Cybrids with mtDNA K Haplogroups Express Elevated Levels of Complement Inhibitors and Decreased Levels of Complement Activator Genes

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, (ΔΔC_T = 1.28±0.36, 2.4-fold, p=0.0012); CD59 (ΔΔC_T = 0.98±0.25, 1.98-fold, p=0.004); and CFI (ΔΔC_T = 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 (ΔΔC_T = -1.48±0.49, 0.4-fold, p=0.014); and CFP (ΔΔC_T = 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 (ΔΔC_T = -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.