Solving the Unsolved: The Role of Fluorescence In situ Hybridization in the Era of Advanced Clinical Genomics

Abstract

Recent advances in genomic technologies, including chromosomal microarray analysis (CMA), next-generation sequencing, optical genome mapping, and long-read sequencing, have revolutionized the detection and characterization of genetic disorders. These approaches provide high-resolution identification of sequence-level and copy number variants, substantially improving diagnostic yield. However, several clinically significant chromosomal abnormalities, particularly balanced rearrangements, subtle terminal translocations, insertional events, low-level mosaicism, and complex chromosomal reorganizations, often remain difficult to resolve and are expensive to detect with sequencing technologies alone. Fluorescence in situ hybridization (FISH) is a molecular cytogenetic technique that uses fluorescently labeled DNA probes to visualize specific chromosomal regions on metaphase chromosomes or interphase nuclei. The technique provides direct positional and structural information (at low resolution) and remains valuable for identifying deletions, duplications, translocations, aneuploidies, and complex chromosomal rearrangements. In this study, we present ten cases in which conventional cytogenetic and/or genomic investigations identified or suggested chromosomal abnormalities but did not completely resolve their structural organization. Targeted FISH, particularly whole-chromosome painting, was subsequently used in conjunction with conventional karyotyping, CMA, and other molecular investigations to further characterize these abnormalities. These cases include subtle terminal translocations, insertional translocations, and complex chromosomal rearrangements that required direct visualization of chromosomal architecture for accurate interpretation. We also demonstrate the utility of targeted FISH as an important confirmatory and complementary technique to conventional karyotyping and for validating genomic findings, and highlight clinical scenarios in which sequencing technologies are cumbersome and expensive. Our findings emphasize that, despite rapid advances in genomic technologies, targeted FISH remains a valuable complementary tool in modern cytogenomic diagnostics, particularly for resolving structurally complex and balanced chromosomal abnormalities.