Clin Genet 2014: 86: 521–529 Printed in Singapore. All rights reserved

© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd CLINICAL GENETICS doi: 10.1111/cge.12391

Original Article

Olfaction evaluation and correlation with brain atrophy in Bardet-Biedl syndrome Braun J.-J., Noblet V., Durand M., Scheidecker S., Zinetti-Bertschy A., Foucher J., Marion V., Muller J., Riehm S., Dollfus H., Kremer S. Olfaction evaluation and correlation with brain atrophy in Bardet-Biedl syndrome. Clin Genet 2014: 86: 521–529. © John Wiley & Sons A/S. Published by John Wiley & Sons Ltd, 2014 Bardet-Biedl syndrome (BBS) is a well-recognized ciliopathy characterized by cardinal features namely: early onset retinitis pigmentosa, polydactyly, obesity, hypogonadism, renal and cognitive impairment. Recently, disorders of olfaction (anosmia, hyposmia) have been also described in BBS patients. Moreover, morphological brain anomalies have been reported and prompt for further investigations to determine whether they are primary or secondary to peripheral organ involvement (i.e. visual or olfactory neuronal tissue). The objective of this article is to evaluate olfactory disorders in BBS patients and to investigate putative correlation with morphological cerebral anomalies. To this end, 20 BBS patients were recruited and evaluated for olfaction using the University of Pennsylvania Smell Identification Test (UPSIT). All of them underwent a structural magnetic resonance imaging (MRI) scan. We first investigated brain morphological differences between BBS subjects and 14 healthy volunteers. Then, we showed objective olfaction disorders in BBS patients and highlight correlation between gray matter volume reduction and olfaction dysfunction in several brain areas. Conflict of interest

The authors declare that they have no conflict of interest.

J.-J. Brauna,† , V. Nobletb,† , M. Durandc , S. Scheideckerc , A. Zinetti-Bertschyd,e , J. Foucherb,d , V. Marionf , J. Mullerb,g,h , S. Riehmi , H. Dollfusc,f and S. Kremerb,j a Service

ORL-CCF, Hôpitaux Universitaires de Strasbourg, Strasbourg, France , b Laboratoire ICUBE, UMR CNRS 7357, Université de Strasbourg, Strasbourg, France , c Service de Génétique Médicale, Centre de Référence pour les Affections Rares en Génétique Ophtalmologique (CARGO), Hôpitaux Universitaires de Strasbourg, Strasbourg, France , d Pôle de Psychiatrie et Santé Mentale, Hôpitaux Universitaires de Strasbourg, Fédération de Médecine Translationnelle de Strasbourg (FMTS) , e Laboratoire de Neuropsychologie cognitive et physiopathologie de la schizophrénie, INSERM U1114, Fédération de Médecine Translationnelle de Strasbourg (FMTS) , f Laboratoire de Génétique Médicale, INSERM U1112, Fédération de Médecine Translationnelle de Strasbourg (FMTS), Université de Strasbourg, Strasbourg, France , g Laboratoire de Diagnostic Génétique, Hôpitaux Universitaires de Strasbourg, Strasbourg, France , h Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), UMR 7104 and INSERM U964 , i Service de Radiologie 1, Hôpitaux Universitaires de Strasbourg, Fédération de Médecine Translationnelle de Strasbourg (FMTS) , and j Service de Radiologie 2, Hôpitaux Universitaires de Strasbourg, Fédération de Médecine Translationnelle de Strasbourg (FMTS), Université de Strasbourg, Strasbourg, France † J. J. B.

and V. N. have equally contributed to this work. Key words: Bardet-Biedl syndrome – brain atrophy – MRI – olfaction

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Braun et al. Corresponding author: Dr Stéphane Kremer, Service de Radiologie 2, Hôpital de Hautepierre, Avenue Molière, CHU de Strasbourg, 67098 Strasbourg, France. Tel.: +33 3 88 12 83 91; Fax: +33 3 88 12 71 18; e-mail: [email protected] Received 20 December 2013, revised and accepted for publication 27 March 2014

Bardet-Biedl syndrome (BBS) is a rare autosomal recessive disease characterized by cardinal features such as polydactyly, obesity, hypogonadism, renal and cognitive impairment, and in which neurosensory disorders including early retinitis pigmentosa and dysosmia can also be displayed. To date 19 BBS genes have been identified (1). Although the clinical description of the syndrome is well-defined, the detailed physiopathogenesis that underpins the condition at the level of the brain remains to be specified. From a pathophysiological point of view, this syndrome is related to disorders in primary cilia, ciliogenesis and planar cell polarity. Cilia are involved in the exploration of the internal and external environment through chemo-, photo- and mechanosensing functions. In addition to retina (photoreceptor cell) and cochlea (kinocilium), such sensory cilia are found in olfactory cells (2). Disorders of olfaction (anosmia, hyposmia) have been recently described in BBS patients (3–6) and warrant further investigation. The ciliary disorders of olfactory neurons could explain the dysosmia observed in BBS patients. Tadenev et al. showed 47% of complete or partial anosmia in 19 BBS patients using the Brief Smell ID Test (12 odors) (6). Iannacone et al. reported two cases of anosmia in BBS4 patients using the University of Pennsylvania Smell Identification Test (UPSIT) technique (Sensonics Inc., Haddon Heights, NJ) (4). More recently Brinckman et al. reported a series of 20 subjects among 42 BBS patients with olfaction evaluation by the UPSIT (3): six mild hyposmia, six moderate hyposmia, two severe hyposmia and six anosmia. Finally, for another ciliopathy, namely the Leber Congenital Amaurosis, Papon et al. described olfactory disorders in three of seven patients using the Brief Smell ID Test, which were associated with ultrastructural ciliary abnormalities (7). Moreover, olfactory impairments have been described in KO mice deleted for the BBS1, BBS4 (5) and BBS8 (6) genes. These disorders are associated with: (i) a loss of BBS proteins with reduced ciliary edge or a total loss of cilia in olfactory cells, (ii) a disruption of the dendritic microtubule network with retention of olfactory ciliary proteins in dendrites and cell bodies and (iii) aberrant axonal connections to the olfactory bulb (6). As opposed to the visual or auditory acuity, the exploration of the olfaction and its disorders remains difficult, as it requires the cooperation of the subject and is deeply

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related to its olfactory education and memory. Many olfactometry techniques have been described in order to objectify the reality of dysosmia and to characterize olfactory impairments. These tests face many issues: the calibration of the olfactory stimulus (choice of odorants, dosimetry of olfactory stimuli, sniffing, and retrovelar component of olfaction which is usually unexplored), the possible interaction between the trigeminal component and the purely olfactory component of a given stimulus and the dependence of the olfactory discrimination and identification ability with the ‘olfactory culture’ of patients. Thus, among the psychophysical methods, that are the only ones used in clinical practice, we can distinguish the following techniques (8–12): (1) Determination of the odor threshold corresponding to the lowest concentration of an odorant molecule that can be detected with a great inter- and intra-individual variability (ascending, descending or random thresholds) (2) Suprathreshold evaluation of the olfaction based on the perception and qualitative identification of odors, which are sometimes difficult even for a normosmic subject, thus requiring the use of identification tests with multiple choice in order to limit bias in the response such as the UPSIT. More recently, magnetic resonance imaging (MRI) cerebral volumetric studies have showed that alteration of sensory input (vision, audition, and olfaction) induces gray matter (GM) and white matter (WM) remodeling (13–16). It has been shown that hyposmia induces secondary GM volume loss in the insular cortex, anterior cingulate cortex, orbitofrontal cortex, cerebellum, fusiform gyrus, precuneus, middle temporal gyrus and piriform cortex associated to WM volume loss underneath the insular cortex, in the cerebellum and middle frontal gyrus (17). Most of the affected WM areas correspond to fibertracts that are connected to the affected GM areas (17). Similar GM alterations have been reported in anosmia but to a larger extent affecting additionally nucleus accumbens with adjacent subcallosal gyrus, middle cingulate cortex, hippocampus and parahippocampal gyrus, supramarginal gyrus (18). Moreover, additionally to the importance of olfactory deficit, disease duration

Olfaction evaluation and correlation is also correlated to the extent of the observed GM atrophy (17). The affected cortex belongs to olfaction cerebral processing primary and secondary areas as showed with functional (19) or morphological (17, 18, 20) MRI studies. But on the contrary to what is reported in audition or vision deficit, no GM or WM increase is observed in olfaction impairment (13–15). GM increase in audition or vision deficit may reflect compensatory experience-dependent plasticity in the spared modalities (15, 17). This phenomenon has not been described in olfactory deficit probably because remaining senses does not compensate olfactory loss. Moreover, there is no direct evidence yet for active remodeling in BBS secondary to peripheral sensory degeneration. The objective of this article is to evaluate olfactory disorders in BBS patients and to investigate putative correlation with morphological cerebral alteration. To this end, brain morphological differences between BBS subjects and healthy volunteers are firstly studied. Materials and methods Participants

Table 1. Description of the Bardet-Biedl syndrome (BBS) cohort Patients Age Gender Genotype 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

26 17 24 44 23 27 27 20 24 39 37 38 22 27 36 18 25 22 39 27

F M M F M M F F M M M M M M F M M F M F

Olfaction

BBS4 Anosmia BBS1 Anosmia BBS1 Mild hyposmia BBS1 Mild hyposmia BBS1 Severe hyposmia BBS1 Severe hyposmia BBS1 Mild hyposmia BBS10 Severe hyposmia BBS5 Severe hyposmia BBS5 Mild hyposmia BBS10 Anosmia ND Severe hyposmia BBS1 Mild hyposmia BBS9 Anosmia BBS17 Severe hyposmia BBS3 Mild hyposmia BBS10 Anosmia BBS12 Anosmia BBS12 Anosmia BBS10 Anosmia

Vision VIQ 4 1 1 4 0 2 0 1 4 2 4 6 0 4 4 1 6 2 4 1

68.5 57.5 60.5 66 62 64 93 51.5 93 71.5 90 62 94.5 102 ND 88.5 102 51.5 68.5 62

F, female; M, male; Olfaction, University of Pennsylvania Smell Identification Test evaluation; Vision, class 0–6; VIQ, verbal intelligence quotient.

This study received ethical approval from CPP ‘EST IV’ (Strasbourg, France) 10 June 2008 for the study PHRC National Bardet Biedl 2007 IDRCB 2007-A00868-45. Twenty BBS patients [7 female/13 male, age: 28.1 (17–44 years)] were recruited through the Clinical Trial Center, University Hospital of Strasbourg, France. Eligible participants had a clinical diagnosis of BBS, confirmed in all cases by genetic analysis of known BBS genes. Patient’s inclusion was performed between 25 August 2009 and 14 January 2013. Details on the BBS patient characteristics are provided in Table 1. For quantitative analyses, the BBS group was compared to a group of 14 healthy subjects without olfactory or visual impairment [9 female/5 male, age: 35.9 (28–51 years)]. Exclusion criteria for the comparison group were foreign metallic body, neurologic and psychiatric illness, claustrophobia, patient recently included in another medical study, pregnancy, minor patient and patient with legal guardianship.

10 odors in three trials with randomized order of the 10 odors taking into account the proportion of correct responses for the three trials. Levels of olfaction were obtained by summing up the proportions of correct responses for the 10 odors: 7–10 normosmia, 3–6 hyposmia and 0–3 anosmia. For the UPSIT, identification of the odors was made using the technique of multiple forced choices of 40 odors contained in microcapsules and adapted to the country of origin (UPSIT French version). Olfactory disorders are classified using the following rule on the UPSIT scores: normosmia 34–40 for men and 35–40 for women; mild hyposmia 26–33 for men and 26–34 for women; severe hyposmia 19–25 for both men and women and anosmia 6–18 (8, 9, 19). For the statistical correlation analyses, the degree of olfaction alteration was quoted using a score ranging from 0 to 3; 0: anosmia, 1: severe hyposmia, 2: mild hyposmia and 3: normosmia.

Olfactory evaluation

Visual evaluation

A senior Ear Nose Throat (ENT) specialist (J. J. B.) evaluated all BBS patients according to the following protocol: (i) clinical evaluation of olfaction and its dysfunction, (ii) ENT examination with nasal endoscopy and (iii) olfactometry using two different psychophysical methods, namely a suprathreshold evaluation of the olfaction and the UPSIT (Sensonics Inc., Haddon Heights, NJ) (8, 9, 12). The suprathreshold evaluation of the olfaction (normosmia, anosmia, and hyposmia) was based on perception and identification of odors such as vanilline, anethol, eucalyptol, thymol, coumarine, phenylethylic alcohol and on evaluation of the trigeminal sensitivity of the nasal mucosa with ether, ammonia (8). The scoring of the suprathreshold olfaction test was performed after evaluation of the perception and the identification of

Visual evaluation was performed by a senior ophthalmologist (H. D.). Visual impairment can be classified in different classes according to the International Classification of Functioning, Disability and Health (ICF), Geneva World Health Organization 2001, taking into account the keenness of vision (KV) of the best eye with the best visual correction: class 1: 3/10 > KV ≥ 1/10; class2: 1/10 > KV ≥ 1/20; class 3: 1/20 > KV ≥ 1/60; class 4: 1/60 > KV ≥ perception of the light; class 5: no perception of the light; class 6 : KV unknown. Verbal intelligence quotient evaluation

The estimated verbal intelligence quotient (VIQ) has been calculated on the basis of the administration of four

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Braun et al. subscales from the Wechsler Adult Intelligence Scale (WAIS-III, (22)): Vocabulary, Similarities, Arithmetic and Digit span, following the Warrington procedure (21, 22). Levels of intelligence are divided as such according to the score: IQ < 69 mental deficiency, 70 < IQ < 79 limited intelligence, 80 < IQ < 89 normal weak intelligence, 90 < IQ < 109 average intelligence, 110 < IQ < 119 normal strong intelligence, 120 < IQ < 129 superior intelligence, IQ > 130 very superior intelligence. Image acquisition

Each subject underwent a high-resolution anatomical MRI scan on a clinical 3T MR scanner (MAGNETOM Verio, Siemens Healthcare, Erlangen, Germany) using the T1-weighted SPACE (Sampling Perfection with Application optimized Contrasts using different flip angle Evolution) sequence with the following acquisition parameters: repetition time = 4000 ms, echo time = 383 ms, inversion time = 380 ms, flip angle = 120∘ , field of view = 25.6 cm, matrix = 512 × 512, 176 slices of 1 mm. SPACE is a single slab TurboSE sequence optimized for 3D data acquisition with slab selective variable excitation pulse. Image analysis Global brain atrophy estimation

Quantitative analysis of brain atrophy was performed using the SIENAX algorithm (23) provided in the FSL-v5.0 (FMRIB’s Software Library, http:// www.fmrib.ox.ac.uk/fsl/) software (23). This method estimates total brain, GM and WM tissue volumes from a single image by taking into account the skull size to normalize the volume in order to reduce head size-related variability between subjects. The comparison of total brain, GM and WM tissue volumes between groups was performed using the general linear model (GLM) while introducing age and gender as covariates (Table 2). Voxel-based morphometry

Focal GM atrophy was investigated using the voxelbased morphometry (VBM) framework provided in SPM12b (Statistical Parametric Mapping, http://www. fil.ion.ucl.ac.uk/spm/). VBM is a technique that identifies cerebral volume changes on a voxel-by-voxel basis from structural MRI data. It provides the opportunity to correlate variables of interest to regional cerebral Table 2. Global brain volume comparison between Bardet-Biedl syndrome (BBS) and controls (unilateral test) BBS (in mm3 )

Controls (in mm3 )

p value

Total brain 1,497,830 ± 64,007 1,543,205 ± 161,819 0.20 volume Gray matter 719,799 ± 67,822 762,052 ± 101,700 0.06 White matter 778,031 ± 56,146 781,153 ± 75,323 0.28

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volume. VBM analyses included image pre-processing and statistical analyses. Anatomical MRI images are spatially pre-processed in the following way: all T1 structural images are bias corrected, segmented using an extension of the unified segmentation procedure (24) that includes six classes of tissue. Spatial normalization is then performed using DARTEL algorithm (25). First, a study-specific template was created using GM images of all subjects. Second, this template was normalized to Montreal Neurological Institute (MNI) space. Third, the individual deformation field that permits to normalize each GM image to the template was computed and applied to each GM image and modulated to preserve the total amount of GM volume. A Gaussian kernel (FWHM: 8 mm) was then applied to modulated GM images and entered in the statistical analysis. For display purpose, the deformation fields were also applied to the T1 structural images to compute the mean structural image. Group comparison and statistical correlations between local GM volume and olfactory scores are then investigated using the GLM. Age and total amount of GM were included as nuisance covariates in both statistical analyses. Statistical analyses were only performed on voxels belonging to a mask built from the mean GM image of all subjects after DARTEL realignment thresholded at the level of 0.3. A statistical threshold of p < 0.001 without multiple comparison correction and with a cluster spatial extend of 75 voxels was considered in all analyses. The visualization of the results and the identification of the detected brain regions were done using Xjview (http://www. alivelearn.net/xjview8/). Results Behavioral results

Clinical evaluation with nasal endoscopy did not find any incidental nasal- or sinus-related medical or surgical conditions or other disorders associated with olfactory dysfunction. None of the patients complained about olfaction impairment like patients with congenital anosmia. Olfaction evaluation was in concordance between the two psychophysical methods used in this study and revealed anosmia in eight BBS patients (one BBS1, one BBS4, one BBS9, three BBS10, two BBS12), severe hyposmia in six patients (two BBS1, one BBS5, one BBS10, one BBS17, one BBS genetic analysis in progress) and mild hyposmia in six patients (four BBS1, one BBS5, two BBS3). No BBS patient presented normosmia. The mean age of the patients was 27.6 years for the anosmia group, 28 years for the severe hyposmia group and 29.1 for the mild hyposmia group. There was no significant statistical correlation between age and the degree of olfaction alteration (p = 0.810, r = 0.057). According to the levels of intelligence, the cohort of patients could be divided in four groups (i) 11 patients with mental deficiency (5 anosmia, 4 severe hyposmia and 2 mild hyposmia), (ii) 6 patients with average intelligence (3 anosmia, 1 severe hyposmia and 2 mild

Olfaction evaluation and correlation

Fig. 1. Local gray matter (GM) comparison between Bardet-Biedl syndrome (BBS) and controls: GM atrophy areas (blue) and hypertrophy areas (red) (minimal cluster size of 75 voxels and a threshold of p < 0.001, uncorrected).

hyposmia), (iii) 1 patient with limited intelligence (mild hyposmia) and (iv) 1 patient with normal weak intelligence (mild hyposmia) (21, 22). One patient refused IQ evaluation. No significant statistical correlation between IQ evaluation and the degree of olfaction alteration can be exhibited (p = 0.761, r = 0.075). Visual impairment seems to be more frequent and severe in patients with anosmia and severe hyposmia compared to patients with mild hyposmia. Indeed, when considering the international classification of visual impairment (class 0 to 5) as a measure of visual impairment severity (patients of class 6 being discarded as their KV is unknown), we found a mean value of 2.86 for the anosmia group, 2.20 for the severe hyposmia group

and 1.33 for the mild hyposmia group. Nonetheless, this correlation is not statistically significant (p = 0.091, r = −0.41). Brain atrophy results

Global volumetry comparison showed total GM volume reduction in Bardet-Biedl patients as compared to volunteers, but not statistically significant (p = 0.06). Total brain volume and WM volume were not significantly different between both groups (Table 2). However, local GM volume comparisons showed reduced GM volume in both cerebellar hemispheres, in both occipital and parietal lobes and in both parahippocampal gyrus and in

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Braun et al. Table 3. Local gray matter comparison between Bardet-Biedl syndrome (BBS) and controls (minimal cluster size of 75 voxels and a threshold of p < 0.001, uncorrected) Region Atrophied areas in BBS Right parahippocampal gyrus and uncus Left parahippocampal gyrus Right parahippocampal gyrus Right and left occipital lobe (lingual gyrus, calacarine, cuneus), right and left cerebellum, vermis, right and left parietal lobe (precuneus), right parahippocampal gyrus Right precuneus Hypertrophied areas in BBS Right frontal lobe (inferior frontal gyrus, middle frontal gyrus, frontal sup orb, frontal inf orb) Right medial frontal gyrus, right and left gyrus rectus Right and left anterior cingulate, right and left olfactory Left superior frontal gyrus, middle frontal gyrus Right superior frontal gyrus Left inferior parietal lobule Left middle frontal gyrus Right precentral gyrus, middle frontal gyrus

Table 4. Correlation between gray matter volume and olfaction dysfunction (minimal cluster size of 75 voxels and a threshold of p < 0.001, uncorrected) Region Right parahippocampal gyrus, amygdala, hippocampus Left middle temporal gyrus Right inferior parietal lobule, superior and middle temporal gyrus

MNIx MNIy MNIz Cluster size t score 18

−12

587

6

−48 −58.5 1.5

170

4.92

336

5.47

51

−6

−48 22.5

the right uncus and increase of GM in both frontobasal regions and frontal lobes, in left inferior parietal lobule and in both anterior cingulate regions (Table 3; Fig. 1). Correlation between GM volume reduction and olfaction dysfunction was highlighted in left middle temporal gyrus, and right hippocampus, amygdala, para hippocampus, middle and superior temporal gyrus and inferior parietal lobule (Table 4; Fig. 2). Discussion Behavioral results

Olfaction deficiency has been pointed in various recent studies as an additional clinical feature in BBS, an emblematic ciliopathy. In this study, olfaction evaluation revealed olfaction deficits in all BBS patients (eight anosmia, six severe hyposmia and six mild hyposmia). No BBS patient presented normosmia. None of the BBS participants in the current study complained about any

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MNIx

MNIy

MNIz

Cluster size t score

21 −25.5 24 6

6 −24 −24 −54

−28.5 −25.5 −25.5 1.5

132 341 164 6331

−4.23 −4.89 −4.6 −7.89

10.5

−78

43.5

315

−4.63

19.5

39

−24

562

5.15

9 7.5 −24 25.5 −42 −33 34.5

42 22.5 46.5 39 −42 15 −9

−13.5 −7.5 12 22.5 46.5 46.5 55.5

416 685 141 217 624 84 433

4.59 5.94 4.41 4.41 6.14 5.1 6.75

subjective limitations in their smell-identification ability like patients with congenital anosmia. While Tadenev et al. showed only 47% of complete or partial anosmia in 19 BBS patients with the Brief Smell ID Test (6), Brinckman et al. presented similar results to our study with the UPSIT for 20 BBS patients with anosmia or hyposmia (3). There was no clear correlation or relationship between olfaction impairment, age or IQ. Nevertheless the patients with anosmia had the lowest IQ level compared to the patients with mild hyposmia but the number of patients does not allow statistical conclusions. This fact can also be partially explained by the necessity of the patient’s cooperation and the potential influence of the olfactory education and memory for olfaction evaluation. Interestingly visual impairment was more frequent and severe in the series of patients with anosmia and severe hyposmia compared to the series of patients with mild hyposmia. There was also no statistically significant correlation between visual and olfaction impairments. Note that similar observations have been made in another condition related to ciliopathies, namely the Usher syndrome (26). Brain atrophy results

Ciliopathies manifest with multiple sensorial deficiencies (mainly retinal dystrophy and anosmia) that impact the life of the patients and also raise questions about their secondary consequences on the brain, taking into account that primary lesions may be occurring too. Comparison between BBS patients vs healthy volunteers

In this study, the 20 BBS patients have reduced total GM volume as compared to healthy volunteers but preserved WM and total brain volume preservation in

Olfaction evaluation and correlation

Fig. 2. Correlation between gray matter volume and olfaction dysfunction (minimal cluster size of 75 voxels and a threshold of p < 0.001, uncorrected).

accordance with literature data (27). We showed reduced GM volume in different cerebral areas, namely both cerebellar hemispheres, both occipital and parietal lobes and both parahippocampal gyrus. These results are only partially in accordance with previous published data by Baker et al., who showed GM reduction in a midline orbitofrontal region (gyrus rectus, orbitofrontal gyrus, extending toward the olfactory tract on the left) and bilaterally in temporal pole areas (more extensive on left than right). At lower statistical threshold these changes were bilateral (27). The different registration scheme in the VBM analysis and the number of included subjects (20 BBS patients in our study and 10 in Baker’s study) may explain these differences. But our results and the results of Baker et al. are very different from the data of Keppler-Noreuil et al.,

who showed preserved GM volume, with increased cerebral cortex volume in only the occipital lobe and reduced GM in the subcortical regions of the brain, including the caudate, putamen and thalamus, but not in the cerebellum (27, 28). It should be noted that in Keppler-Noreuil’s study, the same MRI scanner was not used for BBS patients and healthy volunteers (28). Moreover, in this last study children and adults have been included, the two youngest patients aged 4 and 8 years. These methodological issues could perhaps explain the results differences. The tissue loss in the cortex of parietal and occipital region could be secondary to visual impairment of BBS patients, as it has been shown in early and late acquired blindness (14, 15, 29). Interestingly, we report for the first time reduction of GM volume in both cerebellar hemispheres. Cerebellar

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Braun et al. abnormalities are a well-known hallmark in various syndromes related to ciliopathies such as Joubert syndrome. Cerebellar vermis hypoplasia has been seldom reported in the literature in BBS patients and we show herein that it may be a much more prominent feature (30, 31). Nonetheless, patients did not present obvious cerebellar ataxia or other cerebellar neurological features. We did not find basal ganglia abnormalities, although bilateral caudate reduction has been reported in BBS mouse models (32). Moreover, reduced hippocampal volume has been reported using region-of-interest measurements technique, and could perhaps be induced by hippocampal dysgenesis (27, 33). We could show only right uncus volume reduction using VBM analysis but outlined both parahippocampal gyrus volume reductions. We showed increase of GM volume in both frontobasal regions and frontal lobes, in left inferior parietal lobule and in both anterior cingulate regions. Areas of regional GM enlargement have been previously reported in the occipital lobe (27, 28) The elevation of GM could be explained by brain compensation mechanisms with cortical reorganization (13–16, 34–36). GM remodeling has been previously reported in totally blind subjects, with increase in GM volume found in left middle occipital and middle frontal gyrus and right entorhinal cortex (14, 15). To summarize, there are many brain regions that are altered in BBS patients as compared to healthy control. These regions may be linked with several impairments related to the pathology, such as visual impairment, but also other sensory alterations such as olfaction deficits. Moreover BBS patients present cognitive impairment in 11 cases in our study in accordance with Brinckman et al., who also showed impaired fine motor function in addition (3). Thus, these modifications of the neurobehaviorial profile could also contribute to brain modifications (3). Nonetheless, due to the large number of confounders, it is difficult to untangle what brain modification is specifically in connection with each behavioral impairment. Correlation with olfactory impairments

Olfaction alterations were associated to cerebral modifications. We showed correlation between GM volume reduction and olfaction dysfunction in left middle temporal gyrus, and right hippocampus, amygdala, parahippocampus, middle and superior temporal gyrus and inferior parietal lobule. These results are in accordance with the studies of Bitter et al. (17, 18), although the registration method they used in their VBM analysis was less accurate than the DARTEL algorithm. Moreover GM volume reduction in cortical olfactive areas has been already been reported in BBS patients by Baker et al., who showed GM reduction in a midline orbitofrontal region (gyrus rectus, orbitofrontal gyrus, extending toward the olfactory tract on the left) and bilaterally in temporal pole areas (more extensive on left than right), but also in the region of the olfactory bulb (extending into medial orbitofrontal cortex) (27). Finally, it has to be noticed that this correlation analysis might suffer from some confounders, such as IQ

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impairment, which may have an impact on both olfactory evaluation and GM alteration. Conclusion

This study shows that anosmia or hyposmia can be considered as a cardinal and constant feature in BBS. We showed objective olfaction disorders in BBS patients and show associated brain GM alterations. But brain modifications appear to be plurifactorial. Indeed, BBS patients present modifications in visual and olfactory areas but also in other brain regions. Further studies on patients with other ciliopathies would be highly interesting as the clinical manifestation may be overlooked totally. Gene therapy has proven efficient action on murine models of various ciliopathies (2) and could be tested efficiently in clinical research in ciliopathy patients. Acknowledgement The authors acknowledge the Inserm, Centre d’Investigation Clinique de Strasbourg, CIC-P 1002, CHRU de Strasbourg, F67091 Strasbourg, France for their help in the study management. This work was funded by the study PHRC National Bardet Biedl 2007 IDRCB 2007-A00868-45.

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