Abstract

Inherited retinal diseases are a group of clinically and genetically heterogeneous disorders for which a significant number of cases remain genetically unresolved. Increasing knowledge on underlying pathogenic mechanisms with precise phenotype–genotype correlation is, however, critical for establishing novel therapeutic interventions for these yet incurable neurodegenerative conditions. We report phenotypic and genetic characterization of a large family presenting an unusual autosomal dominant retinal dystrophy. Phenotypic characterization 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 Protein 2B, which co-segregates with the disease in this large family and lies within the 24.6 Mb interval 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 amyloid β precursor protein, critical in Alzheimer disease physiopathology. Interestingly, two distinct ITM2B mutations, both resulting in a longer protein product, had already been reported in two large autosomal dominant 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 genes involved in inherited retinal dystrophies.

INTRODUCTION

Inherited retinal dystrophies are a heterogeneous group of disorders including stationary conditions such as congenital stationary night blindness (CSNB) characterized by post-phototransduction dysfunction or degenerative processes such as rod–cone and cone–rod dystrophies. This clinical heterogeneity is mirrored by genetic heterogeneity (https://sph.uth.edu/Retnet/, last accessed date on September 16, 2013). Despite the increasing number of identified genes recognized for their role in retinal physiology and pathology, the underlying genetic defect(s) remains unknown in a number of cases. Increasing knowledge in pathogenic mechanisms and their associated phenotype–genotype correlation is de facto critical for a better understanding of these heterogeneous disorders, even for rare subgroups. It may not only provide important insights into the complexity of retinal physiology or neurophysiology in general but also help for accurate counselling and, moreover, for the establishment of innovative therapeutic interventions in these yet incurable conditions. Recently, techniques of next-generation sequencing (NGS) have proved their efficiency to increase chances of deciphering unknown genetic defect in this context (1).

We applied these techniques to decipher the underlying gene defect in a large dominant pedigree with 14 members affected by a clinically novel retinal dystrophy dominated by inner retinal dysfunction and ganglion cell abnormalities. Applying whole-exome sequencing and subsequent haplotype analysis, we identified a missense mutation in ITM2B, encoding Integral Membrane Protein 2B, which co-segregates with the disease, and further investigated expression of this gene in the retina.

RESULTS

Clinical characterization

The reported family includes at least 14 affected family members spanning three generations, with two deceased, suffering from visual symptoms segregating as a dominant trait (Fig. 1, proband with a single arrow). Eleven affected and eight unaffected subjects were available for genetic testing. Detailed clinical examination was performed on affected family members ranging in age from 46 to 73 years (average 54) (Table 1). Onset of symptoms appeared between 25 and 40 years (average 36). Light sensitivity was the first sign, reported by all subjects, followed by progressive loss of central vision. Visual acuity varied from 20/25 to 20/400 (average 20/50). Visual fields showed decreased central retinal sensitivity with preservation of peripheral visual field. Fundus examination, fundus autofluorescence imaging and spectral domain optical coherence tomography (SD-OCT) revealed macular changes associated with optic disc pallor, hyper-reflectivity of ganglion cell and nerve fibre layers (Fig. 2A) with loss of optic nerve fibres (Fig. 2B). Full-field electroretinogram (Fig. 2C) showed inner retinal dysfunction in all cases: all affected subjects displayed an absent or very reduced b-wave in response to a dim flash of 0.01 cd s m−2 under dark-adapted (scotopic) conditions; stimulation with a bright flash (3 and 12 0.01 cd s m−2) under scotopic conditions revealed a normal a-wave, reflecting normal photoreceptor function, dominated under dark adaptation by rod function, but a severely reduced b-wave, reflecting post-receptoral dysfunction with the typical electronegative waveform. Oscillatory potentials, originating at the inner retinal level, were undetectable on older affected subjects and decreased in amplitude with a simplified wave shape in younger subjects, in keeping with inner retinal dysfunction. Light-adapted (photopic) responses, testing the cone system function, were relatively preserved for both 3 cd s m−2 single flash and 30 Hz Flicker stimulation in younger subjects, whereas older affected subjects showed additional photopic response abnormalities with decreased amplitudes and delayed implicit time (Fig. 2C). Since photopic responses are driven by cone photoreceptors but represent responses generated at the inner retinal level, this could suggest either a worsening of inner retinal dysfunction affecting the cone pathway or direct cone photoreceptor dysfunction. None of the affected subject displayed the abnormal wave form seen in selective ON-bipolar cell dysfunction [i.e. an a-wave with a broaden trough and a sharply arising b-wave characterizing the complete form of CSNB (2)]. To further investigate whether ON- and/or OFF-bipolar cell function was affected, long-duration stimulation recordings applying an orange flash stimulus on a green background to document ON and OFF responses from the L/M-cone systems (3) were attempted. Traces obtained by such stimuli were variable, partially linked to the photophobia present in most affected subjects. When reproducible, results suggested both ON- and OFF-pathway dysfunction with decrease in the amplitude of both the b- and d-wave, respectively (Fig. 2D). In addition, pattern ERG, performed to document macular and ganglion cell function (4), revealed reduced amplitudes for both P50 and N95 components in all patients in keeping with proximal macular dysfunction (Fig. 2E). In this context, selective ganglion cell function could not specifically be assessed.

Table 1.

Clinical characteristics of affected family members

Patient Age at the time of testing Age of onset Sex Symptoms BCVA
OD/OS
refraction 
Colour vision Kinetic visual field Fundus examination FAF SD-OCT 
III.13 73 Around 40 Mild photophobia
Decreased vision 
20/100; 20/250
−1.75 (−1.75) 95°
−3.75 (−0.5) 70° 
OD normal
OS mild tritanopia 
Relative central scotoma within 10 central degrees with normal peripheral isopter Temporal optic disc pallor; mild retinal vessel narrowing; foveal changes Hyper-autofluorescent ring around the macular region; hypo-autofluorescence within the ring with subtle foveal hyper-autofluorescence Hyper-reflectivity within the foveal ONL; small drusenoid changes in the foveal region; hyper-reflectivity of the inner retina 
III.15 72 Around 40 Mild photophobia
Decreased vision 
20/250; 20/400
+1.25 (−1.75) 130°
+1 (−1) 80° 
OD and OS mild deuteranopia Relative central scotoma within 20 central degrees with normal peripheral isopter Pale optic disc; mild retinal vessel narrowing; foveal changes Hyper-autofluorescent ring around the macular region; hypo-autofluorescence within the ring with subtle foveal hyper-autofluorescence Hyper-reflectivity within the foveal ONL; hyper-reflectivity and thinning of the inner retina 
IV.11 47 Photophobia at around 30
Decreased vision at age 35 
Mild photophobia
Decreased vision 
20/63; 20/80
+1 (−0.25) 125°
−0.75 (−0.25) 85° 
OD and OS normal Relative central scotoma within the 5 central degree; blind spot exclusion; normal peripheral isopter Temporal pale optic disc; subtle foveal changes Mild hyper-autofluorescent ring around the macular region; subtle hypo-autofluorescence within the ring Hyper-reflectivity within the foveal ONL; hyper-reflectivity of the inner retina 
IV.12 47 Photophobia since age 25
Decreased vision at age 35 
Mild photophobia
Decreased vision 
20/50; 20/63
+0.50
+0.75 
OD and OS normal Relative central scotoma within the 5 central degree; blind spot exclusion; normal peripheral isopter Temporal pale optic disc; subtle foveal changes Mild hyper-autofluorescent ring around the macular region; subtle hypo-autofluorescence within the ring Hyper-reflectivity within the foveal ONL; hyper-reflectivity of the inner retina 
IV.13 46 Photophobia around 30 and then decreased vision at age 33 Mild photophobia
Decreased vision 
20/80; 20:63
−4.50 (−0.50) 100°
−4 (−0.75) 85° 
OD and OS normal Relative central scotoma within the 5 central degree; normal peripheral isopter Temporal pale optic disc; subtle foveal changes Mild hyper-autofluorescent ring around the macular region; subtle hypo-autofluorescence within the ring
Peripheral autofluorescence abnormalities 
Hyper-reflectivity within the foveal ONL; hyper-reflectivity of the inner retina 
IV.15 51 Around 40 Mild photophobia
Decreased vision 
20/25; 20/63
Plano
+0.50 
OD mild deuteranopia
OS moderate dyschromatopsia without preferential axis 
Centrocoecal scotoma with normal peripheral isopter Temporal pale optic disc; subtle foveal changes Hyper-autofluorescence in the posterior pole with decreased autofluorescence within the macular region and a fascicular inferior hypo-autofluorescence in the right eye Hyper-reflectivity within the foveal ONL; hyper-reflectivity of the inner retina; thinning of the outer retina in the paramacular area 
IV.22 50 Around 40 Mild photophobia
Decreased vision 
20/100; 20/125
−3.50 (−0.75) 110°
−4.25 (−0.75) 65° 
OD and OS normal Relative central scotoma within the 15 central degree; normal peripheral isopter Subtle temporal pale optic disc; subtle foveal changes Hyper-autofluorescent ring around the macular region; hypo-autofluorescence within the ring with subtle foveal hyper-autofluorescence Hyper-reflectivity within the foveal ONL; hyper-reflectivity of the inner retina 
IV.27 50 Night vision problem and photophobia at age 38 Night blindness
Mild photophobia
Decreased vision 
20/100; 20/100
−2 (−1) 110°
−2 (1;50) 60° 
OD and OS normal Relative central scotoma within the 15 central degree; normal peripheral isopter Subtle temporal pale optic disc; subtle foveal changes Hyper-autofluorescent ring around the macular region; hypo-autofluorescence within the ring Hyper-reflectivity within the foveal ONL; hyper-reflectivity of the inner retina 
IV.28 49 Around 40 Night blindness
Mild photophobia
Decreased vision 
20/50; 20/50
−2.50 (−1.25) 65°
−2.50 (−1.25) 60° 
OD and OS deuteranopia Relative central scotoma within the 10 central degree; normal peripheral isopter Subtle temporal pale optic disc; subtle foveal changes Subtle hyper-autofluorescent ring around the macular region; hypo-autofluorescence within the ring Hyper-reflectivity within the foveal ONL; hyper-reflectivity of the inner retina 
Patient Age at the time of testing Age of onset Sex Symptoms BCVA
OD/OS
refraction 
Colour vision Kinetic visual field Fundus examination FAF SD-OCT 
III.13 73 Around 40 Mild photophobia
Decreased vision 
20/100; 20/250
−1.75 (−1.75) 95°
−3.75 (−0.5) 70° 
OD normal
OS mild tritanopia 
Relative central scotoma within 10 central degrees with normal peripheral isopter Temporal optic disc pallor; mild retinal vessel narrowing; foveal changes Hyper-autofluorescent ring around the macular region; hypo-autofluorescence within the ring with subtle foveal hyper-autofluorescence Hyper-reflectivity within the foveal ONL; small drusenoid changes in the foveal region; hyper-reflectivity of the inner retina 
III.15 72 Around 40 Mild photophobia
Decreased vision 
20/250; 20/400
+1.25 (−1.75) 130°
+1 (−1) 80° 
OD and OS mild deuteranopia Relative central scotoma within 20 central degrees with normal peripheral isopter Pale optic disc; mild retinal vessel narrowing; foveal changes Hyper-autofluorescent ring around the macular region; hypo-autofluorescence within the ring with subtle foveal hyper-autofluorescence Hyper-reflectivity within the foveal ONL; hyper-reflectivity and thinning of the inner retina 
IV.11 47 Photophobia at around 30
Decreased vision at age 35 
Mild photophobia
Decreased vision 
20/63; 20/80
+1 (−0.25) 125°
−0.75 (−0.25) 85° 
OD and OS normal Relative central scotoma within the 5 central degree; blind spot exclusion; normal peripheral isopter Temporal pale optic disc; subtle foveal changes Mild hyper-autofluorescent ring around the macular region; subtle hypo-autofluorescence within the ring Hyper-reflectivity within the foveal ONL; hyper-reflectivity of the inner retina 
IV.12 47 Photophobia since age 25
Decreased vision at age 35 
Mild photophobia
Decreased vision 
20/50; 20/63
+0.50
+0.75 
OD and OS normal Relative central scotoma within the 5 central degree; blind spot exclusion; normal peripheral isopter Temporal pale optic disc; subtle foveal changes Mild hyper-autofluorescent ring around the macular region; subtle hypo-autofluorescence within the ring Hyper-reflectivity within the foveal ONL; hyper-reflectivity of the inner retina 
IV.13 46 Photophobia around 30 and then decreased vision at age 33 Mild photophobia
Decreased vision 
20/80; 20:63
−4.50 (−0.50) 100°
−4 (−0.75) 85° 
OD and OS normal Relative central scotoma within the 5 central degree; normal peripheral isopter Temporal pale optic disc; subtle foveal changes Mild hyper-autofluorescent ring around the macular region; subtle hypo-autofluorescence within the ring
Peripheral autofluorescence abnormalities 
Hyper-reflectivity within the foveal ONL; hyper-reflectivity of the inner retina 
IV.15 51 Around 40 Mild photophobia
Decreased vision 
20/25; 20/63
Plano
+0.50 
OD mild deuteranopia
OS moderate dyschromatopsia without preferential axis 
Centrocoecal scotoma with normal peripheral isopter Temporal pale optic disc; subtle foveal changes Hyper-autofluorescence in the posterior pole with decreased autofluorescence within the macular region and a fascicular inferior hypo-autofluorescence in the right eye Hyper-reflectivity within the foveal ONL; hyper-reflectivity of the inner retina; thinning of the outer retina in the paramacular area 
IV.22 50 Around 40 Mild photophobia
Decreased vision 
20/100; 20/125
−3.50 (−0.75) 110°
−4.25 (−0.75) 65° 
OD and OS normal Relative central scotoma within the 15 central degree; normal peripheral isopter Subtle temporal pale optic disc; subtle foveal changes Hyper-autofluorescent ring around the macular region; hypo-autofluorescence within the ring with subtle foveal hyper-autofluorescence Hyper-reflectivity within the foveal ONL; hyper-reflectivity of the inner retina 
IV.27 50 Night vision problem and photophobia at age 38 Night blindness
Mild photophobia
Decreased vision 
20/100; 20/100
−2 (−1) 110°
−2 (1;50) 60° 
OD and OS normal Relative central scotoma within the 15 central degree; normal peripheral isopter Subtle temporal pale optic disc; subtle foveal changes Hyper-autofluorescent ring around the macular region; hypo-autofluorescence within the ring Hyper-reflectivity within the foveal ONL; hyper-reflectivity of the inner retina 
IV.28 49 Around 40 Night blindness
Mild photophobia
Decreased vision 
20/50; 20/50
−2.50 (−1.25) 65°
−2.50 (−1.25) 60° 
OD and OS deuteranopia Relative central scotoma within the 10 central degree; normal peripheral isopter Subtle temporal pale optic disc; subtle foveal changes Subtle hyper-autofluorescent ring around the macular region; hypo-autofluorescence within the ring Hyper-reflectivity within the foveal ONL; hyper-reflectivity of the inner retina 

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, outer nuclear layer.

Figure 1.

Family pedigree revealing autosomal dominant segregation of the disease. Affected individuals are presented with filled symbols, unaffected with white symbols; square symbols represent male and round symbols, female; deceased individuals are presented with a slash; subjects who underwent ophthalmic evaluation in our centre are marked with an asterisk; mutation segregation is shown on the pedigree as ‘[=];[=]’ carrying both normal allele and ‘[M];[=]’ 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 included in whole-exome sequencing are marked with arrows (i.e. IV.1 and IV.12).

Figure 1.

Family pedigree revealing autosomal dominant segregation of the disease. Affected individuals are presented with filled symbols, unaffected with white symbols; square symbols represent male and round symbols, female; deceased individuals are presented with a slash; subjects who underwent ophthalmic evaluation in our centre are marked with an asterisk; mutation segregation is shown on the pedigree as ‘[=];[=]’ carrying both normal allele and ‘[M];[=]’ 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 included in whole-exome sequencing are marked with arrows (i.e. IV.1 and IV.12).

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 colour fundus photograph shows subtle changes, including temporal pallor of the optic disc and very mild changes in the macula; (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) 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, respectively. (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.

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 colour fundus photograph shows subtle changes, including temporal pallor of the optic disc and very mild changes in the macula; (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) 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, respectively. (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.

None of the affected subjects had specific systemic diseases, and no cases of dementia had been reported in the family, including individuals aged >50 years, suggesting a retinal-restricted disease. Furthermore, all subjects appear well temporally and spatially oriented during examination, and Mini-Mental State Examination was normal in the proband.

Genetic studies

Direct Sanger sequencing of coding and flanking regions of genes implicated in dominant post-photoreceptor dysfunction (RHO, PDE6B, GNAT1 and TRPM1) did not reveal pathogenic changes. Further genetic analysis of proband's DNA applying NGS targeted towards exons of all genes implicated in retinal diseases (5) did not reveal pathogenic variants co-segregating with the disease. Whole-exome analysis applied to proband IV.27, one affected IV.12 and one unaffected IV.1 cousin (see arrows on Fig. 1) followed by stringent filtering of genetic variants reduced the number of variants from 118 777 single-nucleotide polymorphisms (SNPs) to 19 and from 12 714 insertion and deletions (indels) to 8. Of these, the only gene already involved in retinal dystrophies was IMPG2 (MIM#*607056) (6), for which NGS and whole-exome sequencing detected a heterozygous 3 base pair deletion on affected subjects. However, family co-segregation study excluded the causality of this variant. Eight of the 19 SNPs were selected for co-segregation analysis after bioinformatic pathogenic prediction. These variants were located in the following genes: OMP (MIM#*164340), ITM2B (MIM#*603904), CC2D1A (MIM#*610055), RFX1 (MIM#*600006), OR7A5, C2orf44, FAM184A and OPLAH (MIM#*614243) (Supplementary Material, Table S1). Only the change located in exon 6 of ITM2B (c.782A>C, p.Glu261Ala) co-segregated with the phenotype. This variant was not found in 380 control alleles, is predicted to be probably damaging and is well conserved (Supplementary Material, Fig. S1). It was not found in public sequence repositories including the Human Gene Mutation Database (http://www.hgmd.cf.ac.uk, last accessed date on September 16, 2013) or the Leiden Open Variation Database (www.lovd.nl). Haplotype analysis using 16 custom microsatellite from chromosome 13q on 19 family members identified crossovers between markers D13S1272 and D13S275 leading to an interval of 24.6 Mb including ITM2B with the same haplotype co-segregating in affected subjects (Supplementary Material, Fig. S2).

The 24.6 Mb interval contains 74 protein-coding genes. Among these, 52 were reported to be expressed in the eye on Unigene, but only RB1 and ITM2B had previously been associated with an ocular disease, namely retinoblastoma and cataract with amyloid angiopathy and dementia, respectively. Only exon 1 of RB1 was poorly covered by whole-exome sequencing within the 24.6 Mb and was further Sanger-sequenced. None of the coding regions and exon boundaries of the 74 genes were carrying a variant that co-segregates with the phenotype in the family besides the (c.782A>C, p.Glu261Ala) change in ITM2B.

Residue number 261 lies within a secreted peptide consisting of 23 residues obtained after ITM2B cleavage by furin or furin-like convertase (7,8). There is no reliable three-dimensional structure of this secreted peptide, but it is predicted to form beta–loop–beta structures. Substitution of a negatively charged glutamic acid for a non-polar alanine would most probably induce instability of this predicted structure.

Sanger sequencing of coding and flanking exonic regions of ITM2B (RefSeq NM_021999.4) did not reveal additional pathogenic variants in a panel of 95 subjects with a partially resembling phenotype (11 cases of progressive autosomal dominant cone dystrophy and 84 cases with post-photoreceptoral dysfunction–CSNB) (Supplementary Material, Table S2).

Expression and immunolocalization studies

The EST profile on Unigene shows ubiquitous expression of ITM2B including the eye, and the mouse retinal gene expression profile database exhibits expression in all retinal cell types (9). In-house rd1 mouse expression database revealed an increase in Itm2b transcript with photoreceptor degeneration suggesting its expression in inner retinal cells (Fig. 3A). A similar expression profile was found with APP (amyloid β precursor protein), known to directly interact with ITM2B (Fig. 3A) (11–13). Real-time PCR experiments confirmed high expression of ITM2B in human retina (ΔCT ITM2B-ACTIN = −1.16) (Fig. 3B). RNA in situ hybridization revealed Itm2b mRNA in inner nuclear and ganglion cell layers (Fig. 3C). Application of anti-ITM2B monoclonal antibody to mouse retinal section revealed a punctuate immunostaining within ganglion cell, inner plexiform and inner nuclear layers which overlapped with the anti-APP immunostaining (Fig. 4A and B). Ganglion cell localization was further confirmed on flat mount human retina, with anti-ITM2B polyclonal antibody immunostaining being seen in BRN3A-positive cells (Fig. 4C).

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 mice on identical genetic backgrounds were hybridized to the mouse genome 430 2.0 array (Affymetrix, High Wycombe, UK). The expression profiles are 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. Interestingly, 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 (ΔCT ITM2B-ACTIN = −1.16) compared with lymphocytes (ΔCT ITM2B-ACTIN = 0.46) and HEK293 cells (ΔCT ITM2B-ACTIN = 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.

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 mice on identical genetic backgrounds were hybridized to the mouse genome 430 2.0 array (Affymetrix, High Wycombe, UK). The expression profiles are 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. Interestingly, 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 (ΔCT ITM2B-ACTIN = −1.16) compared with lymphocytes (ΔCT ITM2B-ACTIN = 0.46) and HEK293 cells (ΔCT ITM2B-ACTIN = 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.

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 = 40 µm). (B) Immunohistochemistry of mouse retinal section reveals strong labelling of ganglion cell and fainter labelling of inner nuclear layers (scale bar = 50 µm). (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 = 40 µm).

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 = 40 µm). (B) Immunohistochemistry of mouse retinal section reveals strong labelling of ganglion cell and fainter labelling of inner nuclear layers (scale bar = 50 µm). (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 = 40 µm).

In vitro expression studies on wild-type and ITM2B mutant constructs

Immunolocalization before and after cell fixation and permeabilization revealed both membrane and intracellular labelling (Supplementary Material, Fig. S3) with no difference between the normal and the three-mutant constructs, suggesting that pathogenic mechanism does not involve cellular mislocalization.

DISCUSSION

The autosomal dominant phenotype we report here, combining inner retinal dysfunction, ganglion cell abnormalities with progressive loss of vision, has never been described before. This clinical picture is distinct from other causes of inner retinal dysfunction of genetic origin (reviewed in 14). These include Schubert Bornschein types of CSNB, with the complete form being characterized by selective ON-bipolar pathway dysfunction and the incomplete form with loss of ON- and OFF-bipolar function. Both CSNB types are congenital and not classically associated with progressive decreased vision and ganglion cell loss as presented in the current report. Similarly, X-linked retinoschisis, snowflake vitreoretinal degeneration, autosomal dominant neovascular inflammatory vitreoretinopathy (15), Muller cell sheen dystrophy or systemic disorders such as Batten disease manifest distinct ophthalmic abnormalities. We could not find a similar clinical presentation while investigating the phenotypic database of our reference centre for rare diseases, currently including 5000 individuals with inherited retinal dystrophies, and inquiring in other specialized centres in France and Europe (presentation of the phenotype at the French Ophthalmology Society and at the Moorfields Eye Hospital). This may suggest the rare occurrence of this unusual phenotype. This is also the first report of a genetic variant in ITM2B associated with a degenerative process restricted to the retina.

Interestingly, mutations in ITM2B have previously been reported in Familial British Dementia [FBD, or cerebral amyloid angiopathy ITM2B-related type 1 (CAA-ITM2B1) OMIM#176500] (7), Familial Danish Dementia [FDD, or cerebral amyloid angiopathy ITM2B-related type 2 (CAA-ITM2B2) OMIM#117300] (16) and two Alzheimer disease (AD)-like autosomal dominant dementia with cerebral amyloid deposits: FBD is characterized by progressive dementia starting with memory loss around age 45 followed by progressive cerebellar ataxia, spastic tetraparesis and death around age 60 but no reported ocular abnormalities (17). Nevertheless, due to the worsening of the neurological status, ocular abnormalities may have been undermined in the late stages of the disease. On the other hand, in FDD, also known as heredopathia ophthalmo-oto-encephalica, bilateral cataract is the first manifestation between age 20 and 30 followed by progressive neurosensory deafness, severe by age 45, cerebellar ataxia with paranoid psychosis and dementia 10 years later and death in the fifth decade (18). Other ocular abnormalities are reported in relation with amyloid angiopathy including retinal ischaemia and neovascular complications (19). None of these vascular, ocular or neurological findings were present in our family. Two distinct variants in ITM2B have been identified underlying FBD and FDD, a T>A transversion at the stop codon (rs104894417; c.799T>A; p.Stop267Argext*11) and a decamer duplication at the 3′ end (c.795-796insTTTAATTTGT; p.Ser266Pheext*11), respectively, both mutants resulting in an 11-amino-acid-longer protein product (7,16).

ITM2B, also known as BRI or BRI2, is located on 13q14.2, comprises 6 exons and encodes a 266-amino acid protein of unclear function belonging to the single-pass Integral Membrane type 2 protein family. ITM2B mRNA is ubiquitously expressed (7,20,21) but to date no information on its expression or role within the retina is available. Our study suggests its localization in inner retinal and ganglion cells, where it may interact with APP (Figs 3 and 4). The physiological role of ITM2B remains unclear. It is known to be proteolytically cleaved in three distinct locations. These include an extracellular furin-like convertase cleavage in C-terminus releasing a 23-amino acid secreted fragment, with the remaining membrane-bound part containing a BRICHOS domain (7,8,22). The exact role of the BRICHOS domain itself is unknown and at least three functions have been suggested, including the targeting of proteins to the secretory pathway, a role of intramolecular chaperone and a facilitation for intracellular protease processing (23). In addition, studies have emphasized ITM2B direct interaction with APP, its key role as a modulator of APP processing and as an inhibitor of amyloid β (Aβ) oligomerization, a major component of amyloid plaques in FBD, FDD and AD (11,12,24–27): the 23-amino acid secreted fragment would critically inhibit Aβ aggregation, and the membrane-bound fragment of ITM2B may control cleavage of APP and, therefore, Aβ genesis. Furthermore, ITM2B is able to promote Aβ degradation by increasing the expression of the secreted form of the protease, insulin-degrading enzyme (28). It was also suggested that ITM2B may play a role in neurite outgrowth (29), a function also attributed to APP (30). All together, these data outline the key role of ITM2B in Aβ metabolism and its relevance as a therapeutic target for AD and AD-like dementias.

In case of FBD and FDD, the mutations would result in a longer secreted peptide (34 instead of 23 amino acid), respectively, called ABri (7) and ADan (16). These two peptides, which contain a disulphide bond, adopt a β-sheet structure in solution and are able to form oligomers and fibrils, a major component of amyloid lesions found in FBD and FDD that may initiate cell death. These peptides may also be able to form ion permeable channels in lipid biolayers, leading to cytotoxic effects (31). The longer length of ABri and ADan may also hamper their inhibitory role on Aβ oligomerization (31,32). Furthermore, Tau triplets were found in brain extracts of FBD subjects as in AD and may also have a role in the disease process (33).

Pathogenic mechanism(s) associated with the ITM2B missense change we identified is unknown but is most likely distinct from disease mechanisms associated with FBD and FDD. Our findings suggest a strong phenotype–genotype correlation with a longer peptide underlying the dementia phenotype, whereas a missense change would be responsible for the retina-restricted phenotype. Our in vitro studies show no difference in membrane localization between the normal and the three-mutant constructs, suggesting that pathogenic mechanism does not involve cellular mislocalization (Supplementary Material, Fig. S3). The change of a glutamic acid for an alanine may disturb the beta–loop–beta structure of the 23-amino acid secreted peptide and, therefore, modify protein interaction with Aβ or other unknown partners. The subtle changes induced by the p.Glu261Ala mutation are less likely to lead to oligomer formations or if so, this phenomenon would be restricted to inner retinal layers, and the hyper-reflectivity seen on SD-OCT may represent such aggregation. Of note, ganglion cell loss is reported in AD (34) but abnormal diffuse reflectivity of inner retinal layers has never been reported. Interestingly, APP knock-out mice display inner retinal dysfunction (13), which could also suggest that such dysfunction in our family may be mediated through APP/mutated-ITM2B-disturbed interaction.

The exact cellular origin of progressive abnormalities in photopic responses remains unclear. Since photopic responses are cone photoreceptor-driven but represent a summation of the visual signal, it could represent a progressive cone degeneration, supported by macular dysfunction seen with pattern ERG and abnormal macular autofluorescence. Alternatively, it could be a progressive degeneration at the inner retinal level affecting both ON- and OFF-bipolar cell pathways as seen in incomplete CSNB. Mechanism(s) leading to this late-onset cone or cone ON-/OFF-pathway degeneration is also unclear. If progressive cone degeneration is considered, it may be an indirect phenomenon since we failed to find ITM2B expression in photoreceptors. In particular, it could represent a toxic effect either through Aβ, already shown to induce photoreceptor cell death after intravitreal injection (35), or through 23-amino acid secreted mutated peptide aggregation. If progressive involvement of cone ON- and OFF-bipolar pathway is considered, it is also unclear whether it is a specific and direct cellular toxicity upon bipolar cells or whether this progressive inner retinal dysfunction is an indirect mechanism that may involve material deposition with the inner retina. The abnormal hyper-reflectivity seen on SD-OCT may support this later hypothesis.

Our study provides compelling evidence for an ITM2B missense mutation as being the candidate defect underlying a novel retinal dystrophy. Further mechanistic studies are, however, needed to establish causality and associated disease mechanisms. Elucidating underlying pathogenic mechanism(s) in this family affected with a novel phenotype would lead to a better understanding of ITM2B physiopathology in neurological disorders using the retina as a unique model and may help in designing innovative therapeutic targets for AD and AD-like dementia. Our study also provides a new candidate in ITM2B, to be added to the list of genes involved in inherited retinal diseases.

MATERIALS AND METHODS

Clinical examination

Research procedures were conducted in accordance with institutional guidelines and the Declaration of Helsinki. Each affected subject included in the genetic study underwent full ophthalmic examination as described before (3,4,36). Mini-Mental State Examination was performed to evaluate cognitive function on proband (37).

Molecular genetics and bioinformatic analyses

Informed consent was obtained from all patients and their family members. Sanger sequencing, targeted NGS and whole-exome sequencing were performed followed by stringent filtering of genetic variants as previously reported (5,10,36). In brief, for whole-exome sequencing, exons of DNA samples were captured and investigated as shown before (10) using in-solution enrichment methodology (SureSelect Human All Exon Kits Version 3, Agilent, Massy, France), NGS (Illumina HISEQ, Illumina, San Diego, CA, USA), image analysis and base calling using Real Time Analysis Pipeline version 1.9 with default parameters (Illumina). The bioinformatic analysis of sequencing data was based on a pipeline [Consensus Assessment of Sequence and Variation (CASAVA) 1.8, Illumina] which performs alignment, calls the SNPs based on the allele calls and read depth and detects variants (SNPs and indels). Genetic variation annotation was realized by an in-house pipeline (IntegraGen, Evry, France) and results were provided per sample in tabulated text files. For filtering, exonic and splice variants were selected on the basis of their heterozygosity in affected subjects and absence in the unaffected siblings, in dbSNP 137, HapMap (38), the 1000 Genome Project (39) and the Exome Variant Server (http://evs.gs.washington.edu/EVS/, last accessed date on September 16, 2013). Sequence conservation, PolyPhen2 (Polymorphism Phenotyping, http://genetics.bwh.harvard.edu/pph2/, last accessed date on September 16, 2013) and SIFT (Sorting Intolerant From Tolerant; http://sift.bii.a-star.edu.sg/, last accessed date on September 16, 2013) software were used to predict the pathogenic nature of sequence alterations. Haplotype analysis was performed using 16 custom microsatellite markers and a fluorescently labelled universal primer [method adapted from de Arruda et al. (40)] from chromosome 13q on 19 family members (Supplementary Material, Fig. S1). Coding and flanking exonic regions of ITM2B was analysed by Sanger sequencing (RefSeq NM_021999.4, primer sequences and PCR conditions available upon request). Sequence conservation, PolyPhen2 and SIFT software were used to predict the pathogenic nature of sequence alterations.

Expression analysis

Expression profile for the eye and retina was investigated using three databases: Unigene, the mouse retinal gene expression profile from Siegert et al. (9) and the in-house rd1 mouse expression database [the rd1 mouse, carrying Pde6b mutations, is a naturally occurring retinitis pigmentosa model leading to a complete loss of all rod photoreceptors by post-natal day 36, and preserved inner retina (41)]. Real-time PCR experiments on commercially available human retina cDNA (Clontech, Saint-Germain-en-Laye, France), lymphocyte and HEK293 cell cDNA were performed for ITM2B (MIM#*603904), using ACTB (MIM#*102630) as a control (primers available upon request).

RNA in situ hybridization studies on mouse retinas

RNA in situ hybridization on mouse retina was performed using a riboprobe encompassing exons 2 to 6 of the Itm2b mouse mRNA (RefSeq NM_008410.2) as described before (42) (details available upon request).

Immunohistochemistry

Immunolocalization of ITM2B, BRN3A and APP in mouse and human retina was studied on 20 µm-thick gelatine-embedded coronal eye cryosections and whole-mount retina as described before (10). Human retina specimens were obtained from the Minnesota Lions Eye Bank with due consent in accordance with the Declaration of Helsinki. Antibody dilutions are as follows: 1/50 dilution for polyclonal rabbit anti-ITM2B (HPA029292, Sigma-Aldrich, St Quentin Fallavier, France), 1/100 dilution for monoclonal mouse anti-ITM2B (SAB1402472, Sigma-Aldrich), 1/200 dilution for polyclonal rabbit anti-APP HPA001462 (Sigma-Aldrich), 1/200 dilution for monoclonal mouse anti-BRN3A (Millipore, Molsheim, France), localized in ganglion cells and 1/600 for secondary fluorescent antibodies (Alexa Fluor 488-conjugated and CY3, Invitrogen, Courtabœuf, France) and nuclei counterstained with 4′,6-diamidino-2-phenylindole (Euromedex, Souffelweyersheim, France) or a mouse peroxidase-coupled secondary antibodies (Jackson ImmunoResearch, West Grove, PA, USA) using a peroxidase substrate kit (kit VIP peroxydase Vector, Cliniscience, Nanterre, France).

In vitro expression studies on wild-type and ITM2B mutant constructs

Subcellular immunolocalization of the normal and three mutated ITM2B variants [c.799T>A; p.Stop267Argext*11 identified in the British Dementia Family (7), c.795-796insTTTAATTTGT; p.Ser266Pheext*11 identified in the Danish Dementia family (16) and c.782A>C, p.Glu261Ala from our study] was analysed under live-cell condition and after permeabilization as described before (43).

WEB RESOURCES

Databases used to predict the pathogenic character of a sequence alteration, expression and protein function:

SUPPLEMENTARY MATERIAL

Supplementary Material is available at HMG online.

FUNDING

The project was supported by GIS-maladies rares (C.Z.), Retina France (part of the 100-Exome Project) (I.A., J.-A.S. and C.Z.), Foundation Voir et Entendre (C.Z.), Foundation Fighting Blindness (FFB) grant CD-CL-0808-0466-CHNO (I.A. and the CIC503, recognized as an FFB centre), FFB grant C-CMM-0907-0428-INSERM04, Ville de Paris and Region Ile de France and by the French State programme ‘Investissements d'Avenir’ managed by the Agence Nationale de la Recherche (LIFESENSES: ANR-10-LABX-65).

ACKNOWLEDGEMENTS

The authors are grateful to the family described in this study; to Gaël Orieux in assisting with immunostaining, to Stéphane Fouquet for his support on confocal microscopy, to Dominique Santiard-Baron and Christine Chaumeil for their help in DNA collection; to the clinical staff.

Conflict of Interest statement. None declared.

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Supplementary data