Disruption of the ProSAP2 gene in a t(12;22)(q24.1;q13.3) is associated with the 22q13.3 deletion syndrome

Abstract

The terminal 22q13.3 deletion syndrome is characterized by severe expressive-language delay, mild mental retardation, hypotonia, joint laxity, dolichocephaly, and minor facial dysmorphisms. We identified a child with all the features of 22q13.3 deletion syndrome. The patient's karyotype showed a de novo balanced translocation between chromosomes 12 and 22, with the breakpoint in the 22q13.3 critical region of the 22q distal deletion syndrome [46, XY, t(12;22)(q24.1;q13.3)]. FISH investigations revealed that the translocation was reciprocal, with the chromosome 22 breakpoint within the 22q subtelomeric cosmid 106G1220 and the chromosome 12q breakpoint near STS D12S317. Using Southern blot analysis and inverse PCR, we located the chromosome 12 breakpoint in an intron of the FLJ10659 gene and located the chromosome 22 breakpoint within exon 21 of the human homologue of the ProSAP2 gene. Short homologous sequences (5-bp, CTG[C/A]C) were found at the breakpoint on both derivative chromosomes. The translocation does not lead to the loss of any portion of DNA. Northern blot analysis of human tissues, using the rat ProSAP2 cDNA, showed that full-length transcripts were found only in the cerebral cortex and the cerebellum. The FLJ10659 gene is expressed in various tissues and does not show tissue-specific isoforms. The finding that ProSAP2 is included in the critical region of the 22q deletion syndrome and that our proband displays all signs and symptoms of the syndrome suggests that ProSAP2 haploinsufficiency is the cause of the 22q13.3 deletion syndrome. ProSAP2 is a good candidate for this syndrome, because it is preferentially expressed in the cerebral cortex and the cerebellum and encodes a scaffold protein involved in the postsynaptic density of excitatory synapses.

Figure 1

A (top), Cut-out of normal chromosomes 12 and 22, aligned with their homologue translocation derivatives der(12) and der(22) in G-banding at a resolution of ⩾550 bands. A (bottom), Corresponding high-resolution G-banding ideograms of chromosomes 12 (green) and 22 (red) and their derivatives der(12) (green with a small red portion) and der(22) (red with a green portion). Arrowheads indicate the breakpoints in subbands 12q24.1 and 22q13.3. The reciprocal exchange of chromosome material involves a larger region from chromosome 12 than from chromosome 22 (see ideogram); no 22q material is visible on der(12), when examined by cytogenetics or FISH with a 22-painting library. B, Results of FISH with the 22q subtelomeric probe 106G1220 (VYSIS). The normal chromosome 22 (arrow) and both der(12) and der(22) (arrowheads) show a signal demonstrating the translocation's reciprocity, with the chromosome 22 breakpoint within probe 106G1220.

Figure 2

A, Genomic structure of human ProSAP2 and FLJ10659 genes. The left-to-right orientation of both maps is from centromere to telomere. Sizes of exons (boxes) and introns are drawn to scale. Vertical arrows indicate the breakpoint’s location on both chromosomes. Horizontal lines (A, B, and C) indicate the positions and sizes of the restriction fragments isolated from cosmid n85a3 and used as Southern hybridization probes. B, Southern blot analysis. Genomic DNA (10-μg) from the proband (P), his mother (M), and father (F), digested with EcoRV (V), EcoRI (E), or HindIII (H), was loaded in each lane. C, Selective amplification of der(12) and der(22) in the proband. Genomic DNA from the proband and two normal subjects, as well as a negative control sample that contained no DNA, was amplified with primer pairs spanning the breakpoints on chromosome 12, 22, der(12) (12/22), and der(22) (22/12). D, Alignment of chromosome 12, 22, der(12) and der(22) sequences at the breakpoint. Chromosome 12–derived sequence is shown in lowercase letters, and chromosome 22–derived sequence is shown in uppercase letters. Breakpoints are indicated by arrowheads. The CTG(C/T)C sequence at the breakpoint on both chromosome 12 and 22 is shown in bold. The orientation of sequences is as in panel A.

Figure 3

A, Expression of ProSAP2 and FLJ10659. Commercial northern blot analysis was performed using 2 μg per lane of poly-A+ RNA from human tissues (panels a and c), and human brain tissues (panels b and d). The filters were hybridized to rat ProSAP2 cDNA (panels a and b) or IMAGE clone 3450646 (panels c and d). Filters a and b were exposed for 2 w, and filters c and d were exposed for 40 h. B, PCR analysis of human ProSAP2 expression. Groups of exons spanning most of the gene’s coding region (exons 1/4, 10/12, 10/16, 16/21, and 21/22) were amplified from human brain (b), cerebellum (c), and heart (h) cDNAs using a nested PCR protocol. Negative control amplifications (−) were always included. Human β-actin cDNA was used as a control, and amplified at similar levels in all tissues. Some products (exons 1/4, 10/12, and 21/22) were visible after the primary amplification (PCR 1), but others (exons 10/16 and 16/21) required a secondary amplification (PCR 2). All amplified fragments were isolated and sequenced. The smallest fragment (200 bp) from the secondary amplification of exons 10/12 from brain tissue does not contain exon 11. The 540-bp fragment from the secondary amplification of exon 10/16 from brain tissue does not contain exons 11 and 12, but the 600-bp fragment from heart in the same panel was amplified from genomic DNA. The 128-bp fragment from the primary amplification of exons 21/22 from brain tissue does not contain exon 21b. All other fragments have the expected sequence.