Development and application of a next-generation-sequencing (NGS) approach to detect known and novel gene defects underlying retinal diseases

  • Isabelle Audo1, 2, 3, 4, 5Email author,

    Affiliated with

    • Kinga M Bujakowska1, 2, 3,

      Affiliated with

      • Thierry Léveillard1, 2, 3,

        Affiliated with

        • Saddek Mohand-Saïd1, 2, 3, 4,

          Affiliated with

          • Marie-Elise Lancelot1, 2, 3,

            Affiliated with

            • Aurore Germain1, 2, 3,

              Affiliated with

              • Aline Antonio1, 2, 3, 4,

                Affiliated with

                • Christelle Michiels1, 2, 3,

                  Affiliated with

                  • Jean-Paul Saraiva6,

                    Affiliated with

                    • Mélanie Letexier6,

                      Affiliated with

                      • José-Alain Sahel1, 2, 3, 4, 7, 8,

                        Affiliated with

                        • Shomi S Bhattacharya1, 2, 3, 5, 9 and

                          Affiliated with

                          • Christina Zeitz1, 2, 3Email author

                            Affiliated with

                            Orphanet Journal of Rare Diseases20127:8

                            DOI: 10.1186/1750-1172-7-8

                            Received: 12 September 2011

                            Accepted: 25 January 2012

                            Published: 25 January 2012

                            Abstract

                            Background

                            Inherited retinal disorders are clinically and genetically heterogeneous with more than 150 gene defects accounting for the diversity of disease phenotypes. So far, mutation detection was mainly performed by APEX technology and direct Sanger sequencing of known genes. However, these methods are time consuming, expensive and unable to provide a result if the patient carries a new gene mutation. In addition, multiplicity of phenotypes associated with the same gene defect may be overlooked.

                            Methods

                            To overcome these challenges, we designed an exon sequencing array to target 254 known and candidate genes using Agilent capture. Subsequently, 20 DNA samples from 17 different families, including four patients with known mutations were sequenced using Illumina Genome Analyzer IIx next-generation-sequencing (NGS) platform. Different filtering approaches were applied to identify the genetic defect. The most likely disease causing variants were analyzed by Sanger sequencing. Co-segregation and sequencing analysis of control samples validated the pathogenicity of the observed variants.

                            Results

                            The phenotype of the patients included retinitis pigmentosa, congenital stationary night blindness, Best disease, early-onset cone dystrophy and Stargardt disease. In three of four control samples with known genotypes NGS detected the expected mutations. Three known and five novel mutations were identified in NR2E3, PRPF3, EYS, PRPF8, CRB1, TRPM1 and CACNA1F. One of the control samples with a known genotype belongs to a family with two clinical phenotypes (Best and CSNB), where a novel mutation was identified for CSNB. In six families the disease associated mutations were not found, indicating that novel gene defects remain to be identified.

                            Conclusions

                            In summary, this unbiased and time-efficient NGS approach allowed mutation detection in 75% of control cases and in 57% of test cases. Furthermore, it has the possibility of associating known gene defects with novel phenotypes and mode of inheritance.

                            Keywords

                            NGS retinal disorders diagnostic tool.

                            Background

                            Inherited retinal disorders affect approximately 1 in 2000 individuals worldwide [1]. Symptoms and associated phenotypes are variable. In some groups the disease can be mild and stationary such as in congenital stationary night blindness (CSNB) or achromatopsia (ACHM), whereas other disorders are progressive leading to severe visual impairment such as in rod-cone dystrophies, also known as retinitis pigmentosa (RP) or cone and cone-rod dystrophies. The heterogeneity of these diseases is reflected in the number of underlying gene defects. To date more than 150 genes have been implicated in different forms of retinal disorders http://​www.​sph.​uth.​tmc.​edu/​Retnet/​home.​htm and yet in a significant proportion of patients the disease causing mutation could not be identified, suggesting additional novel genes that remain to be discovered. Furthermore, recent studies have outlined that distinct phenotypes can be related to the dysfunction of the same gene [24]. Furthermore, there may be additional phenotype-genotype associations that are still not recognized. The state-of-the-art phenotypic characterization including precise family history and functional as well as structural assessment (i.e. routine ophthalmic examination, perimetry, color vision, full field and multifocal electroretinography (ERG), fundus autofluorescence (FAF) imaging and optical coherence tomography (OCT)) allows targeted mutation analysis for some disorders. However, in most cases of inherited retinal diseases, similar phenotypic features can be due to a large number of different gene defects.

                            Various methods can be used for the identification of the corresponding genetic defect. All these methods have advantages and disadvantages. Sanger sequencing is still the gold-standard in determining the gene defect, but due to the heterogeneity of the disorders it is time consuming and expensive to screen all known genes. Mutation detection by commercially available APEX genotyping microarrays (ASPER Ophthalmics, Estonia) [5, 6] allows the detection of only known mutations. In addition, a separate microarray has been designed for each inheritance pattern, which tends to escalate the costs especially in simplex cases, for which inheritance pattern cannot be predetermined. Indirect methods with single nucleotide polymorphism (SNP) microarrays for linkage and homozygosity mapping are also powerful tools, which has proven its reliability in identifying novel and known gene defects [712]. However, in case of homozygosity mapping the method can only be applied to consanguineous families or inbred populations. To overcome these challenges, we designed a custom sequencing array in collaboration with a company (IntegraGen, Evry, France) to target all exons and part of flanking sequences for 254 known and candidate retinal genes. This array was subsequently applied through NGS to a cohort of 20 patients from 17 families with different inheritance pattern and clinical diagnosis including RP, CSNB, Best disease, early-onset cone dystrophy and Stargardt disease.

                            Methods

                            Clinical investigation

                            The study protocol adhered to the tenets of the Declaration of Helsinki and was approved by the local Ethics Committee (CPP, Ile de France V). Informed written consent was obtained from each study participant. Index patients underwent full ophthalmic examination as described before [13]. Whenever available, blood samples from affected and unaffected family members were collected for co-segregation analysis.

                            Previous molecular genetic analysis

                            Total genomic DNA was extracted from peripheral blood leucocytes according to manufacturer's recommendations (Qiagen, Courtaboeuf, France). DNA samples from some patients with a diagnosis of RP were first analyzed and excluded for known mutations by applying commercially available microarray analysis (arRP and adRP ASPER Ophthalmics, Tartu, Estonia). In some cases, pathogenic variants in EYS, C2orf71, RHO, PRPF31, PRPH2 and RP1 were excluded by direct Sanger sequencing of the coding exonic and flanking intronic regions of the respective genes [1317]. Conditions used to amplify PRPH2 can be provided on request.

                            Molecular genetic analysis using NGS

                            A custom-made SureSelect oligonucleotide probe library was designed to capture the exons of 254 genes for different retinal disorders and candidate genes according to Agilent's recommendations (Table 1). These genes include 177 known genes underlying retinal dysfunction (http://​www.​sph.​uth.​tmc.​edu/​retnet/​sum-dis.​htm, October 2010, Table 1) and 77 candidate genes associated with existing animal models and expression data (Table 2). The eArray web-based probe design tool was used for this purpose https://​earray.​chem.​agilent.​com/​earray. The following parameters were chosen for probe design: 120 bp length, 3× probe-tiling frequency, 20 bp overlap in restricted regions, which were identified by the implementation of eArray's RepeatMasker program. A total of 27,430 probes, covering 1177 Mb, were designed and synthesized by Agilent Technologies (Santa Clara, CA, USA). Sequence capture, enrichment, and elution were performed according to the manufacturer's instructions (SureSelect, Agilent). Briefly, three μg of each genomic DNA were fragmented by sonication and purified to yield fragments of 150-200 bps. Paired-end adaptor oligonucleotides from Illumina were ligated on repaired DNA fragments, which were then purified and enriched by six PCR cycles. 500 ng of the purified libraries were hybridized to the SureSelect oligo probe capture library for 24 h. After hybridization, washing, and elution, the eluted fraction underwent 14 cycles of PCR-amplification. This was followed by purification and quantification by qPCR to obtain sufficient DNA template for downstream applications. Each eluted-enriched DNA sample was then sequenced on an Illumina GAIIx as paired-end 75 bp reads. Image analysis and base calling was performed using Illumina Real Time Analysis (RTA) Pipeline version 1.10 with default parameters. Sequence reads were aligned to the reference human genome (UCSC hg19) using commercially available software (CASAVA1.7, Illumina) and the ELANDv2 alignment algorithm. Sequence variation annotation was performed using the IntegraGen in-house pipeline, which consisted of gene annotation (RefSeq), detection of known polymorphisms (dbSNP 131, 1000 Genome) followed by mutation characterization (exonic, intronic, silent, nonsense etc.). For each position, the exomic frequencies (homozygous and heterozygous) were determined from all the exomes already sequenced by IntegraGen and the exome results provided by HapMap project.
                            Table 1

                            Known retinal disease genes

                            Number

                            Gene name

                            1

                            ABCA4

                            2

                            ABCC6

                            3

                            ADAM9

                            4

                            AHI1

                            5

                            AIPL1

                            6

                            ALMS1

                            7

                            ARL6

                            8

                            ARMS2

                            9

                            ATXN7

                            10

                            BBS10

                            11

                            BBS12

                            12

                            BBS2

                            13

                            BBS4

                            14

                            BBS5

                            15

                            BBS7

                            16

                            BBS9

                            17

                            BEST1

                            18

                            C1QTNF5

                            19

                            C2

                            20

                            C2orf71

                            21

                            C3

                            22

                            CA4

                            23

                            CABP4

                            24

                            CACNA1F

                            25

                            CACNA2D4

                            26

                            CC2D2A

                            27

                            CDH23

                            28

                            CDH3

                            29

                            CEP290

                            30

                            CERKL

                            31

                            CFB

                            32

                            CFH

                            33

                            CHM

                            34

                            CLN3

                            35

                            CLRN1

                            36

                            CNGA1

                            37

                            CNGA3

                            38

                            CNGB1

                            39

                            CNGB3

                            40

                            CNNM4

                            41

                            COL11A1

                            42

                            COL2A1

                            43

                            COL9A1

                            44

                            CRB1

                            45

                            CRX

                            46

                            CYP4V2

                            47

                            DFNB31

                            48

                            DMD

                            49

                            DPP3

                            50

                            EFEMP1

                            51

                            ELOVL4

                            52

                            ERCC6

                            53

                            EYS

                            54

                            FAM161A

                            55

                            FBLN5

                            56

                            FSCN2

                            57

                            FZD4

                            58

                            GNAT1

                            59

                            GNAT2

                            60

                            GPR98

                            61

                            GRK1

                            62

                            GRM6

                            63

                            GUCA1A

                            64

                            GUCA1B

                            65

                            GUCY2D

                            66

                            HMCN1

                            67

                            HTRA1

                            68

                            IDH3B

                            69

                            IMPDH1

                            70

                            IMPG2

                            71

                            INPP5E

                            72

                            INVS

                            73

                            IQCB1

                            74

                            JAG1

                            75

                            KCNJ13

                            76

                            KCNV2

                            77

                            KLHL7

                            78

                            LCA5

                            79

                            LRAT

                            80

                            LRP5

                            81

                            MERTK

                            82

                            MFRP

                            83

                            MKKS

                            84

                            MKS1

                            85

                            MTND1

                            86

                            MTND6

                            87

                            MT-AP6

                            88

                            MTND2

                            89

                            MTND5

                            90

                            MTND4

                            91

                            MYO7A

                            92

                            NDP

                            93

                            NPHP1

                            94

                            NPHP3

                            95

                            NPHP4

                            96

                            NR2E3

                            97

                            NRL

                            98

                            NYX

                            99

                            OAT

                            100

                            OFD1

                            101

                            OPA1

                            102

                            OPA3

                            103

                            OPN1LW

                            104

                            OPN1MW

                            105

                            OPN1Sw

                            106

                            OTX2

                            107

                            PANK2

                            108

                            PAX2

                            109

                            PCDH15

                            110

                            PCDH21

                            111

                            PDE6A

                            112

                            PDE6B

                            113

                            PDE6C

                            114

                            PDE6G

                            115

                            PDZD7

                            116

                            PEX1

                            117

                            PEX2

                            118

                            PEX7

                            119

                            PGK1

                            120

                            PHYH

                            121

                            PITPNM3

                            122

                            PRCD

                            123

                            PROM1

                            124

                            PRPF3

                            125

                            PRPF31

                            126

                            PRPF8

                            127

                            PRPH2

                            128

                            RAX2

                            129

                            RB1

                            130

                            RBP3

                            131

                            RBP4

                            132

                            RD3

                            133

                            RDH12

                            134

                            RDH5

                            135

                            RGR

                            136

                            RGS9

                            137

                            RGS9BP

                            138

                            RHO

                            139

                            RIMS1

                            140

                            RLBP1

                            141

                            ROM1

                            142

                            RP1

                            143

                            RP1L1

                            144

                            RP2

                            145

                            RP9

                            146

                            RPE65

                            147

                            RPGR

                            148

                            RPGRIP1

                            149

                            RPGRIP1L

                            150

                            RS1

                            151

                            SAG

                            152

                            SDCCAG8

                            153

                            SEMA4A

                            154

                            SLC24A1

                            155

                            SNRNP200

                            156

                            SPATA7

                            157

                            TEAD1

                            158

                            TIMM8A

                            159

                            TIMP3

                            160

                            TLR3

                            161

                            TLR4

                            162

                            TMEM126A

                            163

                            TOPORS

                            164

                            TREX1

                            165

                            TRIM32

                            166

                            TRPM1

                            167

                            TSPAN12

                            168

                            TTC8

                            169

                            TTPA

                            170

                            TULP1

                            171

                            UNC119

                            172

                            USH1C

                            173

                            USH1G

                            174

                            USH2A

                            175

                            VCAN

                            176

                            WFS1

                            177

                            ZNF513

                            Table 2

                            Candidate genes for retinal disorders

                            Number

                            Gene name

                            Reason

                            References

                            1

                            ADCY1

                            diff. Expression rd1 mouse

                            Chalmel et al., manuscript in preparatiom

                            2

                            ANKRD33

                            diff. Expression rd1 mouse

                            Chalmel et al., manuscript in preparatiom

                            3

                            ANXA2

                            Promotion of choroidal neovascularization

                            [36]

                            4

                            ARL13B

                            Cilia protein, mutations lead to Joubert Syndrome

                            [37]

                            5

                            BMP7

                            Regulation of Pax 2 in mouse retina

                            [38]

                            6

                            BSG

                            -

                            Thierry Leveillard personal commmunication

                            7

                            CAMK2D

                            diff. Expression rd1 mouse

                            Chalmel et al., manuscript in preparatiom

                            8

                            CCDC28B

                            Modifier for BBS

                            [39, 40]

                            9

                            CLCN7

                            Cln7-/- mice severe osteopetrosis and retinal degeneration

                            [41]

                            10

                            COL4A3

                            Alport syndrome, with eye abnormalities

                            [42, 43]

                            11

                            COL4A4

                            Alport syndrome, with eye abnormalities

                            [42, 44]

                            12

                            COL4A5

                            Alport syndrome, with eye abnormalities

                            [42, 45]

                            13

                            CUBN

                            -

                            Personal communication Renata Kozyraki

                            14

                            CYP1B1

                            glaucoma

                            [46]

                            15

                            DOHH

                            diff. Expression rd1 mouse

                            Chalmel et al., manuscript in preparatiom

                            16

                            DSCAML1

                            diff. Expression rd1 mouse

                            Chalmel et al., manuscript in preparatiom

                            17

                            ESRRB

                            diff. Expression rd1 mouse

                            Chalmel et al., manuscript in preparatiom

                            18

                            FIZ1

                            Interactor of NRL

                            [47]

                            19

                            GJA9

                            diff. Expression rd1 mouse

                            Chalmel et al., manuscript in preparatiom

                            20

                            GNAZ

                            diff. Expression rd1 mouse

                            Chalmel et al., manuscript in preparatiom

                            21

                            GNGT1

                            diff. Expression rd1 mouse

                            Chalmel et al., manuscript in preparatiom

                            22

                            GPR152

                            diff. Expression rd1 mouse

                            Chalmel et al., manuscript in preparatiom

                            23

                            HCN1

                            diff. Expression rd1 mouse

                            Chalmel et al., manuscript in preparatiom

                            24

                            HEATR5A

                            diff. Expression rd1 mouse

                            Chalmel et al., manuscript in preparatiom

                            25

                            HIST1H1C

                            Expressed in retina

                            Expression databases

                            26

                            IMPG1

                            diff. Expression rd1 mouse

                            Chalmel et al., manuscript in preparatiom

                            27

                            INSL5

                            diff. Expression rd1 mouse

                            Chalmel et al., manuscript in preparatiom

                            28

                            KCNB1

                            diff. expression rd1 mouse

                            Chalmel et al., manuscript in preparatiom

                            29

                            KCTD7

                            Expressed in retina

                            Expression databases

                            30

                            LASS4

                            diff. expression rd1 mouse

                            Chalmel et al., manuscript in preparatiom

                            31

                            LRIT2

                            diff. expression rd1 mouse

                            Chalmel et al., manuscript in preparatiom Rd1 mouse

                            32

                            LRP2

                            -

                            Personal communication Renata Kozyraki

                            33

                            MAB21L1

                            diff. expression Rd1 mouse

                            Chalmel et al., manuscript in preparatiom

                            34

                            MAP2

                            diff. expression rd1 mouse

                            Chalmel et al., manuscript in preparatiom

                            35

                            MAS1

                            Degeneration of cones due to expression of Mas1

                            [48]

                            36

                            MAST2

                            diff. expression rd1 mouse

                            Chalmel et al., manuscript in preparatiom

                            37

                            MPP4

                            diff. expression rd1 mouse

                            Chalmel et al., manuscript in preparatiom

                            38

                            MYOC

                            glaucoma

                            [49]

                            39

                            NDUFA12

                            diff. expression rd1 mouse

                            Chalmel et al., manuscript in preparatiom

                            40

                            NEUROD1

                            BETA2/NeuroD1 -/- mouse: photoreceptor degeneration

                            [50]

                            41

                            NOS2

                            glaucoma

                            [51]

                            42

                            NXNL1

                            Rod-derived cone viability factor

                            [52]

                            43

                            NXNL2

                            Rod-derived cone viability factor 2

                            [53]

                            44

                            OPN1MW2

                            Cone opsin, medium-wave-sensitive2

                            [54]

                            45

                            OPTN

                            glaucoma

                            [55]

                            46

                            PFKFB2

                            diff. expression rd1 mouse

                            Chalmel et al., manuscript in preparatiom

                            47

                            PIAS3

                            Rod photoreceptor development

                            [56]

                            48

                            PKD2L1

                            Diff. expression in human retinal detachment

                            Delyfer et al. 2011 submitted

                            49

                            PLEKHA1

                            Age-related macular degeneratiom

                            [57]

                            50

                            PPEF2

                            diff. expression rd1 mouse

                            Chalmel et al., manuscript in preparatiom

                            51

                            RAB8A

                            Interacts with RPGR, role in cilia biogenesis and maintenance

                            [58]

                            52

                            RABGEF1

                            diff. expression rd1 mouse

                            Chalmel et al., manuscript in preparatiom

                            53

                            RCVRN

                            diff. expression rd1 mouse

                            Chalmel et al., manuscript in preparatiom

                            54

                            RGS20

                            diff. expression rd1 mouse

                            Chalmel et al., manuscript in preparatiom

                            55

                            RNF144B

                            diff. expression rd1 mouse

                            Chalmel et al., manuscript in preparatiom

                            56

                            RORB

                            Rod photoreceptor development in mice

                            [59]

                            57

                            RXRG

                            Retinoic acid receptor, highly expressed in the eye

                            Expression databases

                            58

                            SGIP1

                            diff. expression rd1 mouse

                            Chalmel et al., manuscript in preparatiom

                            59

                            SLC16A8

                            Altered visual function in ko-mice

                            [60]

                            60

                            SLC17A7

                            diff. expression rd1 mouse

                            Chalmel et al., manuscript in preparatiom

                            61

                            STAM2

                            diff. expression rd1 mouse

                            Chalmel et al., manuscript in preparatiom

                            62

                            STK35

                            diff. expression rd1 mouse

                            Chalmel et al., manuscript in preparatiom

                            63

                            STX3

                            diff. expression rd1 mouse

                            Chalmel et al., manuscript in preparatiom

                            64

                            SV2B

                            diff. expression rd1 mouse

                            Chalmel et al., manuscript in preparatiom

                            65

                            TBC1D24

                            diff. expression rd1 mouse

                            Chalmel et al., manuscript in preparatiom

                            66

                            THRB

                            Essential for M-cone development in rodents

                            [61]

                            67

                            TMEM216

                            Cilia protein, mutations lead to Joubert and Meckel syndrome

                            [62]

                            68

                            TMEM67

                            Cilia protein, mutations lead to Joubert

                            [63]

                            69

                            TRPC1

                            diff. expression rd1 mouse

                            diff. expression Rd1 mouse

                            70

                            UHMK1

                            diff. expression rd1 mouse

                            diff. expression Rd1 mouse

                            71

                            VSX1

                            Stimulator for promoter NXNL1

                            [64]

                            72

                            VSX2

                            Stimulator for promoter NXNL1

                            [64]

                            73

                            WDR17

                            diff. expression rd1 mouse

                            diff. expression Rd1 mouse

                            74

                            WDR31

                            diff. expression Nxnl1-/- mouse

                            [65]

                            75

                            WISP1

                            diff. expression rd1 mouse

                            Chalmel et al., manuscript in preparatiom

                            76

                            XIAP

                            Protects photoreceptors in animal models of RP

                            [66]

                            77

                            ZDHHC2

                            diff. expression Rd1 mouse

                            Chalmel et al., manuscript in preparatiom

                            Investigation of annotated sequencing data

                            We received the annotated sequencing data in the form of excel tables. On average 946 SNPs and 83 insertions and deletions were identified for each sample (Figure 1). By using the filtering system, we first investigated variants (nonsense and missense mutations, intronic variants located +/- 5 apart from exon), which were absent in dbSNP and NCBI databases http://​ncbi.​nlm.​nih.​gov/​. In the absence of known gene defects or putative pathogenic variants (see below) in the first step, we selected known genes, which were previously clinically associated including variants present in dbSNP and NCBI databases (Figure 1). Each predicted pathogenic variant was confirmed by Sanger sequencing.
                            http://static-content.springer.com/image/art%3A10.1186%2F1750-1172-7-8/MediaObjects/13023_2011_370_Fig1_HTML.jpg
                            Figure 1

                            Flow chart of variant analysis. IntegraGen provided the results in form of excel tables. For each sample on average 946 SNPs and 83 inDels were detected, of which 11 represent missense, nonsense or putative splice site mutations, which were absent in dbSNB, NCBI and 1000 genome databases. Of those 1-5 variants were predicted to be pathogenic. In case where none of the variants were predicted to be pathogenic, dbSNB, NCBI and 1000 genome databases were included to detect mutations referenced with an rs-number. Co-segregation analysis was performed in families with putative pathogenic variants.

                            Assessment of the pathogenicity of variants

                            Following criteria were applied to evaluate the pathogenic nature of novel variations identified by NGS: 1) stop/frameshift variants were considered as most likely to be disease causing; 2) co-segregation in the family; 3) absence in control samples; 4) for missense mutations amino acid conservation was studied in the UCSC Genome Browser http://​genome.​ucsc.​edu/​ across species from all different evolutionary branches. If the amino acid residue did not change it was considered as "highly conserved". If a different change was seen in fewer than five species and not in the primates then it was considered as "moderately conserved" and if a change was present in 5-7, it was considered as "weakly conserved", otherwise the amino acid residue was considered as "not conserved", 5) pathogenicity predictions with bioinformatic tools (Polyphen: Polymorphism Phenotyping, http://​genetics.​bwh.​harvard.​edu/​pph/​ and SIFT: Sorting Intolerant From Tolerant, http://​blocks.​fhcrc.​org/​sift/​SIFT.​html) if at least one of the program predicted the variant to be possibly damaging, it was considered to be pathogenic; 6) presence of the second mutant allele in the case of autosomal recessive inheritance. Mutations were described according to the HGVS website http://​www.​hgvs.​org/​mutnomen. In accordance with this nomenclature, nucleotide numbering reflects cDNA numbering with +1 corresponding to the A of the ATG translation initiation codon in the reference sequence. The initiation codon is codon 1. The correct nomenclature for mutation was checked applying Mutalyzer http://​www.​lovd.​nl/​mutalyzer/​.

                            Results

                            The overall sequencing coverage of the captured regions was 98.4% and 90.4% for a 1× and a 10× coverage respectively. The overall sequencing depth was > 120×. The number of reference and variant sequences detected by NGS, reflected the correct zygosity state of the variant; on average if 50% of the sequences represented the variant, then a heterozygous state was called, while if 100% of the sequences represented the variant, then a homozygous or hemizygous state was annotated by IntegraGen.

                            Validation of the novel genetic testing tool for retinal disorders

                            To validate the novel genetic testing tool for retinal disorders, we used four DNA samples from families, in which we had previously identified different types of mutations by Sanger sequencing: one 1 bp duplication and one 1 bp deletion in PRPF31 and missense mutations in TRPM1 and BEST1 (Table 3). Three of the four mutations were detectable by NGS, whereas the deletion in PRPF31 was not identified. To validate if this was due to a technical problem of deletion detection in general or low coverage at this position, the sequencing depth was investigated in detail. Indeed the coverage at this position reflected by the mean depth was only ~1-6 for all samples. This indicates that although the coverage in general was very good, specific probes used here need to be redesigned to improve the capture for specific exons.
                            Table 3

                            Patients with known mutations used to validate the novel genetic approach for retinal disorders

                            Index

                            Phenotype

                            Gene

                            Mutation

                            Allele State

                            Read reference NGS

                            Read variant NGS

                            Mutation detected by NGS

                            Mean depth

                            CIC00034, F28

                            adRP

                            PRPF31

                            c.666dup

                            p.I223YfsX56

                            het

                            11

                            13

                            yes

                            21.3-22.5

                            CIC00140, F108

                            adRP

                            PRPF31

                            c.997delG

                            p.E333SfsX5

                            het

                            -

                            -

                            no

                            5.0-5.2

                            CIC00238, F165

                            arCSNB

                            TRPM1

                            c.1418G > C

                            p.R473P

                            homo

                            0

                            38

                            yes

                            36.7

                            CIC00707, F470

                            Best and adCSNB see Table 5

                            BEST1

                            c.73C > T

                            p.R25W

                            het

                            40

                            38

                            yes

                            99.4

                            Detection of known and novel mutations

                            Some of the patients from the 14 families with no known gene defect were previously excluded for known mutations using microarray analysis and by Sanger sequencing in the known genes EYS, C2orf71, RHO, PRPF31, PRPH2 and RP1. Other samples were never genetically investigated. In four DNA samples known mutations were detected (Table 4) from three different families with autosomal dominant (ad) or recessive (ar) RP. All mutations co-segregated with the phenotype (Figure 2). In seven samples, novel mutations in known genes were identified. These mutations co-segregated with the phenotype from five different families with adCSNB, x-linked incomplete CSNB, adRP, arRP and x-linked RP (Table 5, Figures 3 and 4). One of the cases from these five families was also used as a control for Best disease carrying a known BEST1 mutation (Table 3). In addition to the Best phenotype, ERG-responses of this patient resembled those of complete CSNB, i.e. showing selective ON-bipolar pathway dysfunction. This phenotype was independent of the Best phenotype (Figure 3). The most likely disease causing mutation detected by NGS was a novel heterozygous TRPM1 mutation (Table 4, Figure 3).
                            Table 4

                            Detection of known mutations by using the novel genetic approach for retinal disorders

                            Index

                            Phenotype

                            Pre-screening

                            Gene

                            Mutation

                            Allele State

                            Read reference NGS

                            Read variant NGS

                            Reference

                            Mutation verified by Sanger and co-segregation

                            CIC00019, F16

                            adRP

                            Linkage, RHO, PRPF31, PRPH2, RP1

                            PRPF3

                            c.1481C > T

                            p.T494M

                            het

                            25

                            22

                            [67]

                            yes

                            CIC0000893, F574

                            adRP

                            RHO, PRPF31, PRPH2, RP1

                            NR2E3

                            c.166G > A

                            p.G56R

                            het

                            5

                            3

                            [68]

                            yes

                            CIC000128, F100

                            arRP, consang.

                            -

                            EYS

                            c.408_423del p.N137VfsX24

                            homo

                            -

                            179

                            [13, 69]

                            yes

                            CIC0000943, F100

                            arRP, consang

                            -

                            EYS

                            c.408_423del p.N137VfsX24

                            homo

                            0

                            193

                            [13, 69]

                            yes

                            http://static-content.springer.com/image/art%3A10.1186%2F1750-1172-7-8/MediaObjects/13023_2011_370_Fig2_HTML.jpg
                            Figure 2

                            Detection of known mutations by NGS in 254 retinal genes. The index patient 19 of family 16 with adRP revealed the p.T494M mutations in PRPF3, which co-segregates with the phenotype. Two family members never clinically investigated from the last generation (984 and 1167 carrying a question mark) were reported to be not affected but carried the mutation. They may develop the phenotype at a later stage. In addition variability of the phenotype of this mutation was documented [35]. Two patients, 128 and 943 of family 100 with arRP from Jewish origin revealed the known EYS mutation p.N137VfsX24, which was found in all screened affected family members. The index patient 893 of family 574 showed the previously described NR2E3 p.G56R mutation, which co-segregated with the phenotype.

                            Table 5

                            Detection of novel mutations by using the novel genetic approach for retinal disorders

                            Index

                            Phenotype

                            Pre-screening

                            Gene

                            Mutation

                            Allele State

                            Read reference NGS

                            Read variant NGS

                            Mutation verified by Sanger and co-segregation

                            Conservation

                            Polyphen

                            Sift

                            CIC00707,

                            F470

                            adCSNB and Best see Table 3

                            RHO, PDE6B, GNAT1

                            TRPM1

                            c.1961A > C

                            p.H654P

                            het

                            39

                            38

                            yes

                            moderately conserved

                            possibly damaging

                            tolerated

                            CIC000348, F232

                            adRP, mild

                            RHO, PRPF31, PRPH2, RP1, adRP chip

                            PRPF8

                            c.6992A > G

                            p.E2331G

                            het

                            13

                            10

                            yes

                            moderately conserved

                            possibly damaging

                            affect protein function

                            CIC000346, F232

                            adRP

                            -

                            PRPF8

                            c.6992A > G

                            p.E2331G

                            het

                            5

                            9

                            yes

                            moderately conserved

                            possibly damaging

                            affect protein function

                            CIC000347, F232

                            as

                            adRP

                            -

                            PRPF8

                            c.6992A > G

                            p.E2331G

                            het

                            15

                            17

                            yes

                            moderately conserved

                            possibly damaging

                            affect protein function

                            CIC04240,

                            F2025

                            arRP, consang., detailed clinic in [70]

                            RS1

                            CRB1

                            c.2219C > T

                            p.S740F

                            homo

                            2

                            194

                            yes

                            highly conserved

                            probably damaging

                            affect protein function

                            CIC00199,

                            F146

                            adRP or x-linked RP with affected carrier

                            RHO, PRPF31, PRPH2, RP1, adRP chip

                            RPGR

                            c.248-2A > G

                            splice defect

                            hetero

                            30

                            22

                            yes

                            conserved

                            splice site

                            n.a.

                            n.a.

                            CIC04094,

                            F1915

                            icCSNB

                            -

                            CACNA1F

                            c.973C > T

                            p.Q325X

                            hemi

                            0

                            28

                            yes

                            n.a.

                            n.a.

                            n.a.

                            http://static-content.springer.com/image/art%3A10.1186%2F1750-1172-7-8/MediaObjects/13023_2011_370_Fig3_HTML.jpg
                            Figure 3

                            Best disease and CSNB co-segregating in one family. a) Sanger and NGS detected in all patients with Best disease a BEST1 mutation. b) NGS detected in all patients with a cCSNB phenotype a novel TRPM1 mutation. c) Fundus colour photographs (above) and fundus autofluorescence (below) of patient 707 showing multiple yellow deposits within the posterior pole which are hyper autofluorescent d) Electro-oculogram of patient 707 showing no slight rise after illumination in keeping with the diagnosis of Best disease e) Full Field Electroretinogram of patient 707 showing ON-bipolar cell pathway dysfunction in keeping with the diagnosis of cCSNB.

                            http://static-content.springer.com/image/art%3A10.1186%2F1750-1172-7-8/MediaObjects/13023_2011_370_Fig4_HTML.jpg
                            Figure 4

                            Detection of novel mutations using NGS in 254 retinal genes. Novel mutations in PRPF8, CRB1, RPGR and CACNA1F co-segregated in affected and asymptomatic carriers with the adRP, arRP, x-linked dominant and X-liked icCSNB phenotypes respectively. Asymptomatic individuals are marked with a question mark.

                            Unsolved cases

                            In six of the 14 families with Stargardt disease, adRP, adCD with postreceptoral defects, arRP, early onset arCD with macrocephaly and mental retardation described in affected sister and x-linked cCSNB, the disease associated mutations remain to be elucidated or validated (Table 6, Figure 5).
                            Table 6

                            Patients with unsolved genotype and unlikely disease causing mutations

                            Index

                            Phenotype

                            Pre-screening

                            Gene

                            Mutation

                            Allele State

                            Read reference NGS

                            Read variant NGS

                            Mutation verified by Sanger and co-segregation

                            Comment

                            CIC03282,

                            F1388

                            Stargardt

                            ABCA4 microarray

                            ABCA4

                            c.1268A > G

                            p.H423R

                            het

                            77

                            61

                            yes

                            but reported as polymorphism

                            [71]

                                

                            c.6764G > T

                            p.S2255I

                            no additional variants in lower covered exons

                            het

                            2

                            7

                            yes

                            but reported as polymorphism

                            [72]

                               

                            CFH

                            c.3482C > A

                            p.P1161Q

                            het

                            77

                            52

                            yes

                            conserved, probably damaging

                                

                            c.1204C > T

                            p.H402Y

                            het

                            94

                            87

                            yes

                            AMD

                            CIC01269, F761

                            adRP

                            -

                            RP1L1

                            c.5959C > T

                            p.Q1987X

                            het

                            145

                            150

                            yes, did not co-segregate

                            pass to whole exome sequencing

                            CIC01312,

                            F795

                            adCD with post-receptoral defects

                            RHO, PDE6B,

                            GNAT1 adRP chip

                            CUBN

                            c.127C > T

                            p.R43X

                            het

                            139

                            102

                            yes, did not co-segregate

                            pass to whole exome sequencing

                               

                            CUBN

                            c.9340G > A

                            p.G3114S

                            het

                            61

                            44

                            yes, did not co-segregate

                             
                               

                            GUCY2D

                            c.1499C > T

                            p.P500L

                            het

                            41

                            34

                            yes, did not co-segregate

                             
                               

                            TRPM1

                            c.3904T > C

                            p.C1302R

                            het

                            102

                            99

                            yes, did not co-segregate

                             

                            CIC03225,

                            F1362

                            arRP consang.

                            arRP chip

                            PROM1

                            c.314A > G

                            p.Y105C

                            het

                            120

                            115

                            yes, but no additional mutation

                            no homo, no compound hets, pass to whole exome sequencing

                               

                            GUCY2D

                            c.2917G > A

                            p.V973L

                            het

                            6

                            2

                            false positive, not found by Sanger

                             
                               

                            DSCAML1

                            c.592C > T

                            p.R198C

                            het

                            70

                            81

                            yes, but no additional mutation

                             
                               

                            TBC1D24

                            c.641G > A

                            p.R214H

                            het

                            27

                            12

                            yes, but no additional mutation

                             
                               

                            TMEM67

                            c.1700A > G

                            p.Y567C

                            het

                            80

                            58

                            yes, but no additional mutation

                             

                            CIC04757

                            F2364

                            Index and affected sister early onset arCD, macro-cephaly and mental retardation in affected sister consang.

                            -

                            IMPG2

                            c.3439C > T

                            p.P1147S

                            homo

                            0

                            140

                            no

                            Polyphen and Sift benign, not conserved

                               

                            PKD2L1

                            c.1027C > T

                            p.R343C

                            het

                            63

                            68

                              
                                

                            c.1202T > G

                            p.V401G

                            het

                            25

                            19

                             

                            appeared also het in 11 of our samples

                            appeared also het in affected sister but no other mutation in less covered exons

                               

                            DFNB31

                            c.1943C > A p.S648Y

                            het

                            7

                            7

                            yes

                            affected sister also both variants but both come from father, no other variant in lower covered region.

                                

                            c.2644C > A

                            p.R882S

                            het

                            27

                            14

                            yes

                             
                               

                            EYS

                            c.7597A > G

                            p.K2533E

                            het

                            151

                            149

                            yes

                            Affected sister does not carry this variant

                               

                            RPGRIP1

                            c.2417C > T

                            p.T806I

                            het

                            138

                            132

                            no

                            not conserved

                            CIC04152, F1955

                            male x-linked cCSNB, has affected nephew

                            NYX

                            TRPM1

                            c.470C > T

                            p.S157F

                            het

                            118

                            130

                            yes, no other het mutation.

                            x-linked inheritance and phenotype verification

                            Index patients and respective gene defect are highlighted in bold. In some cases also family members were used for NGS.

                            http://static-content.springer.com/image/art%3A10.1186%2F1750-1172-7-8/MediaObjects/13023_2011_370_Fig5_HTML.jpg
                            Figure 5

                            Detection of novel mutation by using NGS in 254 retinal genes. Family 795 reveals autosomal dominant cone dystrophy with post-receptoral defects. Four putative disease causing mutations were investigated on the basis of co-segregation. However, none of them co-segregated in all affected family members with the phenotype and thus are not considered to be disease causing. Individuals marked with a star were clinically investigated, patients with a question mark are asymptomatic and patients with a plus sign show high myopia.

                            Discussion

                            By using NGS in 254 known and candidate genes we were able to detect known and novel mutations in 57% of families tested. In order to achieve this goal, we applied a rigorous protocol (Figure 1). To our knowledge, this is the first report using NGS to investigate all inherited retinal disorders at once. In a study restricted to adRP, Bowne and co-workers used a similar approach including 46 known and candidate genes for adRP [18]. All their cases had previously been screened and excluded for most of the known genes underlying adRP. The authors were able to identify known or novel mutations in five out of 21 cases in genes not included in a pre-screening [18]. This added five patients to their adRP cohort with known gene defects, indicating that 64% of their patients show known mutations with new genes still to be discovered in the remaining 36%. The current study provides a more exhaustive tool, since it incorporates screening of 254 genes implicated in various retinal disorders of different inheritance patterns and additional candidate genes for these phenotypes. With this approach a cohort of both pre-screened and unscreened samples, was investigated. The mutation detection rate of 57% is high and was never obtained before by high throughput screening methods. Furthermore, this approach is probably less time consuming and expensive than existing methods such as direct sequencing of all known genes or microarray analysis. Of note however is one of the variants detected with the NGS approach (i.e. p.V973L exchange in GUCY2D), which was not confirmed by direct Sanger sequencing, suggesting the possibility of false positive using the high throughput screening. Verification by direct Sanger sequencing of most likely pathogenic variants is therefore essential to validate NGS data, although the false positive rate is assumed to be low (in our study 1/28 verified sequence variants represented a false positive).

                            Overall, the study of 20 subjects from 17 families by NGS showed that most of the targeted regions are well covered (more than 98%). However, some of the regions showed a lower coverage (GC-rich regions) or were not captured (repetitive regions). This was for instance the case for two genes underlying cCSNB, (i.e. NYX and GRM6) and the repetitive region of ORF15 of RPGR. For GC-rich regions the capture design could be improved in the future by modifying NGS chemistry, as it was successfully achieved for Sanger sequencing using different additives, which improved the amplification and subsequent sequencing. If repetitive regions like ORF15 of RPGR remain problematic for sequencing by NGS, direct Sanger sequencing of these targets might be the first screening of choice; in particular for disorders caused only by a few gene defects such as CSNB, and xl-RP.

                            By applying NGS sequencing to our retinal panel, known and novel mutations were detected in different patients. We believe that our diagnostic tool is particularly important for heterogeneous disorders like RP, for which many gene defects with different prevalence have been associated to one phenotype. It also allows the rapid detection of novel mutations in minor genes which are often not screened as a priority by direct Sanger sequencing. This was the case in our study for three individuals from one family with adRP in which NGS detected a novel PRPF8 mutation in both affected and one unaffected family member (Table 4, Figure 4). In this family, the RP phenotype is mild and therefore it is possible that the unaffected member may develop symptoms later in life or alternatively it may be a case of incomplete penetrance as reported for another splicing factor gene, PRPF31 and recently for PRPF8 as well [1922]. Interestingly, a novel TRPM1 mutation was identified in a patient with adCSNB, a gene previously only associated with arCSNB [2326]. This is the first report of a TRPM1 mutation co-segregating with ad Schubert-Bornschein type complete CSNB. Since the location of this mutation is not different compared to other mutations leading to arCSNB, it is not quite clear how TRPM1 mutations might lead to either ad or arCSNB. Functional investigations are needed to validate the pathogenicity of this variant. Furthermore, this finding suggests that TRPM1 heterozygous mutation carriers from arCSNB families should be investigated by electroretinography to determine whether they display similar retinal dysfunction as in affected members of the presented adCSNB family. Detection of a novel RPGR splice site mutation in family 146 presented a challenge. The actual disease causing change was concealed under a wrongly annotated rs62638633, which had previously been clinically associated to RP by a German group http://​www.​ncbi.​nlm.​nih.​gov/​sites/​varvu?​gene=​6103&​rs=​62638633, (personal communication, Markus Preising). These observations indicate that the stringent filtering we applied initially can mask those referenced disease causing variants. Bearing this in mind one can still first investigate unknown variants, but should then examine dbSNP for referenced variants either described to be disease causing, having a low minor allele frequency or present in interesting candidate genes. An accurate discrimination of non-pathogenic polymorphisms versus disease causing polymorphism in SNP databases is warranted to resolve this challenge.

                            In six families from the investigated cohort the disease causing mutations still remain to be identified. In the Stargardt patient with no pathogenic ABCA4 mutations two variants in CFH were detected, one of which (rs1061170) had previously been reported to predispose to age related macular degeneration (AMD) [2729]. The second CFH change is a novel variant, affecting a highly conserved residue, not found in NGS data from the other 19 samples and never associated with a disease. The variants co-segregated in the only available family members, which were the patient's parents. Apart from the association with AMD, CFH mutations have been previously associated with renal diseases, the most common being membranoproliferative glomerulonephritis and hemolytic uremic syndrome, which can be also associated with an eye phenotype [30, 31]. No renal dysfunction was present in our patient. To validate if the two variants identified in CFH are indeed disease causing, the DNA samples from other available family members for co-segregation analysis as well as characterization of functional consequences of the novel variant are needed. One patient with complete CSNB had an affected nephew and thus x-linked inheritance was assumed. However, neither Sanger nor NGS detected a mutation in the only known x-linked gene, NYX, causing cCSNB. To exclude recessive inheritance TRPM1 and GRM6 were investigated in detail. Indeed the patient carried a novel heterozygous TRPM1 variant, which affects a highly conserved amino acid and was not identified in the other 19 samples investigated here (Table 6). However, direct Sanger sequencing of lower covered regions did not identify a second mutation in this gene. Similarly no mutations in GRM6 were identified. These findings outline the need for additional family members to determine, through co-segregation, the pathogenicity of the numerous variants identified by NGS. This was also true for two other families with nonsense mutations in CUBN (Fam795) and RP1L1 (Fam761) (Table 6). The nonsense mutation in CUBN, co-segregated with the phenotype in most of the family members (Figure 5). Had we not had access to additional family members, we might have retained this gene defect as the underlying cause for adCD and considered CUBN as a new gene involved in adCD. None of the other putatively pathogenic mutations identified in CUBN, TRPM1 and GUCY2D co-segregated with the phenotype in this family (Table 6, Figure 5). RP1L1 was already a candidate for adRP [32] but was previously associated with occult macular dystrophy [33]. In our study, this variant did not co-segregate with the phenotype in other affected family members (data not shown).

                            This NGS study ended with six genetically unresolved families, which can be further investigated with whole exome sequencing. Although, no clear information about the actual percentage of missing gene defects underlying each group of inherited retinal disorders exists, previous studies have reported that in many cases the genetic cause still needs to be determined [18, 34]. Whole exome sequencing approaches allow the detection of both, novel and known gene defects, but also generate numerous variants and therefore require the inclusion of more than one DNA sample for each family to rapidly exclude non-pathogenic variants. Due to the higher costs of exome sequencing for one sample compared to targeted sequencing, we propose to initially perform targeted sequencing in the index patient and proceed only after exclusion of a known gene defect to whole exome sequencing.

                            Conclusions

                            In summary, our diagnostic tool is an unbiased time efficient method, which not only allows detecting known and novel mutations in known genes but also potentially associates known gene defects with novel phenotypes. This genetic testing tool can now be applied to large cohorts of inherited retinal disorders and should rapidly deliver the prevalence of known genes and the percentage of cases with missing genetic defect for underlying forms of retinal disorders.

                            List of abbreviations

                            ad: 

                            autosomal dominant

                            ar: 

                            autosomal recessive

                            as: 

                            asymptomatic

                            het: 

                            heterozygous

                            homo: 

                            homozygous

                            hemi: 

                            hemizygous

                            not noted; consang: 

                            consanguinity was reported

                            n.a.: 

                            not applicable

                            CSNB: 

                            congenital stationary night blindness

                            RP: 

                            retinitis pigmentosa

                            Declarations

                            Acknowledgements

                            The authors are grateful to the families described in this study, Dominique Santiard-Baron and Christine Chaumeil for their help in DNA collection and to clinical staff. The project was financially supported by GIS-maladies rares (CZ), Agence Nationale de la Recherche (ANR, SSB), Foundation Voir et Entendre and BQR, Foundation Fighting Blindness (IA, FFB Grant # CD-CL-0808-0466-CHNO and the CIC503 recognized as an FFB center, FFB Grant # C-CMM-0907-0428-INSERM04), Ville de Paris and region Ile de France.

                            Authors’ Affiliations

                            (1)
                            INSERM, U968
                            (2)
                            UMR_7210, CNRS
                            (3)
                            UMR_S 968, Department of Genetics, Institut de la Vision, UPMC Univ Paris 06
                            (4)
                            INSERM-DHOS CIC 503, Centre Hospitalier National d’Ophtalmologie des Quinze-Vingts
                            (5)
                            UCL-Institute of Ophthalmology
                            (6)
                            IntegraGen SA, Genopole CAMPUS 1 bat G8 FR-91030 EVRY
                            (7)
                            Fondation Ophtalmologique Adolphe de Rothschild
                            (8)
                            Académie des Sciences-Institut de France
                            (9)
                            Department of Celular Therapy and Regenerative Medicine, Andalusian Molecular Biology and Regenerative Medicine Centre (CABIMER), Isla de Cartuja

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