Study design and populations

We performed a rare-variant gene-burden study using DNA derived from blood samples obtained for age-related macular degeneration patients from clinical trials for ranibizumab (NCT00061594/NCT00056836 [DAWN], NCT00891735 [HARBOR], NCT02960828 [SAVE], FU and PALMER), ranibizumab Port Delivery System (PDS) (NCT03677934 [ARCHWAY], NCT02510794 [LADDER]), faricimab (NCT03823300 [LUCERNE], NCT03823287 [TENAYA]), lampalizumab (NCT02247479 [CHROMA], NCT01229215 [MAHALO], NCT02479386 [PROXIMA A], NCT02399072 [PROXIMA B], NCT02247531 [SPECTRI], NCT02288559 [LAMPA Dosing Study]), and the National Eye Institute (NEI) studies (NCT00000145 [AREDS1], NCT00345176 [AREDS2]) (Supplementary Table 1). These study populations were selected for inclusion on the basis of available phenotypic information and DNA availability for WGS. All AMD patients and non-AMD controls in this study were aged 50 years or older and were of European ancestry, which were determined by comparison with samples from the International Haplotype Map (HapMap) Project. Family members presented in Fig. 1 were evaluated at NEI (NCT02077894).

Samples and data for controls in the study population were obtained from clinical trial studies of asthma, autoimmune disease, Crohn’s disease, chronic obstructive pulmonary disease, cancer, inflammatory bowel disease, interstitial lung disease, idiopathic pulmonary fibrosis, rheumatoid arthritis, systemic lupus erythematosus and ulcerative colitis.

Study population ethics statements

All research in this study was conducted in accordance with the Declaration of Helsinki. The Roche/Genentech data used in this study were generated from clinical trial participants who signed informed consent forms approved by the ethics committee or IRB responsible for the country or site where the trial’s participants donated samples for research. Informed consent included use of these data for genetics research. Before execution of the study, an internal Genentech team of informed consent form experts reviewed the forms from all the studies to ensure appropriate use of the samples.

For both the AREDS and AREDS2, institutional review board approval was obtained at each site and written informed consent was obtained from all participants. The research was conducted under the tenets of the Declaration of Helsinki and, for the AREDS2, complied with the Health Insurance Portability and Accountability Act.

A population used in this study consisted of patients enrolled in the FinnGen Biobank cohort. Patients and control subjects in FinnGen provided informed consent for biobank research, based on the Finnish Biobank Act. Alternatively, separate research cohorts, collected prior the Finnish Biobank Act came into effect (in September 2013) and start of FinnGen (August 2017), were collected based on study-specific consents and later transferred to the Finnish biobanks after approval by Fimea (Finnish Medicines Agency), the National Supervisory Authority for Welfare and Health. Recruitment protocols followed the biobank protocols approved by Fimea. The Coordinating Ethics Committee of the Hospital District of Helsinki and Uusimaa (HUS) statement number for the FinnGen study is Nr HUS/990/2017.

The FinnGen study is approved by Finnish Institute for Health and Welfare (permit numbers: THL/2031/6.02.00/2017, THL/1101/5.05.00/2017, THL/341/6.02.00/2018, THL/2222/6.02.00/2018, THL/283/6.02.00/2019, THL/1721/5.05.00/2019, THL/1524/5.05.00/2020, and THL/2364/14.02/2020), Digital and population data service agency (permit numbers: VRK43431/2017-3, VRK/6909/2018-3, VRK/4415/2019-3), the Social Insurance Institution (permit numbers: KELA 58/522/2017, KELA 131/522/2018, KELA 70/522/2019, KELA 98/522/2019, KELA 138/522/2019, KELA 2/522/2020, KELA 16/522/2020, Findata THL/2364/14.02/2020 and Statistics Finland (permit numbers: TK-53-1041-17 and TK/143/07.03.00/2020 (earlier TK-53-90-20).

The Biobank Access Decisions for FinnGen samples and data utilized in FinnGen Data Freeze 7 include: THL Biobank BB2017_55, BB2017_111, BB2018_19, BB_2018_34, BB_2018_67, BB2018_71, BB2019_7, BB2019_8, BB2019_26, BB2020_1, Finnish Red Cross Blood Service Biobank 7.12.2017, Helsinki Biobank HUS/359/2017, Auria Biobank AB17-5154 and amendment #1 (August 17 2020), Biobank Borealis of Northern Finland_2017_1013, Biobank of Eastern Finland 1186/2018 and amendment 22 § /2020, Finnish Clinical Biobank Tampere MH0004 and amendments (21.02.2020 & 06.10.2020), Central Finland Biobank 1-2017, and Terveystalo Biobank STB 2018001.

DNA analysis

The WGS data for the study population was generated to a read depth of 30X using the HiSeq platform (Illumina X10, San Diego, CA, USA) processed using the Burrows-Wheeler Aligner (BWA) / Genome Analysis Toolkit (GATK) best practices pipeline. WGS short reads were mapped to hg38 / GRCh38 (GCA_000001405.15), including alternate assemblies, using BWA version 0.7.9a-r786 to generate BAM files. All WGS data was subject to quality control and checked for concordance with SNP fingerprint data collected before sequencing. After filtering for genotypes with a GATK genotype quality greater than 90, samples with heterozygote concordance with SNP chip data of less than 75% were removed. Sample contamination was determined with VerifyBamID software, and samples with a freemix parameter of more than 0.03 were excluded. Joint variant calling was done using the GATK best practices joint genotyping pipeline to generate a single variant call format (VCF) file. The called variants were then processed using ASDPEx to filter out spurious variant calls in the alternate regions.

Quality control—patient DNA samples

Prior to genotype QC, 15,438 samples were eligible for inclusion. Samples were excluded if the call rate was less than 90% (n = 5). Identity-by-descent analysis was used to detect and filter out relatedness in the dataset. Samples were excluded if PI_HAT was 0.4 or higher (n = 102). Samples were removed if they showed excess heterozygosity with more than three SDs of the mean (n = 222). Ancestry was assigned using an ancestry threshold of 0.7 obtained from the supervised analysis with ADMIXTURE 1.334 using samples from phase 3 of the 1000 Genomes Project as reference samples labeled by their superpopulation. Due to sample sizes, this analysis was restricted to those with EUR ancestry and the remaining ancestry groups were removed (n = 760). This resulted in cohorts comprised of controls (n = 8294) and advanced AMD cases (n = 6055) diagnosed with geographic atrophy (GA; n = 2366) and/or macular neovascularization (MNV; n = 3689) in the study population.

Quality control—genotype

Sample genotypes were set to missing if the Genotype Quality score was less than 20 and SNPs were removed if the missingness was higher than 5%. SNPs were filtered out if the significance level for the Hardy-Weinberg equilibrium test was less than 5×10-8. The allele depth balance test was performed to test for equal allele depth at heterozygote carriers using a binomial test; SNPs were excluded if the p value was less than 1×10-5.

Statistical analysis—WGS

The SKAT-O algorithm using the R package SKAT35 (https://CRAN.R-project.org/package=SKAT) was used to assess the cumulative effect of rare variants (MAF < 1%). Tests conducted in the analysis were two-sided. Variants were annotated using SnpEff and SnpSift annotation softwares36 as well as the Ensembl Variant Effect Predictor37. Variants were included in the gene burden analysis if they were classified as missense variants with a PolyPhen38 score that was predicted to be damaging (>=0.5) or if they were high impact variants (stop gain, stop loss, frameshift, start loss, and canonical splicing). The rare variant gene burden test was adjusted for age, sex and genetic ancestry. In addition, METAL software was used to perform a meta-analysis of summary statistics for 34 AMD susceptibility loci from the International AMD Genomics Consortium (IAMDGC) with summary statistics from this study6.

Since samples from other disease areas were used as controls, we used a genotype-on-phenotype reverse regression11 to test for association with the non-AMD controls. We calculated the posterior probability (PP) that a gene burden score is also associated with each individual disease area included in the control cohort; genes passing the 0.6 PP threshold in any of the control cohorts disease areas were flagged and excluded from the results. The SLC16A8 variant enrichment in AMD cohort was analyzed using Fisher’s exact test (two tailed) in Supplementary Data 2.

Variant classification

Variant classification was performed according to the ACMG/AMP variant interpretation framework with the following modifications13. PVS1 was applied to frameshift and canonical splicing variants, by downgrading c.102_109del p.(Phe35fs) for the presence of Methionine at a.a. 47, and c.1449_1459del p.(Ser483fs) for being located at the last exon and C-terminal to the basolateral sorting signal at a.a. 451-48439. PM2 was applied to variants with gnomAD v3.1.2 popmax AF < 0.0005, the threshold was 22 folds less than the maximum credible population allele frequency threshold determined based on the prevalence (~20%) of late-stage AMD among people greater than 95 years old40, the maximum allelic contribution by the SLC16A8 c.214+1 G > C (2.8%) in our AMD cohort, and penetrance of 50% (20%*2.8%/50% = 0.0112). The maximum credible population allele frequency threshold at a penetrance of 10% was 0.056 (20%*2.8%/10%), thus gnomAD v3.1.2 popmax AF of 0.05 was used for BS1 cut-off. As the only statistically-significantly enriched variant in our AMD cohort was c.214+1 G > C with gnomAD popmax AF of 0.0096, variants with gnomAD popmax AF > 0.01 and not enriched in AMD cohort (OR of allele count < 1.2) were assigned BS1_P. PP3/BP4 was applied for missense variants based on AlphaMissense prediction14.

Drosophila orthologs

Subsets of human genes were prioritized and a list of corresponding orthologs were generated using the DRSC Integrative Ortholog Prediction Tool (DIOPT) which uses a ranking system of low, moderate, and high orthologs for each gene queried. Screenings focused on high ranked orthologs for all genes. Due to the replication of SLC16A8 with Fritsche et al.6 study, one of the top-scoring moderately ranked orthologs (sln) was prioritized for screening at the time of DIOPT queries. All other human genes with only low or moderately ranked Drosophila orthologs were not included in fly screens. Genes with no readily available Drosophila RNAi stocks were also excluded from functional screens (methods; see Supplementary Table 2).

Drosophila stocks

Flies crosses were initially cultured on standard cornmeal food at 18 °C to minimize potential GAL4 mediated developmental eye defects. Then male offspring were transferred to 25 °C at 65% humidity with a 12 h light/dark cycle post-eclosion for RNAi screenings using a fluorescent microscope at days 1, 7, and/or 14. An eye-specific conditional Gal4-UAS expression system was used to conditionally express UAS-linked transgenes for RNAi screenings of candidate genes and controls. Furthermore, an EGFP reporter under the control of the rhodopsin 1 promoter (ninaE-EGFP) was used to specifically label photoreceptors #1-6 towards fluorescent imaging of fly screens.

The following fly lines were obtained from the Bloomington Drosophila Stock Center (BDSC)18: L.GMR-GAL4 (8605), ninaE-EGFP (8601), rsg7RNAi (28574), haspRNAi (65101), luciferaseRNAi (31603), mCherryRNAi (35785).

The following fly lines were obtained from the Vienna Drosophila RNAi Center (VDRC)19: slnRNAi (109464, 4607, and 4609), rsg7RNAi (101733), hasp RNAi (101250).

The following fly lines were obtained from the Jasper lab stock: UAS-tdTomato and LacZRNAi.

Drosophila sample preparation

A small drop of clear nail polish was applied on either standard microscope slides when using the 4X objective. Alternatively, nail polish was applied on small 35 mm diameter petri dishes mounted on top of microscope slides (to hold in place for microscope stage) for use with a 40X water immersion objective. The 4X objective was used for fluorescent deep pseudopupil (DPP) visualization and the 40X water immersion objective for optic neutralization of the cornea (ONC) visualization techniques21. Each CO2 anesthetized fly was then placed carefully on top of each drop of nail polish in sets of 5–10 male flies per slide/petri dish for phenotypic readouts and then discarded after screenings. The heads were carefully arranged with forceps to ensure eyes were perpendicular to the plane of the objectives. Females were not used for screenings. An Olympus BX61 fluorescent upright with low mag 4X objective (UPlanSApo) and high mag 40X water dipping objective (LUMPlanFL-N) were used with standard FITC and Texas Red filters21. Only ~10–20 photoreceptor units in the approximate center of view, rather than in the peripheral regions, can be directly visualized due to curvature of the fly compound eye after imaging with 40X immersion objective for ONC visualization. All Drosophila images were acquired and pseudocolored using SlideBook 6 software and processed for brightness, contrast, and labeled using Microsoft Powerpoint 2021 and Adobe Photoshop CC 2019 Software. Drosophila deep pseudopupil graphic images were prepared using GraphPad Prism Software v9.

Drosophila screening

For all crosses (n = 10 flies per timepoint, per fly line) offspring were screened for EGFP expression patterns of photoreceptors and ommatidia morphology at days 1, 7, and, 14 with a fluorescent low mag 4X objective by DPP. If a phenotype was detected, then a more low-throughput, fluorescent 40X immersion objective was used to further validate eye phenotypes directly by ONC. To account for potential phenotypic variability, multiple negative control screens were carried out (L.GMR-GAL4; ninaE-EGFP crossed to LacZRNAi, luciferaseRNAi, mCherryRNAi, or UAS-tdTom) and a mean loss of DPP below 70% (n = 10 flies per line, per timepoint) was then determined to be a plausible phenotype for each RNAi line screened. For each gene screened, attempts were made to prioritize one VDRC and one BDSC RNAi line if available. Parents were rotated into new tubes with food every 3-4 days at 18 C. Then male offspring were collected for aging at 25 C and imaging was prioritized for days 1 and 14. For negative controls or lines with DPP loss below the 70% threshold screens were also acquired at day 7 by microscopy.

Mouse Models

Animal experiments were conducted in accordance with protocols approved by the Genentech Institutional Animal Care and Use Committee and with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. CRISPR mediated deletion of Slc16a8 exons 1-6 (4298 bp deletion) was outsourced and carried out by Taconic Inc. The founder Slc16a8 KO line was backcrossed to the original C57BL/6 J WT littermates used for CRISPR deletions to generate Slc16a8 Hets, and then these Slc16a8 Het littermates were bred to each other to eventually generate 10-week old Slc16a8 WT (n = 8; 4 male and 4 female), Het (n = 13; 10 male and 3 female), and KOs (n = 6; 3 male and 3 female) for subsequent ERG, OCT, and histological analyses. All animals were housed in pathogen-free animal facility at Genentech with 12/12 h of light/dark cycle prior to the start of constant light exposure.

Randomization and blinding

Randomization and blinding was used whenever possible. During CLE, mice were randomly assigned to a light box in the room, regardless of gender or genotype. For ERG and OCT measurements, mice were assessed in random order and the operator did not have knowledge of the mouse’s gender or genotype. For data analysis, ERGs and OCTs were marked in random order and the scientist had no knowledge of the gender or genotype associated with a given ERG waveform or OCT image.

Data exclusions

One WT mouse was excluded from analysis because they died inadvertently between baseline and day 7. Additionally, one eye from a mouse from the heterozygous group at baseline had a waveform in which the c-wave could not be confidently marked and thus was excluded from analysis. Finally, one mouse from the WT group at day 7 obtained cataracts before the OCT measurements so these datapoints were excluded.

Constant light exposure (CLE)

See Natoli et al. for more information regarding CLE methods24. Briefly, age matched Slc16a8 -WT, Het, and KO animals were placed 2 per plastic box with free access to food and water. The mice were exposed to constant (24 hr) 100 K lux natural white LEDs for 7 days and pupils were dilated twice per day with 1% atropine sulfate. Immediately following 7 days of constant light exposure, mice were dark-adapted overnight in preparation for subsequent functional measurements.

Electroretinogram recordings (ERGs)

The Celeris ERG system (Diagnosys) was used to perform full-field scotopic ERGs at baseline and after CLE. A-, b-, and c-wave amplitudes were measured in order to determine photoreceptor, bipolar, and RPE cell function, respectively. Slc16a8 + /+ (WT, n = 8), Slc16a8 + /− (HET, n = 13), and Slc16a8−/− (KO, n = 6) mice were dark-adapted overnight and then anesthetized with an intraperitoneal (IP) injection of a Ketamine (75–80 mg/kg) and Xylazine (7.5–15 mg/kg) in 200-300 uL of pharmaceutical grade saline. Mouse body temperature was maintained at 37 °C using a homeothermic plate connected to its control unit and pupils were dilated with 1% tropicamide under dark conditions. Mice were placed on a platform and a reference electrode inserted subcutaneously through the forehead and a ground electrode inserted at the base of the tail. ERGs from both eyes were recorded simultaneously. Gonak hypermellose ophthalmic demulcent solution was placed on the cornea to establish an electrical contact between the cornea and the electrode, and to protect eyes from drying during the experiment. Electrodes with light stimulator simultaneously placed on both eyes. In scotopic conditions, retinas were stimulated with a flash intensity of 150 cd.s/m2. Recorded signals were bandpass-filtered at 0.15–1000 Hz and sampled at 2 kHz. Espion v6.59.9 software (Diagnosys) was used to record the waveforms of each response. Responses to 3 flashes of light stimulation were averaged. All of the recorded waveforms were then analyzed using custom Matlab software (MathWorks) and the amplitudes were marked as follows: a-wave amplitude measured from the baseline to the trough of the a-wave while b-wave amplitude from the trough of the a-wave to the peak of the b-wave. C-wave amplitude was recorded from the trough following the b-wave to the c-wave peak. Data was then analyzed using GraphPad (GraphPad Prism Software v10, Boston, MA). For comparisons between groups (genotypes) within timepoints (baseline and day 7) a mixed-effects analysis with a Sidak post-hoc was used. For percent change comparisons a one-way ANOVA with a Sidak post-hoc was used.

Optical coherence tomography (OCT)

Immediately following ERG recordings, retinal morphology and thickness were examined by spectral domain optical coherence tomography (SD-OCT) scans using a Bioptigen Envisu R machine (Leica Microsystems, IL, USA). Mice were still anesthetized from ERG recordings (by intraperitoneal injection of ketamine (75-80 mg/kg body weight) and xylazine (15 mg/kg body weight)). Pupils were dilated with drops of Tropicamide Ophthalmic Solution USP 1% (Akorn). Drops of Systane lubricant eye drop (Alcon) were applied bilaterally to prevent corneal dehydration during procedure. Mouse body temperature was again maintained at 37 C using a homeothermic plate. Total retina thickness was defined as the width from the nerve fiber layer to the RPE/choroid layer on the cross-sectional images with an algorithm. Data were exported to the Matlab software (MathWorks) and analyzed. Data was then analyzed using GraphPad (GraphPad Prism Software v10, Boston, MA). For comparisons between groups (genotypes) within timepoints (baseline and day 7) a mixed-effects analysis with a Sidak post-hoc was used. For percent change comparisons a one-way ANOVA with a Sidak post-hoc was used.

H&E histology

Mice were euthanized via C02 overdose. Formalin-fixed, paraffin-embedded (FFPE) mouse eyes were sectioned in the sagittal plane at 4 um thickness and stained with hematoxylin and eosin (H&E). Slides were examined by a pathologist blinded to the treatment groups. Also see Weber et al Scientific Reports 2022 for more methods information41.