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Danli Xing, Deepika Malik, Payam Falatoonzadeh, Shari Atilano, Marilyn Chwa, S Michal Jazwinski, Miceli Michael, Baruch Kuppermann, Cristina Kenney; Comparison Study of EFEMP1 Gene Expression Levels in Human ARPE-19 Cybrid Models Having Mitochondrial DNA of Haplogroups H, J, K or L. Invest. Ophthalmol. Vis. Sci. 2013;54(15):5013.
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© ARVO (1962-2015); The Authors (2016-present)
EFEMP1 (Epidermal Growth Factor-containing fibulin-like extracellular matrix protein) is a gene expressed in retinal pigment epithelium (RPE) cells and is one of the high-risk genes associated with Age-related Macular Degeneration (AMD). EFEMP1 suppresses VEGFA-induced angiogenesis, and mutations in this gene are associated with drusen formation. Histology of AMD eyes shows that EFEMP1 protein can be found between the RPE cell layer and drusen. It has been suggested that misfolded EFEMP1 protein accumulates within RPE cells causing altered cellular function and inflammation. The aim of this study is to assess the baseline expression of the EFEMP1 gene in a human ARPE-19 cybrid cytoplasmic hybrid (cybrid) model where the cells have identical nuclei, but contain mitochondrial DNA haplogroup H, J, K or L. Haplogroup H and J are of Northern-European descent, however, J generally has more risk of developing AMD while H is protective. Haplogroup K, of Central-European descent, has some risk, and haplogroup L, of African descent, has the least predisposition to developing AMD.
Human ARPE-19 cells that were depleted of mitochondria (Rho0) were fused with platelets from individuals with known H, J, K or L haplogroups to create unique cybrid cell lines. RNA was extracted from individual cybrids and cDNA was synthesized. EFEMP1 gene expression levels were measured using Q-PCR and analyzed for changes in gene expression levels relative to cybrid H.
The expression levels of the EFEMP1 gene were found to be significantly lower in Cybrids J, K and L when compared to cybrid H: cybrid J= 0.25 fold, p=0.0001; cybrid L= 0.4 fold, p=0.008; and cybrid K= 0.17 fold, p=0.003.
This study demonstrates that human ARPE-19 cybrids with identical nuclei but variable mitochondrial DNA haplogroups have differentially expressed levels of the EFEMP1 gene which is involved in VEGF-A induced angiogenesis. Cybrids J, K and L showed significantly lower levels of EFEMP1 gene expression as compared to cybrid H in the following order H > L > J > K. This finding may help us in further understanding the pathogenesis of AMD with regards to differential risks amongst populations
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