Multiexon deletion alleles of ATF6 linked to achromatopsia

1Department of Ophthalmology, Shiley Eye Institute, and 2Department of Pathology, UCSD, San Diego, California, USA. 3Department of Ophthalmology, Stanford University, Stanford, California, USA. 4Developmental Neurobiology Unit, Okinawa Institute of Science and Technology Graduate University, Okinawa, Japan. 5Department of Cellular Biology, University of Georgia, Athens, Georgia, USA. 6Department of Ophthalmology and Visual Sciences, Medical College of Wisconsin, Milwaukee, Wisconsin, USA. 7Departments of Ophthalmology and Pathology and Cell Biology, Vagelos College of Physicians and Surgeons, Columbia University, New York, New York, USA. 8Department of Pathology, Stanford University, Stanford, California, USA. 9Department of Molecular Medicine, The Scripps Research Institute, San Diego, California, USA. 10Institute for Ophthalmic Research, Centre for Ophthalmology, University of Tübingen, Tübingen, Germany. 11Sorbonne Université, INSERM, CNRS, Institut de la Vision, Paris, France. 12CHNO des Quinze-Vingts, DHU Sight Restore, INSERM-DGOS CIC1423, Paris, France. 13Department of Ophthalmology, UCSF, San Francisco, California, USA. 14VA Palo Alto Healthcare System, Palo Alto, California, USA.

ATF6 encodes a glycosylated 670-amino acid type 2 ER transmembrane protein (14,15). The ATF6 protein consists of a luminal ER stress-sensing domain coupled across the ER membrane to a cytosolic basic leucine zipper (bZIP) domain transcription factor. It is an important regulator of the unfolded protein response (UPR), an intracellular signal transduction mechanism that prevents ER stress and ensures ER homeostasis (16). In response to ER stress, monomeric ATF6 protein migrates from the ER to the Golgi apparatus where it Achromatopsia (ACHM) is an autosomal recessive disease that results in severe visual loss. Symptoms of ACHM include impaired visual acuity, nystagmus, and photoaversion starting from infancy; furthermore, ACHM is associated with bilateral foveal hypoplasia and absent or severely reduced cone photoreceptor function on electroretinography. Here, we performed genetic sequencing in 3 patients from 2 families with ACHM, identifying and functionally characterizing 2 mutations in the activating transcription factor 6 (ATF6) gene. We identified a homozygous deletion covering exons 8-14 of the ATF6 gene from 2 siblings from the same family. In another patient from a different family, we identified a heterozygous deletion covering exons 2 and 3 of the ATF6 gene found in trans with a previously identified ATF6 c.970C>T (p.Arg324Cys) ACHM disease allele. Recombinant ATF6 proteins bearing these exon deletions showed markedly impaired transcriptional activity by qPCR and RNA-Seq analysis compared with WT-ATF6. Finally, RNAscope revealed that ATF6 and the related ATF6B transcripts were expressed in cones as well as in all retinal layers in normal human retina. Overall, our data identify loss-of-function ATF6 disease alleles that cause human foveal disease.
is cleaved in the transmembrane domain by Golgi-resident site 1 and site 2 proteases, releasing its cytosolic bZIP transcription factor domain (17,18). The liberated ATF6 bZIP transcription factor domain then enters the nucleus and upregulates target genes that include ER chaperones, such as BiP/Grp78 and protein-folding enzymes (14,19,20). Thus, ATF6 signaling helps cells survive ER stress by increasing the cell's protein-folding capability.
To date, 11 different ATF6 disease alleles have been identified in patients with ACHM (8,9,11,12,21), containing missense, nonsense, and indel mutations or splice-site changes. In this paper, we identified and characterized 2 additional multiexon-spanning ATF6 disease alleles in patients with ACHM. Consistent with prior analyses of other ATF6 ACHM alleles, we found that recombinant ATF6 proteins with these large deletions show severely impaired transcriptional activity. The data support the hypothesis that intact ATF6 transcription factor function is necessary for cone photoreceptor function and survival (8,21). Interestingly, we found, using the RNAscope assay, that ATF6 was expressed in cones and throughout the retinal layers. Thus, defects in ATF6 may also have an important role in visual processing of the retina.

Results
Multiexon deletions in ATF6 found in patients with ACHM. Eleven ATF6 photoreceptor disease alleles have been previously identified in patients with ACHM or cone-rod dystrophy; these alleles contain single-nucleotide changes (i.e., missense, nonsense, and splice site mutations), small deletions, or duplications that disrupt ATF6 production or function (Table 1) (8,9,11,12,21). In the current study, we identified 2 ATF6 mutations that delete large fragments of the ATF6 gene, leading to loss of multiple exons (Table 1).
In 2 siblings from family A, we identified a homozygous deletion, c.909+1_1720-1del, that leads to the loss of exons 8-14 (Table 1 and Figure 1A). The exact breakpoint is defined as NG_029773.1:g.58488_115797de-linsAGAGCTC; NG_029773:1(ATF6_v001):c.1008_1719+13728delinsAGAGCTC. Segregation in both parents using breakpoint PCR analysis showed that both parents are heterozygous carriers of the deletion ( Figure 1A). In another patient from family B, we studied a heterozygous deletion, c.82+1_248-1del, that leads to the loss of exons 2 and 3 (Table 1 and Figure 1A) (10). The patient has a second heterozygous mutation, c.970C>T;p.Arg324Cys, previously reported in other patients with ACHM (8). The parents subsequently underwent genetic testing and were found to be heterozygous carriers of either the previously characterized c.970C>T, (p.Arg324Cys) ATF6 allele or the c.82+1_248-1del ATF6 allele, respectively (Figure 1A). The parents from both families A and B had no visual defects. Furthermore, parents reported no consanguinity. At early infancy, all patients presented reduced visual acuity, nystagmus, and photophobia.
Both novel mutations (i.e., exons 8-14 deletion and exons 2-3 deletion) in ATF6 affect domains of the ATF6 transcriptional activity ( Figure 1B). If the deletion flanking exons are spliced directly onto each other, both deletions in the mRNA are in-frame. The exon 8-14 deletion removed 270 amino acid residues, including the bZIP domain, the transmembrane domain, and most of the luminal domain of ATF6 ( Figure  1B). When exons 2 and 3 were deleted, 55 amino acid residues were removed, leading to removal of part of the transcriptional activator domain of ATF6 ( Figure 1B).
The patient with the c.82+1_248-1del ATF6 allele also carries a SNP in ATF6 (rs1058405) that introduces a Met67Val amino acid change in the protein ( Figure 1B, M67V). Methionine and valine are aliphatic, nonreactive amino acids with similar molecular structures, suggesting that this conservative substitution is neither pathogenic to the ATF6 conformation nor detrimental to its function. Furthermore, this variant is widespread in the general population, with an allele frequency of 2.31 × 10 -1 (https://gnomad.broadinstitute.org/gene/ENSG00000118217?dataset=gnomad_r2_1), making it unlikely to be a disease-causing variant. For comparison, the allele frequency of the pathogenic c.970C>T;(p.Arg324Cys) ATF6 allele is 2.13 × 10 -5 (https://gnomad.broadinstitute.org/gene/ENSG00000118217?dataset=gnomad_r2_1). However, prior biochemical experiments reported functional differences for the M67V ATF6 variant (22).  (Figure 2A). The WT-ATF6 cytosolic domain served as the positive control and N267*, a previously identified ACHM-linked mutation that does not possess any ATF6 transcriptional activity, served as a negative control (21).
To determine the transcriptional activity on the ATF6 variants, we first investigated the protein expression levels of BiP/GRP78 (one of the most highly induced transcriptional targets of ATF6, refs. Clinical findings in the patient with exons 2 and 3 of the ATF6 gene deleted in this mutation. The patient with the deletion of exons 2-3 in the ATF6 allele is a 7-year-old boy, who, starting in infancy, presents nystagmus, impaired visual acuity, and photophobia. Fundus examination demonstrated crowded optic discs in both eyes and mild macular atrophy ( Figure 4A). Fundus autofluorescence imaging showed mild foveal hyperautofluorescence ( Figure 4B). Spectral domain-optical coherence tomography showed severe foveal hypoplasia in the right ( Figure 4C, top) and left ( Figure 4C, bottom) eyes and focal disruption in the inner segment ellipsoid layer at the site of the presumed foveal pit ( Figure 4C, arrowheads). In addition, the photopic full-field (light-adapted 3.0) electroretinogram (ERG) recordings showed defective (barely detectable) amplitudes for the a-wave and b-wave responses (Table 2) in the patient with the deletion of exons 2-3 in ATF6. These findings are consistent with prior imaging studies of ATF6-associated patients with ACHM (10).
Expression of ATF6 and ATF6B mRNA in human retina. ATF6 is a member of the bZIP transcription factors and is encoded as 2 isoforms, ATF6 and ATF6B (8). Other ACHM disease genes are specifically expressed in cones and are all functional components of the phototransduction cascade. To determine the localization and expression of ATF6 and ATF6B in the human retina, and especially in the cones, we performed triple-labeling experiments using RNA oligo probes specific for the mRNA of human retinal cone arrestin-3 (Arr3), ATF6, and ATF6B in vertical cross sections of the human adult retina. Human retinal Arr3 is a cone specific marker (28)(29)(30). Figure 5 shows an example of a vertical section of the midperiphery of the human retina triple labeled with Arr3, ATF6, and ATF6B. In situ hybridization results showed that the mRNA probe for Arr3 is found in the cell bodies and outer segments of cones; furthermore, mRNA probes for ATF6 and ATF6B are observed in all retinal layers. In a merged figure, ATF6 and ATF6B mRNA puncta were localized within the cell bodies that are labeled with Arr3 mRNA in the outer nuclear layer. In addition, ATF6 and ATF6B puncta were localized in the outer plexiform layer, inner nuclear layer, inner plexiform layer, and ganglion cell layer. Our data show that ATF6 was not only expressed in cones, but also widely expressed in all the retinal layers.

Discussion
ATF6 mutations have previously been found in patients with ACHM or cone-rod dystrophy and those patients carried single-nucleotide changes (i.e., missense, nonsense, and splice site mutations), small deletions, and small duplications of the gene (8,9,11,12). These mutations lead to loss of ATF6 function by damaging the amino-terminal transcriptional activator/bZIP DNA-binding domain of ATF6 or impairing the carboxyl-terminal ER to Golgi intracellular protein trafficking domain of ATF6 (8,21). Here, we study two new larger genomic deletions of ATF6 in 3 patients with ACHM that lead to multiexon deletions within ATF6. The deletion c.909+1_1720-1del in the ATF6 gene identified in the affected siblings removes the in-frame bZip, transmembrane, and luminal domains of ATF6 (exon 8-14 deletion, p.I304_R573del, Figure 1B). The deletion c.82+1_248-1del in the ATF6 gene identified in the affected single patient removes an in-frame part of the transcriptional activation domain (exons 2-3 deletion, p.D28_T82del, Figure 1B) The 2 isoforms of ATF6 share significantly conserved bZIP domains and DNA-binding domains; however, the ATF6 isoform is better studied than ATF6B biochemically and physiologically (31)(32)(33)(34)(35). In the current study, we examined the expression of both ATF6 and ATF6B mRNA by in situ hybridization in the human retina and RNAscope assay. The RNAscope assay is based on the branched DNA method described in previous studies (36)(37)(38)(39). RNAscope in situ hybridization decreases nonspecific hybridization and increases signal amplification by using a Z probe, which has 25 bp of oligonucleotides that are complementary to a region of the ATF6 and ATF6B mRNA. We observed ATF6 and ATF6B mRNA expression   HerpUD1, and Chop) and an XBP1 transcriptional target gene (ERdj4) were measured by real-time quantitative PCR and normalized to mRNA levels in untransfected control samples. Data are shown as mean ± SEM; 3 sets of independent experiments were performed. Data in B were analyzed by 1-way ANOVA and showed significance at **P < 0.01; *** P < 0.001; ****P < 0.0001. on the cone photoreceptors and in all the retinal layers of the human retina by RNAscope in situ hybridization. The mRNA expression of ATF6 and ATF6B described in the current study is consistent with previously performed immunohistochemistry studies in the mouse retina, which also show ATF6 expression in all the retinal layers (9,12). Furthermore, several in vitro studies show that ATF6 expression in inner retinal cells (e.g., retinal ganglion cells) is induced after ER stress (40)(41)(42). Together with our current results, these findings suggest that ATF6 plays an essential role in regulating ER stress in all retinal neurons. The reasons why loss of ATF6 preferentially causes a cone defect are unclear. One possibility is that other retinal cells have additional mechanisms for dealing with ER stress not found in cones. Another possibility is that cones undergo unique forms of ER stress that specifically require ATF6 function.
In recent years, structurally distinct small molecules have been identified that can activate or inhibit ATF6 or other parallel signal transduction pathways of the UPR to help cells tolerate ER stress (27,(43)(44)(45)(46). Our functional studies support that ATF6 mutations linked to retinal development disease arise from loss of ATF6 transcriptional activity, and, in turn, this is caused by defects at distinct steps of ATF6 activation (8,21). Based on these findings, ATF6-activating compounds may be helpful to correct the vision loss disease arising from ATF6 mutations in patients. In particular, in patients carrying loss-of-function ATF6 alleles arising from ER retention of the ATF6 protein, the activator of ATF6 (N-(2-hydroxy-5-methylphenyl)-3-phenylpropanamide [AA147]) (27), may be helpful to restore activity through promotion of ATF6 ER-to-Golgi trafficking. Alternatively, small molecules that induce UPR transcription factors with similar downstream transcriptional programs to ATF6, such as XBP1, may be helpful to compensate for loss of ATF6 (27,47).

Methods
Patients. Patients were clinically evaluated at 2 centers (Institute de la Vision, Paris, France, and Medical College of Wisconsin). For RNAscope in situ hybridization, adult human eye samples were collected with appropriate consent from the San Diego Eye bank (product code V0180).
Clinical evaluation. Spectral-domain optical coherence tomography, fundus, and autofluorescence (Heidelberg Engineering) imaging were performed using standard methods using the Heidelberg Spectralis (8). Light-adaptation thresholds full-field ERG was performed on all patients using the International Society for Clinical Electrophysiology of Vision standard protocols using JET disposable contact electrodes (48).
Genetic analysis and screening of ATF6. The patient with the deletion of exons 2-3 in ATF6 and his father underwent genetic testing using Retinal Dystrophy Panel Plus (Blueprint Genetics), which covers 266 genes known to cause inherited retinal diseases discovered by high-throughput sequencing and deletion/duplication analysis. The siblings with the deletion of exons 8-14 in ATF6 underwent genetic testing at the Institute of Ophthalmology, Tuebingen, Germany, by a step-wise procedure analyzing the genes known to be associated with ACHM by PCR and Sanger sequencing as previously described (8). The exact breakpoint was  (21). ATF6 plasmids were transiently transfected into HEK293 cells, using Lipofectamine 2000 (Invitrogen). HEK293 cells expressing WT or mutant ATF6 were lysed in SDS lysis buffer (2% SDS in PBS) and phosphatase inhibitor (Thermo Scientific). Protein concentrations of the total cell lysates were determined by BCA protein assay (Pierce). Equal amounts of protein were applied onto 4%-15% Mini-PROTEAN TGX precast gels (Bio-Rad) and analyzed by immunoblot. The following antibodies and dilutions were used: anti-FLAG at 1:5,000 (catalog A8592, MilliporeSigma), anti-BiP/GRP78 at 1:1,000 (catalog GTX113340, GeneTex Inc.), and anti-HSP90 at 1:5,000 (catalog GTX101448, GeneTex Inc.). After overnight incubation with primary antibody, membranes were washed in TBS with 0.1% Tween-20, followed by incubation of a horseradish peroxidase-coupled secondary antibody (catalog 7074, Cell Signaling). Immunoreactivity was detected using the Super-Signal West chemiluminescent substrate (Pierce).
qRT-PCR analysis. HEK293 cells expressing WT and mutant ATF6 were lysed, and total RNA was collected using the RNeasy mini kit, according to manufacturer's instructions (Qiagen). The mRNA was reverse-transcribed using the iScript cDNA Synthesis Kit (Bio-Rad). For quantitative PCR analysis, cDNA was used as template in SYBR Green qPCR Supermix (Bio-Rad). Primers used include human BIP/GRP78 (5′-GCCTGTATTTCTAGACCTGCC-3′ and 5′-TTCATCTTGCCAGCCAGTTG-3′), human HERPUD1 (5′-AACGGCATGTTTTGCATCTG-3′ and 5′-GGGGAAGAAAGGTTC-CGAAG-3′), human CHOP (5′-ACCAAGGGAGAACCAGGAAACG-3′ and 5′-TCACCATTCG-  International Society for Clinical Electrophysiology of Vision standard ERG recordings for the patient with deletion of exons 2-3 and a healthy control individual. The full-field flash strength (in cd·s/m 2 ) after light adaptation (photopic response) for the right eye (OD) and left eye (OS). The amplitude for a-wave and b-wave was not within the normal ranges under photopic condition in the patient.
RNAscope assay. In situ RNA hybridization was performed using the RNAscope assay (Advanced Cell Diagnostics) according to the manufacturer's recommendations and previous study (38). Briefly, the anterior segment and crystalline lens were removed, and the eye cups were fixed with 4% paraformaldehyde and processed for cryostat sections (20 μm). The vertical sections were subjected to RNAscope Multiplex Fluorescent Assay. The sections were incubated with pretreatment 2 (RNAscope Target Retrieval Reagents, Advanced Cell Diagnostics) for 30 minutes at 40°C in a HybEZ oven (HybEZ Hybridization System, Advanced Cell Diagnostics), followed by pretreatment 3 (RNAscope ProteaseIII, Advanced Cell Diagnostics) containing protease for 30 minutes at 40°C. After pretreatment 3, the sections were washed with 5× deionized water and incubated with the prewarmed mRNA target probes (ATF6-C1, 555291; Arr3-C2, 486461; or ATF6B-C3, 555301 [Advanced Cell Diagnostics]) for 2 hours at 40°C in the HybEZ oven. No-probe controls were performed in parallel (Supplemental Figure 1; supplemental material available online with this article; https://doi.org/10.1172/jci.insight.136041DS1). The sections were processed in wash buffer for 5 minutes and then incubated with Amp1 for 30 minutes at 40°C and washed with washing buffer for 5 minutes. The procedure was repeated for Amp2, Amp3, HRP-C1 (Cy3), HRP-C2 (FITC), and HRP-C3 (Cy5). After washing with washing buffer for 5 minutes, the sections were coverslipped with Vectashield-DAPI mounting medium (Vector Laboratories). The images of sections were saved and processed with the Leica application suitex software under a Leica SP8 confocal microscopy and then were adjusted using Adobe Photoshop 7.0. Photoshop adjustments were carried out equally across sections. According to Advanced Cell Diagnostics, each mRNA molecule hybridized to a probe appears as separate small puncta. Vertical section triple labeled with Arr3 (green), ATF6 (red), and ATF6B (white) mRNA in normal human retina. Nuclei were labeled with DAPI (blue) to identify the nuclear layers of the retina. The mRNA probe for Arr3 was found in the cell bodies and inner segments of cones, and the mRNA probes for ATF6 (red) and ATF6B (white) were observed in all the retinal layers. In the merged image, ATF6 and ATF6B mRNA puncta were localized within the cell bodies that are labeled with Arr3 in the outer nuclear layer (ONL). Three sets of independent experiments were performed (n = 2 retinas). Expression of ATF6 and ATF6B puncta was localized in the ONL, outer plexiform layer (OPL), inner nuclear layer (INL), inner plexiform layer (IPL), and ganglion cell layer (GCL). Scale bar: 10 μm.
Statistics. Data are expressed as mean ± SEM. Comparisons of multiple groups were performed using 1-way ANOVA. All in vitro studies were done with at least 3 sets of independent experiments. GraphPad Prism was used for all analyses. A P value of less than 0.05 was considered significant.
Study approval. This study was approved by the Institutional Review Boards of UCSD, the Medical College of Wisconsin, the University Tuebingen, and Columbia University and conducted in accordance with the tenets of the Declaration of Helsinki and NIH guidelines. Patients and parents of the patients provided informed consent for the study.

Author contributions
EJL, WCJC, and JHL conceived and designed the study. EJL, WCJC, HK, CXB, JMDG, LC, DSK, KK, and RLW performed the biochemical, transcriptomic, and RNAscope experiments. DLC, SHT, RRM, SRL, IA, ATM, and JC performed the clinical imaging. BB and SK performed the genetic analyses. All authors contributed to data analyses. EJL and JHL wrote the manuscript, and all authors contributed to manuscript editing.