Intrachromosomal insertion as a diagnostic challenge: a hidden structural rearrangement causing recurrent duplication...

Intrachromosomal insertion is a rare form of structural chromosomal rearrangement that often cannot be accurately delineated by conventional G-banding, making it difficult to predict reproductive outcomes. In clinical practice, such insertions are often misinterpreted as inversions or remain undetected, leading to recurrent segmental imbalances in offspring. We aimed to characterize an unresolved structural rearrangement identified in a family and to clarify its reproductive implications through advanced cytogenetic and molecular analyses.

Cytogenetic and molecular studies were conducted in a family where the proband exhibited a 17.8 Mb duplication at 9q21.31-q22.33. Although G-banding suggested a parental structural abnormality, its configuration could not be precisely defined. Subsequent preimplantation genetic testing for structural rearrangements (PGT-SR) using shallow whole-genome sequencing was performed on embryos, and further structural characterization was achieved through fluorescence in situ hybridization (FISH) and nanopore long-read sequencing.

PGT-SR identified recurrent segmental imbalances involving the same region as in the proband, including four duplications and one deletion among 13 embryos. FISH and long-read sequencing demonstrated that the paternal rearrangement represented an intrachromosomal inverted insertion, described as ins(9)(q34.13q22.33q21.31). The father was phenotypically normal but transmitted unbalanced gametes generated by recombination between the insertion and original sites, leading to recurrent chromosomal abnormalities.

This case highlights the potential of intrachromosomal insertions, although balanced in carriers, to cause recurrent segmental duplications or deletions in offspring. Comprehensive analysis using FISH and long-read sequencing is essential for accurate diagnosis, appropriate genetic counseling, and informed reproductive decision-making.