Molecular Cytogenetics BioMed Central

Autism spectrum disorders have been associated with maternally derived duplications that involve the imprinted region on the proximal long arm of chromosome 15. Here we describe a boy with a chromosome 15 duplication arising from a 3:1 segregation error of a paternally derived translocation between chromosome 15q13.2 and chromosome 9q34.12, which led to trisomy of chromosome 15pter-q13.2 and 9q34.12-qter. Using array comparative genome hybridization, we localized the breakpoints on both chromosomes and sequence homology suggests that the translocation arose from non-allelic homologous recombination involving the low copy repeats on chromosome 15. The child manifests many characteristics of the maternally-derived duplication chromosome 15 phenotype including developmental delays with cognitive impairment, autism, hypotonia and facial dysmorphisms with nominal overlap of the most general symptoms found in duplications of chromosome 9q34. This case suggests that biallelically expressed genes on proximal 15q contribute to the idic(15) autism phenotype.


Background
The clinical association of Wilms' tumor, aniridia, genitourinary abnormalities and mental retardation (WAGR) is a contiguous gene deletion syndrome caused by a deletion on the short arm of chromosome 11. The syndrome is caused by haploinsufficiency for the PAX6 gene (causing aniridia) and the WT1 gene (predisposing Wilms' tumor, genital abnormalities and nephropathies). Aniridia is clinically required for the diagnosis [1]. Most WAGR patients are mentally retarded to some extent, and obesity has occasionally been noted, however the genetic causes for these traits have not been elucidated [2][3][4][5]. Recently Xu et al hypothesised that the SLC1A2, PRRG4 and BDNF genes might contribute to the abnormal mental development [6].
Potocki-Shaffer syndrome (PSS) is another gene deletion syndrome caused by a deletion on chromosome 11, but more proximal (11p11.2) than the WAGR deletion. The syndrome is characterized by foramina parietalia per-manga, multiple exostoses and in some cases craniofacial dysostosis and mental retardation. Haploinsufficiency for EXT2 and ALX4 explains exostoses and parietal foramina respectively [7][8][9].
We present here a patient with an 8.5 Mb deletion on chromosome 11 located in the area between the WAGR and PSS deletions (11p13-p12).

Case presentation
A 15-year-old boy was referred to us for cytogenetic studies. He was the first child of unrelated parents. Pregnancy and delivery at term (41 weeks of gestation, birth weight 3220 g and length 50 cm) were normal and uneventful.
The patient is mildly to moderately mentally retarded and attends special school. His face is dysmorphic with a depressed nasal bridge, folded ears (especially on the right side), and a maxillary overbite and bilaterally down slanting eyes with ptosis (figure 1). Further ophthalmologic examination revealed unilateral cataract, astigmatism and myopia (right eye).
In the neonatal period cryptorchidism and hypospadias were noted. Upon surgery the testes were found atrophic as well. Nine years old he began to have seizures that were treated medically. After medication a weight gain from the 75 centile to just beneath the 97 centile (weight-for-height ratio) was noted.
Cytogenetic analyses were performed after obtaining informed consent. Conventional cytogenetic preparations were made from PHA-stimulated peripheral blood. At first a normal male karyotype 46,XY was found, but upon reexamination using R-banding with a quality corresponding to approximately 550 bands an 11p deletion was revealed with some uncertainty. Comparative genomic hybridization (chromosome CGH) showed a 11p13 deletion. Fluorescence in situ hybridization (FISH) analysis was carried out using BACs and fosmid clones (CHORI BACPAC resource http://bacpac.chori.org/order.php). The positions of relevant probes are shown in figure 2. The clones for FISH analysis were labeled with biotin using a nick-translation kit following the manufacturer's protocol (Roche Molecular Biochemicals). The probes were preannealed with Cot1 DNA in hybridization mix, denatured for 5 minutes at 75°C, and added to the denatured chromosomal slides. Hybridization was carried out overnight. Signals were detected using 2-3 rounds of amplification with FITC (fluoresceinisothiocyanate) conjugated avidin and anti-avidin antibodies. The chromosomal slides were counterstained with propidium iodide and DAPI. The chromosomes were viewed using Leica FISH station Q550CW using the DMRXA microscope equipped with appropriate filters. A minimum of 20 metaphases was analyzed. MLPA analysis was performed using the P219 kit from mrc-Holland following manufacturer's instructions. The positions of relevant MLPA probes are shown in figure 2.
FISH analysis using probe B2.1 (WT1, 11p14.1 [10]) showed deletion of Wilms' tumor-locus while FISH analysis using probe FAT5 (aniridia-locus, PAX6 gene, 11p14.1 [10]) showed signals from both chromosomes 11 ( figure 3). A further mapping of the deletion with FISH analysis using BAC and fosmid clones revealed a deletion of approximately 8.5 Mb. This is the maximum size as it is measured from the distal point of the probe juxtaposed to the distal probe deleted (i.e. BAC clone RP10-83G3 juxtaposed to deleted BAC clone RP1-319D17) to the proximal point of the probe juxtaposed to the proximal probe deleted (i.e. fosmid clone G248P8673G5 juxtaposed to deleted fosmid clone G248P89483C8). Thus the distal breakpoint mapped between position 31,803,008 (BAC clone RP10-83G3 not deleted) and position 31,922,410 Patient presenting with depressed nasal bridge, maxillary overbite and bilaterally down slanting eyes with ptosis

Conclusion
The patient presented in this work shows mental retardation, unilateral cataract, bilateral ptosis, genital abnormalities, seizures and a dysmorphic face. It is uncertain whether the obesity observed is a side effect of the treatment for seizures or if it is part of the syndrome. The deletion breakpoints were mapped using FISH and MLPA analyses. The distal breakpoint was mapped between the PAX6 and RCN1 genes, while the proximal breakpoint was mapped to lie either within or proximal to the LRRC4C gene (Figure 2). The deletion encompasses a total of 44 genes or open reading frames, including the WT1 gene, which explains the genital abnormalities observed. As PAX6 is left intact the cataract observed cannot be explained by a deletion of this gene. One explanation may be that regulatory elements of PAX6 is deleted as such elements have been demonstrated [11]. Regulatory elements, located 5' to the Pax6 gene important for lens induction have been identified in mouse [12,13]. These upstream elements were not involved in the deletion in the patient described here, and it is purely speculative that others could exist. Another explanation is that the cataract is due to other genes involved in the deletion or causes unrelated to the deletion.
McGaughran et al. reported a case with a cytogenetic visible 11p deletion (del11(p11.2p14) [14]. This patient exhibited features of both WAGR as well as PSS; developmental delay but no seizures. Bremond-Gignac et al described a further case with 11p deletion encompassing EXT2, ALX4, WT1 and PAX6 genes showing features of both WAGR and PSS [15]. In addition, the patient showed obesity. Recently Xu et al. reported 31 WAGR cases and identified the genes deleted in each case using oligonucleotide array-CGH [6]. Three of these cases had seizures. One patient had an intact PAX6 gene, however, ophthalmologic findings of the patient were not described since the scope of the paper was the mental retardation and autism observed in WAGR patients. Xu et al hypothesizes that SLC1A2 and BDNF contributes to the autism and mental retardation [6]. In the patient presented here the SLC1A2 gene was deleted however the BDNF gene was intact.