Mechanisms for nonrecurrent genomic rearrangements associated with CMT1A or HNPP: rare CNVs as a cause for missing heritability

Abstract

Genomic rearrangements involving the peripheral myelin protein gene (PMP22) in human chromosome 17p12 are associated with neuropathy: duplications cause Charcot-Marie-Tooth disease type 1A (CMT1A), whereas deletions lead to hereditary neuropathy with liability to pressure palsies (HNPP). Our previous studies showed that >99% of these rearrangements are recurrent and mediated by nonallelic homologous recombination (NAHR). Rare copy number variations (CNVs) generated by nonrecurrent rearrangements also exist in 17p12, but their underlying mechanisms are not well understood. We investigated 21 subjects with rare CNVs associated with CMT1A or HNPP by oligonucleotide-based comparative genomic hybridization microarrays and breakpoint sequence analyses, and we identified 17 unique CNVs, including two genomic deletions, ten genomic duplications, two complex rearrangements, and three small exonic deletions. Each of these CNVs includes either the entire PMP22 gene, or exon(s) only, or ultraconserved potential regulatory sequences upstream of PMP22, further supporting the contention that PMP22 is the critical gene mediating the neuropathy phenotypes associated with 17p12 rearrangements. Breakpoint sequence analysis reveals that, different from the predominant NAHR mechanism in recurrent rearrangement, various molecular mechanisms, including nonhomologous end joining, Alu-Alu-mediated recombination, and replication-based mechanisms (e.g., FoSTeS and/or MMBIR), can generate nonrecurrent 17p12 rearrangements associated with neuropathy. We document a multitude of ways in which gene function can be altered by CNVs. Given the characteristics, including small size, structural complexity, and location outside of coding regions, of selected rare CNVs, their identification remains a challenge for genome analysis. Rare CNVs may potentially represent an important portion of "missing heritability" for human diseases.

Figure 1

Oligonucleotide aCGH Analysis of Simple Nonrecurrent Genomic Rearrangements of 17p12 Associated with CMT1A or HNPP Above, for reference, is a horizontal line showing the 1.4 Mb common CMT1A- or HNPP-associated rearrangement region and its flanking sequences on human chromosome 17 with cytogenetic bands depicted above and Megabase (Mb) genomic coordinates (NCBI build 36) below. Locations of the PMP22 gene, the distal and proximal CMT1A-REPs, and LCR17pA are shown. The black horizontal bar shows the location and size of common recurrent 17p12 rearrangements (both deletion and duplication) associated with CMT1A or HNPP. To the left are laboratory identification numbers of the subjects with nonrecurrent 17p12 rearrangements. The green (loss), black (no change), and red (gain) dots show the relative intensities (deviation from the horizontal line of log₂Ratio = zero) and genomic locations of the oligonucleotide probes employed in our aCGH assay. The regions that lack unique probes correspond to the LCRs. The regions with copy number gains are indicated in red horizontal bars, and the losses are shown in green. Related subjects: deletions B273.1 and B273.2; duplications C3159 and C4316. The blue vertical lines indicate the location of the PMP22 gene. The arrow indicates the copy number change caused by known polymorphism in the control DNA (NA15510).

Figure 2

CMT1A-Associated 17p12 Duplications Exclusive of the PMP22 Coding Region but Involving the Potential Upstream Regulatory Sequence (A) Two duplications (red bars) of the upstream region of PMP22 were identified in subject SPR1 and related subjects SD11 and SD14. The SRO involves the TEKT3 and CDRT4 genes, whereas PMP22 is intact and has normal copy number. Both the noncoding exons (1A and 1B) and coding exons (2 to 5) are shown. Three ultra CNSs associated with conserved human transcription factor binding sites were present in SRO. (B) The information of CNS size, lod score, and transcription factor binding site was provided. Details are provided in Figures S2 and S3.

Figure 3

Oligonucleotide aCGH Analysis Revealed Complex Genomic Rearrangements in 17p12 (A) Subjects C1292 and C2405 are related (C2405 is the sister of C1292's maternal grandfather). Two proposed FoSTeS events (FoSTeS × 2) consistent with this complex rearrangement were shown. (B) In C3011, the 17p CGH array (4 × 44k format) revealed additional duplications in 17p11.2 that is not covered by the CMT1A array (8 × 15k format). Rearrangement abbreviations: nml, normal; dup, duplication. This complex rearrangement can be alternatively interpreted as duplication-normal-duplication-normal-duplication or duplication-normal-duplication that is accompanied by an inversion polymorphism mediated by NAHR between two inverted LCRs (distal and proximal SMS-REPs).

Figure 4

Oligonucleotide aCGH Analysis of Exonic Rearrangements of PMP22 The deleted regions are shadowed. Below, the locations of the PMP22 gene and its coding exons (2 to 5) are shown. Exon 4 is deleted in subject SPR2. Both subjects A23 and A29 have deletions of exons 1–3.