Characterization of Potocki-Lupski syndrome (dup(17)(p11.2p11.2)) and delineation of a dosage-sensitive critical interval that can convey an autism phenotype

Abstract

The duplication 17p11.2 syndrome, associated with dup(17)(p11.2p11.2), is a recently recognized syndrome of multiple congenital anomalies and mental retardation and is the first predicted reciprocal microduplication syndrome described--the homologous recombination reciprocal of the Smith-Magenis syndrome (SMS) microdeletion (del(17)(p11.2p11.2)). We previously described seven subjects with dup(17)(p11.2p11.2) and noted their relatively mild phenotype compared with that of individuals with SMS. Here, we molecularly analyzed 28 additional patients, using multiple independent assays, and also report the phenotypic characteristics obtained from extensive multidisciplinary clinical study of a subset of these patients. Whereas the majority of subjects (22 of 35) harbor the homologous recombination reciprocal product of the common SMS microdeletion (~3.7 Mb), 13 subjects (~37%) have nonrecurrent duplications ranging in size from 1.3 to 15.2 Mb. Molecular studies suggest potential mechanistic differences between nonrecurrent duplications and nonrecurrent genomic deletions. Clinical features observed in patients with the common dup(17)(p11.2p11.2) are distinct from those seen with SMS and include infantile hypotonia, failure to thrive, mental retardation, autistic features, sleep apnea, and structural cardiovascular anomalies. We narrow the critical region to a 1.3-Mb genomic interval that contains the dosage-sensitive RAI1 gene. Our results refine the critical region for Potocki-Lupski syndrome, provide information to assist in clinical diagnosis and management, and lend further support for the concept that genomic architecture incites genomic instability.

Figure 1.

Identification of the common duplication and differentiation from unusual-sized duplications by PFGE analysis. An ∼1.1-Mb novel junction (jct) fragment was predicted for the common duplication after the genomic DNA was digested with NotI, was separated by PFGE, and was probed with a genomic fragment from the CLP gene. An asterisk (*) indicates the subjects who harbor this junction fragment. Junction fragment data from subject 1192 was reported elsewhere. Subject 2555 is shown in both gel segments. The ∼1.1-Mb junction fragment was not observed in the parents, which is consistent with a de novo event. A junction fragment was not observed in 13 of the 35 subjects, indicating that those 13 have unusual-sized duplications.

Figure 2.

A, Array CGH analysis of patients 2571 and 2543. A combined result from two dye-swap experiments was presented with normalized log₂(Cy3/Cy5) ratios of patient versus control for each individual clone plotted on the Y-axis and represented by dots with SD. Patient 2571 has the common duplication spanning a region from the distal to the proximal SMS-REP. Patient 2543 harbors a duplicated genomic region smaller in size than the common recurrent dup(17)(p11.2p11.2). The size of the duplicated genomic interval is ∼1.3 Mb, spanning from the distal SMS-REP to a site telomeric to the middle SMS-REP. Since patient 2543 exhibits the key phenotypic features of PLS, this ∼1.3-Mb duplicated interval represents the critical region. The 14 genes, including RAI1, contained within this critical interval are listed. The regions of the critical duplication and the common duplication are indicated by thick horizontal lines. The BACs in the vicinity of the breakpoints are indicated by short black lines. Note that one clone, CTD-457L16, located in a CNV region between the middle and the proximal SMS-REPs showed increased copy number. B and C, FISH analyses of interphase nuclei, which detected the breakpoints in subject 2543. BAC probes used in FISH analysis are indicated by red or green circles. In panel A, the probes are differentially labeled to give a green signal for RP11-209J20 and a red signal for RP11-416I2. In panel B, the presence of three red signals (RP11-416I2) and two green signals (RP11-209J20) indicate that the distal breakpoint maps within the distal SMS-REP. In panel C, the presence of three red signals (RP11-258F1) and two green signals (RP11-189D22) indicate that the proximal breakpoint maps distal to the middle SMS-REP. D–H, Common dup(17)(p11.2p11.2) detected by a FISH assay. D, Schematic representation of a dual-color interphase FISH assay developed to screen for common versus unusual duplications. A map of chromosome 17p11.2 with the placement of FISH probes is above. The SMS-REP–flanking clones are differentially labeled and detected with green and red dyes, respectively. The presence of three red signals indicates that the breakpoints map between RP11-209J20 and RP11-416I2 (E) and between CTD-2010G8 and RP11-98L14 (F). G and H, FISH analysis of interphase nuclei of patient 2555 to confirm the common duplication. The presence of three red signals (RP11-416I2 and CTD-2010G8) and two green signals (RP11-209J20 and RP11-98L14) indicates that the breakpoints map within the distal (G) and proximal SMS-REPs (H). Cen = centromere; dup = duplicated chromosome 17; nl = normal chromosome 17.

Figure 3.

Pedigrees and haplotypes of 18 Houston families (HOU). Patient numbers are given below the blackened symbols representing the affected children in the pedigree. A, Eight families with common recurrent proximal 17p duplications. B, Ten families with uncommon nonrecurrent proximal 17p duplications. To the left of each pedigree is a list of microsatellite markers used for genotyping. The sizes of each allele are written below each family member. The dotted lines outline alleles inherited by the patients from the parent of origin.

Figure 4.

FISH analyses of interphase nuclei for mapping of breakpoints. Above is a map of proximal 17p, with the locations of FISH probes indicated by arrows. A–F, Centromeric breakpoints in patients 2488 (A and B), 2362 (C and D), and 2211 (E and F) mapped within the pericentromeric region. The probes used are listed below interphase nuclei. The presence of one signal in addition to the normal two signals indicates duplication in that region. Duplication was observed for BAC probe RP11-746M1 in 17p11.2. FISH using centromeric probe D17Z1 and BAC probe RP11-22N2 in 17q11.1 indicated no duplication in either the centromere or 17q. G, FISH with the proximal SMS-REP flanking clones CTD-2010G8 (red) and RP11-98L14 (green), mapping the proximal breakpoint in patient 2440 in the proximal SMS-REP. H–J, Two separate duplications in proximal 17p in patient 2153, confirmed by FISH. Duplicated chromosome 17 is indicated by arrows. Cen = centromere.

Figure 5.

Determination of duplicated regions by array CGH analysis. A diagram of proximal 17p and important architectural features are shown at the top with the centromere (white circle), SMS-REPs (gradient boxes), and CMT1A-REPs (white boxes) marked. Horizontal arrows depict orientation of the LCRs. Normalized log₂(Cy3/Cy5) ratios of patient versus control for each individual clone are plotted on the Y-axis. The patients are arranged by size of duplication.

Figure 6.

Summary of breakpoint mapping in 13 patients with uncommon duplications of 17p11.2. Top, Schematic representation of the short arm of chromosome 17 with LCRs depicted. The clones used in the array analysis are depicted just below the diagram of the LCRs and are ordered from the telomere (left) to the centromere (right). The positions of some individual large insert clones are indicated by dashed lines. Below the clones are 13 horizontal bars labeled with individual patient numbers. Blue depicts regions with normal dosage, red represents duplicated genomic segments, and green represents deletion. The sizes of the duplicated or deleted segments are labeled just above each subject bar. The asterisk (*) adjacent to patients 621, 2153, and 2337 indicates that these subjects harbor a complex chromosomal rearrangement including the proximal 17p duplication. Cen = centromere.

Figure 7.

Facial features of PLS. A–J, Patients with the common duplication. A, Patient 2555, at age 22 mo. B, Patient 2306, at age 33 mo. C, Patient 2167, at age 3 years 3 mo. D, Patient 1671, at age 3 years 9 mo. E, Patient 1579, at age 4 years 2 mo. F, Patient 2414, at age 9 years 6 mo. G, Patient 1006, at age 14 years 2 mo. H, Patient 1618, at age 14 years 6 mo. I, Patient 990, at age 16 years 1 mo. J, Patient 1913, at age 13 years. Shared features include a broad forehead, gentle down-slant of the palpebral fissures, and relatively long nasal tip. Younger patients have a triangular face with prominence to the angle of the jaw and micrognathia. A more oval-shaped face and larger chin is seen in older individuals. Patients 1579 (E) and 1913 (J) were microcephalic and hyperteloric. Patient 1913 has more pronounced facial dysmorphisms, including down-slanting palpebral fissures, low-set and posteriorly rotated ears, and broad mouth. An interesting feature shared by most patients is an asymmetric smile that is seen in multiple photographs taken of each patient in the GCRC and in photographs shared by their parents. K, Patient 2543, who harbors a small (∼1.3-Mb) duplication, at age 8 years and 1 mo. L, Patient 2211, who harbors a large (8.2-Mb) duplication, at age 4 years and 10 mo. Physical features seen in these patients are very similar to those of patients with the common duplication.