Identification of uncommon recurrent Potocki-Lupski syndrome-associated duplications and the distribution of rearrangement types and mechanisms in PTLS

Abstract

Nonallelic homologous recombination (NAHR) can mediate recurrent rearrangements in the human genome and cause genomic disorders. Smith-Magenis syndrome (SMS) and Potocki-Lupski syndrome (PTLS) are genomic disorders associated with a 3.7 Mb deletion and its reciprocal duplication in 17p11.2, respectively. In addition to these common recurrent rearrangements, an uncommon recurrent 5 Mb SMS-associated deletion has been identified. However, its reciprocal duplication predicted by the NAHR mechanism had not been identified. Here we report the molecular assays on 74 subjects with PTLS-associated duplications, 35 of whom are newly investigated. By both oligonucleotide-based comparative genomic hybridization and recombination hot spot analyses, we identified two cases of the predicted 5 Mb uncommon recurrent PTLS-associated duplication. Interestingly, the crossovers occur in proximity to a recently delineated allelic homologous recombination (AHR) hot spot-associated sequence motif, further documenting the common hot spot features shared between NAHR and AHR. An additional eight subjects with nonrecurrent PTLS duplications were identified. The smallest region of overlap (SRO) for all of the 74 PTLS duplications examined is narrowed to a 125 kb interval containing only RAI1, a gene recently further implicated in autism. Sequence complexities consistent with DNA replication-based mechanisms were identified in four of eight (50%) newly identified nonrecurrent PTLS duplications. Our findings of the uncommon recurrent PTLS-associated duplication at a relative prevalence reflecting the de novo mutation rate and the distribution of 17p11.2 duplication types in PTLS reveal insights into both the contributions of new mutations and the different underlying mechanisms that generate genomic rearrangements causing genomic disorders.

Figure 1

Identification of Two 5 Mb Uncommon Recurrent PTLS-Associated Duplications High-resolution oligonucleotide aCGH analyses revealed the recurrent SMS- and PTLS-associated rearrangements mediated by NAHR between the LCRs on 17p. The following abbreviations are used: deletion, del; duplication, dup. The green (loss), black (no change), and red (gain) dots show the relative intensities in log₂ratio (deviation from horizontal line of zero) and genomic locations of the oligonucleotide probes employed in our aCGH assay. The regions that lack unique probes correspond to the LCRs. The 3.7 Mb common rearrangements are mediated by the distal and proximal SMS-REPs depicted on the horizontal black lines representing proximal chromosome 17p, whereas the recombinations between LCR17pA and LCR17pD lead to the 5 Mb uncommon recurrent rearrangements. The regions with copy number gains are indicated with red horizontal bars, and the losses are shown in green. The known copy number polymorphisms are indicated by arrows.

Figure 2

Recombination Hot Spot Analyses of Uncommon Recurrent PTLS-Associated Duplications (A) The PCR assay of the recombination hot spot in LCR17pA (blue) and LCR17pD (yellow). The ∼630 bp crossover interval fragment (D-A) of the recombinant LCR17pD and LCR17pA in the 5 Mb uncommon recurrent PTLS duplication was amplified by using LCR-specific primers (DF, forward primers in LCR17pD; AR, reverse primers in LCR17pA). This assay is positive in PTLS subjects 2808 and 2959 but negative in their parents and the control (NA 15510). (B) Sequence analysis of the recombination hot spot in LCR17pA and LCR17pD. The paralogous sequence variations (PSVs) between these two LCRs are represented by the capital letters between colored bars. The polymorphic nucleotide (SNP) is shown by the lowercase letter. In the PCR assay with the LCR-specific primers (DF2 and AR2), the recombination hot spot has been narrowed from 124 kb down to 570 bp, within which the 13-mer recombination hot spot sequence motif (CCNCCNTNNCCNC, black) was found. Asterisks indicate the newly investigated uncommon recurrent duplications (red) and deletion (green) whose strand exchanges have been further located within smaller intervals by sequencing analysis: 46 bp in subject 2808 and 359 bp in subjects 2959 and 547. The crossover intervals of our previously reported uncommon recurrent SMS deletions (green, no asterisk) are also shown.

Figure 3

Size Distribution of the PTLS-Associated Duplication Types The size and genomic content of 74 PTLS-associated 17p rearrangements are shown. Duplicated intervals are shown as red horizontal bars. Above, for reference, is a 17p karyogram and a horizontal line showing the short arm of chromosome 17 with cytogenetic bands depicted above and Mb genomic coordinates below. The open circle denotes the centromere. To the left are laboratory identification numbers. The recurrent duplications, both common and uncommon (shown in red), include: (1) the 3.7 Mb common type between distal and proximal SMS-REPs and (2) the 5 Mb uncommon type between LCR17pA and LCR17pD, shown between black vertical dashed lines. Out of these 50 common PTLS duplications, 25 have been described previously. In this study, 25 common PTLS duplications are newly reported, including 2578, 2581, 2592, 2597, 2603, 2634, 2671, 2692, 2708, 2718, 2724, 2728, 2738, 2745, 2748, 2758, 2767, 2789, 2948, 2950, 2953, 2956, 2962, 2995, and 2998. The nonrecurrent PTLS rearrangements have variable duplication sizes (from 0.41 to ∼13.3 Mb). Twelve out of 22 nonrecurrent PTLS rearrangements have complexities, i.e., additional duplication, triplication (blue), and/or deletion (green). The smallest region of overlap (SRO; gray vertical bar) of all of these 74 PTLS rearrangements is 125 kb, which includes the RAI1 gene only. The previous studies ascertaining the PTLS-associated rearrangements by conventional cytogenetic or molecular assays are cited.

Figure 4

Facial Features of the PTLS Subjects with Uncommon Recurrent Duplication and Small Duplication Types (A) Subject 2808 (uncommon recurrent), age 40 years. (B) Subject 2959 (uncommon recurrent), age 7 years. (C) Subject 2811 (small), age 3 years. (D) Subject 2933 (small), age 8 years. The facial features are not strikingly dysmorphic, yet these subjects have similar facial features, including broad forehead, mildly down-slanting palpebral fissures, hypoplastic alae nasi with a long nasal tip that extends below the level of the nares, a relatively smooth philtrum, and mild micrognatia. Ears are normally set and formed, and the head circumference is normal in each subject. Epicanthal folds are present in subjects 2959 and 2811 (unilaterally).