Characterization of GUCA1A-associated dominant cone/cone-rod dystrophy: low prevalence among Japanese patients with inherited retinal dystrophies

GUCA1A gene variants are associated with autosomal dominant (AD) cone dystrophy (COD) and cone-rod dystrophy (CORD). GUCA1A-associated AD-COD/CORD has never been reported in the Japanese population. The purpose of this study was to investigate clinical and genetic features of GUCA1A-associated AD-COD/CORD from a large Japanese cohort. We identified 8 variants [c.C50_80del (p.E17VfsX22), c.T124A (p.F42I), c.C204G (p.D68E), c.C238A (p.L80I), c.T295A (p.Y99N), c.A296C (p.Y99S), c.C451T (p.L151F), and c.A551G (p.Q184R)] in 14 families from our whole exome sequencing database composed of 1385 patients with inherited retinal diseases (IRDs) from 1192 families. Three variants (p.Y99N, p.Y99S, and p.L151F), which are located on/around EF-hand domains 3 and 4, were confirmed as “pathogenic”, whereas the other five variants, which did not co-segregate with IRDs, were considered “non-pathogenic”. Ophthalmic findings of 9 patients from 3 families with the pathogenic variants showed central visual impairment from early to middle-age onset and progressive macular atrophy. Electroretinography revealed severely decreased or non-recordable cone responses, whereas rod responses were highly variable, ranging from nearly normal to non-recordable. Our results indicate that the three pathogenic variants, two of which were novel, underlie AD-COD/CORD with progressive retinal atrophy, and the prevalence (0.25%, 3/1192 families) of GUCA1A-associated IRDs may be low among Japanese patients.

In this study, we identified rare GUCA1A variants from our whole exome sequencing (WES) database composed of IRD patients from a large Japanese cohort. The purpose of this study was to investigate the pathogenicity of rare GUCA1A variants and clinical and genetic features of GUCA1A-associated IRDs.

Results
Genetic analysis. We Table S1). According to the American College of Medical Genetics and Genomics (ACMG) criteria, three variants (p.Y99N, p.Y99S, and p.L151F) from three families (NTMC 244, JIKEI 136, and JIKEI 215) were confirmed as "pathogenic" (Fig. 1A,B). Two (p.Y99N and p.Y99S) of the 3 variants were novel, while the remaining variant (p.L151F) was previously reported as the cause of AD-CORD 16,19 . The 3 variants co-segregated with the disease and were located on/around EF-hand domain 3 or 4, which is essential for cytosolic Ca 2+ /Mg 2+ binding (Fig. 1C). In silico programs predicted severe damage to GCAP-1 (Supplementary Table S1), and each affected patient was diagnosed with COD/CORD. In our WES analysis, no other pathogenic variant, which is listed in the RetNet database, was found in the 3 families (NTMC 244, JIKEI 136, and JIKEI 215). In contrast, the other 5 variants (p.E17VfsX22, p.F42I, p.D68E, p.L80I, and p.Q184R) identified in 11 families were classified as "uncertain significance" according to the ACMG criteria (Supplementary Table S1). All 5 variants were located outside of EF-hand domains 3 and 4 ( Fig. 1C) and did not segregate with the disease; therefore, these variants were considered to be "non-pathogenic". clinical study. In 3 families with the pathogenic GUCA1A variants (p.Y99S, p.Y99N, and p.L151F), a total of 9 affected patients were clinically investigated (Fig. 1A). The examined age ranged from 15 to 69 years old (mean age: 43.0 years, standard deviation: 18.3 years). Clinical findings of the 9 affected patients are summarized in Table 1. onset, initial symptoms, and visual acuity. The age of onset ranged from 3 to 30 years old (mean age: 14.6 years, standard deviation: 9.6 years). Initial symptoms were available from 9 patients and included reduced visual acuity (9/9, 100%), photophobia (3/9, 33.3%), and central visual field loss (1/9; 11.1%). The decimal best-corrected visual acuity of the nine patients ranged from 0.01 to 0.8 at the examined age (Table 1).
Fundus photographs, FAF, and OCT findings. Each examination showed variable degrees of retinal abnormalities (Fig. 2) and we classified the abnormalities into early, middle, and advanced stages according to the severity of macular atrophy. Patient F2: III-1 was classified into early stage and exhibited discoloration and slight hyper-autofluorescence limited at the fovea in fundus and FAF images and preserved hyper-reflectivity of the ellipsoid zone (EZ) with the foveal bulge in OCT images. Two patients (F3: II-1 and F3: II-2) were classified into middle stage and exhibited almost normal fundus appearance or slight macular atrophy in fundus photographs, hyper-autofluorescent ring around the fovea in FAF images, and diffusely decreased or disrupted EZ without the foveal bulge in OCT images. Patient F2: II-1 was also classified into middle stage and exhibited macular atrophy in fundus photographs, hypo-autofluorescent area corresponding to retinal atrophy with hyper-autofluorescence around the area in FAF images, and diffusely decreased or disrupted EZ without the foveal bulge in OCT images. Five patients (F1: II-2, F1: I-2, F2: II-2, F2: I-1, and F3: I-1) were classified into advanced stage and exhibited retinal atrophy at the macula or posterior pole in fundus photographs, loss of autofluorescent area corresponding to retinal atrophy with hyper-autofluorescence around the area in FAF images, and disrupted EZ and thinning of the outer retina corresponding to the retinal atrophic area with hypo-reflectivity of the EZ outside the area in OCT images.
The atrophic areas were apparently more enlarged in older patients (i.e., 15-year-old patient F2: III-1 at the early stage, mean age of 33.3 years in the middle stage, and mean age of 54.4 years in the advanced stage). In addition, the enlargement pattern of the atrophic area was different between family 1 and the other 2 families. Retinal atrophy in family 1 extended over the optic disc and inferior arcade whereas that in families 2 and 3 showed elliptical enlargement within the vascular arcade. The other six patients were examined using GP. Two patients with middle stage abnormalities showed central scotoma of V-4e isopter in the right eye (F3: II-2) and central scotoma of I-4e isopter in three eyes (both eyes of F3: II-1 and the left eye of F3: II-2). One patient with middle stage abnormality and two patients with advanced stage abnormalities showed central scotoma of V-4e isopter in all eyes (F2: II-1, F1: II-2, and F2: II-2), and one patient with advanced stage abnormality showed central visual field loss in both eyes (F1: I-2). The areas of central scotomas were consistent with the lesions of retinal atrophy.
Four (F1: II-2, F2: II-2, F3: II-1, and F3: II-2) of 5 patients, whose retinal atrophy was limited to the posterior pole, showed preserved peripheral visual fields. In contrast, patients F2: II-1 and F1: I-2 exhibited constriction of the peripheral visual field and an island limited to the inferior area.

Discussion
In this study, eight rare GUCA1A variants, three of which were pathogenic, were identified in our large Japanese cohort. Furthermore, we demonstrated clinical features of nine COD/CORD patients from three unrelated Japanese families with two novel variants (p.Y99S and p.Y99N) and one known variant (p.L151F).
To date, 19 GUCA1A missense and 3 in-frame deletion/insertion variants in heterozygous states have been reported as causes of AD-MD and AD-COD/CORD in HGMD Professional (2019.3) [14][15][16][17][18]20,[23][24][25][26][27][28][29][30][31][32][33] . Most reported variants (18/22, 81.8%) were concentrated within or around EF-hand domains 3 and 4, which are essential for cytosolic Ca 2+ /Mg 2+ binding 4-6 . Amino acid residues Y99 and L151 are also located on EF-hand 3 helix E and EF-hand 4 loop, respectively (Fig. 1C). Regarding the two novel variants (p.Y99S and p.Y99N), a different variant (p.Y99C) at the same position has been reported as "pathogenic" and leads to COD/ CORD 14,34 . The variant p.L151F has also been reported as a cause of CORD 16,19 . In contrast, the other five variants (p.E17VfsX22, p.F42I, p.D68E, p.L80I, and p.Q184R) identified in the present study, which resulted in "uncertain significance" in the ACMG criteria, did not co-segregate with the disease. Previously, only five missense variants (p.L50I, p.L84F, p.G86R, p.E89K, and p.L176F) located outside EF-hand domains 3 and 4 have been reported as "pathogenic", 14,15,18,23,33 and one variant (p.L50I) has been concluded as "non-pathogenic" by subsequent studies 25,35 . Experimental studies have clarified that most GUCA1A variants within or around EF-hand domains 3 and 4 lead to the constitutive activation of RetGC by mechanisms of either a dominant-negative effect or gain of function, not haploinsufficiency 9,19,23,27,30,31,36 . While, recent studies have also demonstrated that the mutated GCAP-1 (p.L84F and p.L176F), located outside EF-hand domains 3 and 4, showed a significantly high affinity for Mg 2+ by altered tertiary structure or conformational changes, resulting in stabilizing the RetGC-activating state 36,37 . Certainly, all reported GUCA1A variants are heterozygous missense or in-frame insertion/deletion variants, but not truncated variants, compatible with the mechanisms of dominant-negative effect/gain of function. Regarding the reported p.Y99C variant 34 , the mutated GCAP-1 disrupts the N-terminal helix of the helix-loop-helix conformation of the EF-hand domain 3, severely affecting Ca 2+ binding at the site 9,10 . Further, the p.Y99C mutant activates RetGC at low Ca 2+ concentration, similar to wild-type GCAP-1, but remains active even at high Ca 2+ concentration, when wild-type GCAP-1 normally inhibits the target 9,10,13 . A similar pathomechanism whereby RetGC activity cannot be suppressed even at high cytosol Ca 2+ concentration may be considered to be CORD caused by our novel variants (p.Y99N and p.Y99S). While it is demonstrated that the p.L151F mutant showed decreased Ca 2+ affinity and significantly lower thermal stability compared to the wild-type protein 38 . Among the five non-pathogenic GUCA1A variants identified in this study, p.E17VfsX22, a truncated variant, might be considered to be a haploinsufficiency mechanism, whereas the other four missense variants were located outside EF-hand domains 3 or 4. However, we did not perform any biochemical or biophysical experiment using the four missense mutatns (p.F42I, p.D68E, p.L80I, and p.Q184R). Further, complex pathogenic mechanisms have been reported about mutated GCAP-1 outside EF-hand domains 3 and 4 18,36,37 . Although functional influence by the mutated GCAP-1 was not determined experimentally, all five GUCA1A variants (p.E17VfsX22, p.F42I, p.D68E, p.L80I, and p.Q184R) did not co-segregate with the disease; some family members with the variants were unaffected, concluding that the 5 variants were non-pathogenic.
All reported GUCA1A-associated phenotypes are classified into AD-MD or AD-COD/CORD with progressive macular atrophy 14,15,19,23,25,32 . In our study, the retinal atrophic areas were apparently more enlarged in older patients. In regard to cone ERG, although previous studies have shown that a variable degree of responses is seen ranging from preserved cone responses in AD-MD to severely decreased cone responses in AD-COD/ CORD 14,19,23,25,32 , all 8 patients examined exhibited severely decreased or non-recordable cone responses (Fig. 3). In contrast, the findings of rod ERG showed a variable degree of responses ranging from nearly normal to non-recordable (Fig. 3), consistent with data from previous studies 14,15,19,20,[23][24][25]30,31 . It is unclear why cone photoreceptors are initially and predominantly affected in GUCA1A-associated COD/CORD. In rod photoreceptors of mice, both GCAP-1 and GCAP-2 (an isoform of GCAP-1) are expressed, whereas GCAP-1 is predominantly expressed in cone photoreceptors 39 . Thus, GCAP-2 could compensate for dysfunction of GCAP-1 caused by GUCA1A variants in human rod photoreceptors as pointed out previously 40 . Another possibility is that GCAP-2 reduces an abnormal RetGC activity through competition with mutated GCAP-1 in which wild-type versus mutated GCPA-1 might function at different ratios between cone and rod photoreceptors. Taken  www.nature.com/scientificreports www.nature.com/scientificreports/ function gradually deteriorates and leads to COD/CORD in patients with heterozygous pathogenic GUCA1A variants. Clinical features of our 9 patients were consistent with previous studies as CORD with progressive macular atrophy; however, the centrifugal extension pattern of retinal atrophy was different between the families (Fig. 2). Unlike elliptical enlargement of retinal atrophy in families 2 and 3 and previous studies [11][12][13] , retinal atrophy in family 1 extended over the optic disc and inferior arcade. This unique pattern of retinal atrophy might be a characteristic finding of the p.Y99S variant or be influenced by secondary (genetic or environmental) factors. In addition, the older patient (F1: I-2) with p.Y99S showed non-recordable responses not only in cone ERG but also in rod and combined ERG, and peripheral visual field loss except for the inferior area. To our knowledge, this is the first report of such a case. The clinical course of GUCA1A-associated dystrophies typically exhibits progressive cone dysfunction with macular atrophy, accompanied by later rod dysfunction. Although it is not clarified whether GUCA1A-associated dystrophies ultimately lead to entire loss of rod function in addition to loss of cone function, our finding of patient F1: I-2 suggests that these dystrophies might lead to severe loss of both rod and cone function as the result of continuous progression.   www.nature.com/scientificreports www.nature.com/scientificreports/ In this study, our cohort of IRDs revealed the prevalence (0.25%, 3/1192 families) of GUCA1A-associated IRDs was lower than that (0.7%, 7/1000 families) of previous large US cohort study 21 . Our study suggests that GUCA1A-associated IRDs in Japanese population may be rarer compared with US population. In fact, any IRDs associated with GUCA1A variants have never been reported in the Japanese population. Further investigations will need to be undertaken in order to clarify the prevalence of GUCA1A-associated IRDs in each population. www.nature.com/scientificreports www.nature.com/scientificreports/ In conclusion, we identified eight rare GUCA1A variants including three pathogenic variants from a large Japanese cohort. Our results indicated that the three pathogenic variants underlie AD-COD/CORD with progressive retinal atrophy, and the prevalence of GUCA1A-associated IRDs may be low among Japanese patients with IRDs.

Molecular genetic study.
Patients with IRDs were studied from the genotype-phenotype database of the Japan Eye Genetics Consortium (http://www.jegc.org/) 41,42 . We examined our in-house WES database of 1385 IRD patients and 682 of their family members from 1192 Japanese families. In fact, WES with targeted analysis of the IRD genes listed in the RetNet database (https://sph.uth.edu/retnet/home.htm) was performed.The details of the WES methodology are described elsewhere 41,43 . We selected patients with IRDs who had rare GUCA1A variants in at least in one allele with a frequency of less than 1% in the Human Genetic Variation Database (http://www.hgvd.genome.med.kyoto-u.ac.jp/index.html) and a total frequency of less than 1.0% of the genome Aggregation Database (http://gnomad.broadinstitute.org). Subsequently, the pathogenicity of identified GUCA1A variants was evaluated according to the standards and guidelines of the ACMG 44 . Lastly, we evaluated the damage to GCAP-1 by using three in silico programs [PolyPhen2 (http://genetics.bwh.harvard.edu/pph2/),