Genomic basis of aromatase excess syndrome: recombination- and replication-mediated rearrangements leading to CYP19A1 overexpression

Abstract

Context: Genomic rearrangements at 15q21 have been shown to cause overexpression of CYP19A1 and resultant aromatase excess syndrome (AEXS). However, mutation spectrum, clinical consequences, and underlying mechanisms of these rearrangements remain to be elucidated.

Objective: The aim of the study was to clarify such unsolved matters.

Design, setting, and methods: We characterized six new rearrangements and investigated clinical outcome and local genomic environments of these rearrangements and of three previously reported duplications/deletions.

Results: Novel rearrangements included simple duplication involving exons 1-10 of CYP19A1 and simple and complex rearrangements that presumably generated chimeric genes consisting of the coding region of CYP19A1 and promoter-associated exons of neighboring genes. Clinical severities were primarily determined by the copy number of CYP19A1 and the property of the fused promoters. Sequences at the fusion junctions suggested nonallelic homologous recombination, nonhomologous end-joining, and replication-based errors as the underlying mechanisms. The breakpoint-flanking regions were not enriched with GC content, palindromes, noncanonical DNA structures, or known rearrangement-associated motifs. The rearrangements resided in early-replicating segments.

Conclusions: These results indicate that AEXS is caused by duplications involving CYP19A1 and simple and complex rearrangements that presumably lead to the usage of cryptic promoters of several neighboring genes. Our data support the notion that phenotypes depend on the dosage of CYP19A1 and the characteristics of the fused promoters. Furthermore, we show that the rearrangements in AEXS are generated by both recombination- and replication-mediated mechanisms, independent of the known rearrangement-inducing DNA features or late-replication timing. Thus, AEXS represents a unique model for human genomic disorders.

Figure 1.

Copy-number analyses in cases 1–6. A, Schematic representation of the normal genomic structure around CYP19A1. The arrows indicate genomic positions and transcriptional direction of genes (5′→3′). For CYP19A1, the dark and light blue lines denote the genomic regions for noncoding exons 1 and coding exons 2–10, respectively. Genomic positions refer to Human Genome Database (hg19, build 37). Only genes around the fusion junctions are shown. B, CGH analyses in the six cases. The black, red, and green dots denote signals indicative of the normal, increased (>+0.5) and decreased (<−1.0) copy-numbers, respectively.

Figure 2.

Fine genomic structures of the rearrangements. A, Schematic representation of the normal genomic structure. Arrowheads indicate the positions and the directions (5′→3′) of PCR primers utilized in this study (P1–P12). The open and color-painted boxes denote noncoding and coding exons, respectively. The sizes of the exons, introns, and primers are not drawn to scale. B, Schematic representation of the rearrangements and the DNA sequences at the fusion junctions. The red, blue, and green areas indicate duplications, deletions, and inversions, respectively. P1–P12 indicate the same PCR primers as shown in panel A. The fusion junctions of case 3 were not characterized. For case 4, the precise genomic position of the duplication remains to be clarified.

Figure 3.

Schematic representation of the 11 rearrangements. Cases 1, 2, and 4–6 are from the present study, and patients from families A–F, patient 2, and patients from family 2 have been reported previously (1, 2, 5). The genomic abnormalities of case 3 were not characterized. The arrows indicate the positions and transcriptional direction of CYP19A1 and its neighboring genes (5′→3′). Only genes around the fusion junctions are shown. The red, blue, and green lines indicate duplications, deletions, and inversions, respectively. For CYP19A1, the dark and light blue lines denote the genomic regions for the noncoding and coding exons, respectively. The inversions of family 2 and patient 2 may be complex rearrangements because copy-number analyses have not been performed in these cases.