Molecular diagnosis of DMD-related dystrophinopathies in patients with non-representative phenotypes by using a broad neuromuscular next generation sequencing (NGS) panel
Laboratory Genetics and Genomics
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Primary Categories:
- Laboratory Genetics
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Secondary Categories:
- Laboratory Genetics
Introduction:
X-linked DMD-related dystrophinopathies include Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), and DMD-associated dilated cardiomyopathy (DCM). The clinical manifestations of dystrophinopathies can vary from asymptomatic, increased creatine phosphokinase (CK) in serum to severe, progressive DMD/BMD, or dilated cardiomyopathy. As deletions of DMD exons account for approximately 60%-70% of pathogenic variants in individuals with DMD/BMD, deletion/duplication analysis of the DMD gene has been the initial testing strategy, followed by sequence analysis if no copy number variant (CNV) is identified. This traditional testing strategy is time-consuming and costly. In addition, males with the BMD phenotype and most females carrying a heterozygous variant may not exhibit the distinct features that would initiate targeted DMD gene testing. For these reasons, we developed a broad neuromuscular NGS panel that can test a broad spectrum of neuromuscular diseases including individuals with less severe clinical presentations of DMD-related dystrophinopathies.
Methods:
The neuromuscular NGS panel includes the DMD gene and 233 other genes known to be associated with neuromuscular disorders. This NGS panel was developed on an exome sequencing backbone at the Vanderbilt Clinical Genomic Laboratory, Vanderbilt University Medical Center (VUMC). Briefly, the whole exome was captured using IDT X-gen Exome (Integrated DNA Technologies) followed by sequencing on the Illumina NovaSeq 6000 platform. The NGS sequencing data was analyzed using Case Analyzer (Genosity, LabCorp) followed by CNV analysis via NxClinical (Bionano Genomics, Inc). Clinically significant CNVs were confirmed by multiplex ligation-dependent probe amplification (MLPA) assay.
Results:
Among 70 cases with clinical indications of neuromuscular disorders ordered by VUMC neurologists, there were 4 pathogenic/likely pathogenic variants detected in the DMD gene in 5 patients that resulted in a definitive molecular diagnosis. Two of the four variants are predicted to disrupt canonical splice sites (c.1812+1G>A, c.3603+2_3603+3insTA) and were identified by regular NGS analysis. The other two variants involve multiple exon deletions (del exons 49-51, del exons 47-52) detected by CNV analysis. The CNV variants were further confirmed independently by MLPA assay.
Conclusion:
Our neuromuscular NGS panel combined with CNV analysis has efficiently detected both typical sequence variants and multi-exon deletions in the DMD gene in undiagnosed patients. For patients with non-representative phenotypes of dystrophinopathies or asymptomatic, early stage DMD/BMD patients without a clinical diagnosis, this multifaceted NGS panel approach performed as a single-step assay is more suitable than the single DMD gene testing strategy.
X-linked DMD-related dystrophinopathies include Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), and DMD-associated dilated cardiomyopathy (DCM). The clinical manifestations of dystrophinopathies can vary from asymptomatic, increased creatine phosphokinase (CK) in serum to severe, progressive DMD/BMD, or dilated cardiomyopathy. As deletions of DMD exons account for approximately 60%-70% of pathogenic variants in individuals with DMD/BMD, deletion/duplication analysis of the DMD gene has been the initial testing strategy, followed by sequence analysis if no copy number variant (CNV) is identified. This traditional testing strategy is time-consuming and costly. In addition, males with the BMD phenotype and most females carrying a heterozygous variant may not exhibit the distinct features that would initiate targeted DMD gene testing. For these reasons, we developed a broad neuromuscular NGS panel that can test a broad spectrum of neuromuscular diseases including individuals with less severe clinical presentations of DMD-related dystrophinopathies.
Methods:
The neuromuscular NGS panel includes the DMD gene and 233 other genes known to be associated with neuromuscular disorders. This NGS panel was developed on an exome sequencing backbone at the Vanderbilt Clinical Genomic Laboratory, Vanderbilt University Medical Center (VUMC). Briefly, the whole exome was captured using IDT X-gen Exome (Integrated DNA Technologies) followed by sequencing on the Illumina NovaSeq 6000 platform. The NGS sequencing data was analyzed using Case Analyzer (Genosity, LabCorp) followed by CNV analysis via NxClinical (Bionano Genomics, Inc). Clinically significant CNVs were confirmed by multiplex ligation-dependent probe amplification (MLPA) assay.
Results:
Among 70 cases with clinical indications of neuromuscular disorders ordered by VUMC neurologists, there were 4 pathogenic/likely pathogenic variants detected in the DMD gene in 5 patients that resulted in a definitive molecular diagnosis. Two of the four variants are predicted to disrupt canonical splice sites (c.1812+1G>A, c.3603+2_3603+3insTA) and were identified by regular NGS analysis. The other two variants involve multiple exon deletions (del exons 49-51, del exons 47-52) detected by CNV analysis. The CNV variants were further confirmed independently by MLPA assay.
Conclusion:
Our neuromuscular NGS panel combined with CNV analysis has efficiently detected both typical sequence variants and multi-exon deletions in the DMD gene in undiagnosed patients. For patients with non-representative phenotypes of dystrophinopathies or asymptomatic, early stage DMD/BMD patients without a clinical diagnosis, this multifaceted NGS panel approach performed as a single-step assay is more suitable than the single DMD gene testing strategy.