Patients with the 22q13.3 deletion syndrome have a common phenotype characterized by developmental delay, including gross motor delays and absent or delayed speech, hypotonia, and craniofacial dysmorphic features. Clinical features of the patients in the present study are similar to that of previous reports () except for the lower prevalence of hypotonia in the present report. As established in previous reports, the present data also suggest that developmental delay is an almost universal feature of the 22q13.3 deletion syndrome. Behavioral problems such as impaired social skills, persistent mouthing, and chewing of nonfood items, inattentiveness, and hyperactivity were found in 12 of 13 (92%) patients. Sleep disturbances were also seen in 6 of 13 (46%) of the patients. A common association of the 22q13.3 deletion syndrome with autism spectrum disorders has begun to emerge, which is also supported in this cohort although only five patients received a formal neuropsychological evaluation. The first reported association between autism and 22q13.3 deletion was published in a 14-year-old girl [Goizet et al., 2000
Autism spectrum disorders are etiologically heterogeneous and are associated with a recognizable cause in only 10% of patients, most commonly with fragile X syndrome, tuberous sclerosis, and cytogenetically detectable chromosomal anomalies. Mutations in the neuroligin genes (NLGN3
) on the X chromosome [Jamain et al., 2003
] and SHANK3
on chromosome 22q13 [Durand et al., 2007
] are monogenic causes of autism spectrum disorders. In one study of 400 unrelated subjects with autism spectrum disorder, one de novo mutation and two gene deletions of the SHANK3
gene were discovered, contributing 0.75% causality to this cohort [Moessner et al., 2007
]. However, in another recent study by Philippe et al. 
autism was suspected in seven of eight patients with the 22q13.3 deletion syndrome. These patients did not fulfill DSM-IV criteria for autism. Their relationship pattern, language development, and nature of repetitive behaviors were distinct from autism. In the patients described here, two of five (40%) patients who underwent neuropsychological evaluations were diagnosed with autism. This is similar to previous studies [Cusmano-Ozog et al., 2007
Two of the patients reported here were found to have a ring chromosome 22 (Patients 11 and 12). r(22) is assumed to arise from breakage and subsequent fusion of both chromosome arms to generate a ring chromosome with concomitant loss of terminal short and long arm sequences. A review of 17 patients with r(22) showed that clinical features are similar in patients with simple 22q13 deletion and r(22) patients when they are of similar ages and similarly sized deletions [Luciani et al., 2003
]. The features common in both conditions are global developmental delay with severe speech delay, hypotonia, macrotia, epicanthal folds, and small toenails. Patient 12 showed a degree of growth failure which is similar to previously published reports of patients with r(22). These patients are known to exhibit growth failure rather than accelerated growth as seen in 22q13.3 deletion syndrome [Hunter et al., 1977
; Jeffries et al., 2005
Historically, FISH and chromosome G-banding have been commonly used to demonstrate the deletions in this syndrome [Doheny et al., 1997
]. Koolen et al. 
used aCGH on nine patients with subtelomeric deletions of 22q and reported deletion sizes ranging from 3.3 to 8.4 Mb. At that time, they were unable to demonstrate a deletion–phenotype correlation. They used a tiling resolution array with an average clone spacing of 100 kb. However, minimal coverage across the most terminal segment of 22q precluded identification of disruptions within or in the immediate vicinity of the SHANK3
gene, the haploinsufficiency of which has been established to be associated with the neurological phenotype of patients with the 22q13.3 deletion syndrome. It is clear that aCGH analysis offers higher resolution compared to microscopic cytogenetics for molecular characterization of patients with this phenotype.
All the patients described here had terminal deletion of the 22q13 region and in all of them, either all or a part of the SHANK3
gene was found to be deleted. The deletion sizes ranged from 95 kb (Patient 1) to 8.55 Mb (Patient 12). The two patients with the smallest deletion had distinct phenotypes with Patient 1 showing more severe behavioral issues compared to Patient 2. This is an example of the difficulty in making genotype–phenotype correlations in patients with the 22q13.3 deletion syndrome. One hypothesis is that epigenetic factors may play a role in the clinical features of this syndrome. Alternatively, sequence variations in alleles on the nondeleted chromosome may have qualitative and/or quantitative effects on their expression that is unmasked by deletion of their paired allele [Ching et al., 2005
]. Three patients (Patients 7–9) had a similar sized ~5.5 Mb deletion. All three patients had feeding problems while two of three had behavioral issues, seizures, and recurrent infections. As more such patients are reported with exact deletion sizes and breakpoints, a genotype–phenotype correlation could emerge, possibly delineating the role of other genes contributing to the 22q13.3 phenotype. A recent report by Wilson et al. 
of two patients with interstitial deletion of the 22q13 region with intact SHANK3
indicates that haploinsufficiency for other 22q13 genes could also have major effects on cognitive and language development. More studies are required to explore the interaction of these proximal genes and their role in the neurological phenotype of this syndrome.
Approximately 90 genes are deleted in the largest sized deletion found in Patient 12, whereas the smallest deletion segment contained the three genes, SHANK3, ACR, and RABL2B (). Some of the genes (besides SHANK3) responsible for clinically significant disorders that are present in this interval include ARSA (metachromatic leukodystrophy), TYMP (mitochondrial neuro-gastrointestinal encephalomyopathy), MLC1 (megalencephalic leukoencephalopathy with subcortical cysts), ALG12 (congenital disorders of glycosylation 1g), ATXN10 (cerebellar ataxia), UPK3A (renal adysplasia), and SULT4A1 (susceptibility to schizophrenia).
We hypothesize that there may be as yet uncharacterized genes in the vicinity of SHANK3
, SNP, or other variants within the regulatory sequences of SHANK3
itself, or modifier effects of unlinked genes that qualitatively or quantitatively affect its expression. If the deletion breakpoint is located within these regulatory genes, expression of SHANK3
could be altered leading to a clinical phenotype. Point mutations in SHANK3
, just as in the case of the neuroligin genes, are rare in patients with autism. Micro-deletions and disruptions of SHANK3
, however, provide the most convincing evidence for a role in autism. It is possible that similar microdeletions will identify additional genes that may contribute to the autism phenotype [Jacquemont et al., 2006
]. Such additional candidates may include genes that are involved in the growth and development of the pre- and postsynaptic compartments.
In summary, this study illustrates the use of aCGH in delineating deletion breakpoints in the 22q13.3 deletion syndrome and highlights the utility of aCGH as a diagnostic tool for analysis of patients with developmental delay, unexplained hypotonia, and autism spectrum disorder with or without craniofacial dysmorphism. We anticipate that as more patients are diagnosed with the 22q13.3 deletion syndrome using aCGH, a clearer picture of genotype–phenotype correlation may emerge based on the variety of the deleted genes and the differing sizes of the deletions.