Researchers have developed a targeted long-read sequencing (LRS) approach optimized for routine, non-high-molecular-weight (non-HMW) DNA that may improve characterization of X-linked genetic defects by simultaneously assessing genomic variants and methylation changes.
Published in the journal Clinical Chemistry, the study evaluated an adaptive sampling (AS)–based protocol in 20 families with X-linked genetic variants previously identified through chromosomal microarray analysis (CMA) and/or low-pass genome sequencing.
A closer look at X-linked variants
X-linked disorders can be challenging to characterize because they may involve structural variants, single-nucleotide variants, small insertions and deletions, and methylation changes associated with X-chromosome inactivation (XCI). In females, skewed XCI can preferentially silence one X chromosome, potentially influencing disease severity.
The researchers optimized the AS protocol by modifying DNA fragmentation and size selection, library construction, and sequencing. The resulting approach achieved a minimum 15-fold enrichment of target regions, compared with 8.1-fold enrichment reported for the standard AS protocol.
The optimized LRS detected all X-linked variants that had been identified previously. It also revealed additional findings in 7 of the 20 families, or 35% of cases.
More information from the same assay
Those additional findings contributed to variant reclassification in three families, representing 15% of the study population. The reclassifications were enabled by resolving structural-variant breakpoints and determining gene methylation status.
The approach also allowed researchers to assess haplotypes and methylation status for individual genes. According to the study, this capability could provide additional information when evaluating XCI in females with X-linked genomic changes, including cases involving de novo deletions or meiotic crossover.
The researchers noted that methylation findings may depend on the tissue used for DNA analysis because XCI can exhibit tissue-specific patterns.
The study had several limitations, including its small sample size and a requirement for at least 3.6 μg of input DNA. The authors also noted that the assay currently functions primarily as a confirmatory test for known variants or for patients suspected of having X-linked genetic defects.
The researchers concluded that the optimized approach "enabled the comprehensive characterization of genomic and methylation changes," adding that it "shows strong potential for broader application in other disease settings."
Note: This news summary was generated by AI based on a published press release, followed by a review from human editors.
