**4. Animal models for human 16p11.2 CNVs**

Mouse models were generated through a chromosome engineering approach for the study of human 16p11.2 deletion and duplication CNVs [29]. It was observed that ~50% of mice harboring the deletion CNV die shortly after birth, while duplication mice sur‐ vive to adulthood, suggesting that the deletion CNV results in a more severe phenotype than the duplication [29]. A similar phenomenon has been observed in other genomic disorders caused by reciprocal CNVs, including Smith-Magenis and Potocki-Lupski syn‐ dromes [15]. Expression of the genes within the 16p11.2 region corresponds to gene dos‐ age in four brain regions that may be relevant for autism, including the olfactory bulbs, cortex, cerebellum, and brainstem [29].

Another study was able to identify homologs of 21 of the known 16p11.2 human genes in the zebrafish genome by family tree comparisons [58]. These genes were then targeted for loss of function studies by injecting antisense morpholino oligonucleotides into early em‐ bryos [58]. Interestingly ~79% of the genes tested by this method were required for proper brain, eye, or nervous system development, and two of the genes were determined to be dosage-sensitive, with abnormal phenotypes present with a ~50% reduction in gene expres‐ sion [58]. The results of this study suggest that at least two genes, aldolase a (*aldoaa*) and ki‐ nesin family member 22 (*kif22*), are highly dosage-sensitive and are required for proper brain function, making them likely candidates for future studies of the ASD associated with

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These two studies indicate that while *kctd13*, *kif22*, and *aldoaa* are all potentially interesting dosage-sensitive candidate genes, further investigation is needed to determine whether these genes act together in a epistatic manner to contribute to the full neurological pheno‐ type, whether they modify each other via *cis* interactions, or whether other genes or genetic elements in the 16p11.2 locus are also contributing to the phenotype. The current data, which could not have been obtained without these important studies in model organisms, cannot distinguish between these possibilities, but they provide a starting point for research into the function of these genes and the molecular pathways underpinning the phenotypes

Chromosome 15q11-13 is enriched for LCRs, providing a mechanism for LCR-mediated NAHR, and generating a series of recurrent breakpoints along this chromosome. As a result, interstitial deletion or duplication of this region is common. LCRs can also mediate triplica‐ tions, or, alternatively, the presence of supernumerary isodicentric chromosome 15 (idic(15)) can lead to crossing over between these LCRs, and ultimately, duplication of the region. A bipartite imprinting center lies at this locus and directs the expression of a number of genes, resulting in a tissue-specific parent-of-origin effect. As a result, many of the phenotypes

Paternally- or maternally-inherited deletions of human chromosome 15q11-13 result in Prader-Willi syndrome (PWS) or Angelman syndrome (AS), respectively. Alternatively, these disorders can be caused by uniparental disomy (UPD), or by balanced translocations involving this region. Less frequently, imprinting errors, leading to aberrant methylation of the PWS imprinting center can also cause PWS, and mutations or deletions in the gene *UBE3A* can cause AS [11, 59]. The critical region for AS lies 35 kb telomeric to the PWS criti‐ cal region [60]. PWS is characterized by intellectual disability, hypotonia, hyperphagia, obe‐ sity, compulsive and repetitive behaviors, skin picking, tantrums, irritability. In addition, congenital abnormalities are often observed, including hypogonadism, facial dysmorphism, and small hands and feet, among others. PWS can also be associated with psychosis, mood

caused by these structural rearrangements also display parent-of-origin effects.

CNV of this region [58].

associated with 16p11.2 CNVs.

disorders, and ASD [61].

**5. Prader-Willi and Angelman syndromes**

In-cage neurobehavioral phenotypes were assessed in these mice to determine what, if any, affect these CNVs had on autistic-like behaviors. As expected, deletion mice displayed the most abnormal phenotypes, while duplication mice had fewer and milder symptoms. Inter‐ estingly, reciprocal phenotypes were sometimes observed for mice harboring reciprocal CNVs. For example, the amount of time spent resting in the cage was lower in deletion mice but higher in duplication mice relative to controls, indicating that 16p11.2 CNVs affect the rate and timing of specific behaviors in a dosage-dependent manner. Deletion mice dis‐ played an abnormal ceiling-climbing behavior where they demonstrated marked stereotypic and nonprogressive motor behaviors, similar to what is often observed in patients with au‐ tism or patients with lateral hypothalamic and nigrostriatal lesions in the brain. These ab‐ normal behaviors were accompanied by volumetric and morphological changes in several brain regions, including the lateral hypothalamus. Importantly, the difference between dele‐ tion mice and duplication mice was greater than that between deletion mice and controls, indicating that these effects are reciprocal or opposing in nature.

No significant abnormal social behavior was observed in these animal models in the 3 chamber test for sociability, indicating either that these animals do not display social abnor‐ malities, or that further investigation into the social behavior of these animals is required. Indeed, with the subtle nature of many social interactions in rodents, it is quite possible that social abnormalities exist in these mice but have not yet been described. It is also distinctly possible that the 'in-cage' environment does not elicit a social deficit that might perhaps be observed in the wild or natural environment of the animal. An extensive battery of tests for social behavior will be required to rule out the possibility of further abnormalities.

Many of the genes mapped to the altered region have unknown function, and therefore, unknown significance or contribution to the disease phenotype. In order to further delin‐ eate the function of the dosage-sensitive genes within the common duplication/deletion region, zebrafish models were generated [57, 58]. The first study aimed to investigate the diametric head size phenotypes linked to this locus, as in addition to ASD, deletion is known to result in macrocephaly, and duplication gives rise to microcephaly [36, 57]. In this study, zebrafish were utilized for an *in vivo* overexpression screen, which identified the gene *KCTD13* as the likely candidate for the neurodevelopmental phenotypes associ‐ ated with CNVs at 16p11.2. Interestingly, this gene was also one of the 5 genes found in a minimal critical deletion interval for ASD [54]. Overexpression of this gene in zebrafish resulted in microcephaly, while the reciprocal reduced expression of this locus by mor‐ pholino oligonucelotides resulted in macrocephaly, thereby mirroring the phenotypes seen in humans harboring CNVs at this locus [57]. Further study revealed that the func‐ tion of this gene is likely conserved across species, and it is required to maintain the proliferative status of cortical progenitor cells in mice [57]. Furthermore, this gene is af‐ fected in a complex genomic rearrangement identified in a patient with autism [57]. Tak‐ en together, these results indicate that *KCTD13* is a likely candidate for further study of the neurological phenotypes associated with CNV at this locus.

Another study was able to identify homologs of 21 of the known 16p11.2 human genes in the zebrafish genome by family tree comparisons [58]. These genes were then targeted for loss of function studies by injecting antisense morpholino oligonucleotides into early em‐ bryos [58]. Interestingly ~79% of the genes tested by this method were required for proper brain, eye, or nervous system development, and two of the genes were determined to be dosage-sensitive, with abnormal phenotypes present with a ~50% reduction in gene expres‐ sion [58]. The results of this study suggest that at least two genes, aldolase a (*aldoaa*) and ki‐ nesin family member 22 (*kif22*), are highly dosage-sensitive and are required for proper brain function, making them likely candidates for future studies of the ASD associated with CNV of this region [58].

These two studies indicate that while *kctd13*, *kif22*, and *aldoaa* are all potentially interesting dosage-sensitive candidate genes, further investigation is needed to determine whether these genes act together in a epistatic manner to contribute to the full neurological pheno‐ type, whether they modify each other via *cis* interactions, or whether other genes or genetic elements in the 16p11.2 locus are also contributing to the phenotype. The current data, which could not have been obtained without these important studies in model organisms, cannot distinguish between these possibilities, but they provide a starting point for research into the function of these genes and the molecular pathways underpinning the phenotypes associated with 16p11.2 CNVs.
