Whole-Exome Sequencing Identifies LRIT3 Mutations as a Cause of Autosomal-Recessive Complete Congenital Stationary Night Blindness
2012; Elsevier BV; Volume: 92; Issue: 1 Linguagem: Inglês
10.1016/j.ajhg.2012.10.023
ISSN1537-6605
AutoresChristina Zeitz, Samuel G. Jacobson, Christian Hamel, Kinga M. Bujakowska, Marion Neuillé, Elise Orhan, Xavier Zanlonghi, Marie‐Elise Lancelot, Christelle Michiels, Sharon Schwartz, Béatrice Bocquet, Aline Antonio, Claire Audier, Mélanie Letexier, Jean‐Paul Saraiva, Tien D. Luu, Florian Sennlaub, Hoan Nguyen, Olivier Poch, Hélène Dollfus, Odile Lecompte, Susanne Kohl, José‐Alain Sahel, Shomi S. Bhattacharya, Isabelle Audo,
Tópico(s)Cellular transport and secretion
ResumoCongenital stationary night blindness (CSNB) is a clinically and genetically heterogeneous retinal disorder. Two forms can be distinguished clinically: complete CSNB (cCSNB) and incomplete CSNB. Individuals with cCSNB have visual impairment under low-light conditions and show a characteristic electroretinogram (ERG). The b-wave amplitude is severely reduced in the dark-adapted state of the ERG, representing abnormal function of ON bipolar cells. Furthermore, individuals with cCSNB can show other ocular features such as nystagmus, myopia, and strabismus and can have reduced visual acuity and abnormalities of the cone ERG waveform. The mode of inheritance of this form can be X-linked or autosomal recessive, and the dysfunction of four genes (NYX, GRM6, TRPM1, and GPR179) has been described so far. Whole-exome sequencing in one simplex cCSNB case lacking mutations in the known genes led to the identification of a missense mutation (c.983G>A [p.Cys328Tyr]) and a nonsense mutation (c.1318C>T [p.Arg440∗]) in LRIT3, encoding leucine-rich-repeat (LRR), immunoglobulin-like, and transmembrane-domain 3 (LRIT3). Subsequent Sanger sequencing of 89 individuals with CSNB identified another cCSNB case harboring a nonsense mutation (c.1151C>G [p.Ser384∗]) and a deletion predicted to lead to a premature stop codon (c.1538_1539del [p.Ser513Cysfs∗59]) in the same gene. Human LRIT3 antibody staining revealed in the outer plexiform layer of the human retina a punctate-labeling pattern resembling the dendritic tips of bipolar cells; similar patterns have been observed for other proteins implicated in cCSNB. The exact role of this LRR protein in cCSNB remains to be elucidated. Congenital stationary night blindness (CSNB) is a clinically and genetically heterogeneous retinal disorder. Two forms can be distinguished clinically: complete CSNB (cCSNB) and incomplete CSNB. Individuals with cCSNB have visual impairment under low-light conditions and show a characteristic electroretinogram (ERG). The b-wave amplitude is severely reduced in the dark-adapted state of the ERG, representing abnormal function of ON bipolar cells. Furthermore, individuals with cCSNB can show other ocular features such as nystagmus, myopia, and strabismus and can have reduced visual acuity and abnormalities of the cone ERG waveform. The mode of inheritance of this form can be X-linked or autosomal recessive, and the dysfunction of four genes (NYX, GRM6, TRPM1, and GPR179) has been described so far. Whole-exome sequencing in one simplex cCSNB case lacking mutations in the known genes led to the identification of a missense mutation (c.983G>A [p.Cys328Tyr]) and a nonsense mutation (c.1318C>T [p.Arg440∗]) in LRIT3, encoding leucine-rich-repeat (LRR), immunoglobulin-like, and transmembrane-domain 3 (LRIT3). Subsequent Sanger sequencing of 89 individuals with CSNB identified another cCSNB case harboring a nonsense mutation (c.1151C>G [p.Ser384∗]) and a deletion predicted to lead to a premature stop codon (c.1538_1539del [p.Ser513Cysfs∗59]) in the same gene. Human LRIT3 antibody staining revealed in the outer plexiform layer of the human retina a punctate-labeling pattern resembling the dendritic tips of bipolar cells; similar patterns have been observed for other proteins implicated in cCSNB. The exact role of this LRR protein in cCSNB remains to be elucidated. Congenital stationary night blindness (CSNB) is a clinically and genetically heterogeneous group of retinal disorders caused by mutations in genes implicated in the phototransduction cascade or in retinal signaling from photoreceptors to adjacent bipolar cells.1Zeitz C. Molecular genetics and protein function involved in nocturnal vision.Expert Rev. Ophthalmol. 2007; 2: 467-485Crossref Scopus (40) Google Scholar Most of the individuals affected by CSNB show a characteristic electroretinogram (ERG) in which the b-wave amplitude is smaller than the a-wave amplitude in the dark-adapted bright-flash condition.2Schubert G. Bornschein H. [Analysis of the human electroretinogram].Ophthalmologica. 1952; 123: 396-413Crossref PubMed Scopus (161) Google Scholar This electronegative waveform can be divided in two subtypes, incomplete CSNB (icCSNB) (CSNB2A [MIM 300071] and CSNB2B [MIM 610427]) and complete CSNB (cCSNB) (CSNB1A [MIM 310500], CSNB1B [MIM 257270],3Miyake Y. Yagasaki K. Horiguchi M. Kawase Y. Kanda T. Congenital stationary night blindness with negative electroretinogram. A new classification.Arch. Ophthalmol. 1986; 104: 1013-1020Crossref PubMed Scopus (377) Google Scholar CSNB1C [MIM 613216]), and CSNB1E [MIM 614565]). icCSNB is characterized by a reduced rod b-wave and substantially reduced cone responses, indicative of both ON and OFF bipolar cell dysfunction. icCSNB has been associated with mutations in CACNA1F (MIM 300110), CABP4 (MIM 608965), and CACNA2D4 (MIM 608171), genes coding for proteins important for the continuous glutamate release at the photoreceptor synapse.1Zeitz C. Molecular genetics and protein function involved in nocturnal vision.Expert Rev. Ophthalmol. 2007; 2: 467-485Crossref Scopus (40) Google Scholar cCSNB is characterized by a drastically reduced rod-b-wave response due to ON bipolar cell dysfunction, as well as specific cone ERG waveforms.4Audo I. Robson A.G. Holder G.E. Moore A.T. The negative ERG: Clinical phenotypes and disease mechanisms of inner retinal dysfunction.Surv. Ophthalmol. 2008; 53: 16-40Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar cCSNB has been associated with mutations in NYX (MIM 300278), GRM6 (MIM 604096), TRPM1 (MIM 603576), and GPR179 (MIM 614515), genes expressed and localized in ON bipolar cells. To date, more than 300 mutations have been identified in the genes underlying icCSNB and cCSNB.5Bech-Hansen N.T. Naylor M.J. Maybaum T.A. Pearce W.G. Koop B. Fishman G.A. Mets M. Musarella M.A. Boycott K.M. Loss-of-function mutations in a calcium-channel alpha1-subunit gene in Xp11.23 cause incomplete X-linked congenital stationary night blindness.Nat. Genet. 1998; 19: 264-267Crossref PubMed Scopus (426) Google Scholar, 6Zeitz C. Labs S. Lorenz B. Forster U. Uksti J. Kroes H.Y. De Baere E. Leroy B.P. Cremers F.P. Wittmer M. et al.Genotyping microarray for CSNB-associated genes.Invest. Ophthalmol. Vis. Sci. 2009; 50: 5919-5926Crossref PubMed Scopus (34) Google Scholar, 7Strom T.M. Nyakatura G. Apfelstedt-Sylla E. Hellebrand H. Lorenz B. Weber B.H. Wutz K. Gutwillinger N. Rüther K. Drescher B. et al.An L-type calcium-channel gene mutated in incomplete X-linked congenital stationary night blindness.Nat. Genet. 1998; 19: 260-263Crossref PubMed Scopus (394) Google Scholar, 8Bech-Hansen N.T. Naylor M.J. Maybaum T.A. Sparkes R.L. Koop B. Birch D.G. Bergen A.A. Prinsen C.F. Polomeno R.C. Gal A. et al.Mutations in NYX, encoding the leucine-rich proteoglycan nyctalopin, cause X-linked complete congenital stationary night blindness.Nat. Genet. 2000; 26: 319-323Crossref PubMed Scopus (281) Google Scholar, 9Pusch C.M. Zeitz C. Brandau O. Pesch K. Achatz H. Feil S. Scharfe C. Maurer J. Jacobi F.K. Pinckers A. et al.The complete form of X-linked congenital stationary night blindness is caused by mutations in a gene encoding a leucine-rich repeat protein.Nat. Genet. 2000; 26: 324-327Crossref PubMed Scopus (207) Google Scholar, 10Dryja T.P. McGee T.L. Berson E.L. Fishman G.A. Sandberg M.A. Alexander K.R. Derlacki D.J. Rajagopalan A.S. Night blindness and abnormal cone electroretinogram ON responses in patients with mutations in the GRM6 gene encoding mGluR6.Proc. Natl. Acad. Sci. USA. 2005; 102: 4884-4889Crossref PubMed Scopus (195) Google Scholar, 11Zeitz C. van Genderen M. Neidhardt J. Luhmann U.F. Hoeben F. Forster U. Wycisk K. Mátyás G. Hoyng C.B. Riemslag F. et al.Mutations in GRM6 cause autosomal recessive congenital stationary night blindness with a distinctive scotopic 15-Hz flicker electroretinogram.Invest. Ophthalmol. Vis. Sci. 2005; 46: 4328-4335Crossref PubMed Scopus (120) Google Scholar, 12Zeitz C. Kloeckener-Gruissem B. Forster U. Kohl S. Magyar I. Wissinger B. Mátyás G. Borruat F.X. Schorderet D.F. Zrenner E. et al.Mutations in CABP4, the gene encoding the Ca2+-binding protein 4, cause autosomal recessive night blindness.Am. J. Hum. Genet. 2006; 79: 657-667Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 13Wycisk K.A. Zeitz C. Feil S. Wittmer M. Forster U. Neidhardt J. Wissinger B. Zrenner E. Wilke R. Kohl S. Berger W. Mutation in the auxiliary calcium-channel subunit CACNA2D4 causes autosomal recessive cone dystrophy.Am. J. Hum. Genet. 2006; 79: 973-977Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 14Audo I. Kohl S. Leroy B.P. Munier F.L. Guillonneau X. Mohand-Saïd S. Bujakowska K. Nandrot E.F. Lorenz B. Preising M. et al.TRPM1 is mutated in patients with autosomal-recessive complete congenital stationary night blindness.Am. J. Hum. Genet. 2009; 85: 720-729Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar, 15Li Z. Sergouniotis P.I. Michaelides M. Mackay D.S. Wright G.A. Devery S. Moore A.T. Holder G.E. Robson A.G. Webster A.R. Recessive mutations of the gene TRPM1 abrogate ON bipolar cell function and cause complete congenital stationary night blindness in humans.Am. J. Hum. Genet. 2009; 85: 711-719Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, 16van Genderen M.M. Bijveld M.M. Claassen Y.B. Florijn R.J. Pearring J.N. Meire F.M. McCall M.A. Riemslag F.C. Gregg R.G. Bergen A.A. Kamermans M. Mutations in TRPM1 are a common cause of complete congenital stationary night blindness.Am. J. Hum. Genet. 2009; 85: 730-736Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, 17Riazuddin S.A. Shahzadi A. Zeitz C. Ahmed Z.M. Ayyagari R. Chavali V.R. Ponferrada V.G. Audo I. Michiels C. Lancelot M.E. et al.A mutation in SLC24A1 implicated in autosomal-recessive congenital stationary night blindness.Am. J. Hum. Genet. 2010; 87: 523-531Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 18Audo I. Bujakowska K. Orhan E. Poloschek C.M. Defoort-Dhellemmes S. Drumare I. Kohl S. Luu T.D. Lecompte O. Zrenner E. et al.Whole-exome sequencing identifies mutations in GPR179 leading to autosomal-recessive complete congenital stationary night blindness.Am. J. Hum. Genet. 2012; 90: 321-330Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 19Peachey N.S. Ray T.A. Florijn R. Rowe L.B. Sjoerdsma T. Contreras-Alcantara S. Baba K. Tosini G. Pozdeyev N. Iuvone P.M. et al.GPR179 is required for depolarizing bipolar cell function and is mutated in autosomal-recessive complete congenital stationary night blindness.Am. J. Hum. Genet. 2012; 90: 331-339Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 20Audo I. Bujakowska K.M. Léveillard T. Mohand-Saïd S. Lancelot M.E. Germain A. Antonio A. Michiels C. Saraiva J.P. Letexier M. et al.Development and application of a next-generation-sequencing (NGS) approach to detect known and novel gene defects underlying retinal diseases.Orphanet J. Rare Dis. 2012; 7: 8Crossref PubMed Scopus (134) Google Scholar, 21O'Connor E. Allen L.E. Bradshaw K. Boylan J. Moore A.T. Trump D. Congenital stationary night blindness associated with mutations in GRM6 encoding glutamate receptor MGluR6.Br. J. Ophthalmol. 2006; 90: 653-654Crossref PubMed Scopus (27) Google Scholar, 22Wang Q. Gao Y. Li S. Guo X. Zhang Q. Mutation screening of TRPM1, GRM6, NYX and CACNA1F genes in patients with congenital stationary night blindness.Int. J. Mol. Med. 2012; 30: 521-526PubMed Google Scholar, 23Xu X. Li S. Xiao X. Wang P. Guo X. Zhang Q. Sequence variations of GRM6 in patients with high myopia.Mol. Vis. 2009; 15: 2094-2100PubMed Google Scholar, 24Sergouniotis P.I. Robson A.G. Li Z. Devery S. Holder G.E. Moore A.T. Webster A.R. A phenotypic study of congenital stationary night blindness (CSNB) associated with mutations in the GRM6 gene.Acta Ophthalmol. (Copenh.). 2012; 90: e192-e197Crossref PubMed Scopus (29) Google Scholar Mutations in many genes associated with CSNB have been identified through a candidate gene approach comparing the human phenotype to similar phenotypes observed in knockout or naturally occurring animal models,10Dryja T.P. McGee T.L. Berson E.L. Fishman G.A. Sandberg M.A. Alexander K.R. Derlacki D.J. Rajagopalan A.S. Night blindness and abnormal cone electroretinogram ON responses in patients with mutations in the GRM6 gene encoding mGluR6.Proc. Natl. Acad. Sci. USA. 2005; 102: 4884-4889Crossref PubMed Scopus (195) Google Scholar, 11Zeitz C. van Genderen M. Neidhardt J. Luhmann U.F. Hoeben F. Forster U. Wycisk K. Mátyás G. Hoyng C.B. Riemslag F. et al.Mutations in GRM6 cause autosomal recessive congenital stationary night blindness with a distinctive scotopic 15-Hz flicker electroretinogram.Invest. Ophthalmol. Vis. Sci. 2005; 46: 4328-4335Crossref PubMed Scopus (120) Google Scholar, 12Zeitz C. Kloeckener-Gruissem B. Forster U. Kohl S. Magyar I. Wissinger B. Mátyás G. Borruat F.X. Schorderet D.F. Zrenner E. et al.Mutations in CABP4, the gene encoding the Ca2+-binding protein 4, cause autosomal recessive night blindness.Am. J. Hum. Genet. 2006; 79: 657-667Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 13Wycisk K.A. Zeitz C. Feil S. Wittmer M. Forster U. Neidhardt J. Wissinger B. Zrenner E. Wilke R. Kohl S. Berger W. Mutation in the auxiliary calcium-channel subunit CACNA2D4 causes autosomal recessive cone dystrophy.Am. J. Hum. Genet. 2006; 79: 973-977Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 14Audo I. Kohl S. Leroy B.P. Munier F.L. Guillonneau X. Mohand-Saïd S. Bujakowska K. Nandrot E.F. Lorenz B. Preising M. et al.TRPM1 is mutated in patients with autosomal-recessive complete congenital stationary night blindness.Am. J. Hum. Genet. 2009; 85: 720-729Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar, 15Li Z. Sergouniotis P.I. Michaelides M. Mackay D.S. Wright G.A. Devery S. Moore A.T. Holder G.E. Robson A.G. Webster A.R. Recessive mutations of the gene TRPM1 abrogate ON bipolar cell function and cause complete congenital stationary night blindness in humans.Am. J. Hum. Genet. 2009; 85: 711-719Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, 16van Genderen M.M. Bijveld M.M. Claassen Y.B. Florijn R.J. Pearring J.N. Meire F.M. McCall M.A. Riemslag F.C. Gregg R.G. Bergen A.A. Kamermans M. Mutations in TRPM1 are a common cause of complete congenital stationary night blindness.Am. J. Hum. Genet. 2009; 85: 730-736Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, 25Nakamura M. Sanuki R. Yasuma T.R. Onishi A. Nishiguchi K.M. Koike C. Kadowaki M. Kondo M. Miyake Y. Furukawa T. TRPM1 mutations are associated with the complete form of congenital stationary night blindness.Mol. Vis. 2010; 16: 425-437PubMed Google Scholar but techniques using massively parallel sequencing of all human exons have recently been successful in identifying mutations in genes underlying heterogeneous diseases, including Leber congenital amaurosis and, more recently, CSNB.18Audo I. Bujakowska K. Orhan E. Poloschek C.M. Defoort-Dhellemmes S. Drumare I. Kohl S. Luu T.D. Lecompte O. Zrenner E. et al.Whole-exome sequencing identifies mutations in GPR179 leading to autosomal-recessive complete congenital stationary night blindness.Am. J. Hum. Genet. 2012; 90: 321-330Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 26Sergouniotis P.I. Davidson A.E. Mackay D.S. Li Z. Yang X. Plagnol V. Moore A.T. Webster A.R. Recessive mutations in KCNJ13, encoding an inwardly rectifying potassium channel subunit, cause leber congenital amaurosis.Am. J. Hum. Genet. 2011; 89: 183-190Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 27Bamshad M.J. Ng S.B. Bigham A.W. Tabor H.K. Emond M.J. Nickerson D.A. Shendure J. Exome sequencing as a tool for Mendelian disease gene discovery.Nat. Rev. Genet. 2011; 12: 745-755Crossref PubMed Scopus (1262) Google Scholar Thus, to rapidly identify the gene defect of another autosomal-recessive-cCSNB-affected family (family A, Figure 1A), previously excluded for known cCSNB-associated gene defects, we sequenced the index nonconsanguineous affected female after whole-exome enrichment (IntegraGen, Evry, France). Research procedures were conducted in accordance with institutional guidelines and the Declaration of Helsinki; institutional-review-board approvals were obtained from the participating universities and the national Ministries of Health of each participating center. Prior to genetic testing, informed consent was obtained from all CSNB-affected individuals and their family members. Ophthalmic examinations were performed on all subjects: a full-field ERG incorporated the International Society for Clinical Electrophysiology of Vision standards and methodology previously described.28Marmor M.F. Fulton A.B. Holder G.E. Miyake Y. Brigell M. Bach M. International Society for Clinical Electrophysiology of VisionISCEV Standard for full-field clinical electroretinography (2008 update).Doc. Ophthalmol. 2009; 118: 69-77Crossref PubMed Scopus (837) Google Scholar, 29Aleman T.S. Lam B.L. Cideciyan A.V. Sumaroka A. Windsor E.A. Roman A.J. Schwartz S.B. Stone E.M. Jacobson S.G. Genetic heterogeneity in autosomal dominant retinitis pigmentosa with low-frequency damped electroretinographic wavelets.Eye (Lond.). 2009; 23: 230-233Crossref PubMed Scopus (13) Google Scholar, 30Jacobson S.G. Yagasaki K. Feuer W.J. Román A.J. Interocular asymmetry of visual function in heterozygotes of X-linked retinitis pigmentosa.Exp. Eye Res. 1989; 48: 679-691Crossref PubMed Scopus (91) Google Scholar Exons of DNA samples were captured and investigated as shown before with in-solution enrichment methodology (SureSelect Human All Exon Kits version 3, Agilent, Massy, France) and next-generation sequencing (NGS) (Illumina HISEQ, Illumina, San Diego, CA, USA). Image analysis and base calling were performed with Real Time Analysis software (Illumina).18Audo I. Bujakowska K. Orhan E. Poloschek C.M. Defoort-Dhellemmes S. Drumare I. Kohl S. Luu T.D. Lecompte O. Zrenner E. et al.Whole-exome sequencing identifies mutations in GPR179 leading to autosomal-recessive complete congenital stationary night blindness.Am. J. Hum. Genet. 2012; 90: 321-330Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar Genetic-variation annotations were performed by an in-house pipeline (IntegraGen), and results were provided per sample or family in tabulated text files. After very stringent criteria were used for excluding variants observed in dbSNP 132, HapMap, 1000 Genomes Project, and internal (IntegraGen) variant-detection databases, the results were further filtered so that only compound heterozygous or homozygous variants in coding regions remained. This allowed us to reduce the number of variants from 4,267 to 0 indels and from 50,807 SNPs to 21 homozygous variants in 13 genes and 30 compound heterozygous variants in 10 genes. To determine the most likely disease-causing gene defect for this cCSNB-affected family, we investigated missense changes with bioinformatic tools to predict the pathogenicity of the mutations and the conservation of affected amino acid residues (PolyPhen-2,31Adzhubei I.A. Schmidt S. Peshkin L. Ramensky V.E. Gerasimova A. Bork P. Kondrashov A.S. Sunyaev S.R. A method and server for predicting damaging missense mutations.Nat. Methods. 2010; 7: 248-249Crossref PubMed Scopus (9293) Google Scholar SIFT,32Ng P.C. Henikoff S. SIFT: Predicting amino acid changes that affect protein function.Nucleic Acids Res. 2003; 31: 3812-3814Crossref PubMed Scopus (4074) Google Scholar KD4v,33Luu T.D. Rusu A. Walter V. Linard B. Poidevin L. Ripp R. Moulinier L. Muller J. Raffelsberger W. Wicker N. et al.KD4v: Comprehensible Knowledge Discovery System for Missense Variant.Nucleic Acids Res. 2012; 40: W71-W75Crossref PubMed Scopus (25) Google Scholar and the USCS Human Genome Browser). Those genes harboring the selected variants were assessed for eye and retinal expression with the use of the UniGene database, the retinal gene-expression profile database provided by Siegert et al.,34Siegert S. Cabuy E. Scherf B.G. Kohler H. Panda S. Le Y.Z. Fehling H.J. Gaidatzis D. Stadler M.B. Roska B. Transcriptional code and disease map for adult retinal cell types.Nat. Neurosci. 2012; 15 (S1–S2): 487-495Crossref PubMed Scopus (195) Google Scholar and the in-house rd1 mouse expression database (courtesy of Thierry Leveillard). On the basis of these criteria, the only selected variants were compound heterozygous mutations (c.983G>A [p.Cys328Tyr] and c.1318C>T [p.Arg440∗]) in exon 4 of LRIT3 (RefSeq accession number NM_198506.3), which encodes leucine-rich-repeat (LRR), immunoglobulin-like, and transmembrane-domain 3 (LRIT3) (Figures 1A–1C; family A). For the c.983G>A variant, the G and A were found 46× and 53×, respectively, and for c.1318C>T, the C and T were present 70× and 54×, respectively, indicating that both variants were present heterozygously. Both variants were absent in more than 340 control chromosomes, and only c.983G>A (p.Cys328Tyr) was found at a very low frequency (1 out of 10,757 alleles, indicating that only one person was heterozygous for this substitution) in the National Heart, Lung, and Blood Institute (NHLBI) Exome Sequencing Project Exome Variant Server (EVS, 15.06.2012). The cystein at amino acid position 328 is highly conserved; only alpaca show a different amino acid residue (Phe) (USCS Human Genome Browser 15.06.2012) (Table 1). PolyPhen-2, SIFT, and KD4v predicted this variant to be probably damaging (Table 1). The second mutation, c.1318C>T (p.Arg440∗), was absent in all publically available databases, including the EVS. Cosegregation analysis revealed that the unaffected father and brother were heterozygous for the p.Cys328Tyr substitution. Subsequent Sanger sequencing of seven fragments covering the four exons and flanking intronic regions of LRIT3 (RefSeq NM_198506.3) (detailed conditions will be communicated on request) in 89 individuals (with cCSNB and unclassified CSNB) of various ethnic origins and from different clinical centers in Europe, the United States, Canada, Israel, and India (CSNB study group) detected one additional cCSNB-affected person, who carried compound heterozygous disease-causing mutations (c.1151C>G [p.Ser384∗] and c.1538_1539del [p.Ser513Cysfs∗59]) in exon 4 (Figures 1A–1C; family B). Both variants cosegregated with the phenotype, were absent in more than 370 control chromosomes, and were not described in the current EVS database (Figure 1A, family B). The frequencies of LRIT3 polymorphisms found in our individuals with CSNB are provided in Table S1, available online. On the basis of all of the above evidence, we conclude that mutations in LRIT3 lead to cCSNB.Table 1LRIT3 Mutations and Predicted Functional Consequences in Individuals with cCSNBIndex Affected IndividualsOriginExonNucleotide MutationAllele StateProtein AlterationFrequencyConservation and PredictionFamily B: CH6126 (II.1)France4c.1151C>Gheterozygousp.Ser384∗0/380 allelestruncated protein4c.1538_1539delheterozygousp.Ser513Cysfs∗590/376 allelestruncated proteinFamily A: 19018 (II.I)USA4c.1318C>Theterozygousp.Arg440∗0/348 allelestruncated protein4c.983G>Aheterozygousp.Cys328Tyr0/380 alleles; 1 out of 10,757 alleles in the Exome Variant Server databasePolyPhen-2: predicted to be probably damaging with a score of 1.000 (sensitivity: 0.00; specificity: 1.00)SIFT: predicted to be damaging with a score of 0.00; conserved except in alpaca (Phe)KD4v: predicted to be deleterious Open table in a new tab In family A, used for the whole-exome NGS approach (Figure 1A, family A), the index case (II.1) had visual blurring and night-vision disturbances from childhood. When she was 4 years old, spectacles for myopia were prescribed and strabismus surgery was performed in the right eye. A diagnosis was not specifically made, but the individual recalls being told that she had a progressive blinding disease with high myopia. Laser treatment of retinal tears occurred when she was 25 years old. When she was 45 years of age, an ERG was taken for the first time (Figure 1A, family A). The ERG features were those of cCSNB: undetectable responses to a dim flash under dark-adapted conditions (rod, Figure 1A, family A), a negative waveform in the mixed rod-cone response in the dark-adapted state, and an unusual square-shape appearance of the a-wave in the cone ERG1Zeitz C. Molecular genetics and protein function involved in nocturnal vision.Expert Rev. Ophthalmol. 2007; 2: 467-485Crossref Scopus (40) Google Scholar, 14Audo I. Kohl S. Leroy B.P. Munier F.L. Guillonneau X. Mohand-Saïd S. Bujakowska K. Nandrot E.F. Lorenz B. Preising M. et al.TRPM1 is mutated in patients with autosomal-recessive complete congenital stationary night blindness.Am. J. Hum. Genet. 2009; 85: 720-729Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar, 18Audo I. Bujakowska K. Orhan E. Poloschek C.M. Defoort-Dhellemmes S. Drumare I. Kohl S. Luu T.D. Lecompte O. Zrenner E. et al.Whole-exome sequencing identifies mutations in GPR179 leading to autosomal-recessive complete congenital stationary night blindness.Am. J. Hum. Genet. 2012; 90: 321-330Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar (29 Hz, Figure 1A, family A). In the detectable ERGs, amplitudes were abnormally reduced, a result that might be associated with high myopia.35Westall C.A. Dhaliwal H.S. Panton C.M. Sigesmun D. Levin A.V. Nischal K.K. Héon E. Values of electroretinogram responses according to axial length.Doc. Ophthalmol. 2001; 102: 115-130Crossref PubMed Scopus (94) Google Scholar Visual acuities were 20/80 (−26.00 D sphere) in the right eye and 20/30 (−27.00 D sphere) in the left eye. Eye pressures were normal, and the fundus appearance was that of myopia (1 Hz, Figure 1A). Other than undergoing eye-muscle and laser surgery (see above), the index case had arthroscopic knee surgery in her 40s. In family B, the affected person was diagnosed with high myopia at the age of 2 years. Prominent night blindness was also noticed at that age, and later on, visual acuity was found to be decreased. She had no photoaversion or loss in the peripheral visual field. At the time of presentation, 9 years of age, visual acuities were 20/40 (−7.00 D sphere) in the right eye and 20/50 (−8.00 D sphere) in the left eye. Lenses were transparent, and the fundus appearance was that of myopia with a tilted optic disc. Fundus autofluorescence was normal. An optical-coherence-tomography-3 scan of the maculae disclosed a normal photoreceptor layer. Light sensitivity was found moderately decreased over the whole visual field. She had an electronegative ERG mixed rod-cone response in the dark-adapted state (Figure 1A, family B). In addition, there were moderately reduced cone 30 Hz flicker responses, an atypical waveform in the light-adapted state (1 Hz, Figure 1A, family B), and no ERG responses to dim stimuli in the dark-adapted state (rod, Figure 1A, family B); all of these findings are compatible with the diagnosis of cCSNB (Figure 1A, family B). To date, only little information is available on the expression, localization, and function of LRIT3. It maps to chromosomal region 4q25 and contains four exons, the first of which was only recently identified and codes for a protein with 679 amino acids.36Kim S.D. Liu J.L. Roscioli T. Buckley M.F. Yagnik G. Boyadjiev S.A. Kim J. Leucine-rich repeat, immunoglobulin-like and transmembrane domain 3 (LRIT3) is a modulator of FGFR1.FEBS Lett. 2012; 586: 1516-1521Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar An available expressed-sequence-tag (EST) profile (from the UniGene EST Profile Viewer) indicates that the gene is expressed in the brain and the eye. Real-time-PCR experiments and subsequent Sanger sequencing of amplified real-time-PCR products confirmed the expression of LRIT3 in the human retina (commercially available cDNA from Clontech, Saint-Germain-en-Laye, France) by giving a signal of ΔCT = 6.33 (CT LRIT3 = 24.96) in relation to β-actin (ACTB [MIM 102630]) (CT ACTB = 18.63) (primers Table S2). To immunolocalize the exact location of LRIT3 in the retina, we used a validated human LRIT3 antibody (Figures S1 and S2) in human retina from a cryosectioned eye and performed immunostainings. LRIT3 localization could be detected in the outer plexiform layer (OPL) (Figures 2A and 2B , green). Immunofluorescence was analyzed with a confocal microscope (FV1000 fluorescent, Olympus, Hamburg, Germany). Colocalization studies with an antibody against a mouse Goα (Millipore, Molsheim, France), a specific ON bipolar cell marker (Figures 2A and B, red), demonstrated that human LRIT3 antibody reveals a characteristic synaptic punctate labeling at the dendrites of depolarizing bipolar cells. Comparing this immunostaining with the immunostaining from other molecules implicated in cCSNB, we conclude that the dotted punctate labeling (arrows) represents multiple rod bipolar cell dendritic tips that invaginate a rod spherule and that the innermost punctate labeling organized in rows (arrowheads) represents labeling of cone bipolar cell tips that invaginate the foot of a cone pedicle (Figure 2B).37Vardi N. Duvoisin R. Wu G. Sterling P. Localization of mGluR6 to dendrites of ON bipolar cells in primate retina.J. Comp. Neurol. 2000; 423: 402-412Crossref PubMed Scopus (177) Google Scholar, 38Pearring J.N. Bojang Jr., P. Shen Y. Koike C. Furukawa T. Nawy S. Gregg R.G. A role for nyctalopin, a small leucine-rich repeat protein, in localizing the TRP melastatin 1 channel to retinal depolarizing bipolar cell dendrites.J. Neurosci. 2011; 31: 10060-10066Crossref PubMed Scopus (67) Google Scholar Fainter
Referência(s)