September 2016
Volume 57, Issue 12
Open Access
ARVO Annual Meeting Abstract  |   September 2016
Cybrids with mtDNA K Haplogroups Express Elevated Levels of Complement Inhibitors and Decreased Levels of Complement Activator Genes
Author Affiliations & Notes
  • Lillian Choi
    Ophthalmology, Gavin Herbert Eye Inst, UC Irvine, Irvine, California, United States
  • Kunal Thaker
    Ophthalmology, Gavin Herbert Eye Inst, UC Irvine, Irvine, California, United States
  • Shari R Atilano
    Ophthalmology, Gavin Herbert Eye Inst, UC Irvine, Irvine, California, United States
  • Marilyn Chwa
    Ophthalmology, Gavin Herbert Eye Inst, UC Irvine, Irvine, California, United States
  • Cristina M Kenney
    Ophthalmology, Gavin Herbert Eye Inst, UC Irvine, Irvine, California, United States
  • Footnotes
    Commercial Relationships   Lillian Choi, None; Kunal Thaker, None; Shari Atilano, None; Marilyn Chwa, None; Cristina Kenney, None
  • Footnotes
    Support  This work was supported by Discovery Eye Foundation, Polly and Michael Smith Foundation, Iris and B. Gerald Cantor Foundation, Beckman Initiative for Macular Research, Max Factor Family Foundation.
Investigative Ophthalmology & Visual Science September 2016, Vol.57, 5874. doi:
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      Lillian Choi, Kunal Thaker, Shari R Atilano, Marilyn Chwa, Cristina M Kenney; Cybrids with mtDNA K Haplogroups Express Elevated Levels of Complement Inhibitors and Decreased Levels of Complement Activator Genes. Invest. Ophthalmol. Vis. Sci. 2016;57(12):5874.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : Haplogroups are defined by single nucleotide polymorphisms (SNPs) in mtDNA that have accumulated over time and represent different geographic origins of populations. H haplogroup represents the most common European haplogroup, and K haplogroup is commonly found in Ashkenazi Jews. The purpose of this study was to compare transmitochondrial cybrids, possessing identical nuclei but either H or K haplogroup mtDNA, and measure the expression levels of complement inhibitors and activators.

Methods : Cybrid Cultures: Platelets were isolated by a series of centrifugation steps and fused with ARPE-19 cells deficient in mtDNA (Rho0). DNA was isolated and mtDNA haplogroups were determined by PCR, restriction enzyme digestions and sequencing. SNPs defining the H haplogroup were C7028T and A73G. K defining SNPs were G9055A and A12308G.
Quantitative PCR Analyses: Q-PCR was performed using primers for CFH, a major inhibitor for the alternative complement pathway; CD59, an inhibitor for the complement membrane attack complex (MAC); CD55/DAF, regulator of the complement cascade; CFI, an inhibitor that inactivates C4b and C3b; CFHR4, a CFH-related protein that enhances C3b inactivation; and CFP, a positive regulator of alternative pathway activation.

Results : Although all cybrids have identical nuclei, the K cybrids showed increased expression of the complement inhibitors CFH, (ΔΔCT = 1.28±0.36, 2.4-fold, p=0.0012); CD59 (ΔΔCT = 0.98±0.25, 1.98-fold, p=0.004); and CFI (ΔΔCT = 1.15±0.49, 2.2-fold, p=0.025) compared to H cybrids. Gene expression levels were lower in K cybrids for complement activators CFHR4 (ΔΔCT = -1.48±0.49, 0.4-fold, p=0.014); and CFP (ΔΔCT = 0.49±0.26, 0.7-fold, p=0.078) compared to H cybrids. The expression level for CD55/DAF was similar in H and K cybrids (ΔΔCT = -0.23±0.32, 0.85-fold, p=0.49). The H cybrids were assigned a fold value of 1.

Conclusions : Complement inhibitors CFH, CD59, and CFI showed elevated expression levels in K cybrids compared to H cybrids. However, the complement activators, CFP and CFHR4, showed decreased levels of expression in the K cybrids. These data suggest that cells with the K mtDNA haplogroup may have lower levels of complement activation compared to H cybrids, which may contribute to differences in disease susceptibilities found in these populations.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.

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