A novel complex genomic rearrangement affecting the KCNJ2 regulatory region causes a variant of Cooks syndrome.

Cooks syndrome (CS) is an ultrarare limb malformation due to in tandem microduplications involving KCNJ2 and extending to the 5' regulatory element of SOX9. To date, six CS families were resolved at the molecular level. Subsequent studies explored the evolutionary and pathological complexities of the SOX9-KCNJ2/Sox9-Kcnj2 locus, and suggested a key role for the formation of novel topologically associating domain (TAD) by inter-TAD duplications in causing CS. Here, we report a unique case of CS associated with a de novo 1;17 translocation affecting the KCNJ2 locus. On chromosome 17, the breakpoint mapped between KCNJ16 and KCNJ2, and combined with a ~ 5 kb deletion in the 5' of KCNJ2. Based on available capture Hi-C data, the breakpoint on chromosome 17 separated KCNJ2 from a putative enhancer. Gene expression analysis demonstrated downregulation of KCNJ2 in both patient's blood cells and cultured skin fibroblasts. Our findings suggest that a complex rearrangement falling in the 5' of KCNJ2 may mimic the developmental consequences of in tandem duplications affecting the SOX9-KCNJ2/Sox9-Kcnj2 locus. This finding adds weight to the notion of an intricate role of gene regulatory regions and, presumably, the related three-dimensional chromatin structure in normal and abnormal human morphology.

A next generation sequencing gene panel for use in the diagnosis of anorexia nervosa.

PURPOSE: The aim of this study was to increase knowledge of genes associated with anorexia nervosa (AN) and their diagnostic offer, using a next generation sequencing (NGS) panel for the identification of genetic variants. The rationale underlying this test is that we first analyze the genes associated with syndromic forms of AN, then genes that were found to carry rare variants in AN patients who had undergone segregation analysis, and finally candidate genes intervening in the same molecular pathways or identified by GWAS or in mouse models. METHODS: We developed an NGS gene panel and used it to screen 68 Italian AN patients (63 females, 5 males). The panel included 162 genes. Family segregation study was conducted on available relatives of probands who reported significant genetic variants. RESULTS: In our analysis, we found potentially deleterious variants in 2 genes (PDE11A and SLC25A13) associated with syndromic forms of anorexia and predicted deleterious variants in the following 12 genes: CD36, CACNA1C, DRD4, EPHX2, ESR1, GRIN2A, GRIN3B, LRP2, NPY4R, PTGS2, PTPN22 and SGPP2. Furthermore, by Sanger sequencing of the promoter region of NNAT, we confirmed the involvement of this gene in the pathogenesis of AN. Family segregation studies further strengthened the possible causative role of CACNA1C, DRD4, GRIN2A, PTGS2, SGPP2, SLC25A13 and NNAT genes in AN etiology. CONCLUSION: The major finding of our study is the confirmation of the involvement of the NNAT gene in the pathogenesis of AN; furthermore, this study suggests that NGS-based testing can play an important role in the diagnostic evaluation of AN, excluding syndromic forms and increasing knowledge of the genetic etiology of AN. LEVEL OF EVIDENCE: Level I, experimental study.

Adult-onset glutaric aciduria type I: rare presentation of a treatable disorder.

Glutaric aciduria type I (GA1; OMIM #231670) is an autosomal recessively inherited and treatable disorder characterized by the accumulation and irregular excretion of glutaric acid due to a defect in the glutaryl-CoA dehydrogenase enzyme involved in the catabolic pathways of L-lysine, L-hydroxylysine, and L-tryptophan. Glutaryl-CoA dehydrogenase is encoded by the GCDH gene (OMIM #608801), and several mutations in this gene are known to result in GA1. GA1 usually presents in the first 18-36 months of life with mild or severe acute encephalopathy, movement disorders, and striatal degeneration. Few cases of adult-onset GA1 have been described so far in the literature, often with non-specific and sometimes longstanding neurological symptoms. Since a preventive metabolic treatment is available, neurologists must be aware of this rare but likely underdiagnosed presentation, especially when typical neuroimaging features are identified. Here, we describe 35-year-old presenting with headache and subjective memory problems. There was no history of dystonic movement disorders. Neurological examination and neurocognitive tests were normal. Brain MRI scan revealed white matter abnormalities associated with subependymal nodules and mild frontotemporal hypoplasia suggestive of glutaric aciduria type 1 (GA1). Genetic testing confirmed the presence of homozygous c.1204C > T (p.R402W) variant in the GCDH gene, inherited from heterozygous parents.

A novel p.(Glu111Val) missense mutation in GUCA1A associated with cone-rod dystrophy leads to impaired calcium sensing and perturbed second messenger homeostasis in photoreceptors.

Guanylate Cyclase-Activating Protein 1 (GCAP1) regulates the enzymatic activity of the photoreceptor guanylate cyclases (GC), leading to inhibition or activation of the cyclic guanosine monophosphate (cGMP) synthesis depending on its Ca2+- or Mg2+-loaded state. By genetically screening a family of patients diagnosed with cone-rod dystrophy, we identified a novel missense mutation with autosomal dominant inheritance pattern (c.332A>T; p.(Glu111Val); E111V from now on) in the GUCA1A gene coding for GCAP1. We performed a thorough biochemical and biophysical investigation of wild type (WT) and E111V human GCAP1 by heterologous expression and purification of the recombinant proteins. The E111V substitution disrupts the coordination of the Ca2+ ion in the high-affinity site (EF-hand 3, EF3), thus significantly decreasing the ability of GCAP1 to sense Ca2+ (∼80-fold higher Kdapp compared to WT). Both WT and E111V GCAP1 form dimers independently on the presence of cations, but the E111V Mg2+-bound form is prone to severe aggregation over time. Molecular dynamics simulations suggest a significantly increased flexibility of both the EF3 and EF4 cation binding loops for the Ca2+-bound form of E111V GCAP1, in line with the decreased affinity for Ca2+. In contrast, a more rigid backbone conformation is observed in the Mg2+-bound state compared to the WT, which results in higher thermal stability. Functional assays confirm that E111V GCAP1 interacts with the target GC with a similar apparent affinity (EC50); however, the mutant shifts the GC inhibition out of the physiological [Ca2+] (IC50E111V ∼10 µM), thereby leading to the aberrant constitutive synthesis of cGMP under conditions of dark-adapted photoreceptors.

FOXC2 Disease Mutations Identified in Lymphedema Distichiasis Patients Impair Transcriptional Activity and Cell Proliferation.

FOXC2 is a member of the human forkhead-box gene family and encodes a regulatory transcription factor. Mutations in FOXC2 have been associated with lymphedema distichiasis (LD), an autosomal dominant disorder that primarily affects the limbs. Most patients also show extra eyelashes, a condition known as distichiasis. We previously reported genetic and clinical findings in six unrelated families with LD. Half the patients showed missense mutations, two carried frameshift mutations and a stop mutation was identified in a last patient. Here we analyzed the subcellular localization and transactivation activity of the mutant proteins, showing that all but one (p.Y109*) localized to the nucleus. A significant reduction of transactivation activity was observed in four mutants (p.L80F, p.H199Pfs*264, p.I213Tfs*18, p.Y109*) compared with wild type FOXC2 protein, while only a partial loss of function was associated with p.V228M. The mutant p.I213V showed a very slight increase of transactivation activity. Finally, immunofluorescence analysis revealed that some mutants were sequestered into nuclear aggregates and caused a reduction of cell viability. This study offers new insights into the effect of FOXC2 mutations on protein function and shows the involvement of aberrant aggregation of FOXC2 proteins in cell death.

Aldo-Keto Reductase 1C1 (AKR1C1) as the First Mutated Gene in a Family with Nonsyndromic Primary Lipedema.

Lipedema is an often underdiagnosed chronic disorder that affects subcutaneous adipose tissue almost exclusively in women, which leads to disproportionate fat accumulation in the lower and upper body extremities. Common comorbidities include anxiety, depression, and pain. The correlation between mood disorder and subcutaneous fat deposition suggests the involvement of steroids metabolism and neurohormones signaling, however no clear association has been established so far. In this study, we report on a family with three patients affected by sex-limited autosomal dominant nonsyndromic lipedema. They had been screened by whole exome sequencing (WES) which led to the discovery of a missense variant p.(Leu213Gln) in AKR1C1, the gene encoding for an aldo-keto reductase catalyzing the reduction of progesterone to its inactive form, 20-α-hydroxyprogesterone. Comparative molecular dynamics simulations of the wild-type vs. variant enzyme, corroborated by a thorough structural and functional bioinformatic analysis, suggest a partial loss-of-function of the variant. This would result in a slower and less efficient reduction of progesterone to hydroxyprogesterone and an increased subcutaneous fat deposition in variant carriers. Overall, our results suggest that AKR1C1 is the first candidate gene associated with nonsyndromic lipedema.

Expanding the Clinical and Genetic Spectrum of RAB28-Related Cone-Rod Dystrophy: Pathogenicity of Novel Variants in Italian Families.

The small Ras-related GTPase Rab-28 is highly expressed in photoreceptor cells, where it possibly participates in membrane trafficking. To date, six alterations in the RAB28 gene have been associated with autosomal recessive cone-rod dystrophies. Confirmed variants include splicing variants, missense and nonsense mutations. Here, we present a thorough phenotypical and genotypical characterization of five individuals belonging to four Italian families, constituting the largest cohort of RAB28 patients reported in literature to date. All probands displayed similar clinical phenotype consisting of photophobia, decreased visual acuity, central outer retinal thinning, and impaired color vision. By sequencing the four probands, we identified: a novel homozygous splicing variant; two novel nonsense variants in homozygosis; a novel missense variant in compound heterozygous state with a previously reported nonsense variant. Exhaustive molecular dynamics simulations of the missense variant p.(Thr26Asn) in both its active and inactive states revealed an allosteric structural mechanism that impairs the binding of Mg2+, thus decreasing the affinity for GTP. The impaired GTP-GDP exchange ultimately locks Rab-28 in a GDP-bound inactive state. The loss-of-function mutation p.(Thr26Asn) was present in a compound heterozygosis with the nonsense variant p.(Arg137*), which does not cause mRNA-mediated decay, but is rather likely degraded due to its incomplete folding. The frameshift p.(Thr26Valfs4*) and nonsense p.(Leu13*) and p.(Trp107*) variants, if translated, would lack several key structural components necessary for the correct functioning of the encoded protein.

Genetic tests in lymphatic vascular malformations and lymphedema.

Syndromes with lymphatic malformations show phenotypic variability within the same entity, clinical features that overlap between different conditions and allelic as well as locus heterogeneity. The aim of this review is to provide a comprehensive clinical genetic description of lymphatic malformations and the techniques used for their diagnosis, and to propose a flowchart for genetic testing. Literature and database searches were performed to find conditions characterised by lymphatic malformations or the predisposition to lymphedema after surgery, to identify the associated genes and to find the guidelines and genetic tests currently used for the molecular diagnosis of these disorders. This search allowed us to identify several syndromes with lymphatic malformations that are characterised by a great heterogeneity of phenotypes, alleles and loci, and a high frequency of sporadic cases, which may be associated with somatic mutations. For these disorders, we found many diagnostic tests, an absence of harmonic guidelines for molecular diagnosis and well-established clinical guidelines. Targeted sequencing is the preferred method for the molecular diagnosis of lymphatic malformations. These techniques are easy to implement and have a good diagnostic success rates. In addition, they are relatively inexpensive and permit parallel analysis of all known disease-associated genes. The targeted sequencing approach has improved the diagnostic process, giving patients access to better treatment and, potentially, to therapy personalised to their genetic profiles. These new techniques will also facilitate the prenatal and early postnatal diagnosis of congenital lymphatic conditions and the possibility of early intervention.

Variant discovery in patients with Mendelian vascular anomalies by next-generation sequencing and their use in patient clinical management.

OBJECTIVE: An accurate "molecular" diagnosis and classification of similar but distinct diseases is sometime challenging but often crucial for the definition of the appropriate patient medical management and treatment as well as for genetic counseling and risk assessment in families. The advent of next-generation sequencing (NGS), which analysed all known disease-associated genes in parallel in a cost- and time-effective manner, eased this process of disease definition and also for vascular anomalies that are a heterogeneous group of vascular tumors and congenital circulatory malformations and often characterized by overlapping phenotypes. METHODS: We designed a NGS-based screening of the 25 currently most prevalent genes identified in patients with vascular anomalies with Mendelian inheritance and applied this panel to study the DNA of 150 patients affected with vascular anomalies for autosomal recessive and autosomal dominant variants and to analyse the paired blood and DNA from intralesional biopsy specimens in 17 patients for somatic unbalance. Results were confirmed with Sanger sequencing. RESULTS: We identified 14 pathogenic variants in 13 of 150 patients. Eight variants were previously reported as a disease-causing variant, and six were new. In 55 additional probands we detected 75 variants with unknown significance. Moreover, a previously reported somatic variant was detected in five of 17 available tissue biopsy specimens. CONCLUSIONS: Our results show that many genes can cause a wide variety of syndromic and nonsyndromic disorders, confirming that genetic testing by NGS is the approach of choice to diagnose heritable vascular anomalies, especially, but not only, when an intralesional biopsy specimen is available. The identification of the causative genes and the possibility of tracing somatic variants in tissues provide important information about etiology, patient clinical management, and treatment, and it could highlight otherwise unsuspected clinical situations.

MLL-AF6 fusion oncogene sequesters AF6 into the nucleus to trigger RAS activation in myeloid leukemia.

A rare location, t(6;11)(q27;q23) (MLL-AF6), is associated with poor outcome in childhood acute myeloid leukemia (AML). The described mechanism by which MLL-AF6, through constitutive self-association and in cooperation with DOT-1L, activates aberrant gene expression does not explain the biological differences existing between t(6;11)-rearranged and other MLL-positive patients nor their different clinical outcome. Here, we show that AF6 is expressed in the cytoplasm of healthy bone marrow cells and controls rat sarcoma viral oncogene (RAS)-guanosine triphosphate (GTP) levels. By contrast, in MLL-AF6-rearranged cells, AF6 is found localized in the nucleus, leading to aberrant activation of RAS and of its downstream targets. Silencing MLL-AF6, we restored AF6 localization in the cytoplasm, thus mediating significant reduction of RAS-GTP levels and of cell clonogenic potential. The rescue of RAS-GTP levels after MLL-AF6 and AF6 co-silencing confirmed that MLL-AF6 oncoprotein potentiates the activity of the RAS pathway through retention of AF6 within the nucleus. Exposure of MLL-AF6-rearranged AML blasts to tipifarnib, a RAS inhibitor, leads to cell autophagy and apoptosis, thus supporting RAS targeting as a novel potential therapeutic strategy in patients carrying t(6;11). Altogether, these data point to a novel role of the MLL-AF6 chimera and show that its gene partner, AF6, is crucial in AML development.

CBFA2T3-GLIS2 fusion transcript is a novel common feature in pediatric, cytogenetically normal AML, not restricted to FAB M7 subtype.

Pediatric cytogenetically normal acute myeloid leukemia (CN-AML) is a heterogeneous subgroup of myeloid clonal disorders that do not harbor known mutations. To investigate the mutation spectrum of pediatric CN-AML, we performed whole-transcriptome massively parallel sequencing on blasts from 7 CN-AML pediatric patients. In 3 patients we identified a recurrent cryptic inversion of chromosome 16, encoding a CBFA2T3-GLIS2 fusion transcript. In a validation cohort of 230 pediatric CN-AML samples we identified 17 new cases. Among a total of 20 patients with CBFA2T3-GLIS2 fusion transcript out of 237 investigated (8.4%), 10 patients (50%) did not belong to the French-American-British (FAB) M7 subgroup. The 5-year event-free survival for these 20 children was worse than that for the other CN-AML patients (27.4% vs 59.6%; P = .01). These data suggest that the presence of CBFA2T3-GLIS2 fusion transcript is a novel common feature of pediatric CN-AML, not restricted to the FAB M7 subtype, predicting poorer outcome.

Sox4 cooperates with CREB in myeloid transformation.

The cAMP response element-binding protein (CREB) is a nuclear transcription factor that is critical for normal and neoplastic hematopoiesis. Previous studies have demonstrated that CREB is a proto-oncogene whose overexpression promotes cellular proliferation in hematopoietic cells. Transgenic mice that overexpress CREB in myeloid cells develop a myeloproliferative disease with splenomegaly and aberrant myelopoiesis. However, CREB overexpressing mice do not spontaneously develop acute myeloid leukemia. In this study, we used retroviral insertional mutagenesis to identify genes that accelerate leukemia in CREB transgenic mice. Our mutagenesis screen identified several integration sites, including oncogenes Gfi1, Myb, and Ras. The Sox4 transcription factor was identified by our screen as a gene that cooperates with CREB in myeloid leukemogenesis. We show that the transduction of CREB transgenic mouse bone marrow cells with a Sox4 retrovirus increases survival and self-renewal of cells in vitro. Furthermore, leukemic blasts from the majority of acute myeloid leukemia patients have higher CREB, phosphorylated CREB, and Sox 4 protein expression. Sox4 transduction of mouse bone marrow cells results in increased expression of CREB target genes. We also demonstrate that CREB is a direct target of Sox4 by chromatin immunoprecipitation assays. These results indicate that Sox4 and CREB cooperate and contribute to increased proliferation of hematopoietic progenitor cells.

MicroRNA-34b promoter hypermethylation induces CREB overexpression and contributes to myeloid transformation.

MicroRNA-34b down-regulation in acute myeloid leukemia was previously shown to induce CREB overexpression, thereby causing leukemia proliferation in vitro and in vivo. The role of microRNA-34b and CREB in patients with myeloid malignancies has never been evaluated. We examined microRNA-34b expression and the methylation status of its promoter in cells from patients diagnosed with myeloid malignancies. We used gene expression profiling to identify signatures of myeloid transformation. We established that microRNA-34b has suppressor ability and that CREB has oncogenic potential in primary bone marrow cell cultures and in vivo. MicroRNA-34b was found to be up-regulated in pediatric patients with juvenile myelomonocytic leukemia (n=17) and myelodysplastic syndromes (n=28), but was down-regulated in acute myeloid leukemia patients at diagnosis (n=112). Our results showed that hypermethylation of the microRNA-34b promoter occurred in 66% of cases of acute myeloid leukemia explaining the low microRNA-34b levels and CREB overexpression, whereas preleukemic myelodysplastic syndromes and juvenile myelomonocytic leukemia were not associated with hypermethylation or CREB overexpression. In paired samples taken from the same patients when they had myelodysplastic syndrome and again during the subsequent acute myeloid leukemia, we confirmed microRNA-34b promoter hypermethylation at leukemia onset, with 103 CREB target genes differentially expressed between the two disease stages. This subset of CREB targets was confirmed to associate with high-risk myelodysplastic syndromes in a separate cohort of patients (n=20). Seventy-eight of these 103 CREB targets were also differentially expressed between healthy samples (n=11) and de novo acute myeloid leukemia (n=72). Further, low microRNA-34b and high CREB expression levels induced aberrant myelopoiesis through CREB-dependent pathways in vitro and in vivo. In conclusion, we suggest that microRNA-34b controls CREB expression and contributes to myeloid transformation from both healthy bone marrow and myelodysplastic syndromes. We identified a subset of CREB target genes that represents a novel transcriptional network that may control myeloid transformation.

Characterization of a Novel Frameshift Mutation Within the TRPS1 Gene Causing Trichorhinophalangeal Syndrome Type 1 in a Kindred Cypriot Family.

Trichorhinophalangeal syndrome (TRPS) is an extremely rare autosomal dominant multisystem disorder characterized by craniofacial and skeletal abnormalities. Three subtypes of TRPS have been described: TRPS type I, TRPS type II, and TRPS type III. Mutations in the TRPS1 gene can cause both TRPS type I and TRPS type III. Therefore, the genotype-phenotype correlation is crucial to determine the subtype. The current family study from Cyprus involves affected patients from 4 generations who presented with alopecia, unoperated umbilical hernia, caput quadratum, long philtrum, depressed nasal bridge, frontal bossing, pes planus, beaked nose, and some deformities in hands and feet. Sequence analysis of the TRPS1 gene revealed a novel c.2854_2858del (p.Asn952ArgfsTer2) frameshift variant leading to a premature stop codon. To the best of our knowledge, we report here the first case of a Turkish Cypriot family of 4 generations with a novel frameshift mutation leading to truncated protein in the TRPS1 gene causing TRPS type I clinical phenotype. Overall, as the genotype and phenotype correlation in TRPSI is still uncertain and complex, the present outcome can enhance our knowledge of this complicated, rare, and severe genetic disorder.

CDH5, a Possible New Candidate Gene for Genetic Testing of Lymphedema.

Background: Expressed by endothelial cells, CDH5 is a cadherin involved in vascular morphogenesis and in the maintenance of vascular integrity and lymphatic function. The main purpose of our study was to identify distinct variants of the CDH5 gene that could be associated with lymphatic malformations and predisposition for lymphedema. Methods and Results: We performed Next Generation Sequencing of the CDH5 gene in 235 Italian patients diagnosed with lymphedema but who tested negative for variants in known lymphedema genes. We detected six different variants in CDH5 five missense and one nonsense. We also tested available family members of the probands. For family members who carried the same variant as the proband, we performed lymphoscintigraphy to detect any lymphatic system abnormalities. Variants were modeled in silico. The results showed that CDH5 variants may contribute to the onset of lymphedema, although further in vitro studies are needed to confirm this hypothesis. Conclusions: Based on our findings, we propose CDH5 as a new gene that could be screened in patients with lymphedema to gather additional evidence.

A Multi-Gene Panel to Identify Lipedema-Predisposing Genetic Variants by a Next-Generation Sequencing Strategy.

Lipedema is a disabling disease characterized by symmetric enlargement of the lower and/or upper limbs due to deposits of subcutaneous fat, that is easily misdiagnosed. Lipedema can be primary or syndromic, and can be the main feature of phenotypically overlapping disorders. The aim of this study was to design a next-generation sequencing (NGS) panel to help in the diagnosis of lipedema by identifying genes specific for lipedema but also genes for overlapping diseases, and targets for tailored treatments. We developed an NGS gene panel consisting of 305 genes potentially associated with lipedema and putative overlapping diseases relevant to lipedema. The genomes of 162 Italian and American patients with lipedema were sequenced. Twenty-one deleterious variants, according to 3 out of 5 predictors, were detected in PLIN1, LIPE, ALDH18A1, PPARG, GHR, INSR, RYR1, NPC1, POMC, NR0B2, GCKR, PPARA in 17 patients. This extended NGS-based approach has identified a number of gene variants that may be important in the diagnosis of lipedema, that may affect the phenotypic presentation of lipedema or that may cause disorders that could be confused with lipedema. This tool may be important for the diagnosis and treatment of people with pathologic subcutaneous fat tissue accumulation.

Psychomotor Delay in a Child with FGFR3 G380R Pathogenic Mutation Causing Achondroplasia.

Achondroplasia (ACH) is a hereditary disorder of dwarfism that is caused by the aberrant proliferation and differentiation of chondrocyte growth plates. The common findings of macrocephaly and facial anomalies accompany dwarfism in these patients. Fibroblast growth factor receptor 3 ( FGFR3 ) gene mutations are common causes of achondroplasia. The current study presents a case of 2-year-old male child patient presenting with phenotypic characteristics of ACH. The interesting finding of the case is the presence of psychomotor delay that is not very common in these patients. Clinical exome sequencing analyzing 4.813 disease causing genes revealed a de novo c.1138G > A mutation within the FGFR3 gene. In conclusion, the mutation confirms the clinical diagnosis of ACH, and it seems to be causing the psychomotor delay in this patient.

Recessive multiple epiphyseal dysplasia and Stargardt disease in two sisters.

BACKGROUND: The rapid spread of genome-wide next-generation sequencing in the molecular diagnosis of rare genetic disorders has produced increasing evidence of multilocus genomic variations in cases with a previously well-characterized molecular diagnosis. Here, we describe two patients with a rare combination of skeletal abnormalities and retinal dystrophy caused by variants in the SLC26A2 and ABCA4 genes, respectively, in a family with parental consanguinity. METHODS: Next-generation sequencing and Sanger sequencing were performed to obtain a molecular diagnosis for the retinal and skeletal phenotypes, respectively. RESULTS: Genetic testing revealed that the sisters were homozygous for the p.(Cys653Ser) variant in SLC26A2 and heterozygous for the missense p.(Pro68Leu) and splice donor c.6386+2C>G variants in ABCA4. Segregation analysis confirmed the carrier status of the parents. CONCLUSION: Despite low frequency of occurrence, the detection of multilocus genomic variations in a single disease gene-oriented approach can provide accurate diagnosis even in cases with high phenotypic complexity. A targeted sequencing approach can detect relationships between observed phenotypes and underlying genotypes, useful for clinical management.

Potential role of microbiome in Chronic Fatigue Syndrome/Myalgic Encephalomyelits (CFS/ME).

Chronic Fatigue Syndrome/Myalgic Encephalomyelitis (CFS/ME) is a severe multisystemic disease characterized by immunological abnormalities and dysfunction of energy metabolism. Recent evidences suggest strong correlations between dysbiosis and pathological condition. The present research explored the composition of the intestinal and oral microbiota in CFS/ME patients as compared to healthy controls. The fecal metabolomic profile of a subgroup of CFS/ME patients was also compared with the one of healthy controls. The fecal and salivary bacterial composition in CFS/ME patients was investigated by Illumina sequencing of 16S rRNA gene amplicons. The metabolomic analysis was performed by an UHPLC-MS. The fecal microbiota of CFS/ME patients showed a reduction of Lachnospiraceae, particularly Anaerostipes, and an increased abundance of genera Bacteroides and Phascolarctobacterium compared to the non-CFS/ME groups. The oral microbiota of CFS/ME patients showed an increase of Rothia dentocariosa. The fecal metabolomic profile of CFS/ME patients revealed high levels of glutamic acid and argininosuccinic acid, together with a decrease of alpha-tocopherol. Our results reveal microbial signatures of dysbiosis in the intestinal microbiota of CFS/ME patients. Further studies are needed to better understand if the microbial composition changes are cause or consequence of the onset of CFS/ME and if they are related to any of the several secondary symptoms.