View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by DSpace@MIT Human Molecular Genetics, 2017, Vol. 26, No. 19 3699–3712 doi: 10.1093/hmg/ddx255 Advance Access Publication Date: 7 July 2017 Original Article ORIGINAL ARTICLE The 16p11.2 homologs fam57ba and doc2a generate certain brain and body phenotypes Jasmine M. McCammon1, Alicia Blaker-Lee1, Xiao Chen2 and Hazel Sive1,2,* 1Whitehead Institute for Biomedical Research, Cambridge, MA 02142, USA and 2Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA *To whom correspondence should be addressed at: 455 Main Street, Cambridge, MA 02142, USA. Tel: 617 2588242; Fax: 617 2585578; Email: [email protected] Abstract Deletion of the 16p11.2 CNV affects 25 core genes and is associated with multiple symptoms affecting brain and body, including seizures, hyperactivity, macrocephaly, and obesity. Available data suggest that most symptoms are controlled by haploinsufficiency of two or more 16p11.2 genes. To identify interacting 16p11.2 genes, we used a pairwise partial loss of function antisense screen for embryonic brain morphology, using the accessible zebrafish model. fam57ba, encoding a ceramide synthase, was identified as interacting with the doc2a gene, encoding a calcium-sensitive exocytosis regulator, a ge- netic interaction not previously described. Using genetic mutants, we demonstrated that doc2aþ/À fam57baþ/À double heterozy- gotes show hyperactivity and increased seizure susceptibility relative to wild-type or single doc2aÀ/À or fam57baÀ/À mutants. Additionally, doc2aþ/À fam57baþ/À double heterozygotes demonstrate the increased body length and head size. Single doc2aþ/À and fam57baþ/À heterozygotes do not show a body size increase; however, fam57baÀ/À homozygous mutants show a strongly increased head size and body length, suggesting a greater contribution from fam57ba to the haploinsufficient interaction be- tween doc2a and fam57ba. The doc2aþ/À fam57baþ/À interaction has not been reported before, nor has any 16p11.2 gene previ- ously been linked to increased body size. These findings demonstrate that one pair of 16p11.2 homologs can regulate both brain and body phenotypes that are reflective of those in people with 16p11.2 deletion. Together, these findings suggest that dysregulation of ceramide pathways and calcium sensitive exocytosis underlies seizures and large body size associated with 16p11.2 homologs in zebrafish. The data inform consideration of mechanisms underlying human 16p11.2 deletion symptoms. Introduction body function, including autism spectrum disorders (ASDs), in- Identifying multigenic contribution and relevant genetic inter- tellectual disability, developmental delay, language impair- actions contributing to neurodevelopmental disorders is a huge ment, seizures, macrocephaly, obesity, hypotonia, motor challenge facing the field. In these disorders, physical comorbid- impairment, and gastrointestinal problems (2–10). ities are frequently associated with mental health disorders and The 16p11.2 CNV affects 25 core genes, and we considered compound challenges faced by people with these diagnoses. four models to account for the connection between loss of func- Multigenic regions associated with neurodevelopmental disor- tion in core genes and specific phenotypes or symptoms. First, a ders include copy number variants (CNVs). Loss of one copy of single gene from 16p11.2 could be linked to all symptoms the 16p11.2 is the CNV is highly prevalent, occurring in 1 in 2000 (Fig. 1A). This model is not supported by the extensive sequenc- people (1), and is linked to many symptoms affecting brain and ing analysis that has been done for people with autism and Received: December 20, 2016. Revised: June 26, 2017. Accepted: June 29, 2017 VC The Author 2017. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/ licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact [email protected] 3699 Downloaded from https://academic.oup.com/hmg/article-abstract/26/19/3699/3932275 by M I T Libraries user on 10 July 2018 3700 | Human Molecular Genetics, 2017, Vol. 26, No. 19 single genes to mediate 16p11.2 deletion phenotypes, of eight mouse mutants for single homologs, only one (PPP4C) had a haploinsufficient phenotype: retarded growth (26). Some homo- zygous mouse mutants for other 16p11.2 homologs showed phenotypes with potential relevance to CNV deletion symp- toms, including Doc2aÀ/À mice, which exhibit defects in synaptic transmission (27), and Coro1aÀ/À and Mapk3À/À mice, which have some immune deficiencies (28,29). Other mouse studies on 16p11.2 homologs have deleted the entire syntenic interval, and therefore do not address single gene activity (30–32). After com- plete loss-of-function (LOF) in single genes using antisense techniques, we previously found that 22 of 25 homologs were active during early zebrafish development, but only two (aldoaa and kif22) showed haploinsufficient effects on early brain mor- phology (33). A small 16p11.2 screen in Drosophila found that a homolog for KIF22 mediated axonal branching patterns (34). A single study has reported that suppressing and overexpress- ing kctd13 has reciprocal effects on head size in zebrafish, and the suggestion was made that this reflected the opposing effects Figure 1. Possible hypotheses for gene-phenotype relationships in multi-symp- of 16p11.2 CNV deletion and duplication on people (35). Overall, tomatic CNV disorders. (A) A single gene accounts for all of the associated symp- between human genetics and animal data, there are few single toms. (B) The interaction of two or more genes accounts for all of the associated 16p11.2 genes or homologs that regulate distinct symptoms symptoms. (C) Single genes account for a single symptom each. (D) Two or more genes interact to affect one symptom each. These interacting genes can be in after single copy gene deletion, and only a few of the symptoms different and overlapping sets. There are also additional models that would be associated with 16p11.2 CNVs can be associated with composites of the last two hypotheses. single genes. Based on these considerations, we explored the fourth possi- bility outlined above (Fig. 1D), that multiple genes interact to im- schizophrenia. No statistically significant variants for any of the pact each 16p11.2 deletion symptom. We used the zebrafish as a 25 single genes from this region have been recovered (11–15), tool to address the most parsimonious hypothesis that two making it unlikely that a single gene could account for all the 16p11.2 genes interact to elicit each phenotype. We have previ- other associated symptoms as well. Second, two or more genes ously noted the value of the zebrafish as a tool to study neurode- from the 16p11.2 region interact and mediate all symptoms velopmental disorders (23–25,36). Genetic manipulations are (Fig. 1B). Explorations for smaller internal CNVs associated with effective, while the wide range of assays and rapid embryonic de- autism have not recovered significant hits in the 16p11.2 inter- velopment in zebrafish means several phenotypes can be studied val (16–18), but these would not be able to account for multiple simultaneously. Although zebrafish cannot demonstrate human- critical genes if they are not syntenic. Third, single genes from specific phenotypes associated with 16p11.2 CNVs, their genes this region may each mediate a distinct symptom (Fig. 1C). are orthologous to human disease genes (37). This facilitates the Extensive sequence analysis on large samples from people with discovery of the biology that triggers pathological mechanisms. autism has not revealed statistically significant variants for any Beginning with the simplest interaction hypothesis of just two of the 25 single genes from this region (11,12,14,15). However, genes involved in symptomatology, we employed an antisense single genes have been implicated in some of the other symp- pair-wise partial LOF screen and double heterozygote lines. Our toms associated with 16p11.2 CNVs. PRRT2 has been associated, data identify unpredicted 16p11.2 homolog interactions and new as heterozygous or homozygous mutations, with paroxysomal gene functions underlying brain and body phenotypes. These neurological disorders, benign familial infantile seizures, febrile data support the hypothesis that two or more 16p11.2 genes in- convulsions, and epileptic seizures (19). A single case of epilepsy teract to mediate distinct symptoms, and identify lipid metabo- was found in a person with a KIF22 heterozygous mutation (20). lism and calcium-sensitive exocytosis as pivotal elements in TBX6 is a candidate for vertebral anomalies associated with certain 16p11.2 deletion symptoms. 16p11.2 CNVs as hypomorphic/null compound mutations (21,22). However, overall, there are not many single gene associ- ations from the 16p11.2 interval, and these cannot account for Results the full spectrum of symptoms associated with the CNV. A subset of interacting 16p11.2 pairs affect early brain Therefore, we consider a fourth model where interaction of two development and other phenotypes or more genes each mediates one symptom (Fig. 1D). These could include either distinct or overlapping gene groups that in- We were particularly interested in genetic