Distinct cerebellar foliation anomalies in a CHD7 haploinsufficient mouse model of CHARGE syndrome

Mutations in the gene encoding the ATP dependent chromatin‐remodeling factor, CHD7 are the major cause of CHARGE (Coloboma, Heart defects, Atresia of the choanae, Retarded growth and development, Genital‐urinary anomalies, and Ear defects) syndrome. Neurodevelopmental defects and a range of neurological signs have been identified in individuals with CHARGE syndrome, including developmental delay, lack of coordination, intellectual disability, and autistic traits. We previously identified cerebellar vermis hypoplasia and abnormal cerebellar foliation in individuals with CHARGE syndrome. Here, we report mild cerebellar hypoplasia and distinct cerebellar foliation anomalies in a Chd7 haploinsufficient mouse model. We describe specific alterations in the precise spatio‐temporal sequence of fissure formation during perinatal cerebellar development responsible for these foliation anomalies. The altered cerebellar foliation pattern in Chd7 haploinsufficient mice show some similarities to those reported in mice with altered Engrailed, Fgf8 or Zic1 gene expression and we propose that mutations or polymorphisms in these genes may modify the cerebellar phenotype in CHARGE syndrome. Our findings in a mouse model of CHARGE syndrome indicate that a careful analysis of cerebellar foliation may be warranted in patients with CHARGE syndrome, particularly in patients with cerebellar hypoplasia and developmental delay.

, has led to significant progress in elucidating the developmental and molecular genetic mechanisms underlying specific phenotypes associated with CHARGE syndrome (Layman, Hurd, & Martin, 2010). However, CHARGE syndrome is characterized by significant variability in incidence and severity of specific abnormalities, which does not correlate with the nature of CHD7 mutation (Basson & van Ravenswaaij-Arts, 2015;Bergman, Janssen et al., 2011;Jongmans et al., 2008). These observations implicate other genetic or non-genetic factors, or even stochastic effects as modifiers of disease severity. As CHD7 co-factors and target genes are highly context-dependent, the identity of these disease modifiers are likely to be distinct for each of the different phenotypes associated with CHARGE syndrome (Basson & van Ravenswaaij-Arts, 2015).
Chromatin immunoprecipitation and subsequent sequencing (CHIP-seq) studies demonstrate that CHD7 is recruited preferentially to distal gene regulatory elements, or enhancers, implying a role for CHD7 in enhancer-regulated gene transcription (Engelen et al., 2011;Schnetz et al., 2009;Zentner et al., 2010). In vitro studies have provided direct experimental evidence for the ATP-dependent nucleosome remodeling activity of CHD7. The introduction of CHARGE syndrome-associated mutations in the ATP-dependent chromatin remodeling and chromodomains of CHD7, tested in nucleosome remodeling assays, provided proof that chromatin remodeling activity is central to the pathogenesis of CHARGE syndrome (Bouazoune & Kingston, 2012). We recently reported changes in DNA accessibility at thousands of putative gene regulatory elements in Chd7-deficient cerebellar neuron progenitors, providing in vivo evidence that altered nucleosome remodeling underlies specific phenotypes associated with CHD7 deficiency (Whittaker et al., 2017).
Our studies on mouse mutants with either constitutive (gene-trap) or conditional loss-of-function Chd7 mutations have identified two temporally distinct roles for CHD7 during cerebellar development.
During early to mid-gestation, CHD7 is essential for the maintenance of high levels of Fgf8 expression in the mid-hindbrain organizer (Yu et al., 2013). Diminished FGF signaling contributes specifically to hypoplasia of the cerebellar vermis (Yu et al., 2013). During the periand early postnatal stages of development, Chd7 is highly expressed in granule cell progenitors (GCps) on the surface of the cerebellar anlage, where it regulates cerebellar growth by controlling the proliferation, differentiation, and survival of this cell population (Whittaker et al., 2017).
Growth and foliation of the cerebellum are closely linked processes during perinatal development. Both are driven by GCp proliferation and therefore, disruption in GCp expansion usually affects both cerebellar size and the degree of foliation. Mutant mouse models with reduced cerebellar GCp proliferation, such as those with altered SHH signaling, exhibit cerebellar hypoplasia with a simplified pattern of foliation. Importantly, despite a marked reduction in cerebellar size, cardinal lobes, and therefore the presence and position of basic folia are retained, meaning that reduced cerebellar growth alone is insufficient to cause an alteration in the precise temporal and spatial sequence of fissure formation (Corrales, Blaess, Mahoney, & Joyner, 2006). The process of foliation is initiated by the formation of specific multicellular anchor points at the position of prospective fissures, identified by indentations on the surface of the developing cerebellum (Sudarov & Joyner, 2007). These fissures serve to divide the cerebellum into specific lobes across its anterior-posterior extent.
Specific fissures appear in a stereotypical pattern at specific developmental time points in a highly regulated manner (Sudarov & Joyner, 2007). At completion of this process, the mammalian cerebellum is divided into ten cardinal lobules in the vermis (I-X) and four in the hemispheres (Simplex, CrusI, CrusII, Paramedian) (Larsell, 1952).
Although the exact molecular mechanisms that determine the position and timing of fissure formation have not been elucidated, it is clear that these features are under genetic control. The cerebellum is partitioned into zones, each with its own distinct gene expression patterns that likely pre-figure and represent important functional subdivisions (Sillitoe & Joyner, 2007). The curious cerebellar phenotypes of spontaneous mouse mutants like meander tail (mea/mea) were the first to suggest that cerebellar regionalization was genetically determined. In this mouse, cellular disorganization is restricted to the anterior vermis up to the junction between lobules VI and VII (Norman, Fletcher, & Heintz, 1991;Ross, Fletcher, Mason, Hatten, & Heintz, 1990). Since then, specific genes that control foliation have been identified. For example, mutation of the Engrailed (En) genes, which encode homeobox transcription factors, alters the sequence, and position of fissure formation leading to changes in the shape and location of intervening folia (Cheng et al., 2010;Sudarov & Joyner, 2007).
Our previous analysis of cerebellar structure in Chd7 heterozygous mice did not detect overt cerebellar vermis hypoplasia (Yu et al., 2013).
However, only a small number of animals were examined in that study.
Here, we report an analysis of a larger group of animals, which revealed mild, but significant, cerebellar hypoplasia and altered cerebellar foliation in approximately 65% (n = 12/18) of Chd7 heterozygous mice.

| Histology
All samples were dissected in ice-cold PBS, fixed overnight in 4% paraformaldehyde (PFA) at 4°C, dehydrated and embedded in paraffin wax. Serial, sagittal sections were cut (10 µm) and left to dry overnight at 42°C.

| Structural MRI
Adult mice (∼P60) were terminally anesthetized and intracardially perfused with 30 ml of 0.1 M PBS containing 10 U/ml heparin and 2 mM ProHance (Bracco Diagnostics Inc., Montreal, Quebec, Canada), a Gadolinium contrast agent, followed by 30 ml of 4% paraformaldehyde (PFA) containing 2 mM ProHance (Cahill et al., 2012;Spring, Lerch, & Henkelman, 2007). Perfusions were performed at a rate of approximately 60 ml/hr. After perfusion, the brain and remaining skull structures were incubated in 4% PFA + 2 mM ProHance overnight at 4°C and transferred to 0.1 M PBS containing 2 mM ProHance and 0.02% sodium azide for at least 1 month prior to MRI scanning (de Guzman, Wong, Gleave, & Nieman, 2016). A multi-channel 7.0 Tesla MRI scanner (Agilent Inc., Palo Alto, CA) was used to image the brains within skulls. Sixteen custom-built solenoid coils were used to image the brains in parallel Lerch, Sled, & Henkelman, 2011). Parameters used in the anatomical MRI scans: T2-weighted 3D fast spin-echo sequence, with a cylindrical acquisition of k-space, and with a TR of 350 ms, and TEs of 12 ms per echo for 6 echoes, 2 averages, field-of-view of 20 × 20 × 25 mm 3 and matrix size = 504 × 504 × 630 giving an image with 0.040 mm isotropic voxels (Spencer Noakes, Henkelman, & Nieman, 2017). The current scan time required for this sequence is ∼14 hr. To visualize and compare any changes in the mouse brains the images were linearly (6 parameter followed by a 12 parameter) and non-linearly registered together, and then iteratively linearly and non-linearly aligned to each other to create a population atlas representing the average anatomy of the entire study sample. At completion of this registration, all scans had been deformed into alignment with each other in an unbiased fashion. As with typical deformation based morphometry, this allows for analysis of the deformations required to register the anatomy of each individual mouse into the final atlas space Nieman, Flenniken, Adamson, Henkelman, & Sled, 2006). The Jacobian determinants, as calculated through this analysis process, were used as measures of volume at each voxel and compared across genotypes.

| Statistics
For MRI data, volumetric changes were calculated on a regional and a voxel-wise basis. Regional volumes were determined using a preexisting classified MRI atlas encompassing 159 different structures throughout the brain (Dorr, Lerch, Spring, Kabani, & Henkelman, 2008;Steadman et al., 2014;Ullmann, Watson, Janke, Kurniawan, & Reutens, 2013). Statistical analyses were applied comparing the absolute and relative volume of these 159 different regions and on a voxel-wise basis in the brains of control and Chd7 gt/+ mice. Multiple comparisons were controlled for using the False Discovery Rate (Genovese, Lazar, & Nichols, 2002).
Volumetric analyses revealed a 9% reduction in mean total brain volume in Chd7 gt/+ mice compared to controls ( Figure 1a). Mean cortical volume was likewise reduced by 9.7% (Figure 1b), in keeping with the general reduction in brain size. Mean cerebellar volume was reduced by approximately 12% in Chd7 gt/+ mice, compared to Chd7 +/+ controls ( Figure 1c). Voxel-wise, volumetric comparisons ( Figure 1d) revealed the most significant (FDR < 0.05) changes in the posterior vermis (16% reduction in lobule VIII) and anterior hemisphere (15% reduction in simplex lobule) of the cerebellum (Figures 1e and 1f).
To examine whether these volumetric changes in specific cerebellar areas were related to specific changes in cerebellar foliation, cerebella were examined on sagittal MRI slices. The assessment of images from the cerebellar vermis (Figure 2a-d) identified a subtle foliation anomaly in 64% (n = 7/11) of Chd7 gt/+ mice (Figures 2c and 2d), with the remaining 36% (n = 4/11) of mutants ( Figure 2b) exhibiting a normal foliation structure. The severity of the observed phenotype appeared to differ between FIGURE 1 Brain, cortex, and cerebellar hypoplasia in Chd7 haploinsufficient mice. (a) Absolute brain volumes (mm 3 ) of Chd7 +/+ (WT, n = 13) and Chd7 gt/+ (HET, n = 11) adult mouse brains determined by high-resolution structural MRI. (b) Total cortical volumes (mm 3 ) of WT and HET adult mouse brains determined by high-resolution structural MRI. (c) Total cerebellar volumes (mm 3 ) of WT and HET adult mouse brains determined by high-resolution structural MRI. (d) MRI images in the sagittal plane showing mid-sagittal (a) and lateral (b) views of the brain, anterior to the right, with brain regions with significant (FDR < 0.05) volumetric differences between HET and WT mice colored according to the color scale. Note the hypoplastic regions in the posterior cerebellar vermis (lobule VIII) and anterior cerebellar hemispheres (Simplex lobule). (e) Lobule VIII volumes (mm 3 ) of WT and HET adult mouse brains determined by high-resolution structural MRI. (f) Simplex lobule volumes (mm 3 ) of WT and HET adult mouse brains determined by high-resolution structural MRI individual mice. Mildly affected mice (n = 3, Figure 2c) had a deeper prepyramidal (Ppy) fissure and correspondingly shallower secondary (Sec) fissure, such that lobule VIII was located in a more posterior position. This phenotype was even more striking in the severely affected group (n = 4, Figure 2d), in which a marked reduction in the size of lobule VIII was accompanied by a more pronounced posterior shift of this lobule. Cerebellar hypoplasia also appeared to be more pronounced in the severely affected group, an observation that was supported by volumetric analysis, which showed a reduction in mean cerebellar volume of 17% in this group compared to wild type (Table 1).
To visualize the foliation abnormalities at higher resolution, a careful histological analysis was performed on sections from Chd7 gt/+ and Chd7 +/+ cerebella at P21 (Figure 2e fissure that separates the simplex lobule from Crus I (CI), appeared to be shallower in some mutants, leading to partial "fusion" of these lobules in the anterior cerebellar hemispheres (Figures 2k,2l,2o,and 2p). This hemisphere-specific foliation defect was identified in the same mice exhibiting foliation defects in the vermis and the overall penetrance of this foliation phenotype in the hemispheres is therefore also 67% (n = 12/18).

FIGURE 2
Subtle cerebellar foliation anomalies in Chd7 haploinsufficient mice. (a-d) Representative mid-sagittal MRI images of cerebella from Chd7 +/+ (WT) and Chd7 gt/+ (HET) mice, anterior to the left. Cerebellar lobules are labeled with Roman numerals according to Inouye and Oda (1980). Images from HET cerebella with normal foliation, subtle posterior shift of lobule VIII accompanied by deeper Ppy (broken purple line) and shallower Sec (broken orange line) fissures (mild), and most pronounced foliation phenotype characterized by small and posteriorly shifted lobule VIII associated with shallow Sec fissure (severe) are shown. (e-h) Representative Cresyl violet-stained sagittal sections demonstrating the foliation patterns in (a-d) at higher resolution. (i-l) Representative sagittal MRI images of lateral cerebella from Chd7 +/+ (WT) and Chd7 gt/+ (HET) mice. Cerebellar lobules are labeled with Roman numerals as above. Images from HET cerebella with normal foliation (HET-normal), and shallower Sp (broken red line) fissures (HET-mild and HET-severe) are shown. (m-p) Representative Cresyl violet-stained sagittal sections demonstrating the foliation patterns in (i-l) at higher resolution. Purple shading outlines the simplex lobule (m-p). Ppy, prepyramidal; Sec, secondary; Sp, superior posterior; S, simplex; CI, CrusI; CII, CrusII; PM, paramedian. Scale bar = 1 mm To visualize these foliation anomalies across the whole cerebellum, representative histological sections along the medial-lateral extent of Chd7 gt/+ and Chd7 +/+ cerebella were compared (Figure 3).
The alterations in Ppy and Sec fissures, with associated hypoplasia of lobule VIII (false colored in pink in Figure 3) can clearly be seen in midsagittal sections from both Chd7 gt/+ mutants (Figures 3b and 3c). This phenotype was present along the entire vermis (Figure 3d-i) and paravermis (Figure 3j-l). The shallower Sp fissure and hypoplastic  simplex lobule "fused" to CI can be discerned in the hemispheres from both groups (Figure 3m-r).
Together, these analyses showed that Chd7 haploinsufficiency in mice is associated with mild cerebellar hypoplasia, and identified incompletely penetrant roles for CHD7 in regulating cerebellar foliation in the vermis and hemispheres. The specific foliation anomalies observed in these mutants appeared to be responsible for the hypoplasia of the VIIIth and simplex lobules.
3.2 | CHD7 coordinates the precise temporal sequence of fissure formation during perinatal development In control mice, the fissures developed in a highly coordinated and reproducible temporal series as previously described (Sudarov & Joyner, 2007). At E18.5, the three cardinal fissures that are initiated a day earlier, the preculminate (Pc), primary (Pr), and secondary ( (Figures 4d and 4f). We therefore concluded that the fissure beginning to form in the mutant cerebella was the prepyramidal fissure that was being initiated at a more posterior position than normal (Figure 4b). The Sec fissure, which normally forms in the centre of the Fgf5 expression domain in the posterior vermis (Figure 4e), was absent in the mutants (Figure 4f).
These abnormalities in fissure formation was detected in 60% (n = 3/5) of the Chd7 gt/+ mice analyzed at E18.5 and P0, which represents a similar frequency to the foliation defects observed in adult heterozygous Chd7 mutants.
By following fissure formation in histological sections at postnatal stages, we confirmed a pronounced delay in formation of the posterolateral and Sec fissures. The posterolateral fissure first became evident in affected Chd7 haploinsufficient mice by P1 (Figure 4h), >2 days later than its formation in control mice at E18.5 (Figure 4a). By P2, when the secondary fissure is already prominent in controls (Figure 4i), these mutants still completely lacked any signs of this fissure ( Figure 4j). The more posterior position of the Ppy fissure in mutants, compared to controls was also evident at P2 (Figures 4i and 4j). By P7, a Sec fissure was present in the mutants (Figure 4l), indicating that this fissure forms between P2 and P7, several days later than controls where this fissure starts forming at E17.5. Taken together, this analysis shows that formation of the preculminate, primary, secondary, and posterolateral fissures is delayed in some Chd7 haploinsufficient mice, while the prepyramidal fissure is shifted to a more posterior position.
We These abnormalities in the hemispheres were present in 60% of Chd7 gt/+ brains examined, in agreement with the incidence of foliation defects observed in adults.
Overall, these data demonstrate that foliation defects in Chd7 haploinsufficient mice result from abnormalities in the timing and position of fissure formation during cerebellar development. The regulation of the precise temporal sequence of fissure formation in the perinatal cerebellum by CHD7 represents a previously unidentified role for this chromatin-remodeling factor in cerebellar foliation.

| DISCUSSION
Here, we report the first small-scale, unbiased structural brain MRI study of a Chd7 haploinsufficient mouse model. Several intriguing WHITTAKER ET AL.

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observations were made, which included a general reduction in brain size in these mutants that appears to affect several brain regions. These findings warrant further examination, including a careful longitudinal study of brain growth to determine to what extent brain anomalies are related to the general growth retardation typical of CHARGE syndrome. Quantitative structural MRI imaging in CHARGE syndrome patients will be important to identify specific brain regions most sensitive to CHD7 haploinsufficiency during human brain development, as altered growth trajectories and volumetric changes in specific brain regions may underlie specific neuropsychiatric symptoms associated with CHARGE syndrome. Previous studies have shown that cerebellar foliation occurs during perinatal development and is driven by extensive proliferation of cerebellar GCps (Sudarov & Joyner, 2007). The analysis of GCpspecific Chd7 conditional mutant mice has identified important roles for CHD7 in regulating GCp apoptosis, proliferation and differentiation in the perinatal cerebellum (Feng et al., 2017;Whittaker et al., 2017).
However, a reduction in GCp expansion is by itself not sufficient to explain the cerebellar foliation defects described here. Although some fissures and folia show a general delay in their formation, which could be accounted for by reduced GCp proliferation, our data are consistent with a specific role for CHD7 in regulating the precise position and timing of fissure formation, which ultimately alters the position, size and shape of specific folia.

| Specific genetic pathways controlling cerebellar foliation may be disrupted in CHARGE syndrome
The wide range of phenotypes associated with CHARGE syndrome is thought to primarily be the consequence of tissue-specific dysregulation of gene transcription during development (Basson & van Ravenswaaij-Arts, 2015;Engelen et al., 2011;Schnetz et al., 2009;Schnetz et al., 2010). It has been proposed that mutations or variants in genes that are regulated by or otherwise interact with CHD7 may contribute to, or modify, the phenotypic outcome of a mutation in CHD7 (Basson, 2014). Mouse models have provided compelling evidence in support of this hypothesis, linking CHD7 to many important developmental pathways. For instance, Fgf8 interacts with Chd7 during early cerebellar vermis development (Yu et al., 2013), and Chd7 interacts with Tbx1 during arch vessel development (Randall et al., 2009). As cerebellar foliation is under genetic control (see section 1), it seems likely that genes controlling this process may be regulated by CHD7 and interact with Chd7.
Interestingly, our recent RNA-seq analysis of Chd7-deficient cerebellar GCps showed that En1 expression was upregulated in these cells, implicating CHD7 as a potential repressor of En1 expression (Whittaker et al., 2017). It is tempting to speculate that dysregulated En1 expression contributes to the specific foliation anomalies we have identified in Chd7-deficient mice. A role for CHD7 in regulating Engrailed gene expression would also be consistent with its conserved function as a Trithorax family member. Trithorax proteins regulate the expression of homeobox genes involved in regional identity and patterning during development (Schuettengruber, Martinez, Iovino, & Cavalli, 2011;Yu et al., 2013).
We previously showed that Fgf8 expression in the isthmic organizer is reduced in Chd7 gt/+ embryos (Yu et al., 2013). We have also reported that Fgf8 is expressed in the late embryonic (E16.5) and early postnatal cerebellum at the site of the developing secondary fissure (Yaguchi et al., 2009). Sato and Joyner have shown that Fgf8 deletion from ∼E12 of development results in mice with a posteriorly located lobule VIII (Sato & Joyner, 2009), the identical foliation change we report here in Chd7 gt/+ mice. These findings suggest the intriguing possibility that a prolonged reduction in FGF8 signaling in Chd7 gt/+ embryos may also contribute to this specific foliation anomaly, in addition to its established role during early cerebellar development.
The cerebellar vermis anomalies identified in CHARGE syndrome patients share some phenotypic similarities with Dandy Walker malformation (DWM), the most frequent congenital cerebellar malformation in the human population (Barkovich, Millen, & Dobyns, 2009;Yu et al., 2013). Specifically, documented neuroanatomical malformations in CHARGE syndrome patients include cerebellar vermis hypoplasia with anti-clockwise rotation away from the brainstem and a large posterior fossa (Yu et al., 2013). and Simplex and CrusI lobules are fused (Aruga, Inoue, Hoshino, & Mikoshiba, 2002). It will be of interest to quantify Zic gene expression in the anterior cerebellar hemispheres of Chd7 gt/+ mice to establish whether Zic expression in the developing anterior cerebellar hemisphere is sensitive to Chd7 haploinsufficiency.
The phenotypic variability we report in Chd7 gt/+ mice suggests that this aspect of CHARGE syndrome can be modeled in a mouse model. The maintenance of these mice on an F1, rather than single inbred genetic background, may certainly contribute to higher levels of phenotypic variation (Keane et al., 2011). In addition, Chd7 gt/+ embryos may be more susceptible to unknown, stochastic effects on developmental gene expression.

| Functional consequences of foliation anomalies in CHARGE syndrome
It is worth considering whether the relatively mild cerebellar anomalies contributes to specific neurological or psychiatric aspects of CHARGE syndrome. A couple of pertinent findings suggest the possibility that WHITTAKER ET AL.
| 473 lobule VIII-specific foliation defects may indeed contribute to some of the deficits in motor coordination and learning frequently associated with CHARGE syndrome (Admiraal & Huygen, 1997;Bergman, Janssen et al., 2011;Sanlaville & Verloes, 2007). Lobule VIII is active during sensorimotor tasks (Stoodley & Schmahmann, 2009;Stoodley, Valera, & Schmahmann, 2012) and abnormal motor learning and function have been reported in En2 mutant mice, which have cerebellar vermis foliation defects similar to Chd7 gt/+ mice (Cheh et al., 2006;Gerlai, 1996;Joyner et al., 1991;Millen et al., 1994). However, to date, a correlation between motor dysfunction in CHARGE patients and cerebellar pathology has not been established. While cerebellar hypoplasia in CHARGE syndrome could influence gait or motor learning, there is a high prevalence (94% of CHARGE patients) of semicircular canal anomalies leading to vestibular dysfunction (Abadie et al., 2000). The vestibular apparatus is important in psychomotor development and therefore, it remains difficult to discern the precise contribution of cerebellar hypoplasia to the difficulties in postural and axial motor control in these patients. Previous studies imply that vestibular dysfunction alone is not responsible for gait abnormalities in patients and therefore further investigation of cerebellar contribution is necessary (Abadie et al., 2000;Wiener-Vacher, Amanou, Denise, Narcy, & Manach, 1999).
The cerebellar hemispheres have been implicated in higher cognitive processes (Kelly & Strick, 2003;Stoodley & Schmahmann, 2009;Stoodley & Schmahmann, 2010;Stoodley et al., 2012) and thus, the foliation defects identified in the anterior cerebellum tentatively suggest a connection between these cerebellar defects and intellectual disability and autistic phenotypes associated with CHARGE syndrome.
Separately, the relationship between cerebellar anomalies and autism is well established and in contrast to other brain regions, gross and microscopic changes in the cerebellum are most frequently associated with autism (Becker & Stoodley, 2013). However, we previously reported that cerebellar hypoplasia and foliation defects in Chd7 GCpspecific conditional mouse mutants, alone were not sufficient to lead to social deficits (Whittaker et al., 2017). The contribution of cerebellar dysfunction to autistic phenotypes in patients also remains unclear.
In conclusion, we have identified mild cerebellar hypoplasia and distinct cerebellar foliation anomalies in Chd7 gt/+ mice. Our findings imply a specific function for CHD7 in controlling the spatiotemporal initiation of cerebellar fissures and show that normal fissure formation requires bi-allelic Chd7 expression, consistent with the haploinsufficient nature of CHARGE syndrome. The incomplete penetrance and expressivity of these phenotypes are also consistent with the observed phenotypic spectrum of CHARGE syndrome. As with other phenotypes, the identification of possible genetic and environmental modifiers that interact with Chd7 to regulate cerebellar foliation will be of significant interest.