Biallelic Mutations in GNB3 Cause a Unique Form of Autosomal-Recessive Congenital Stationary Night Blindness
2016; Elsevier BV; Volume: 98; Issue: 5 Linguagem: Inglês
10.1016/j.ajhg.2016.03.021
ISSN1537-6605
AutoresAjoy Vincent, Isabelle Audo, Erika Tavares, Jason T. Maynes, Anupreet Tumber, Tom Wright, Shuning Li, Christelle Michiels, Christel Condroyer, H. Robson MacDonald, R Verdet, José‐Alain Sahel, Christian Hamel, Christina Zeitz, Elise Héon, Eyal Banin, Béatrice Bocquet, Elfride De Baere, Ingele Casteels, Sabine Defoort‐Dhellemmes, Isabelle Drumare, Christoph Friedburg, Irène Gottlob, Samuel G. Jacobson, Ulrich Kellner, Robert K. Koenekoop, Susanne Kohl, Bart P. Leroy, Birgit Lorenz, Rebecca J. McLean, Françoise Meire, Isabelle Meunier, Francis L. Munier, Thomy de Ravel, Charlotte Reiff, Saddek Mohand‐Saïd, Dror Sharon, Daniel F. Schorderet, Sharon Schwartz, Xavier Zanlonghi,
Tópico(s)Retinal Diseases and Treatments
ResumoCongenital stationary night blindness (CSNB) is a heterogeneous group of non-progressive inherited retinal disorders with characteristic electroretinogram (ERG) abnormalities. Riggs and Schubert-Bornschein are subtypes of CSNB and demonstrate distinct ERG features. Riggs CSNB demonstrates selective rod photoreceptor dysfunction and occurs due to mutations in genes encoding proteins involved in rod phototransduction cascade; night blindness is the only symptom and eye examination is otherwise normal. Schubert-Bornschein CSNB is a consequence of impaired signal transmission between the photoreceptors and bipolar cells. Schubert-Bornschein CSNB is subdivided into complete CSNB with an ON bipolar signaling defect and incomplete CSNB with both ON and OFF pathway involvement. Both subtypes are associated with variable degrees of night blindness or photophobia, reduced visual acuity, high myopia, and nystagmus. Whole-exome sequencing of a family screened negative for mutations in genes associated with CSNB identified biallelic mutations in the guanine nucleotide-binding protein subunit beta-3 gene (GNB3). Two siblings were compound heterozygous for a deletion (c.170_172delAGA [p.Lys57del]) and a nonsense mutation (c.1017G>A [p.Trp339∗]). The maternal aunt was homozygous for the nonsense mutation (c.1017G>A [p.Trp339∗]). Mutational analysis of GNB3 in a cohort of 58 subjects with CSNB identified a sporadic case individual with a homozygous GNB3 mutation (c.200C>T [p.Ser67Phe]). GNB3 encodes the β subunit of G protein heterotrimer (Gαβγ) and is known to modulate ON bipolar cell signaling and cone transducin function in mice. Affected human subjects showed an unusual CSNB phenotype with variable degrees of ON bipolar dysfunction and reduced cone sensitivity. This unique retinal disorder with dual anomaly in visual processing expands our knowledge about retinal signaling. Congenital stationary night blindness (CSNB) is a heterogeneous group of non-progressive inherited retinal disorders with characteristic electroretinogram (ERG) abnormalities. Riggs and Schubert-Bornschein are subtypes of CSNB and demonstrate distinct ERG features. Riggs CSNB demonstrates selective rod photoreceptor dysfunction and occurs due to mutations in genes encoding proteins involved in rod phototransduction cascade; night blindness is the only symptom and eye examination is otherwise normal. Schubert-Bornschein CSNB is a consequence of impaired signal transmission between the photoreceptors and bipolar cells. Schubert-Bornschein CSNB is subdivided into complete CSNB with an ON bipolar signaling defect and incomplete CSNB with both ON and OFF pathway involvement. Both subtypes are associated with variable degrees of night blindness or photophobia, reduced visual acuity, high myopia, and nystagmus. Whole-exome sequencing of a family screened negative for mutations in genes associated with CSNB identified biallelic mutations in the guanine nucleotide-binding protein subunit beta-3 gene (GNB3). Two siblings were compound heterozygous for a deletion (c.170_172delAGA [p.Lys57del]) and a nonsense mutation (c.1017G>A [p.Trp339∗]). The maternal aunt was homozygous for the nonsense mutation (c.1017G>A [p.Trp339∗]). Mutational analysis of GNB3 in a cohort of 58 subjects with CSNB identified a sporadic case individual with a homozygous GNB3 mutation (c.200C>T [p.Ser67Phe]). GNB3 encodes the β subunit of G protein heterotrimer (Gαβγ) and is known to modulate ON bipolar cell signaling and cone transducin function in mice. Affected human subjects showed an unusual CSNB phenotype with variable degrees of ON bipolar dysfunction and reduced cone sensitivity. This unique retinal disorder with dual anomaly in visual processing expands our knowledge about retinal signaling. Congenital stationary night blindness (CSNB) is a heterogeneous group of non-progressive inherited retinal disorders that follow autosomal-dominant, autosomal-recessive, or X-linked patterns of inheritance.1Zeitz C. Robson A.G. Audo I. Congenital stationary night blindness: an analysis and update of genotype-phenotype correlations and pathogenic mechanisms.Prog. Retin. Eye Res. 2015; 45: 58-110Crossref PubMed Scopus (205) Google Scholar CSNB results from defects in visual signal transduction either within rod photoreceptors or in rod and cone bipolar pathways.1Zeitz C. Robson A.G. Audo I. Congenital stationary night blindness: an analysis and update of genotype-phenotype correlations and pathogenic mechanisms.Prog. Retin. Eye Res. 2015; 45: 58-110Crossref PubMed Scopus (205) Google Scholar Full-field electroretinogram (ERG) testing is an essential tool to diagnose CSNB and helps to localize the functional deficit to photoreceptor or bipolar-cell signaling.1Zeitz C. Robson A.G. Audo I. 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Eye Res. 2015; 45: 58-110Crossref PubMed Scopus (205) Google Scholar Identification of new gene defects will enable better understanding of visual signal processing within the photoreceptors and from photoreceptors to bipolar cells. The study protocol followed the tenets of the Declaration of Helsinki and was approved by the institutional ethics review board of each participating hospital or university. Prior informed consent was obtained from all participating members and parents (on behalf of children). Family A, with three affected individuals, was identified at the Hospital for Sick Children, Toronto (Figure 1A). Affected members had mildly reduced vision and normal fundus appearance; childhood-onset night blindness was present in the proband (III-2) and maternal aunt (II-5). Mutational analysis of the proband did not identify any pathogenic variant in 17 genes known to be mutated in CSNB or in 114 additional genes known to be mutated in retinal dystrophies. 11 members of the family were recruited. Detailed eye examinations and ISCEV standard ERG testing was performed in ten members (except I-1).2McCulloch D.L. Marmor M.F. Brigell M.G. Hamilton R. Holder G.E. Tzekov R. Bach M. ISCEV Standard for full-field clinical electroretinography (2015 update).Doc. Ophthalmol. 2015; 130: 1-12Crossref PubMed Scopus (985) Google Scholar, 30Marmor 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 (836) Google Scholar LA ERG with long-duration white flashes (150 ms and 200 ms; 250 cd.m−2; to analyze cone ON and OFF pathways) was performed in the three affected members.31Dragas R. Westall C. Wright T. Changes in the ERG d-wave with vigabatrin treatment in a pediatric cohort.Doc. Ophthalmol. 2014; 129: 97-104Crossref PubMed Scopus (12) Google Scholar Whole-exome capture and sequencing (WES) was performed in two trios (two affected subjects and their unaffected parents; Figure 1A) at The Center for Applied Genomics, Toronto as previously described (see the Supplemental Material and Methods).32Vincent A. Forster N. Maynes J.T. Paton T.A. Billingsley G. Roslin N.M. Ali A. Sutherland J. Wright T. Westall C.A. et al.FORGE Canada ConsortiumOTX2 mutations cause autosomal dominant pattern dystrophy of the retinal pigment epithelium.J. Med. Genet. 2014; 51: 797-805Crossref PubMed Scopus (28) Google Scholar The filtering steps used in the WES analysis in the pedigree are summarized in Table S1. Under the assumption of autosomal-recessive inheritance, genes carrying ≥two rare non-synonymous coding variants, splicing variants, or indels were prioritized; only 13 genes were shared among III-2 and II-5. WES data from the unaffected parents and sibling were sequentially used to filter through shared rare variants. A single variant in guanine nucleotide-binding protein subunit beta-3 (GNB3 [GenBank: NM_002075.3] or Gβ3 [MIM: 139130]) was shared between III-2 and II-5. This variant, c.1017G>A in exon 10 and predicted to lead to nonsense mutation p.Trp339∗, was homozygous in II-5 and heterozygous in III-2. Individual III-2 also carried a second heterozygous variant in GNB3; this variant, c.170_172delAGA in exon 4, was predicted to cause an in-frame deletion, p.Lys57del. Both the c.170_172delAGA and c.1017G>A variants were confirmed by Sanger sequencing (conditions available on request) and segregated with disease phenotype in the family (Figure 1A). Neither variant was reported in any public databases, including the Exome Aggregation Consortium (ExAC) Browser, the NHLBI ESP Exome Variant Server, and the 1000 Genomes Browser (release 14).33Abecasis G.R. Altshuler D. Auton A. Brooks L.D. Durbin R.M. Gibbs R.A. Hurles M.E. McVean G.A. 1000 Genomes Project ConsortiumA map of human genome variation from population-scale sequencing.Nature. 2010; 467: 1061-1073Crossref PubMed Scopus (5936) Google Scholar GNB3 mutational analysis was performed with Sanger sequencing (conditions available on request) in 58 additional CSNB cases; most individuals were excluded for mutations in genes associated with CSNB. The samples originated from different centers in Europe, the United States, Canada, and Israel. A presumably homozygous missense variant, c.200C>T in exon 4 of GNB3 and leading to p.Ser67Phe, was identified in a sporadic case subject (IV-3, family B; Figure 1B) clinically investigated at the Centre National de Références Maladies Sensorielles Génétiques in Montpellier (this individual was excluded for mutations in all known genes associated with CSNB). This extremely rare variant was found at a low frequency in the ExAC (MAF = 0.00004) and ESP (MAF = 0.0002) databases, but never in a homozygous state. In view of consanguineous ancestry, we determined this mutation to be most likely homozygous in the subject. Co-segregation could not be performed because the parents were deceased and no other family members were available for testing. Hence, the possibility of a heterozygous deletion involving the entirety of GNB3 cannot be completely excluded. Geneious v.8.1.8 was used to align the amino acid sequences of GNB3 and GNB proteins in available species.34Kearse M. Moir R. Wilson A. Stones-Havas S. Cheung M. Sturrock S. Buxton S. Cooper A. Markowitz S. Duran C. et al.Geneious Basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data.Bioinformatics. 2012; 28: 1647-1649Crossref PubMed Scopus (12563) Google Scholar The amino acid residues p.Lys57 and p.Trp339 in GNB3 are highly conserved across vertebrates, including zebrafish; p.Ser67Phe is conserved in most vertebrates (except frogs; Figure 1C). Residues p.Lys57 and p.Trp339 are conserved in all human GNB paralogs and in GNB proteins across a wide range of taxonomic groups (fungi, plants, and single-celled eukaryotes; Figures 1C and 1D). The p.Ser67 residue is conserved in all human paralogs, except GNB5, and is moderately conserved in GNB proteins present in other taxonomic groups. The mutant p.Phe67 is not seen in any paralog or homolog. All three variants, p.Lys57del, p.Trp339∗, and p.Ser67Phe, were predicted by MutationTaster to be disease causing with probabilities >0.99. Residues p.Lys57, p.Trp339, and p.Ser67 had PhylopPvert-100 average scores of 7.3, 9.6, and 4.1, respectively (strong conservation). The PhyloPMam average conservation score was 2.83 for p.Trp339 (strong conservation); p.Lys57 and p.Ser67 had scores of 2.22 and 2.46, respectively (moderate conservation). GNB3 located on chromosome 12p13.31 encodes the β subunit of the G protein heterotrimer (Gαβγ); it is expressed in the retina and has been previously associated with a partial form of cCSNB in mice (Gnb3−/−).35Dhingra A. Ramakrishnan H. Neinstein A. Fina M.E. Xu Y. Li J. Chung D.C. Lyubarsky A. Vardi N. Gβ3 is required for normal light ON responses and synaptic maintenance.J. Neurosci. 2012; 32: 11343-11355Crossref PubMed Scopus (47) Google Scholar, 36Ansari-Lari M.A. Muzny D.M. Lu J. Lu F. Lilley C.E. Spanos S. Malley T. Gibbs R.A. A gene-rich cluster between the CD4 and triosephosphate isomerase genes at human chromosome 12p13.Genome Res. 1996; 6: 314-326Crossref PubMed Scopus (88) Google Scholar GNB3 is a WD40 protein made up of seven highly conserved repeating units (∼40 amino acid motifs) usually ending with Trp-Asp (WD).37Clapham D.E. Neer E.J. G protein beta gamma subunits.Annu. Rev. Pharmacol. Toxicol. 1997; 37: 167-203Crossref PubMed Scopus (701) Google Scholar WD40 repeat motifs act as sites for protein-protein interaction, and WD40 proteins regulate cellular function, including transmembrane signaling.38Neer E.J. Schmidt C.J. Nambudripad R. Smith T.F. The ancient regulatory-protein family of WD-repeat proteins.Nature. 1994; 371: 297-300Crossref PubMed Scopus (1291) Google Scholar Residues Lys57 and Ser67 involve WD40 repeat 1, and Trp339 involves WD40 repeat 7. To determine the pathological basis of the identified mutations, we created a homology model of GNB3 from the known GNB1 (Gβ1) amino-acid sequence (shares 83% identity and 97% amino acid similarity with GNB3) structure by using Phyre2 software with a confidence score of 100 (Figures 1E and 1F).39Sondek J. Bohm A. Lambright D.G. Hamm H.E. Sigler P.B. Crystal structure of a G-protein beta gamma dimer at 2.1A resolution.Nature. 1996; 379: 369-374Crossref PubMed Scopus (707) Google Scholar, 40Kelley L.A. Mezulis S. Yates C.M. Wass M.N. Sternberg M.J. The Phyre2 web portal for protein modeling, prediction and analysis.Nat. Protoc. 2015; 10: 845-858Crossref PubMed Scopus (6080) Google Scholar The Gβ1 subunit forms a seven-bladed β propeller, typical of WD40 repeat proteins, and the entire sequence of GNB3 was fit onto this structure. Despite being at the extreme C terminus of the GNB3 protein chain, Trp339 occupies an important position in the β-propeller structure, buried between the first and last β sheets that constitute the propeller. If a stop codon is introduced in place of Trp339, both Trp339 and Asn340 would be removed, creating a significant distortion of the circular propeller structure and exposing normally buried residues (Figures 1G and 1H). The β-propeller fold is commonly used to facilitate protein-protein interactions and the identified p.Trp339∗ would pathologically affect the ability of GNB3 to form important interactions within the G protein signaling complex. The residue Ser67 lies at the top ridge of the overall β-barrel structure of GNB3 with two potential hydrogen-bonding partners Ala322 and Asp323 (Figure 1I). The p.Ser67Phe mutation would remove the ability to participate in both hydrogen bonds, disrupting the structure of the top of the barrel where other protein-protein interactions occur. Interestingly, the region of the β barrel disrupted by p.Ser67Phe is the same region disrupted by p.Trp339∗, potentially indicating that this ridge or geographic region of GNB3 forms interactions important for cellular function. The other variant, p.Lys57del, lies within a surface loop on the GNB3 structure. To determine how this mutation might affect Gβ protein interactions with regulators of G protein signaling (RGS), we used the X-ray crystal structure of a Gβ5:RGS complex (PDB: 2PBI).41Cheever M.L. Snyder J.T. Gershburg S. Siderovski D.P. Harden T.K. Sondek J. Crystal structure of the multifunctional Gbeta5-RGS9 complex.Nat. Struct. Mol. Biol. 2008; 15: 155-162Crossref PubMed Scopus (86) Google Scholar Lys57 forms a conserved interaction with an aspartate in the RGS protein (Figures 1J and 1K), and loss of the Lys57 residue would cause disruption of bonding between GNB3 and cognate Gβ binding partners, inhibiting the formation of effective G protein complexes. The ocular phenotype of the four affected individuals is summarized in Table S2. Childhood-onset night blindness was observed in three subjects. Photophobia and color vision deficits were noted only in middle aged subjects (II-5, family A and IV-3, family B). None had nystagmus and all subjects had a best-corrected visual acuity of 20/30 or better. Mild myopia was observed in one; two had hyperopia. All had normal fundus and visual fields. Central retinal thickness and retinal lamination were normal (spectral domain-optical coherence tomography) in all subjects; Gnb3-null mice also showed normal retinal morphology.35Dhingra A. Ramakrishnan H. Neinstein A. Fina M.E. Xu Y. Li J. Chung D.C. Lyubarsky A. Vardi N. Gβ3 is required for normal light ON responses and synaptic maintenance.J. Neurosci. 2012; 32: 11343-11355Crossref PubMed Scopus (47) Google Scholar, 42Nikonov S.S. Lyubarsky A. Fina M.E. Nikonova E.S. Sengupta A. Chinniah C. Ding X.Q. Smith R.G. Pugh Jr., E.N. Vardi N. Dhingra A. Cones respond to light in the absence of transducin β subunit.J. Neurosci. 2013; 33: 5182-5194Crossref PubMed Scopus (18) Google Scholar The visual parameters remained stable on follow-up in all cases; the proband from family B had 47 years of follow-up. These findings are consistent with the diagnosis of CSNB.43Bijveld M.M. Florijn R.J. Bergen A.A. van den Born L.I. Kamermans M. Prick L. Riemslag F.C. van Schooneveld M.J. Kappers A.M. van Genderen M.M. Genotype and phenotype of 101 dutch patients with congenital stationary night blindness.Ophthalmology. 2013; 120: 2072-2081Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar All seven unaffected members in family A had normal eye examinations and normal ERGs (similar to the control eye data provided in Figure 2E). The ERG phenotype in all affected subjects is shown in Figure 2 and the Supplemental Data (including Table S3). Rod ERG findings suggested partial (sibling pair, p.Lys57del and p.Trp339∗; Figures 2A and 2B) or severe rod ON bipolar dysfunction (II-5, family A, p.Trp339∗ homozygous and IV-3, family B, p.Ser67Phe homozygous; Figures 2C and 2D). The single flash and 30 Hz LA ERGs were normal in the sibling pair. The single flash LA 3.0 ERG a-wave showed normal amplitudes, but implicit times were markedly delayed in two subjects (24 ms in II-5, family A and 31 ms in IV-3, family B [normal range: 14–17 ms]; Figures 2C and 2D). The subsequent b-wave showed reduced amplitudes and delayed implicit times (36 ms in II-5, family A and 49 ms in IV-3, family B [normal range 27–30 ms]). The LA 30 Hz flicker ERG implicit times were also delayed in these two individuals. The LA ERG findings in these two subjects are peculiar and reminiscent of the functional abnor
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