Benjamin Cogné

Xenia Latypova

Lokuliyanage Dona

Samudita Senaratne

Ludovic Martin

Daniel C Koboldt

Georgios Kellaris

Lorraine Fievet

Guylène Le Meur

Dominique Caldari

Dominique Debray

Mathilde Nizon

Eirik Frengen

Sara J Bowne

Lives Consortium

Elizabeth L Cadena

Stephen P Daiger

Kinga Bujakowska

Eric A Pierce

Michael Gorin

Nicholas Katsanis

Stéphane Bézieau

Simon M Petersen-Jones

Laurence M Occelli

Leslie A Lyons

Laurence Legeai-Mallet

Lori S Sullivan

Dr Erica E Davis
email: eridavis@luriechildrens.org

Dr Bertrand Isidor
email: bertrand.isidor@chu-nantes.fr

Mutations in the Kinesin-2 Motor KIF3B Cause an Autosomal-Dominant Ciliopathy

Keywords: KIF3B, primary cilia, retinopathy, hepatic fibrosis, kinesin, whole exome sequencing, zebrafish, feline genetics

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

MAIN TEXT

Kinesin family (KIF) genes encode a superfamily of microtubule-based molecular motors that transport intracellular cargo. Pathogenic variants in KIF-encoding loci have been associated with at least 14 distinct Mendelian disorders in humans, transmitted in both dominant or recessive inheritance fashions [START_REF] Reilly | Loss of function mutations in KIF14 cause severe microcephaly and kidney development defects in humans and zebrafish[END_REF] . One unifying feature of autosomal dominant KIF-related disorders is the primary impact on the central nervous system as evidenced by phenotypes such as intellectual disability (KIF1A, MIM: 614255); cortical dysplasia (KIF2A, MIM: 615411); spastic paraplegia (KIF5A, MIM: 604187), and microcephaly (KIF11, MIM: 152950). KIF genes mutated in autosomal recessive disorders also impair neurological development, and have been reported in X-linked intellectual disability (KIF4A, MIM: 300521) or syndromic ciliopathies such as Acrocallosal syndrome (KIF7, MIM: 200990) and Meckel syndrome (KIF14, MIM: 616258).

Kinesin-2 subfamily motors, not currently associated with human disease phenotypes, have an established role in ciliogenesis. These macromolecular complexes ensure anterograde trafficking by ATP-dependent movement from the ciliary base toward the growing end of microtubules. In the context of kinesin-2 ablation, model organisms such as mouse or zebrafish display stunted or absent ciliary formation, leading to broad impairment of primary ciliary functions during vertebrate development [START_REF] Goetz | The primary cilium: a signalling centre during vertebrate development[END_REF] .

Protein synthesis does not occur in the cilium; the formation and maintenance of this organelle depends on intraflagellar transport (IFT) of protein cargo along the ciliary axoneme. KIF3A-KIF3B or KIF3A-KIF3C heterodimers and the non-motor kinesin-associated protein (KAP), encoded by KIFAP3, operate as the main effectors of kinesin-2 driven trafficking, together with homodimeric KIF17. Ciliopathies, defined as clinical entities caused by defects in ciliary structure or function, share common phenotypic features; these include obesity, hepatic fibrosis, retinitis pigmentosa (RP), cystic kidney disease, polydactyly and intellectual disability. [START_REF] Badano | The ciliopathies: an emerging class of human genetic disorders[END_REF] Here, we report affected individuals from a small nuclear family and an unrelated six-generation pedigree who harbor heterozygous nonsynonymous KIF3B variants and share partially overlapping ciliopathy clinical features. Individual 1 (family A, II-1; Table 1) is a male born from nonconsanguineous parents of northern European ancestry. At birth, postaxial hexadactyly of both hands and the right foot were observed. At 12 months, he presented with failure to thrive and hepatosplenomegaly. An abdominal ultrasound performed at 18 months showed a dysmorphic liver with dilatation of intrahepatic biliary ducts. A liver biopsy showed micronodular cirrhosis with modification of global hepatic architecture by annular fibrosis and persistence of peripheric biliary neoductules. Cardiac ultrasound showed a bicuspid aortic valve. At 24 months of age, he had esophageal varices, and thrombocytopenia. At 4 years of age, because of suggestive clinical features of a ciliopathy, we performed an ophthalmological exam. The parents only reported their child to have difficulties moving at night. Fundus examination was normal (Figure 1Aa), but the fundus autofluorescence showed a modification of the peripheral autofluorescence and hyperautofluorescent perimacular points. Electroretinograms showed alteration of the flicker and the scotopic responses (Figure S1). Spectral Domain Optical Coherence Tomography (SD-OCT) analysis showed a normal foveal profile (Figure 1Ac,d). At last ophthalmological examination at the age of 5 years, fundus examination showed retinal thinning with an increase in the visibility of the choroidal vascularization on the retinal periphery and on the temporal macula.

To investigate the genetic etiology of this individual's clinical presentation, we consented the parent-child trio for all subsequent research procedures in accordance with ethical guidelines of Nantes University Hospital (Figure 2A). Array comparative genomic hybridization on the family A proband sample was negative, ruling out the possibility of large copy number variants. Next, we performed exome sequencing of the proband and achieved an average mean target coverage of 86X (Supplemental Methods). We retained variants with at least 9 reads and with a variant read frequency over 20 percent impacting exonic sequences or splice sites (+/-10bp from the junction) and with an allele frequency <0.5% in 1000 genomes, genome aggregation database (gnomAD, 123,136 exomes and 15,496 whole genome sequences; accessed on 11/10/2018) and in a local database of 952 exomes.

We retained 280 rare variants (Table S1), including 13 that matched an X-linked or a recessive inheritance (homozygous, hemizygous or at least 2 rare variants in the same gene) and 267 that matched a dominant inheritance (single heterozygous variant). We did not identify deleterious variants in any disease-associated gene, and selected 88 variants of uncertain significance that were within an established human genetic disease-associated gene in either OMIM or Orphanet for further consideration (Table S1).

The constellation of clinical phenotypes observed in Individual 1 was suggestive of a ciliary disorder. We therefore overlaid the rare exome sequencing variant set with 303 ciliary genes from the SYSCILIA Gold Standard (SCGSv.1) [START_REF] Van Dam | The SYSCILIA gold standard (SCGSv1) of known ciliary components and its applications within a systems biology consortium[END_REF] . We identified five rare heterozygous nonsynonymous variants that intersected with the SCGSv.1 dataset (Table S1). Five variants were heterozygous alleles in genes already causally implicated in recessive ciliopathies. However, one variant in KIF3B (GenBank ID: NM_004798.4, c.748G>C, p.Glu250Gln) was absent from gnomAD and predicted to be deleterious by SIFT [START_REF] Kumar | Predicting the effects of coding non-synonymous variants on protein function using the SIFT algorithm[END_REF] , PolyPhen-2 [START_REF] Adzhubei | A method and server for predicting damaging missense mutations[END_REF] and Mutation Taster [START_REF] Schwarz | MutationTaster evaluates disease-causing potential of sequence alterations[END_REF] . Given the important role of KIF3B in anterograde IFT in cilia, and localization of this variant to the conserved kinesin motor domain (Figure 2B,C; Figure S2, S3), we tested familial segregation by Sanger sequencing and confirmed that the variant was de novo.

We excluded false paternity and maternity by analysis of 11 microsatellite markers.

Through the data sharing platform, GeneMatcher 8 , we became aware of another rare KIF3B variant that had been identified as a possible cause of RP. This second variant (GenBank ID:NM_004798.4, c.1568T>C, p.Leu523Pro) was found in two samples from a cohort of over 200 families with autosomal dominant RP ascertained in an ongoing study at the University of Texas Health Science Center in Houston. [START_REF] Sullivan | Prevalence of disease-causing mutations in families with autosomal dominant retinitis pigmentosa: a screen of known genes in 200 families[END_REF] The two females were originally thought to be unrelated but the identification of rare variants during exome sequencing led to the eventual discovery that they were cousins (family B: IV-4 and V-4; Figure 2A; Table 1; Supplementary Methods). They are part of an extended Ashkenazi Jewish family that has RP segregating through at least six generations with over 19 affected individuals reported (Figure 2A). There is no evidence of consanguinity in either branch of the family and male-to-male transmission of the disease excludes an X-linked mode of inheritance.

Affected members of family B exhibit classic symptoms of RP, with an average age of onset in the first decade. We performed detailed clinical assessment for five individuals (IV-1, IV-3, V-4, VI-2, and VI-1 [individuals 2-6]; Table 1; Figure 1B). Two members of the family have postaxial polydactyly (IV-8 and V-7), and one individual is reported to have kidney dysfunction (V-I; Table 1).

Given their symptoms evocative of a ciliopathy, targeted genetic testing had been done previously on known genes for Bardet-Biedl syndrome (MIM: 209900) and dominant forms of inherited retinal degeneration. No likely pathogenic variants were identified, leading to additional testing by exome sequencing.

Bioinformatic analysis of exome data from family B was done using MendelScan as described, [START_REF] Koboldt | Exome-based mapping and variant prioritization for inherited Mendelian disorders[END_REF] resulting in 28 rare variants shared between both individuals (Table S2). The majority of shared variants were present in multiple individuals in gnomAD, especially Ashkenazi Jewish (n=23 variants with allele frequency >0.05%; 50,040 individuals). The KIF3B variant was the only segregating variant absent in gnomAD. We performed segregation analysis of the four variants with the lowest overall incidence in gnomAD (KIF3B, PCNXL3, OR4D2 and TOB1) in an additional seven family members from the extended pedigree using bidirectional Sanger sequencing (six affected, one unaffected; Figure 2A). The KIF3B c.1568T>C, p.Leu523Pro variant was the sole change that segregated with RP in the pedigree. To confirm that the KIF3B variant and the disease are not likely to be co-segregating by chance, we calculated a LOD score (logarithm of the odds) for the 14 informative meioses observed across the extended family. The LOD score of 4.2 (odds ratio 16,384:1) is strong statistical evidence supporting the KIF3B change as the cause of disease in the family. p.Leu523Pro is located in the coiled coil domain of KIF3B at a position conserved throughout vertebrates (Figure 2B,C, Figure S2,S3), is predicted to be pathogenic by SIFT, [START_REF] Kumar | Predicting the effects of coding non-synonymous variants on protein function using the SIFT algorithm[END_REF] PolyPhen-2 [START_REF] Adzhubei | A method and server for predicting damaging missense mutations[END_REF] and Mutation Taster 7 and is absent from all available databases, including gnomAD.

To characterize the cellular phenotypes induced by the dominantly-inherited KIF3B variants identified in humans, we evaluated primary cilia length in skin fibroblasts isolated from individual 1 (family A; Supplementary Methods). We cultured fibroblasts under standard protocols of serum deprivation for 24 h to promote primary cilium elongation [START_REF] Pampliega | Functional interaction between autophagy and ciliogenesis[END_REF][START_REF] Stoetzel | A mutation in VPS15 (PIK3R4) causes a ciliopathy and affects IFT20 release from the cis-Golgi[END_REF][START_REF] Rozycki | The fate of the primary cilium during myofibroblast transition[END_REF] . We fixed case and age-matched control cells, and immunolabeled γ-tubulin and ARL13b as markers of the basal body and ciliary axoneme, respectively (Figure 3A; Table S3). We first noted that the proportion of ciliated cells from individual 1 was similar to those from the age-matched control (>90%; n=66/72 and n=147/153 fibroblasts/condition isolated from age-matched control and individual 1, respectively; Figure 3B).

Next, we found that the primary cilia length was increased significantly in fibroblasts from individual 1 compared to age-matched control fibroblasts (36% average increase in case versus control cilia; p<0.0001; Figure 3C).

To validate the cilia length abnormalities observed in primary fibroblasts from individual 1, we evaluated the effect of KIF3B variants from both families in a ciliated context using hTERT-RPE1 cells, a commonly used immortalized retinal pigment epithelial cell line. We transfected C-terminally myc-tagged vectors encoding either WT or variant KIF3B (p.Glu250Gln; p.Leu523Pro or p.Val435Ile

[negative control, rs41288638; 230/276,748 alleles in gnomAD]), fixed cells at 24 hours posttransfection, performed immunostaining with γ-tubulin and ARL13B, and measured cilia length (Supplemental Methods; Tables S3,S4). We observed no significant differences in primary cilia length between cells transfected with empty vector, KIF3B-WT-myc or KIF3B-p.Val435Ile-myc constructs (Figure 3D,E). However, we detected a significant increase in primary cilia length for both case-associated variants compared to WT KIF3B (9% increase in p.Glu250Gln cells vs control, p<0.01; and 13% increase in p.Leu523Pro cells versus control, p<0.0001; Figure 3D,E). Notably, immunoblotting of myc-tag in whole-cell protein lysates isolated from transiently transfected HEK293 cells expressing KIF3B-myc variant constructs showed no significant differences in KIF3B protein levels compared to the KIF3B-WT-myc transfected condition (Supplemental Methods; Figure S4; Table S3).

To investigate further the stability of KIF3B protein harboring p.Glu250Gln or p.Leu523Pro, we monitored protein levels in a time course study after exposure to a protein synthesis inhibitor (Supplementary Methods; Table S3). We transiently transfected HEK293 cells with control and mutant KIF3B-myc constructs, and treated cells with cycloheximide (50 µm for 2 and 4 hours before protein harvest). Consistent with our initial results (Figure S4), we found no significant difference in KIF3B-myc protein levels across conditions exposed to cycloheximide for up to 4 hours (Figure 3F-4J). These data support the notion that KIF3B protein harboring p.Glu250Gln or p.Leu523Pro do not undergo rapid degradation. Together, the cilia length and protein stability data indicate that primary cilia are markedly altered in the context of both KIF3B p.Glu250Gln and p.Leu523Pro variants but not for the KIF3B p.Val435Ile variant, supporting the specificity of this organellar defect.

To establish further a functional link between altered ciliary structure and overlapping human phenotypes observed in individuals with KIF3B variants, we considered prior knowledge from in vivo models of KIF3B dysfunction. Kif3b mouse models harboring a gene-trapped locus deleting a portion of exon 1 are embryonic lethal in homozygosity [START_REF] Nonaka | Randomization of left-right asymmetry due to loss of nodal cilia generating leftward flow of extraembryonic fluid in mice lacking KIF3B motor protein[END_REF][START_REF] Takeda | Leftright asymmetry and kinesin superfamily protein KIF3A: new insights in determination of laterality and mesoderm induction by kif3A-/-mice analysis[END_REF][START_REF] Alsabban | Kinesin Kif3b mutation reduces NMDAR subunit NR2A trafficking and causes schizophrenia-like phenotypes in mice[END_REF] . A Cre-loxP conditional knockout mouse showed that loss of Kif3b results in a rapid photoreceptor degeneration associated with opsin mislocalization to rod inner segments, but Kif3b -/+ animals are viable and display no detectable retinal phenotypes [START_REF] Jimeno | Kinesin-2 and photoreceptor cell death: requirement of motor subunits[END_REF] .

Additionally, kif3b -/-zebrafish models carrying an NM_001100145.1: c.1105C>T, p.Gln369* encoding mutation display either absent or significantly reduced cilia length in multiple tissue types [START_REF] Zhao | Kinesin-2 family in vertebrate ciliogenesis[END_REF] , contrary to our observations from KIF3B mutant primary fibroblasts with elongated cilia (Figure 3A,B).

Moreover, as part of ongoing work to identify the molecular basis of clinically relevant traits in the domestic cat, we identified a nonsynonymous KIF3B change, ENSFCAT00000022266:c.1000G>A, p.Ala334Thr as the likely cause of a recessive progressive retinal atrophy in the Bengal cat [START_REF] Ofri | Characterization of an Early-Onset, Autosomal Recessive, Progressive Retinal Degeneration in Bengal Cats[END_REF] (Supplemental Note). We performed a genome-wide association study on 98 Bengal cats to localize an associated region on cat chromosome A3 (Supplementary Methods; Figure 4A; Table S5), and overlaid these data with whole genome sequencing data from a trio of cats from the Bengal research colony (Table S6). The KIF3B p.Ala334Thr-encoding change was the sole variant that segregated with disease; and is predicted to impair the kinesin motor domain (Figure S5). Importantly, we observed rhodopsin mislocalization to photoreceptor inner segments in KIF3B -/-mutant kittens as early as 8 weeks of age, consistent with structural and functional photoreceptor degeneration [START_REF] Ofri | Characterization of an Early-Onset, Autosomal Recessive, Progressive Retinal Degeneration in Bengal Cats[END_REF] (Figure 4B; Supplementary Note; Supplementary Methods). Together, our in vitro data and in vivo observations from recessive KIF3B vertebrate models argue against a haploinsufficiency model for the affected humans in our study.

To pursue the possibility of a dominant pathogenic effect for the p.Glu250Gln and p.Leu523Pro changes, we introduced mutant mRNAs into the developing zebrafish (Supplementary Methods; Table S4). The zebrafish genome encodes a single KIF3B ortholog (GRcZ11), for which the encoded protein (NP_001093615.1) has 78% identity and 87% similarity to that of human (NP_004789.1). Importantly, ablation of each of kif3b and its cognate kinesin-2 effectors encoded by kif3a and kif3c have been characterized previously in the zebrafish retina and perform similar trafficking functions to mammalian counterparts [START_REF] Zhao | Kinesin-2 family in vertebrate ciliogenesis[END_REF] . We and others have shown that alteration of numerous genes involved in retinal degeneration can give rise to microphthalmia in zebrafish [START_REF] Liu | Putative digenic inheritance of heterozygous RP1L1 and C2orf71 null mutations in syndromic retinal dystrophy[END_REF][START_REF] Li | A mutation in ZNF513, a putative regulator of photoreceptor development, causes autosomal-recessive retinitis pigmentosa[END_REF][START_REF] Shu | Knockdown of the zebrafish ortholog of the retinitis pigmentosa 2 (RP2) gene results in retinal degeneration[END_REF] . As an initial test of the impact of either missense change on KIF3B function, we injected 100 pg WT or mutant KIF3B mRNA into embryos at the one-to-four cell stage and evaluated them live at 3 days post-fertilization (dpf) for external optic area in lateral bright field images using automated imaging and image analysis platforms (Supplementary Methods). We found no significant differences in eye size for clutches injected with p.Leu523Pro-or p.Val435Ile-encoding mRNA versus either WT or uninjected controls (Figure S6A,B). However, we observed marked lethality induced by p.Glu250Gln

and assessed the effects of progressively decreasing amounts of mutant mRNA (50 pg, 25 pg, and 10 pg). The highest non-lethal doses resulted in a significant, dose-dependent, and reproducible reduction in eye area (19% reduction vs WT-injected at 50 pg; p<0.0001; Figure S6C), an observation that persisted when normalized to larval body length to control for any possible developmental delay (Figure S6D). Co-injection of a 2:1 stoichiometric ratio of 100 pg WT with 50 pg p.Glu250Gln mRNA restored eye size to normal (not significantly different from equivalent doses of either WT or p.Glu250Gln alone; Figure S6A,B), supporting the specificity of p.Glu250Gln effect on eye morphology.

We hypothesized that the observed microphthalmia was caused by excessive cell death in the retina. In zebrafish and other vertebrates, the developing retina undergoes several waves of apoptosis which are necessary for the establishment of the retinal neuron networks. The first wave arises around 3 dpf, slows down at 5 dpf and the second wave takes place at 7dpf [START_REF] Biehlmaier | Onset and time course of apoptosis in the developing zebrafish retina[END_REF] . We evaluated apoptosis in retinal sections at 5 dpf by counting terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) positive cells (Supplementary Methods). We found no significant differences between larvae injected with either WT, p.Val435Ile-or p.Leu523Pro-encoding mRNA compared to uninjected controls. However, we observed a modest but significant increase in apoptotic cells in larvae injected with p.Glu250Gln-encoding mRNA (50 pg; p<0.05; Figure S7A,B) which is concordant with the microphthalmia phenotype observed in this condition (Figure S6).

In the zebrafish retina, Kif3b plays a role in photoreceptor formation and function through the connecting cilium that is specific to low light intensity (scotopic) detection facilitated by rod cells [START_REF] Zhao | Kinesin-2 family in vertebrate ciliogenesis[END_REF] .

Further, kif3b mutants have documented defects in rhodopsin transport, consistent with other kinesin-2 mutants such as kif3a -/-18 . To determine whether zebrafish larvae with heterologous expression of mutant human KIF3B display defects in photoreceptor integrity, we evaluated rhodopsin localization in retinal sections at 5 dpf (Supplementary Methods; Table S3). We found no detectable differences between larvae injected with either WT or p.Val435Ile-encoding mRNA compared to uninjected controls (Figure 5A,B,C). However, we observed a significant increase of rhodopsin in the rod inner segment for p.Leu523Pro (100 pg; p<0.001), and to a greater extent, p.Glu250Gln-encoding (50 pg; p<0.0001), versus WT mRNA (repeated with similar results, investigator masked to experimental condition; Figure 5A,B,C). Consistent with the eye area studies, co-injection of 100 pg WT human KIF3B mRNA rescued the rhodopsin mislocalization defects of both variants such that they were indistinguishable from uninjected controls (Figure 5A,C)

We next wondered whether the observed rhodopsin mislocalization was coincident with altered cilia length in zebrafish with ectopic expression of KIF3B mutant mRNAs. To paint photoreceptor connecting cilia, we performed antibody staining of transverse cryosections from 5 dpf larvae with two ciliary markers, acetylated α-tubulin and IFT52, and measured length of structures with double labeling (Supplementary Methods; Figure 5D; Table S3). We found no significant differences in connecting cilium length between larvae injected with either WT or p.Val435Ile encoding mRNA compared to uninjected controls (Figure 5E,F). However, we observed a significant elongation of connecting cilia for p.Leu523Pro-(100 pg; p<0.0001) and p.Glu250Gln-encoding (50 pg; p<0.0001), versus WT mRNA (repeated with similar results; Figure 5E,F). We detected no differences in length measurements when acetylated α-tubulin and IFT52 signal was assessed individually, arguing against sequestration of IFT-B components as the molecular mechanism underpinning the long cilia phenotype (Figure S8). In aggregate, these data reinforce the relevance of KIF3B function to photoreceptor integrity and opsin trafficking in rod photoreceptors and support the pathogenicity of p.Glu250Gln and p.Leu523Pro.

The molecular motor is essential for kinesin complexes to move along microtubules in an ATP-dependent manner [START_REF] Lee | De novo mutations in the motor domain of KIF1A cause cognitive impairment, spastic paraparesis, axonal neuropathy, and cerebellar atrophy[END_REF] and multiple dominant pathogenic amino acid substitutions in kinesin genes have been identified in this domain. The Glu250 position is an ultra-conserved residue in 41/42 human kinesin motor domain protein sequences, except KIF26B (Figure S2) and vertebrate KIF3B orthologs (Figure 2C). This residue was substituted by a lysine in KIF1A (NM_001244008.1: c.757G>A, p.Glu253Lys) in two individuals with neuroanatomical phenotypes described by Lee et al. [START_REF] Lee | De novo mutations in the motor domain of KIF1A cause cognitive impairment, spastic paraparesis, axonal neuropathy, and cerebellar atrophy[END_REF] Notably, individuals carrying this variant exhibited a severe phenotype, consistent with the more acute presentation of individual 1 (family A) in our study. Five cases with substitutions of the equivalent residue in KIF5C by a Lys or Val were also reported. [START_REF] Michels | Mutations of KIF5C cause a neurodevelopmental disorder of infantile-onset epilepsy, absent language, and distinctive malformations of cortical development[END_REF] Rice et al. observed that when the glutamic acid was substituted by alanine (p.Glu236Ala), the ATPase activity of the kinesin was reduced at least 1,000-fold [START_REF] Rice | A structural change in the kinesin motor protein that drives motility[END_REF] . Hence, this variant might act in a dominant manner by altering the ability of the KIF3A/B heterodimer to move along the ciliary microtubules, disorganizing ciliogenesis and thus causing the augmented primary cilia length of the fibroblasts of individual 1 (Figure 3).

Kittens homozygous for the KIF3B mutation showed a rapid loss of photoreceptors. KIF3B

immunolabeling showed that initially the mutant protein was detectable in the region of inner and outer segments; however, with progressive degeneration of both layers and loss of photoreceptor nuclei Kif3b immunoreactivity was no longer detectable (Figure 4B). This loss of immunoreactivity was accompanied by pronounced mislocalization of rhodopsin. Abnormal trafficking of opsin to the outer segment with retention in the inner segment and cell body has been reported in a number of retinal degeneration conditions including RP in humans and animal models [START_REF] Adamian | Rod and cone opsin mislocalization in an autopsy eye from a carrier of X-linked retinitis pigmentosa with a Gly436Asp mutation in the RPGR gene[END_REF][START_REF] Concepcion | Q344ter mutation causes mislocalization of rhodopsin molecules that are catalytically active: a mouse model of Q344ter-induced retinal degeneration[END_REF][START_REF] Gao | Progressive photoreceptor degeneration, outer segment dysplasia, and rhodopsin mislocalization in mice with targeted disruption of the retinitis pigmentosa-1 (Rp1) gene[END_REF][START_REF] Nishimura | Bbs2-null mice have neurosensory deficits, a defect in social dominance, and retinopathy associated with mislocalization of rhodopsin[END_REF][START_REF] Mowat | Tyrosine capsid-mutant AAV vectors for gene delivery to the canine retina from a subretinal or intravitreal approach[END_REF][START_REF] Beltran | Gene therapy rescues photoreceptor blindness in dogs and paves the way for treating human X-linked retinitis pigmentosa[END_REF][START_REF] Hollingsworth | Defective trafficking of rhodopsin and its role in retinal degenerations[END_REF] . The KIF3B p.Ala334Thr variant in the Bengal cat might not totally ablate protein function resulting in a recessive retinal phenotype, in contrast to previously reported zebrafish and mouse recessive KIF3B mutants that are embryonic lethal [START_REF] Nonaka | Randomization of left-right asymmetry due to loss of nodal cilia generating leftward flow of extraembryonic fluid in mice lacking KIF3B motor protein[END_REF][START_REF] Takeda | Leftright asymmetry and kinesin superfamily protein KIF3A: new insights in determination of laterality and mesoderm induction by kif3A-/-mice analysis[END_REF][START_REF] Alsabban | Kinesin Kif3b mutation reduces NMDAR subunit NR2A trafficking and causes schizophrenia-like phenotypes in mice[END_REF][START_REF] Feng | The cytoplasmic tail of rhodopsin triggers rapid rod degeneration in kinesin-2 mutants[END_REF][START_REF] Zhao | Kinesin-2 family in vertebrate ciliogenesis[END_REF] . Further studies are required to investigate impact of p.Ala334Thr on cilia length and KIF3B protein stability in the feline retina.

The ciliopathies are known for exhibiting variable expressivity of endophenotypes [START_REF] Davis | The ciliopathies: a transitional model into systems biology of human genetic disease[END_REF] , and this phenomenon is exemplified by the two pedigrees in our study and the Bengal cat. Inter-and intrafamilial variability within this disease spectrum has been attributed to allelism at the same locus [START_REF] Braun | Ciliopathies. Cold Spring Harb[END_REF] ;

second-site modification in trans [START_REF] Kousi | Genetic modifiers and oligogenic inheritance[END_REF] ; and for dominant disorders, variable expression of the wild-type allele [START_REF] Rivolta | Variation in retinitis pigmentosa-11 (PRPF31 or RP11) gene expression between symptomatic and asymptomatic patients with dominant RP11 mutations[END_REF] . Two different KIF3B protein domains are impacted by each of the deleterious variants characterized by this study (kinesin motor, family A vs coiled coil domain, family B), leading us to speculate that allele effect could be sufficient to drive phenotype differences between the two families.

Our in vivo assessment of rhodopsin trafficking in the zebrafish retina supports this posit (Figure 5).

However, we cannot exclude the possibility that the family A proband has an exacerbated syndromic phenotype due to genetic interaction with additional variation in other loci that interact either directly or indirectly with KIF3B. We recognize that a limitation of this study is the fact that only coding regions were queried for variation; whole genome sequencing will be required to obtain a complete list of contributory sites that determine phenotype in both families.

In conclusion, our study adds to the repertoire of ciliary genes that result in human pathology and characterize at clinical and molecular levels a ciliopathy hallmarked by RP, polydactyly and liver fibrosis likely caused by an alteration of kinesin-2 driven transport. Most ciliopathies, especially those caused by IFT motor or raft proteins, are autosomal recessive disorders [START_REF] Afzelius | A human syndrome caused by immotile cilia[END_REF][START_REF] Huynh Cong | A homozygous missense mutation in the ciliary gene TTC21B causes familial FSGS[END_REF][START_REF] Bujakowska | Mutations in IFT172 cause isolated retinal degeneration and Bardet-Biedl syndrome[END_REF] . However, we add another exception to this paradigm by reporting humans who harbor dominant de novo or inherited heterozygous variants placing them in the class of dominant ciliopathies inclusive of conditions such as polycystic kidney disease (MIM: 173900) and orofacial digital syndrome (MIM: 311200). (A) Representative confocal images of primary human fibroblasts from the family A proband. Cells were methanol-fixed and immunostained with anti-ARL13b rabbit polyclonal antibody (red) and antiγ-tubulin mouse IgG1 monoclonal antibody (green), and mounted with DAPI-Fluoromount G (blue) as markers of the primary cilium, basal body and nuclei, respectively. Scale bars: 10 µm (top row) and 1 µm (bottom row). See Table S3 for details about antibodies used.

Figure 1 .

 1 Figure 1. Individuals harboring nonsynonymous KIF3B variants display retinal phenotypes. (A) Ophthalmological examination of individual 1 (family A) who harbors a de novo c.748G>C; p.Glu250Gln KIF3B variant. a. Ophthalmological fundus imaging of the right and the left eyes; b. Fundus autofluorescence imaging of the right and the left eyes; c. Spectral domain optical tomography of the right fovea. Scale: 1 mm. d. Spectral domain optical tomography of the left fovea. Scale: 1 mm. Refraction was performed under cyploplegia with chlorhydrate of cyclopentolate 0.5% (Alcon, Rueil-Malmaison, France). Optical coherence tomography (OCT) analysis and the autofluorescence fundus imaging were conducted with an OCT spectal domain (Heidelberg Engineering, Spectralis HRA-OCT). (B) Ophthalmological examination of individual VI-2 (family B) who harbors a c.1569T>C; p.Leu523Pro KIF3B variant. White arrows indicate retinal pigments characteristic of retinitis pigmentosa.

Figure 2 .F1QN54;

 2 Figure 2. Heterozygous nonsynonymous variants in KIF3B segregate with ciliopathy phenotypes in dominant pedigrees. (A) Pedigrees of families A and B with segregation of KIF3B variants. Filled circles and squares represent affected female or male individuals, respectively; unfilled circles and squares represent unaffected female or male individuals; respectively. Deceased individuals are indicated by diagonal lines. Exome sequencing was performed for individuals marked with an arrow. Individual genotypes at the KIF3B locus are indicated in blue. All affected individuals in family B carried the KIF3B c.1568T>C, p.Leu523Pro encoding variant (IV-1, IV-3, IV-4, IV-6, V-4, V-7, VI-1, VI-2) but the unaffected individual (V-3) was WT. (B) Schematic of KIF3B gene organization and protein domains. Untranslated exons, white boxes; translated exons, black boxes; red arrows and arrow heads, KIF3B variants identified in humans; Kinesin motor, coiled coil and globular domains (UniProtKB, PROSITE annotations, GenBank: NP_004789.1) are indicated. (C) Conservation of 40 amino acid blocks impacted by nonsynonymous changes shown with a multiple sequence alignment (Clustal W v1.81) of 25 species sorted by pairwise identity. Red boxes, variant residues; blue shading of amino acids from dark to light represents most to least conserved, respectively. UniProtKB identifiers: Homo sapiens, O15066; Macaca mulatta, F6S877; Gorilla gorilla, G3RAF7; Mustela putorius furo, M3Z2F0; Canis lupus familiaris, E2QUS2; Loxodonta africana, G3T0G8; Callithrix jacchus, F7IBN6; Ailuropoda melanoleuca, G1M429; Felis catus, A0A2I2UKW2; Sus scrofa, F1S519; Bos taurus, F1N020; Mus musculus, Q61771; Ovis aries, W5NZV7; Rattus norvegicus, D3ZI07; Monodelphis domestica, F6RWN1; Sarcophilus harrisii, G3WA27; Cavia aperea, ENSCAPP00000010080 (ensembl, UniProtKB identifier not available); Gallus gallus, Q5F423; Xenopus tropicalis, F6R640; Oryctolagus cuniculus, G1U1D0; Danio rerio, F1QN54; Oryzias latipes, H2LAE9; Ciona intestinalis, F7B875; Drosophila melanogaster, P46867; Saccharomyces cerevisiae, P28742.

Figure 3 .

 3 Figure 3. KIF3B p.Glu250Gln and p.Leu523Pro variants increase primary cilia length.

ACKNOWLEDGEMENTS

We are grateful to the families who participated in this study. We thank Kevin Adams, Westley Heydeck, Julien Philippe, for technical support and advice. We thank Brian Perkins for the IFT52 antibody, and his assistance with zebrafish antibody staining protocols. We also thank Stefan Scholz and Elisabet Teixido from UFZ as well as Tobias Kießling from "Scientific Software Solutions" (www.tks3.com) for use and assistance with FishInspector software. This work was supported by US National Institutes of Health grants HD042601 (N.K.), GM121317 (N.K.), DK072301 (N.K. and E.E.D.) and EY007142 (S.P.D), and by grants from the Foundation Fighting Blindness USA (S.P.D.) and the William Stamps Farish Fund (S.P.D.). N.K. is a Distinguished Valerie and George D. Kennedy Professor. N.K. is a paid consultant for and holds significant stock of Rescindo Therapeutics, Inc. This project was funded in part previously by the National Center for Research Resources R24 RR016094 and the Office of Research Infrastructure Programs OD R24OD010928, the Phyllis and George Miller Feline Health Fund, Center for Companion Animal Health, School of Veterinary Medicine, University of California -Davis (2007-38-FM, 2008-08-F); the Winn Feline Foundation (MT07-012, W12-022) Cat Health Network (D12FE-509) (LAL). We appreciate the dedicated assistance of Bengal cat breeders for their participation in the research. SMPJ is funded by the Myers-Dunlap Endowment at Michigan State University. This work was supported by grants from the National Eye Institute [R01EY012910 (EAP), R01EY026904 (KMB/EAP) and P30EY014104 (MEEI core support)], and the Foundation Fighting Blindness (EGI-GE-1218-0753-UCSD, KMB/EAP).

DECLARATION OF INTEREST

The authors declare no competing interests.

Report

13

(B) Frequency of ciliated cells in primary skin fibroblasts from individual 1 (family A) and a matched control.

(C) Quantification of the primary cilia length measured on cells from a matched human control (4.55 ± 0.11µm, n=82) and individual 1 (family A) primary fibroblasts (6.20 ± 0.08µm, n=141). For panels B and C, six random images were assessed for each of control and affected; two independent replicates. (D) Representative confocal images of hTERT-RPE1 cells transiently transfected with pCS2 + -KIF3B-myc vectors and immunostained with anti-ARL13b (red) and anti-γ-tubulin (green) and mounted with DAPI-Fluoromount G as markers of the primary cilium, basal body and nuclei, respectively. Scale bars: 10 µm (top row) and 1 µm (bottom row). (A) Manhattan plot of GWAS for cat progressive retinal atrophy. After Bonferroni correction, several SNPs on cat chromosome A3 suggest a significant association with Bengal cat progressive retinal atrophy (Table S5). Cat chromosome A3 has genes homologous to human chromosome 20.

(B) Morphological and cellular changes as analyzed by immunohistochemistry. Left panel, anti-Kif3b antibody labels the region of the photoreceptor inner segment (IS) in wild type cats. Similar labeling was detectable in sections from 8-week old KIF3B homozygous mutants, but not in sections from 20week old mutant kittens. See Table S3 for details of antibodies used. Central panel, combination staining of the cone markers peanut agglutinin (PNA; cone sheath) with cone ML opsin (MLO). Cone morphology in KIF3B mutants at 8 weeks appears relatively normal but by 20 weeks there was distortion and stunting of outer segments with reduced ML opsin labeling. By 34 weeks of age only short residual PNA labeling material remained with reduced ML opsin labeling. The outer nuclear layer (ONL) was progressively thinned. Right panel, labeling with a rhodopsin marker (RetP1).

Report 14

Rhodopsin is mislocalized in mutants to the inner segment as early as 8-weeks of age then mislocalized to the outer nuclear layer cells bodies and synaptic terminals by 20-weeks. The rod inner and outer segments (OS) also showed some degeneration with disease progression. Statistical comparisons were performed with a non-parametric one-way ANOVA followed by Tukey's multiple comparison (GraphPad PRISM software; version 7.0c). ****p<0.0001, ***p<0.001; ns, not significant; WT, wild type; p.Glu250Gln and p.Leu523Pro are variants identified in cases; p.Val435Ile is a negative control (rs41288638; 230/276,748 alleles in gnomAD). See Table S3 for antibodies used.


Sabrina Mechaussier

Basamat Almoallem

Christina Zeitz

Kristof Van Schil

Laila Jeddawi

Jo Van Dorpe

Alfredo Dueñas

Christel Condroyer

Olivier Pelle

Michel Polak

Kristof Van Schil

Jo Van Dorpe

Alfredo Dueñas Rey

Nathalie Boddaert

Nadia Bahi-Buisson

Mara Cavallin

Jean-Louis Bacquet

Alexandra Mouallem-Bézière

Olivia Zambrowski

José Alain Sahel

Isabelle Audo

Josseline Kaplan

Jean-Michel Rozet

Elfride De Baere
email: elfride.debaere@ugent.

Isabelle Perrault
email: isabelle.perrault@inserm.fr

Loss of Function of RIMS2 Causes a Syndromic Congenital Cone-Rod Synaptic Disease with Neurodevelopmental and Pancreatic Involvement

Keywords: RIMS2, synaptic membrane exocytosis gene, congenital cone-rod synaptic disorder (CRSD), syndromic, neurodevelopmental or pancreatic involvement, stationary versus degenerative retinal disease, differential diagnosis, exome sequencing

scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Retinal rod and cone cells are photosensitive neurons that possess ribbon synapses to provide rapid and sustained transmission of graded light responses to second-order neuron bipolar and horizontal cells that shape the visual message for the cortex [START_REF] Mercer | The dynamic architecture of photoreceptor ribbon synapses: Cytoskeletal, extracellular matrix, and intramembrane proteins[END_REF] . Genetic alterations affecting synaptic transmission from photoreceptors to bipolar cells manifest in congenital stationary night blindness (CSNB, [MIM:PS310500]). It is a rare but also overlooked retinal disorder with an estimated prevalence of at least 1:40,000 (drawn from solved cases in a French cohort). This form of CSNB can be subdivided in complete (cCSNB) and incomplete (iCSNB) forms [START_REF] Zeitz | Congenital stationary night blindness: An analysis and update of genotype-phenotype correlations and pathogenic mechanisms[END_REF] . While cCSNB affects mainly proteins located post-synaptically at the dendritic tips of ON-bipolar cells, iCSNB affects mainly proteins located at the synapse of photoreceptor cells [START_REF] Zeitz | Congenital stationary night blindness: An analysis and update of genotype-phenotype correlations and pathogenic mechanisms[END_REF] . Affected individuals with iCSNB are characterized by both ON-and OFF-bipolar cell dysfunction as shown in the electroretinogram (ERG) [START_REF] Zeitz | Congenital stationary night blindness: An analysis and update of genotype-phenotype correlations and pathogenic mechanisms[END_REF] . Pathogenic variants in CACNA1F (MIM:300110) [START_REF] Strom | An L-type calcium-channel gene mutated in incomplete X-linked congenital stationary night blindness[END_REF][START_REF] Bech-Hansen | Loss-of-function mutations in a calcium-channel α1-subunit gene in Xp11.23 cause incomplete X-linked congenital stationary night blindness[END_REF] , CABP4 (MIM:608965) [START_REF] Zeitz | Mutations in CABP4, the Gene Encoding the Ca2+-Binding Protein 4, Cause Autosomal Recessive Night Blindness[END_REF] and CACNA2D4 (MIM:608171) [START_REF] Wycisk | Mutation in the Auxiliary Calcium-Channel Subunit CACNA2D4 Causes Autosomal Recessive Cone Dystrophy[END_REF] were identified in affected individuals with iCSNB [START_REF] Zeitz | Congenital stationary night blindness: An analysis and update of genotype-phenotype correlations and pathogenic mechanisms[END_REF] . Of note, stable low vision, nystagmus and more importantly photophobia may be major symptoms in affected individuals with variants in CACNA1F, CABP4, and CACNA2D4 in addition to phenotypic variability exceptionally leading to mildly progressive inherited retinal disorders (IRD) [START_REF] Zeitz | Congenital stationary night blindness: An analysis and update of genotype-phenotype correlations and pathogenic mechanisms[END_REF] and thus the term iCSNB may be misleading [START_REF] Khan | CABP4 Mutations Do Not Cause Congenital Stationary Night Blindness[END_REF][START_REF] Riemslag | Visually impaired children: "coming to better terms[END_REF] . Therefore, we use the term congenital cone-rod synaptic disorder (CRSD).

The ocular presentation in infants may be reminiscent of Leber congenital amaurosis (LCA, [MIM:PS204000]), a degenerative disease of the retina that is a leading cause of childhood blindness, with an estimated prevalence of 1:30,000 [START_REF] Kaplan | Leber congenital amaurosis: from darkness to spotlight[END_REF] . Occasionally, LCA can be the earliest manifestation of syndromic disease, such as ciliopathies [START_REF] Waters | Ciliopathies: an expanding disease spectrum[END_REF] , neuro-metabolic disorders [START_REF] Casteels | Leber Congenital Amaurosis -Differential Diagnosis, Ophthalmological and Neuroradiological Report of 18 Patients[END_REF] or tubulinopathies [START_REF] Luscan | Mutations in TUBB4B Cause a Distinctive Sensorineural Disease[END_REF] . ERG is a critical test for early differential diagnosis. ERG responses are undetectable in keeping with extremely severe rod and cone dysfunction in LCA. On the other side, ERG traces reveal generalized inner retinal dysfunction with both ON-and OFF-bipolar cell dysfunction with normal photoreceptor function manifested with an electronegative waveform in response to a bright flash under dark adaptation and severely reduced and delayed light adapted responses in CRSD. However, ERG recordings may be challenging in young children this may contribute to a misdiagnosis between ultra-rare CRSD and more prevalent LCA.

Here, a genomic study of unresolved cases with IRD revealed bi-allelic loss-of-function variants in Regulating Synaptic Membrane Exocytosis 2 (RIMS2 [MIM:606630]) in seven cases from four unrelated families of Senegalese, French and Saudi-Arabian origin, with an initial diagnosis of CSNB or LCA. Consistent with a role of RIMS2 in regulating synaptic membrane exocytosis in photoreceptors, brain and pancreas and with its localization in human tissues, we demonstrated syndromic CRSD with neurodevelopmental and possible pancreatic involvements in individuals with bi-allelic mutations in RIMS2.

MATERIALS AND METHODS

Subjects

This study involved seven affected subjects (three females and four males) and five healthy relatives from four unrelated families with early-onset IRD. and Ghent University Hospital Ethics Committee (B670201734438). Genomic DNA was extracted from peripheral blood by standard procedures.

Genetic analysis

The notation of the variants was based on RIMS2α transcript NM_00134848.1, corresponding to the variant X5 transcript for RefSeq accession number XM_033203201 (version XM_033203201.1).

Gene panel testing and exome sequencing (ES)

. Families 1 and 2 underwent testing with a small IRD panel followed by exome sequencing (ES). Genomic DNA libraries were generated from DNA (F1:III-3, F1:III-5, F2:I-1, F2:I-2, F2:II-2) sheared with a Covaris S2 Ultrasonicator using SureSelect XT Library PrepKit (Agilent). Regions of interest (ROI) were captured using the Agilent Sure Select All Exon V5 kit (Agilent) and sequenced on an Illumina HiSeq2500 HT system (Illumina). Data analysis was performed with Paris Descartes University/Imagine Institute Bioinformatics core facilities (Imagine pipeline, POLYWEB) [START_REF] Gerber | Recessive and Dominant De Novo ITPR1 Mutations Cause Gillespie Syndrome[END_REF] .

Whole-genome homozygosity mapping and ES.

DNA from F3:II-1 and F3:II-2 of Family 3 were genotyped using the HumanCytoSNP-12 BeadChip platform (Illumina). The genotypes were evaluated for runs of homozygosity (ROH) >1Mb using PLINK software [START_REF] Purcell | PLINK: A Tool Set for Whole-Genome Association and Population-Based Linkage Analyses[END_REF] integrated in in-house Software ViVar [START_REF] Sante | ViVar: A Comprehensive Platform for the Analysis and Visualization of Structural Genomic Variation[END_REF] . Resulting ROH were ranked according to their length and number of consecutive homozygous single-nucleotide polymorphisms (SNPs) [START_REF] Coppieters | Identity-by-descent-guided mutation analysis and exome sequencing in consanguineous families reveals unusual clinical and molecular findings in retinal dystrophy[END_REF] . For ES, exome enrichment and sequencing were performed using the Agilent SureSelect Human All exon v5/v6 kit followed by paired-end sequencing on a HiSeq2000 (2x100 cycles). The CLC Genomics Workbench version 9.0.1 (CLCBio) was used for read mapping against the human genome reference (NCBI build37/hg19 version), post mapping duplicate read removal, coverage analysis, and quality-based variant calling using Alamut (Visual version 2.7.2; interactive biosoftware). S1A andS1B). RIMS2 variants identified by ES were confirmed and segregation analysis was performed in all available family members by Sanger sequencing (Figure 1A).

Targeted testing of

RIMS2 expression studies in human tissues

Data mining in human adult single-cell (sc) retinal transcriptional datasets [START_REF] Lukowski | A single-cell transcriptome atlas of the adult human retina[END_REF] . Data was processed for evaluating RIMS2 expression at the sc level. The expression matrix derived from pooling 3 donor neural retina samples was retrieved and imported into R (v.3.6.2) using the Seurat sc analysis package (v3.1.4) [START_REF] Satija | Spatial reconstruction of single-cell gene expression data[END_REF] . Pre-processing and quality control was conducted to remove outlier cells. We filtered out cells that had unique (gene) feature counts less than 200 or over 2,500 and that expressed >5% mitochondrial counts. The dataset was subsequently normalized employing the built-in global-scaling normalization method LogNormalize. Prior to dimensional reduction, the data was subjected to scaling, and heterogeneity associated with number of unique molecular identifiers (UMIs) per cell and mitochondrial contamination was regressed out. After quality control pre-processing, a total of 15,635 cells were kept and clustered using the K-nearest neighbor graph method implemented in Seurat after PCAreduction. The non-linear dimensional reduction technique UMAP (Uniform Manifold Approximation and Projection) [START_REF] Becht | Dimensionality reduction for visualizing single-cell data using UMAP[END_REF] was applied to visualize and explore the dataset. As input, the same principal components from the clustering analysis were used. Markers associated with major neural retina cell populations were used to assess RIMS2 expression at sc level.

Data mining in human adult bulk retinal transcriptional datasets. Expression levels by

transcripts per million (TPM) were retrieved from postmortem retina samples characterized in Ratnapriya et al. [START_REF] Ratnapriya | Retinal transcriptome and eQTL analyses identify genes associated with age-related macular degeneration[END_REF] . A total of 453 samples which passed quality control were firstly considered. In order to avoid introducing confounding variables in the downstream analysis, only donor retinas without age-related macular degeneration were selected, resulting in 105 individual samples. Gene set selection: TPM values were further filtered for a set of candidate genes which included all genes reported to cause IRD (RetNet) and genes identified in synaptic vesicle pools and/or with pre-/post-synaptic curated annotations (SynGO) [START_REF] Koopmans | SynGO: An Evidence-Based, Expert-Curated Knowledge Base for the Synapse[END_REF] . A total of 379 genes were eventually considered. Normalization/stabilization and statistics: prior to examining correlations in the expression of the candidate genes, the set was further filtered by both mean expression and variance. To remove potential noise, 25% genes with lowest mean expression across all samples were firstly filtered out. The set was then subjected to a variance-stabilizing transformation to correct for mean-variance dependency [START_REF] Russo | CEMiTool: a Bioconductor package for performing comprehensive modular co-expression analyses[END_REF][START_REF] Zwiener | Transforming RNA-Seq Data to Improve the Performance of Prognostic Gene Signatures[END_REF] , which resulted in a total of 289 genes retained. We then examined the expression of RIMS2 and several synaptic genes (CACNA1F, CABP4, CACNA2D4, SV2B). For each gene pair, Spearman's correlations were computed along with pairwise p-values adjusted for multiple comparisons (Holm's method). A matrix correlation plot was then generated for visualization.

Expression analysis. RT-qPCR was performed using total RNA of fetal brain (22 weeks), retina (16 weeks) and head of the pancreas (25 weeks) (RNeasy Mini Kit, Qiagen), for cDNA synthesis random hexamer anchored oligo(dT) primers were used (Verso cDNA kit, Life Technologies ThermoFisher Scientific). RIMS2 expression was measured by amplifying 138, 78 and 89 bp fragments (Table S1C). Real-time PCR amplification and normalization were performed as described [START_REF] Gerard | AON-mediated Exon Skipping Restores Ciliation in Fibroblasts Harboring the Common Leber Congenital Amaurosis CEP290 Mutation[END_REF] . Data were analyzed using Realplex software (Eppendorf).

Immunohistochemistry. Human retinal, brain and pancreatic tissues used for immunohistochemistry were fixed in 10% neutral buffered formaldehyde and embedded in paraffin. Staining for RIMS2 was performed on 3-μm-thick sections using an automatic immunostainer (BenchMark Ultra, Ventana Medical Systems, Tucson, AZ). The rabbit polyclonal antibody anti-RIMS2 (1:25; Antibodies-Online, ABIN1003091) was used and visualization was achieved with the OptiView Amplification Kit (Ventana Medical Systems). Heat-induced epitope retrieval was performed using Cell Conditioning 2 (Ventana Medical Systems).

Functional analysis of RIMS2 nonsense variants

Site-directed mutagenesis. Constructs were made for the following variants: c.1595C>G, c.2884C>T, c.3126G>A, and c.3508C>T using mutagenesis via inverse PCR with Phusion polymerase using vector pcDNA3.1/(+)-N-(K)DYK-RIMS2α (RIMS2α: NM:00134848.1; 10277bp) (Genscript) as a template. Primers can be found in Table S1D. The amplified product was digested with DpnI to avoid re-ligation of original non-mutated DNA. Constructs were amplified in Escherichia coli TOP10 chemically competent cells, and inserts were sequenced to assess the mutagenesis and the rest of the sequence.

Western blot analysis in HEK293 cells. 5×10 5 cells/well in 6-well plates were co-transfected with wild-type or mutant FLAG-tagged RIMS2 plasmids (1.6µg; Genscript) and the pCAGGS-GFP plasmid (400ng; Clontech) using the FuGene HD transfection reagent according to the manufacturer's protocol (Promega). Proteins were prepared using RIPA lysis buffer (Life Technologies ThermoFisher Scientific). 50µg of total proteins were resolved by Mini-ProteanTGX Stain Free 4-15% gel according to the supplier's recommendations (BioRad). Proteins were transferred to a PVDF membrane using a RTA transfer kit (BioRad) which was probed with the following primary antibodies: polyclonal goat IgG anti-DDDDK tag (1:5,000; Abcam) and monoclonal mouse IgG1κ anti-GFP (1:2,000 ; Sigma Aldrich); and rabbit anti-goat IgG-HRP (1:2,000; Abcam) and rabbit anti-mouse IgG-HRP (1:2,000; Abcam) secondary antibodies. Blots were developed using the Clarity Western ECL and ChemiDoc XRS+ Imaging System (BioRad). Western blot images were acquired and analyzed using Image Lab Software 3.0.1 build 18 (BioRad). The abundance of FLAG relative to GFP was estimated by densitometry using the Image Lab Software 3.0.1 build 18.

A t-Student test was performed.

Insulin secretion assay in MIN6B1 cells. Clone B1 derived from Mouse INsulinoma pancreatic 6 β-cells (MIN6 B1

) were seeded at 3×10 6 cells/well in 6-well plates and cotransfected with wild-type or mutant FLAG-tagged RIMS2 plasmids (2µg, Genscript) and the pCAGGS-GFP plasmid (1µg; Clontech) using the FuGene HD transfection reagent according to the manufacturer's protocol (Promega). GFP-expressing cells were sorted on a BD FACS Aria II SORP cell sorter (BD Biosciences) from cell pools. GFP-MIN6 B1 cells were seeded at 5×10 4 cells/well in 96-well plates. After 1 hour of DMEM Glutamax I medium (5mM of glucose), medium was replaced by DMEM Glutamax I medium (25mM of glucose) Concentration of insulin in the medium was determined after 25 minutes of incubation using Mouse INSULIN ELISA kit (Invitrogen ThermoFisher Scientific). Absorbance was measured immediately at 450 nm and 550 nm by a VICTOR X4 2030 multilabel plate reader (PerkinElmer). The significance of variations among samples was determined using one-way ANOVA with post hoc Tukey's test thanks to the Prism6 software.

RESULTS

Identification of bi-allelic RIMS2 variants in seven cases from four unrelated families

Exome datasets were generated for affected siblings F1:III-3 and F1:III-5, F3:II-1 and F3:II-2 and index case F2:II-2 and his unaffected parents F2:I-1 and F2:I-2 (Figure 1A). A total of seven and two candidate genes were found in Family 1 and Family 2 respectively, applying stringent filtering (Table S2). This strategy revealed bi-allelic RIMS2 variants (RefSeq XM_033203201) in the two families: c.3126G>A p.Trp1042* in F1:III-3 and F1:III-5 (homozygous) and c.2884C>T, p.Arg962* in trans with c.4363+1G>A, p.? in F2:II-2.

Similarly, homozygosity mapping combined with ES in Family 3 identified a homozygous RIMS2 variant c.3508C>T, p.Arg1170* in the largest 25Mb autozygous region in F3:II-1 and F3:II-2, being the most plausible candidate variant (Table S2). Of note, no LOD score could be calculated due to the small size of the family. Subsequent targeted RIMS2 testing of prescreened affected individuals with iCSNB identified two affected individuals with a homozygous RIMS2 variant: c.1595C>G, p.Ser532* in F4:II-1 and c.3126G>A p.Trp1042* in an affected individual that retrospectively appeared to be related to Family 1 (F1:III-3) (Figure 1B and1C). Co-segregation of the RIMS2 variants with the disease was confirmed (Figure 1A).

Clinical re-evaluation of affected individuals

The complete lack of Rims2 in the mouse has been reported to cause behavioral anomalies, defective synaptic visual signal transmission from photoreceptors to secondary order retinal neurons consistent with iCSNB and insulin resistance [START_REF] Löhner | Analysis of RIM Expression and Function at Mouse Photoreceptor Ribbon Synapses[END_REF] . In Family 1, the index case F1:III-5 and her elder affected sister F1:III-3, born to consanguineous parents from Senegal, were reported to have manifested nystagmus, poor visual function and altered ERGs around birth.

Upon ophthalmological evaluation, the sisters, as of now aged 23 and 31 years with myopic correction, displayed features of inner retinal dysfunction, with a preserved a-wave to a bright flash under dark adapted conditions (originating from photoreceptor hyperpolarization) but a severely reduced b-wave (originating from ON-bipolar depolarization) and severely reduced and delayed light adapted responses in keeping with cone ON-and OFF bipolar cell dysfunction (Figure 2A). Funduscopy showed optic disc pallor, moderate vascular attenuation, normal macular area, and a normal mid-peripheral and peripheral aspect with no pigmentary migration (Figure 2B). Spectral domain optical coherence tomography (SD-OCT) of the macular region (Figure 2C) and of the retinal nerve fiber layer (RNFL) (Figure 2D) revealed inner retinal thinning throughout the posterior pole, a disruption of the foveal ellipsoid line in F1:III-5 and a temporal RNFL loss, respectively. These data are consistent with a cone-rod synaptic disorder with additional foveal changes. Disc pallor, inner retinal changes and thinned retinal vessels have to be interpreted in the context of high myopia. The eldest sister (F1:III-3) had a known history of persistent elevated blood glucose since the age of 29 years, and fasting blood glucose testing confirmed the diagnosis of insulin dependent diabetes mellitus (Table 1). Clinical neurologic assessment and neuroimaging of the youngest affected sister F1:III-5 demonstrated autistic features with unremarkable cerebral anomalies with the exception of temporal optic nerve atrophy. She displayed marked lipoatrophy with facial sparing but the metabolic workup was unremarkable, in particular there was no resistance to insulin or blood glucose homeostasis alteration. Neither were lipid homeostasis and liver function altered. Her affected son F1:IV-4 born to a first-cousin marriage presented at 13 months of age with severe visual dysfunction, marked neurodevelopmental delay (poor language, mild ataxia, general movement disorganization) with dysmorphic corpus callosum at brain Magnetic Resonance Imaging (MRI) (Figure 2E). The metabolic workup was unremarkable (Table 1). The young individual IV-4 in Family 1 is born to a double consanguineous marriage. As the familial RIMS2 pathogenic variant in IV-4 was found by targeted RIMS2 testing and not by WES, we cannot rule out that dysmorphology of the corpus callosum is attributed to a homozygous pathogenic variant in another gene due to this parental consanguinity.

Affected individual F2:II-2 was born at term after an uneventful pregnancy. He was first seen in ophthalmology at 2.5 months for pendular nystagmus, strabismus and photophobia. Upon examination, he presented with the inability to follow lights or objects, hyperopia and a normal fundus. At the age of 7 months, the poor quality of ERG recording leads to a presumed diagnosis of LCA. At 2 years, the child manifested behavioral problems and inability to walk without assistance. In addition to nystagmus and photophobia, the ophthalmologist noted some albinoid characteristics, including fair ashy hairs, peripheral iris transillumination and pale fundus. Furthermore, bilateral active pupillary reflexes were present, questioning the initial ERG results and diagnosis of LCA. ERG recordings could not be repeated at that time however due to high agitation. Genotype-directed clinical reexamination was conducted at the age of 5.5-6 years. ERG recordings under general anesthesia revealed an electronegative wave form under limited dark adaptation and barely detectable photopic responses consistent with iCSNB (Figure 2A). Funduscopy showed optic nerve pallor and a normal mid-peripheral and peripheral aspect (Figure 2B). Neuropediatric assessment showed autistic features including stereotypies and obsessive-compulsive disorder (OCD). Neuroimaging using MRI displayed no brain anomalies. Blood glucose homeostasis dosage was in the normal range (Table 1).

The two affected sibling of Family 3 (F3:II-1 and F3:II-2) born to consanguineous Saudi Arabian parents, displayed poor visual acuity reaching light perception in both eyes, photophobia, pendular nystagmus, the absence of pupillary response and oculo-digital signs.

Retinal imaging of both siblings showed waxy pallor of the optic discs and attenuated retinal vessels (Figure 2B). Both siblings displayed autistic behavior with a variable degree of severity: while the female younger sibling F3:II-2 (3 years) could produce simple speech, the male sibling F3:II-1 (4 years) was not capable of speech and showed aggressive and anxious behavior (Table 1), not allowing ERG recordings.

Affected individual F4:II-1 of Family 4 was born to consanguineous Senegalese parents. He was first referred to the electrophysiology unit when he was 2.5 years of age for photophobia, nystagmus and poor vision. He was a premature (born at 33-week post amenorrhea) twin baby, his fraternal twin brother being exempt of any relevant medical and ophthalmic history beside the prematurity and a mild ataxia developed while starting to walk at 14 months of age. F4:II-1 had been diagnosed with developmental delay with delayed speech and walking acquisition which was resolving upon rehabilitation. After thorough psychomotor examination, a diagnosis of attention disorder with hyperactivity within the spectrum of autistic behavior had been diagnosed with no intellectual disability. His metabolic work-up was otherwise normal. F3:II-1 was myopic with a visual acuity around 20/200 for both eye at age 7 (Table 1). Fundus examination revealed a mild optic disc pallor and some vessel attenuation that may be attributed to the myopia (Figure 2B). SD-OCT of the macula was normal (Figure 2C). Fundus autofluorescence was normal. ERG recordings revealed an electronegative waveform to a bright flash in dark adapted conditions as well as delayed and reduced responses in light adaptation, leading to a diagnosis of iCSNB. The child manifested neurodevelopmental delay with speech difficulties but no learning disability in elementary school, an ataxic gait and a certain degree of hyperactive autistic behavior (Table 1).

RIMS2 is present in human retina, brain and pancreas

Consistent with the neuro-ophthalmo-metabolic phenotype of the knock-out model, Rims2 is detected in mouse retinal, brain and pancreatic tissues (BioGPS; biogps.org). According to the human retinal transcriptome dataset of the Ocular Genomics Institute, the Human Protein Atlas, and BioGPS human RIMS2 is equally detected in these tissues. Data mining of sc transcriptional datasets of human adult neural retina showed that RIMS2 is predominantly expressed in rod photoreceptor clusters (Figure S1A), with an expression pattern similar to the one of CABP4 (Figure S1B). Next, the expression patterns of RIMS2, CACNA1F, CABP4, CACNA2D4 and SV2B in human adult bulk retinal transcriptional datasets were assessed for coordinated correlation, reasoning that this could provide insight into potential regulatory interactions. A matrix correlation plot (Figure S1C) showed RIMS2 expression to be anticorrelated with CACNA1F (rho=-0.3435, p=0.002) and to be correlated with SV2B (rho=0.6565, p<0.0001). The latter is known to be localized in synaptic vesicles where it may function in the regulation of vesicle trafficking and exocytosis.

In addition to a promoter located in the 5'UTR region driving the longest RIMS2α transcript, the gene has two internal alternative promoters yielding shorter transcripts (RIMS2β and RIMS2γ) (Figure 1B). RT-qPCR analysis using primers specific for each of the three isoforms applied to fetal human tissues, showed by far the highest expression of all transcripts in the brain compared to other tissues. In the retina, the 3 transcripts seemed equally expressed. In the pancreas RIMS2γ had the highest expression followed by RIMS2α then RIMS2β whereas RIMS2α was undetectable in fibroblasts with a predominant expression of RIMS2γ in these non-neuronal non-secretory cells (Figure 3). RIMS2 protein localization was assessed in adult human retina, brain and pancreas by immunostaining using an antibody recognizing both RIMS2α and RIMS2β isoforms but not RIMS2γ. Strong and specific RIMS2 immunostaining was observed in the outer plexiform retinal layer (Figure 4A), cerebellar cortical neurons, more specifically in Purkinje cells (Figure 4B), and in pancreatic Langerhans islets (Figure 4C). This localization is in agreement with the spectrum of oculo-cerebro-pancreatic anomalies in bi-allelic mutant RIMS2 cases.

In silico predictions and in vitro functional characterization of RIMS2 variants

Effect of a splice variant. The c.4363+1G>A splice variant found in Family 2 is the only one out of five unique RIMS2 variants that is expected to affect the three transcripts. In silico predictions suggest an abolished consensus donor splice-site leading to skipping of the adjacent exon, a frameshift and the introduction of a premature termination codon (c.4251_4363del, p.Asn1417Lysfs*2). The insertion of a stop codon in the shortest RIMS2γ transcript may likely result in nonsense-mediated decay (NMD) and in the absence of this isoform. The mutant RIMS2α and RIMS2β transcripts however may escape NMD and lead to truncated isoforms (Figure S2).

Protein levels of nonsense variants.

The four RIMS2 nonsense variants however are predicted to produce normal RIMS2γ and truncated RIMS2α and RIMS2β proteins. Consistent with this, Western blot analysis of lysates from HEK293 cells overexpressing individually the wild-type, p.Ser532*, p.Arg962*, p.Trp1042* and p.Arg1170* FLAG-tagged RIMS2α cDNAs, using FLAG antibody, revealed a ~210 kDa protein and truncated isoforms of expected ~60, ~120, ~130 and ~150 kDa sizes, respectively (Figure 5A). The wild-type and truncated FLAG-tagged proteins were produced with the same intensity (Figure 5B). No endogenous RIMS2α or RIMS2β were detected in lysates from untransfected HEK293 cells using primary antibodies polyclonal rabbit IgG anti-RIMS2 (1:1000; Synaptic Systems Cat.No. 140 303) and monoclonal mouse IgG anti-β-actin (1:4000; Abcam); and goat antirabbit IgG-HRP (1:2000; Abcam) and goat anti-mouse IgG-HRP (1:2000; Abcam) secondary antibodies (Figure S3).

Insulin secretion in MIN6B1 cells.

We investigated the ability to promote insulin secretion in MIN6B1 cells of these truncated proteins that lack all or most of the domain that interacts with RIM-binding proteins 1 and 2 (RIMBP1/2). We overexpressed the pCAGGS-GFP plasmid alone or in combination with the wild-type or mutant FLAG-tagged RIMS2 constructs and measured the accumulation of insulin in the culture medium upon glucose stimulation. The amount of insulin in the culture medium from cells transfected with pCAGGS-GFP alone or in combination with the wild-type FLAG-tagged RIMS2α constructs was in the same range, suggesting that overexpression of wild-type RIMS2α does not affect insulin secretion. In contrast, we observed a reproducible reduction of insulin accumulation in cells transfected with each of the three mutant constructs, supporting that truncation of all or most of the RIMBP1/2 binding domain alters insulin secretion in this cellular system (Figure 6).

DISCUSSION

The human visual system continues to develop after birth, mainly in the first years of life. The differential diagnosis of early-onset IRD, both stationary (CSNB) or degenerative (LCA), may be challenging in the first months of life mainly due to the inability to respond verbally to visual testing, the absence of apparent fundus changes, the difficulties in cooperation for testing especially in case of behavioral problems that may lead to unreliable ERG recordings and the presence of a nystagmus, hampering high-resolution retinal imaging without general anesthesia. The identification of the underlying genetic defect can accelerate an early differential diagnosis between a stationary and degenerative retinal disease, as there is no genetic overlap.

Here, we demonstrated bi-allelic loss-of-function of RIMS2 in a phenotype characterized by syndromic CRSD with neurodevelopmental and pancreatic involvement, consistent with a role of RIMS2 in regulating synaptic membrane exocytosis in photoreceptors, brain and pancreas. Regarding the retinal phenotype, visual acuity seems low in our cases with RIMS2associated CRSD and would be on the lower functional spectrum of the high phenotypic variability spectrum of iCSNB, characterized a post-photoreceptor defect affecting both the cone and rod signalling pathways 2 . Indeed, in a series of 60 affected individuals carrying mutation in CACNA1F, Boycott et al. report a high variability in visual acuity in correlation with the presence of a nystagmus [START_REF] Boycott | Clinical variability among patients with incomplete X-linked congenital stationary night blindness and a founder mutation in CACNA1F. Can[END_REF] . In addition, for some of the index cases of our study, behavioural disturbances made it difficult to have an accurate measurement of visual acuity which may have been underestimated. In addition, one of the affected individuals presented here had foveal changes that may also hampered visual acuity. Similar changes have been reported in association with CABP4 mutations [START_REF] Smirnov | Retinal findings in a patient of French ancestry with CABP4-related retinal disease[END_REF] . These changes were only present once in our report and further studies are needed to document the frequency of this occurrence in RIMS2associated CRSD. Furthermore, on inner retinal thinning was also found in our series which may also contribute to poor visual acuity. This finding has also been reported in association with iCSNB [START_REF] Chen | Autofluorescence imaging and spectral-domain optical coherence tomography in incomplete congenital stationary night blindness and comparison with retinitis pigmentosa[END_REF] .

RIMS2 is one of the Rab3-interacting molecules or RIMs, which are multi-domain scaffolding proteins that were first described as putative presynaptic active zone proteins in the retinal photoreceptors with an essential role in neurotransmitter release [START_REF] Acuna | How to Make an Active Zone: Unexpected Universal Functional Redundancy between RIMs and RIM-BPs[END_REF][START_REF] Wang | Genomic definition of RIM proteins: evolutionary amplification of a family of synaptic regulatory proteins☆☆Sequence data from this article have been deposited with the GenBank Data Library under Accession Nos. AF548738 and AF548739[END_REF][START_REF] Heidelberger | Synaptic transmission at retinal ribbon synapses[END_REF] . In vertebrates, RIMs are encoded by four regulating synaptic membrane exocytosis genes (RIMS1-4) [START_REF] Wang | Genomic definition of RIM proteins: evolutionary amplification of a family of synaptic regulatory proteins☆☆Sequence data from this article have been deposited with the GenBank Data Library under Accession Nos. AF548738 and AF548739[END_REF] .

While Rims1α -/-and Rims2α -/-mice are viable, the ablation of both genes causes postnatal mortality due to a defective neurotransmitter release despite preserved synaptic structure and exocytosis ability [START_REF] Schoch | Redundant functions of RIM1α and RIM2α in Ca2+-triggered neurotransmitter release[END_REF][START_REF] Schoch | RIM1α forms a protein scaffold for regulating neurotransmitter release at the active zone[END_REF] . Synapses of double mutants contain active zones and release neurotransmitters, but cannot mediate normal Ca 2+ -triggered release [START_REF] Schoch | Redundant functions of RIM1α and RIM2α in Ca2+-triggered neurotransmitter release[END_REF][START_REF] Schoch | RIM1α forms a protein scaffold for regulating neurotransmitter release at the active zone[END_REF] . The RIMS2associated phenotype in human cases is consistent with the known function of the mouse ortholog in photoreceptor ribbon synapse, conventional chemical synapse and synaptic vesicle exocytosis and with its localization in the outer retinal plexiform layer [START_REF] Löhner | Analysis of RIM Expression and Function at Mouse Photoreceptor Ribbon Synapses[END_REF] , in brain synapses [START_REF] Schoch | Redundant functions of RIM1α and RIM2α in Ca2+-triggered neurotransmitter release[END_REF] and in pancreatic β cells [START_REF] Yasuda | Rim2α Determines Docking and Priming States in Insulin Granule Exocytosis[END_REF] .

RIMS2 is the predominant large RIM isoform present at photoreceptor ribbon synapses.

RIMS2 plays an important role in maintaining the normal synaptic connection at the ribbon synapse of the photoreceptors [START_REF] Mercer | The dynamic architecture of photoreceptor ribbon synapses: Cytoskeletal, extracellular matrix, and intramembrane proteins[END_REF][START_REF] Löhner | Analysis of RIM Expression and Function at Mouse Photoreceptor Ribbon Synapses[END_REF] . This can be achieved via several interactions required for synaptic vesicle docking and priming [START_REF] Acuna | How to Make an Active Zone: Unexpected Universal Functional Redundancy between RIMs and RIM-BPs[END_REF] . The majority of photoreceptor-RIMS2 lacks the Nterminal zinc finger and part of RAB3A-binding domain suggesting that photoreceptor synaptic transmission does not depend on full-length RIMSα [START_REF] Löhner | Analysis of RIM Expression and Function at Mouse Photoreceptor Ribbon Synapses[END_REF] . The role of RIMS2 at photoreceptor ribbon synapses is different from RIM function in most other types of chemical synapses [START_REF] Mittelstaedt | RIM proteins and their role in synapse function[END_REF] . At the photoreceptor ribbon synaptic active zone, RIMS2 is not essential for vesicle priming [START_REF] Löhner | Analysis of RIM Expression and Function at Mouse Photoreceptor Ribbon Synapses[END_REF] or for Ca 2+ channel clustering but it does act as a Ca 2+ channel modulator [START_REF] Grabner | RIM1/2-Mediated Facilitation of Cav1.4 Channel Opening Is Required for Ca2+-Stimulated Release in Mouse Rod Photoreceptors[END_REF] .

The influence of Rims1/2 on synaptic processes at rod terminals was assessed by generating a conditional double knockout mouse model in rod photoreceptors as early as three weeks of Interestingly, a RIMS1 variant was found to co-segregate with autosomal dominant cone-rod dystrophy (CORD7 [MIM:603649]) in a British family, implicating a gene with a synaptic function in an IRD [START_REF] Kelsell | Localization of a Gene (CORD7) for a Dominant Cone-Rod Dystrophy to Chromosome 6q[END_REF][START_REF] Warwick | Retinitis pigmentosa and bilateral cystoid macular oedema in a patient heterozygous for the RIM1 mutation previously associated with cone-rod dystrophy 7[END_REF][START_REF] Johnson | Genomic organisation and alternative splicing of human RIM1, a gene implicated in autosomal dominant cone-rod dystrophy (CORD7)[END_REF] . In a mouse model, carrying the same Rims1 variant as the human CORD7 (p.Arg655His), the mutant was shown to modify Rims1 function in regulating voltage dependent Ca 2+ channel currents [START_REF] Miki | Mutation associated with an autosomal dominant cone-rod dystrophy CORD7 modifies RIM1-mediated modulation of voltage-dependent Ca2+ channels[END_REF] . Rims1 variant on different presynaptic voltagedependent calcium channels (VDCCs) may eventually lead to CORD and enhanced cognitive abilities. Moreover, RIMS1 has been associated with ASD by two independent studies [START_REF] Dong | De Novo Insertions and Deletions of Predominantly Paternal Origin Are Associated with Autism Spectrum Disorder[END_REF][START_REF] Krumm | Excess of rare, inherited truncating mutations in autism[END_REF] .

Regarding RIMS2, a copy number variant (CNV), more specifically a duplication of 277 kb (Chr8_104901578-105178819), was found in an individual affected with autosomal dominant retinitis pigmentosa (adRP [MIM:268000]). Although this CNV was found to be absent in genomic databases, the role of this duplication in the pathogenesis of adRP is still a matter of debate especially since no segregation analysis could be performed in the rest of the family, including the affected father [START_REF] Huang | Genome-Wide Detection of Copy Number Variations in Unsolved Inherited Retinal Disease[END_REF] .

RIMS2 or RIMS1 variants have not been associated with metabolic dysfunction before. Yet, RIMS2 has been identified by yeast two-hybrid performed on pancreatic β cells to determine docking and priming states in insulin granule exocytosis [START_REF] Yasuda | Rim2α Determines Docking and Priming States in Insulin Granule Exocytosis[END_REF] . Consistently, Rims2 -/-mice display a phenotype which consists of anomalies of photoreceptor synaptic transmission [START_REF] Löhner | Analysis of RIM Expression and Function at Mouse Photoreceptor Ribbon Synapses[END_REF] , deficit in maternal behavior [START_REF] Schoch | Redundant functions of RIM1α and RIM2α in Ca2+-triggered neurotransmitter release[END_REF][START_REF] Schoch | RIM1α forms a protein scaffold for regulating neurotransmitter release at the active zone[END_REF] and insulin resistance [START_REF] Yasuda | Rim2α Determines Docking and Priming States in Insulin Granule Exocytosis[END_REF] . This retino-neuro-metabolic phenotype is reminiscent of the RIMS2-associated disease we report here. Whether abnormal glucose homeostasis is an invariable feature remains to be confirmed. Evidence of insulin resistance in the eldest affected individual of our cohort suggests that this could an age-related feature, requiring a close follow-up of metabolic functions.

In summary, bi-allelic loss-of-function variants of RIMS2 have been shown to cause a previously unreported syndromic iCSNB, manifesting as a CRSD with neurodevelopmental disease and occasional anomalies of glucose homeostasis. The identification of a syndromic stationary congenital IRD has a major impact on the differential diagnosis of syndromic congenital IRD, which has been exclusively linked with degenerative IRD before. Finally, our study implicates a photoreceptor synaptic gene with syndromic disease.

LEGENDS TO FIGURES

LEGENDS TO TABLES

  Families 1 and 2 were investigated at Imagine -Necker -Enfants Malades University Hospital. Affected individual III-3 of Family 1 (F1:III-3 in Figure 1A) was also investigated at the Centre Hospitalier National d'Ophtalmologie des Quinze-Vingts. Family 3 was clinically investigated at the Pediatric Ophthalmology Division, Dhahran Eye Specialist Hospital, Dhahran, Saudi Arabia and Family 4 at the Centre Hospitalier National d'Ophtalmologie des Quinze-Vingts. Family 1, admitted for LCA, consists of an inbred pedigree from Senegal with several consanguinity loops and three affected individuals over two generations, i.e. two adult sisters and the child of one of them born to a first-cousin marriage. Family 2, referred for LCA, comprises a simplex case and his two unrelated parents of French origin. Family 3, includes an affected sib ship born to a first-cousin marriage from Saudi Arabia, and Family 4, with a clinical diagnosis of CSNB, consists of a simplex case originating from first degree cousins, mother being Franco-Senegalese and father from Senegalese descent (Figure 1A). All individuals or legal representatives consented with the study, which received approval from the Institutional Review Boards Comité de Protection des Personnes Ile de France II (Necker), Ile de France V (Project number 06693, N•EUDRACT 2006-A00347-44, 11 December 2006, Quinze-Vingts)

  RIMS2. Forty-six affected individuals diagnosed with iCSNB and 133 diagnosed with LCA underwent RIMS2 testing either by Sanger sequencing (Big Dye Terminator v3.1 Kit, Applied Biosystems) or targeted next-generation sequencing (Nextera XT DNA Library Prep kit, MiSeq, Illumina) (Table

Figure 1 :

 1 Figure 1: Bi-allelic RIMS2α variants in the four families with corresponding variant

Figure 2 :

 2 Figure 2: Clinical presentation of affected individuals. (A) Representative

Figure 3 :

 3 Figure 3: Expression analysis of RIMS2 mRNA in fetal human tissues by quantitative

Figure 4 :

 4 Figure 4: Immunohistochemistry of RIMS2 protein in adult human brain, pancreas and

Figure 5 :

 5 Figure 5: Western blot analysis of HEK293 cells overexpressing wild-type and mutant

Figure 6 :

 6 Figure 6: Insulin secretion level of MIN6 B1 cells overexpressing wild-type and mutant

Figure 7 :

 7 Figure 7: Schematic and simplified drawing of protein complexes present at the

Table 1 : Summary of clinical features of seven affected individuals from four families with RIMS2 variants

 1 . Ophthalmologic, neurologic and metabolic investigations in all affected individuals. Abbreviations used: CF, count fingers; LP, light perception; ERG, electroretinogram; RE, right eye; LE, left eye; BE, both eyes; SD-OCT, spectral domain optical coherence tomography; RNFL-SD-OCT, spectral domain optical coherence tomography measuring the retinal nerve fiber layer thickness; MRI, magnetic resonance imaging; OCD, obsessive-compulsive disorders; NA, not available; NP, not performed.

			Individual	F1:III-3	F1:III-5	F1:IV-4
			Age (years)	33	25	2
			Origin	Senegal	Senegal	Senegal
			Nystagmus	+	+	Erratic ocular movement
			Photophobia	+	+	+
			Night blindness	-	-	-
	Ophthalmologic features	Visual acuity Refractive error ERG electronegativity Fundus	RE : 20/250 LE : 20/320 RE : -2 (1.75 )90° LE : -1 Present -Optic disc pallor -No peripheral pigmentary migration, greyish retina	RE : 20/3200 LE : 20/320 RE : -0.75 (1.25) 20° LE : -2.50 (-2.25) 170° Present -Temporal optic disc pallor -No peripheral pigmentary migration -Retinal thinning at the expense of inner	NP RE : +2.25 LE : +3 NP -Temporal optic disc pallo -No peripheral pigmentar
			Macular SD-OCT	Retinal thinning at the expense of inner retina	retina -Retrofoveal focal ellipsoid zone	NP
					disruption
			RNFL-SD-OCT	NA (Optic nerve dysversion)	Temporal RNFL loss	NP
			Autofluorescence	Normal	Normal	NP
	Neurologic features	Neurological examination MRI	NP Normal	Autistic behavior Normal	-Neurodevelopmental del -General movement disor -Ataxia manifestations -Poor language Dysmorphic corpus callosu
	Metabolic	features	Fasting blood glucose Blood glucose	Insulin dependent diabetes mellitus Hyperglycemia: 1.26 g/L	Normal: 0.94 g/L Hyperglycemia: 1.13 g/L	NP Hyperglycemia: 1.33 g/L

ACKNOWLEDGEMENTS

We are grateful to the families for their participation in the study. We thank Dr. Raphael Roduit for MIN6 cells and Dr. Julien Diana for MIN6 B1 cells. This work was supported by grants from the "Fondation de France Berthe Fouassier" (N° Engt 00079330) to S.M, Retina France, UNADEV-AVIESAN ITMO MNP (R16073KS) and VISIO to I.P and J.-M.R, Ghent University Special Research Fund (BOF15/GOA/011; BOF20/GOA/023), Hercules Foundation (AUGE/13/023), Ghent University Hospital Innovation Fund NucleUZ and European Union's Horizon 2020 research and innovation programme Marie Sklodowska-Curie Innovative Training Networks (ITN) StarT (grant No. 813490) to E.D.B. E.D.B. is a senior clinical investigator of the Research Foundation-Flanders (FWO) (1802215N; 1802220N), A.D.R. is Early Starting Researcher of StarT (grant No. 813490). DNA samples from the "Centre Hospitalier National d'Ophtalmologie des Quinze-Vingts" originate from NeuroSensCol DNA bank for research in neuroscience (PI: J.-A.S., co-PI I.A., partner with Centre Hospitalier National d'Ophtalmologie des Quinze-Vingts, INSERM, and CNRS), Fondation Voir et Entendre (C.Z.), Prix Dalloz for "La recherche en ophtalmologie" (C.Z.), Ville de Paris and Region Ile de France, LABEX LIFESENSES (ANR-10-LABX-65) supported by French state funds managed by the Agence Nationale de la Recherche within the Investissements d'Avenir program (ANR-11-IDEX-0004-0), IHU FOReSIGHT (ANR-18-IAHU-0001) supported by French state funds managed by the Agence Nationale de la Recherche within the Investissements d'Avenir program (C.Z., I.A., J.-A.S.). E.D.B., C.Z., I.A., J.-A.S., J.-M.R. are members of ERN-EYE project which is co-funded by the Health Program of the European Union under the Framework Partnership Agreement No 739534-'ERN-EYE'. RetNet, https://sph.uth.edu/retnet/ Ocular Genomics Institute, https://oculargenomics.meei.harvard.edu/ The Human Protein Atlas, www.proteinatlas.org/ BioGPS, biogps.org/ OMIM, https://omim.org/

age. Deletion of Rims1/2 in mouse rods (called Rims1/2 double knock-out) showed a dramatic loss of Ca 2+ influx through Cav1.4 channels and an associated reduction in evoked release [START_REF] Grabner | RIM1/2-Mediated Facilitation of Cav1.4 Channel Opening Is Required for Ca2+-Stimulated Release in Mouse Rod Photoreceptors[END_REF] . Both Rims1/2 isoforms were shown to be expressed in the retina as early as three weeks of age. However, it has been shown that Rims1-specific antibody supports an absence of Rims1 protein from photoreceptors and their ribbon synapses in the outer plexiform layer and that Rims1α does not contribute to the regulation of exocytosis at the cone photoreceptors ribbon synapse [START_REF] Löhner | Analysis of RIM Expression and Function at Mouse Photoreceptor Ribbon Synapses[END_REF] . Five weeks later at eight weeks, Rims2 was shown to be present exclusively in the outer plexiform layer of wild type mice while it was absent in the knockout mice. It was concluded that Rims2 potently enhances the influx of Ca 2+ into rod terminals through Cav1.4 channels, which is vitally important for the release of vesicles from the rod ribbon possibly through direct or indirect modulation of the channel [START_REF] Grabner | RIM1/2-Mediated Facilitation of Cav1.4 Channel Opening Is Required for Ca2+-Stimulated Release in Mouse Rod Photoreceptors[END_REF][START_REF] Hibino | RIM Binding Proteins (RBPs) Couple Rab3-Interacting Molecules (RIMs) to Voltage-Gated Ca2+ Channels[END_REF][START_REF] Kaeser | RIM Proteins Tether Ca2+ Channels to Presynaptic Active Zones via a Direct PDZ-Domain Interaction[END_REF] . As Rims2 has many partners at the photoreceptor synapse (synthesized in Figure 7) [START_REF] Mercer | The dynamic architecture of photoreceptor ribbon synapses: Cytoskeletal, extracellular matrix, and intramembrane proteins[END_REF][START_REF] Acuna | How to Make an Active Zone: Unexpected Universal Functional Redundancy between RIMs and RIM-BPs[END_REF][START_REF] Mittelstaedt | RIM proteins and their role in synapse function[END_REF][START_REF] Hibino | RIM Binding Proteins (RBPs) Couple Rab3-Interacting Molecules (RIMs) to Voltage-Gated Ca2+ Channels[END_REF][START_REF] Kaeser | RIM Proteins Tether Ca2+ Channels to Presynaptic Active Zones via a Direct PDZ-Domain Interaction[END_REF][START_REF] Schmitz | EF hand-mediated Ca2+-and cGMP-signaling in photoreceptor synaptic terminals[END_REF][START_REF] Tom Dieck | Ribbon synapses of the retina[END_REF][START_REF] Tom Dieck | Molecular dissection of the photoreceptor ribbon synapse[END_REF][START_REF] Rizo | Mechanism of neurotransmitter release coming into focus: Mechanism of Neurotransmitter Release[END_REF][START_REF] Wahl-Schott | Switching off calcium-dependent inactivation in L-type calcium channels by an autoinhibitory domain[END_REF] that have not been linked with any human or animal phenotype so far, the genes encoding them merit consideration in genomic studies of children with congenital IRD.

Regarding the brain, Rims2, like Rims1, was shown to be expressed as early as three weeks of age in whole brain lysates. Analysis of double knockout mice has demonstrated expression of the two paralogs in overlapping but distinct patterns throughout the brain [START_REF] Schoch | Redundant functions of RIM1α and RIM2α in Ca2+-triggered neurotransmitter release[END_REF][START_REF] Schoch | RIM1α forms a protein scaffold for regulating neurotransmitter release at the active zone[END_REF] . Here, we showed localization of RIMS2 in human adult cerebellar cortical neurons, more specifically in Purkinje cells. Interestingly, involvement of RIMS2 in neurological disease, more specifically in autism spectrum disease (ASD), is supported by a genome-wide association study in affected individuals with Asperger syndrome (ASPG [MIM:608638]), revealing a significant association with the RIMS2-associated SNP rs2080610 [START_REF] Salyakina | Variants in several genomic regions associated with asperger disorder[END_REF] . A study on alternative splicing in neural tissues showed that one of the RIMS2 transcripts contains a highly-conserved microexon which is neuron-specific [START_REF] Irimia | A Highly Conserved Program of Neuronal Microexons Is Misregulated in Autistic Brains[END_REF] . RIMS2 has been listed as an ASD-associated gene in a recently developed ASD database [START_REF] Krishnan | Genome-wide prediction and functional characterization of the genetic basis of autism spectrum disorder[END_REF] .

DECLARATION OF INTERESTS

The authors declare no competing interests.