Background
3q29 deletion syndrome (3q29Del) is a rare (~ 1 in 30,000) [
1,
2] genomic disorder characterized by a 1.6 Mb typically
de novo deletion on chromosome 3 [
3‐
5]. The interval contains 21 distinct protein-coding genes, 3 antisense transcripts, 1 long noncoding RNA, and 1 microRNA. Our understanding of the syndrome phenotype continues to evolve. Initial reports found developmental delay/intellectual disability universal among 3q29 deletion carriers, though some case reports have since identified individuals without cognitive impairment [
6]. The 3q29 deletion is associated with a 20 to 40-fold increased risk for schizophrenia (SZ), with multiple replication studies supporting this association [
7‐
11]. Case reports also indicate other neuropsychiatric phenotypes may exist, including attention-deficit/hyperactivity disorder (ADHD) and bipolar disorder [
3,
4,
12‐
16]. Previous work by our team examining self-report data from 44 individuals with 3q29Del revealed a high prevalence (~ 20%) of generalized anxiety disorder [
5]. Further, case reports have long suggested an association with autism spectrum disorder (ASD), and studies with large sample sizes indicate that the 3q29 deletion may confer a 19-fold increased risk for ASD (
p = 0.001) [
17,
18].
The range of neuropsychiatric manifestations in 3q29Del is consistent with other genomic disorders. For example, the 22q11.2 deletion has a well-known association with schizophrenia but is also associated with intellectual disability (ID), ASD, anxiety, mood disorders, and ADHD [
19,
20]. A similar constellation of phenotypes, including ASD, ADHD, ID, SZ, and anxiety, has been identified in 16p11.2 deletion and duplication syndromes [
21,
22], 7q11.23 duplication syndrome [
23], and 1q21.1 deletion syndrome [
24]. Thus, risk for multiple neuropsychiatric phenotypes appears to be a feature common to many genomic disorders, including 3q29 deletion syndrome.
The present study aims to improve the current understanding of 3q29 deletion-associated neuropsychiatric and neurodevelopmental phenotypes, and ASD in particular, by examining data from comprehensive, standardized questionnaires in the largest cohort of individuals with 3q29Del ever assembled. Developing a clearer and more comprehensive picture of 3q29 deletion-associated phenotypes will aid in management of the syndrome for both families and clinicians, which may in turn improve long-term outcomes. Additionally, a careful description of the phenotypic spectrum of 3q29Del provides a basis for cross-disorder comparison between genomic disorders, which may ultimately create inroads for identifying common mechanisms underlying 3q29Del and similar CNV disorders.
Discussion
Previous studies have found enrichment of the 3q29 deletion in large samples ascertained based on clinical ASD diagnosis [
17,
18]. We have approached the association of 3q29Del with ASD from a different angle; by ascertaining subjects with 3q29Del and investigating the prevalence of reported ASD diagnosis and ASD-related phenotypes, the current study complements the existing literature, providing additional evidence for the 3q29 deletion as a genetic risk factor for ASD. Notably, the male:female ratio of self-reported ASD diagnosis in our study population is 2:1. This is a reduction from the 4:1 male bias observed in idiopathic ASD in the general population. A substantial reduction in male bias in ASD prevalence has been observed in studies of other CNVs and single-gene mutations; a recent study has shown that as the severity of a mutation increases, the sex ratio in ASD prevalence approaches 1:1 [
52]. Taken together, this suggests that the 3q29 deletion is approaching the severe end of the spectrum of ASD-associated mutations.
We have shown that compared to typically developing children, our 3q29Del sample is significantly enriched for ASD features and other behavioral problems, irrespective of a clinical ASD diagnosis. This finding is particularly concerning; while individuals with 3q29Del who have an ASD diagnosis tend to score higher on symptomology scales overall, 3q29Del individuals without an ASD diagnosis still score significantly higher than typically developing children. This indicates several possible explanations: (a) an enrichment for ASD features or social disability that falls short of diagnostic criteria, (b) possible undiagnosed ASD in our study population, or (c) non-specificity of the SRS, and potentially SCQ, for phenotypes other than ASD, such as anxiety. The possibility of undiagnosed ASD in our study population is aligned with anecdotal reports from parents of our study participants, where they have reported concerns about atypical social development that do not appear to have been addressed using gold-standard ASD evaluations. Based on the elevated symptomology scores in our study population, the substantially increased risk for ASD associated with the 3q29 deletion, and the apparent severity of the 3q29 deletion, our data suggest that gold-standard ASD evaluations should be the recommended standard of care for individuals diagnosed with 3q29Del. If implemented, this practice would enable patients to gain access to early interventions, treatments, and therapeutic programs that are known to improve later outcomes.
Based on the SRS sub-scales, participants with 3q29Del display a strikingly different behavioral profile as compared to a study of children with idiopathic ASD [
43]. Male and female 3q29Del individuals show substantially less impaired social motivation in the context of an otherwise typical ASD profile, with the most severe deficits in the Restricted Interests and Repetitive Behaviors domain. This profile is also observed when dividing scores for 3q29Del participants based on reported ASD diagnosis. This qualitative difference from idiopathic ASD may serve as an inroad to therapeutic interventions in 3q29Del, as well as an investigative inroad to a distinct subtype of ASD. Because social motivation appears to be relatively well-preserved in 3q29Del, this suggests that therapies such as cognitive-behavioral therapy to teach social skills and effective strategies for social interaction may be particularly successful in this patient population.
Some facets of the difference in ASD features between 3q29Del and idiopathic ASD are recapitulated by the scores on the Withdrawn sub-scale of the CBCL and ABCL. Previous studies utilizing the CBCL in idiopathic ASD have found that mean scores for participants with ASD are in the borderline range, with over 50% of subjects scoring in the borderline or clinical range [
53,
54]. While 3q29Del participants generally, as well as males and females separately, score significantly higher than controls, their mean score is still in the normal range (Additional file
1: Figure S2A and B). However, 60% of 3q29Del participants reporting an ASD diagnosis score in the borderline or clinical range (Additional file
1: Figure S2C and Table S10), which is in line with what is expected based on studies of idiopathic ASD [
53,
54]. This is in conflict with the relatively well-preserved social motivation in 3q29Del individuals with ASD identified in our analysis of the SRS sub-scales and suggests that a more refined analysis is merited to identify the true degree of social disability in this population.
We tested the hypothesis that the score inflation observed in our 3q29Del study subjects may be due to the high prevalence of developmental delay or congenital heart defects [
5]. Our available data do not support this hypothesis and instead reveal that social disability is equally distributed in our study population. Lack of direct measures of intellectual disability, and errors or missing data in self-report measures, may obscure this relationship; however, numerous studies of the relationship between ID and ASD in genomic disorders suggests that when the population is stratified by the presence of a specific genetic variant, the association between these two phenotypes diminishes. A large study of several genetic disorders showed that the prediction of genetic diagnosis based on ADI-R scores was not confounded by IQ [
55], a study of 7q11.23 duplication found that IQ was not significantly associated with ASD status [
56], and multiple studies of 22q11.2 deletion have shown that IQ is not significantly associated with SRS score, ASD severity, and ASD status [
57‐
59]. A question ripe for future investigation is the potential role for microcephaly in the ASD-related phenotypes observed in 3q29Del. Microcephaly, ASD, and ID are associated with the 16p11.2 duplication [
21]; microcephaly has been shown to be associated with ASD and ID in probands with pathogenic CNVs [
60]; and children with “complex autism”, defined as ASD with microcephaly and/or dysmorphology, have significantly lower cognitive function than children with “essential autism” [
61]. Reports have shown a high prevalence of microcephaly in 3q29Del [
3,
4,
12]; however, this question was not probed in the current study due to the high rate (> 50%) of 3q29Del participants responding “Unsure” to the medical history questionnaire regarding their child’s head circumference at birth, rendering this data unreliable. Ongoing studies with direct evaluation of study subjects [
44] will address these questions.
While this study is the most comprehensive study of behavioral phenotypes in 3q29Del to date, it is not without limitations. All of the data used in the present study were collected from questionnaires completed by the parents and guardians of individuals with 3q29Del, which introduces several potential sources of bias. Some studies have questioned the validity and reliability of parent-report data [
62]; however, a recent study in Williams syndrome patients has shown that parents are more accurate in predicting their child’s social behaviors than the child themselves [
63]. The responses to the medical and demographic questionnaire are more likely to include error due to the fact that the data is retrospective. By limiting our study to only a few key points in the medical history (heart defects, age at walking, and ID/ASD diagnosis), we aimed to reduce recall errors; however, we only had proxies for ID, rather than direct evaluation of cognitive ability. Further, the sample sizes for our stratified analyses were small, rendering them underpowered; while the differences between males and females were not statistically significant, males do score higher than females on all measures. Studies with larger sample size will be better able to assess the importance of and estimate the true effect size of any difference between males and females. Additionally, there is likely ascertainment bias within our sample. First, our sample of 93 individuals with 3q29Del is 87.1% white, indicating that we are not adequately reaching minority populations. Second, parents that register their children and complete in-depth questionnaires are likely to be highly motivated, possibly because their children experience significant morbidity—a potential indication that we are sampling from the extreme of the phenotypic distribution of 3q29Del. Thus, scores on the standardized questionnaires, as well as rates of heart defects and clinical neuropsychiatric diagnoses, may be higher in our study sample than in the general 3q29Del population. Additionally, the odds ratios calculated for the increased risk for ASD associated with the 3q29 deletion may also be overestimated, due to the combined effects of self-report data and ascertainment bias; however, if this increased risk is replicated using gold-standard diagnostic measures, it could provide valuable insight into possible sex-specific effects of the deletion. Finally, the lack of observed association between congenital heart defects and neurodevelopmental outcomes may be obscured by the high rate of patent ductus arteriosus in 3q29 deletion syndrome [
5], which is a relatively mild heart defect; however, the low number of participants with different types of heart defects rendered analyses to assess their associations with neurodevelopment underpowered (Additional file
1: Table S11). Ongoing studies by the Emory 3q29 Project (
http://genome.emory.edu/3q29/), including direct in-person patient evaluations [
44] aim to address some of the weaknesses of the present work by performing comprehensive gold-standard evaluations by expert clinicians.
While direct in-person evaluations are the ideal method to corroborate the findings of this study, the low population frequency of the 3q29 deletion and geographic dispersal of our study population (Additional file
1: Figure S3) renders this approach infeasible for a large number of study subjects. However, a small number of 3q29 deletion study subjects have been directly assessed as part of the Emory 3q29 Project (
http://genome.emory.edu/3q29/). We confirm high concordance between registry-leveraged data and gold-standard direct evaluation, as all participants qualifying for an ASD diagnosis based on gold-standard evaluation have clinically significant scores on the SRS and all participants reporting an ASD diagnosis qualified for an ASD diagnosis after gold-standard assessment by the Emory 3q29 Project team (Additional file
1: Table S12). Notably, one participant that did not report a prior diagnosis of ASD received an ASD diagnosis after assessment by our team, supporting our hypothesis that ASD may be underdiagnosed in the 3q29Del population. Five additional participants with a clinically significant SRS score did not qualify for an ASD diagnosis, suggesting that the SRS is not selectively identifying children with ASD in participants with 3q29Del, possibly due to the high rates of reported anxiety in our study population. However, this comparison does suggest that our analysis, though based on self-report data, reveals valid conclusions about behavioral phenotypes in 3q29 deletion syndrome. For genetic syndromes with low population frequencies, data collection through remote means such as online patient registries remains a valuable phenotyping tool.
While the current understanding of the 3q29 deletion is still evolving, there are more well-understood CNV disorders that can be used as a comparison point to determine whether the social disability phenotypes described in this study are distinct to 3q29Del. These include Williams Syndrome (WS, or the 7q11.23 deletion), the reciprocal 7q11.23 duplication, 16p11.2 deletion and duplication, Smith-Magenis Syndrome (SMS), and 22q11.2 deletion. WS is typically associated with hyper-sociability [
64], and patients with WS show more problems with social cognition than with pro-social behaviors [
65], similar to what we have observed in our population of individuals with 3q29Del. However, the prevalence of restricted interests and repetitive behaviors appears to be lower in WS as compared to 3q29Del [
65], and the mean SRS sub-scale Social Motivation score indicates enhanced social motivation in WS as compared to 3q29Del (WS mean T-score = 55.24, 3q29Del mean T-score = 62.1,
p = 0.0005) [
66]. Studies of the reciprocal 7q11.23 duplication showed that parent-reported ASD symptomology via standardized questionnaires was higher than ASD features as assessed by gold-standard instruments, that some probands had been diagnosed with ASD based on delayed speech and social anxiety but did not qualify for ASD via gold-standard measures, that substantially more males than females qualified for an ASD diagnosis, and that 7q11.23 duplication probands were indistinguishable from children with idiopathic ASD on measures of ASD severity and diagnosis status [
56,
67,
68]. This is qualitatively different from our 3q29Del population; all of the participants with a prior ASD diagnosis who were later assessed by the Emory 3q29 Project team had their diagnosis confirmed using gold-standard measures (Additional file
1: Table S12), the male:female ratio in our sample is 2:1, and we see significant differences between 3q29Del cases and idiopathic ASD [
43] on the SRS Social Motivation sub-scale.
Similar to 7q11.23 duplication, ASD probands with 16p11.2 deletion or duplication were indistinguishable from idiopathic ASD probands [
68]; probands with 16p11.2 deletion also have a significantly higher mean SRS score as compared to 3q29Del (16p11.2 mean T-score = 77.8, 3q29Del mean T-score = 71.8,
p = 0.003) [
22], and males with 16p11.2 deletion are at increased risk for ASD compared to females and are overrepresented when cases are ascertained based on neurodevelopmental disorders [
69,
70], indicating a different sex-based ASD risk as compared to 3q29Del. A study of 16p11.2 duplication probands found that scores on the SRS Social Motivation sub-scale were not significantly different from controls and that ASD cases had specific impairments in social cognition and communication [
71]; 3q29Del cases score significantly higher than controls on the SRS Social Motivation sub-scale and do not have substantially higher scores on the Social Cognition or Social Communication sub-scales relative to the other SRS sub-scales.
A recent study of SMS showed that female probands scored higher than males on SRS sub-scales and the sex ratio of ASD was reversed, with more females than males qualifying for a diagnosis [
72], which we do not observe in our 3q29Del study population. Finally, studies of 22q11.2 deletion show some similarities with 3q29Del, including SRS total scores that are not significantly different, high levels of ASD features in the absence of ASD diagnosis, and a male:female ASD ratio of approximately 1:1 [
19,
58,
59,
73]; however, 22q11.2 deletion probands have a significantly lower mean ASSQ score as compared to 3q29Del (22q11.2 mean = 11, 3q29Del mean = 22.2,
p = 0.00004), and 3q29Del cases have significantly higher scores on several CBCL/ABCL sub-scales (Additional file
1: Table S13) [
74,
75]. Taken together, this evidence suggests that while the ASD features in 3q29Del reported in this study share some characteristics with other CNV disorders, the complete constellation of symptoms is discrete from previously described genomic syndromes.
There are significant strengths of this study as compared to previous studies of 3q29Del. First, this is the largest cohort of individuals with 3q29Del ever assembled. This is a critical step in capturing the true phenotypic spectrum associated with the 3q29 deletion. Our use of standardized questionnaires allowed for comparison between ASD features present in 3q29Del and those reported in idiopathic ASD and ASD in other CNV disorders. Additionally, our online patient registry allows for remote data collection, which has enabled us to expand our sample size. This study has shown that high-quality, comprehensive medical history and symptomology data can be collected through an online patient registry, effectively reducing the patient-ascertainment burden associated with studying rare disorders. Taken together, these attributes make the present study an excellent complement to previously published case reports on individuals with 3q29Del; by capturing a larger patient base with systematic assessments, we are able to more accurately measure the presence of a variety of neuropsychiatric and neurodevelopmental phenotypes associated with the 3q29 deletion. The findings reported here indicate that comprehensive neuropsychiatric and neurodevelopmental assessments with gold standard tools are merited for individuals diagnosed with 3q29Del, and that such assessments should be the standard of care for this patient population.
Acknowledgements
We gratefully acknowledge our study population, the 3q29 deletion community, for their participation and commitment to research. This manuscript is currently available on the bioRxiv preprint server (
https://doi.org/10.1101/386243).
We also acknowledge the contributions of the members of the Emory 3q29 Project: Hallie Averbach, Emily Black, Gary J Bassell, T Lindsey Burrell, Grace Carlock, Tamara Caspary, Joseph F Cubells, David Cutler, Paul A Dawson, Roberto Espana, Michael J Gambello, Katrina Goines, Henry R Johnston, Sookyong Koh, Elizabeth J Leslie, Longchuan Li, Bryan Mak, Tamika Malone, Trenell Mosley, Derek Novacek, Ryan Purcell, Timothy Rutkowski, Rossana Sanchez, Jason Schroeder, Esra Sefik, Brittney Sholar, Sarah Shultz, Nikisha Sisodiya, Steven Sloan, Elaine F Walker, Stephen T Warren, David Weinshenker, Zhexing Wen, and Mike Zinsmeister.