Genomic disorders are often caused by recurrent copy number variations (CNVs), with nonallelic homologous recombination (NAHR) as the underlying mechanism. Recently, several microhomology-mediated repair mechanisms—such as microhomology-mediated end-joining (MMEJ), fork stalling and template switching (FoSTeS), microhomology-mediated break-induced replication (MMBIR), serial replication slippage (SRS), and break-induced SRS (BISRS)—were described in the etiology of non-recurrent CNVs in human disease. In addition, their formation may be stimulated by genomic architectural features. It is, however, largely unexplored to what extent these mechanisms contribute to rare, locus-specific pathogenic CNVs. Here, fine-mapping of 42 microdeletions of the FOXL2 locus, encompassing FOXL2 (32) or its regulatory domain (10), serves as a model for rare, locus-specific CNVs implicated in genetic disease. These deletions lead to blepharophimosis syndrome (BPES), a developmental condition affecting the eyelids and the ovary. For breakpoint mapping we used targeted array-based comparative genomic hybridization (aCGH), quantitative PCR (qPCR), long-range PCR, and Sanger sequencing of the junction products. Microhomology, ranging from 1 bp to 66 bp, was found in 91.7% of 24 characterized breakpoint junctions, being significantly enriched in comparison with a random control sample. Our results show that microhomology-mediated repair mechanisms underlie at least 50% of these microdeletions. Moreover, genomic architectural features, like sequence motifs, non-B DNA conformations, and repetitive elements, were found in all breakpoint regions. In conclusion, the majority of these microdeletions result from microhomology-mediated mechanisms like MMEJ, FoSTeS, MMBIR, SRS, or BISRS. Moreover, we hypothesize that the genomic architecture might drive their formation by increasing the susceptibility for DNA breakage or promote replication fork stalling. Finally, our locus-centered study, elucidating the etiology of a large set of rare microdeletions involved in a monogenic disorder, can serve as a model for other clustered, non-recurrent microdeletions in genetic disease.
Genomic disorder is a general term describing conditions caused by genomic aberrations leading to a copy number change of one or more genes. Copy number changes with the same length and clustered breakpoints for a group of patients with the same disorder are named recurrent rearrangements. These originate mostly from a well-studied mechanism, namely nonallelic homologous recombination (NAHR). In contrast, non-recurrent rearrangements vary in size, have scattered breakpoints, and can originate from several different mechanisms that are not fully understood. Here we tried to gain further insight into the extent to which these mechanisms contribute to non-recurrent rearrangements and into the possible role of the surrounding genomic architecture. To this end, we investigated a unique group of patients with non-recurrent deletions of the FOXL2 region causing blepharophimosis syndrome. We observed that the majority of these deletions can result from several mechanisms mediated by microhomology. Furthermore, our data suggest that rare pathogenic microdeletions do not occur at random genome sequences, but are possibly guided by the surrounding genomic architecture. Finally, our study, elucidating the etiology of a unique cohort of locus-specific microdeletions implicated in genetic disease, can serve as a model for the formation of genomic aberrations in other genetic disorders.