A trio of recent publications in the journal NEURON reports the presence of hundreds of diverse de novo gene mutations indicating that autism spectrum disorder (ASD) may be a disease of genomic instability, with a significant environmental component. Altered double strand break formation and repair pathways (DSB) may be a commonality among the diverse genetic mutations that have been documented in ASD. US birthyear changepoints in AD are apparent in 1980, 1988 and 1996, coinciding with the switch to or introduction of childhood vaccines contaminated with human endogenous retrovirus K (HERVK) and human fetal DNA fragments. The HERVK and human fetal DNA contaminants could contribute to the genomic instability of ASD demonstrated by de novo mutations.
Objectives:
Human fetal DNA introduced with vaccines may be taken up via cellular DNA uptake receptors or may spontaneously penetrate cell membranes that have become permeable during inflammatory reactions. This foreign DNA could be integrated into the host genome during double strand break repair. In this study we demonstrate foreign DNA uptake in human cells and genomic integration by incubating the cells with Cy3-labeled human Cot1 (placental) DNA fragments.
Methods:
Human Cot1 DNA labeled with Cy3 was incubated with human cell lines for 24-48 hours. Suspension cell lines included U937 (monocytic leukemia) and HL-60 (promyelocytic leukemia), loosely adherent NCCIT (teratocarcinoma), and adherent cell lines were HFF1 (human foreskin fibroblast), BE (2)-C (neuroblastoma), M059J and M059K (glioblastoma). Cy3 labeled DNA uptake was viewed under fluorescent microscope. Genomic DNA was purified from U937 cells, and the amount of Cy3 labeled human cot1 DNA was calculated from the relative fluorescent unit (RFU) of Cy3 in the U937 DNA measured using spectrofluorimetry.
Results:
Spontaneous cellular and nuclear DNA uptake was evident in HFF1 and U937. Spontaneous cellular uptake was seen in NCCIT. DNA uptake in BE (2)-C, M059J, and M059K was not measurable because of high auto fluorescence of the cells. No Cy3 signal was observed in HL-60. The amount of labeled Cy3 human Cot1 DNA incorporation in U937 genomic DNA was 0.0111 +/- 0.0034pg (n=12) per cell in 24 hours, which was approximately 0.167% of total U937 genomic DNA.
Table: DNA uptake in Various Cell lines
|
Spontaneous Cellular uptake |
Spontaneous Nuclear uptake |
Genomic DNA incorporation |
Cellular Uptake With Permeabilization |
HFF1 |
Yes |
Yes |
ND |
Yes |
NCCIT |
Yes |
ND |
ND |
ND |
BE(2)-C |
No |
No |
No |
No |
M059K |
No |
No |
No |
No |
M059J |
No |
No |
No |
No |
U937 |
Yes |
Yes |
Yes |
Yes |
HL60 |
No |
No |
No |
No |
Conclusions:
This study demonstrates that primitive short DNA fragments (50-300 bp) are spontaneously taken up by HFF-1, U937 and NCCIT cells and inserted into the genome of the monocytic leukemia cell line U937. Hence, vaccines containing residual HERVK and human fetal DNA fragments may contribute to the genomic instability observed in ASD.