Microhomology-Mediated Mechanisms Underlie Non-Recurrent Disease-Causing Microdeletions of the FOXL2 Gene or Its Regulatory Domain

Hannah Verdin,Björn Menten,Diane Beysen,James R Lupski,Barbara D'Haene,Pablo Lapunzina,Elfride De Baere,Tom Sante,Julian Nevado,Claudia M B Carvalho,Yana Novikova,Nancy B Spinner

doi:10.1371/journal.pgen.1003358

Abstract

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.

Highlights

Copy number variations (CNVs) are defined as DNA segments that are present at a variable copy number in comparison with a reference genome such as a deletions, duplications or insertions [1,2]
Based on the arraybased comparative genomic hybridization (aCGH) and quantitative PCR (qPCR) analyses, long-range PCR was performed for 35 deletions of which 22 resulted in a specific junction product
The FOXL2 encompassing deletions ranged from 1.4 kb to 5.51 Mb while the regulatory deletions ranged from 7.4 kb to 3.02 Mb, including one complex deletion consisting of two deletions interspersed with a segment without copy number variation

Summary

Introduction

Copy number variations (CNVs) are defined as DNA segments that are present at a variable copy number in comparison with a reference genome such as a deletions, duplications or insertions [1,2]. Many of the identified CNVs represent benign polymorphic variants; CNVs can lead to a genetic disease when for instance a dosage-sensitive gene is affected Such genetic diseases caused by genomic rearrangements are defined as genomic disorders [12,13,14,15]. The genomic rearrangements causing these disorders can be recurrent sharing a common interval and size, and having clustered breakpoints in multiple different subjects. These rearrangements are mostly the result of nonallelic homologous recombination (NAHR) between low-copy repeats (LCRs) or segmental duplications (SDs), a recombination-based mechanism [16]. Several mechanisms causing non-recurrent genomic rearrangements have been proposed such as (i) non-replicative repair mechanisms: non-homologous end-joining (NHEJ) [17], microhomology mediated end-joining (MMEJ) [18] and NAHR between repetitive elements (for example, Alu or L1) [19,20]; and (ii) replicative-based repair mechanisms: fork stalling and template switching (FoSTeS) [21], microhomology-mediated

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Discussion

Conclusion