Isabelle Audo
email: isabelle.audo@inserm.fr

Saddek Mohand-Said

Elise Boulanger-Scemama

Xavier Zanlonghi

Christel Condroyer

Vanessa Démontant

Fiona Boyard

Aline Antonio

Cécile Méjécase

Said El

José-Alain Sahel

Christina Zeitz
email: christina.zeitz@inserm.fr

Alain Sahel

MERTK mutation update in inherited retinal diseases

Keywords: inherited retinal dystrophy, MERTK, mutation spectrum, mutation prevalence ORCID

MER tyrosine kinase (MERTK) encodes a surface receptor localized at the apical membrane of the retinal pigment epithelium. It plays a critical role in photoreceptor outer segment internalization prior to phagocytosis. Mutations in MERTK have been associated with severe autosomal recessive retinal dystrophies in the RCS rat and in humans. We present here a comprehensive review of all reported MERTK disease causing variants with the associated phenotype. In addition, we provide further data and insights of a large cohort of 1,195 inherited retinal dystrophies (IRD) index cases applying state-of-the-art genotyping techniques and summarize current knowledge. A total of 79 variants have now been identified underlying rod-cone dystrophy and cone-rod dystrophy including 11 novel variants reported here. The mutation spectrum in MERTK includes 33 missense, 12 nonsense, 12 splice defects, 12 small deletions, 2 small insertion-deletions, 3 small duplications, and 2 exonic and 3 gross deletions. Altogether, mutations in MERTK account for ∼2% of IRD cases with a severe retinal phenotype. These data are important for current and future therapeutic trials including gene replacement therapy or cell-based therapy.

BACKGROUND

Inherited retinal diseases (IRD) are a clinically and genetically heterogeneous group of disorders characterized by progressive photoreceptor degeneration [START_REF] Hartong | Retinitis pigmentosa[END_REF]. Its prevalence is estimated from one out of 3,500 to one out of 5,000 worldwide with variable ages of onset and degrees of severity. So far, close to 250 genes have been implicated in cases of IRD (https://sph.uth.edu/retnet/) following all modes of Mendelian or mitochondrial inheritance with rare cases of digenism. Most of these genes encode proteins involved in various cellular functions essential for photoreceptor or retinal pigment epithelium (RPE) homeostasis [START_REF] Wright | Photoreceptor degeneration: Genetic and mechanistic dissection of a complex trait[END_REF]. The most common form of IRD is rodcone dystrophy (RCD), also known as retinitis pigmentosa (RP) (MIM# 268000), which manifests with night blindness, progressive visual field constriction and possible loss of central vision due to gradual dysfunction, and loss of rod photoreceptors followed by cone photoreceptors. On the other hand, cone and cone-rod dystrophies (CCRD) (e.g., MIM# 602093, 304020, and 120970) start by a decreased vision, photophobia with possible secondary night blindness, and peripheral visual field abnormalities leading to blindness. Of note, the distinction between RCD and CCRD is sometime clinically difficult, especially in advanced or severe cases, and there are overlapping genetic mechanisms, mutations for some genes leading to both phenotypes [START_REF] Boulanger-Scemama | Next-generation sequencing applied to a large French cone and cone-rod dystrophy cohort: Mutation spectrum and new genotype-phenotype correlation[END_REF]. There is currently no cure for IRD and most of them result in severe visual impairment or blindness with however promising clinical trials or therapeutic perspectives advocating for a better clinical and genetic characterization [START_REF] Thompson | Advancing therapeutic strategies for inherited retinal degeneration: Recommendations from the Monaciano Symposium[END_REF].

MERTK (MER proto-oncogene, tyrosine kinase) (MIM# 604705) is one of the mutated genes in IRD. It is localized in 2q14.1 [START_REF] Weier | Assignment of protooncogene MERTK (a.k.a. c-mer) to human chromosome 2q14.1 by in situ hybridization[END_REF] and comprises 19 exons. It encodes the tyrosine-protein kinase Mer, a 999-amino-acid transmembrane protein (Uniprot # Q12866) belonging to the TAM (TYRO3/AXL/MER) receptor kinase family, a family of homeostatic regulators for the mature immune, reproductive, hematopoietic, vascular, and nervous systems mainly through its phagocytic function of apoptotic cells [START_REF] Lemke | Biology of the TAM receptors[END_REF]. MERTK is highly expressed in monocyte/macrophages, ovary, prostate, testis, lung, and kidney as well as in epithelial cells including the RPE. In the RPE, it plays a critical role for shed photoreceptor outer segment (POS) engulfment prior to phagocytosis, a circadian process essential for photoreceptor homeostasis [START_REF] Feng | Mertk triggers uptake of photoreceptor outer segments during phagocytosis by cultured retinal pigment epithelial cells[END_REF][START_REF] Kevany | Phagocytosis of retinal rod and cone photoreceptors[END_REF]. MERTK functional domains, common to the TAM receptors, include two immunoglobulin-like-C2 (Ig-like C2 type 1 from amino acid (aa) residue 81 to 186 and Ig-like C2 type 2 aa residue 197 to 273) and two fibronectin type III (FN-III from aa residue 286 to 381 and 386 to 484) domains in its extracellular portion and an intracellular region with a highly conserved kinase domain (aa residue 587 to 858) including the KW(I/L)A(I/L)ES motif characteristic of the TAM receptors (Figure 1) [START_REF] Strick | Focus on molecules: MERTK[END_REF]. In addition, the kinase domain contains three autophosphorylation sites , which are critical for its full activity [START_REF] Ling | Identification of the major autophosphorylation sites of Nyk/Mer, an NCAM-related receptor tyrosine kinase[END_REF]. MERTK exists as a membrane-bound receptor and as a soluble form [START_REF] Sather | A soluble form of the Mer receptor tyrosine kinase inhibits macrophage clearance of apoptotic cells and platelet aggregation[END_REF] through its cleavage at proline 485 by ADAM17 (MIM# 603639), at least in murine macrophages and RPE cells [START_REF] Law | Cleavage of Mer tyrosine kinase (MerTK) from the cell surface contributes to the regulation of retinal phagocytosis[END_REF][START_REF] Thorp | Shedding of the Mer tyrosine kinase receptor is mediated by ADAM17 protein through a pathway involving reactive oxygen species, protein kinase Cdelta, and p38 mitogen-activated protein kinase (MAPK)[END_REF]. This soluble form would act as a negative feedback loop limiting POS binding to RPE cell surface prior to phagocytosis [START_REF] Nandrot | Retinal pigment epithelial cells use a MerTK-dependent mechanism to limit the phagocytic particle binding activity of 𝛼v𝛽5 integrin[END_REF]. Phagocytosis of POS is a complex process which is now at least partially understood [START_REF] Mazzoni | Understanding photoreceptor outer segment phagocytosis: Use and utility of RPE cells in culture[END_REF]. The exposure of phosphatidyl serines (PS) at the surface of POS is the triggering stimulus. On one side, PS are recognized by the complex formed by the milk fat globule-EGF-factor 8 (MFG-E8) (MIM# 602281) bound to 𝛼v𝛽5 integrin (MIM# 193210, MIM# 147561), CD36 (MIM# 173510) and possibly other unknown partners. MFG-E8/𝛼v𝛽5 recognition leads to the protein tyrosine kinase 2 (MIM# 600758) (previously known as focal adhesion kinase) activation at the apical membrane of the RPE and to its autophosphorylation. This process is important for MERTK activation [START_REF] Nandrot | Loss of synchronized retinal phagocytosis and age-related blindness in mice lacking alphavbeta5 integrin[END_REF]. On the other side, MERTK indirectly interacts with PS through its ligands, Gas6/Protein S (MIM# 600441, MIM# 176880) which results in MERTK autophosphorylation at the three sites abovementioned and in cellular actin remodeling, through myosin light chain 2 (MIM# 160781). MERTK is then involved in POS engulfment through an interaction with its ligands. POS are subsequently internalized and degraded (reviewed in [START_REF] Kevany | Phagocytosis of retinal rod and cone photoreceptors[END_REF].

MERTK was first associated with retinal degeneration after the identification of a deletion causing the naturally occurring autosomal recessive (ar) retinal degeneration of the Royal College of Surgeon (RCS) rat [START_REF] D'cruz | Mutation of the receptor tyrosine kinase gene Mertk in the retinal dystrophic RCS rat[END_REF][START_REF] Nandrot | Homozygous deletion in the coding sequence of the c-mer gene in RCS rats unravels general mechanisms of physiological cell adhesion and apoptosis[END_REF], a retinal dystrophy model secondary to a phagocytic defect of the RPE [START_REF] Bok | The role of the pigment epithelium in the etiology of inherited retinal dystrophy in the rat[END_REF][START_REF] Dowling | Inherited retinal dystrophy in the rat[END_REF][START_REF] Edwards | Defective phagocytosis of isolated rod outer segments by RCS rat retinal pigment epithelium in culture[END_REF][START_REF] Herron | Retinal dystrophy in the rat-a pigment epithelial disease[END_REF][START_REF] Lavail | Influence of eye pigmentation and light deprivation on inherited retinal dystrophy in the rat[END_REF][START_REF] Li | Inherited retinal dystrophy in the RCS rat: Prevention of photoreceptor degeneration by pigment epithelial cell transplantation[END_REF].

Shortly after this discovery, mutations in MERTK were identified in patients with ar RP [START_REF] Gal | Mutations in MERTK, the human orthologue of the RCS rat retinal dystrophy gene, cause retinitis pigmentosa[END_REF]. More recently, an overexpression of MERTK was found in a canine progressive retinal atrophy, from a Swedish Vallhund breed, suggesting a gain of function mutation which still needs to be molecularly characterized [START_REF] Ahonen | Increased expression of MERTK is associated with a unique form of canine retinopathy[END_REF]. An ongoing clinical trial with gene augmentation in patients with IRD linked to MERTK mutations in Saudi Arabia (NCT01482195) [START_REF] Ghazi | Treatment of retinitis pigmentosa due to MERTK mutations by ocular subretinal injection of adeno-associated virus gene vector: Results of a phase I trial[END_REF] as well as interesting perspectives in cell therapy [START_REF] Zarbin | Cell-based therapy for degenerative retinal disease[END_REF] are an incentive to conduct comprehensive studies to prepare patients for these therapies. In the present manuscript, we provide a comprehensive review on reported variants in MERTK associated with IRD, add further data generated by the screening of a large French cohort of more than 1000 IRD index cases, deliver new information on prevalence and discuss phenotype-genotype correlations.

MATERIAL AND METHODS

Literature search

Literature search was performed on Pubmed, with a last check on March 12th, 2018, in order to collect all reported variants on MERTK (NM_006343.2) in association with IRD. Additional databases were queried such as The Human Gene Mutation Database (HGMD R Professional 2017.4, last queried on March 12th, 2018), Leiden Open Variation Database (LOVD V.3.0), last queried on March 12th, 2018, Retina International Mutation Database (https://www. retina-international.org/files/sci-news/mertkmut.htm, last updated in March 2004). For each of the reported variants, we collected information on phenotype/genotype correlation, familial segregation and documented minor allele frequency (MAF) as well as assessed functional impact through in silico pathogenic predictions tools.

Novel MERTK variants

In addition to the comprehensive literature search, we analyzed a large French cohort of 1,195 unrelated subjects with IRD. Each patient underwent a full ophthalmic examination as described elsewhere [START_REF] Audo | EYS is a major gene for rod-cone dystrophies in France[END_REF]. After this clinical evaluation, patients and family members were asked to donate a blood sample for DNA extraction and further genetic studies. Written informed consent was obtained from each subject after explanation of the study and its potential outcomes. The study protocol adhered to the tenets of the Declaration of where A of the ATG initiation codon is 1. The nonsense and frameshift mutations appear in red, gross deletions appear in purple, missense variants in blue and the splice site change in green. Novel variants are circled in black. The protein is a transmembrane protein and its structure includes: in the extracellular portion, two immunoglobulin-like-C2 (Ig-like C2 type 1 and type 2) from amino acid (aa) residue 81 to 186 and residue 197 to 273, respectively, two fibronectin type III domains (FN-III in blue from aa residue 286 to 381 and 386 to 484) and an intracellular region with a highly conserved kinase domain (in yellow from aa residue 587 to 858). The respective residues are written below the protein schematic analysis on a commercial chip (ASPER Ophthalmics, Tartu, Estonia) [START_REF] Koenekoop | Genetic testing for retinal dystrophies and dysfunctions: Benefits, dilemmas and solutions[END_REF] was initially applied to 186 index patients with IRD. Samples with no identified pathogenic variant as well as additional new index cases were subsequently investigated through a targeted nextgeneration sequencing (NGS) as described previously [START_REF] Audo | Development and application of a nextgeneration-sequencing (NGS) approach to detect known and novel gene defects underlying retinal diseases[END_REF][START_REF] Boulanger-Scemama | Next-generation sequencing applied to a large French cone and cone-rod dystrophy cohort: Mutation spectrum and new genotype-phenotype correlation[END_REF]. Guidelines from the American College of Medical Genetics and Genomics were used for variant classification [START_REF] Richards | Standards and guidelines for the interpretation of sequence variants: A joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology[END_REF]. Functional impact was assessed using in silico prediction tools (i.e. Align GVGD (https://agvgd.hci. utah.edu/agvgd_input.php) [START_REF] Tavtigian | Comprehensive statistical study of 452 BRCA1 missense substitutions with classification of eight recurrent substitutions as neutral[END_REF], Mutation Taster (https://www.mutationtaster.org/) [START_REF] Schwarz | Mutation-Taster2: Mutation prediction for the deep-sequencing age[END_REF], Sorting Intolerant From Tolerant SIFT (https:// sift.jcvi.org/) [START_REF] Kumar | Predicting the effects of coding non-synonymous variants on protein function using the SIFT algorithm[END_REF], Polymorphism Phenotyping v2 Polyphen-2 (https://genetics.bwh.harvard.edu/pph2/) [START_REF] Adzhubei | A method and server for predicting damaging missense mutations[END_REF], Splice Site Prediction by Neural Network, NNSPLICE, https://www.fruitfly.org/seq_tools/splice.html, [START_REF] Reese | Improved splice site detection in Genie[END_REF]; Human Splicing Finder v.2.4.1, HSF, https://www.umd.be/HSF/#, [START_REF] Desmet | Human Splicing Finder: An online bioinformatics tool to predict splicing signals[END_REF]

MERTK VARIANTS UNDERLYING IRD

A total of 68 genetic changes were found in the literature underlying IRD. These variants span the entire gene and therefore impact on all functional domains (Figure 1 and Table 1). In addition, the screening of a large cohort of IRD followed at our center (1,095 RCD and 100 CRD) identified 11 novel changes (Figure 1 [START_REF] Li | Detection of variants in 15 genes in 87 unrelated Chinese patients with Leber congenital amaurosis[END_REF] Variant mentioned in italic are those considered as nonpathogenic based on allele frequency, functional assay or only found at the single heterozygous state; NMD, nonsense-mediated mRNA decay; RP, retinitis pigmentosa; RCD, rod-cone dystrophy; CRD, cone-rod dystrophy; LCA, Leber congenital amaurosis: in the phenotypic column we mentioned the phenotype reported in the respective paper. RCD is most of the time considered as a synonymous for RP, with the symptom at onset characterized by night blindness followed by peripheral visual field constriction and secondary loss of vision; on the contrary, CRD usually starts by central retinal involvement with decreased central vision, photophobia and secondary loss of peripheral vision; this phenotypic distinction is however somewhat artificial, sometime based only on retrospective data when patients present with advance retinal disease; het, heterozygous; ho, homozygous.

unique except two: the known c.2189+1C>T splice variant [START_REF] Ebermann | Truncating mutation of the DFNB59 gene causes cochlear hearing impairment and central vestibular dysfunction[END_REF] was found in four independent families including three of North African descent and may represent a founder effect in this population and the large exonic deletion encompassing exon 3 to exon 19 that we reported earlier [START_REF] Boulanger-Scemama | Next-generation sequencing applied to a large French cone and cone-rod dystrophy cohort: Mutation spectrum and new genotype-phenotype correlation[END_REF], which was found in three unrelated cases including one from African descent (Burkina Faso) and 2 from French descent. Novel mutations include five missense changes, one 2-bp-insertion-deletion also resulting in an amino acid substitution. Three of these amino acid substitutions are located within the Ig-like C2 type 2 domain, one within the first FN-III domain and two within the kinase domain (Figure 1). Amino acid residues involved by these mutations are highly conserved (Figure 2) and the changes induced by the amino acid substitutions are predicted to be pathogenic by prediction programs (Table 1). We found one novel splice site variant (c.2079+2T>G), the sequence being conserved across species (Figure 2). This change is predicted to affect the wild type donor site most probably affecting splicing of intron 15 [START_REF] Desmet | Human Splicing Finder: An online bioinformatics tool to predict splicing signals[END_REF]. This may result in the continuation of protein translation after the lysine encoded by the last codon of exon 15 with the addition of 8 amino acid residues before reaching a stop codon. An alternative cryptic site in intron 15 could also be unmasked with also part of intron 15 being translated. In both instances, this would results in a truncation of the protein missing part of the kinase domain or in nonsense-mediated mRNA decay (NMD). Additional novel changes include one 1-bp-deleletion, one 2-bp-deletion, one 1-bp-duplication, and one 4-bp-duplication, all resulting either in a frameshift with premature stop codon and a truncated protein product or NMD. Our findings therefore add 11 novel changes to the 68 variants reported in the literature for a total of 79 reported variants (Table 1) spanning the entire coding and exon-flanking region (Figure 1).

All mutations identified by our comprehensive literature review and described herein, were submitted to the existing Locus Specific Databases (LSDB) on MERTK (https://www.LOVD.nl/ MERTK).

PREVALENCE DATA

The screening of our cohort including 1,095 RCD and 100 CRD index cases lead to the identification of 25 affected subjects belonging to 20 unrelated families (Supp. Figure S2) who carried likely pathogenic mutations in MERTK including 21 cases of RCD and four cases of CRD.

Therefore, mutations in MERTK would account for a prevalence of ∼1.7% IRD families in this large cohort with nearly 4% in the CRD subgroup. These numbers are comparable with previous reports with prevalence in the literature ranging from 1% [START_REF] Huang | Genotype-phenotype correlation and mutation spectrum in a large cohort of patients with inherited retinal dystrophy revealed by next-generation sequencing[END_REF][START_REF] Strick | Focus on molecules: MERTK[END_REF][START_REF] Tschernutter | Clinical characterisation of a family with retinal dystrophy caused by mutation in the Mertk gene[END_REF], ∼2% [START_REF] Ge | NGS-based molecular diagnosis of 105 eyeGENE((R)) probands with retinitis pigmentosa[END_REF], 3% [START_REF] Patel | Expanding the clinical, allelic, and locus heterogeneity of retinal dystrophies[END_REF] and up to 30% in the Faroe Islands with a common 91 kb founder deletion encompassing exons 1 to 7 [START_REF] Ostergaard | A novel MERTK deletion is a common founder mutation in the Faroe Islands and is responsible for a high proportion of retinitis pigmentosa cases[END_REF]. Of note, the 4% prevalence in our CRD should be taken with caution as the distinction between RCD and CRD is often difficult especially in advanced cases and may have been made retrospectively in some cases. This higher prevalence may only reflect the early macular involvement characterizing IRD associated with MERTK mutations.

CLINICAL CHARACTERISTICS OF PATIENTS CARRYING MERTK MUTATIONS

Mutations in MERTK underlying IRD was first reported by [START_REF] Gal | Mutations in MERTK, the human orthologue of the RCS rat retinal dystrophy gene, cause retinitis pigmentosa[END_REF], following the genetic characterization underlying retinal degeneration in the RCS rat [START_REF] D'cruz | Mutation of the receptor tyrosine kinase gene Mertk in the retinal dystrophic RCS rat[END_REF]. The associated IRD phenotype is usually severe characterized by an early onset of the disease and rapid macular involvement. The screening of our large IRD cohort (i.e., 1,095 RCD and 100 CRD, with a distinction made retrospectively based on the initial symptoms) identified 25 patients with MERTK mutation whose clinical findings are summarized in Supp.

Table S2 with representative retinal images presented in Figure 3. The 25 patients carrying mutations in MERTK were examined including 13 male and 12 female patients, with an age at time of examination ranging from 13 to 60 with an average of 28.76 and a median of 25.

They all experienced onset of symptoms, consisting primarily of night vision disturbances, before 18 with a majority of them during their early teens. Of note, none of the patients had nystagmus, which was only reported once in association with MERTK mutations [START_REF] Mchenry | MERTK arginine-844-cysteine in a patient with severe rod-cone dystrophy: Loss of mutant protein function in transfected cells[END_REF]. They all presented with severe retinal abnormalities and early macular involvement consistent with previous reports on IRD associated with MERTK mutations [START_REF] Charbel Issa | Characterisation of severe rod-cone dystrophy in a consanguineous family with a splice site mutation in the MERTK gene[END_REF][START_REF] Ebermann | Truncating mutation of the DFNB59 gene causes cochlear hearing impairment and central vestibular dysfunction[END_REF][START_REF] Gal | Mutations in MERTK, the human orthologue of the RCS rat retinal dystrophy gene, cause retinitis pigmentosa[END_REF][START_REF] Jinda | A novel start codon mutation of the MERTK gene in a patient with retinitis pigmentosa[END_REF][START_REF] Ksantini | Homozygous mutation in MERTK causes severe autosomal recessive retinitis pigmentosa[END_REF]Mackay et al., 2010;[START_REF] Mchenry | MERTK arginine-844-cysteine in a patient with severe rod-cone dystrophy: Loss of mutant protein function in transfected cells[END_REF][START_REF] Thompson | Retinal dystrophy due to paternal isodisomy for chromosome 1 or chromosome 2, with homoallelism for mutations in RPE65 or MERTK, respectively[END_REF][START_REF] Tschernutter | Clinical characterisation of a family with retinal dystrophy caused by mutation in the Mertk gene[END_REF].

Visual acuity ranges from light perception to 20 out of 32 for the youngest patient. Color vision was abnormal in all tested patients (24 out of 25) with 32 eyes showing severe dyschromatopsia, 10 eyes with tritan, two with deutan, and one with protan defects. Visual fields were constricted in all patients with a majority (92%) reduced to the 20 central degrees or below at the binocular III4e stimulus. All patients had none-detectable full field and multifocal ERG responses in keeping with severe generalized retinal dysfunction for both the rod and cone system with additional severe macular dysfunction. In this respect, the distinction between RCD (21 patients) and CRD (4 patients) could not be made based on functional assessment but mainly on retrospective elements, patients reporting initial decreased central vision, possible photophobia followed by night vision disturbances as inaugural symptoms, and macular atrophy, being classified as CRD. This distinction is however questionable and combined photoreceptor dystrophy could in these cases with severe functional involvement be a better term.

On fundus examination, 20 out of 25 patients had waxy pallor of the optic discs (e.g., Figure 3A andB), almost all had narrowed retinal vessels (e.g., Figure 3A, B, and D but not C), 13 out of 25 had a beaten-bronze appearance with very few pigment migrations (e.g., Figure 3A andC) among which three had subtle white dots in the retinal periphery (e.g., Figure 3A andC) with one also in the macular area. Along with the early onset macular involvement, the F I G U R E 2 Evolutionary conservation of likely pathogenic novel missense and splice-site variants identified by our group beaten-bronze appearance of the fundus and the presence of white dots have been reported before [START_REF] Charbel Issa | Characterisation of severe rod-cone dystrophy in a consanguineous family with a splice site mutation in the MERTK gene[END_REF][START_REF] Ebermann | Truncating mutation of the DFNB59 gene causes cochlear hearing impairment and central vestibular dysfunction[END_REF][START_REF] Jinda | A novel start codon mutation of the MERTK gene in a patient with retinitis pigmentosa[END_REF][START_REF] Ksantini | Homozygous mutation in MERTK causes severe autosomal recessive retinitis pigmentosa[END_REF]Mackay et al., 2010). These white dots are not specific of IRD with MERTK mutations since they have also been reported in association with TULP1, LRAT, RLBP1, RPE65, or RHO mutations among others [START_REF] Lewis | Tubby-like protein 1 homozygous splice-site mutation causes early-onset severe retinal degeneration[END_REF][START_REF] Littink | A homozygous frameshift mutation in LRAT causes retinitis punctata albescens[END_REF][START_REF] Mataftsi | Novel TULP1 mutation causing leber congenital amaurosis or early onset retinal degeneration[END_REF][START_REF] Schatz | Fundus albipunctatus associated with compound heterozygous mutations in RPE65[END_REF][START_REF] Souied | Retinitis punctata albescens associated with the Arg135Trp mutation in the rhodopsin gene[END_REF]. Some of them may by hyperautofluorescent and hyper-reflective on SD-OCT, located at the level of the outer retina (see below).

Short-wave length fundus autofluorescence revealed patchy loss in the periphery for all patients. In addition, none of the patients display the classical ring of increased fundus autofluorescence reported in patients with RCD and preserved macular function [START_REF] Robson | Pattern ERG correlates of abnormal fundus aut-ofluorescence in patients with retinitis pigmentosa and normal visual acuity[END_REF]. Instead, all patients had abnormal autofluorescence at the macula with two distinct patterns (Figure 3): 14 out of 25 patients had foveal increase of autofluorescence (e.g., Figure 3A), whereas the rest had loss of autofluorescence in the macula (e.g., Figure 3C andD).

In in Figure 3A andC). In addition, 24 out of 25 patients had evidence of hyper-reflective dots (e.g., zoom in Figure 3A andC) in the choroid which has, to our knowledge, never been noticed before although they are visible on SD-OCT figures from recent papers [START_REF] Charbel Issa | Characterisation of severe rod-cone dystrophy in a consanguineous family with a splice site mutation in the MERTK gene[END_REF][START_REF] Jinda | A novel start codon mutation of the MERTK gene in a patient with retinitis pigmentosa[END_REF]. Some of the dots located in the outer retina may correspond to the hyperautofluorescent dots on FAF seen on some of the patients and maybe to the occasional white dots seen on fundus examination. These dots may represent outer segment debris, RPE cell migration or/and phagocytes (i.e., macrophages and activated microglia) also seen in RCS rats [START_REF] Dowling | Inherited retinal dystrophy in the rat[END_REF][START_REF] Lavail | The interphotoreceptor matrix in rats with inherited retinal dystrophy[END_REF] and Mertk knockout mice [START_REF] Duncan | An RCS-like retinal dystrophy phenotype in mer knockout mice[END_REF]. In keeping with this, a recent study applying two photon microscopy to various Mertk-/-mutant models suggested that microglial cells could be recruited to transport byproducts of the phototransduction cascade to the RPE and be involved in the elimination of dying photoreceptor cells [START_REF] Palczewska | Receptor MER tyrosine kinase proto-oncogene (MERTK) is not required for transfer of bis-retinoids to the retinal pigmented epithelium[END_REF]. Macrophages may also account for the [START_REF] Ge | NGS-based molecular diagnosis of 105 eyeGENE((R)) probands with retinitis pigmentosa[END_REF], Bietti crystalline dystrophy, independently of crystal location [START_REF] Saatci | Spectral domain optical coherence tomographic findings of bietti crystalline dystrophy[END_REF], and RCD [START_REF] Kuroda | Intraretinal hyperreflective foci on spectral-domain optical coherence tomographic images of patients with retinitis pigmentosa[END_REF]. In the latter, [START_REF] Kuroda | Intraretinal hyperreflective foci on spectral-domain optical coherence tomographic images of patients with retinitis pigmentosa[END_REF] suggested that these hyper-reflective dots were associated with disease progression. However, no genotype was available in this publication therefore the occurrence of outer retinal hyperreflective dots in correlation with specific gene defects in IRD remains to be established. If they indeed represent phagocytic cells, these dots may have some incidence in a putative inflammatory response to treatment such as cell therapy.

Altogether, the association with early onset IRD with macular involvement associated with a bronze-beaten appearance, several white spots on fundoscopy, abnormal macular FAF, lack of preservation of outer retinal hyper-reflective bands with hyper-reflective dots in the outer retina and the choriocapilaris may be suggestive but not specific of a MERTK-related IRD in a sporadic or autosomal recessive case.

Although the present cohort is one of the largest reported in the literature on patients with retinal dystrophy related with MERTK mutations, we were not able to draw any specific conclusion on phenotype/genotype correlations, most likely due to the diversity of the mutation spectrum in a yet limited number of patients.

Intriguingly, patients from two distinct families (F751 and F3928)

were carrying the same large deletion. Individuals for F751 had a CRD phenotype with early onset decreased vision and photophobia whereas the individual from F3928 had more a RCD phenotype.

In addition, one of the patients with the most severe phenotype (CIC00239) was carrying two missense mutations. There may be additional factors, genetic or environmental that may also impact on the phenotype as suggested by [START_REF] Vollrath | Tyro3 modulates Mertk-associated retinal degeneration[END_REF]. Larger cohort studies and natural history of the disease may help better define such correlations.

Our work expands the mutation spectrum of MERTK in IRD which spans the entire coding and splice regions of the gene. It sets basis for therapeutic intervention aiming at gene augmentation [START_REF] Ghazi | Treatment of retinitis pigmentosa due to MERTK mutations by ocular subretinal injection of adeno-associated virus gene vector: Results of a phase I trial[END_REF][START_REF] Lavail | Gene Therapy for MERTK-Associated Retinal Degenerations[END_REF] or cell therapy [START_REF] Jones | Cell-based therapeutic strategies for replacement and preservation in retinal degenerative diseases[END_REF] for this genetic group linked to mutations in a selectively RPEexpressed gene. Mutations in MERTK are however associated with early onset retinal degeneration with rapid macular involvement urging for early diagnosis and intervention in strategies aiming at rescuing photoreceptors. Natural history studies with longitudinal followup may help better define therapeutic strategies and window of intervention.

  Helsinki and was approved by the local ethics committee (CPP, Comité de Protection des Personnes Ile de France V, project number 06693, N • EUDRACT 2006-A00347-44).The DNA of patients with a presumed diagnosis of autosomal recessive RCD and cone-rod dystrophy (CRD) was assessed. Microarray

F

  I G U R E 1 Distribution of the 68 MERTK variants reported in the literature (sources: pubmed search up to the March 12, 2018) and the 11 novel variants identified by our group in the gene and protein. Nucleotide numbering is based on cDNA sequence of MERTK (RefSeq NM_006343.2)

  addition, 12 out of 25 patients had fine hyperautofluorescent dots including eight with increased autofluorescence of the fovea (e.g., Figure 3B). Consistently, patients with loss of macular autofluorescence had severe outer central retinal thinning on SD-OCT unlike those with increased foveal autofluorescence. Of note, four out of 25 patients presented with mild epiretinal membranes but no obvious vitreomacu-lar tractions on SD-OCT requiring surgery. Besides these four patients, we did not notice the wrinkled appearance of the inner retina on fundus examination reported by Charbel Issa et al. (2009) associated to a peculiar wave-like appearance of the innermost neurosensory retina on SD-OCT. None of the patients with MERTK mutations had preservation of the outer retinal hyper-reflective bands (i.e., external limiting membrane, ellipsoid, and interdigitation zones). They all display hyperreflective dots above the RPE/Bruch's membrane complex (e.g., zoom

F

  Retinal images illustrating the phenotypic variability of inherited retinal diseases associated with MERTK mutations. A: Left eye of CIC00568: color fundus photograph (left) reveals a moderately pale optic disc; narrowed retinal vessels, a beaten-bronze appearance of the retinal periphery with only very few bone spicules; fundus autofluorescence (FAF) imaging (middle) shows a foveal increase of autofluorescence surrounded by a patchy loss of autofluorescence and some hyperautofluorescent dots; Spectral domain-optical coherence tomography (SD-OCT) (right with close-up) reveals a thinning of the outer nuclear layer, hyper-reflective dots above the RPE/Bruch complex and within the choroid (white arrows) with no visible external limiting membrane (ELM), ellipsoid zone (EZ), and interdigitation zone (IDZ). B: Right eye of CIC04456: color fundus photograph (top) reveals a pale optic disc, narrowed retinal vessels, an albinoid fundus with very few bone spicules and cystoid macular changes; FAF imaging (bottom left) shows severe diffused patchy loss of FAF including the macula with fine hyperAF dots; SD-OCT (bottom right) reveals intraretinal cysts with no visible ELM, EZ, or IDZ associated with hyper-reflective dots above the RPE/Bruch complex and in choroid (white arrows). C: Left eye of CIC03112: color fundus photograph (top left) reveals a well colored optic nerve, no narrowed retinal vessels, beaten-bronze appearance in the periphery with only few bone spicules, pigment clumping in the macula; FAF imaging (top right) shows severe diffused patchy loss of FAF including the macula with fine hyperautofluorescent dots; SD-OCT (bottom with close-up) reveals a severe outer retinal thinning with hyperreflective dots above a thinned RPE/Bruch complex and in choroid (white arrows). D: Right eye of CIC04949: color fundus photograph (top left) reveals a well colored optic disc, narrowed retinal vessels and diffuse pigment clumping; FAF imaging (top right) shows a diffuse patchy loss of FAF including the fovea; SD-OCT (bottom) reveals severe thinning of the outer retina with some hyper-reflective dots above the thinned RPE/Bruch complex but a thinned choroid with no obvious dots hyperflective dots seen in the choroid. These hyper-reflective dots are not specific of IRD linked to MERTK mutations. They have also been reported in association with Stargardt disease

Exon/ intron cDNA (NM_ 006343.2) Protein change Protein domain involved/putative functional consequence Status Phenotype if speci- fied Country of origin RS # MAF in ExAC/gnomAD if available Remarks and pathogenic prediction for missense changes Reference of the first report

  Variants in MERTK reported in the literature and novel variant identified in a large French cohort in association with inherited retinal dystrophies

			Reference of the first Reference of the first	Ostergaard, Duno, Reference of the first report Batbayli, Reference of the first Reference of the first report Reference of the first report Vilhelmsen, and report (Eisenberger et al., (Khan et al., 2017) Rosenberg (2011) 2013) (Wang et al., 2014a) Novel Reference of the first report (Eisenberger et al., 2013) Reported only as a Reference of the first report Novel (Mendez-Vidal et al., 2014) Reference of the first report single het in a patient Jinda et al. (2016) with two pathogenic Novel Reference of the first report (Eisenberger et al., Reference of the first report report (Ellingford et al., 2016) Reference of the first report (Siemiatkowska et al., 2011) report (McHenry et al., 2004) Reference of the first report (Tschernutter et al., 2006) Reference of the first report (Ge et al., 2015) Reference of the first report (Eisenberger et al., 2013) Reported only as single het in a patient with ho pathogenic mutation on RPGRIP1 Tada et al. (2006) mutations in EYS (Eisenberger et al., 2013) Reference of the first report (McHenry et al., 2004) who performed functional analysis and considered it likely benign	2013) (Xu et al., 2014)	(Oishi et al., 2014) (Gal et al., 2000)			(Xu et al., 2014) (Patel et al., 2015) who considered it as (Aldahmesh et al., (Wang et al., 2014b) (Siemiatkowska et al., 2011)	(Tschernutter et al., (Yang et al., 2018) (Li et al., 2011) (Oishi et al., 2014) (Shahzadi et al., 2010) 2006) less likely pathogenic with additional more pathogenic variant as (Lukovic et al., 2015) 2009) (Coppieters et al., 2012) Novel (Al-Khersan et al., 2017) (Ellingford et al., considered this variant a disease cause 2016) as most likely not (Gal et al., 2000) (Ge et al., 2015) (McHenry et al., (Tada et al., 2006)	disease causing since (Xu et al., 2014) the p.Arg20Ser (Coppieters et al., (rs35898499) has a (Mackay et al., 2010) MAF of 0.05467 and (Wang et al., 2014a) the residue is poorly (Srilekha et al., 2015) Novel 2014) 2004)	conserved (Patel et al., 2015) (Huang et al., 2015)	(Evans et al., 2017) (Mackay et al., 2010) 2012) (Gal et al., 2000) (Collin et al., 2011) (Coppieters et al.,	Novel	Novel Novel (Ellingford et al., (Xu et al., 2014) Novel Novel (Ksantini, Lafont, Bocquet, Meunier, (Boulanger-Scemama 2016) (Xu et al., 2014) et al., 2015) (Tada et al., 2006) (Eisenberger et al., (Riera et al., 2017) Demonstrated to 2013) (Mandal et al., 2005) affect splicing on (Ebermann et al., & Hamel, 2012) (Collin et al., 2011)	(Mackay et al., 2010) N o v e l 2013) with small amount (Eisenberger et al., patient's mRNA of normal length 2007) (Patel et al., 2015) (Consugar et al., 2015) (Xu et al., 2014) who mRNA	(Wang et al., 2014b) who considered it 2012) (Wang et al., 2014b) (Continues) (O'Sullivan et al., pathogenicity (Continues) questioned its (Continues) as pathogenic (Continues) (Continues) (Continues) (Continues) (Continues) (Continues) (Continues) (Continues) (Continues) (Continues)	(Continues) (Continues) (Continues)
	Remarks and Remarks and	-pathogenic prediction for Remarks and pathogenic missense changes prediction for Remarks and Remarks and pathogenic prediction for missense changes Remarks and pathogenic prediction for missense changes missense changes Align GVGD class Align GVGD class C65; SIFT -deleterious (score C35; SIFT delerious (score 0); Mutation 0); Mutation Taster -disease causing (P Taster disease causing (P Align GVGD class C0; value = 1); value SIFT Deleterious Polyphen2 = 0.997) Polyphen2 (score: 0.04); probably damaging probably damaging Mutation Taster -with a score of 1 with a score of polymorphism (P (sensitivity: 0.00; 0.988 (sensitivity: value: 1); specificity: 1.00) 0.73; specificity: Polyphen2 possibly and conserved 0.96) and damaging with a residue conserved residue score of 0.910 -Reported only as a Align GVGD class C0; -single het but partial SIFT tolerated (score sequencing of the 0.38); Mutation exons Taster polymorphism Align GVGD class C45; (P value = 1); SIFT deleterious Polyphen2 benign (score 0); Mutation with a score of 0 Taster disease (sensitivity: 1.00; -specificity: 0.00) causing (P value = 1); Polyphen2 probably with poor -damaging with a conservation score of 1 (sensitivity: (polymorphic residue 0.00; specificity: with a Thr present in 1.00) and conserved Opossum) residue AA nonpolar neutral changed to AA polar neutral Align GVGD class C65, SIFT deleterious score 0, no prediction on Mutation Taster; Polyphen2, probably damaging, P value = 1, (sensitivity: 0.00; specificity: 1.00) --AA able to form Remarks and pathogenic prediction for missense changes Align GVGD class C55; SIFT deleterious (score 0); Mutation Taster disease causing (P value = 1); Polyphen2 probably damaging with a score of 1 (sensitivity: 0.00; specificity: 1.00) and conserved residue Align GVGD class C15; SIFT deleterious (score 0); Mutation Taster disease causing (P value = 0.939); Polyphen2 probably damaging with a score of 0.999 (sensitivity: 0.14; specificity: 0.99) and conserved residue except in Lizard (Thr residue) -Remarks and pathogenic prediction for missense changes Both AA are polar neutral Align GVGD class C0; SIFT deleterious score 0.03; mutation taster disease causing P value = 0.995; Polyphen2 possibly damaging with a score of 0.472 (sensitivity: 0.89; specificity:0.90) ---prediction for missense changes ---Align GVGD class C35; SIFT deleterious (score 0); Mutation Taster disease causing (P value = 1) probably damaging with a score of 1 (sensitivity: 0; specificity: 1) and conserved residue Remarks and pathogenic prediction for missense changes ----Remarks and pathogenic prediction for missense changes Align GVGD class C15; SIFT deleterious (score 0); Mutation Taster disease causing (P value = 1); Polyphen2 probably damaging with a score of 1 (sensitivity: 0.00; specificity: 1.00) and conserved residue -AA nonpolar neutral changed to AA polar Remarks and pathogenic pathogenic prediction for missense changes Align GVGD class C0; SIFT deleterious (score 0); Mutation Taster disease causing (P value = 1); Polyphen2 probably damaging with a score of 1 (sensitivity: 0.00; specificity: 1.00) and conserved residue Align GVGD class C0; SIFT deleterious (score 0); Mutation Taster disease causing (P value = 1); Polyphen2 probably damaging with a score of 1 (sensitivity: 0.00; specificity: 1.00) and conserved residue -Remarks and pathogenic prediction for missense changes ----AA polar positive -AA change from disulfide bound changed to an -nonpolar neutral to aromatic AA --Predicted to affect splicing -neutral Align GVD class C0; SIFT deleterious Align GVGD class C65; polar negative Align GVGD class Align GVGD class C0; score 0, mutation SIFT deleterious Align GVGD class C25; (score 0); Mutation SIFT deleterious Taster disease (score 0); Mutation -causing (P value = 1); Taster disease Polyphen2 probably causing (P value damaging with a = 1); Polyphen2 probably score of 1 damaging with a (sensitivity: 0.00; score of 1 specificity: 1.00) and -conserved residue (sensitivity: 0.00; specificity: 1.00) and Align GVGD class C65; SIFT deleterious score 0; mutation taster disease causing P value = 1; Polyphen2 probably damaging with a score of 1.000 (sensitivity: 0.00; specificity: 1.00) conserved residue C65; SIFT deleterious score 0; mutation taster disease causing P value = 1; Polyphen2 probably damaging with a score of 1.000 (sensitivity: 0.00; specificity: 1.00) --SIFT tolerated (score 0.29); Mutation Taster disease causing (P value = 1) Polyphen2 benign with a score of 0.444 (sensitivity: 0.89; specificity: 0.90) and conserved residue Predicted to affect -taster disease causing P value = 1, Polyphen2 probably damaging with a score of 1.000 ((sensitivity: 0.00; specificity: 1.00) Align GVD Class C0; SIFT deleterious (score 0); Mutation Taster disease causing (P value = 1); Polyphen2 probably damaging with a score of 1 (sensitivity: 0.00; specificity: 1.00) and conserved residue changed to an AA polar neutral Align GVGD class C15; SIFT deleterious score 0; mutation taster disease causing P value = 1; Polyphen2 probably damaging with a score of 1 (sensitivity: 0; specificity:1) pathogenic prediction for missense changes --Align GVGD class C35; SIFT deleterious (score 0); Mutation Taster disease causing (P value = 1); Polyphen2 probably damaging with a score of 1 (sensitivity: 0.00; specificity: 1.00) and conserved residue --Remarks and pathogenic prediction for missense changes -Align GVGD class C65; SIFT deleterious (score 0); Mutation Taster disease causing (P value = 1); Polyphen2 probably damaging with a score of 1 (sensitivity: 0.00; specificity: 1.00) and conserved residue Align GVGD class C25; SIFT deleterious (score 0); Mutation Taster disease causing (P value = 1); Polyphen2 probably damaging with a score of 1 (sensitivity: 0.00; specificity: 1.00) and conserved residue -Remarks and pathogenic prediction for missense changes Align GVGD class C35; SIFT deleterious (score 0); Mutation Taster disease causing (P value = 1); Polyphen2 probably damaging with a score of 1 (sensitivity: 0.00; specificity: 1.00) and conserved residue Align GVGD class C55; SIFT deleterious (score 0); Mutation Taster disease causing (P value = 1); Polyphen2 probably damaging with a score of 1 (sensitivity: 0.00; specificity: 1.00) and conserved residue -Remarks and pathogenic prediction for missense changes Align GVGD class C65; SIFT deleterious (score 0); Mutation Taster disease causing (P value = 1); Polyphen2 probably damaging with a score of 1 (sensitivity: 0.00; specificity: 1.00) and conserved residue -Align GVGD class C15; SIFT deleterious (score 0.01); Mutation Taster disease causing (P value = 1); Polyphen2 probably damaging with a score of 1 (sensitivity: 0.00; specificity: 1.00) and conserved residue Align GVGD class C0; SIFT deleterious (score 0.02); Mutation Taster disease causing (P value = 1); Polyphen2 probably damaging with a score of 1 (sensitivity: 0.00; specificity: 1.00) and conserved residue Remarks and pathogenic prediction for missense changes Align GVGD class 0; SIFT deleterious (score 0.02); Mutation Taster polymorphism (P value = 1); Polyphen2 probably damaging with a score of 0958 (sensitivity: 0.78; specificity: 0.95) and poorly conserved residue) Align GVGD class C0; SIFT tolerated (score: 0.19); Mutation Taster polymorphism (P value = 1) Polyphen2 benign with a score of 0 (sensitivity: 1.00; specificity: 0.00) with same change in nonhuman primate Align GVGD class C0; SIFT tolerated (score: 0.7); Mutation Taster polymorphism (P value = 1) Polyphen2 benign with a score of 0.001 (sensitivity: 0.99; specificity: 0.15) and low conservation with same change in dog	splicing
		RS # MAF in RS # MAF in	-RS # MAF in ExAC/gnomAD if available ExAC/gnomAD if RS # MAF in RS # MAF in ExAC/gnomAD if available RS # MAF in ExAC/gnomAD if available available rs772421550 -ExAC 0.00007 -gnomAD -RS # MAF in ExAC/gnomAD if available rs199779970 ExAC 0.0004284 gnomAD 0.0003861, RS # MAF in ExAC/gnomAD if available rs34943572, ExAC 0.0002553 gnomAD 0.0003210 0.00004329, only -het alleles (12 only het alleles found once ho in 'other' populations rs755593299 including 10 in available -rs776644374 ExAC or dbSNP no MAF ExAC no MAF European gnomAD gnomAD non-Finish) 0.000004064, only 0.00001872, only 4 rs527236083 one het allele in het allele ExAC no MAF European gnomAD non-Finish 0.000004061 with rs140065278 only 1 het allele ExAC 0.0000082 -RS # MAF in ExAC/gnomAD if available -rs786205534 ExAC no MAF gnomAD rs527236134 ExAC no MAF rs552509122 -ExAC no MAF gnomAD No MAF in ExAC or gnomAD no MAF 0.000008124, only gnomAD but one 2 het alleles in East other variant for the Asians same codon -0.00007577, only het alleles -rs781442827 rs35898499 ExAC 0.00003295 c.60A > T, gnomAD 0.00004692, p.(Arg20Ser) -ExAC 0.05467 -only het alleles gnomAD no MAF rs730880273 ExAC no MAF gnomAD 0.00006456, only het alleles RS # MAF in ExAC/gnomAD if available -No MAF in ExAC or gnomAD but one other variant c.1787-2A > T reported only in gnomAD with a rs786205533 gnomAD 0.03604 with rs761251269 MAF of ExAC no MAF 218 ho cases ExAC 0.00000082 0.000004061, only gnomAD no MAF gnomAD one het alleles -0.000004063, only RS # MAF in ExAC/gnomAD if ExAC/gnomAD if available rs775460185 ExAC 0.00000082 gnomAD 0.000004061, only one het allele rs543947091 ExAC no MAF gnomAD 0.000004061, only one het allele rs119489105 ExAC 0.00004945 gnomAD one het allele --0.00005686, only RS # MAF in ExAC/gnomAD if available ----rs749472520 ---rs746108786 ExAC 0.00000082 --gnomAD -rs527236084 0.000004062, only het allele -ExAC 0.00008237, gnomAD 0.000004061, only one het allele in ExAC no MAF rs781470358 gnomAD ExAC 0.0000033 0.00001219, only gnomAD 0.00003612, -only het alleles (10 rs777350533 het alleles one het allele -European non-Finish ExAC/gnomAD if available rs541717028 ExAC 0.0000247 gnomAD 0.00002165, only het alleles -ExAC no MAF gnomAD 0.000004061, only het alleles No MAF in ExAC or gnomAD but one other change in the same codon (c.2179C > G, p.Arg727Gly, rs746238212, not found in ExAC, gnomAD 0.000004061, only one het allele rs371956016ExAc 0.000008237gno-mAD 0.00002165, only het alleles including 7 in ExAC or dbSNP no rs774577413 European non-Finish) MAF ExAC 0.00003296 gnomAD gnomAD 0.000008122, two 0.00002889, only het allele in -European het alleles -RS # MAF in ExAC/gnomAD if available -ExAC no MAF gnomAD 0.000004066, only one het allele -rs786205535 ExAC no MAF gnomAD no MAF RS # MAF in ExAC/gnomAD if available ExAC no MAFgnomAD 0.00002445, only het alleles rs387907314 ExAC 0.000008246 gnomAD no MAF ExAC no MAF gnomAD 0.000004061, only one het allele RS # MAF in ExAC/gnomAD if available rs141361084 ExAC 0.0000247 gnomAD 0.00006903, only het allele -rs746291728 ExAC 0.00001650 gnomAD 0.000008123, only het alleles -RS # MAF in ExAC/gnomAD if available rs2230516 ExAC 0.001837 gnomAD 0.001984 with one ho case rs149131360 ExAC 0.0000095 gnomAD 0.00009022 with only het alleles rs201460398 ExAC 0.0000033 gnomAD 0.00003251, only het alleles	non-Finish
				Country of origin Country of origin	Founder mutation in Country of origin the Faroe Island Country of origin Country of origin which would Country of origin Germany British cohort represent 30% of France Not specified (US and RP in this island! Country of origin Germany Country of origin Madagascar Spanish Country of origin Canadian cohorts) Country of origin Not specified (British cohort) Country of origin Indonesia US cohort Country of origin Middle East Country of origin Not specified (US cohort) Thailand North Africa Country of origin Germany Recurrent mutation in Saudi Arabia Japan Germany (Patel et al., 2015) Country of origin Saudi Arabia Country of origin US cohort	China	Japanese Unspecified	(Germany for Gal	et al.?)	China Saudi Arabia Saudi Arabia Not specified (US cohort) Indonesia	Pakistan Chinese China Japanese Pakistan Spain Unspecified, (Belgium cohort) Lebanon Not specified (US cohort) Japan	Not specified (British	Not specified Not specified (US cohort) US cohort	China (Germany?) cohort)	Turkey	Not specified (US and India Middle East France	Saudi Arabia Canadian cohorts) China	Newfoundland, (Germany for Gal cohort) Unspecified The Netherlands Unspecified, (Belgium China et al.?) Canada France	France France Not specified (British China Albania Russia Morocco	Japan Italy Spain China France cohort) The Netherlands	Not specified (US Morocco cohort)	Turkey	Caucasian France Saudi Arabia Not specified (US	Not specified (US cohort)	cohort)	Unspecified (British	Not specified (US cohort)	cohort)
		Phenotype Phenotype	RP Phenotype if speci-fied if speci-Phenotype Phenotype if speci-fied Phenotype if speci-fied fied RP/RCD IRD not CRD RP speci-fied Phenotype if speci-fied RP Phenotype if speci-fied RCD fied RP Phenotype if speci-RP RCD fied Phenotype if speci-fied RP/RCD Phenotype if speci-if speci-fied RP/RCD Phenotype if speci-fied RP/RCD if speci-fied CRD Phenotype if speci-fied RCD with early RP RP/RCD onset Phenotype if speci-fied RP/RCD Phenotype if speci-fied LCA Phenotype if speci-fied CRD	RP macu-lar	RP/RCD RP involve-	ment		RP RP/RCD RP/RCD RP/RCD RP/RCD	RP RP LCA RP/RCD RP RP/RCD LCA RCD RP/RCD RP/RCD	RP/RCD	RP/RCD RP CRD	RP	RP/RCD	RP/RCD RD RP RP Early onset RP RCD	RP/RCD RP LCA	RP/RCD LCA	RCD	RCD CRD RP/RCD RP RCD RCD RP/RCD	RP RP RP/RCD RP CRD RP/RCD	RP CRD		LCA	Early RCD RP/RCD CRD	onset RP/RCD RD	RP	RP/RCD
	, Table 1, Supp Figure S1, and Supp. Table S1). Altogether, the mutation spectrum includes 33 missense mutations among which 4 may affect splicing and 13 are of questionable pathogenicity (based on allele frequency >0.005, func-tional assays or only found at the single heterozygous state, presented in italic in Table 1), 12 nonsense mutations, 12 splice site defects, 12 small deletions, 2 small insertion-deletions, 3 small duplication, 2 large exonic deletions, and 3 gross deletions. The screening of our large IRD cohort identified a total of 20 distinct mutations among which 11 are novel. The mutation spectrum in our cohort includes 3 small deletions, 2 small duplications, 1 large exonic deletion, 2 splice defects, 9 missense and 3 nonsense changes. All the mutations were 1 c.60del p.(Ala21Leufs*43) Premature stop codon leading to a shorter protein product with only the N-terminus fragment or NMD Putative compound het with c.1796G > A; p.(Gly599Glu) Familial segregation was not available IVS1 c.61+1G > A Splicing defect Exon skipping leading to a shorter protein product with only the N-terminus fragment or NMD Compound het with c.1951C > T; p.(Arg651*) IVS1 c.62-1G > A Splicing defect Exon skipping leading to a shorter protein product with only the N-terminus fragment or NMD Compound het with c.1296+1G > A of Ig-like C2 type I or NMD 2 c.325A > T p.(Lys109*) Premature stop codon leading to a shorter protein product with only the N-terminus fragment and part of Ig-like C2 type I or NMD Ho 2 c.343T > G p.(Cys115Gly) or NMD 2 c.390G > A p.(Trp130*) Premature stop codon leading to a shorter protein product with only the N-terminus fragment and part of Ig-like C2 type I or NMD 3-19 119 kb deletion -Most likely absence of protein product 3 c.500G > A p.(Arg167His) Ig-like C2-type 1 domain 4 c.665G > A p.(Gly222Asp) Ig-like C2-type 2 domain domain or NMD fragment and part of Ig-like C2 type I of Ig-like C2 type 2 the N-terminus fragment and part product with part product with only the N-terminus shorter protein shorter protein product with only codon leading to a codon leading to a shorter protein domain 1 c.59G > C p.(Arg20Thr) Signal peptide Ho 2 c.308_309del p.(Ile103Asnfs*4) Premature stop Ho codon leading to a 4 fragment or NMD 2 c.370C > T p.(Gln124*) Premature stop c.640T > G p.(Phe214Val) Ig-like C2-type 2 4 c.718G > T p.(Glu240*) Premature stop 4 c.721C > T p.(Gln241*) Premature stop codon leading to a shorter protein product with part of Ig-like C2 type 2 domain or NMD 5 c.785G > T p.(Cys262Phe) Ig-like C2-type 2 domain domain the N-terminus 5 product with only c.814G > A p.Val272Met Ig-like C2-type 2 6 25 kb deletion including ex6-8 -Most likely an absence of protein product 6 c.933_935 delinsTT p.(Pro313Argfs*15) Premature stop codon leading to a shorter protein product with part of the first FN-III domain or NMD 7 c.985_988dup p.(Gly330Glufs*8) Premature stop codon leading to a shorter protein product with part of the first FN-III domain or NMD 7 c.992_993del p.(Ser331Cysfs*5) Premature stop codon leading to a shorter protein product with part of the first FN-III domain or NMD 8 ∼9 kb deletion Shorter protein product or NMD 8 c.1179dup p.(Leu394Serfs*3) Premature stop codon leading to a shorter protein product with part of the second FN-III domain or NMD 8 c.1186G > T p.(Glu396*) Premature stop codon leading to a shorter protein product with part of the second FN-III domain or NMD IVS8 c.1296+1G > A Splicing defect Exon skipping leading to a shorter protein product with part of the second FN-III domain or NMD 9 c.1335_1336del p.(Ala446Serfs*28) Premature stop codon leading to a shorter protein product with part of the second FN-III domain or NMD 9 c.1441C > T p.(Pro481Ser) At the end of the second FN-III domain 9 c.1450G > A p.(Gly484Ser) Most likely affect splicing with exon skipping leading to a shorter protein product with the absence of the transmembrane domain and the kinase domain or NMD IVS10 c.1604+2T > G Splicing defect Exon skipping leading to a shorter protein product missing the kinase domain or NMD IVS10 c.1605-2A > G Splicing defect Exon skipping leading to a shorter protein product missing the kinase domain or NMD IVS11 c.1690+1G > A Splicing defect Exon skipping leading to a shorter protein product missing the kinase domain or NMD IVS11 c.1691-1G > A Splicing defect Exon skipping leading to a shorter protein product missing the kinase domain or NMD 12 c.1744_ 1751delinsT p.(Ile582*) Shorter protein product missing the kinase domain or NMD IVS12 c.1787-2A > C Splicing defect Exon skipping leading to a shorter protein product missing the kinase domain or NMD 13 c.1796G > A p.(Gly599Glu) Kinase domain 14 c.1893C > G p.(Ile631Met) Kinase domain 14 c.1951C > T p.(Arg651*) Premature stop codon leading to a shorter protein product with a truncated kinase domain or NMD 15 c.1961G > T p.Gly654Val Exon 15 skipping as demonstrated from patient's mRNA which may lead to an abnormal kinase domain IVS15 c.2079+2T > G 16 c.2105dup 16 c.2163T > A 16 c.2179C > T 16 c.2180G > A IVS16 c.2189+1G > T 17 c.2194C > T 17 NMD shorter protein p.Gln124* NMD kinase domain or codon leading to a c.370C > T domain or NMD 2 c.225del p.(Gly76Glufs*3) Premature stop Compound het with of the first FN-III shorter protein fragment or NMD product with part codon leading to a the N-terminus 1 c.35T > C p.(Leu12Pro) Signal peptide Ho product with only codon leading to a shorter protein 10 c.1530del p.(Cys510Trpfs*5) Premature stop product missing the 12 1 c.3G > A p.(Met1?) Start codon loss Ho codon leading to a shorter protein delinsAA 2 c.92_98del p.(Pro31Argfs*31) Premature stop Ho domain 4 product intron 006343.2) Protein change consequence Status 2 c.345C > G p.(Cys115Trp) Ig-like C2 type I c.634A > C p.(Thr212Pro) Ig-like C2-type 2 domain intron absence of protein Exon/ cDNA (NM_ functional intron TA B L E 1 1-7 91 kb deletion -Most likely an Ho Protein domain involved/putative Exon/ cDNA (NM_ 006343.2) Protein change Protein domain involved/putative functional consequence Exon/ intron cDNA (NM_ 006343.2) Protein change Protein domain involved/putative functional consequence TA B L E 1 (Continued) Exon/ cDNA (NM_ 006343.2) Protein change Protein domain involved/putative functional consequence 4 c.665_666 p.(Gly222Glu) Ig-like C2-type 2 domain TA B L E 1 (Continued) Exon/ intron cDNA (NM_ 006343.2) Protein change Protein domain involved/putative functional consequence 5 c.791C > G p.(Ala264Gly) Ig-like C2-type 2 domain TA B L E 1 (Continued) Exon/ intron cDNA (NM_ 006343.2) Protein change Protein domain involved/putative functional consequence 7 c.986A > G p.(Asn329Ser) First FN-III domain TA B L E 1 (Continued) Exon/ intron cDNA (NM_ 006343.2) Protein change Protein domain involved/putative functional consequence IVS8 c.1297-2A > G Splicing defect Exon skipping leading to a shorter protein product with part of the second FN-III domain or NMD 9 c.1301_1302del p.(Glu434Alafs*40) Premature stop TA B L E 1 (Continued) Exon/ intron cDNA (NM_ 006343.2) Protein change Protein domain involved/putative functional consequence TA B L E 1 (Continued) Exon/ intron cDNA (NM_ 006343.2) Protein change Protein domain involved/putative functional consequence c.1786G > A p.(Gly596Arg) Most likely affect splicing with exon skipping leading to a shorter protein product missing the kinase domain or TA B L E 1 (Continued) Exon/ intron cDNA (NM_ 006343.2) Protein change Protein domain involved/putative functional consequence 13 c.1866G > C p.(Lys622Asn) Kinase domain May affect splicing TA B L E 1 (Continued) Exon/ intron cDNA (NM_ 006343.2) Protein change Protein domain involved/putative functional consequence 15 1732-bp deletion p.Gly654Alafs*41 or NMD Premature stop codon leading to a shorter protein product with a truncated kinase domain or NMD 15 TA B L E 1 (Continued) Exon/ intron cDNA (NM_ 006343.2) 16 c.2164C > T 16 c.2171del TA B L E 1 (Continued) Exon/ intron cDNA (NM_ 006343.2) 17 c.2214del c.2219C > T 17 c.2237A > G 17 c.2262C > G TA B L E 1 (Continued) Exon/ intron cDNA (NM_ 006343.2) 17 c.2287C > A 17 c.2302G > A 17 c.2323C > T 18 c.2377del TA B L E 1 (Continued) Exon/ intron cDNA (NM_ 006343.2) 18 c.2435A > C IVS18 c.2487-2A > G 19 c.2530C > T 19 c.2531G > C Exon/ intron 19 19 19 TA B L E 1 (Continued) cDNA (NM_ 006343.2) c.2593C > T c.2726G > A c.2873C > T c.2070_2074del p.(Gly691Lysfs*42) Premature stop codon leading to a shorter protein product with truncated kinase domain or NMD Splice defect Most likely exon skipping leading to a shorter protein product with a truncated kinase domain or NMD p.(Phe703Valfs*32) Premature stop codon leading to a shorter protein product with truncated kinase domain or NMD p.(His721Gln) Kinase domain Protein change Protein domain involved/putative functional consequence p.(Arg722*) Premature stop codon leading to a shorter protein product with truncated kinase domain or NMD p.(Leu724*) Premature stop codon leading to a shorter protein product with truncated kinase domain or NMD p.(Arg727*) Premature stop codon leading to a shorter protein product with a truncated kinase domain or NMD p.(Arg727Gln) Kinase domain Splicing defect with p.(His694Valfs*4) Exon 16 skipping leading to a shorter protein product with a truncated kinase domain or NMD p.(Arg732*) Premature stop codon leading to a shorter protein product with a truncated kinase domain or NMD Protein change Protein domain involved/putative functional consequence p.(Ala740Val) p.(Lys746Arg) p.(Tyr754*) Protein change p.(Pro763Thr) p.(Ala768Thr) p.(Arg775*) p.(Ile793*) Protein change p.(Tyr812Ser) Splicing defect p.Arg844Cys p.(Arg844Pro) Protein change p.Arg865Trp p.(Arg909His) p.(Pro958Leu) p.(Cys738Trpfs*32) Premature stop codon leading to a shorter protein product with a truncated kinase domain or NMD Kinase domain Kinase domain Premature stop codon leading to a shorter protein product with a truncated kinase domain or NMD Protein domain involved/putative functional consequence Kinase domain Kinase domain Premature stop codon leading to a shorter protein product with a truncated kinase domain or NMD Premature stop codon leading to a shorter protein product with a truncated kinase domain or NMD Protein domain involved/putative functional consequence Kinase domain Premature stop codon leading to a shorter protein product with a truncated kinase domain or NMD Kinase domain Kinase domain Protein domain involved/putative functional consequence Kinase domain C-terminus C-terminus Ig-like C2 type I domain Only het change Status Compound het with c.1530del; p.(Ala446Serfs*28) Compound het with c.225del p.(Gly76Glufs*3) Ho Ho Single het Status Compound het with c.345C > G, p.(Cys115Trp) Single het Compound het with c.665_666delinsAA; p.(Gly222Glu) Phase confirmed by familial segregation Status Compound het with c.665G > A; p.(Gly222Asp) Phase confirmed by familial segregation ho Ho Ho Status Single het Compound het but not specified with which variant Ho Compound het with c.2180G > A p.(Arg727Gln) Compound het with c.1450G > A p.(Gly484Ser) Phase confirmed by familial segregation Status Ho Ho Ho Ho Ho Compound het with no specified variant in Xu et al., 2014 and with c.62-1G > A in Wang et al., 2014a Status Ho Ho Ho Single het Ho Status Compound het with c.345C > G (p.Cys115Trp) Ho on presumed uniparental paternal disomy Ho Ho Ho Ho Status Ho Ho Putative compound het with c.60del; p.(Ala21Leufs*43) Familial segregation was not available Status Ho Single het Single het in Gal et al. but compound het with c.61+1G > A in Mackay et al., 2010 Ho Status Ho Ho Putative ompound het with c.1951C > T p.(Arg651*) Familial segregation was not available Putative compound het with c.345C > G; p.(Cys115Trp) Familial segregation was not available Ho Status In cis with c.2593C > T p.Arg865Trp; Compound het with p.Arg844Cys And also reported with c.2219C > T p.(Ala740Val) Compound het with c.1296+1G > A Compound het with c.2530C > T p.Arg844Cys Ho Ho Ho Status Ho Compound het with c.2164C > T p.(Arg722*) Single het Ho Status Compound het with c.390G > A; p.(Trp130*) Ho Ho Ho Status Single het Ho Compound Hz with complex allele p.(Arg722*) p.Arg865Trp And also reported with c.2179C > T p.(Arg727*) Ho on possible uniparental disomy Status Complex allele in cis with p.Arg722* and trans with p.Arg844Cys Single het Single het

TA B L E 1 (Continued) TA B L E 1 (Continued) TA B L E 1 (Continued)

ACKNOWLEDGMENTS

The authors thank the patients and their family participating in the study and the clinical staff from the CIC1423. DNA samples incorporated in this study were obtained from the NeuroSensCol DNA bank, for research in neuroscience (PI: JA Sahel, co-PI I Audo, partner with CHNO des Quinze-Vingts, Inserm and CNRS).

Funding information

Contract grant sponsors: Fondation Voir et Entendre; LABEX LIFESENSES (ANR-10-LABX-65); Agence Nationale de la Recherche within the Investissements d'Avenir program (ANR-11-IDEX-0004-0); Foundation Fighting Blindness center grant (C-C-CL-0912-0600-INSERM01, GE-0912-0601-INSERM02); Prix de la Fondation de l'OEil; Ministère de l'Enseignement Supérieur et de la Recherche (MESR).

Communicated by Jürgen Horst

DISCLOSURE STATEMENT

The authors declare no conflict of interest.

SUPPORTING INFORMATION

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