Gonadotropin-Releasing Hormone Receptor (GnRHR) and Hypogonadotropic Hypogonadism.

Human sexual and reproductive development is regulated by the hypothalamic-pituitary-gonadal (HPG) axis, which is primarily controlled by the gonadotropin-releasing hormone (GnRH) acting on its receptor (GnRHR). Dysregulation of the axis leads to conditions such as congenital hypogonadotropic hypogonadism (CHH) and delayed puberty. The pathophysiology of GnRHR makes it a potential target for treatments in several reproductive diseases and in congenital adrenal hyperplasia. GnRHR belongs to the G protein-coupled receptor family and its GnRH ligand, when bound, activates several complex and tissue-specific signaling pathways. In the pituitary gonadotrope cells, it triggers the G protein subunit dissociation and initiates a cascade of events that lead to the production and secretion of the luteinizing hormone (LH) and follicle-stimulating hormone (FSH) accompanied with the phospholipase C, inositol phosphate production, and protein kinase C activation. Pharmacologically, GnRHR can be modulated by synthetic analogues. Such analogues include the agonists, antagonists, and the pharmacoperones. The agonists stimulate the gonadotropin release and lead to receptor desensitization with prolonged use while the antagonists directly block the GnRHR and rapidly reduce the sex hormone production. Pharmacoperones include the most recent GnRHR therapeutic approaches that directly correct the misfolded GnRHRs, which are caused by genetic mutations and hold serious promise for CHH treatment. Understanding of the GnRHR's genomic and protein structure is crucial for the most appropriate assessing of the mutation impact. Such mutations in the GNRHR are linked to normosmic hypogonadotropic hypogonadism and lead to various clinical symptoms, including delayed puberty, infertility, and impaired sexual development. These mutations vary regarding their mode of inheritance and can be found in the homozygous, compound heterozygous, or in the digenic state. GnRHR expression extends beyond the pituitary gland, and is found in reproductive tissues such as ovaries, uterus, and prostate and non-reproductive tissues such as heart, muscles, liver and melanoma cells. This comprehensive review explores GnRHR's multifaceted role in human reproduction and its clinical implications for reproductive disorders.

Stbd1 promotes glycogen clustering during endoplasmic reticulum stress and supports survival of mouse myoblasts.

Imbalances in endoplasmic reticulum (ER) homeostasis provoke a condition known as ER stress and activate the unfolded protein response (UPR) pathway, an evolutionarily conserved cell survival mechanism. Here, we show that mouse myoblasts respond to UPR activation by stimulating glycogenesis and the formation of α-amylase-degradable, glycogen-containing ER structures. We demonstrate that the glycogen-binding protein Stbd1 is markedly upregulated through the PERK signalling branch of the UPR pathway and is required for the build-up of glycogen structures in response to ER stress activation. In the absence of ER stress, Stbd1 overexpression is sufficient to induce glycogen clustering but does not stimulate glycogenesis. Glycogen structures induced by ER stress are degraded under conditions of glucose restriction through a process that does not depend on autophagosome-lysosome fusion. Furthermore, we provide evidence that failure to induce glycogen clustering during ER stress is associated with enhanced activation of the apoptotic pathway. Our results reveal a so far unknown response of mouse myoblasts to ER stress and uncover a novel specific function of Stbd1 in this process, which may have physiological implications during myogenic differentiation.This article has an associated First Person interview with the first author of the paper.

Muscle-derived exosomes encapsulate myomiRs and are involved in local skeletal muscle tissue communication.

Exosomes are extracellular vesicles that are released from most cell types encapsulating specific molecular cargo. Exosomes serve as mediators of cell-to-cell and tissue-to-tissue communications under normal and pathological conditions. It has been shown that exosomes carrying muscle-specific miRNAs, myomiRs, are secreted from skeletal muscle cells in vitro and are elevated in the blood of muscle disease patients. The aim of this study was to investigate the secretion of exosomes encapsulating the four myomiRs from skeletal muscle tissues and to assess their role in inter-tissue communication between neighboring skeletal muscles in vivo. We demonstrate, for the first time, that isolated, intact skeletal muscle tissues secrete exosomes encapsulating the four myomiRs, miR-1, miR-133a, miR-133b, and miR-206. Notably, we show that the sorting of the four myomiRs within exosomes varies between skeletal muscles of different muscle fiber-type composition. miR-133a and miR-133b downregulation in TA muscles caused a reduction of their levels in neighboring skeletal muscles and in serum exosomes. In conclusion, our results reveal that skeletal muscle-derived exosomes encapsulate the four myomiRs, some of which enter the blood, while a portion is used for the local communication between proximal muscle tissues. These findings provide important evidence regarding novel pathways implicated in skeletal muscle function.

Circulating small RNA signatures differentiate accurately the subtypes of muscular dystrophies: small-RNA next-generation sequencing analytics and functional insights.

Muscular dystrophies are a group of rare and severe inherited disorders mainly affecting the muscle tissue. Duchene Muscular Dystrophy, Myotonic Dystrophy types 1 and 2, Limb Girdle Muscular Dystrophy and Facioscapulohumeral Muscular Dystrophy are some of the members of this family of disorders. In addition to the current diagnostic tools, there is an increasing interest for the development of novel non-invasive biomarkers for the diagnosis and monitoring of these diseases. miRNAs are small RNA molecules characterized by high stability in blood thus making them ideal biomarker candidates for various diseases. In this study, we present the first genome-wide next-generation small RNA sequencing in serum samples of five different types of muscular dystrophy patients and healthy individuals. We identified many small RNAs including miRNAs, lncRNAs, tRNAs, snoRNAs and snRNAs, that differentially discriminate the muscular dystrophy patients from the healthy individuals. Further analysis of the identified miRNAs showed that some miRNAs can distinguish the muscular dystrophy patients from controls and other miRNAs are specific to the type of muscular dystrophy. Bioinformatics analysis of the target genes for the most significant miRNAs and the biological role of these genes revealed different pathways that the dysregulated miRNAs are involved in each type of muscular dystrophy investigated. In conclusion, this study shows unique signatures of small RNAs circulating in five types of muscular dystrophy patients and provides a useful resource for future studies for the development of miRNA biomarkers in muscular dystrophies and for their involvement in the pathogenesis of the disorders.

Identification of exosomal muscle-specific miRNAs in serum of myotonic dystrophy patients relating to muscle disease progress.

Myotonic dystrophy type 1 (DM1) is the most common form of adult-onset muscular dystrophy, which is characterised by progressive muscle wasting and the discovery of reliable blood-based biomarkers could be useful for the disease progress monitoring. There have been some reports showing that the presence of specific miRNAs in blood correlates with DM1. In one of these, our group identified four muscle-specific miRNAs, miR-1, miR-133a, miR-133b and miR-206, which correlated with the progression of muscle wasting observed in DM1 patients. The levels of the four muscle-specific miRNAs were elevated in the serum of DM1 patients compared to healthy participants and were also elevated in the serum of progressive muscle wasting DM1 patients compared to disease-stable DM1 patients. The aim of this work was to characterise the ontology of these four muscle-specific miRNAs in the blood circulation of DM1 patients. Here we show that the four muscle-specific miRNAs are encapsulated within exosomes isolated from DM1 patients. Our results show for the first time, the presence of miRNAs encapsulated within exosomes in blood circulation of DM1 patients. More interestingly, the levels of the four exosomal muscle-specific miRNAs are associated with the progression of muscle wasting in DM1 patients. We propose that exosomal muscle-specific miRNAs may be useful molecular biomarkers for monitoring the progress of muscle wasting in DM1 patients. There has been a growing interest regarding the clinical applications of exosomes and their role in prognosis and therapy of various diseases and the above results contribute towards this way.

The Spectrum of Genetic Defects in Congenital Adrenal Hyperplasia in the Population of Cyprus: A Retrospective Analysis.

Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency (21-OHD) is caused by mutations in the CYP21A2 gene. The study refers to CAH patients of Greek-Cypriot ancestry between years 2007 and 2018. One hundred and twenty patients with various degrees of CAH were categorized and genotyped. The patients were categorized in 4 mutation groups based on their clinical and biochemical findings. The majority of patients (85.0%) belonged to the non-classic (NC)-CAH form and the disorder was more often diagnosed in females (71.7%). The most severe classic salt-wasting (SW) form was identified in 11 neonates (9.2%). Seven (5.8%) children were also identified with the simple virilizing (SV) form and a median presentation age of 5 years [interquartile range (IQR) 3.2-6.5]. In the 240 nonrelated alleles, the most frequent mutation was p.Val281Leu (60.0%) followed by c.655 A/C>G (IVS2-13A/C>G) (8.8%), p.Pro453Ser (5.8%), DelEx1-3 (4.6%), p.Val304Met (4.6%), and p.Gln318stop (4.2%). Other less frequent mutations including rare deletions were also identified. Following our recent report that the true carrier frequency of CYP21A2 in Greek-Cypriots is 1:10, this study reports that the CAH prevalence is predicted around 1.7 cases per 10 000 people. Therefore, the up-to-date 120 CAH patients identified by our group make only the 6.9% of the ones estimated (approximately 1750) to exist in the Greek Cypriot population. The compiled data from a coherent population such as the Greek-Cypriot could be valuable for the antenatal diagnosis, management and genetic counselling of the existing and prospect families with CAH.

MyoD transcription factor induces myogenesis by inhibiting Twist-1 through miR-206.

Twist-1 is mostly expressed during development and has been previously shown to control myogenesis. Because its regulation in muscle has not been fully exploited, the aim of this project was to identify micro (mi)RNAs in muscle that regulate Twist-1. miR-206, one of the most important muscle-specific miRNAs (myomiRs), was identified as a possible regulator of Twist-1 mRNA. Luciferase assays and transfections in human foetal myoblasts showed that Twist-1 is a direct target of miR-206 and that through this pathway muscle cell differentiation is promoted. We next investigated whether MyoD, a major myogenic transcription factor, regulates Twist-1 because it is known that MyoD induces expression of the miR-206 gene. We found that forced MyoD expression induced miR-206 upregulation and Twist-1 downregulation through binding to the miR-206 promoter, followed by increased muscle cell differentiation. Finally, experiments were performed in muscle cells from subjects with congenital myotonic dystrophy type 1, in which myoblasts fail to differentiate into myotubes. MyoD overexpression inhibited Twist-1 through miR-206 induction, which was followed by an increase in muscle cell differentiation. These results reveal a previously unidentified mechanism of myogenesis that might also play an important role in muscle disease.

In silico analysis of a novel MKRN3 missense mutation in familial central precocious puberty.

BACKGROUND: The onset of puberty is influenced by the interplay of stimulating and restraining factors, many of which have a genetic origin. Premature activation of the GnRH secretion in central precocious puberty (CPP) may arise either from gain-of-function mutations of the KISS1 and KISS1R genes or from loss-of-function manner mutations of the MKRN3 gene leading to MKRN3 deficiency. OBJECTIVE: To explore the genetic causes responsible for CPP and the potential role of the RING finger protein 3 (MKRN3) gene. DESIGN AND PATIENTS: We investigated potential sequence variations in the intronless MKRN3 gene by Sanger sequencing of the entire 507 amino acid coding region of exon 1 in a family with two affected girls presented with CPP at the age of 6 and 5·7 years, respectively. RESULTS: A novel heterozygous g.Gly312Asp missense mutation in the MKRN3 gene was identified in these siblings. The imprinted MKRN3 missense mutation was also identified as expected in the unaffected father and followed as expected an imprinted mode of inheritance. In silico analysis of the altered missense variant using the computational algorithms Polyphen2, SIFT and Mutation Taster predicted a damage and pathogenic alteration causing CPP. The pathogenicity of the alteration at the protein level via an in silico structural model is also explored. CONCLUSION: A novel mutation in the MKRN3 gene in two sisters with CPP was identified, supporting the fundamental role of this gene in the suppression of the hypothalamic GnRH neurons.

miR-186 inhibits muscle cell differentiation through myogenin regulation.

The complex process of skeletal muscle differentiation is organized by the myogenic regulatory factors (MRFs), Myf5, MyoD, Myf6, and myogenin, where myogenin plays a critical role in the regulation of the final stage of muscle differentiation. In an effort to investigate the role microRNAs (miRNAs) play in regulating myogenin, a bioinformatics approach was used and six miRNAs (miR-182, miR-186, miR-135, miR-491, miR-329, and miR-96) were predicted to bind the myogenin 3'-untranslated region (UTR). However, luciferase assays showed only miR-186 inhibited translation and 3'-UTR mutagenesis analysis confirmed this interaction was specific. Interestingly, the expression of miR-186 mirrored that of its host gene, ZRANB2, during development. Functional studies demonstrated that miR-186 overexpression inhibited the differentiation of C2C12 and primary muscle cells. Our findings therefore identify miR-186 as a novel regulator of myogenic differentiation.

Familial Mediterranean fever associated with MEFV mutations in a large cohort of Cypriot patients.

Familial Mediterranean fever (FMF) is caused by mutations in the MEFV gene and the spectrum of mutations among Greek-Cypriots with FMF-related symptoms was examined. Sequence analysis for exons 2, 3, 5, and 10 of the MEFV gene was performed in a cohort of 593 patients. A total of 70 patients carried mutations in the homozygote or compound heterozygote state, 128 were identified with one MEFV mutation and 395 had no mutations. Of the 268 identified alleles, p.Val726Ala (27.61%) was the most frequent followed by p.Met694Val (19.40%). The missense mutations p.Arg761His (3.73%) and p.Ala744Ser (2.24%) were identified as the rarest. An interesting finding is the high frequency (18.28%) of the complex p.Phe479Leu-p.Glu167Asp that was identified in 49 of the mutated alleles. The MEFV genotypes did not follow a binomial distribution and proved not to satisfy the HWE (P < 0.001). The high percentage (66.61%) of patients with unidentified mutations could be due to mutations in the rest of the coding or noncoding MEFV gene or due to mutations in other genes that are also causing Hereditary Recurrent Fevers. Results from this work indicate the high incidence of FMF in Cyprus and describe the spectrum of the mutations which occur in the country.

Special Issue--Towards Understanding the Mechanisms and Curing of Muscular Dystrophy Diseases.

Muscular dystrophies are a heterogeneous group of inherited diseases with different molecular basss, but sharing similar clinical features and dystrophic changes.

Genetic diagnosis of endocrine disorders in Cyprus through the Cyprus Institute of Neurology and Genetics: an ENDO-ERN Reference Center.

The report covers the current and past activities of the department Molecular Genetics-Function and Therapy (MGFT) at the Cyprus Institute of Neurology and Genetics (CING), an affiliated Reference Center for the European Reference Network on Rare Endocrine Conditions (Endo-ERN).The presented data is the outcome of > 15 years long standing collaboration between MGFT and endocrine specialists from the local government hospitals and the private sector. Up-to-date > 2000 genetic tests have been performed for the diagnosis of inherited rare endocrine disorders. The major clinical entities included Congenital Adrenal Hyperplasia (CAH) due to pathogenic variants in CYP21A2 gene and Multiple Endocrine Neoplasia (MEN) type 2 due to pathogenic variants in the RET proto-oncogene. Other rare and novel pathogenic variants in ANOS1, WDR11, FGFR1, RNF216, and CHD7 genes were also found in patients with Congenital Hypogonadotropic Hypogonadism. Interestingly, a few patients with Disorders of Sexual Differentiation (DSD) shared rare pathogenic variants in the SRD5A2, HSD17B3 and HSD3B2 while patients with Glucose and Insulin Homeostasis carried theirs in GCK and HNF1A genes. Lastly, MGFT over the last few years has established an esteemed diagnostic and research program on premature puberty with emphasis on the implication of MKRN3 gene on the onset of the disease and the identification of other prognosis biomarkers.As an Endo-ERN member MGFT department belongs to this large European network and holds the same humanistic ideals which aim toward the improvements of health care for patients with rare endocrine conditions in respect to improved and faster diagnosis.

Salt-wasting congenital adrenal hyperplasia phenotype as a result of the TNXA/TNXB chimera 1 (CAH-X CH-1) and the pathogenic IVS2-13A/C > G in CYP21A2 gene.

BACKGROUND: Genetic diversity of mutations in the CYP21A2 gene is the main cause of the monogenic congenital adrenal hyperplasia (CAH) disorder. On chromosome 6p21.3, the CYP21A2 gene is partially overlapped by the TNXB gene, the two residing in tandem with their highly homologous corresponding pseudogenes (CYP21A1P and TNXA), which leads to recurrent homologous recombination. METHODS AND RESULTS: In the present study, the genetic status of an ethnic Greek-Cypriot family, with a female neonate that was originally classified as male and manifested the salt-wasting (SW) form, is presented. Genetic defects in the CYP21A2 and TNXB genes were investigated by Sanger sequencing multiplex ligation-dependent probe amplification (MLPA) and a real-time PCR assay. The neonate carried in compound heterozygosity the TNXA/TNXB chimeric gene complex (termed CAH-X CH-1) that results in a contiguous CYP21A2 and TNXB deletion and in her second allele the pathogenic IVS2-13A/C > G (c.655A/C > G) in CYP21A2. CONCLUSIONS: The classic SW-CAH due to 21-hydroxylase (21-OH) deficiency may result from various complex etiological mechanisms and, as such, can involve the formation of monoallelic TNXA/TNXB chimeras found in trans with other CYP21A2 pathogenic variants. This is a rare case of CAH due to 21-hydroxylase deficiency, which elucidates the role of the complex RCCX CNV structure in the development of the disease. Identification of the correct CAH genotypes for a given phenotype is of considerable value in assisting clinicians in prenatal diagnosis, appropriate treatment, and genetic counseling.

RET Proto-Oncogene Variants in Patients with Medullary Thyroid Carcinoma from the Mediterranean Basin: A Brief Report.

Multiple endocrine neoplasia type 2 (MEN2) is an autosomal dominant (AD) condition with very high penetrance and expressivity. It is characterized into three clinical entities recognized as MEN2A, MEN2B, and familial medullary thyroid carcinoma (FMTC). In both MEN2A and MEN2B, there is a manifestation of multicentric tumor formation in the major organs such as the thyroid, parathyroid, and adrenal glands where the RET proto-oncogene is expressed. The FMTC form differs from MEN2A and MEN2B, since medullary thyroid carcinoma (MTC) is the only feature observed. In this present brief report, we demonstrate a collection of RET proto-oncogene genotype data from countries around the Mediterranean Basin with variable characteristics. As expected, a great extent of the Mediterranean RET proto-oncogene genotype data resemble the data reported globally. Most interestingly, higher frequencies are observed in the Mediterranean region for specific pathogenic RET variants as a result of local prevalence. The latter can be explained by founder effect phenomena. The Mediterranean epidemiological data that are presented herein are very important for domestic patients, their family members' evaluation, and ultimately their treatment.

The pathogenic p.Gln319Ter variant is not causing congenital adrenal hyperplasia when inherited in one of the duplicated CYP21A2 genes.

Objective: The study aimed to identify the pathogenic status of p.Gln319Ter (NM_000500.7: c.955C>T) variant when inherited in a single CYP21A2 gene (bimodular RCCX haplotype) and to discriminate between a non-causing congenital adrenal hyperplasia (CAH) allele when inherited in a duplicated and functional CYP21A2 gene context (trimodular RCCX haplotype). Methods: 38 females and 8 males with hyperandrogenemia, previously screened by sequencing and identified as carriers for the pathogenic p.Gln319Ter, were herein tested by multiplex ligation-dependent probe amplification (MLPA) and a real-time PCR Copy number Variation (CNV) assay. Results: Both MLPA and real-time PCR CNV analyses confirmed a bimodular and pathogenic RCCX haplotype with a single CYP21A2 in 19/46 (41.30%) p.Gln319Ter carriers and who in parallel all shared elevated 17-OHP levels. The remaining 27 individuals that also carried the p.Gln319Ter exhibited low 17-OHP levels as a result of their carriership of a duplicated CYP21A2 with a trimodular RCCX haplotype. Interestingly, all of these individuals also carried in linkage disequilibrium with p.Gln319Ter two single nucleotide polymorphisms, the c.293-79G>A (rs114414746) in intron 2 and the c.*12C>T (rs150697472) in the 3'-UTR. Therefore, these variants can be used to distinguish between pathogenic and non-pathogenic genomic contexts of the c.955T (p.Gln319) in the genetic diagnosis of congenital adrenal hyperplasia (CAH). Conclusion: The employed methodologies identified a considerable number of individuals with non-pathogenic p.Gln319Ter from the individuals that typically carry the pathogenic p.Gln319Ter in a single CYP21A2. Therefore, it is extremely important the detection of such haplotypes for the prenatal diagnosis, treatment and genetic counseling in patients with CAH.

Selective Delivery to Cardiac Muscle Cells Using Cell-Specific Aptamers.

In vivo SELEX is an advanced adaptation of Systematic Evolution of Ligands by Exponential Enrichment (SELEX) that allows the development of aptamers capable of recognizing targets directly within their natural microenvironment. While this methodology ensures a higher translation potential for the selected aptamer, it does not select for aptamers that recognize specific cell types within a tissue. Such aptamers could potentially improve the development of drugs for several diseases, including neuromuscular disorders, by targeting solely the proteins involved in their pathogenesis. Here, we describe our attempt to utilize in vivo SELEX with a modification in the methodology that drives the selection of intravenously injected aptamers towards a specific cell type of interest. Our data suggest that the incorporation of a cell enrichment step can direct the in vivo localization of RNA aptamers into cardiomyocytes, the cardiac muscle cells, more readily over other cardiac cells. Given the crucial role of cardiomyocytes in the disease pathology in DMD cardiomyopathy and therapy, these aptamers hold great potential as drug delivery vehicles with cardiomyocyte selectivity.

Molecular modelling of novel ADCY3 variant predicts a molecular target for tackling obesity.

Severe early­onset obesity is mainly attributed to single gene variations of the hypothalamic leptin­melanocortin system, which is critical for controlling the balance between appetite and energy expenditure. Adenylate cyclase 3 (ADCY3), a transmembrane enzyme localized in primary neuronal cilia, is a key genetic candidate, which appears to have an essential role in regulating body weight. The present study aimed to identify ADCY3 genetic variants in severely obese young patients of Greek­Cypriot origin by genomic sequencing. Apart from previously reported variants, the novel and probably pathogenic variant c.349T>A, causing a p.Leu117Met substitution within one of the two pseudo­symmetric halves of the transmembrane part of the protein, was reported. Molecular modelling analysis used to delineate bonding interactions within the mutated protein structure strongly suggested a change in interactive forces and energy levels affecting the pseudo­twofold symmetry of the transmembrane domain of the protein and probably its catalytic function. These results support the involvement of ADCY3 in the pathology of the disease and point towards the requirement of defining protein function and evaluating the clinical significance of the detected variants.

Methylation status of hypothalamic Mkrn3 promoter across puberty.

Makorin RING finger protein 3 (MKRN3) is an important factor located on chromosome 15 in the imprinting region associated with Prader-Willi syndrome. Imprinted MKRN3 is expressed in hypothalamic regions essential for the onset of puberty and mutations in the gene have been found in patients with central precocious puberty. The pubertal process is largely controlled by epigenetic mechanisms that include, among other things, DNA methylation at CpG dinucleotides of puberty-related genes. In the present study, we investigated the methylation status of the Mkrn3 promoter in the hypothalamus of the female mouse before, during and after puberty. Initially, we mapped the 32 CpG dinucleotides in the promoter, the 5'UTR and the first 50 nucleotides of the coding region of the Mkrn3 gene. Moreover, we identified a short CpG island region (CpG islet) located within the promoter. Methylation analysis using bisulfite sequencing revealed that CpG dinucleotides were methylated regardless of developmental stage, with the lowest levels of methylation being found within the CpG islet region. In addition, the CpG islet region showed significantly lower methylation levels at the pre-pubertal stage when compared with the pubertal or post-pubertal stage. Finally, in silico analysis of transcription factor binding sites on the Mkrn3 CpG islet identified the recruitment of 29 transcriptional regulators of which 14 were transcriptional repressors. Our findings demonstrate the characterization and differential methylation of the CpG dinucleotides located in the Mkrn3 promoter that could influence the transcriptional activity in pre-pubertal compared to pubertal or post-pubertal period. Further studies are needed to clarify the possible mechanisms and effects of differential methylation of the Mkrn3 promoter.

Serum miRNAs as biomarkers for the rare types of muscular dystrophy.

Muscular dystrophies are a group of disorders that cause progressive muscle weakness. There is an increasing interest for the development of biomarkers for these disorders and specifically for Duchene Muscular Dystrophy. Limited research however, has been performed on the biomarkers' development for the most rare muscular dystrophies, like the Facioscapulohumeral Muscular Dystrophy, Limb-Girdle Muscular Dystrophy and Myotonic Dystrophy type 2. Here, we aimed to identify novel serum-based miRNA biomarkers for these rare muscular dystrophies, through high-throughput next-generation RNA sequencing. We identified many miRNAs that associate with muscular dystrophy patients compared to controls. Based on a series of selection criteria, the two best candidate miRNAs for each of these disorders were chosen and validated in a larger number of patients. Our results showed that miR-223-3p and miR-206 are promising serum-based biomarkers for Facioscapulohumeral Muscular Dystrophy type 1, miR-143-3p and miR-486-3p for Limb-Girdle Muscular Dystrophy type 2A whereas miR-363-3p and miR-25-3p associate with Myotonic Dystrophy type 2. Some of the identified miRNAs were significantly elevated in the serum of the patients compared to controls, whereas some others were lower. In conclusion, we provide new evidence that certain circulating miRNAs may be used as biomarkers for three types of rare muscular dystrophies.