The current study identified FXS-specific changes in miRNAs among Iranian blood samples. We identified twenty-five differentially expressed miRNAs sequenced in blood of individuals with FXS compared to the controls, and we found minor downregulation of miR-125a-5p. The main finding of this study is that levels of three miRNAs (i.e., hsa-miR-532-5p, hsa-miR-652-3p and hsa-miR-4797-3p) were significantly upregulated in FXS group versus healthy controls while levels of six miRNAs (i.e., hsa-miR-191-5p, hsa-miR-181-5p, hsa-miR-26a-5p, hsa-miR-30e-5p, and hsa-miR-186-5p, hsa-miR-4797-5p) exhibited significant downregulation in FXS patients compared to controls; and these dysregulations were confirmed by RT-qPCR.
MicroRNAs regulate mRNAs at the post-transcriptional level and therefore affecting protein translation . Changed miRNA expression patterns epigenetically affect almost every aspect of CNS function (i.e., in neurogenesis, synaptogenesis and neuronal migration) and its development [20–22]. For instance, miR-532, is reliably expressed in the human brain, localized as distinct granules in distal axons and growth cones, and proposed to play a role in axon growth and guidance . ZFHX3 gene, among 5 target genes of the hsa-miR-532-5p from the MiRTarBase microRNA Targets dataset, encodes a transcription factor that regulates neuronal differentiation . Hence, unsurprisingly, in many neuropsychiatric disorders it has been demonstrated that the dysregulation of miRNAs is associated with changes in behaviour, learning, and memory . Understanding the miRNA-mediated translational regulation mechanism(s) whereby FMRP modulate the translation of its mRNA ligands would help in understanding of the molecular pathogenesis of FXS and also of converging mechanisms shared by FXS and its related disorders . FXS is a well-known monogenic cause of autism spectrum disorder (ASD) ; list of other related disorders to FXS may include but not limited to FXTAS, Rett syndrome, Down syndrome, attention deficit hyperactivity disorder (ADHD), and schizophrenia.
FXS is the first neurodevelopmental disease found to be linked to the dysfunction of miRNA pathway . The in vivo evidence of miRNA involvement in FXS pathogenesis was first provided in a study of the zebrafish model by identifying and isolating numerous miRNAs, including miR-fmr1-27 and miR-fmr1-42 in this model . Subsequent studies in the Fmr1 KO mouse models found that disruption of the regulating of miR-125a, miR-125b, and miR-132 causes early neural development and synaptic physiology  and that there is an interaction between miR-34b, miR-340, and miR-148a with the Met 3′ UTR of the FMR1 gene . Moreover, by isolating mesenchymal stem cells from peripheral blood and differentiating these cells into neuronal cells, Fazeli et al.  recently analyzed the expression of miR-510 by qPCR method. The authors reported an enhanced expression of miR-510, located on chromosome X in the 27.3Xq region, flanking to a fragile X site, in the female carriers of FMR1 full mutation .
There are twelve brain miRNAs identified to interact with FMRP in mouse brain including, miR-125a, miR-125b, and miR-132 . To the best of our knowledge, there is only one published study on miRNA expression profiling in FXS patients using deep sequencing . In a most recent study Putkonen et al.  showed upregulation of miR-125a in urine from children with FXS. The investigators did not examine differential miRNA expression changes in FXS blood samples or the correlations of miR-125a levels in urine with those of in the cell-free circulation (i.e., in serum and plasma) and other body fluids . In our study, we found a minor downregulation of miR-125a-5p in blood of individuals with FXS. One preliminary hypothesis for this finding is that due to urinary secretion or of miR-125a-5p, its blood level expression decreased similar to what happens for blood-urine balance of electrolytes.
In line with our results in Alvarez-Mora MI study on FXTAS patients using deep sequencing, the authors also observed a slight but not significant reduction of miR-125a-5p in blood of FXTAS patients . Mundalil Vasu et al. , found thirteen differentially expressed serum miRNAs in individuals with autism spectrum disorder (ASD) compared to the controls and miR-125a was not among the dysregulated miRNAs.
Even though larger studies are needed to confirm our results and investigate the effect of other miRNAs, the changes in miRNAs seen among our patients provide evidence that these miRNAs could have roles in developmental processes, nervous system homeostasis, and the function of nerve cells in those with FXS. Our finding demonstrates significant involvement of hsa-miR-30e-5p in FXS, which was found to be the most significantly upregulated miRNA in FXS patients compared with controls. miR-30 family play a major regulating role in the tissue and organ development and the pathogenesis of various clinical diseases . Several studies have shown that hsa-miR-30e-5p among other miRNAs might be associated with the onset and progression of Parkinson's disease and schizophrenia [36–38]. Sun et al.  observed significant increase in expression of has-miR-30e in both plasma samples and peripheral blood mononuclear cells (PBMC) samples amongst schizophrenia patients.
Our results also showed deregulated hsa-miR-191-5p. Although, as far as we are aware of, no evidence for hsa-miR-191-5p contribution to FXS has been reported so far, alterations in expression level of hsa-miR-191-5p has earlier been found in patients with neuropsychiatric disorders sharing genetic overlap with FXS, including ASD, ADHD, schizophrenia, bipolar disorder, and major depressive disorder [40–43].
Moreover, in concordance with our results, the association between autistic traits and X-linked SNPs in the gene family linked with FXS, is likely to be owing to a disruption in the recognition between has-miR-181 and the corresponding seed match sequences in these genes ; miR-181d and FMRP cooperatively regulate the axon elongation process [10, 45]. Altered expression pattern for miR-181 and miR-191 in hippocampal neuron development has shown to occur .
In one recent study, 13 miRNAs were differentially expressed in maternal plasma samples from pregnant women with fetal Down syndrome versus healthy control subjects; among the others, hsa-miR-191 was upregulated and hsa-miR30e downregulated . In another study, miR-26b-5p, miR-185-5p, and miR-191-5p were identified as potential biomarkers for ADHD in peripheral blood mononuclear cells . Altered expression of miR-26a and miR-26b have been shown in peripheral blood of major depressive patients during antidepressant therapy, in Alzheimer's disease and in Parkinson's disease . Finally, hsa-miR-532-5p and hsa-miR-652-3p have been shown to be upregulated in schizophrenia [48, 49] .
It is noteworthy that despite large number of miRNAs associated with FXS and its related disorders that have been identified in multiple expression studies, only a few miRNAs are common between various studies. This discrepancy can be explained in part by the polygenic and complex nature of neuropsychiatric disorders . The expression profiles of the miRNAs in our study confirm some existing findings but conflict with others. For example, our result regarding expression level of has-miR-30e in FXS patients is consistent Sun et al.  report that miR-30e was upregulated in PBMCs from patients with schizophrenia. In contrast, Perkins et al.  have found that miR-30e is downregulated in the prefrontal cortex of subjects with schizophrenia compared with healthy subjects. The exact reason for these conflicting results remains to be determined but it may be because of differences in screening standards (i.e., patients' ethnicity, geographical region, and screening criteria), techniques used for miRNA detection and profiling, and experimental design. Furthermore, as previously mentioned by Alvarez-Mora et al. , it is documented that the expressions of miRNAs are tissue-specific and/or temporally regulated which may partially explain the differences seen between the findings of different studies.
Several limitations to our study should be addressed. First, the sample size is relatively small. Despite this, it is the first evidence of altered miRNA expressions in blood samples from FXS patients. Second, although patients consisted of individuals from all across the country, they were only Iranian in origin. Third, we examined miRNA expression changes in non-neuronal cells due to the fact that neuronal tissue is not easily accessible. However, it has been shown that miRNA expression changes in the peripheral circulation are highly correlated with those of neuronal tissue from patients with various neuropsychiatric disorders .