North Carolina Macular Dystrophy Is Caused by Dysregulation of the Retinal Transcription Factor PRDM13

Abstract

Purpose: To identify specific mutations causing North Carolina macular dystrophy (NCMD).

Design: Whole-genome sequencing coupled with reverse transcription polymerase chain reaction (RT-PCR) analysis of gene expression in human retinal cells.

Participants: A total of 141 members of 12 families with NCMD and 261 unrelated control individuals.

Methods: Genome sequencing was performed on 8 affected individuals from 3 families affected with chromosome 6-linked NCMD (MCDR1) and 2 individuals affected with chromosome 5-linked NCMD (MCDR3). Variants observed in the MCDR1 locus with frequencies <1% in published databases were confirmed using Sanger sequencing. Confirmed variants absent from all published databases were sought in 8 additional MCDR1 families and 261 controls. The RT-PCR analysis of selected genes was performed in stem cell-derived human retinal cells.

Main outcome measures: Co-segregation of rare genetic variants with disease phenotype.

Results: Five sequenced individuals with MCDR1-linked NCMD shared a haplotype of 14 rare variants spanning 1 Mb of the disease-causing allele. One of these variants (V1) was absent from all published databases and all 261 controls, but was found in 5 additional NCMD kindreds. This variant lies in a DNase 1 hypersensitivity site (DHS) upstream of both the PRDM13 and CCNC genes. Sanger sequencing of 1 kb centered on V1 was performed in the remaining 4 NCMD probands, and 2 additional novel single nucleotide variants (V2 in 3 families and V3 in 1 family) were identified in the DHS within 134 bp of the location of V1. A complete duplication of the PRDM13 gene was also discovered in a single family (V4). The RT-PCR analysis of PRDM13 expression in developing retinal cells revealed marked developmental regulation. Next-generation sequencing of 2 individuals with MCDR3-linked NCMD revealed a 900-kb duplication that included the entire IRX1 gene (V5). The 5 mutations V1 to V5 segregated perfectly in the 102 affected and 39 unaffected members of the 12 NCMD families.

Conclusions: We identified 5 rare mutations, each capable of arresting human macular development. Four of these strongly implicate the involvement of PRDM13 in macular development, whereas the pathophysiologic mechanism of the fifth remains unknown but may involve the developmental dysregulation of IRX1.

Figure 1

Retinal images spanning 30 years from the left eye of an affected member of Family A: color fundus photograph (A), red free fundus photograph (B), early phase fluorescein angiogram (C) and late phase fluorescein angiogram (D) at age 6; color fundus photographs at ages 8 (E), 10 (F), 11 (G) and 33 years (H); optical coherence tomogram at age 33 years (I, J). This patient has been previously reported (Table 1). Stereo images of panels B–D are provided in Supplemental Figure 2 (available at http://aaojournal.org).

Figure 2

Discovery of NCMD-causing variants in MCDR1. The critical region of MCDR1 was narrowed to 883kb by a single unaffected recombinant individual (Supplemental Figure 1J, asterisk, available at http://aaojournal.org). Genome sequencing revealed 14 rare variants (violet vertical bars) across this region, one of which has never been observed in normal individuals (V1). This novel variant falls within a DNAse hypersensitivity site (pink) upstream of the PRDM13 gene (green) that was later found to include other rare variants in NCMD families (V2 and V3). Additionally, a 123kb tandem duplication containing the PRDM13 gene (yellow – V4) was discovered in one NCMD family.

Figure 3

Using normal human iPSCs to model retinal development. A–D: Immunocytochemical analysis of iPSC derived eyecup-like structures targeted against F-actin (Phalloidin), SOX2, PAX6, OTX2, HuC/D and recoverin (RCVRN). After 30 days of differentiation (D30) polarized neural epithelia (A, F-Actin - green) comprised of proliferating cells (A, Ki67 - red) positive for the early retinal progenitor cell markers SOX2 (B, green), PAX6 (B, red) and OTX2 (B, white) are present. After 60 days of differentiation, PAX6 (C, red) expression is restricted to OTX2 negative presumptive RPE while OTX2 (C, white) is restricted to PAX6 negative photoreceptor precursor cells. After 100 days of differentiation, eyecups are laminated with HuC/D-positive (D, green) ganglion cell like neurons in the inner layer and recoverin-positive (D, Red) photoreceptor precursor cells in the outer layer. Insets depict individual fluorescent channels. A & D: 40X magnification. B & C: 20X magnification.

Figure 4

Retinal expression of PRDM13 is developmentally regulated. RT-PCR analysis of iPSCs after 0, 30, 60 and 100 days of retinal differentiation using primers targeted against the retinal lineage markers PAX6, s-Opsin, and Rhodopsin, and genes within the MCDR1 locus, PRDM13 and CCNC. As iPSCs progress from a pluripotent state to immature PAX6-expressing retinal progenitor cells to mature s-Opsin-expressing cone and rhodopsin-expressing rod photoreceptor cells, PRDM13 expression decreases (PRDM13 iPSC-L1, iPSC-L2 and iPSC-L3). iPSC-L1 – Control iPSC line 1. iPSC-L2 - Control iPSC line 2. iPSC-L3 - Control iPSC line 3.

Figure 5

Discovery of the NCMD-causing variant in MCDR3. A: Using whole genome sequencing, a 900kb duplication (yellow – V5) containing the gene IRX1 (green) was found in a family mapped to MCDR3. B: RT-PCR of developing iPSC-derived photoreceptor precursor cells revealed that unlike PRDM13, IRX1 expression is consistent across all developmental time points tested.