Identification of plasma microRNAs as a biomarker of sporadic Amyotrophic Lateral Sclerosis.
BACKGROUND
Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease, which leads to the loss of upper and lower motor neurons, with a currently unknown etiology. Specific biomarkers could help in early detection and diagnosis, and could also act as indicators of disease progression and therapy effectiveness. MicroRNAs (miRNAs) are small (18-25 nucleotides), single-stranded non-coding RNA molecules that play important regulatory roles in animals and plants by targeting mRNAs for cleavage or translational repression, and are essential for nervous system development. Many of the genes associated with genetic ALS have pathological biological pathways related to RNA metabolism, and their pathogenesis may be affecting the maturing processes of miRNA.


RESULTS
We compared miRNA from the plasma of sALS patients and healthy controls using two cohorts; a discovery cohort analyzed with microarray (16 sALS patients and ten healthy controls) and a validation cohort confirmed with qPCR (48 sALS patients, 47 healthy controls and 30 disease controls). We measured the total amount of extracted RNA along with a spike-in control that ensured the quality of our quantification. A percentage of the 10-40 nt RNAs extracted from the total RNA showed a significant increase in ALS patients. There was a negative correlation between total RNA concentration and disease duration from onset to end point. Three of the miRNAs were up-regulated and six were down-regulated significantly in the discovery cohort. Since an internal control is required as a sample stability indicator of both the patients and controls in microarray analysis, we selected the miRNA showing the smallest dispersion and equivalency between the two groups' mean value, and decided to use hsa-miR-4516. We found hsa-miR-4649-5p to be up-regulated, and hsa-miR-4299 to be down-regulated, where each was not influenced by clinical characteristics. EPHA4, a target gene linked to the nervous system which has also been reported to be a disease modifier of ALS, is the common and most notable target gene of hsa-miR-4649-5p and hsa-miR-4299.


CONCLUSIONS
We have shown the relationship circulating plasma miRNA has with both healthy controls and diseased patients. Hsa-miR-4649-5p and hsa-miR-4299 have the potential to be ALS diagnosis biomarkers.
Department of Neurology, Hokkaido University Graduate School of Medicine, North 15 West 7, Kita-ku, Sapporo, Hokkaido, 060-8638, Japan. ikukotak@med.hokudai.ac.jp.
Ikuko
I
Takahashi
Department of Neurology, Hokkaido University Graduate School of Medicine, North 15 West 7, Kita-ku, Sapporo, Hokkaido, 060-8638, Japan. hama@pop.med.hokudai.ac.jp.
Yuka
Y
Hama
Department of Neurology, Hokkaido University Graduate School of Medicine, North 15 West 7, Kita-ku, Sapporo, Hokkaido, 060-8638, Japan. mmasaaki@huhp.hokudai.ac.jp.
Masaaki
M
Matsushima
Department of Neurology, Hokkaido University Graduate School of Medicine, North 15 West 7, Kita-ku, Sapporo, Hokkaido, 060-8638, Japan. hirotani@med.hokudai.ac.jp.
Makoto
M
Hirotani
Department of Neurology, Hokkaido University Graduate School of Medicine, North 15 West 7, Kita-ku, Sapporo, Hokkaido, 060-8638, Japan. tkanoh76@med.hokudai.ac.jp.
Takahiro
T
Kano
Department of Neurology, Obihiro Kosei General Hospital, West 6, South 8, Obihiro, Hokkaido, 080-0016, Japan. hohzen@f1.octv.ne.jp.
Hideki
H
Hohzen
Department of Neurology, Hokkaido University Graduate School of Medicine, North 15 West 7, Kita-ku, Sapporo, Hokkaido, 060-8638, Japan. yabe@med.hokudai.ac.jp.
Ichiro
I
Yabe
Department of Neurology, Hokkaido University Graduate School of Medicine, North 15 West 7, Kita-ku, Sapporo, Hokkaido, 060-8638, Japan. jutsumi@pop.med.hokudai.ac.jp.|Cancer Institute, Japanese Foundation for Cancer Research, 3-8-31, Ariake, Koto, Tokyo, 135-8550, Japan. jutsumi@pop.med.hokudai.ac.jp.
Jun
J
Utsumi
Department of Neurology, Hokkaido University Graduate School of Medicine, North 15 West 7, Kita-ku, Sapporo, Hokkaido, 060-8638, Japan. h-isasak@med.hokudai.ac.jp.
Hidenao
H
Sasaki
D000368:Aged; D000690:Amyotrophic Lateral Sclerosis / Q000097:blood*; D015415:Biomarkers / Q000097:blood*; D016022:Case-Control Studies; D015331:Cohort Studies; D015536:Down-Regulation / Q000235:genetics; D005260:Female; D020869:Gene Expression Profiling; D006801:Humans; D008297:Male; D035683:MicroRNAs / Q000097:blood* / Q000235:genetics; D008875:Middle Aged; D020411:Oligonucleotide Array Sequence Analysis; D010300:Parkinson Disease / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D015203:Reproducibility of Results; D013997:Time Factors; D015854:Up-Regulation / Q000235:genetics
D015415:Biomarkers; D035683:MicroRNAs