Balanced chromosomal insertions as the mechanism of recurrent familial microstructural abnormalities: detailed analyses using long-read whole-genome sequencing.

Chromosomal insertions are a type of structural abnormality. While individuals with balanced insertions are typically asymptomatic, their offspring may have unbalanced abnormalities. We report two families with recurrent microstructural chromosomal abnormalities. To investigate the mechanisms, we performed chromosomal microarray (CMA), fluorescence in situ hybridization (FISH), and long-read whole-genome sequencing. In Family A, a duplication of 13q31.2-q33.1 was found in a proband with developmental and epileptic encephalopathy. A reciprocal deletion was detected in a fetus during a subsequent pregnancy. FISH confirmed an interchromosomal insertion involving chromosome 10. Long-read sequencing in the carrier parent revealed two split fragments of the inserted segment, one in inverted orientation. In Family B, a recurrent 1p36 interstitial deletion was associated with intellectual disability. FISH showed no abnormalities in the parents, but long-read sequencing of a suspected carrier revealed an intrachromosomal insertion of the 1p36 segment in inverted orientation. Breakpoint analysis showed minimal deletions or fragment overlaps in both families, indicating chromoanasynthesis as the likely mechanism. Although not routinely required for diagnosis of insertions, long-read sequencing can reveal hidden structural changes and clarify insertion mechanisms, as demonstrated in this study.