The aim of this study was to evaluate a serially ascertained sample of children and adults with SHANK3 deletion or mutation and provide an overview of diagnostic, medical and psychological presentations to guide future research on this syndrome. The results present a picture of SHANK3 deficiency as a disorder characterized by ID, ASD, predominance of gross motor impairments including gait disturbance and hypotonia, and severe speech delays. Approximately half of the sample also reported a history of abnormal EEG findings and gastroesophageal reflux disease on medical record review.
Patterns of dysmorphic features were not observed in our sample. While all patients presented with at least one dysmorphic feature, the most common feature, large fleshy hands, was only present in 53%. It is also important to note that hand size was within height expectations for the sample and, as such, this feature may represent appearance rather than actual size differences. Other commonly observed dysmorphic features included bulbous nose, long eyelashes and ear anomalies. Our sample also did not present with a pattern of accelerated growth reported in previous studies [21, 22, 33, 49]. These results are consistent with a recent study suggesting that the majority of children with SHANK3 deficiency have normal growth .
The integration of careful clinical evaluation, caregiver report and direct structured observations used in autism research has been shown to increase the validity of ASD diagnoses . To date, published studies have not utilized standard research methods for assessing the presence of ASD in SHANK3 deficiency. Using the recommended diagnostic methodology, our study found rates of approximately 84% for all ASDs, with rates of 75% for strictly defined autistic disorder and 9.3% for subthreshold symptoms consistent with ASD on the DSM-IV. Rates of autism in our sample were higher than studies relying only on clinical judgment or parent report, which report rates ranging from 26%  to 54% . By contrast, studies utilizing at least one standardized assessment tool report rates comparable to the present study. Using the Childhood Autism Rating Scale , an investigator rated instrument, Phelan et al.  found 94% of their sample met criteria for an ASD and Jeffries and colleagues  found a rate of ASD of 85% using the Social Communication Questionnaire , a caregiver report screening measure.
Autism-specific diagnostic evaluation tools have been reported to possess limited specificity in individuals with severe ID [35, 46]. Our findings suggest discrepancies between ADOS-G and ADI-R results for 13 out of 32 participants. In 4 out of 13 patients, the ADI-R yielded an autism classification when both ADOS-G and clinical diagnosis did not support the presence of ASD. In 8 out of 13 cases, the ADI-R did not yield an autism classification as a result of subthreshold levels of repetitive behaviors. Overall, the ADOS-G and DSM-IV clinical diagnoses likely provide a more representative picture of the social communication and repetitive behavior profiles presenting in children with SHANK3 deficiency and ASD. As previously mentioned, the ADI-R has less validity in populations with chronological and mental ages below 18 months. In our sample, the mean language age equivalence scores were below 12 months and motor age equivalence scores were below 21 months, suggesting a level of ID which may present diagnostic challenges when using the ADI-R.
Importantly, social communication impairments, and repetitive behaviors or restricted interests were observed in this sample of individuals with SHANK3 deficiency. The most common social communication impairments included social engagement, reciprocity and play. Our results also indicated a significant association between deletion size and scores on social and communication domains of the ADI-R. This finding suggests that the severity of the behavioral phenotype is associated with larger deletion sizes and runs in contrast to results from Sarasua and colleagues , who found that individuals with smaller deletion sizes were more likely to have an ASD. However, the presence of ASD in that study  was based on parental report and only 26% of the sample was identified as having ASD.
Previous research had suggested a low rate of repetitive behaviors in individuals with 22q13 deletion syndrome . In the repetitive behavior and restricted interest domain of the ADI-R, over half of our sample displayed intense sensory interests and repetitive toy or object play, which are commonly found in individuals with ID alone. However, approximately one-third of the sample also reported compulsive behaviors such as adhering to rigid routines. Further evaluation of repetitive behaviors and restricted interest domains may be an important direction for future research, particularly given the discrepancy between repetitive behaviors reported on the ADI-R and clinical evaluations, as well as the high rates of sensory-related behaviors observed in this sample of individuals with SHANK3 deficiency. Increasing sample size and improving specificity of measurements of the repetitive behaviors will be important in reconciling published reports.
The severity of the phenotype in SHANK3 deficiency is highlighted by the results of the standardized evaluations of cognitive, language and functional skills in this study. No participants used phrase speech on a daily basis, and only five used single words to communicate consistently. Scores from direct testing of language and cognitive skills, as well as parent reports on the Vineland, reflect the severe language disability found in most individuals with SHANK3 deficiency. One of 32 participants had a nonverbal IQ estimate in the average range, with approximately three-quarters of participants performing in the severe to profound range of ID.
The floor effects observed on standardized testing did not allow for comparison of differences within domains (for example, receptive or expressive language). While standardized tests allow comparison of the phenotype relative to general population norms, available tools are hampered by a restricted range in individuals with ID. As such, traditional methods are likely insufficient for the purposes of treatment development or progress evaluations. Development of evaluation tools designed to capture precursors of language and early cognitive signals, such as auditory discrimination and visual attention, are needed in future investigations of SHANK3 deficiency.
One significant pattern of results relates to the prevalence of motor impairments found across evaluation methods. Motor disabilities were identified in the earliest clinical profiles of SHANK3 deficiency and their predominance is supported by findings in our sample. The comprehensive clinical evaluation suggests hypotonia as a dominant, early presenting clinical feature, present in 100% of the 16 children who underwent neurological examinations in our sample and described by parents in 75% of the total 32 patients. Motor impairments due to hypotonia may result in feeding difficulties, which are commonly reported as one of the first signs noticed by families in infancy. The neurological evaluation in this study also found high rates of gait abnormalities. Gross motor impairments were moderately associated with deletion size, albeit slightly below the threshold of significance.
Other common clinical features in our sample included gastroesophageal reflux disease, seizures, sleep disturbance, renal abnormalities, and constipation and/or diarrhea. The presence of recurring ear and upper respiratory tract infections was notable and may reflect poor airway protection and sputum clearance due to low muscle tone in SHANK3 deficiency. It is also possible that SHANK3 may play an important role in immune function. Immunocytochemical analyses in rats have verified the presence of Shank3 proteins in thymic tissue and suggest a role of Shank3 in the coordination of immune cell signal transduction . Previous case reports have also suggested a link between immune dysfunction and SHANK3 deficiency [55, 56]. Our findings suggest an increased prevalence of recurring infections in our sample of patients with SHANK3 deficiency and its relationship to SHANK3 and neighboring genes is an area that should be more carefully explored in the future.
Twenty-eight of our patients had MRIs performed and, among them, 75% had abnormal findings based on clinical reports. The prevalence of structural brain abnormalities in our sample highlights the paucity of data on the abnormal neural systems underlying this syndrome. Understanding how brain structure is affected in SHANK3 deficiency will be important for future studies. Given that little is known about the neurobiology associated with SHANK3 deficiency, identification of specific brain abnormalities will aid in more thorough characterization and may provide a critical link between SHANK3 deficiency and associated behavior.
Significant regression in motor and social skills has also been reported among patients in our sample and in the literature [2, 5, 30, 57, 58]. Isolated reports have emerged recently describing significant medical complications of SHANK3 deficiency in the context of severe cognitive and behavioral regression, including seizure-induced aspiration , renal failure  and pneumonia . In addition, recent reports of psychiatric comorbidity in individuals with SHANK3 deficiency suggest that, as patients age, they may be at increased risk for bipolar disorder [30, 57, 59]. Our results suggest that regression may occur at various ages, across multiple domains, and is often associated with the onset of seizures. Future studies examining the natural history of SHANK3 deficiency should clarify the nature and extent of regression.
Several studies have examined genotype-phenotype correlations in SHANK3 deficiency and results are inconsistent [21, 23, 25, 26, 28, 29, 60]. The genotype-phenotype correlations in this study are consistent with at least two previous reports [28, 29], indicating a correlation between deletion size and more severe phenotypes. In our sample, larger deletion sizes were associated with a greater number of dysmorphic features and medical comorbidities. In addition, a correlation between larger deletion sizes and social communication impairments associated with autism was found in the present study using standardized research administrations of the ADI-R. Nonverbal IQ, fine motor skills, repetitive behaviors and language abilities did not show an association with size of deletion in the 22q13 region. The associations identified in our analysis are limited by several factors, including sample size. However, should a clear pattern of genotype-phenotype correlations emerge in larger samples, it may delineate a role for other genes and pathways in SHANK3 deficiency. Replication of potential associations between deletion size and medical comorbidity, social impairments, and possibly motor skills, would also provide valuable information for medical monitoring and treatment planning.