Purpose To identify specific mutations causing North Carolina Macular Dystrophy (NCMD). Study Design Whole genome sequencing coupled with RT-PCR analysis of gene expression in human retinal cells. Subjects 141 members of 12 families with NCMD and 261 unrelated control individuals. Methods Genome sequencing was performed on eight affected individuals from three families affected with chromosome-6-linked NCMD (MCDR1) and two individuals affected with chromosome-5-linked NCMD (MCDR3). Variants observed in the MCDR1 locus with frequencies of less than 1% in published databases were confirmed using Sanger sequencing. Confirmed variants absent from all published databases were sought in affected individuals from 8 additional MCDR1 families and the 261 controls. RT-PCR analysis of selected genes was performed in stem-cell-derived human retinal cells. Main Outcome Measure Cosegregation of rare genetic variants with disease phenotype. Results Five sequenced individuals with MCDR1-linked NCMD shared a haplotype of 14 rare variants that spanned one megabase of the disease-causing allele. One of these variants (V1) was absent from all published databases and all 261 controls, but was found in five additional NCMD kindreds. This variant lies in a DNase 1 hypersensitivity site (DHS) upstream of both the PRDM13 and CCNC genes. Sanger sequencing of 1000 base pairs centered on V1 was performed in the remaining four NCMD probands and two additional novel single nucleotide variants (V2 in three families and V3 in a single family) were identified in the DHS within 134 base pairs of the location of V1. A complete duplication of the PRDM13 gene was also discovered in a single family (V4). RT-PCR analysis of PRDM13 expression in developing retinal cells revealed marked developmental regulation. Next generation sequencing of two individuals affected with chromosome-5-linked NCMD revealed a 900kb duplication that included the entire IRX1 gene (V5). The five mutations V1–V5 segregated perfectly in the 102 affected and 39 unaffected members of the 12 NCMD families. Conclusion We have identified five rare mutations that are each capable of arresting the development of the human macula. Four of these strongly implicate the involvement of the gene PRDM13 in macular development, while the pathophysiologic mechanism of the fifth remains unknown but may involve the developmental dysregulation of IRX1.

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