Genomic breakpoint analysis facilitates the identification of X-chromosomal inversion among molecularly unsolved cases of Duchenne Muscular Dystrophy
Laboratory Genetics and Genomics
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Primary Categories:
- Laboratory Genetics
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Secondary Categories:
- Laboratory Genetics
Introduction:
Duchenne (DMD) and Becker Muscular Dystrophy (BMD) are X-linked recessive disorders with a frequency of 1 in 5000 male infants caused by pathogenic variants in the dystrophin gene. Copy number variants (CNVs) such as intragenic deletions (65%) and duplications (5-10%) are the most frequent type of variants observed in DMD and BMD followed by approximately 30-35% single‐nucleotide variants and complex rearrangements. In approximately 2-5% of patients, no causative variants in the DMD coding region are detected by conventional methods. Causative variants in these patients include deep intronic variants causing splicing effects or complex structural rearrangements that include balanced rearrangements such as inversions or translocations. A paucity of balanced inversions involving the DMD gene are reported due to the technical limitations of conventional molecular techniques such as MLPA and microarray, and determining the inversion breakpoints is complicated as they usually occur in the large DMD introns and cannot be detected by conventional molecular techniques such as MLPA and microarray. We used breakpoint junction sequence analysis for identifying balanced inversion rearrangements affecting the DMD gene.
Methods:
The Agilent Sureselect DMD targeted sequence capture method was used to enrich the entire DMD gene, which includes all exons and introns from the genomic DNA. DNA was analyzed by next-generation sequencing (NGS) on the Illumina MiSeq or NovaSeq™ 6000 with 2x150 paired-end reads. CNVs within the DMD gene were assessed using BioNano’s NxClinical v6.1 software (El Segundo, CA). Split reads were visualized in Integrative Genomics Viewer v2.8.0.7. Sanger sequencing was performed to confirm the breakpoint junctions wherever possible using case-specific primers across the breakpoints. In one case, Bionano optical genome mapping was also used to confirm the results.
Results:
We present three cases with a large inversion affecting the DMD gene. Analysis of NGS soft-clipped reads was used to discover the complex rearrangement and investigate the breakpoints. Sanger sequencing was used for confirmation of the breakpoints. Case 1 was found to harbor a 92.4 Megabase (Mb) pericentric inversion of chromosome Xp21.2 – Xq25. The inverted region extends from intron 67 in the DMD gene (Xp21.2, chrX:31214006) through Xq25 (chrX:123586570). This inversion disrupts the DMD gene from exon 68 through the end of the gene resulting in a DMD phenotype. Parental testing was not performed. Case 2 was found to harbor a 9.1 Mb paracentric inversion of chromosome Xp21.1-Xp22.11. The inverted region extends from Xp22.11 (chrX: 23028859) through intron 44 in the DMD gene (Xp21.1, chrX:32142959). This inversion disrupts the DMD gene from exon 45 through the end of the gene. This inversion was maternally inherited. Case 3 was found to harbor a 5.9 Mb paracentric inversion of chromosome Xp21.1-Xp21.3. The inverted region extends from Xp21.3 (chrX:26929141) through intron 2 in DMD gene (Xp21.1, 32873783). This inversion disrupts the DMD gene from exon 3 through the end of the gene. This inversion was maternally inherited. RNA seq analysis in these three cases showed aberrant DMD expression.
Conclusion:
In conclusion, breakpoint junction analysis of genomic data facilitated the identification of large balanced inversions that were undiagnosed. The identification of split reads at the breakpoints in DMD patients enhances our understanding of the mechanisms underlying structural rearrangements, thereby facilitating the precise molecular diagnosis of Duchenne muscular dystrophy in these three cases, and helping in clinical evaluation, variant classification, and therapeutics. Our results emphasize the importance of utilizing breakpoint analysis of NGS data or long-read sequencing to identify the complex rearrangements, particularly in cases where a dystrophinopathy was diagnosed clinically and histologically but conventional methods identified no pathogenic variants.
Duchenne (DMD) and Becker Muscular Dystrophy (BMD) are X-linked recessive disorders with a frequency of 1 in 5000 male infants caused by pathogenic variants in the dystrophin gene. Copy number variants (CNVs) such as intragenic deletions (65%) and duplications (5-10%) are the most frequent type of variants observed in DMD and BMD followed by approximately 30-35% single‐nucleotide variants and complex rearrangements. In approximately 2-5% of patients, no causative variants in the DMD coding region are detected by conventional methods. Causative variants in these patients include deep intronic variants causing splicing effects or complex structural rearrangements that include balanced rearrangements such as inversions or translocations. A paucity of balanced inversions involving the DMD gene are reported due to the technical limitations of conventional molecular techniques such as MLPA and microarray, and determining the inversion breakpoints is complicated as they usually occur in the large DMD introns and cannot be detected by conventional molecular techniques such as MLPA and microarray. We used breakpoint junction sequence analysis for identifying balanced inversion rearrangements affecting the DMD gene.
Methods:
The Agilent Sureselect DMD targeted sequence capture method was used to enrich the entire DMD gene, which includes all exons and introns from the genomic DNA. DNA was analyzed by next-generation sequencing (NGS) on the Illumina MiSeq or NovaSeq™ 6000 with 2x150 paired-end reads. CNVs within the DMD gene were assessed using BioNano’s NxClinical v6.1 software (El Segundo, CA). Split reads were visualized in Integrative Genomics Viewer v2.8.0.7. Sanger sequencing was performed to confirm the breakpoint junctions wherever possible using case-specific primers across the breakpoints. In one case, Bionano optical genome mapping was also used to confirm the results.
Results:
We present three cases with a large inversion affecting the DMD gene. Analysis of NGS soft-clipped reads was used to discover the complex rearrangement and investigate the breakpoints. Sanger sequencing was used for confirmation of the breakpoints. Case 1 was found to harbor a 92.4 Megabase (Mb) pericentric inversion of chromosome Xp21.2 – Xq25. The inverted region extends from intron 67 in the DMD gene (Xp21.2, chrX:31214006) through Xq25 (chrX:123586570). This inversion disrupts the DMD gene from exon 68 through the end of the gene resulting in a DMD phenotype. Parental testing was not performed. Case 2 was found to harbor a 9.1 Mb paracentric inversion of chromosome Xp21.1-Xp22.11. The inverted region extends from Xp22.11 (chrX: 23028859) through intron 44 in the DMD gene (Xp21.1, chrX:32142959). This inversion disrupts the DMD gene from exon 45 through the end of the gene. This inversion was maternally inherited. Case 3 was found to harbor a 5.9 Mb paracentric inversion of chromosome Xp21.1-Xp21.3. The inverted region extends from Xp21.3 (chrX:26929141) through intron 2 in DMD gene (Xp21.1, 32873783). This inversion disrupts the DMD gene from exon 3 through the end of the gene. This inversion was maternally inherited. RNA seq analysis in these three cases showed aberrant DMD expression.
Conclusion:
In conclusion, breakpoint junction analysis of genomic data facilitated the identification of large balanced inversions that were undiagnosed. The identification of split reads at the breakpoints in DMD patients enhances our understanding of the mechanisms underlying structural rearrangements, thereby facilitating the precise molecular diagnosis of Duchenne muscular dystrophy in these three cases, and helping in clinical evaluation, variant classification, and therapeutics. Our results emphasize the importance of utilizing breakpoint analysis of NGS data or long-read sequencing to identify the complex rearrangements, particularly in cases where a dystrophinopathy was diagnosed clinically and histologically but conventional methods identified no pathogenic variants.