BJO Online First, published on October 15, 2015 as 10.1136/bjophthalmol-2015-306939 Br J Ophthalmol: first published as 10.1136/bjophthalmol-2015-306939 on 15 October 2015. Downloaded from Clinical science A novel homozygous truncating GNAT1 mutation implicated in retinal degeneration Matthew Carrigan,1 Emma Duignan,2 Pete Humphries,1 Arpad Palfi,1 Paul F Kenna,1,2 G Jane Farrar1 ▸ Additional material is ABSTRACT ‘Nougaret’ form of CSNB, which was hypothesised published online only. To view Background The GNAT1 gene encodes the α subunit to be a result of constitutive activation of transdu- please visit the journal online (http://dx.doi.org/10.1136/ of the rod transducin protein, a key element in the rod cin. A second example of a pathogenic GNAT1 bjophthalmol-2015-306939). phototransduction cascade. Variants in GNAT1 have variant (c.598C>G/p.Gln200Glu) was also been implicated in stationary night-blindness in the past, reported in a Danish dominant CSNB pedigree.4 1School of Genetics and Microbiology, Trinity College but unlike other proteins in the same pathway, it has GNAT1 variants have also been associated with Dublin, Dublin, Ireland not previously been implicated in retinitis pigmentosa. recessive forms of CSNB. A missense variant 2Research Foundation, Royal Methods A panel of 182 retinopathy-associated genes (c.386A>G/p.Asp129Gly) was reported to segre- Victoria Eye & Ear Hospital, was sequenced to locate disease-causing mutations in gate with recessive CSNB in a large, consanguin- Dublin, Ireland patients with inherited retinopathies. eous Pakistani family.5 The Asp129Gly variant Correspondence to Results Sequencing revealed a novel homozygous altered a conserved residue of the protein, and was Dr Matthew Carrigan, Smurfit truncating mutation in the GNAT1 gene in a patient with predicted to be deleterious to function, although Institute of Genetics, Trinity significant pigmentary disturbance and constriction of no functional studies on it were carried out. College Dublin, College Green, visual fields, a presentation consistent with retinitis We report here a patient with retinal degeneration Dublin 2, Ireland; fi [email protected] pigmentosa. This is the rst report of a patient homozygous for a premature stop codon in the homozygous for a complete loss-of-function GNAT1 GNAT1 gene (c.904C>T/p.Gln302*). The GNAT1 Received 27 March 2015 mutation. The clinical data from this patient provide mutation was identified during the course of a next- Revised 9 July 2015 definitive evidence of retinitis pigmentosa with late onset generation sequencing (NGS) study covering the Accepted 1 August 2015 in addition to the lifelong night-blindness that would be Irish patient population with retinal degeneration. expected from a lack of transducin function. This variant causes the elimination of 49 extremely Conclusion These data suggest that some truncating conserved amino acids from the C terminus of the GNAT1 variants can indeed cause a recessive, mild, late- transducin α subunit protein (figure 1), comprising onset retinal degeneration in human beings rather than just under one-sixth of the total protein sequence. just stationary night-blindness as reported previously, This is a much more radical alteration to protein with notable similarities to the phenotype of the Gnat1 structure than that caused by other GNAT1 variants – knockout mouse. previously associated with CSNB.3 5 We have performed an extensive search of the literature and relevant databases, and have not INTRODUCTION located any other record of a patient with two non- Transducin is a heterotrimeric G-protein found on sense GNAT1 alleles. Hence, it is our view that this http://bjo.bmj.com/ the membrane of photoreceptor cells in the retina, represents the first report of a homozygous non- and is a key component of the vertebrate photo- sense GNAT1 mutation in human beings, as well as transduction pathway. On exposure of rod photo- the first observation of retinal degeneration asso- receptor cells to light, the light-sensitive pigment ciated with GNAT1 mutations. rhodopsin is photoisomerised into its active form. This clinical presentation is of particular interest This form activates the G-protein transducin, which as it strongly resembles the phenotype observed in on September 23, 2021 by guest. Protected copyright. − − in turn stimulates cyclic guanosine monophosphate homozygous knockout (Gnat1 / ) mice. Mice with (cGMP)-phosphodiesterase (PDE). The degradation targeted deletions of the GNAT1 gene have few of cGMP causes cGMP-gated ion channels to close, visible changes in gross morphology, but present resulting in hyperpolarisation of the cell membrane with a mild retinal degeneration with age. These and causing the electrical light response.1 mice also show preservation of cone cells, a com- Transducin is composed of three polypeptide plete absence of electrical response from rods as chains, α, β and γ. The α subunit is the active well as a slow loss of rod cells with corresponding guanosine diphosphate (GDP)/guanosine diphos- thinning of the outer nuclear layer,6 features which phate (GTP)-binding component. On activation by closely parallel the clinical presentation in the rhodopsin, GTP/GDP exchange occurs and the α GNAT1 Gln302* patient. subunit dissociates from the βγ subunits; at which point, it becomes capable of binding PDE. The α MATERIALS AND METHODS subunit proteins in rod and cone cells are encoded Patient identification and recruitment To cite: Carrigan M, by the genes GNAT1 and GNAT2, respectively.2 The proband was recruited prospectively at the Duignan E, Humphries P, Variants in GNAT1 have been implicated in con- Research Foundation, The Royal Victoria Eye and et al. Br J Ophthalmol Published Online First: genital stationary night-blindness (CSNB) in the Ear Hospital, Dublin, as part of the Target 5000 [please include Day Month past, although never in retinal degenerations. Dryja study, an ongoing project to identify the molecular Year] doi:10.1136/ et al3 reported a missense variant (NM_000172.3: basis for inherited retinopathies in Irish patients bjophthalmol-2015-306939 c.113G>A/p.Gly38Asp) causing the dominant via NGS. Carrigan M, et al. Br J Ophthalmol 2015;0:1–6. doi:10.1136/bjophthalmol-2015-306939 1 Copyright Article author (or their employer) 2015. Produced by BMJ Publishing Group Ltd under licence. Clinical science Br J Ophthalmol: first published as 10.1136/bjophthalmol-2015-306939 on 15 October 2015. Downloaded from Figure 1 Multiple alignment of the amino acids removed by the Q302* variant against the homologous region in other species, showing extremely high levels of sequence conservation, which implies that it is important to protein function. Clinical assessment Data analysis Best-corrected visual acuity was assessed using revised 2000 Sequence data were demultiplexed and mapped to the human Early Treatment Diabetic Retinopathy Study charts (Precision genome (hg19) using BWAV.0.7.4.9 Duplicate reads were flagged Vision, La Salle, Illinois, USA). Colour vision was examined using Picard V.1.106,10 and downstream analysis and variant using the Lanthony desaturated panel D-157 under standardised calling were performed using the GATK V.2.811 according to the lighting conditions. Goldmann perimetry was used to assess the protocol specified in the GATK Best Practices Workflow.12 peripheral visual fields to the IV4e, I4e and 04e targets. The list of identified variants was annotated with snpEFF13 Following slit lamp biomicroscopy of the anterior segment of and dbNSFP.14 Synonymous and common variants were filtered the eye and Goldmann applanation tonometry, the patient’s out, and the remaining list of rare variants with the potential to pupils were dilated using tropicamide 1% and phenylephrine affect protein sequence was output for manual curation. 2.5%. The subject was dark-adapted for 30 min, and the dark-adapted threshold was then measured using a Goldmann– Weekers dark adaptometer. A full-field electroretinogram (ERG) was performed according to International Society for Clinical RESULTS fi Electrophysiology of Vision (ISCEV) standards8 using a Roland Clinical ndings Consult RETI-port RETIscan. Fundus photography was per- The proband presented at 80 years of age with a 6-month fi formed using a Topcon CRC50DX. Spectral domain optical history of dif culties with steps. He commented on a lifelong coherence tomography was performed using a Cirrus HD-optical history of night-blindness, which had remained unchanged coherence tomography (OCT) (Carl Zeiss Meditec, Germany). throughout his life. There was no family history of night- blindness or day vision problems affecting either his parents, three siblings, ranging in age from 73 to 75 years or his three children aged 38–51 years. There was no history of parental consanguinity. His general health was good. He suffered for 10 years prior to presentation with non-insulin dependent dia- DNA isolation and sequencing betes mellitus, which was well controlled. Following informed consent, blood samples were collected from On examination, the proband’s unaided visual acuity was http://bjo.bmj.com/ patients after clinical assessment. DNA was isolated from 2 mL 6/7.5 in the right eye, which improved to 6/6 with +0.5 ds/ of patient blood using the QIAamp DNA Blood Midi kit +0.5 cx at 165°, and 6/7.5 in the left, which improved to 6/6 (QIAGEN, Valencia, California, USA), according to manufac- with 0.0/+0.5 cx at 165°. Slit lamp examination revealed turer’s recommendations. healthy corneas, normal anterior segments and clear lenses DNA was fragmented for sequencing by ultrasonication in a apart from some peripheral cortical opacities. His vitreous was fi Diagenode Bioruptor (Diagenode, Belgium) for 35 cycles, each normal apart from the presence of a grade 1 cellular in ltrate. on September 23, 2021 by guest. Protected copyright. consisting 30 s of low-intensity sonication followed by a 30 s Intraocular pressures were normal at 14 mm Hg in the right eye pause. Water was replaced and crushed ice added regularly to and 14 mm Hg in the left.