnosis method. Thus, the combination of MLPA, NGS and Sanger sequencing is of great significance for family analysis, gene diagnosis and gene therapy.
Noninvasive prenatal diagnosis (NIPD) of single-gene disorders has recently become the focus of clinical laboratories. However, reports on the clinical application of NIPD of Duchenne muscular dystrophy (DMD) are limited. This study aimed to evaluate the detection performance of haplotype-based NIPD of DMD in a real clinical environment. Twenty-one DMD families at 7–12 weeks of gestation were prospectively recruited. DNA libraries of cell-free DNA from the pregnant and genomic DNA from family members were captured using a custom assay for the enrichment of DMD gene exons and spanning single-nucleotide polymorphisms, followed by next-generation sequencing. Parental haplotype phasing was based on family linkage analysis, and fetal genotyping was inferred using the Bayes factor through target maternal plasma sequencing. Finally, the entire experimental process was promoted in the local clinical laboratory. We recruited 13 complete families, 6 families without paternal samples, and 2 families without probands in which daughter samples were collected. Two different maternal haplotypes were constructed based on family members in all 21 pedigrees at as early as 7 gestational weeks. Among the included families, the fetal genotypes of 20 families were identified at the first blood collection, and a second blood collection was performed for another family due to low fetal concentration. The NIPD result of each family was reported within 1 week. The fetal fraction in maternal cfDNA ranged from 1.87 to 11.68%. In addition, recombination events were assessed in two fetuses. All NIPD results were concordant with the findings of invasive prenatal diagnosis (chorionic villus sampling or amniocentesis). Exon capture and haplotype-based NIPD of DMD are regularly used for DMD genetic diagnosis, carrier screening, and noninvasive prenatal diagnosis in the clinic. Our method, haplotype-based early screening for DMD fetal genotyping via cfDNA sequencing, has high feasibility and accuracy, a short turnaround time, and is inexpensive in a real clinical environment.
Purpose To explore the feasibility of performing rapid prenatal diagnoses of FSHD1 using a combination of Bianano optical mapping and linkage‐based karyomapping. Methods DNA specimens from a family that had been previously diagnosed with FSHD1 using Southern Blot analysis were used for this study. Genetic diagnosis of the proband, fetus chorionic amniotic fluid, and aborted fetal tissue was performed using Bianano optical mapping (BOM) together with linkage‐based karyomapping. Results BOM analysis showed that the proband's 4q35.2 region contained four D4Z4 repeats and the 4qA permissible allele, consistent with the previous FSHD1 diagnosis obtained by Southern Blotting. BOM analysis of the fetus' 4q35.2 region was consistent with that of the proband. Karyomap analysis revealed that the fetus inherited the affected chromosome segment from the proband. After genetic counseling, the couple choose termination of pregnancy, and we performed gene diagnosis of the abortus tissue by BOM. Conclusions Bianano optical mapping can determine the number of D4Z4 repeats and exclude interference of the 10q26.3 homologous region, and in combination with karyomapping, can be used for rapid and accurate prenatal diagnosis of FSHD1.
Purpose Haplotype‐based assay has been proved efficient in noninvasive prenatal testing for various monogenic disorders in singleton pregnancies. However, its application in twin pregnancies is still blank. Here we provide a novel algorithmic approach to noninvasively assess fetal genotypes in a dizygotic twin pregnancy at risk for Duchenne muscular dystrophy (DMD). Methods One pregnant woman carrying a dizygotic twin gestation was recruited as she was a heterozygote of DMD gene duplication and has delivered an affected son. Construction of parental haplotypes was achieved by target sequencing of DNA samples from the parent and the proband. Single nucleotide polymorphisms within target regions were classified into six categories according to parental haplotypes. Individual fetal fractions were calculated using paternal heterozygous. Maternal‐inherited haplotype was deduced using relative haplotype dosage through a two‐step Bayesian model. Results One male fetus with a lower fetal fraction of 4.6% and one female fetus with a higher fetal fraction of 10.1% were observed. The male fetus was predicted to be a DMD pathogenic variant carrier, while the female fetus was predicted to be homozygous normal. Noninvasive prenatal testing (NIPT) results were concordant with the findings of invasive prenatal diagnosis. Conclusions This study is the first report of the use of NIPT for the assessment of DMD in a twin pregnancy. The algorithm provided could expand the use of NIPT to monogenic disorders in dizygotic twin pregnancies.
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