Circulating microRNAs in Fabry Disease

Fabry disease is an X-linked deficiency of the lysosomal hydrolase alpha-galactosidase A (alpha-Gal). This results in an accumulation of globotriaosylceramide (GL-3/Gb3) in a variety of cells with subsequent functional impairment. The continuous progress of FD often leads to decreased quality of life and premature death caused by multi-organic complications. The overall aim of our study was to determine the amount of circulating miRNAs in Fabry patients and to test whether ERT would alter the level of individual circulating miRNAs. We used miRNA sequencing by the HTG EdgeSeq System to identify the circulating miRNA pool from Fabry patients with and without enzyme replacement therapy (n = 6). In total, 296 miRNAs in serum of patients were identified. Among them 9 miRNAs were further evaluated in extra serum samples (n = 31) using real-time qPCR and 6 of them showed significant differential expression. The resulting miRNA pattern may help to better understand mechanisms involved in the beneficial effects of ERT and these new miRNA markers could help to estimate the efficacy of ERT or to identify Fabry patients with specific need for ERT.

Fabry disease (FD) is an X-chromosome linked disorder caused by mutations in gene GLA coding for alpha-galactosidase-A enzyme (alpha-Gal). The enzyme activity deficiency that results in an accumulation of globotriaosylceramide (GL-3/Gb3) in a variety of cells often leads to subsequent functional impairment 1 . The initial manifestations of Fabry disease usually start in adolescence stage of life, including neuropathic pain (acroparesthesia) and abdominal discomfort 2 . The continuous progress of FD results in decreased quality of life and premature death caused by multi-organic complications 3,4 . As a specific treatment, Enzyme replacement therapy (ERT) has been shown to stabilize and reduce many signs and symptoms of Fabry disease [5][6][7] . More recently, oral chaperone therapy was shown to be also effective in selected Fabry patients depending on the underlying gene mutation 8 . Of clinical importance is the fact that early diagnosis and treatment in the disease course may delay or prevent the progression towards irreversible organ dysfunction and the consequent life-threatening complications. This is sometimes difficult due to the high variability of the severity and multi-organ system involvement in Fabry disease 9 . Next to the clinical features, enzyme activity tests and DNA sequencing are available to confirm the diagnosis 10 . Globotriaosylsphingosine (LysoGb3) serves as a useful biomarker to improve the diagnosis of heterozygous Fabry disease for therapeutic evaluation and monitoring 11 . In addition, circulating serum proteins in the blood of Fabry patients may help to get more information about the underlying pathophysiological mechanisms 12 .
Recently, a group of small RNA molecules known as microRNAs (miRNAs) have been proved to play essential roles in the cardiac function 13,14 . Moreover, the expression levels of miRNAs that present in circulating fluid usually differ between healthy and diseased patients. Although the underlying biological function and origin of these circulating molecules remains unclear, miRNAs are becoming potential biomarkers for early stage diagnosis and treatment response 15 . The overall aim of this study was to determine the amount of circulating miRNAs in Fabry patients and to test whether ERT would alter the level of individual circulating miRNAs.

Materials and Methods
We used RNA sequencing technologies to identify a specific miRNA pattern in serum of Fabry patients (Fig. 1). The inclusion criteria for this study were based on a confirmed mutation within the GLA gene and a classical or non-classical/late-onset clinical phenotype. The diagnostic criteria for FD were based on the recent publication by Biegstraaten et al. 16 : a genetically confirmed GLA mutation leading to deficient AGAL activity combined by one or more characteristic FD signs/symptoms, or an increase of plasma lyso-Gb3, or an additional family member with a definite FD diagnosis. Clinical characteristics of recruited patients were summarized in Table 1.
In brief, the HTG EdgeSeq system was first utilized to identify and quantify the expression of regulated miR-NAs directly in serum of 6 Fabry patients with and without ERT. After the bioinformatic analysis of reads data generated from the high-throughput platform, selected miRNA candidates were further evaluated in extra 31 serum samples (Table 1) from 17 patients with ERT and 14 without. Recruited patients for this study or their parents/legal guardian have signed informed consent before participation. The study has been approved by the local ethical committees of the University Hospital of Münster and the University Hospital of Würzburg therefore were performed in accordance with the Helsinki declaration. The HTG EdgeSeq system utilizes a novel target capture and library prep chemistry that enables easy and fast use of next-generation sequencers such as Illumina for transcriptome analysis including miRNAs. The automated extraction-free chemistry of HTG EdgeSeq reduces the input requirement of samples and eliminates biases due to RNA extraction and library preparation. This increases the reproducibility of libraries prepared from raw precious samples such as serum used in this study. The raw read counts data was then generated by combined NGS sequencer for bioinformatic analyses and the selected candidates were validated with a miRNA-specific RT-qPCR method in extra samples as described previously 17 . All experiments were performed according to corresponding manufacturer's protocols or instructions.
MicroRNA Sequencing and quantification. 15 µl serums from each of 6 patients including 3 treated with ERT for more than one year and 3 without ERT were incubated with HTG lysis buffer and Proteinase K (Ambion) at 20 °C for 2 hours. The sample plates were then loaded into an HTG Edgeseq Processor. After the automated preparation process, library were prepared with TruSeq Small RNA Prep kit (Illumina) according to the manufacturer's instruction. Single-end reads of 51 bp in length were then sequenced on an Illumina GAIIx instrument. For expression level quantification, trimmed reads were mapped to the genome reference (hg19) allowing one mismatch and quantified applying Avadis NGS software (v1.4). Reads mapped to multiple locations in the genome were removed from further quantification. Annotation from miRBase v20 were used to designate reference mapped reads to miRNAs. Data normalization and differential expression analysis. A scaling factor for each sample і, is obtained for each gene g and samples m. The scaling factor S i , is the median gene level expression value for each sample-gene count adjusted by the geometric mean over all genes. Note that any genes without expression over all samples are necessarily excluded from this scaling calculation. The formula for the scaling factor for the i th sample can be written as Eq. (1): Where, r gi is the raw count for the i th sample and g th gene.
The scaling factor is then used to modify the original read counts to obtain the normalized count value r gi nor in Eq. (2):

Results
To identify the circulating miRNA pool from Fabry patients, 6 FD patients and 31 FD patients were recruited as screening cohort and validation cohort, respectively. The clinical characteristics of all patients were summarized in Table 1. At the time of visit there is no significant difference between ERT treated and ERT-naïve patients in age (p = 0.23), IVsd (p = 0.32), MSSI score (p = 0.1) and the ratio of mutation types (p = 0.46 by Fisher's exact test), while the lyso-Gb3 and eGFR in ERT treated group were significantly lower than ERT-naïve patients with p = 0.02 and p = 0.01 respectively. Among the ERT-naïve patients visited in our study, 2 out of 3 in screening cohort, and all 14 in validation cohort were treated with ERT afterwards. By using this innovative extraction-free HTG EdgeSeq system and intensive bioinformatical analyses, 296 miRNAs were detected in at least 4 out of 6 serum samples from Fabry patients (Fig. 1b); among them 269 miR-NAs were expressed in both conditions; 145 miRNAs were found to be regulated more than 1.5 fold independent of p-value (Table 2). In addition, the overall expression pattern of the deregulated miRNAs decently distinguishes between the serums of Fabry patients with and without ERT by Hierarchical Clustering and Principal Component Analysis (Fig. 2).
Of interest many miRNAs were detected by the high-throughput approach for which no clear role in biology or pathophysiology has been described yet. However, some miRNAs were already known in the literature. For instance, overexpression of miR-541 promote vascular smooth muscle proliferation and invasion suggesting that lower miR-541 levels might be beneficial in various vascular and pulmonary diseases 22 . Specific inhibition/silencing of miR-21 have been proved to be able to effectively prevent the myocardial and renal fibrosis 14,23 . The miR-17-92 family that comprises miR-17, miR-18a, miR-19a, miR-19b-1, miR-20a, and miR-92a-1 has been implicated in the promotion of cell proliferation and the growth of renal cysts 24 . Reduced levels of miR-26a were observed to be correlated with kidney injury in renal vascular disease and the restored expression could attenuate interstitial fibrosis and tubular apoptosis hence rescuing the renal function 25 .
Since Fabry disease is an X-chromosome linked genetic disorder that affects male patients more severely than female, we made an additional analysis to compare the expression level of candidate miRNAs in 26 serums of male patients (14 with ERT and 12 without). Of interest two additional miRNAs, miR-19a-3p and miR-486-5p were found to be significantly (p < 0.05) down-regulated in male patients with ERT (Fig. 4). These findings are consistent with the facts that female Fabry patients demonstrate more variable symptoms with a wider range of disease severity 26 and suggest that a gender specific miRNA-expression pattern is necessary to develop the optimal markers for female and male patients, respectively.

Discussion
Although efficacy and clinical effects of ERT in patients with Fabry disease have been investigated and reported [5][6][7][8] , less is known about the mechanism and effect on the molecular level. In this study we performed a direct comparison of the miRNA expression pattern between patients with and without ERT that provide novel ideas to unravel the pathway underlying ERT.
To elucidate the putative underlying molecular mechanisms, mirPath 27 was utilized to make pathway enrichment analysis based on top 100 deregulated miRNAs. Of interest, axon guidance and TGF-beta signaling pathways were found to be targeted by the miRNAs (Fig. 5) Continued with decreased neuropathic pain has been reported in FD patient with ERT 28 , the pathogenesis of the peripheral neuropathy correlated with Fabry disease is poorly understood. The predicted functional changes in axon guidance molecules caused by dysregulated miRNAs could affect the neural circuits developments that result in neurological symptoms in FD patients. Renal impairment is often observed in later stage of Fabry disease, which advances to kidney failure causes significant mortality in FD patients. Improvement and slowing of the renal disease progression have been reported after ERT treatment 29 . More recently, proteomic studies demonstrated that VEGF receptor-2 in plasma of patients was significantly higher than controls and decreased after ERT 12 ; increased expression TGF-β1 and VEGF were found to be associated with the renal pathogenesis of Fabry mouse model 30  www.nature.com/scientificreports www.nature.com/scientificreports/ function of TGF-β signaling pathway involved in nephropathy of Fabry disease, which is in general consist with our result from pathway enrichment analysis.
On the other hand, evaluation of the circulating miRNAs as biomarkers have been performed either in the field of kidney disease or Fabry disease. The concentration of circulating miRNAs in plasma including miR-21 and miR-210 were found to be reduced in patients with chronic renal failure, while no correlation was observed between urinary miRNAs and kidney function 31 . In a recent case study of a young Fabry patient without nephropathy manifestations, the expression level of miR-29 and miR-200 were found to be decreased in urinary sediment while the other TGF-β related miRNAs not 32 . Taken together, although TGF-β signalling pathway was suggested to be associated with Fabry nephropathy 12,28 , there is no direct evidence to support the putative involvement of TGF-β regulated miRNAs in ERT treatment.
In our study, a non-biased approach based on high-throughput sequencing were applied instead of knowledge based candidates selection. Although some known TGF-β related miRNAs e.g. miR-29, miR-192 and miR-200  were excluded from further validation due to the extremely low abundance in screening result, our result from pathway enrichment analysis still successfully predicted many miRNAs including miR-21-5p and miR-19a-3p that involved in the TGF-β signalling pathway. Although there were only 6 samples used in the screening step, we have proved the expression changes of miRNA candidates in additional 31 serums. The whole strategy applied in this study is based on a robust but unbiased approach from the technique to the data analysis.
However, the small size of studied population, selection bias (males and females with variable Fabry phenotypes), and the fact that circulating miRNAs from serum could come from various cell types and tissues are obvious limitations of this study. As the objects in this study are diagnosed Fabry patients, and our major aim is to identify miRNA pattern that involved in the beneficial effects of ERT, healthy control group were not included. Future studies including healthy controls could help to increase the specificity of our results to Fabry disease.
In conclusion, the resulting miRNA pattern together with the validated miRNAs are expected to improve the understanding of the mechanisms involved in the beneficial effects of ERT or potentially to identify Fabry patients with specific need for ERT. Further studies are needed in greater patient cohorts and proper controls.

Data availability
All data analysed during this study are included in this article. The datasets generated during the study are available on reasonable request.