Preimplantational Genetic Diagnosis and Mutation Detection in a Family with Duplication Mutation of DMD Gene

Abstract

Duchenne muscular dystrophy (DMD) is an X-linked recessive neuromuscular disease caused by mutation in the DMD gene. A 38-year-old woman was referred to our hospital with her son who was diagnosed with DMD. Multiplex PCR failed to detect DMD mutations in the affected child. The female carrier underwent preimplantation genetic diagnosis by linkage analysis and gender determination. Eight embryos were biopsied after in vitro fertilization. Two healthy embryos determined both as females (E1 and E3) were transferred. Although the paternal allele was absent in E3, it was considered to be a result of allele dropout for the STR-49 marker. Surprisingly, a female and a male baby were delivered at 38 gestational weeks, suggesting that E3 was a male embryo with the allele dropout occurring at the SRY gene. Exon 2 duplication was detected in the DMD patient and the carrier mother using next-generation sequencing and multiple ligation-dependent probe amplification. Next, we verified the duplication of exon 2 by real-time PCR, using a special primer at 3′ of intron 1, very close to exon 2. Finally, we confirmed that both newborns inherited the normal allele, using quantitative real-time PCR and linkage analysis.

Identification of a novel DMD duplication identified by a combination of MLPA and targeted exome sequencing

“…Despite that MLPA is now the most widely used technique for identifying duplication in DMD [8], targeted exome sequencing is prefered when evidence for a diagnosis of DMD is insufficient and when other metabolic diseases have not been ruled out. Furthermore, targeted exome sequencing is capable of detecting subtle mutations in the 79 exons that cannot be detected by MPLA [3]. In our patient, results from targeted exome sequencing were suggestive of a potential duplication in the DMD gene.…”

“…As the most common muscle disease in children, the incidence of DMD is 1 in 3500 live male births [3]. DMD is a progressive neuromuscular disease characterized by pseudohypertrophy in the calf muscle and the Gowers’ sign [4].…”

“…In the case of DMD, deletions found in most patients are followed by duplications, therefore, multiplex PCR, which can detect up to 98% of deletion, was considered as the standard diagnostic method [8]. Nevertheless, multiplex PCR is not the ideal technique for identifying duplications and female carriers due to the presence of a normal copy of the DMD gene [3]. Fortunately, the introduction of a dosimetry method based on multiplex ligation-dependent probe amplification (MLPA) has improved the discovery of large intragenic rearrangements [8].…”

Brazilian consensus on Duchenne muscular dystrophy. Part 1: diagnosis, steroid therapy and perspectives

“…Recommendations regarding prenatal diagnoses of DMD will vary according to each country' s abortion legislation. Preimplantation diagnoses with embryo selection can be offered to women carriers of the DMD mutation (Level of evidence: 4, Class of Recommendation: C) 29,30 . A preimplantation diagnosis is an expensive procedure that is not available in the Unified Health System (Sistema Único de Saude, SUS) of Brazil.…”

Preimplantation genetic haplotyping for six Chinese pedigrees with thalassemia using a single nucleotide polymorphism microarray

“…Preimplantation genetic haplotyping (PGH) technology is a powerful tool for PGD and can detect ADO more accurately by measuring numerous polymorphic markers in DNA segments surrounding the genes of interest. At present, short tandem repeat (STR) markers are used for mapping haplotypes in PGH . However, the STR approach is not only labor‐intensive and time‐consuming but also often less informative because of the limited number and uneven distribution of polymorphic markers in some families.…”