Van de Sompele S , Small KW , Cicekdal MB , Soriano VL , D'haene E , Shaya FS , Agemy S , Van der Snickt T , Rey AD , Rosseel T , Van Heetvelde M , Vergult S , Balikova I , Bergen AA , Boon CJF , De Zaeytijd J , Inglehearn CF , Kousal B , Leroy BP , Rivolta C , Vaclavik V , van den Ende J , van Schooneveld MJ , Gómez-Skarmeta JL , Tena JJ , Martinez-Morales JR , Liskova P , De Baere E .

             embryo development
             eye pigmentation

	Graphical abstract
	Figure 1. Integration of the generated UMI-4C data with publicly available Hi-C data The UMI-4C interaction frequency profiles and domainograms (bottom) for the PRDM13 promoter (left) and IRX1 promoter (right) viewpoints were integrated with Hi-C data from control human retinal organoids (top), demonstrating promoter interactions within the respective TADs.47 Topologically associated domains (TADs) are indicated by blue triangles. Chromosome coordinates are in hg38 annotation.
	Figure 2. Output from the UMI-4C experiment in the PRDM13 locus The UMI-4C data (green) from the PRDM13 promoter viewpoint (right gray bar) illustrate an interaction with PRDM13_cCRE1 (left gray bar), a non-coding region upstream of the promoter (left arrow). Since underlying epigenomic tracks show an overlap of this region with DNase-seq (turquoise) and ATAC-seq (green) peaks, as well as with ChIP-seq profiles of specific histone marks indicative of enhancer activity (H3K4me2, H3K27ac) (yellow) and ChIP-seq profiles of retinal transcription factors (CRX, NRL, OTX2) (orange), this region is a strong cCRE. The reverse UMI-4C experiment using this cCRE as a viewpoint results in a peak around the PRDM13 promoter region (right arrow), confirming this interaction. cCRE, candidate CRE.
	Figure 3. Results from the luciferase assays for the set of fourteen cCREs The bar plot shows, for each cCRE, the fold change of the luciferase reporter level relative to the level of the negative control luciferase vector (fold change = 1). neg, negative; NS, not significant; pos, positive; ∗∗∗p < 0.001.
	Figure 4. Overview of the results from the in vivo transgenic enhancer assays in Xenopus Representative images of EGFP reporter expression in living, transgenic Xenopus tadpoles, driven by SED vector reporter constructs containing non-coding regions of interest. The respective Xenopus species injected and the Nieuwkoop and Faber (NF) stage shown in the pictures are indicated on top of the images, while the injected construct and the stage of injection are indicated left of the images. For each stage, the number of tadpoles displaying the depicted EGFP reporter expression pattern over the total number of analyzed transgenic tadpoles is given. (A and B) Dorsal view of transgenic Xenopus tropicalis embryos upon unilateral injection of the reporter construct containing IRX1_cCRE7. EGFP reporter expression was visible at the neural plate and tube (NP) on the injected side, indicated by the arrow, while no expression was observed on the non-injected side. (C and D) Ventral view of transgenic tadpoles, displaying EGFP reporter expression in the craniofacial cartilage, a derivative of the neural crest (NC), and the eye (E) on the injected side. (E and F) At the same stages, DsRed (positive control) expression was apparent in the myotomes (M) of the tadpoles. (G and H) Detailed view of the eye at NF stage 55 indicates that EGFP reporter expression was maintained on the injected (left) side, while no expression was observed in the non-injected (right) side. (I and J) Lateral and dorsal view of transgenic albino Xenopus laevis tadpoles upon unilateral injection of the reporter construct containing IRX1_cCRE7 in the two-cell stage demonstrated EGFP reporter expression in the eye and brain (B). (K and L) Lateral and dorsal view of transgenic tadpoles injected with the same construct, but in two of the dorsal blastomeres at the four-cell stage, also displayed EGFP reporter expression in the eye and brain. (M and N) Lateral and dorsal view of transgenic tadpoles introduced with the IRX1_cCRE10 reporter construct demonstrated low EGFP reporter expression in the eye and brain. (O–Q) DsRed (positive control) was expressed in the myotomes of the corresponding tadpoles. (R and S) Lateral and dorsal view of transgenic albino Xenopus laevis tadpoles upon injection of the reporter construct containing the PRDM13_cCRE1 region demonstrated EGFP reporter expression in the eye and brain. (T) Lateral view of transgenic tadpoles injected with mutational hotspot-1 (PRDM13_cCRE3) showed no EGFP reporter expression in the eye or brain. (U and V) Lateral and dorsal view of transgenic tadpoles injected with mutational hotspot-2 (PRDM13_cCRE5) demonstrated EGFP reporter expression in the eye and brain. (W) For the five cCREs analyzed using in vivo enhancer detection assays, DNase-seq profiles generated in human embryonic retinal tissue at five different developmental stages are given. In case of IRX1_cCRE7, IRX1_cCRE10, PRDM13_cCRE1, and PRDM13_cCRE7, open chromatin is observed at/until day 103 of development, while PRDM13_cCRE1 is closed exclusively at this period.
	Figure 5. Overview of (likely) pathogenic variants in the PRDM13 locus and IRX1 locus Known and novel SNVs are indicated by red and green bars, respectively. The pathogenic tandem duplications are shown as blue bars, while benign duplications spanning the IRX1 coding region, derived from DGV, are shown as pink bars. The five cCREs analyzed via in vivo enhancer assays in Xenopus are highlighted by grey vertical bars. The DNase-seq track was generated in human embryonic retinal tissue at day 103 of development. Chromosome coordinates are in hg38 annotation. PRDM13 locus, left; IRX1 locus, right; V, variant; DGV, Database of Genomic Variants.
	Figure 6. Overview of the in vitro mutant versus wild-type luciferase assays in ARPE-19 cells (A) In mutational hotspot-1 (PRDM13_cCRE3) upstream of PRDM13, the four known (V1–V3, V12) variants and the V16 variant demonstrate a significant increase of luciferase reporter activity (p < 0.001), relative to the wild-type vector. (B) In contrast, the two known (V10, V11) variants and the V15 variant located in mutational hotspot-2 (PRDM13_cCRE5) upstream of PRDM13 cause a significant decrease of luciferase reporter activity (p < 0.001), relative to the wild-type vector. (C) For three non-coding regions of interest, located in the shared duplicated region of either the PRDM13 or IRX1 locus, the level of luciferase reporter was reduced by half (p < 0.001) when the region was present as tandem duplication, relative to their respective wild-type counterpart, containing the same region of interest as a single insert. neg, negative; pos, positive; ∗∗∗p < 0.001.
	Figure 7. Single-cell transcriptomic analysis of developing human neural retina In particular, data from six embryonic (e) (53, 59, 74, 78, 113, and 132 days) and three adult (25, 50, and 54 years old) human retinal samples are included. (A) UMAP plot of 60,014 human neural retinal cells from all samples, colored based on the ten transcriptionally distinct clusters represented in the key. (B and C) The different retinal samples were grouped into four time points: e50 = d53 and d59, e70 = d74 and d78, e100 = d113 and d132, adult = 3 samples. Violin plots illustrate respective PRDM13 and IRX1 expression in the different retinal cell types for each of these time points. RPCs, retinal precursor cells.
	Figure S1 - Schematic representation of the SED vector The transgenesis internal control cassette is composed of the Cardiac Actin promoter (pink), driving strong expression in the somites, and the DsRed fluorescent protein (red), serving as a control for transgenesis efficiency in vivo in the F0 and the F1 embryos. The enhancer detection cassette contains a Gateway entry site (yellow), the gata2 minimal promoter (light green), and the enhanced green fluorescent protein (EGFP) reporter gene (dark green). EGFP reporter expression can be observed during early development under a fluorescent microscope. Both cassettes are flanked by insulator sequences (purple) to protect them from position effects, and together they are flanked by I-SceI meganuclease recognition sites (blue).
	Figure S2 - Post-dissection expression analysis of human donor retina Output from the qPCR expression analysis of the two dissected retinal samples, compared against a control sample of RPE tissue. The retinal genes CRX, RHO, and OPN1LW are highly expressed in the retinal tissues, while expression of the RPE-specific RPE65 gene is minimal relative to the RPE sample.
	Figure S3 - Overview of identified cCREs of the PRDM13 (A) and IRX1 (B) loci The cCREs are obtained after integration of the generated UMI-4C profiles in the retina-specific multi-omics database. The cCREs were defined as non-coding regions interacting with the PRDM13 or IRX1 promoter while demonstrating overlap with peaks of epigenomics datasets or containing a UCNE or NCMD-associated genetic variants. Known and novel pathogenic SNVs are respectively indicated as red and green bars, while previously reported tandem duplications are indicated as blue bars. cCRE: candidate CRE. UCNE: ultra-conserved noncoding element.
	Figure S4 - Reverse UMI-4C experiment using IRX1_cCRE10 as viewpoint The resulting UMI-4C profile suggests an interaction of this region with the IRX1 promoter, as indicated by the diagonal arrow. However, in the UMI-4C experiment using the IRX1 promoter as viewpoint, the interaction is less evident. The three tandem duplications are displayed as blue bars. The UCNE located in this region is indicated by the horizontal arrow. UCNE: ultra-conserved non-coding element.
	Figure S5 - Overview of the genetic and clinical findings in family F1 (A) Pedigree of family F1. Individuals that were subjected to genetic testing are colored in red or green, respectively indicating whether the heterozygous V15 variant (g.99599064A>G (chr6, hg38)) is present or not. (B) F1-III:1. Fundus BE (top): yellowish-white lesions in the macula, encircled by hypopigmented halos and surrounded by hyperpigmentation. OCT BE (bottom): focal RPE damage and focal neurosensory detachment. (C) F1-IV:1. Fundus BE (top): abnormality of macular pigmentation, drusen, focal RPE atrophy, and gross pigment deposits. OCT BE (bottom): focal RPE damage. RE: focal neurosensory detachment. LE: submacular scarring. (D) F1-IV:2. Fundus BE (top): mild RPE abnormalities with small macular drusen. OCT BE (bottom): mild RPE abnormalities with small macular drusen. (E) F1-II:3. Fundus BE (top): chorioretinal macular atrophy with fibrotic changes more pronounced in the LE. OCT BE (bottom): chorioretinal macular atrophy with fibrotic changes more pronounced in the LE, and vitreofoveal adhesion. This family was previously clinically described by Nekolova et al. (2021).1 LE: left eye, RE: right eye, BE: both eyes.
	Figure S6 - Overview of the genetic and clinical findings in family F2 (A) Pedigree of family F2. Individuals that were subjected to genetic testing are colored in red or green, respectively indicating whether the heterozygous V16 variant (g.99593030G>C (chr6, hg38)) is present or not. (B) F2-III:1 and (C) F2-III:2. Fundus BE: symmetrical coloboma-like macular malformation indicative of grade 3 NCMD. BE: both eyes.
	Figure S7 - Overview of the genetic and clinical findings in family F3 (A) Pedigree of family F3. Individuals that were subjected to genetic testing are colored in red or green, respectively indicating whether the known heterozygous V1 variant (g.99593030G>T (chr6, hg38)) is present or not. (B) F3-III:2. Multicolor (blue, green, and infrared) reflectance images BE: severe bilateral lesions of the RPE affecting the entire macular area, in accordance with grade 3 NCMD (C) F3-III:2. OCT LE: excavated lesion with a temporal shelving edge and subretinal fibrosis. (D) F3-II:1 and (E) F3-II:3. Fundus BE. Mild bilateral yellow specks in the central macula, compatible with grade 1 NCMD are indicated by an arrow. LE: left eye, RE: right eye, BE: both eyes.
	Figure S8 - Visual representation of the results from the in silico assessment of non-coding SNVs using the TRANSFAC database For each of the non-coding SNVs located in the two mutational hotspots upstream of PRDM13, a comparison of the predicted TFBS motifs between the mutant and wild-type sequence is given. The green and red box highlight which TFBS motifs are respectively gained or lost, as a result of the specific nucleotide change. The grey bar highlights the nucleotide change. (A-E) Variant V1, V2, V3, V12, and V16, located in mutational hotspot-1 (PRDM13_cCRE3). (F-H) Variant V10, V11, and V15, located in mutational hotspot-2 (PRDM13_cCRE5).
	Figure S9 - Visualization of the joint point of the eight previously reported NCMD-associated tandem duplications Inserted bases and microhomology are respectively indicated in pink and blue, when present.
	Figure S10 - Single-cell transcriptomic analysis of developing human neural retina UMAP plot showing expression of (A) PRDM13 and (B) IRX1 for four different time points (e50 = d53 and d59, e70 = d74 and d78, e100 = d113 and d132, adult = 3 samples) Plots are scaled to the maximum expression of PRDM13 and IRX1, respectively. (C) Annotation of the ten transcriptionally distinct clusters.

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