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.
The geographic origins of populations can be identified by their maternally inherited mitochondrial DNA (mtDNA) haplogroups. This study compared human cybrids (cytoplasmic hybrids), which are cell lines with identical nuclei but mitochondria from different individuals with mtDNA from either the H haplogroup or L haplogroup backgrounds. The most common European haplogroup is H while individuals of maternal African origin are of the L haplogroup. Despite lower mtDNA copy numbers, L cybrids had higher expression levels for nine mtDNA-encoded respiratory complex genes, decreased ATP turnover rates and lower levels of ROS production, parameters which are consistent with more efficient oxidative phosphorylation. Surprisingly, GeneChip arrays showed that the L and H cybrids had major differences in expression of genes of the canonical complement system (5 genes), dermatan/chondroitin sulfate biosynthesis (5 genes) and CCR3 signaling (9 genes). Quantitative nuclear gene expression studies confirmed that L cybrids had (a) lower expression levels of complement pathway and innate immunity genes and (b) increased levels of inflammation-related signaling genes, which are critical in human diseases. Our data support the hypothesis that mtDNA haplogroups representing populations from different geographic origins may play a role in differential susceptibilities to diseases.
BackgroundMitochondrial dysfunction is associated with the development and progression of age-related macular degeneration (AMD). Recent studies using populations from the United States and Australia have demonstrated that AMD is associated with mitochondrial (mt) DNA haplogroups (as defined by combinations of mtDNA polymorphisms) that represent Northern European Caucasians. The aim of this study was to use the cytoplasmic hybrid (cybrid) model to investigate the molecular and biological functional consequences that occur when comparing the mtDNA H haplogroup (protective for AMD) versus J haplogroup (high risk for AMD).Methodology/Principal FindingsCybrids were created by introducing mitochondria from individuals with either H or J haplogroups into a human retinal epithelial cell line (ARPE-19) that was devoid of mitochondrial DNA (Rho0). In cybrid lines, all of the cells carry the same nuclear genes but vary in mtDNA content. The J cybrids had significantly lower levels of ATP and reactive oxygen/nitrogen species production, but increased lactate levels and rates of growth. Q-PCR analyses showed J cybrids had decreased expressions for CFH, C3, and EFEMP1 genes, high risk genes for AMD, and higher expression for MYO7A, a gene associated with retinal degeneration in Usher type IB syndrome. The H and J cybrids also have comparatively altered expression of nuclear genes involved in pathways for cell signaling, inflammation, and metabolism.Conclusion/SignificanceOur findings demonstrate that mtDNA haplogroup variants mediate not only energy production and cell growth, but also cell signaling for major molecular pathways. These data support the hypothesis that mtDNA variants play important roles in numerous cellular functions and disease processes, including AMD.
Age-related maculopathy (ARM) is a multifactorial disorder known to have a substantial genetic component. The epsilon4 allele of the apolipoprotein E gene (APOE-4) has previously been reported to have a protective effect on ARM risk, while the APOE-2 allele may increase disease risk. This study combined four independent data sets (three US and one European) of Caucasian ARM patients and controls in order to obtain better statistical power to examine the role of APOE in ARM. APOE genotype and allele frequencies were compared for 617 ARM cases and 1260 controls, adjusting for age and sex differences between the two groups via multiple logistic regression. The protective effect of the APOE-4 allele on ARM risk was confirmed (age- and sex-adjusted odds ratio (OR) for APOE-4 carriers 0.54, 95% confidence interval (CI) 0.41-0.70, p < 0.0001). The effect of APOE-4 did not differ significantly between males and females and was observed consistently for both atrophic and neovascular ARM. Evidence for an increased risk of ARM due to the APOE-2 allele was found for men, but not for women (OR for men 1.54, 95% CI 0.97-2.45; OR for women 0.74, 95% CI 0.52-1.06, p = 0.01 for interaction of sex and APOE-2 carrier status). These data confirm that the APOE-4 allele, or an allele in linkage disequilibrium with it, reduces the risk of ARM. They also suggest that the effect of the APOE-2 allele may vary by gender, and that APOE-2 may confer an increased risk only to males.
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