Human Molecular Genetics, 2014, Vol. 23, No. 2 491–501 doi:10.1093/hmg/ddt439 Advance Access published on September 10, 2013 The familial dementia revisited: a missense revealed by whole-exome sequencing identifies ITM2B as a candidate gene underlying a novel autosomal dominant retinal dystrophy in a large family

Isabelle Audo1,2,3,4,5,∗, Kinga Bujakowska1,2,3, Elise Orhan1,2,3, Said El Shamieh1,2,3, Florian Downloaded from https://academic.oup.com/hmg/article/23/2/491/663466 by guest on 01 October 2021 Sennlaub1,2,3, Xavier Guillonneau1,2,3, Aline Antonio1,2,3, Christelle Michiels1,2,3, Marie-Elise Lancelot1,2,3, Melanie Letexier6, Jean-Paul Saraiva6, Hoan Nguyen7, Tien D. Luu7, Thierry Le´veillard1,2,3, Olivier Poch7,He´le`ne Dollfus8,9, Michel Paques1,2,3,4, Olivier Goureau1,2,3, Saddek Mohand-Saı¨d1,2,3,4, Shomi S. Bhattacharya1,2,3,5,10, Jose´-Alain Sahel1,2,3,4,11,12 and Christina Zeitz1,2,3

1INSERM, U968, Paris F-75012, France, 2CNRS, UMR_7210, Paris F-75012, France, 3Department of Genetics, Institut de la Vision, UPMC Univ Paris 06, UMR_S 968, Paris F-75012, France, 4Centre Hospitalier National d’Ophtalmologie des Quinze-Vingts, INSERM-DHOS CIC 503, Paris F-75012, France, 5Department of Genetics, UCL-Institute of Ophthalmology, 11–43 Bath Street, London EC1V 9EL, UK, 6IntegraGen SA, Genopole CAMPUS 1 bat G8, Evry FR- 91030, France, 7Institut de Ge´ne´tique et de Biologie Mole´culaire et Cellulaire, Illkirch Cedex 67404, France, 8Centre de Re´fe´rence pour les Affections Rares en Ge´ne´tique Ophtalmologique, Hoˆpitaux Universitaires de Strasbourg, Strasbourg, France, 9Laboratoire de ge´ne´tique me´dicale, Universite´ de Strasbourg, UMR-S INSERM U1112, Strasbourg, France, 10Andalusian Centre for Molecular Biology and Regenerative Medicine (CABIMER), Isla de Cartuja, Seville, Spain, 11Fondation Ophtalmologique Adolphe de Rothschild, Paris, France and 12Acade´mie des Sciences–Institut de France, Paris 75006, France

Received July 18, 2013; Revised and Accepted September 4, 2013

Inherited retinal diseases are a group of clinically and genetically heterogeneous disorders for which a signifi- cant number of cases remain genetically unresolved. Increasing knowledge on underlying pathogenic mechan- isms with precise phenotype–genotype correlation is, however, critical for establishing novel therapeutic interventions for these yet incurable neurodegenerative conditions. We report phenotypic and genetic charac- terization of a large family presenting an unusual autosomal dominant retinal dystrophy. Phenotypic character- ization revealed a retinopathy dominated by inner retinal dysfunction and ganglion cell abnormalities. Whole- exome sequencing identified a missense variant (c.782A>C, p.Glu261Ala) in ITM2B coding for Integral Membrane 2B, which co-segregates with the disease in this large family and lies within the 24.6 Mb inter- val identified by microsatellite haplotyping. The physiological role of ITM2B remains unclear and has never been investigated in the retina. RNA in situ hybridization reveals Itm2b mRNA in inner nuclear and ganglion cell layers within the retina, with immunostaining demonstrating the presence of the corresponding protein in the same layers. Furthermore, ITM2B in the retina co-localizes with its known interacting partner in cerebral tissue, the

∗To whom correspondence should be addressed at: Department of Genetics, Institut de la Vision, 17, Rue Moreau, Paris 75012, France. Tel: +33 153462542; Fax: +33 153462602; Email: [email protected]

# The Author 2013. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected] 492 Human Molecular Genetics, 2014, Vol. 23, No. 2 b precursor protein, critical in Alzheimer disease physiopathology. Interestingly, two distinct ITM2B , both resulting in a longer protein product, had already been reported in two large autosomal domin- ant families with Alzheimer-like dementia but never in subjects with isolated retinal diseases. These findings should better define pathogenic mechanism(s) associated with ITM2B mutations underlying dementia or retinal disease and add a new candidate to the list of involved in inherited retinal dystrophies.

INTRODUCTION RESULTS

Inherited retinal dystrophies are a heterogeneous group of Clinical characterization disorders including stationary conditions such as congenital The reported family includes at least 14 affected family members stationary night blindness (CSNB) characterized by post- spanning three generations, with two deceased, suffering Downloaded from https://academic.oup.com/hmg/article/23/2/491/663466 by guest on 01 October 2021 phototransduction dysfunction or degenerative processes such from visual symptoms segregating as a dominant trait (Fig. 1, as rod–cone and cone–rod dystrophies. This clinical heterogen- proband with a single arrow). Eleven affected and eight unaf- eity is mirrored by genetic heterogeneity (https://sph.uth.edu/ fected subjects were available for genetic testing. Detailed clin- Retnet/, last accessed date on September 16, 2013). Despite ical examination was performed on affected family members the increasing number of identified genes recognized for their ranging in age from 46 to 73 years (average 54) (Table 1). role in retinal physiology and pathology, the underlying Onset of symptoms appeared between 25 and 40 years genetic defect(s) remains unknown in a number of cases. Increas- (average 36). Light sensitivity was the first sign, reported by ing knowledge in pathogenic mechanisms and their associated all subjects, followed by progressive loss of central vision. phenotype–genotype correlation is de facto critical for a better Visual acuity varied from 20/25 to 20/400 (average 20/50). understanding of these heterogeneous disorders, even for rare Visual fields showed decreased central retinal sensitivity with subgroups. It may not only provide important insights into the preservation of peripheral visual field. Fundus examination, complexity of retinal physiology or neurophysiology in general fundus autofluorescence imaging and spectral domain optical but also help for accurate counselling and, moreover, for the es- coherence tomography (SD-OCT) revealed macular changes tablishment of innovative therapeutic interventions in these yet associated with optic disc pallor, hyper-reflectivity of ganglion incurable conditions. Recently, techniques of next-generation se- cell and nerve fibre layers (Fig. 2A) with loss of optic nerve quencing (NGS) have proved their efficiency to increase chances fibres (Fig. 2B). Full-field electroretinogram (Fig. 2C) showed of deciphering unknown genetic defect in this context (1). inner retinal dysfunction in all cases: all affected subjects dis- We applied these techniques to decipher the underlying gene played an absent or very reduced b-wave in response to a dim defect in a large dominant pedigree with 14 members affected by flash of 0.01 cd s m22 under dark-adapted (scotopic) conditions; a clinically novel retinal dystrophy dominated by inner retinal stimulation with a bright flash (3 and 12 0.01 cd s m22) under dysfunction and ganglion cell abnormalities. Applying whole- scotopic conditions revealed a normal a-wave, reflecting exome sequencing and subsequent haplotype analysis, we iden- normal photoreceptor function, dominated under dark adapta- tified a missense mutation in ITM2B, encoding Integral Mem- tion by rod function, but a severely reduced b-wave, reflecting brane Protein 2B, which co-segregates with the disease, and post-receptoral dysfunction with the typical electronegative further investigated expression of this gene in the retina. waveform. Oscillatory potentials, originating at the inner retinal

Figure 1. Family pedigree revealing autosomal dominant segregation of the disease. Affected individuals are presented with filled symbols, unaffected with white symbols;squaresymbolsrepresentmaleandroundsymbols,female;deceasedindividualsare presentedwitha slash;subjectswho underwent ophthalmicevaluationin our centre are marked with an asterisk; mutation segregation is shown on the pedigree as ‘[¼];[¼]’ carrying both normal allele and ‘[M];[¼]’ heterozygous for the mutant change; the proband IV.27 is marked with a double black arrow and his DNA sample was included in whole-exome sequencing; other subjects includedin whole-exome sequencing are marked with arrows (i.e. IV.1 and IV.12). Table 1. Clinical characteristics of affected family members

Patient Age at Age of onset Sex Symptoms BCVA Colour vision Kinetic visual field Fundus examination FAF SD-OCT the time OD/OS of testing refraction

III.13 73 Around 40 F Mild photophobia 20/100; 20/250 OD normal Relative central scotoma Temporal optic disc Hyper-autofluorescent ring Hyper-reflectivity within

Decreased vision 21.75 (21.75) OS mild tritanopia within 10 central pallor; mild around the macular region; the foveal ONL; small Downloaded fromhttps://academic.oup.com/hmg/article/23/2/491/663466bygueston01October2021 958 degrees with normal retinal vessel hypo-autofluorescence drusenoid changes in 23.75 (20.5) 708 peripheral isopter narrowing; foveal within the ring with subtle the foveal region; changes foveal hyper-reflectivity of hyper-autofluorescence the inner retina III.15 72 Around 40 F Mild photophobia 20/250; 20/400 OD and OS mild Relative central scotoma Pale optic disc; mild Hyper-autofluorescent ring Hyper-reflectivity within Decreased vision +1.25 (21.75) deuteranopia within 20 central retinal vessel around the macular region; the foveal ONL; 1308 degrees with normal narrowing; foveal hypo-autofluorescence hyper-reflectivity and +1(21) 808 peripheral isopter changes within the ring with subtle thinning of the inner foveal retina hyper-autofluorescence IV.11 47 Photophobia at F Mild photophobia 20/63; 20/80 OD and OS normal Relative central scotoma Temporal pale optic Mild hyper-autofluorescent Hyper-reflectivity within around 30 Decreased vision +1(20.25) 1258 within the 5 central disc; subtle foveal ring around the macular the foveal ONL; Decreased vision 20.75 (20.25) degree; blind spot changes region; subtle hyper-reflectivity of at age 35 858 exclusion; normal hypo-autofluorescence the inner retina peripheral isopter within the ring IV.12 47 Photophobia since F Mild photophobia 20/50; 20/63 OD and OS normal Relative central scotoma Temporal pale optic Mild hyper-autofluorescent Hyper-reflectivity within age 25 Decreased vision +0.50 within the 5 central disc; subtle foveal ring around the macular the foveal ONL; Decreased vision +0.75 degree; blind spot changes region; subtle hyper-reflectivity of at age 35 exclusion; normal hypo-autofluorescence the inner retina peripheral isopter within the ring IV.13 46 Photophobia around M Mild photophobia 20/80; 20:63 OD and OS normal Relative central scotoma Temporal pale optic Mild hyper-autofluorescent Hyper-reflectivity within 30 and then Decreased vision 24.50 (20.50) within the 5 central disc; subtle foveal ring around the macular the foveal ONL; decreased vision 1008 degree; normal changes region; subtle hyper-reflectivity of at age 33 24(20.75) 858 peripheral isopter hypo-autofluorescence the inner retina within the ring Peripheral

autofluorescence 2 No. 23, Vol. 2014, Genetics, Molecular Human abnormalities IV.15 51 Around 40 M Mild photophobia 20/25; 20/63 OD mild deuteranopia Centrocoecal scotoma Temporal pale optic Hyper-autofluorescence in the Hyper-reflectivity within Decreased vision Plano OS moderate with normal peripheral disc; subtle foveal posterior pole with the foveal ONL; +0.50 dyschromatopsia isopter changes decreased hyper-reflectivity of without preferential autofluorescence within the inner retina; axis the macular region and a thinning of the outer fascicular inferior retina in the hypo-autofluorescence in paramacular area the right eye IV.22 50 Around 40 M Mild photophobia 20/100; 20/125 OD and OS normal Relative central scotoma Subtle temporal pale Hyper-autofluorescent ring Hyper-reflectivity within Decreased vision 23.50 (20.75) within the 15 central optic disc; subtle around the macular region; the foveal ONL; 1108 degree; normal foveal changes hypo-autofluorescence hyper-reflectivity of 24.25 (20.75) peripheral isopter within the ring with subtle the inner retina 658 foveal hyper-autofluorescence IV.27 50 Night vision M Night blindness 20/100; 20/100 OD and OS normal Relative central scotoma Subtle temporal pale Hyper-autofluorescent ring Hyper-reflectivity within problem and Mild 22(21) 1108 within the 15 central optic disc; subtle around the macular region; the foveal ONL; photophobia at photophobia 22 (1;50) 608 degree; normal foveal changes hypo-autofluorescence hyper-reflectivity of age 38 Decreased vision peripheral isopter within the ring the inner retina IV.28 49 Around 40 F Night blindness 20/50; 20/50 OD and OS deuteranopia Relative central scotoma Subtle temporal pale Subtle hyper-autofluorescent Hyper-reflectivity within Mild 22.50 (21.25) within the 10 central optic disc; subtle ring around the macular the foveal ONL; photophobia 658 degree; normal foveal changes region; hyper-reflectivity of Decreased vision 22.50 (21.25) peripheral isopter hypo-autofluorescence the inner retina 608 within the ring

BCVA, best corrected visual acuity; OD, ocula dextra (right eye); OS, ocula sinistra (left eye); FAF, fundus autofluorescence; SD-OCT, spectral domain optical coherence tomography; F, female; M, male; ONL, 493 outer nuclear layer. 494 Human Molecular Genetics, 2014, Vol. 23, No. 2 Downloaded from https://academic.oup.com/hmg/article/23/2/491/663466 by guest on 01 October 2021

Figure 2. Phenotypic and genetic characteristics of an unusual retinal dystrophy. (A) Ocular fundus abnormalities seen in IV.28 compared with an unaffected subject: (1) the colourfundusphotographshowssubtlechanges,includingtemporalpallorof the optic disc andvery mildchangesin themacula; (2)the fundus autofluorescence imaging reveals a perimacular increase of autofluorescence and a decrease of macular autofluorescence compared with control; (3) SD-OCT reveals a hyper- reflectivity (marked by an asterisk) of the ganglion cell and nerve fibre layers (GCL and NFL, respectively) as well as an irregular reflectivity within the outer nuclear layer (ONL) (arrow). (B) Analysis of the nerve fibre layer (ganglion cell axons) on SD-OCT displays a significant (red areas) or borderline (yellow areas) Human Molecular Genetics, 2014, Vol. 23, No. 2 495 level, were undetectable on older affected subjects and de- nucleotide polymorphisms (SNPs) to 19 and from 12 714 inser- creased in amplitude with a simplified wave shape in younger tion and deletions (indels) to 8. Of these, the only gene already subjects, in keeping with inner retinal dysfunction. Light- involved in retinal dystrophies was IMPG2 (MIM#∗607056) adapted (photopic) responses, testing the cone system function, (6), for which NGS and whole-exome sequencing detected were relatively preserved for both 3 cd s m22 single flash and a heterozygous 3 deletion on affected subjects. 30 Hz Flicker stimulation in younger subjects, whereas older However, family co-segregation study excluded the causality affected subjects showed additional photopic response abnor- of this variant. Eight of the 19 SNPs were selected for co- malities with decreased amplitudes and delayed implicit time segregation analysis after bioinformatic pathogenic predic- (Fig. 2C). Since photopic responses are driven by cone photore- tion. These variants were located in the following genes: ceptors but represent responses generated at the inner retinal OMP (MIM#∗164340), ITM2B (MIM#∗603904), CC2D1A level, this could suggest either a worsening of inner retinal dys- (MIM#∗610055), RFX1 (MIM#∗600006), OR7A5, C2orf44, function affecting the cone pathway or direct cone photoreceptor FAM184A and OPLAH (MIM#∗614243) (Supplementary Ma-

dysfunction. None of the affected subject displayed the abnor- terial, Table S1). Only the change located in exon 6 of ITM2B Downloaded from https://academic.oup.com/hmg/article/23/2/491/663466 by guest on 01 October 2021 mal wave form seen in selective ON-bipolar cell dysfunction (c.782A.C, p.Glu261Ala) co-segregated with the phenotype. [i.e. an a-wave with a broaden trough and a sharply arising This variant was not found in 380 control alleles, is predicted b-wave characterizing the complete form of CSNB (2)]. To to be probably damaging and is well conserved (Supplementary further investigate whether ON- and/or OFF-bipolar cell func- Material, Fig. S1). It was not found in public sequence repositor- tion was affected, long-duration stimulation recordings applying ies including the Human Gene Mutation Database (http://www. an orange flash stimulus on a green background to document ON hgmd.cf.ac.uk, last accessed date on September 16, 2013) or the and OFF responses from the L/M-cone systems (3) were Leiden Open Variation Database (www.lovd.nl). Haplotype attempted. Traces obtained by such stimuli were variable, par- analysis using 16 custom microsatellite from 13q tially linked to the photophobia present in most affected subjects. on 19 family members identified crossovers between markers When reproducible, results suggested both ON- and OFF- D13S1272 and D13S275 leading to an interval of 24.6 Mb in- pathway dysfunction with decrease in the amplitude of both cluding ITM2B with the same haplotype co-segregating in the b- and d-wave, respectively (Fig. 2D). In addition, pattern affected subjects (Supplementary Material, Fig. S2). ERG, performed to document macular and ganglion cell func- The 24.6 Mb interval contains 74 protein-coding genes. tion (4), revealed reduced amplitudes for both P50 and N95 com- Among these, 52 were reported to be expressed in the eye on ponents in all patients in keeping with proximal macular Unigene, but only RB1 and ITM2B had previously been asso- dysfunction (Fig. 2E). In this context, selective ganglion cell ciated with an ocular disease, namely retinoblastoma and cata- function could not specifically be assessed. ract with amyloid angiopathy and dementia, respectively. Only None of the affected subjects had specific systemic diseases, exon1ofRB1 was poorly covered by whole-exome sequencing and no cases of dementia had been reported in the family, includ- within the 24.6 Mb and was further Sanger-sequenced. None of ing individuals aged .50 years, suggesting a retinal-restricted the coding regions and exon boundaries of the 74 genes were disease. Furthermore, all subjects appear well temporally and carrying a variant that co-segregates with the phenotype in spatially oriented during examination, and Mini-Mental State the family besides the (c.782A.C, p.Glu261Ala) change in Examination was normal in the proband. ITM2B. Residue number 261 lies within a secreted peptide consisting of 23 residues obtained after ITM2B cleavage by furin or furin- Genetic studies like convertase (7,8). There is no reliable three-dimensional Direct Sanger sequencing of coding and flanking regions of structure of this secreted peptide, but it is predicted to form genes implicated in dominant post-photoreceptor dysfunction beta–loop–beta structures. Substitution of a negatively char- (RHO, PDE6B, GNAT1 and TRPM1) did not reveal pathogenic ged glutamic acid for a non-polar alanine would most probably changes. Further genetic analysis of proband’s DNA applying induce instability of this predicted structure. NGS targeted towards exons of all genes implicated in retinal Sanger sequencing of coding and flanking exonic regions of diseases (5) did not reveal pathogenic variants co-segregating ITM2B (RefSeq NM_021999.4) did not reveal additional with the disease. Whole-exome analysis applied to proband pathogenic variants in a panel of 95 subjects with a partially re- IV.27, one affected IV.12 and one unaffected IV.1 cousin (see sembling phenotype (11 cases of progressive autosomal dom- arrows on Fig. 1) followed by stringent filtering of genetic var- inant cone dystrophy and 84 cases with post-photoreceptoral iants reduced the number of variants from 118 777 single- dysfunction–CSNB) (Supplementary Material, Table S2).

thinning of this layer compared with normal (green) (OD: oculus dextra, for the right eye; OS: oculus sinistra, for the left eye). (C) Full-field electroretinogram was performed to test global retinal function. Under dark adaptation (scotopic), responses, which are dominated by the rod system, reveal a normal a-wave, in keeping with normal photoreceptor, mainly rod, function, but a reduced b-wave in keeping with inner retinal dysfunction; light-adapted (photopic) responses, which allow cone system function testing, were normal in younger patients and showed additional abnormalities in older subject (III-13 and III-15) in keeping with progressive cone system dysfunction, which could either be a worsening of inner retinal function or direct cone photoreceptor dysfunction. Each trace represents an average of three responses each to five sweeps. (D) Long-duration stimulation recordings applying an orange flash stimulus on a green background to document ON-bipolar and OFF-bipolar responses from the L/M-cone systems suggest both ON- and OFF-pathway dysfunction with both decrease in amplitude of the b- and d-wave, re- spectively. (E) Pattern ERG revealed reduced amplitudes for both P50 and N95 components in all patients in keeping with proximal macular dysfunction. In this context, selective ganglion cell function could not specifically be assessed. 496 Human Molecular Genetics, 2014, Vol. 23, No. 2

Expression and immunolocalization studies In vitro expression studies on wild-type and ITM2B mutant constructs The EST profile on Unigene shows ubiquitous expression of ITM2B including the eye, and the mouse retinal Immunolocalization before and after cell fixation and per- profile database exhibits expression in all retinal cell types (9). meabilization revealed both membrane and intracellular label- In-house rd1 mouse expression database revealed an increase ling (Supplementary Material, Fig. S3) with no difference in Itm2b transcript with photoreceptor degeneration suggesting between the normal and the three-mutant constructs, sug- its expression in inner retinal cells (Fig. 3A). A similar expres- gesting that pathogenic mechanism does not involve cellular sion profile was found with APP (amyloid b precursor mislocalization. protein), known to directly interact with ITM2B (Fig. 3A) (11–13). Real-time PCR experiments confirmed high expres- sion of ITM2B in human retina (DC ¼ 21.16) T ITM2B-ACTIN DISCUSSION (Fig. 3B). RNA in situ hybridization revealed Itm2b mRNA in inner nuclear and ganglion cell layers (Fig. 3C). Application of The autosomal dominant phenotype we report here, combining Downloaded from https://academic.oup.com/hmg/article/23/2/491/663466 by guest on 01 October 2021 anti-ITM2B monoclonal antibody to mouse retinal section inner retinal dysfunction, ganglion cell abnormalities with pro- revealed a punctuate immunostaining within ganglion cell, gressive loss of vision, has never been described before. This inner plexiform and inner nuclear layers which overlapped clinical picture is distinct from other causes of inner retinal dys- with the anti-APP immunostaining (Fig. 4A and B). Ganglion function of genetic origin (reviewed in 14). These include Schu- cell localization was further confirmed on flat mount human bert Bornschein types of CSNB, with the complete form being retina, with anti-ITM2B polyclonal antibody immunostaining characterized by selective ON-bipolar pathway dysfunction being seen in BRN3A-positive cells (Fig. 4C). and the incomplete form with loss of ON- and OFF-bipolar

Figure 3. Transcriptomic analysis of ITM2B within the retina. (A) Expression of Itm2b (1418000_a_at) compared with the expression of App (1420621_a_at, a known partner of Itm2b) in rd1 and wild-type mice during rod degeneration in the rd1 mouse. The rd1 mouse, carrying Pde6b mutations, is a naturally occurring retinitis pigmentosa model leading to a complete loss of rod photoreceptors by post-natal day 36, and preserved inner retina. cDNAs of neural retinas from rd1 and wild-type miceon identicalgenetic backgrounds were hybridized tothe mousegenome4302.0 array(Affymetrix, High Wycombe, UK).The expression profilesare similar from post-natal day 10 onwards for both Itm2b and App probes with an increase compared with wild-type suggesting their expression at least in inner retinal cells. Inter- estingly, the same profile is found for Gpr179 and Nyx, both being expressed in inner retinal cells (10). (B) ITM2B expression in the retina, lymphocytes and HEK293 cells. (1) Agarose gel of the end-point PCR products showing expression of ITM2B in the retina, lymphocytes and HEK293 cells; (2) quantitative real-time PCR, normalized to actin expression, reveals a high expression of Itm2b in the retina (DCT ITM2B-ACTIN ¼ 21.16) compared with lymphocytes (DCT ITM2B-ACTIN ¼ 0.46) and HEK293 cells (DCT ITM2B-ACTIN ¼ 0.16). (C) RNA in situ hybridization in mouse retina reveals expression of Itm2b in the ganglion cell and inner nuclear layers (GCL and INL, respectively). ONL, outer nuclear layer; RPE, retinal pigment epithelium. Human Molecular Genetics, 2014, Vol. 23, No. 2 497 Downloaded from https://academic.oup.com/hmg/article/23/2/491/663466 by guest on 01 October 2021

Figure 4. Immunolocalization of ITM2B. (A) Immunofluorescent labelling of mouse retinal sections reveals the presence of ITM2B within the ganglion cell and inner nuclear layers which co-localize with anti- APP immunolabelling (scale bar ¼ 40 mm). (B) Immunohistochemistry of mouse retinal section reveals strong labelling of ganglion cell and fainter labelling of inner nuclear layers (scale bar ¼ 50 mm). (C) Immunofluorescent labelling of flat mount human retina reveals a localization of ITM2B in the cytoplasm of BRN3A-positive cells suggesting the expression of ITM2B in ganglion cells (scale bar ¼ 40 mm). function. Both CSNB types are congenital and not classically ataxia, spastic tetraparesis and death around age 60 but no associated with progressive decreased vision and ganglion cell reported ocular abnormalities (17). Nevertheless, due to the wor- loss as presented in the current report. Similarly, X-linked reti- sening of the neurological status, ocular abnormalities may have noschisis, snowflake vitreoretinal degeneration, autosomal been undermined in the late stages of the disease. On the other dominant neovascular inflammatory vitreoretinopathy (15), hand, in FDD, also known as heredopathia ophthalmo-oto-ence- Muller cell sheen dystrophy or systemic disorders such as phalica, bilateral cataract is the first manifestation between Batten disease manifest distinct ophthalmic abnormalities. We age 20 and 30 followed by progressive neurosensory deafness, could not find a similar clinical presentation while investigating severe by age 45, cerebellar ataxia with paranoid psychosis the phenotypic database of our reference centre for rare diseases, and dementia 10 years later and death in the fifth decade (18). currently including 5000 individuals with inherited retinal dys- Other ocular abnormalities are reported in relation with trophies, and inquiring in other specialized centres in France amyloid angiopathy including retinal ischaemia and neovascular and Europe (presentation of the phenotype at the French Oph- complications (19). None of these vascular, ocular or neuro- thalmology Society and at the Moorfields Eye Hospital). This logical findings were present in our family. Two distinct variants may suggest the rare occurrence of this unusual phenotype. in ITM2B have been identified underlying FBD and FDD, a T.A This is also the first report of a genetic variant in ITM2B asso- transversion at the stop codon (rs104894417; c.799T.A; ciated with a degenerative process restricted to the retina. p.Stop267Argext∗11) and a decamer duplication at the 3′ end Interestingly, mutations in ITM2B have previously been (c.795-796insTTTAATTTGT; p.Ser266Pheext∗11), respective- reported in Familial British Dementia [FBD, or cerebral ly, both mutants resulting in an 11-amino-acid-longer protein amyloid angiopathy ITM2B-related type 1 (CAA-ITM2B1) product (7,16). OMIM#176500] (7), Familial Danish Dementia [FDD, or cere- ITM2B, also known as BRI or BRI2, is located on 13q14.2, bral amyloid angiopathy ITM2B-related type 2 (CAA-ITM2B2) comprises 6 exons and encodes a 266- protein of OMIM#117300] (16) and two Alzheimer disease (AD)-like unclear function belonging to the single-pass Integral Membrane autosomal dominant dementia with cerebral amyloid deposits: type 2 protein family. ITM2B mRNA is ubiquitously expressed FBD is characterized by progressive dementia starting with (7,20,21) but to date no information on its expression or role memory loss around age 45 followed by progressive cerebellar within the retina is available. Our study suggests its localization 498 Human Molecular Genetics, 2014, Vol. 23, No. 2 in inner retinal and ganglion cells, where it may interact with The exact cellular origin of progressive abnormalities in pho- APP (Figs 3 and 4). The physiological role of ITM2B remains topic responses remains unclear. Since photopic responses are unclear. It is known to be proteolytically cleaved in three distinct cone photoreceptor-driven but represent a summation of the locations. These include an extracellular furin-like convertase visual signal, it could represent a progressive cone degeneration, cleavage in C-terminus releasing a 23-amino acid secreted frag- supported bymacular dysfunctionseenwith pattern ERG andab- ment, with the remaining membrane-bound part containing a normal macular autofluorescence. Alternatively, it could be a BRICHOS domain (7,8,22). The exact role of the BRICHOS progressive degeneration at the inner retinal level affecting domain itself is unknown and at least three functions have both ON- and OFF-bipolar cell pathways as seen in incomplete been suggested, including the targeting of to the secre- CSNB. Mechanism(s) leading to this late-onset cone or cone tory pathway, a role of intramolecular chaperone and a facilita- ON-/OFF-pathway degeneration is also unclear. If progressive tion for intracellular protease processing (23). In addition, cone degeneration is considered, it may be an indirect phenom- studies have emphasized ITM2B direct interaction with APP, enon since we failed to find ITM2B expression in photoreceptors.

its key role as a modulator of APP processing and as an inhibitor In particular, it could represent a toxic effect either through Ab, Downloaded from https://academic.oup.com/hmg/article/23/2/491/663466 by guest on 01 October 2021 of amyloid b (Ab) oligomerization, a major component of already shown to induce photoreceptor cell death after intravi- amyloid plaques in FBD, FDD and AD (11,12,24–27): the treal injection (35), or through 23-amino acid secreted mutated 23-amino acid secreted fragment would critically inhibit Ab ag- peptide aggregation. If progressive involvement of cone ON- gregation, and the membrane-bound fragment of ITM2B may and OFF-bipolar pathway is considered, it is also unclear control cleavage of APP and, therefore, Ab genesis. Further- whether it is a specific and direct cellular toxicity upon bipolar more, ITM2B is able to promote Ab degradation by increasing cells or whether this progressive inner retinal dysfunction is an the expression of the secreted form of the protease, insulin- indirect mechanism that may involve material deposition with degrading enzyme (28). It was also suggested that ITM2B may the inner retina. The abnormal hyper-reflectivity seen on play a role in neurite outgrowth (29), a function also attributed SD-OCT may support this later hypothesis. to APP (30). All together, these data outline the key role of Our study provides compelling evidence for an ITM2B mis- ITM2B in Ab metabolism and its relevance as a therapeutic sense mutation as being the candidate defect underlying a target for AD and AD-like dementias. novel retinal dystrophy. Further mechanistic studies are, In case of FBD and FDD, the mutations would result in a however, needed to establish causality and associated disease longer secreted peptide (34instead of 23amino acid), respective- mechanisms. Elucidating underlying pathogenic mechanism(s) ly, called ABri (7) and ADan (16). These two peptides, which in this family affected with a novel phenotype would lead to a contain a disulphide bond, adopt a b-sheet structure in solution better understanding of ITM2B physiopathology in neurological and are able to form oligomers and fibrils, a major component disorders using the retina as a unique model and may help in of amyloid lesions found in FBD and FDD that may initiate designing innovative therapeutic targets for AD and AD-like de- cell death. These peptides may also be able to form ion perme- mentia. Our study also provides a new candidate in ITM2B,tobe able channels in lipid biolayers, leading to cytotoxic effects added to the list of genes involved in inherited retinal diseases. (31). The longer length of ABri and ADan may also hamper their inhibitory role on Ab oligomerization (31,32). Further- more, Tau triplets were found in brain extracts of FBD subjects MATERIALS AND METHODS as in AD and may also have a role in the disease process (33). Clinical examination Pathogenic mechanism(s) associated with the ITM2B missense change we identified is unknown but is most likely distinct from Research procedures were conducted in accordance with institu- disease mechanisms associated with FBD and FDD. Our findings tional guidelines and the Declaration of Helsinki. Each affected suggest a strong phenotype–genotype correlation with a longer subject included in the genetic study underwent full ophthalmic peptide underlying the dementia phenotype, whereas a missense examination as described before (3,4,36). Mini-Mental State change would be responsible for the retina-restricted phenotype. Examination was performed to evaluate cognitive function on Our in vitro studies show no difference in membrane localization proband (37). between the normal and the three-mutant constructs, suggesting that pathogenic mechanism does not involve cellular mislocaliza- tion (Supplementary Material, Fig. S3). The change of a glutamic Molecular genetics and bioinformatic analyses acid for an alanine may disturb the beta–loop–beta structure Informed consent was obtained from all patients and their family of the 23-amino acid secreted peptide and, therefore, modify members. Sanger sequencing, targeted NGS and whole-exome protein interaction with Ab or other unknown partners. The sequencing were performed followed by stringent filtering of subtle changes induced by the p.Glu261Ala mutation are less genetic variants as previously reported (5,10,36). In brief, for likely to lead to oligomer formations or if so, this phenomenon whole-exome sequencing, exons of DNA samples were captured would be restricted to inner retinal layers, and the hyper- and investigated as shown before (10) using in-solution enrich- reflectivity seen on SD-OCT may represent such aggregation. ment methodology (SureSelect Human All Exon Kits Version Of note, ganglion cell loss is reported in AD (34) but abnormal 3, Agilent, Massy, France), NGS (Illumina HISEQ, Illumina, diffuse reflectivity of inner retinal layers has never been reported. San Diego, CA, USA), image analysis and base calling using Interestingly, APP knock-out mice display inner retinal dysfunc- Real Time Analysis Pipeline version 1.9 with default parameters tion (13), which could also suggest that such dysfunction in our (Illumina). The bioinformatic analysis of sequencing data was family may be mediated through APP/mutated-ITM2B-disturbed based on a pipeline [Consensus Assessment of Sequence and interaction. Variation (CASAVA) 1.8, Illumina] which performs alignment, Human Molecular Genetics, 2014, Vol. 23, No. 2 499 calls the SNPs based on the allele calls and read depth and detects mouse anti-BRN3A (Millipore, Molsheim, France), localized in variants (SNPs and indels). Genetic variation annotation was ganglion cells and 1/600 for secondary fluorescent antibodies realized by an in-house pipeline (IntegraGen, Evry, France) (Alexa Fluor 488-conjugated and CY3, Invitrogen, Courtabœuf, and results were provided per sample in tabulated text files. France) and nuclei counterstained with 4′,6-diamidino-2-pheny- For filtering, exonic and splice variants were selected on the lindole (Euromedex, Souffelweyersheim, France) or a mouse basis of their heterozygosity in affected subjects and absence peroxidase-coupled secondary antibodies (Jackson ImmunoRe- in the unaffected siblings, in dbSNP 137, HapMap (38), search, West Grove, PA, USA) using a peroxidase substrate kit the 1000 Genome Project (39) and the Exome Variant Server (kit VIP peroxydase Vector, Cliniscience, Nanterre, France). (http://evs.gs.washington.edu/EVS/, last accessed date on September 16, 2013). Sequence conservation, PolyPhen2 In vitro expression studies on wild-type and ITM2B (Polymorphism Phenotyping, http://genetics.bwh.harvard.edu/ mutant constructs pph2/, last accessed date on September 16, 2013) and SIFT Subcellular immunolocalization of the normal and three mutated (Sorting Intolerant From Tolerant; http://sift.bii.a-star.edu.sg/, ∗ Downloaded from https://academic.oup.com/hmg/article/23/2/491/663466 by guest on 01 October 2021 last accessed date on September 16, 2013) software were used ITM2B variants [c.799T.A; p.Stop267Argext 11 identified in the British Dementia Family (7), c.795-796insTTTAATTTGT; to predict the pathogenic nature of sequence alterations. Haplo- ∗ type analysis was performed using 16 custom microsatellite p.Ser266Pheext 11 identified in the Danish Dementia family markers and a fluorescently labelled universal primer [method (16) and c.782A.C, p.Glu261Ala from our study] was analysed adapted from de Arruda et al.(40)] from chromosome 13q on under live-cell condition and after permeabilization as described 19 family members (Supplementary Material, Fig. S1). Coding before (43). and flanking exonic regions of ITM2B was analysed by Sanger sequencing (RefSeq NM_021999.4, primer sequences and WEB RESOURCES PCR conditions available upon request). Sequence conserva- Databases used to predict the pathogenic character of a sequence tion, PolyPhen2 and SIFT software were used to predict the alteration, expression and protein function: pathogenic nature of sequence alterations. (i) National Center for Biotechnology Information (NCBI) (http://ncbi.nlm.nih.gov/, last accessed date onSeptember Expression analysis 16, 2013) Expression profile for the eye and retina was investigated using (ii) UniGene (http://www.ncbi.nlm.nih.gov/UniGene/ESTProfile three databases: Unigene, the mouse retinal gene expression Viewer.cgi?Uglist=Hs.643683, last accessed date on profile from Siegert et al.(9) and the in-house rd1 mouse ex- September 16, 2013) pression database [the rd1 mouse, carrying Pde6b mutations, (iii) Online Mendelian Inheritance in Man (http://ncbi.nlm. is a naturally occurring retinitis pigmentosa model leading to a nih.gov//Omim, last accessed date on September 16, complete loss of all rod photoreceptors by post-natal day 36, 2013) and preserved inner retina (41)]. Real-time PCR experiments (iv) USCS Browser (http://genome.ucsc. on commercially available human retina cDNA (Clontech, edu/, last accessed date on September 16, 2013) Saint-Germain-en-Laye, France), lymphocyte and HEK293 (v) GenCards (http://www.genecards.org, last accessed date cell cDNA were performed for ITM2B (MIM#∗603904), using on September 16, 2013) ACTB (MIM#∗102630) as a control (primers available upon (vi) PolyPhen-2 (Polymorphism Phenotyping v2, http:// request). genetics.bwh.harvard.edu/pph2/, last accessed date on September 16, 2013) (vii) SIFT (Sorting Intolerant From Tolerant, http://blocks. RNA in situ hybridization studies on mouse retinas fhcrc.org/sift/SIFT.html, last accessed date on September RNA in situ hybridization on mouse retina was performed using 16, 2013) a riboprobe encompassing exons 2 to 6 of the Itm2b mouse (viii) UniProtKB/Swiss-Prot (www..org, last accessed mRNA (RefSeq NM_008410.2) as described before (42) date on September 16, 2013) (details available upon request). (ix) Exome Variant Server (http://evs.gs.washington.edu/ EVS, last accessed date on September 16, 2013) (x) Human Gene Mutation Database (http://www.hgmd.cf. Immunohistochemistry ac.uk, last accessed date on September 16, 2013) Immunolocalization of ITM2B, BRN3A and APP in mouse and (xi) Leiden Open Variation Database (www.lovd.nl, last human retina was studied on 20 mm-thick gelatine-embedded accessed date on September 16, 2013). coronal eye cryosections and whole-mount retina as described before (10). Human retina specimens were obtained from SUPPLEMENTARY MATERIAL the Minnesota Lions Eye Bank with due consent in accordance with the Declaration of Helsinki. Antibody dilutions are as Supplementary Material is available at HMG online. follows: 1/50 dilution for polyclonal rabbit anti-ITM2B (HPA029292, Sigma-Aldrich, St Quentin Fallavier, France), 1/ ACKNOWLEDGEMENTS 100 dilution for monoclonal mouse anti-ITM2B (SAB1402472, Sigma-Aldrich), 1/200 dilution for polyclonal rabbit anti-APP The authors are grateful to the family described in this study; to HPA001462 (Sigma-Aldrich), 1/200 dilution for monoclonal Gae¨l Orieux in assisting with immunostaining, to Ste´phane 500 Human Molecular Genetics, 2014, Vol. 23, No. 2

Fouquet for his support on confocal microscopy, to Dominique 13. Ho, T., Vessey, K.A., Cappai, R., Dinet, V., Mascarelli, F., Ciccotosto, G.D. Santiard-Baron and Christine Chaumeil for their help in DNA and Fletcher, E.L. (2012) Amyloid precursor protein is required for normal function of the rod and cone pathways in the mouse retina. PLoS One, 7, collection; to the clinical staff. e29892. 14. Arevalo, J.F. and Freeman, W.R. (2000) Corneal endothelial deposits in Conflict of Interest statement. None declared. children positive for human immunodeficiency virus receiving rifabutin prophylaxis for mycobacterium avium complex bacteremia. Am. J. Ophthalmol., 129, 410–411. 15. Mahajan, V.B., Skeie, J.M., Bassuk, A.G., Fingert, J.H., Braun, T.A., FUNDING Daggett, H.T., Folk, J.C., Sheffield, V.C. and Stone, E.M. (2012) Calpain-5 The project was supported by GIS-maladies rares (C.Z.), Retina mutations cause autoimmune uveitis, retinal neovascularization, and photoreceptor degeneration. PLoS Genet., 8, e1003001. France (part of the 100-Exome Project) (I.A., J.-A.S. and C.Z.), 16. Vidal, R., Revesz, T., Rostagno, A., Kim, E., Holton, J.L., Bek, T., Foundation Voir et Entendre (C.Z.), Foundation Fighting Blind- Bojsen-Moller, M., Braendgaard, H., Plant, G., Ghiso, J. et al. (2000) A ness (FFB) grant CD-CL-0808-0466-CHNO (I.A. and the decamer duplication in the 3′ region of the BRI gene originates an amyloid CIC503, recognized as an FFB centre), FFB grant peptide that is associated with dementia in a Danish kindred. Proc. Natl Downloaded from https://academic.oup.com/hmg/article/23/2/491/663466 by guest on 01 October 2021 C-CMM-0907-0428-INSERM04, Ville de Paris and Region Acad. Sci. USA, 97, 4920–4925. 17. Mead, S., James-Galton, M., Revesz, T., Doshi, R.B., Harwood, G., Pan, Ile de France and by the French State programme ‘Investisse- E.L., Ghiso, J., Frangione, B. and Plant, G. (2000) Familial British dementia ments d’Avenir’ managed by the Agence Nationale de la with amyloid angiopathy: early clinical, neuropsychological and imaging Recherche (LIFESENSES: ANR-10-LABX-65). findings. Brain, 123, 975–991. 18. Stromgren, E., Dalby, A., Dalby, M.A. and Ranheim, B. (1970) Cataract, deafness, cerebellar ataxia, psychosis and dementia—a new syndrome. Acta Neurol. Scand., 46 (Suppl. 43), 261+. REFERENCES 19. Bek, T. (2000) Ocular changes in heredo-oto-ophthalmo-encephalopathy. 1. Xuan,J.,Guo,L. andShi,L.(2012)Next-generationsequencingin theclinic: Br. J. Ophthalmol., 84, 1298–1302. promises and challenges. Cancer Lett. doi:pii: S0304-3835(12)00672-6. 20. Pittois, K., Deleersnijder, W. and Merregaert, J. (1998) cDNA sequence 10.1016/j.canlet.2012.11.025. analysis,chromosomalassignmentand expression patternof thegene coding 2. Audo, I., Robson, A.G., Holder, G.E. and Moore, A.T. (2008) The negative for integral membrane protein 2B. Gene, 217, 141–149. ERG: clinical phenotypes and disease mechanisms of inner retinal 21. Pickford, F., Onstead, L., Camacho-Prihar, C., Hardy, J. and McGowan, E. dysfunction. Surv. Ophthalmol., 53, 16–40. (2003) Expression of mBRI2 in mice. Neurosci. Lett., 338, 95–98. 3. Audo, I., Michaelides, M., Robson, A.G., Hawlina, M., Vaclavik, V., 22. Martin, L., Fluhrer, R., Reiss, K., Kremmer, E., Saftig, P. and Haass, C. Sandbach, J.M., Neveu, M.M., Hogg, C.R., Hunt, D.M., Moore, A.T. et al. (2008) Regulated intramembrane proteolysis of Bri2 (Itm2b) by ADAM10 (2008) Phenotypic variation in enhanced S-cone syndrome. Invest. and SPPL2a/SPPL2b. J. Biol. Chem., 283, 1644–1652. Ophthalmol. Vis. Sci., 49, 2082–2093. 23. Sanchez-Pulido, L., Devos, D. and Valencia, A. (2002) BRICHOS: a 4. Holder, G.E., Robson, A.G., Hogg, C.R., Kurz-Levin, M., Lois, N. and Bird, conserved domain in proteins associated with dementia, respiratory distress A.C. (2003) Pattern ERG: clinical overview, and some observations on and cancer. Trends Biochem. Sci., 27, 329–332. associated fundus autofluorescence imaging in inherited maculopathy. Doc. 24. Kim, J., Miller, V.M., Levites, Y., West, K.J., Zwizinski, C.W., Ophthalmol., 106, 17–23. Moore, B.D., Troendle, F.J., Bann, M., Verbeeck, C., Price, R.W. et al. 5. Audo, I., Bujakowska, K.M., Leveillard, T., Mohand-Said, S., Lancelot, (2008) BRI2 (ITM2b) inhibits abeta deposition in vivo. J. Neurosci., 28, M.E., Germain, A., Antonio, A., Michiels, C., Saraiva, J.P., Letexier, M. 6030–6036. et al. (2012) Development and application of a next-generation-sequencing 25. Matsuda, S., Giliberto, L., Matsuda, Y., McGowan, E.M. and D’Adamio, L. (NGS) approach to detect known and novel gene defects underlying retinal (2008) BRI2 Inhibits -peptide precursor protein processing by diseases. Orphanet. J. Rare Dis., 7,8. interfering with the docking of secretases to the substrate. J. Neurosci., 28, 6. Bandah-Rozenfeld, D., Collin, R.W., Banin, E., van den Born, L.I., Coene, 8668–8676. K.L., Siemiatkowska, A.M., Zelinger, L., Khan, M.I., Lefeber, D.J., 26. Peng, S., Fitzen, M., Jornvall, H. and Johansson, J. (2010) The extracellular Erdinest, I. et al. (2010) Mutations in IMPG2, encoding interphotoreceptor domain of Bri2 (ITM2B) binds the ABri peptide (1–23) and amyloid matrix 2, cause autosomal-recessive retinitis pigmentosa. beta-peptide (Abeta1–40): implications for Bri2 effects on processing of Am. J. Hum. Genet., 87, 199–208. amyloid precursor protein and abeta aggregation. Biochem. Biophys. Res. 7. Vidal, R., Frangione, B., Rostagno, A., Mead, S., Revesz, T., Plant, G. and Commun., 393, 356–361. Ghiso, J. (1999) A stop-codon mutation in the BRI gene associated with 27. Matsuda, S., Matsuda, Y., Snapp, E.L. and D’Adamio, L. (2011) Maturation familial British dementia. Nature, 399, 776–781. of BRI2 generates a specific inhibitor that reduces APP processing at the 8. Kim, S.H., Wang, R., Gordon, D.J., Bass, J., Steiner, D.F., Lynn, D.G., plasma membrane and in endocytic vesicles. Neurobiol. Aging, 32, 1400– Thinakaran, G., Meredith, S.C. and Sisodia, S.S. (1999) Furin mediates 1408. enhanced production of fibrillogenic ABri peptides in familial British 28. Kilger, E., Buehler, A., Woelfing, H., Kumar, S., Kaeser, S.A., dementia. Nat. Neurosci., 2, 984–988. Nagarathinam, A., Walter, J., Jucker, M. and Coomaraswamy, J. 9. Siegert, S., Cabuy, E., Scherf, B.G., Kohler, H., Panda, S., Le, Y.Z., Fehling, (2011) BRI2 Protein regulates beta-amyloid degradation by increasing H.J., Gaidatzis, D., Stadler, M.B. and Roska, B. (2012) Transcriptional code levels of secreted insulin-degrading enzyme (IDE). J. Biol. Chem., 286, and disease map for adult retinal cell types. Nat. Neurosci., 15, 487–495, 37446–37457. S1–S2. 29. Choi, S.I., Vidal, R., Frangione, B. and Levy, E. (2004) Axonal transport of 10. Audo, I., Bujakowska, K., Orhan, E., Poloschek, C.M., British and Danish amyloid peptides via secretory vesicles. FASEB J., 18, Defoort-Dhellemmes, S., Drumare, I., Kohl, S., Luu, T.D., Lecompte, O., 373–375. Zrenner, E. et al. (2012) Whole-exome sequencing identifies mutations in 30. Chasseigneaux, S. and Allinquant, B. (2012) Functions of abeta, sAPPalpha GPR179 leading to autosomal-recessive complete congenital stationary and sAPPbeta: similarities and differences. J. Neurochem., 120 (Suppl. 1), night blindness. Am. J. Hum. Genet., 90, 321–330. 99–108. 11. Matsuda, S., Giliberto, L., Matsuda, Y., Davies, P., McGowan, E., Pickford, 31. Ghiso, J., Rostagno, A., Tomidokoro, Y., Lashley, T., Bojsen-Moller, M., F., Ghiso, J., Frangione, B. and D’Adamio, L. (2005) The familial dementia Braendgaard, H., Plant, G., Holton, J., Lal, R., Revesz, T. et al. (2006) BRI2 gene binds the Alzheimer gene amyloid-beta precursor protein and Genetic alterations of the BRI2 gene: familial British and Danish dementias. inhibits amyloid-beta production. J. Biol. Chem., 280, 28912–28916. Brain Pathol., 16, 71–79. 12. Fotinopoulou, A.,Tsachaki,M.,Vlavaki,M.,Poulopoulos,A.,Rostagno,A., 32. El-Agnaf, O.M., Sheridan, J.M., Sidera, C., Siligardi, G., Hussain, R., Haris, Frangione, B., Ghiso, J. and Efthimiopoulos, S. (2005) BRI2 interacts with P.I. and Austen,B.M. (2001) Effect of the disulfidebridge and the C-terminal amyloid precursor protein (APP) and regulates amyloid beta (abeta) extension on the oligomerization of the amyloid peptide ABri implicated in production. J. Biol. Chem., 280, 30768–30772. familial British dementia. Biochemistry, 40, 3449–3457. Human Molecular Genetics, 2014, Vol. 23, No. 2 501

33. Holton, J.L., Ghiso, J., Lashley, T., Rostagno, A., Guerin, C.J., Gibb, G., (2010) Integrating common and rare genetic variation in diverse human Houlden, H., Ayling, H., Martinian, L., Anderton, B.H. et al. (2001) populations. Nature, 467, 52–58. Regional distribution of amyloid-Bri deposition and its association with 39. The 1000 Genomes Project Consortium (2010) A map of human neurofibrillary degeneration in familial British dementia. Am. J. Pathol., genome variation from population-scale sequencing. Nature, 467, 158, 515–526. 1061–1073. 34. Hinton, D.R., Sadun, A.A., Blanks, J.C. and Miller, C.A. (1986) Optic-nerve 40. de Arruda, M.P., Goncalves, E.C., Schneider, M.P., da Silva, A.L. and degeneration in Alzheimer’s disease. N. Engl. J. Med., 315, 485–487. Morielle-Versute, E. (2010) An alternative genotyping method using 35. Jen, L.S., Hart, A.J., Jen, A., Relvas, J.B., Gentleman, S.M., Garey, L.J. and dye-labeled universal primer to reduce unspecific amplifications. Mol. Biol. Patel, A.J. (1998) Alzheimer’s peptide kills cells of retina in vivo. Nature, Rep., 37, 2031–2036. 392, 140–141. 41. Carter-Dawson, L.D., LaVail, M.M. and Sidman, R.L. (1978) Differential 36. Audo, I., Manes, G., Mohand-Said, S., Friedrich, A., Lancelot, M.E., effect of the rd mutation on rods and cones in the mouse retina. Invest. Antonio, A., Moskova-Doumanova, V., Poch, O., Zanlonghi, X., Hamel, Ophthalmol. Vis. Sci., 17, 489–498. C.P. et al. (2010) Spectrum of rhodopsin mutations in French autosomal 42. Di Meglio, T., Nguyen-Ba-Charvet, K.T., Tessier-Lavigne, M., Sotelo, C. dominant rod-cone dystrophy patients. Invest. Ophthalmol. Vis. Sci., 51, and Chedotal, A. (2008) Molecular mechanisms controlling midline 3687–3700. crossing by precerebellar neurons. J. Neurosci., 28, 6285–6294. 37. Folstein, M.F., Folstein, S.E. and McHugh, P.R. (1975) ‘Mini-mental state’. Downloaded from https://academic.oup.com/hmg/article/23/2/491/663466 by guest on 01 October 2021 43. Zeitz, C., Scherthan, H., Freier, S., Feil, S., Suckow, V., Schweiger, S. and A practical method for grading the cognitive state of patients for the clinician. J. Psychiatr. Res., 12, 189–198. Berger, W. (2003) NYX (nyctalopin on chromosome X), the gene mutated in 38. Altshuler, D.M., Gibbs, R.A., Peltonen, L., Altshuler, D.M., Gibbs, R.A., congenital stationary night blindness, encodes a cell surface protein. Invest. Peltonen, L., Dermitzakis, E., Schaffner, S.F., Yu, F., Peltonen, L. et al. Ophthalmol. Vis. Sci., 44, 4184–4191.