Analysis of autism spectrum disorder
This application provides methods to differentiate between subjects with autism spectrum disorder (ASD) and typically developing (TD) or developmentally delayed (DD) subjects using miRNA and/or microbiome levels detected in saliva samples and patient information. The method can be used to monitor the progress of ASD and guide its treatment. RNA-seq, qPCR, or other methods determine counts and abundance of miRNA or microbiomes. MicroRNA and/or microbiome sequencing data are refined by normalization to expression levels or abundance of time-invariant miRNAs and/or microbial RN As to control for time of sample collection or to compensate for circadian fluctuations in these levels. Multivariate logistic regression and nonlinear classification techniques are further used to select a panel of miRNAs and microbiomes that accurately differentiate between subjects with ASD, DD, and TD in subjects with an unknown ASD status. These panels of miRNAs and microbiomes may be developed into a RNA assay kit.
1. A method for detecting or diagnosing an autism spectrum disorder (ASD) comprising
(a) determining abundance or concentration level(s) of one or more micro-RNAs (miRNAs) in a saliva sample taken from a human subject, and
(b) comparing the determined abundance or concentration level(s) of the one or more miRNAs against normal level(s) of the same one or more miRNAs, wherein the normal level is that found in a subject or an average from two or more subjects not having an ASD, and
(c) selecting a subject having an abnormal level of said one or more miRNAs as having, or as being at higher risk for having ASD, and
(d) treating the selected subject with a regimen effective for treating autism, wherein said regimen comprises administering one or more treatment selected from the group consisting of drug therapy, antimicrobial therapy, diet or nutritional therapy, physical therapy, phototherapy, psychotherapy, behavior therapy, or an alternative medical therapy; wherein the one or more miRNAs is at least one selected from the group consisting of miR-502-3p, miR-125a-5p, miR-149-5p, miR-193a-5p, miR-4705, miR-99b-5p, miR-340-5p, miR-183-5p, miR-665, and miR-3074-5p.
2. The method of claim 1 , wherein said miRNA expression levels are normalized to an abundance level, or average abundance level of one or more housekeeping genes whose RNA expression is substantially invariant; and/or adjusted to compensate for differences in age, sex, or genetic background.
3. The method of claim 1 , wherein said one or more miRNAs additionally includes one or more miRNA selected from the group consisting of miR-151a-5p, miR-21-5p, miR-665, miR-25-3p, miR-221-3p, miR-30e-5p, mir-125b-2-3p, and miR-146a-3p.
4. The method of claim 1 , wherein said one or more miRNAs additionally includes one or more miRNA selected from the group consisting of miR-502-5p, miR-125a-3p, miR-149-3p, miR-193-3p, miR-4705-3p, miR-4705-5p, miR-99b-3p, miR-340-3p, miR-183-3p, miR-665-3p, miR-665-5p, and miR-3074-3p.
5. The method of claim 1 , wherein said one or more miRNAs additionally includes one or more miRNA selected from the group consisting of miR-28-3p, miR-148a-5p, miR-151a-3p, miR-125b-5p, miR-130b-3p, miR-92a, let-7d-3p, mir-598, miR-374c-5p, miR-374b-5p, miR-29c-3p, miR-1972, miR-675-3p, miR-7706, miR-500a-3p, miR-374a-S5p, miR-190a-Sp, and let-7e-5p.
6. The method of claim 1 , wherein said one or more miRNAs additionally includes one or more miRNA selected from the group consisting of miR-620, and miR-1277-5p.
7. The method of claim 1 , wherein said one or more miRNAs additionally includes one or more miRNA selected from the group consisting of miR-28-3p, miR-584-5p, let-7a-5p, miR-944, miR-148a-5p, miR-151a-3p, miR-125b-2-3p and miR-7706.
8. The method of claim 1 , wherein said one or more miRNAs additionally includes one or more miRNA selected from the group consisting of miR-620, and miR-1277-5p, miR-620, miR-1277-5p, and miR-193a-5p.
9. The method of claim 1 , wherein said one or more miRNAs additionally includes one or more miRNA selected from the group consisting of miR-28-3p, miR-584-5p, let-7a-5p, miR-944, miR-148a-5p, miR-151a-3p, miR-125b-2-3p and miR-7706, miR-125b-2-3p and miR-7706.
10. The method of claim 1 , wherein said one or more miRNAs additionally includes one or more miRNA selected from the group consisting of mir-374c-5p, miR-29c-3p, or miR-190a-5p, miR-28-3p, miR-148a-5p, miR-151a-3p, miR-125b-5p, miR-92a, mir-1972, and miR-7706.
11. The method of claim 1 , wherein said one or more miRNAs additionally includes one or more miRNA selected from the group consisting of miR-620, miR-1277-5p, let-7a-5p and miR-944.
12. The method of claim 1 , wherein said one or more miRNAs additionally includes one or more miRNA selected from the group consisting of miR-28-3p, miR-584-5p, let-7a-5p, miR-944, miR-148a-5p, miR-151a-3p, miR-125b-2-3p and miR-7706.
13. The method of claim 1 , wherein said one or more miRNAs additionally includes one or more miRNA selected from the group consisting of miR-620, and miR-1277-5p, let-7a-5p, and miR-944.
14. The method of claim 1 , wherein said one or more miRNAs additionally includes one or more miRNA selected from the group consisting of miR-28-3p, miR-584-5p, let-7a-5p, miR-944, miR-148a-5p, miR-151a-3p, miR-125b-2-3p, miR-7706.
15. The method of claim 1 , wherein said one or more miRNAs additionally includes one or more miRNA selected from the group consisting of mir-374c-5p, miR-29c-3p, and miR-190a-5p, miR-130b-3p, let-7d-3p, miR-598, mir374b-5p, miR-675-3p, mir-500a-3p, miR-374a-5p, and let-7d-5p.
16. The method of claim 1 , wherein the saliva sample is taken from the human subject at a particular time of day and the concentration level(s) of miRNAs in said sample are compared to normal miRNA values in saliva taken at the same time of day under otherwise identical conditions.
17. The method of claim 1 , wherein the saliva sample is taken within 1 hour of waking, before brushing or rinsing the mouth, before eating or drinking, and/or before exercise that elevates heart rate.
18. The method of claim 1 , wherein said selecting comprises selecting a subject having abnormal levels of four or more of said miRNAs, and, calculating a Pearson correlation coefficient of said abnormal miRNA levels with likelihood of at least one symptom of an ASD.
19. The method of claim 1 , wherein said selecting comprises selecting a subject having abnormal levels of two or more of said miRNAs, and, calculating a Pearson correlation coefficient of said abnormal miRNA levels with likelihood of at least one symptom of an ASD.