doi:10.1093/brain/aws065 Brain 2012: 135; 1370–1386 | 1370 BRAIN A JOURNAL OF NEUROLOGY

Rhombencephalosynapsis: a malformation associated with incomplete separation of midbrain and forebrain, and a broad spectrum of severity

1 2 1,3 3,4

Gisele E. Ishak, Jennifer C. Dempsey, Dennis W. W. Shaw, Hannah Tully, Downloaded from Margaret P. Adam,2 Pedro A. Sanchez-Lara,5,6 Ian Glass,2 Tessa C. Rue,7 Kathleen J. Millen,2,3 William B. Dobyns2,3 and Dan Doherty2,3

1 Department of Radiology, University of Washington, Seattle Children’s Hospital, Seattle, WA 98105, USA http://brain.oxfordjournals.org/ 2 Division of Genetic Medicine, Department of Paediatrics, University of Washington, Seattle, WA 98195, USA 3 Centre for Integrative Brain Research, Seattle Children’s Research Institute, Seattle, WA 98101, USA 4 Department of Neurology, University of Washington, Seattle, WA 98195, USA 5 Department of Paediatrics, Keck School of Medicine, University of Southern California, Los Angeles, CA 90089, USA 6 Children’s Hospital Los Angeles, Los Angeles, CA 90027, USA 7 Department of Biostatistics, University of Washington, Seattle, WA 98195, USA

Correspondence to: Gisele E. Ishak, Seattle Children’s Hospital,

Department of Radiology, at Cleveland Clinc on May 1, 2012 R-5417, 4800 Sand Point way NE, Seattle WA 98105, USA E-mail: [email protected]

Correspondence may also be addressed to: Dan Doherty, Department of Pediatrics, University of Washington, Box 356320, 1959 NE Pacific St., RR-247, Seattle, WA 98195, USA. E-mail: [email protected]

Rhombencephalosynapsis is a midline brain malformation characterized by missing with apparent fusion of the cerebellar hemispheres. Rhombencephalosynapsis can be seen in isolation or together with other central nervous system and extra-central nervous system malformations. Go´ mez-Lo´ pez-Herna´ndez syndrome combines rhombencephalosynapsis with parietal/temporal alopecia and sometimes trigeminal anaesthesia, towering skull shape and dysmorphic features. Rhombencepha- losynapsis can also be seen in patients with features of vertebral anomalies, anal atresia, cardiovascular anomalies, trachea–oe- sophageal fistula, renal anomalies, limb defects (VACTERL) association. Based on a comprehensive evaluation of neuroimaging findings in 42 patients with rhombencephalosynapsis, we propose a spectrum of severity, ranging from mild (the partial absence of nodulus, anterior and posterior vermis), to moderate (the absence of posterior vermis with some anterior vermis and nodulus present), to severe (the absence of posterior and anterior vermis with some nodulus present), to complete (the absence of the entire vermis including nodulus). We demonstrate that the severity of rhombencephalosynapsis correlates with fusion of the tonsils, as well as midbrain abnormalities including aqueductal stenosis and midline fusion of the tectum. Rhombencephalosynapsis is also associated with multiple forebrain abnormalities including absent olfactory bulbs, dysgenesis of the corpus callosum, absent septum

Received August 26, 2011. Revised January 28, 2012. Accepted January 29, 2012. Advance Access publication March 26, 2012 ß The Author (2012). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: [email protected] Rhombencephalosynapsis neuroimaging Brain 2012: 135; 1370–1386 | 1371 pellucidum and, in rare patients, atypical forms of . The frequent association between rhombencephalosynapsis and aqueductal stenosis prompted us to evaluate brain magnetic resonance images in other patients with aqueductal stenosis at our institution, and remarkably, we identified rhombencephalosynapsis in 9%. Strikingly, subjects with more severe rhombencephalo- synapsis have more severely abnormal neurodevelopmental outcome, as do subjects with holoprosencephaly and patients with VACTERL features. In summary, our data provide improved diagnostic and prognostic information, and support disruption of dorsal– ventral patterning as a mechanism underlying rhombencephalosynapsis.

Keywords: rhombencephalosynapsis; Go´ mez-Lo´ pez-Herna´ ndez syndrome; aqueductal stenosis; holoprosencephaly; hydrocephalus; VACTERL Abbreviations: DTI = diffusion tensor imaging; GLH = Go´ mez-Lo´ pez-Herna´ ndez syndrome; HPE = holoprosencephaly; NOS = not otherwise specified; RES = rhombencephalosynapsis; VACTERL = vertebral anomalies, anal atresia, cardiovascular anomalies, trachea– oesophageal fistula, renal anomalies, limb defects

large majority of patients with RES, and especially GLH, are spor-

Introduction adic, suggesting de novo autosomal dominant mutations as the Downloaded from Rhombencephalosynapsis (RES) is a rare brain malformation most common cause (Romanengo et al., 1997; Toelle et al., defined by midline fusion of the cerebellar hemispheres with par- 2002; Sandalcioglu et al., 2006; Chemli et al., 2007). RES has tial or complete loss of the intervening vermis. RES occurs in been associated with other brain abnormalities (Kepes et al., isolation or in combination with other CNS and extra-CNS malfor- 1969; Michaud et al., 1982; Schachenmayr and Friede, 1982; mations. The most common congenital anomaly syndrome Isaac and Best, 1987; Savolaine et al., 1991; Truwit et al., http://brain.oxfordjournals.org/ associated with RES is Go´ mez-Lo´ pez-Herna´ ndez (GLH) syndrome, 1991; Simmons et al., 1993; Boltenstern et al., 1995; Demaerel which is characterized by RES and scalp alopecia, as well as et al., 1995; Shaw, 1995; Sergi et al., 1997; Utsunomiya et al., abnormal head shape (usually turricephaly or tower skull), low- 1998; Sener, 2000; Jellinger, 2002, 2009; Toelle et al., 2002; set and posteriorly angulated ears and trigeminal anaesthesia in Yachnis, 2002; Boltshauser, 2004; Pavone et al., 2005; Siebert subsets of patients (Sukhudyan et al., 2010). While the prevalence et al., 2005; Chemli et al., 2007; Pasquier et al., 2009), but no of RES is unknown, more than 90 individuals with RES have been large post-natal series exists to determine the spectrum and reported in the literature, including at least 25 with GLH (Gomy prevalence of associated imaging features and neurodevelopmen- et al., 2008; Fernandez-Jaen et al., 2009; Pasquier et al., 2009; tal outcomes. at Cleveland Clinc on May 1, 2012 Poretti et al., 2009; Sukhudyan et al., 2010). The aetiology of RES is unknown. One hypothesis is that RES is caused by dorsal–ventral patterning defects that result in loss of midline and fusion of lateral structures (Sarnat, 2000; Yachnis, Materials and methods 2002). Alternatively, RES could represent loss of anterior cerebellar We identified 10 patients with RES by searching the Seattle Children’s anlage cells destined to become the vermis, or from transform- Hospital imaging database, which contains all clinical MRI reports for ation of these anterior cells to a more posterior and/or ventral the 15 562 patients who had brain MRI studies between 2002 and hemispheric fate. The cerebellar fusion is in some ways compar- mid-2011. After noting a frequent association between RES and con- genital aqueductal stenosis, we reviewed the MRIs for 56 patients with able with holoprosencephaly (HPE) in the forebrain, and indeed, aqueductal stenosis identified in the database and found five additional these two malformations have been reported in multiple patients, patients with RES. We ascertained another 27 patients with RES by suggesting a shared, but unknown, developmental mechanism referral from outside clinicians for a total of 42 patients. At Seattle (Siebert et al., 2005; Pasquier et al., 2009). Understanding the Children’s Hospital, MRIs were performed on Siemens Symphony 1.5 developmental mechanism(s) for RES is hampered by the paucity T, Siemens Avanto 1.5 T or Siemens Trio 3 T scanners. Imaging of known genetic causes and the lack of an animal model. sequences included sagittal and axial T1 spin echo, T2/FLAIR, diffusion

Although Ramocki et al. (2011) recently reported ZIC2 mutations weighted and coronal T2 sequences. T1 MPRAGE (Magnetization in sisters with RES and HPE (Ramocki et al., 2011), the RES diag- Prepared RApid Gradient Echo) was performed instead of spin echo nosis has come into question (Guleria, 2011); therefore, no single on the 3 T magnet. All outside imaging studies included sequences in gene cause of RES has been conclusively reported. Several obser- three planes, with the exception of Subject 27 for whom only limited vations are consistent with a genetic basis for RES: (i) recurrence in images were available. The Institutional Review Boards at Seattle Children’s Hospital, the University of Washington and the University brothers (Pasquier et al., 2009; Ramocki et al., 2011); (ii) affected of Chicago all approved this study. children with non-recurrent chromosomal abnormalities including The imaging studies were evaluated by two paediatric neuroradiol- interstitial deletion of 2q (Truwit et al., 1991), unbalanced ogists and two paediatric neurogeneticists who arrived at consensus translocation of 2p and 10q (Lespinasse et al., 2004), tetrasomy for any structural abnormalities. The infratentorial evaluation included 9p (di Vera et al., 2008), deletion of 7q and duplication of 1p qualitative assessment of the for size (normal or hypoplas- (Pasquier et al., 2009); (iii) parental consanguinity in at least four tic), shape (superior and inferior ectopia), morphology (normal or folial families; and (iv) highly reproducible clinical features. However, a disorganization), patterns of vermian deficiency (nodulus, anterior or 1372 | Brain 2012: 135; 1370–1386 G. E. Ishak et al. posterior vermis deficiency), the posterior fossa for size (normal or outcome severity), while Fisher’s exact test was used to compare small) and the fourth ventricle for shape (normal or abnormal shape non-ordered categorical variables (clinical category) (Kendall, 1938). such as keyhole appearance). On sagittal and axial images, we eval- uated the superior cerebellar peduncles for orientation of their conflu- ence and for the presence of ectopic tissue, the superior medullary velum for thickness and the presence of ectopic tissue, the posterior Results commissure for thickness, the cerebral aqueduct for patency and the As described above, we reviewed imaging studies from 42 subjects pons for size (normal, flat, mild or moderately hypoplastic). On axial, (17 females, 25 males) ranging in age from 2 days to 44 years (at coronal and sagittal images, we evaluated the inferior and superior colliculi (normal, fused across the midline, fused craniocaudally, the the time of the MRI) with a partial or complete absence of the presence of ectopic tissue). cerebellar vermis and fusion of the hemispheres. Thirty-seven of Within the supratentorium, we evaluated for interhemispheric cere- the subjects were diagnosed with RES by their treating clinicians, bral fusion, migrational disorder (the presence of and while an additional five subjects (Subjects 25, 26, 28, 29 and 40) heterotopia), septum pellucidum (present or absent), corpus callosum with a range of RES severity were identified after review of 56 (normal, thinning, agenesis, dysgenesis), fornices (normal or fused), local patients with aqueductal stenosis. mamillary bodies (normal or hypoplastic/absent), pituitary gland (pre- sent or absent), olfactory bulbs (present or absent) and ventricles (normal or dilated). Definitions of key imaging features are listed in

Hindbrain Downloaded from Box 1. Clinical information was collected using a structured intake form Cerebellar fusion and review of medical records. Blinded to the imaging results, two In the literature, RES has been characterized as partial or complete, authors (J.D. and D.D) classified the abnormal neurodevelopmental but the pattern of cerebellar fusion has not been described. In our outcome for each subject as mild, moderate or severe, according to subjects, we found a spectrum of cerebellar fusion in which the the criteria listed in Supplementary Table 1. Based on the literature, we http://brain.oxfordjournals.org/ posterior vermis was most severely involved, followed by the an- divided the subjects into four clinical categories using the following terior vermis and then the nodulus. Based on this observation, we criteria: were able to group all but four of our subjects into the following (i) GLH: subjects with RES and scalp alopecia (usually temporal or categories, ordered by severity (Fig. 2 and Table 1): parietal), with or without trigeminal anaesthesia, abnormal head shape, midface hypoplasia, low-set and/or posteriorly rotated (1) complete (n = 9): absence of all parts of the vermis ears, telecanthus and/or hypertelorism. (Fig. 2A–E); (ii) RES + VACTERL features: subjects with RES plus at least one of: (2) partial–severe (n = 9): absence of the posterior and anterior vertebral, cardiac, limb or renal structural abnormalities, without vermis, with a distinct nodulus still seen although it may be at Cleveland Clinc on May 1, 2012 scalp alopecia. (iii) RES + HPE: subjects with RES plus focal or diffuse interhemi- variably underdeveloped (Fig. 2F–J); spheric cerebral fusion without alopecia or vertebral, cardiac, (3) partial–moderate (n = 8): absence of the posterior vermis limb or renal structural abnormalities. and variable deficiency of the anterior vermis and nodulus (iv) RES NOS (not otherwise specified): subjects who did not fit into (Fig. 2K–O); the categories above, many of whom had RES with abnormal (4) partial–mild (n = 12): fusion of the central part of the ver- head shape, midface hypoplasia, low-set and/or posteriorly mis—mostly involving the posterior vermis—with some an- rotated ears, telecanthus and/or hypertelorism. terior vermis seen above the fusion and posterior vermis and Statistical analysis was performed using SAS, version 9.3 (SAS nodulus seen below the fusion (Fig. 2P–T); Institute). An exact test of Kendall’s tau-b statistic was used to com- (5) atypical (n = 4): absence of the posterior vermis and nodulus pare two ordinal variables (RES severity and neurodevelopmental with some residual anterior vermis.

Box 1 Definition of key imaging features

 Fusion of the cerebellum: continuity of the folia and white matter across the midline without a recognizable transition to vermis and hypoplasia or absence of the posterior cerebellar incisura and vallecula (Fig. 1A and B).  Posterior vermis: midline cerebellar tissue posterior (caudal) to the primary fissure. In complete RES, it is absent. In partial RES, it is present (although frequently hypoplastic) at the position of the uvula, just caudal and dorsal to the nodulus (Fig. 1C and F).  Anterior vermis: midline cerebellar tissue anterior (rostral) to the primary fissure. In complete RES, it is absent. In partial RES, it is variably deficient, being present at the most cranial and ventral aspect of the vermis, just posterior to the superior cerebellar peduncle at the level of junction of the cerebral aqueduct and fourth ventricle (Fig. 1D).  Nodulus: midline cerebellar tissue just caudal to the fastigium. On the coronal image, it is a thumb-like projection from the floor of the fourth ventricle. On axial images, it is typically seen in the same plane as the flocculi, except when this region is distorted by superior ectopia (Fig. 1E and F)  Pons hypoplasia: subjectively small dorsal–ventral dimension of the pons. Rhombencephalosynapsis neuroimaging Brain 2012: 135; 1370–1386 | 1373 Downloaded from http://brain.oxfordjournals.org/ at Cleveland Clinc on May 1, 2012

Figure 1 Typical neuroradiological findings in RES. (A and B) Fusion of the cerebellar hemispheres with continuity of the cerebellar folia and white matter across the midline in Subject 9 (A) and Subject 18 (B). Note the abnormal shape of the fourth ventricle in A. The cerebellum is pear shaped in B, consistent with mild superior herniation. (C) Residual posterior vermis (white arrowhead points to uvula) in Subject 28. (D) Residual anterior vermis in Subject 28 (black arrowhead). (E) Substantial residual nodulus (black arrow) in Subject 18. (F) Hypoplastic residual nodulus (black arrow) in Subject 32. Note the residual posterior vermis (white arrow) that is continuous superiorly with the nodulus (black arrow), as well as ectopic tissue within the cerebral aqueduct at the level of the inferior colliculus. T = Tonsil. (A, C

and D) axial T2;(B) coronal T2;(E and F) sagittal T1. 1374 | Brain 2012: 135; 1370–1386 G. E. Ishak et al. Downloaded from http://brain.oxfordjournals.org/ at Cleveland Clinc on May 1, 2012

Figure 2 Spectrum of severity in RES. (A–E) Complete RES without residual vermis in Subject 17. Note the absent septum pellucidum, aqueductal stenosis and intraventricular cysts (D). (F–J) Severe partial RES in Subject 2. The nodulus (arrow) is present and projects into the 4th ventricle on the coronal image (I) and the tonsils are fused (F). Note the absent septum pellucidum, narrow transverse diameter of the cerebellum and the small posterior fossa. (K–O) Moderate partial RES in Subject 18. The nodulus (arrow) and anterior vermis (black arrowhead) are present. Note the absent septum pellucidum in (N). (P–T) Mild partial RES in Subject 6. The nodulus (arrows) is present but deficient. Anterior (black arrowhead) and posterior (white arrowhead) vermis is also present. The primary fissure is indistinct on the sagittal

images (E, J, O and T). (A–C, K–M and P–R) axial T2 ranging from caudal to rostral; (F–H) axial T1 ranging from caudal to rostral; (D, N and S) coronal T2;(I) coronal T1;(E, J, O and T) sagittal T1. Lower case alphabets in E, J, O and T refer to the level of sections of the corresponding axial images labeled with upper case alphabets.

The nodulus was present in 29/33 subjects with partial RES; how- fusion. The severity of RES did not correlate with the clinical cate- ever, it was severely hypoplastic in Subjects 6, 11, 28, 32 and 37. gories as defined in the ‘Materials and methods’ section (GLH, Portions of the anterior vermis were seen in 24/33 subjects with RES + VACTERL features, RES + HPE and RES NOS). Note that partial RES and portions of the posterior vermis were seen in 12/33 RES + VACTERL features do not indicate that a patient meets criteria subjects. While fusion of the dentate nuclei has been described in for VACTERL, just that they have at least one feature associated with pathology specimens and occasionally by imaging, the dentate nuclei this condition. Although the severe and complete groups had more were not visualized well enough to include in our analysis. Fusion of females, this was not statistically significant, nor was sex associated the tonsils strongly correlated with severity of with clinical category. Rhombencephalosynapsis neuroimaging Brain 2012: 135; 1370–1386 | 1375

Table 1 Extent of cerebellar fusion and cerebellar ectopia in subjects with RES

Subject MRI age Sex Clinical RES NDV NDV Absent Absent Absent Tonsil Ectopiab categorya severity outcome outcome post ant nodulus fusion age vermis vermis 1 2 days F RES NOS Complete Unknown 10 m + + + + S 8 6 days F GLH Complete Mod 3 yrs 6m + + + + À 15 0 days F HPEc Complete Severe 3 yrs + + + ÀÀ 17 5 days M GLH Complete Mild 4 yrs 5m + + + À S 27 19 m M RES NOS Complete Mod 2 yrs + + + NA NA 35 2 days M HPE Complete Severe Deceased + + + + À 36 10 m F VACTERL featuresd Complete Mod 1 yr 7 m + + + + S 38 25 days F RES NOS Complete Mild 13 m + + + + À 41 18 days M GLH Complete Mild 16 m + + + + I 56% Female Totals 9/9 9/9 9/9 6/8 4/8 2 9 m F GLH Severe Mod 3 yrs + + À +B 3 5 m F GLH Severe Mild 22 m + + À +S 5 2 yrs 3 m F GLH Severe Mod 5 yrs 6 m + + À +S

10 26 yrs M RES NOS Severe Mod 29 yrs 2 m + + ÀÀÀ Downloaded from 13 16 m M RES NOS Severe Unknown 2 yrs NA + À +B 20 17 m M RES NOS Severe Severe 6 yrs 6 m + + À +B 21 14 m M VACTERL featurese Severe Severe 3 yrs 10 m + + ÀÀS 26 1 days F RES NOS Severe Mod 5 yrs 9 m + + ÀÀB 31 6 days F GLH Severe Mild 6 yrs 1 m + + À +S http://brain.oxfordjournals.org/ 56% Female Totals 8/9 9/9 0/9 6/9 8/9 12 2 yrs 11 m M GLH Moderate Unknown 4 yrs 6 m + ÀÀ ÀB 14 1 yrs 3 m M HPE MIF Moderate Mod 8 yrs 1 m + ÀÀ +B 18 7 weeks F RES NOS Moderate Mild 8 yrs 3 m + ÀÀ À- 19 4 yrs M VACTERL features Moderate Severe 8 yrs 5 m + ÀÀ ÀS 24 9 m M GLH Moderate Mild 6 yrs 5 m + ÀÀ ÀB 25 3 days M GLHf Moderate Mild 27 m + ÀÀ ÀS 39 3 yrs 10 m M GLH Moderate Mild 4 yrs 6 m + ÀÀ +S 42 17 m M RES NOS Moderate Mild 2 yrs + ÀÀ ÀI at Cleveland Clinc on May 1, 2012 12% Female Totals 8/8 0/8 0/8 2/8 7/8 4 5 weeks M GLH Mild Unknown 5 m ÀÀÀ ÀÀ 6 19 m M GLH Mild Mild 6 yrs 2 m ÀÀÀ ÀÀ 7 2 yrs F GLH Mild Mild 6 yrs 5 m ÀÀÀ ÀÀ 9 6 yrs 9 m M GLH Mild Mild 14 yrs 5 m ÀÀÀ ÀI 11 15 m F RES NOS Mild Mild 6 yrs 11 m ÀÀÀ ÀÀ 16 44 yrs F RES NOS Mild Mild 44 yrs ÀÀÀ ÀÀ 23 10 m M RES NOS Mild Mild 2 yrs ÀÀÀ ÀS 28 3 yrs 3 m M GLH Mild Mod 4 yrs 4 m ÀÀÀ ÀB 29 16 yrs M VACTERL features Mild Mod 17 yrs ÀÀÀ ÀS 30 6 yrs 2 m M Unknown Mild Unknown Unknown ÀÀÀ pS 32 3 yrs 2 m M GLH Mild Mild 5 yrs 2 m ÀÀÀ ÀS 37 3 yrs 9 m F RES NOS Mild Mild 4 yrs ÀÀÀ ÀS 33% Female Totals 0/12 0/12 0/12 1/12 7/12 22 11 yrs 11 m M RES NOSg Atypical Mild 7 yrs 3 m + À +pS 33 6 yrs 6 m F VACTERL features Atypical Severe 12 yrs + À +pS 34 22 m M GLH Atypical Mild 5 yrs 6 m + À ++S 40 6 days F RES NOS Atypical Mod 9 yrs + À ++B

+ = abnormality present; À = abnormality not present; yrs = years; m = month; p = partial; NA = Not Available; NDV = Neurodevelopmental. a See definitions in ‘Materials and methods’ section. b B = both inferior and superior; I = inferior only (Chiari I); S = superior only. c Subject had rib and vertebral anomalies. d Subject had bitemporal hair thinning but not alopecia. e Subject had crossed renal ectopia but not vertebral, rib, heart, or limb malformations. f Subject had mutation-confirmed neurofibromatosis I. g Subject had microtia, corneal scarring and ectopic salivary gland tissue (Adam et al., 2007). 1376 | Brain 2012: 135; 1370–1386 G. E. Ishak et al.

Posterior fossa midbrain abnormalities roughly correlated with the severity of RES The posterior fossa was small in all patients with complete RES, re- (Table 2). In addition, the three subjects with holoprosencephaly flected by a subjectively small transverse diameter and to a lesser and 3/5 subjects with VACTERL features had aqueductal stenosis extent small anterior–posterior diameter. This correlated with the and fused colliculi. severity of the cerebellar hemisphere hypoplasia. In partial RES, the transverse diameter was variably small in 8/9 patients with severe Table 2 Superior cerebellar peduncle and midbrain fusion, 6/8 with moderate fusion, 3/12 with mild fusion and 4/4 with abnormalities correlate with severity of RES atypical fusion. It is unclear whether the small posterior fossa size reflects abnormal development of the bony posterior fossa asso- Subject Abnormal Fused Aqueductal Level of ciated with turricephaly, or is secondary to hydrocephalus (and ex- SCP colliculi stenosis obstruction pansion of the supratentorial calvarium), shunting and subsequent 1 + + ( + ) PC, SC, IC, V, SCP distortion, or a combination of the above. 8 À ++ IC 15 + + + PC, SC, IC, V, SCP Cerebellar size and ectopia 17 + À + SCP Cerebellar hypoplasia was noted predominantly in subjects with 27 NA NA + NA complete RES; however, in subjects with partial RES, the trans- 35 + + + PC, SC, IC, V, SCP 36 ÀÀÀÀ

verse diameter of the cerebellum was small, with preserved or Downloaded from 38 À + + IC, V even increased craniocaudal dimension. We observed ectopia of 41 À + + SC, IC the cerebellum superiorly through the tentorial notch in 25 sub- Totals 4/8 6/8 8/9 jects, resulting in a striking pear shape (Shaw, 1995; Pasquier 3 ÀÀÀÀ et al., 2009) on coronal images (Fig. 1B). Superior ectopia was 2+ À + V, SCP

not associated with the severity of RES. It is also unlikely to rep- 5+ ÀÀ À http://brain.oxfordjournals.org/ resent simple upward herniation due to shunted hydrocephalus, 10 + + + IC since several subjects with no/mild (Subjects 3, 13 + ÀÀ À 5, 9, 22, 24, 30 and 32) and several with unshunted moderate 20 + + + PC, SC, IC, V, SCP ventriculomegaly (Subjects 1, 13, 23 and 33) had superior ectopia. 21 + + + SC, IC, V À In addition, Subjects 25 and 31 had superior ectopia before shunt- 26 + + SC, IC 31 + + + IC, V ing. Subjects 4 (with non-shunted mild/moderate ventriculome- Totals 7/9 5/9 6/9 galy) and 8 (with shunted hydrocephalus) did not have superior 12 ÀÀ+ a ectopia, potentially due to a quadrigeminal plate cistern cyst and a À

14 + + SC, IC at Cleveland Clinc on May 1, 2012 superior posterior fossa cyst, respectively, while Subject 10 had 18 ÀÀÀÀ shunted hydrocephalus without superior ectopia without an obvi- 19 + + + SC, IC, V ous reason. In contrast, superior ectopia was worse after shunting 24 ÀÀÀÀ in Subjects 17, 20, 21, 25, 26 and 28. Inferior cerebellar ectopia 25 + À + SCP was present in 10 subjects. Although cerebellar dysplasia was rare, 39 + ÀÀ À Subjects 5 and 21 had abnormal foliation of the cerebellar hemi- 42 + + + IC, V spheres at the cranial midline and right para-midline, respectively. Totals 4/8 3/8 5/8 4 ÀÀÀÀ Pons 6+ ÀÀ À 7 ÀÀÀÀ The pons was abnormal in only 10 subjects: flat in Subjects 8, 38, 40 9+ ÀÀ À and 41 (who also had severe cerebellar hypoplasia), moderately 11 ÀÀÀÀ hypoplastic in Subjects 31 and 36 and mildly hypoplastic in 16 ÀÀÀÀ Subjects 4, 16, 17 and 20. The pons appeared compressed rather 23 ÀÀÀÀ than hypoplastic in Subjects 12 and 14, possibly due to the small 28 ÀÀ+SC posterior fossa in these subjects. In contrast, Subject 20 also had a 29 À + + SC, IC very small posterior fossa but the pons appeared mildly hypoplastic 30 ÀÀÀÀ rather than compressed. The basilar sulcus of the pons was present in 32 ÀÀÀÀ all but one subject (Subject 1), indicating that the ventral midline was 37 ÀÀÀÀ generally intact. We were unable to consistently evaluate the cranial Totals 2/12 1/12 2/12 nerves due to limitations in the imaging. 22 ÀÀÀÀ 33 ÀÀÀÀ 34 + ÀÀ À Superior cerebellar peduncles and 40 + + + SC, IC midbrain + = abnormality present; À = abnormality not present; (+) = Aqueduct did not We identified superior cerebellar peduncle abnormalities in 19 of appear patent but the ventriculomegaly was mild; IC = inferior colliculus; PC = posterior commissure; SC = superior colliculus; SCP = superior cerebellar 42 subjects and midbrain abnormalities (aqueductal stenosis and peduncle; V = superior medullary velum. fusion of the colliculi) in 22 of 42 subjects. The severity of the a Deformed tectum with midbrain hypoplasia. Rhombencephalosynapsis neuroimaging Brain 2012: 135; 1370–1386 | 1377

Aqueduct and superior cerebellar peduncles except Subject 28, in whom the superior colliculus did not appear Aqueductal stenosis was present in 22/42 subjects and was to be fused, and in Subject 12 who had aqueductal stenosis at the strongly associated with the degree of cerebellar fusion most superior aspect of the aqueduct with a deformed tectum. In (Table 2). Patency of the aqueduct was compromised at the eight subjects (Subjects 1, 2, 15, 20, 21, 31, 19 and 42), the level of the superior (Fig. 3A and B) or inferior colliculus superior medullary velum also appeared thickened, with (Fig. 3C and D), or more caudally at the level of the superior non-visualization or severe narrowing of the aqueduct at that cerebellar peduncles (Fig. 4). All subjects with aqueductal stenosis level (Fig. 3E and F). In four subjects (Subjects 1, 15, 20 and had fusion of the overlying structures at the level of obstruction 35), the aqueductal stenosis was more diffuse, extending from Downloaded from http://brain.oxfordjournals.org/ at Cleveland Clinc on May 1, 2012

Figure 3 Aqueductal stenosis in RES. (A and B) Obstruction at the level of the superior colliculi (white arrow in A and black arrow in B), which are fused across the midline in Subject 29. Note the shunted hydrocephalus and severely dysgenetic corpus callosum with intact rostrum. (C and D) Obstruction at the level of the inferior colliculi (arrowheads), which are fused across the midline in Subject 8. Note the severe hydrocephalus with funnelling of the aqueduct, the retrocerebellar fluid collection and hypoplasia of the cerebellum and pons. (E and F) Obstruction at the junction of the aqueduct and fourth ventricle in Subject 2. The superior medullary velum is thickened (white arrow) and the superior cerebellar peduncles are angled medially. Note the mild inferior cerebellar ectopia in (E). Note the tiny focus of low signal within the superior aspect of the fourth ventricle (adjacent to the white arrow in F) and within the lateral ventricles in E is related to air from recent shunt manipulation. (G–I) Obstruction from the level of the thickened posterior commissure (bracket) to the level of the superior cerebellar peduncles with midline fusion of the superior colliculi (black arrow) in Subject 1. Note partial agenesis of the corpus callosum in (G) and the absence of hydrocephalus, despite complete obliteration of the cerebral aqueduct on imaging. Note also the

severely hypoplastic mammillary bodies in Subjects 8, 2 and 1 (long white arrows in C, E and G). (A, C, E and G) sagittal T1;(B, F and I) axial T1;(D, H) axial T2. 1378 | Brain 2012: 135; 1370–1386 G. E. Ishak et al. Downloaded from http://brain.oxfordjournals.org/ Figure 4 Ectopic cerebellar tissue and superior cerebellar peduncle configuration in RES. (A and B) Subject 34, partial RES. There is anterior vermis (arrows) that extends just posterior to the confluence of the superior cerebellar peduncles. The superior medullary velum is seen superior to the arrow in (B), and inferior to the inferior colliculus. (C and D) Subject 39, partial RES. There is anterior vermis that extends beyond the confluence of the superior cerebellar peduncles into the most caudal aspect of the cerebral aqueduct, reflecting a more severe pattern of ectopic tissue. No associated aqueductal stenosis. The superior medullary velum is not seen in this case, which may relate to the extent of the ectopic cerebellar tissue, in addition to thick slices. (E and F) Subject 17, complete RES. The anterior vermis is not visualized in this case, however, small ectopic tissue is seen at—and obscuring—the confluence of the superior cerebellar peduncles, which appear fused at the midline. This is causing obstruction at the most caudal aspect of the cerebral aqueduct. The superior medullary velum is present superior to the arrow in (F). After shunting, the subject developed an intraventricular cyst (asterisk). at Cleveland Clinc on May 1, 2012

the level of the superior cerebellar peduncle up to the posterior Subjects 2, 17 and 25 (Fig. 4E and F). In Subjects 2 and commissure (Fig. 3G–I). In particular, Subject 15, and to a lesser 25 (and in Subjects 34 and 39 described below), this tissue extent Subject 35, had a mass-like fusion of all of the midbrain appeared to be foliated and continuous with the anterior including the superior cerebellar peduncle, superior medullary vermis, so it could represent ectopic cerebellum. velum, inferior colliculus, superior colliculus—as noted by other (4) Absence of aqueductal stenosis in Subjects 5, 6, 9, 13, 34 authors (Garfinkle, 1996; Pasquier et al., 2009)—and extending and 39, despite ectopic tissue at the convergence of the cranially with fusion of the thalami and hypothalami (Fig. 5A and superior cerebellar peduncles in Subject 34 (Fig. 4A and B) B). We did not observe deficiency of the superior medullary velum and within the aqueduct in Subject 39 (Fig. 4C and D). as described previously (Gross, 1959; Barth, 2008). In 19 subjects, the superior cerebellar peduncles were closer to the midline than normal and angled more medially (Fig. 4). This Tectum abnormal superior cerebellar peduncle configuration was accom- Fusion of the tectum across the midline, defined as the absence of panied by several different patterns of abnormalities of the the two prominences of the superior and/or inferior colliculi on aqueduct. axial images (Fig. 3B, D and I), was present in 16 subjects (Subjects 1, 8, 10, 14, 15, 19–21, 26, 29, 31, 35, 38, 40, 41 (1) Non-visualization of the entire aqueduct, associated with and 42). Craniocaudal fusion of the colliculi was demonstrated thickening of the posterior commissure and superior medul- on sagittal sections in at least four subjects (Subjects 1, 20, 21 lary velum, and midline fusion of the superior and inferior and 40). This may under-represent the true frequency, as cranio- colliculi, in Subjects 1, 15, 20 and 35 (discussed below). caudal fusion may be difficult to identify when the sagittal sections (2) Narrowing of the aqueduct at the level of the tectum in are not thin enough. In contrast, Subject 32 had ectopic tissue Subjects 10, 19, 21, 31, 40 and 42 (Table 2). within the cerebral aqueduct seen on thin cuts at the level of (3) Aqueductal stenosis at the level of the superior cerebellar the inferior colliculus, without collicular fusion, as noted by other peduncles caused by a small focus of ectopic tissue seen at authors (Takano et al., 2010). The absence of ventriculomegaly the convergence of the superior cerebellar peduncles in and funnelling of the aqueduct excluded aqueductal stenosis in Rhombencephalosynapsis neuroimaging Brain 2012: 135; 1370–1386 | 1379 Downloaded from http://brain.oxfordjournals.org/ at Cleveland Clinc on May 1, 2012 Figure 5 Supratentorial abnormalities associated with RES. (A and B) Mass-like fusion of the mesencephalon, diencephalon and hypothalamus (A) with occipital holoprosencephaly (black arrow) and aplasia of the ventricles (B) in Subject 15. (C) Middle interhemi-

spheric fusion (arrowhead) in Subject 14. (D) Absent olfactory bulbs in Subject 17 (white arrows). (A) coronal T2;(B) axial T1;(C) sagittal T1;(D) axial T2.

this subject. Subject 12 had deformed tectum with the absence of abnormalities were seen in subjects without hydrocephalus and fusion of the colliculi and hypoplasia of the midbrain. some of the abnormalities are not typically caused by hydroceph- alus. The septum pellucidum was absent in 26 subjects, including all with obstructive hydrocephalus (Table 3). It was also absent in Forebrain four subjects with ventriculomegaly (three with mild, one with mild/moderate) and three without ventriculomegaly. To Ventriculomegaly and hydrocephalus determine whether the septum forms and later disappears, we Almost half of the subjects (19/42) had obstructive hydrocephalus evaluated prenatal imaging in two subjects with mild ventriculo- due to aqueductal stenosis requiring shunting (Table 3). Of the megaly (Subjects 18 and 23). Subject 18 had a normal septum other subjects, two had moderate, four had mild and 17 had no pellucidum at 22 weeks gestation that was then partially deficient ventriculomegaly. Of note, the aqueduct did not appear patent in at 32 weeks. The ventricles were at worst moderately dilated three subjects; Subject 1 had mild ventriculomegaly and Subjects (16 mm atrial measurement) in utero and only mildly dilated on 15 and 35 did not have visible lateral ventricles, as noted by other post-natal imaging. Subject 23 had a partially absent septum authors (Garfinkle, 1996) (Fig. 5B). The severity of ventriculome- posteriorly on foetal MRI at 18 weeks gestation with moderate galy/hydrocephalus was not strongly correlated with severity of ventriculomegaly (16 mm atrial measurement). A cranial ultra- RES or the clinical diagnosis. sound at birth revealed a partially absent septum with mild prom- inence of the ventricles, but the septum was completely absent on Other forebrain abnormalities MRI by 1 month of age. Although hydrocephalus can account for a number of the fore- The corpus callosum was abnormal in 30/41 subjects with brain imaging abnormalities, our data support a role for primary adequate imaging (Table 4). Most commonly, the corpus callosum forebrain developmental defects as well, since a number the was severely thinned and dysplastic in association with obstructive 1380 | Brain 2012: 135; 1370–1386 G. E. Ishak et al.

Table 3 Forebrain features in subjects with RES

Subject Aqueductal VM Absent Abnormal Abnormal Abnormal Fused Absent stenosis septum anterior temporal mammillary fornices olfactory commissure cortex bodiesa bulbs 1+b ++ À ++ +À 8 + +++ + À ++ ++ 15 + * NA NA NA NA NA À 17 + +++ + À ++ ++ 27 + +++ + NA NA NA NA NA 35 + * NA NA NA NA NA NA 36 ÀÀÀÀ + ÀÀÀ 38 + +++ + + + + + À 41 + +++ + + + + + + Totals 8/9 6/9 6/8 2/6 6/6 5/6 5/6 3/7 3 ÀÀÀÀÀÀÀÀ 2 + +++ + + + + + À 5 ÀÀÀÀÀÀÀÀ

10 + +++ + + + À +NA Downloaded from 13 À ++ ÀÀ + ÀÀÀ 20 + + + + + + + + NA + 21 + +++ + + + À + À 26 + +++ + + + ÀÀÀ 31 + +++ + À ++ À NA http://brain.oxfordjournals.org/ Totals 6/9 7/9 6/9 5/9 7/9 3/9 3/8 1/7 12 + +++ + + À + Single À 14 + +++ + À + À ++ 18 À ++ ÀÀÀ+ À 19 + + + + + + + + NA + 24 ÀÀÀÀ + ÀÀNA 25 + +++ + + + + + À 39 ÀÀ+ ÀÀ++À 42 + +++ + + + ÀÀÀ at Cleveland Clinc on May 1, 2012 Totals 5/8 6/8 7/8 4/8 5/8 4/8 4/7 2/7 4 À ++ + À + À + À 6 ÀÀÀ+ ÀÀ ÀÀ 7 ÀÀÀÀÀÀÀÀ 9 ÀÀ+ À + À + À 11 ÀÀÀ+ ÀÀ ÀNA 16 ÀÀÀNA ÀÀ ÀNA 23 À ++ À + À + À 28 + +++ + + + À ++ 29 + +++ + + + À Single + 30 ÀÀÀÀÀÀÀ+ 32 ÀÀÀÀÀÀÀÀ 37 ÀÀÀÀÀÀÀÀ Totals 2/12 4/12 5/12 4/11 5/12 0/12 4/12 3/10 22 ÀÀÀÀÀÀÀÀ 33 À + ÀÀ + ÀÀÀ 34 ÀÀ+ ÀÀÀ+ À 40 + +++ + + + + + À

NA = not available; single = single unilateral fornix (rather than midline fusion); + = abnormality present; À = abnormality not present; VM = ventriculomegaly. In the column ‘VM’: + = mild ventriculomegaly; + + = moderate ventriculomegaly; + + + = severe or shunted ventriculomegaly; * = absent lateral ventricles. a Hypoplastic aqueduct did not appear patent but the ventriculomegaly was mild. b Aqueduct did not appear patent but the ventriculomegaly was mild. Rhombencephalosynapsis neuroimaging Brain 2012: 135; 1370–1386 | 1381

Table 4 Relationship between ventriculomegaly/hydrocephalus and callosal abnormalities in subjects with RES

Subject Aqueductal VM Corpus callosum phenotype Proposed stenosis mechanism 1(+)a + Diffuse thinning and partial posterior agenesis DM 8 + + + + Diffuse thinning HRD 15 + * Absent DM 17 + + + + Diffuse thinning HRD 27 + + + + NA NA 35 + * Total agenesis DM 36 ÀÀNormal – 38 + + + + Diffuse thinning HRD 41 + + + + Diffuse thinning HRD Totals 8/9 6/9 3 ÀÀDiffuse thinning DM 2 + + + + Diffuse thinning HRD 5 ÀÀNormal – 10 + + + + Absent rostrum with dysgenesis of post corpus callosum DM 13 + + + + Absent rostrum with dysgenesis of post corpus callosum DM Downloaded from 20 + + + + Absent rostrum with dysgenesis of post corpus callosum DM 21 + + + + Absent rostrum with dysgenesis of post corpus callosum DM 26 + + + + Absent rostrum with dysgenesis of post corpus callosum DM 31 + + + + Diffuse thinning HRD

Totals 6/9 7/9 http://brain.oxfordjournals.org/ 12 + + + + Diffuse thinning HRD 14 + + + + Rostrum present with dysgenesis of post corpus callosum HRD 18 À + Mild thinning DM 19 + + + + Absent rostrum with dysgenesis of post corpus callosum DM 24 ÀÀDiffuse thinning and partial posterior agenesis DM 25 + + + + Diffuse thinning HRD 39 ÀÀDiffuse thinning (mild) DM 42 + + + + Diffuse thinning HRD Totals 5/7 6/7 at Cleveland Clinc on May 1, 2012 4 À + + Diffuse thinning DMb 6 ÀÀNormal – 7 ÀÀNormal – 9 ÀÀNormal – 11 ÀÀNormal – 16 ÀÀNormal – 23 À + Diffuse thinning DMb 28 + + + + Absent rostrum with dysgenesis of post corpus callosum DM 29 + + + + Rostrum present with dysgenesis of post corpus callosum HRD 30 ÀÀNormal – 32 ÀÀNormal – 37 ÀÀNormal – Totals 2/12 4/12 22 ÀÀNormal – 33 À + Thickened DM 34 ÀÀDiffuse thinning (mild) DM 40 + + + + Rostrum present with severe dysgenesis of post corpus callosum HRD

DM = developmental malformation; HRD = hydrocephalus-related deformation; NA = not available; + = abnormality present; À = abnormality not present; VM = ventriculomegaly. In the column "VM": + = mild ventriculomegaly; + + = moderate ventriculomegaly; + + + = severe or shunted ventriculomegaly; * = absent lateral ventricles. a Aqueduct did not appear patient but the VM = ventriculomegaly was mild. b Hydrocephalus was not severe enough to explain corpus callosum thinning. hydrocephalus; however, obstructive hydrocephalus does not ex- obstructive hydrocephalus, also supporting the role of a primary plain the absence of the rostrum in six of these subjects, and we developmental defect. hypothesize that they also have a primary defect in callosal devel- The mammillary bodies were not observed and considered to be opment. Similarly, the corpus callosum was variably thin or par- hypoplastic or absent in 13/39 subjects with adequate imaging tially/completely absent in a total of nine subjects without (Fig. 3C, E and G), as noted by other authors (Schachenmayr 1382 | Brain 2012: 135; 1370–1386 G. E. Ishak et al. and Friede, 1982). Subjects 15 and 35 had fusion of the hypo- Neurodevelopmental outcome thalamus including the mammillary bodies (Garfinkle, 1996). The fornices were fused in 18/37 subjects, all of whom also had absent We were able to obtain sufficient clinical information to classify the septum (Fig. 2N). The fornices could not be visualized in five sub- severity of the abnormal neurodevelopmental outcome as mild, jects (Subjects 15, 19, 20, 27 and 35), while a single thinned out moderate or severe in 37 subjects (88%), losing five to follow-up. fornix was seen in two subjects (Subjects 12 and 29); these find- Clinical category was correlated with developmental outcome: the ings could be related to severe distortion due to hydroceph- subjects with RES + HPE had poorer outcomes (two severe, one alus destroying and/or limiting visualization of the fornices. moderate), as did the subjects with RES + VACTERL features Subjects 15 and 35 had the absence of the ventricles, septum (three severe, two moderate), while the subjects with GLH (none and fornices. severe, four moderate, 12 mild) and RES NOS (one severe, four We noted that the anterior commissure was absent or hypo- moderate, eight mild) had less severe outcomes (P-value = 0.0012, plastic in 16/38 subjects: 14 with obstructive hydrocephalus and Fisher’s exact). two without ventriculomegaly, indicating that most anterior com- The severity of RES was also correlated with the severity of the outcome: the subjects with complete RES had poorer outcomes missure abnormalities are secondary to hydrocephalus, rather (two severe, three moderate, three mild), while the subjects with being due to abnormal brain development. Assessment of the an- mild RES had less severe outcomes (none severe, two moderate, terior commissure was challenging in a number of subjects due to eight mild), with the moderate and severe RES subjects falling in immature myelination and/or inadequate imaging quality. Downloaded from between (one-sided P = 0.0028, Kendall tau-b). Of note, each of Similarly, the medial temporal lobes were thin and excessively the components of RES severity (absent posterior vermis, absent an- folded in 25/39 subjects, including 19 with obstructive hydroceph- terior vermis and absent nodulus) did not correlate with the severity alus and six without hydrocephalus. The pituitary gland was un- of neurodevelopmental outcome. The only other imaging features remarkable in all subjects. that correlated with neurodevelopmental outcome were severity of We did not identify cortical abnormalities except in Subjects 14, http://brain.oxfordjournals.org/ ventriculomegaly (P 5 0.0001, Kendall tau-b), aqueductal stenosis 15 and 35 with variants of HPE (see below), Subjects 2 and 20 (P = 0.0054, Fisher’s exact), fused colliculi (P = 0.0087, Fisher’s with bilateral frontal polymicrogyria and heterotopia and Subjects exact) and abnormal temporal cortex (P = 0.016, Fisher’s exact). 7 and 22 with right frontal periventricular heterotopia.

Holoprosencephaly Discussion Subjects 14, 15 and 35 also had abnormal midline continuity of RES is an uncommon malformation of the cerebellum character- posterior cerebral hemispheres associated with dysplastic or even ized by loss of cerebellar midline structures such that the right and at Cleveland Clinc on May 1, 2012 absent lateral ventricles. Subjects 15 and 35 had diencephalon- left cerebellar hemispheres and peduncles are fused. Although par- telencephalosynapsis with an unusual but similar pattern of tial RES and midbrain involvement have been previously reported fusion involving the occipital lobes, thalami, basal ganglia and (Obersteiner, 1916; Gross, 1959; Shaw, 1995; Demaerel et al., hypothalamus with absent or nearly absent ventricles (Fig. 5A 2004; Pavone et al., 2005; Alkan et al., 2009; Pasquier et al., and B), as described by other authors (Garfinkle, 1996). In 2009), we provide the first evidence for an ordered spectrum of Subject 35, fusion of the occipital lobes extended more superiorly RES severity that correlates with the severity of midbrain involve- and anteriorly to involve posterior inferior frontal and parietal ment. Our analysis of brain-imaging studies in 42 previously un- lobes. Both Subjects 15 and 35 had severely dysplastic infolded reported patients provides systematic evidence for the previously cortex in the same regions. The falx was present but deficient in reported associations between RES and hydrocephalus, mesence- the frontal lobes, with separation of the frontal, temporal and phyalosynapsis and holoprosencephaly. In addition, we identified anterior parietal lobes. This is distinct from the known HPE spec- RES in almost 10% of patients with aqueductal stenosis, indicating trum, in which the frontal lobes are more severely affected than that RES is more common than previously appreciated and should the occipital lobes. In contrast, Subject 14 had a much smaller area be specifically evaluated in patients with aqueductal stenosis. of continuity between the posterior frontal lobes across the mid- Strikingly, the clinical categories defined in the literature as well line (Fig. 5C) resembling the ‘middle interhemispheric’ variant of as the severity of RES defined in this work correlate strongly with HPE in location; however, the frontal polymicrogyria and dysplas- neurodevelopmental outcome. tic ventricles differ from other reports of this malformation. Not surprisingly, these subjects all had aqueductal stenosis and sub- stantial midline fusion of the midbrain. Spectrum of imaging severity The olfactory lobes were present in Subjects 14 and 35, but In our series, most patients fit into an ordered spectrum of sever- absent in Subject 15. The olfactory bulbs were also absent in ity, from loss of posterior vermis, to loss of posterior and anterior eight additional subjects (Fig. 5D), possibly representing a form vermis, to loss of posterior and anterior vermis plus nodulus. Prior fruste of telencephalosynapsis. Due to limitations in the clinical studies have not noted the presence of anterior vermis, and this imaging studies, it was not possible to determine whether the may be under-reported due to young age, lack of spatial reso- olfactory bulbs were hypoplastic in six subjects. The olfactory lution and other technical issues. The anterior vermis, if present, is lobes were present and appeared normal in all subjects. usually seen most ventrally and cranially. Careful evaluation of the Rhombencephalosynapsis neuroimaging Brain 2012: 135; 1370–1386 | 1383 dorsal and posterior vermis is required to diagnose the mildest hydrocephalus in 45% of our subjects. In our series, hydrocephalus forms of partial RES. This may indicate a gradient of severity is primarily related to aqueductal stenosis (seen in 52% of patients in more severe dorsally and less severe ventrally. this series) with obstruction at different points along the aqueduct in different subjects. Obstruction may occur more cranial to the aque- Cerebellar and posterior fossa size duct in patients with thalamic fusion (Kepes et al., 1969; Simmons et al., 1993) or possibly distal to the aqueduct in patients with crowd- We report the first association between RES and inferior cerebellar ing of the posterior fossa (with or without ectopia). ectopia (Chiari I malformation) in 24% of the subjects and confirm an Surprisingly, the aqueduct did not appear patent in two subjects association between RES and superior cerebellar ectopia in 60% of with HPE and aventriculy, as well as one subject with mild ven- the subjects. We attribute this to disproportionately small posterior triculomegaly. We hypothesize that the absence of hydrocephalus fossa size relative to cerebellum size. In partial RES, the transverse in these subjects may represent inadequate formation of cerebro- diameter of the cerebellum was variably small with relatively spinal fluid, patency of the aqueduct below the resolution of ima- increased craniocaudal dimension, reflecting the severity of superior ging and/or ectopic routes for cerebrospinal fluid drainage. herniation. In several subjects, superior ectopia was exacerbated by ventriculoperitoneal shunting; however, ectopia was not more common or pronounced in subjects with complete RES, likely be- Rhombencephalosynapsis and other cause the cerebellum was markedly hypoplastic in these subjects. forebrain abnormalities Downloaded from The cause of cerebellar ectopia in the setting of RES is not clear, but could reflect abnormal development of the cerebellum, abnormal In general, the severity of RES did not correlate well with the fre- development of the surrounding meninges and bone, hydrocephalus quency and severity of forebrain imaging abnormalities, most of and shunting, or a combination of these factors. which seem to correlate better with the degree of hydrocephalus, with notable exceptions. The septum pellucidum was absent in all Rhombencephalosynapsis, mesence- subjects with obstructive hydrocephalus implicating a destructive http://brain.oxfordjournals.org/ process in these patients. In contrast, the septum was missing in phalosynapsis and holoprosencephaly 17% of the subjects without obstructive hydrocephalus, and we A variety of midbrain abnormalities have been reported in patients were able to document loss of the septum between mid-gestation with RES, including aqueductal stenosis, midline fusion of the col- and the neonatal period in two subjects without overt hydroceph- liculi (Obersteiner, 1916; Schachenmayr and Friede, 1982; alus. Thus, absent septum is not likely to be due to a destructive Jellinger, 2002; Yachnis, 2002) and abnormal configuration of process in these subjects and may be due to defects in proliferation, the superior cerebellar peduncles (Schachenmayr and Friede, survival and/or fate determination of septal cells. 1982; Shaw, 1995). In line with these prior reports, we observed Although the posterior corpus callosum is strikingly thinned and at Cleveland Clinc on May 1, 2012 midbrain abnormalities that ranged from isolated fusion of inferior dorsally deviated in many patients with RES and hydrocephalus, or superior colliculi, to a mass like fusion of the entire mesenceph- we do not think that this finding is specific for RES, since we have alon, extending from the superior cerebellar peduncles up to the observed it in patients with hydrocephalus but not RES. level of the posterior commissure, reflecting a variable degree of Conversely, the corpus callosum was abnormal in multiple subjects mesencephalosynapsis in 52% of our subjects with RES. Fusion of without hydrocephalus, indicating that primary developmental de- the colliculi occurs both across the midline (involving both superior fects of the corpus callosum occur as well. Furthermore, several and inferior colliculi) or craniocaudally (fusing superior and inferior other forebrain structures (anterior commissure, temporal cortex colliculi). We also observed ectopic (likely cerebellar) tissue asso- and mammillary bodies) were often abnormal in patients with ciated with the inferior colliculus, superior medullary velum and severe hydrocephalus. Therefore, these imaging abnormalities are superior cerebellar peduncles, as previously described by Shaw most often due to deformation and/or destruction from hydro- (1995) and Takano et al. (2010). cephalus and not primarily due to intrinsic defects in the develop- The pattern of forebrain fusion (HPE) in our most severely affected ment of these forebrain structures. subjects was strikingly atypical, predominantly involving the occipital lobes. The absence of the olfactory bulbs in almost 30% of our sub- jects also points to forebrain defects in the HPE spectrum. Based on Limitations these data, RES should be viewed as part of a malformation that This study has several limitations. Despite being the largest RES typically includes the midbrain and sometimes extends to the di- cohort to date, the number of subjects is still quite small, especially encephalon and occasionally telencephalon. In our series, the sever- for the VACTERL and HPE groups. In addition, the cohort is likely ity of RES correlated well with these midbrain and forebrain defects, skewed towards more severely affected subjects with associated likely indicating a shared biological mechanism. abnormalities due to ascertainment bias. This is particularly relevant for estimating the prevalence of findings like aqueductal stenosis in Rhombencephalosynapsis and patients with RES. Due to recruitment of subjects from all over the world, the imaging was performed at different institutions using dif- hydrocephalus ferent protocols resulting in variable imaging quality. Similarly, we Barth (2008) reported that the minimum rate of hydrocephalus from were unable to perform standardized developmental and neuro- his review of the literature was 25%, while we observed obstructive logical assessments, limiting the quality of the outcome data. 1384 | Brain 2012: 135; 1370–1386 G. E. Ishak et al.

Clinical applications features, as well as the subjects with HPE, had more severe mid- brain involvement; however, with the exception of HPE, the clin- Our work also indicates that, although rare, RES may be substan- ical categories cannot be distinguished based on the brain imaging tially more common than previously thought. Prior to this work, features, in agreement with prior publications (Toelle et al., 2002; 10 patients with RES had been identified at our institution. With Poretti et al., 2009). heightened awareness and additional scrutiny of patients RES is a fascinating malformation that differs from all other with aqueductal stenosis, we identified five additional patients vermis hypoplasias, in which the cerebellar hemispheres are sepa- with RES of various degrees of severity. We were unable to evalu- rated rather than fused. Consanguinity and recurrences point to an ate for RES in patients who only had CT scans, so we may have autosomal recessive cause in a minority of families, while the pre- missed additional patients with RES who were not imaged by MRI. dominance of sporadic patients is consistent with de novo domin- Based on our findings and the literature, RES should be specifically ant mutations although a possible association with maternal evaluated by MRI in patients with aqueductal stenosis, absent diabetes has been noted (Garfinkle, 1996; Sergi et al., 1997). septum pellucidum, small cerebellum, superior cerebellar hernia- While the developmental basis of RES remains unknown, a tion and/or the absence of the normal folial pattern of the cere- number of developmental mechanisms have been proposed to ex- bellar vermis on the sagittal image. Additional signs of RES on the plain RES. Numerous studies have shown that defects in dorsal mid- sagittal view include the following: absence of the primary fissure, line signalling lead to malformations of the brainstem and abnormal rounded contour of the fourth ventricle (rather than cerebellum. The mouse cerebellar hypoplasia and human Dandy– Downloaded from triangular contour of fastigium) and deficient indentation of the Walker syndrome phenotypes associated with mutations in Zic1 fourth ventricle by the nodulus. On axial and coronal images, RES and Zic4, Foxc1, as well as the mouse cerebellar phenotype asso- can be identified by continuity of the cerebellar folia and fissures ciated with mutations in Lmx1a, serve as good examples (Millonig across the midline, particularly dorsally. In addition, the posterior et al., 2000; Grinberg et al., 2004; Aldinger et al., 2009; Chizhikov cerebellar incisura and vallecula may be absent, and the dentate et al., 2010). Other studies have shown that defects in dorsal midline http://brain.oxfordjournals.org/ nuclei and superior cerebellar peduncles may be apposed. DTI has signalling can result in HPE, as recently demonstrated with mutations been shown to demonstrate the absence of the transversely ori- of genes in the fibroblast growth factor and bone morphogenetic ented white matter tracts of the vermis and vertical orientation of protein pathways (Shimogori et al., 2004; Fernandes et al., 2007; the tracts in the medial aspect of the fused cerebellum. In add- Geng and Oliver, 2009; Ramocki et al., 2011). Alternatively, several ition, DTI can demonstrate that the deep cerebellar nuclei and the authors (Tan et al., 2005; Gomy et al., 2008; Poretti et al., 2008; superior cerebellar peduncles are oriented more medially; how- Fernandez-Jaen et al., 2009) have proposed that a mouse with a ever, it cannot distinguish close apposition from actual fusion missense mutation in the lysosomal acid phosphatase (Acp2) gene (Widjaja et al., 2006; Merlini et al., 2011). (Mannan et al., 2004) might represent a model for GLH due to cere- RES may be misdiagnosed in patients with Chiari II malforma- at Cleveland Clinc on May 1, 2012 bellar and skin involvement; however, these mice display defective tion (Guntur Ramkumar et al., 2010). Prenatally, cerebellar hypo- hair and cerebellar development without true alopecia or RES. Our plasia and ventriculomegaly, with or without the absence of the analysis of human RES confirms a link between human HPE and RES septum pellucidum, should prompt careful evaluation for hindbrain and shows that both dorsal midbrain- and hindbrain-derived struc- fusion (Litherland et al., 1993; Napolitano et al., 2004). tures are more severely affected than ventral-derived structures. Perhaps most important are the correlations between RES severity These observations collectively suggest that defects of dorsally ex- and clinical diagnosis with neurodevelopmental outcome. The iden- pressed genes cause the combination of HPE and RES, and by exten- tification of RES on brain imaging should prompt additional evalu- sion that defects in related dorsal signalling pathways may cause ations for other malformations, particularly vertebral, rib, renal and other forms of RES. limb abnormalities, as well congenital heart disease. Our data greatly improve the quality of information available for counselling families about prognosis. Patients with HPE, VACTERL features and/or severe to complete RES are at substantially increased risk for moder- Acknowledgements ately to severely abnormal neurodevelopmental outcome. This infor- mation allows families to set appropriate expectations, anticipate The authors thank all of the patients, their families and many needs and develop plans for treatment. In the future, this clinical referring physicians for participating in this study. data may help direct specific genetic testing and/or help with the interpretation of genome-wide genetic testing data. Funding Genetics and pathogenesis NIH (grants KL2-RR025015 to D.D. and T.C.R.); (R01-NS050375 Several clinical categories have been proposed for patients with to W.B.D. and K.J.M.); CHLA-USC Child Health Research Career RES, including GLH, a VACTERL H-like presentation and RES asso- Development Program (K12-HD05954 to P.A.S.), by The Arc of ciated with holoprosencephaly (Gomez, 1979; Lopez-Hernandez, Washington Trust Fund (to D.D.); Harold Amos Faculty 1982; Pasquier et al., 2009). Intriguingly, the severity of RES and Development Program through the Robert Wood Johnson the other brain imaging abnormalities did not correlate with these Foundation (to P.A.S.); NIH/NINDS T32 (Grant 5T32NS051171 clinical subcategories in our cohort. Several subjects with VACTERL to H.T.). Rhombencephalosynapsis neuroimaging Brain 2012: 135; 1370–1386 | 1385

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