Diagnostic Approaches for Structural Variants in Human Disease

Structural variants (SVs), which include deletions, duplications, inversions, insertions, and translocations larger than 50 base pairs, contribute substantially to human genetic diversity and disease. These SVs play a critical role in the rare disease spectrum, including congenital anomalies, neurodevelopmental disorders, as well as infertility and cancer. However, a significant proportion of SVs remain undetected due to limitations of conventional and short-read sequencing-based diagnostic methods. Traditional cytogenetic techniques, such as karyotyping, banding methods, and fluorescence in situ hybridization, remain indispensable for detecting large chromosomal rearrangements and balanced abnormalities, while molecular cytogenetic tools, such as chromosomal microarray analysis, enable high-resolution detection of copy number variations. Despite these advances, balanced and complex rearrangements often escape detection. The advent of next-generation sequencing has improved variant discovery, yet short-read technologies have limited ability to resolve repetitive regions, complex breakpoints, and balanced SVs. Long-read sequencing platforms, such as Pacific Biosciences HiFi and Oxford Nanopore Technologies, have transformed SV detection by enabling precise breakpoint mapping, haplotype phasing, analysis of repeat regions, and identification of complex chromosomal rearrangements, such as chromothripsis. Emerging technologies, such as optical genome mapping, complement sequencing by enabling genome-wide detection of large and cryptic rearrangements. This article reviews the spectrum of SVs, their clinical relevance, and the strengths and limitations of cytogenetic, molecular, and sequencing-based diagnostic technologies, emphasizing the importance of an integrated, multiplatform approach for comprehensive genomic diagnosis.