Matteo Accetturo

HmtDB, a Human Mitochondrial Genomic Resource Based on Variability Studies Supporting Population Genetics and Biomedical Research

BackgroundPopulation genetics studies based on the analysis of mtDNA and mitochondrial disease studies have produced a huge quantity of sequence data and related information. These data are at present worldwide distributed in differently organised databases and web sites not well integrated among them. Moreover it is not generally possible for the user to submit and contemporarily analyse its own data comparing them with the content of a given database, both for population genetics and mitochondrial disease data.ResultsHmtDB is a well-integrated web-based human mitochondrial bioinformatic resource aimed at supporting population genetics and mitochondrial disease studies, thanks to a new approach based on site-specific nucleotide and aminoacid variability estimation. HmtDB consists of a database of Human Mitochondrial Genomes, annotated with population data, and a set of bioinformatic tools, able to produce site-specific variability data and to automatically characterize newly sequenced human mitochondrial genomes. A query system for the retrieval of genomes and a web submission tool for the annotation of new genomes have been designed and will soon be implemented. The first release contains 1255 fully annotated human mitochondrial genomes. Nucleotide site-specific variability data and multialigned genomes can be downloaded. Intra-human and inter-species aminoacid variability data estimated on the 13 coding for proteins genes of the 1255 human genomes and 60 mammalian species are also available. HmtDB is freely available, upon registration, at http://www.hmdb.uniba.it.ConclusionThe HmtDB project will contribute towards completing and/or refining haplogroup classification and revealing the real pathogenic potential of mitochondrial mutations, on the basis of variability estimation.

The pathogenesis of diabetic nephropathy: focus on microRNAs and proteomics

The prevalence of type 2 diabetes mellitus is growing exponentially in Western countries, and the incidence of this condition is today increasing worldwide. Other than for cardiovascular complications, diabetes is particularly challenging for the kidneys health and proper function. Prolonged exposure of the kidneys to hyperglycemia in fact often results in a clinical complication called diabetic glomerulosclerosis, also known as diabetic nephropathy. Diabetic nephropathy represents today the leading cause of end-stage renal disease in Western countries. When left untreated or undiagnosed, diabetic nephropathy is ultimately responsible for the need for dialysis and, in the worst cases, kidney transplantation of the affected individuals. The pathogenesis of diabetic nephropathy has been studied extensively. A great number of metabolites, cytokines, proteins and transcription factors play a role in the accumulation of extracellular matrix and mesangial proliferation in the glomerulus; importantly, these phenotypic alterations are considered the 2 histological hallmarks of diabetic nephropathy. Additional effort is however required to understand the wide network of biochemical pathways that link diabetes to the renal damage in the long run. The integrative analysis of the proteomic and transcriptomic features of body fluids and/or bioptic samples among different categories of patients affected by diabetic nephropathy, if based on the accurate classification of the histopathological changes in the glomerular and tubulointerstitial compartment, could lead to the identification of new early biomarkers. This approach could represent an effective, noninvasive, alternative tool for early diagnosis and intervention.

Complement Modulation of Anti-Aging Factor Klotho in Ischemia/Reperfusion Injury and Delayed Graft Function.

Klotho is an anti‐aging factor mainly produced by renal tubular epithelial cells (TEC) with pleiotropic functions. Klotho is down‐regulated in acute kidney injury in native kidney; however, the modulation of Klotho in kidney transplantation has not been investigated. In a swine model of ischemia/reperfusion injury (IRI), we observed a remarkable reduction of renal Klotho by 24 h from IRI. Complement inhibition by C1‐inhibitor preserved Klotho expression in vivo by abrogating nuclear factor kappa B (NF‐kB) signaling. In accordance, complement anaphylotoxin C5a led to a significant down‐regulation of Klotho in TEC in vitro that was NF‐kB mediated. Analysis of Klotho in kidneys from cadaveric donors demonstrated a significant expression of Klotho in pre‐implantation biopsies; however, patients affected by delayed graft function (DGF) showed a profound down‐regulation of Klotho compared with patients with early graft function. Quantification of serum Klotho after 2 years from transplantation demonstrated significant lower levels in DGF patients. Our data demonstrated that complement might be pivotal in the down‐regulation of Klotho in IRI leading to a permanent deficiency after years from transplantation. Considering the anti‐senescence and anti‐fibrotic effects of Klotho at renal levels, we hypothesize that this acquired deficiency of Klotho might contribute to DGF‐associated chronic allograft dysfunction.

A type I interferon signature characterizes chronic antibody-mediated rejection in kidney transplantation.

Chronic antibody‐mediated rejection (CAMR) represents the main cause of kidney graft loss. To uncover the molecular mechanisms underlying this condition, we characterized the molecular signature of peripheral blood mononuclear cells (PBMCs) and, separately, of CD4+ T lymphocytes isolated from CAMR patients, compared to kidney transplant recipients with normal graft function and histology. We enrolled 29 patients with biopsy‐proven CAMR, 29 stable transplant recipients (controls), and 8 transplant recipients with clinical and histological evidence of interstitial fibrosis/tubular atrophy. Messenger RNA and microRNA profiling of PBMCs and CD4+ T lymphocytes was performed using Agilent microarrays in eight randomly selected patients per group from CAMR and control subjects. Results were evaluated statistically and by functional pathway analysis (Ingenuity Pathway Analysis) and validated in the remaining subjects. In PBMCs, 45 genes were differentially expressed between the two groups, most of which were up‐regulated in CAMR and were involved in type I interferon signalling. In the same patients, 16 microRNAs were down‐regulated in CAMR subjects compared to controls: four were predicted modulators of six mRNAs identified in the transcriptional analysis. In silico functional analysis supported the involvement of type I interferon signalling. To further confirm this result, we investigated the transcriptomic profiles of CD4+ T lymphocytes in an independent group of patients, observing that the activation of type I interferon signalling was a specific hallmark of CAMR. In addition, in CAMR patients, we detected a reduction of circulating BDCA2+ dendritic cells, the natural type I interferon‐producing cells, and their recruitment into the graft along with increased expression of MXA, a type I interferon‐induced protein, at the tubulointerstitial and vascular level. Finally, interferon alpha mRNA expression was significantly increased in CAMR compared to control biopsies. We conclude that type I interferon signalling may represent the molecular signature of CAMR. Copyright

Further phenotypic heterogeneity of CoQ10 deficiency associated with Steroid Resistant Nephrotic Syndrome and novel COQ2 and COQ6 variants.

To the Editor: Genes involved in the coenzyme Q10 biosynthetic pathway (COQ2, COQ6, PDSS1, PDSS2 and ADCK4) are mutated in about 1% of steroid resistant nephrotic syndrome (SRNS) cases and are often associated with neurological symptoms (1, 2). Here, we describe novel phenotypes associated with pathogenic mutations in two genes of the CoQ10 pathway, COQ2 and COQ6, in three patients with SRNS. Written informed consent was obtained from each patient and/or their parents. The study was performed according to the guidelines of the Declaration of Helsinki and the local Ethics Committees of University Hospitals of Foggia and Bari (Italy). One novel homozygous COQ2 variant, p.Gly390Ala (p.Gly340Ala, according to KU877220 GenBank sequence (2)) was identified by Next Generation Sequencing (NGS) (Appendix S1, Supporting information) in two cousins (patients P1 and P2; Fig. 1a; Tables S1 and S2) with SRNS associated with focal segmental glomerulosclerosis (FSGS) lesions and podocyte foot process effacement on renal biopsy (Fig. 1b, panels 1–4; Table S1). The pathogenicity of this variant is supported by its absence in public SNP (Single Nucleotide Polymorphism) database; the segregation with the disease (Fig. S1, panel 1); in silico predictions (Fig. 1c, Table S2); the reduced rate of respiratory growth and CoQ levels of yeast expressing this allele (Fig. 1d); the presence of numerous dysmorphic mitochondria on renal biopsy (Fig. 1b, panels 5 and 6). Remarkably, both patients harboring COQ2 change developed SRNS in adolescence with rapid progression to end-stage renal disease (ESRD) and had only mild neurological symptoms (Table S1). Of note, both cousins received a successful kidney transplant without recurrence of proteinuria and started CoQ10 treatment immediately after the genetic diagnosis. They are currently asymptomatic without any neurological symptoms after a 2-year follow up. Other authors reported patients with inherited COQ2 changes presented with isolated renal symptoms (2), however, to date, the reported age of onset of SRNS in patients carrying COQ2 variants was before the age of 2.5 years (2). To our knowledge, this is the first report describing COQ2 variants in patients with adolescent-onset SRNS and with a clinical spectrum resembling another CoQ10-glomerulopathy caused by mutations in ADCK4 (3). This finding recapitulates what has been observed in the Pdss2 (kd/kd) mice (2) and can be explained by the relatively mild effect of the p.Gly390Ala (p.Gly340Ala) allele, as documented by yeast studies (Fig. 1d). Mutational screening of patient P3 (Fig. 1e; Table S1) revealed a novel homozygous missense change in COQ6 gene: p.Pro261Leu (Fig. 1f; Table S2). The functional effect of this variant is supported by its low MAF in the European population (1:16,683, Table S2); the segregation with the disease (Fig. S1, panel 2); in silico predictions (Fig. 1g; Table S2) and the inability of COQ6 p.Pro261Leu allele to rescue the growth defect of the deleted yeast (Fig. 1h). Few patients with COQ6 changes (12) have been reported so far, they showed SRNS with onset at a median age of 1.2 years and sensorineural deafness (7/12) but some had also encephalopathic features (4). Our patient developed SRNS at 8 months with progressive deterioration of renal function and ESRD at 20 months. Of note, he did not present deafness or encephalopathic features. He is now on peritoneal dialysis treatment, however, CoQ10 treatment was started to prevent neurological symptoms. Interestingly, there was no history of schwannomatosis in the patient’s family, and unless further evidence supporting the link between heterozygous COQ6 mutations and schwannomatosis becomes available, we do not recommend mutational screening of COQ6 gene for schwannomas carriers (5). The most frequent glomerular lesion associated with COQ6 changes is FSGS (4). Remarkably, the analysis of renal biopsy of our patient revealed membranoproliferative glomerulonephritis and C3 deposits (Fig. 1f) usually associated with variants and risk haplotypes of complement-pathway genes, whose analysis did not reveal any variants with MAF (Minor Allele Frequency) <0.01 (data not shown). In summary, this study shows that we should analyze COQ2 and COQ6 genes also in patients with adolescent-onset of SRNS

Interleukin-27 is a potential marker for the onset of post-transplant malignancies

Background Malignancies represent the third leading cause of post-transplant mortality worldwide. The main challenge for transplant physicians is a timely diagnosis of this condition. The aim of the study was to identify a soluble diagnostic marker for monitoring the development of post-transplant malignancies. Methods This is a multicentre, observational, perspective, case-control study. We enrolled 47 patients with post-transplant solid neoplasia. As a control group we employed 106 transplant recipients without a history of neoplasia and matched them with cases for the main demographic and clinical features. We investigated the transcriptomic profiles of peripheral blood mononuclear cells from kidney graft recipients with and without post-transplant malignancies enrolled in two of the participating centres, randomly selected from the whole study population. Microarray results were confirmed by quantitative polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA) in the remaining patients from the same transplant centres and validated in a further independent group enrolled in two different transplant centres. Results We identified 535 differentially expressed genes comparing patients with and without post-transplant malignancies (fold change ≥2.5; false discovery rate <5%). The cancer pathway was closely related to gene expression data, and one of the most down-regulated genes in this pathway was interleukin-27 (IL-27), a cytokine regulating anti-tumour immunity. Quantitative PCR and ELISA confirmed the microarray data. Interestingly, IL-27 plasma levels were able to discriminate patients with post-transplant neoplasia with a specificity of 80% and a sensitivity of 81%. This observation was confirmed in an independent set of patients from two different transplant centres. Conclusions Our data suggest that IL-27 may represent a potential immunological marker for the timely identification of post-transplant neoplasia.

In Vitro Identification of New Transcriptomic and miRNomic Profiles Associated with Pulmonary Fibrosis Induced by High Doses Everolimus: Looking for New Pathogenetic Markers and Therapeutic Targets

The administration of Everolimus (EVE), a mTOR inhibitor used in transplantation and cancer, is often associated with adverse effects including pulmonary fibrosis. Although the underlying mechanism is not fully clarified, this condition could be in part caused by epithelial to mesenchymal transition (EMT) of airway cells. To improve our knowledge, primary bronchial epithelial cells (BE63/3) were treated with EVE (5 and 100 nM) for 24 h. EMT markers (α-SMA, vimentin, fibronectin) were measured by RT-PCR. Transepithelial resistance was measured by Millicell-ERS ohmmeter. mRNA and microRNA profiling were performed by Illumina and Agilent kit, respectively. Only high dose EVE increased EMT markers and reduced the transepithelial resistance of BE63/3. Bioinformatics showed 125 de-regulated genes that, according to enrichment analysis, were implicated in collagen synthesis/metabolism. Connective tissue growth factor (CTGF) was one of the higher up-regulated mRNA. Five nM EVE was ineffective on the pro-fibrotic machinery. Additionally, 3 miRNAs resulted hyper-expressed after 100 nM EVE and able to regulate 31 of the genes selected by the transcriptomic analysis (including CTGF). RT-PCR and western blot for MMP12 and CTGF validated high-throughput results. Our results revealed a complex biological network implicated in EVE-related pulmonary fibrosis and underlined new potential disease biomarkers and therapeutic targets.

Human microRNA expression in sporadic and FAP-associated desmoid tumors and correlation with beta-catenin mutations

Desmoid tumors (DT) are rare, benign, fibroblastic neoplasm with challenging histological diagnosis. DTs can occur sporadically or associated with the familial adenomatous polyposis coli (FAP). Most sporadic DTs are associated with β-catenin gene (CTNNB1) mutations, while mutated APC gene causes FAP disease. microRNAs (miRNAs) are involved in many human carcinogenesis. The miRNA profile was analyzed by microarray in formalin-fixed, paraffin-embedded (FFPE) specimens of 12 patients (8 sporadic, 4 FAP-associated) and 4 healthy controls. One hundred and one mRNAs resulted dysregulated, of which 98 in sporadic DTs and 8 in FAP-associated DTs, 5 were shared by both tumors. Twenty-six miRNAs were then validated by RT-qPCR in 23 sporadic and 7 FAP-associated DT samples matched with healthy controls. The qPCR method was also used to evaluate the CTNNB1 mutational status in sporadic DTs. The correlation between sporadic DTs and miRNA expression showed that miR-21-3p increased in mutated versus wild-type DTs, while miR-197-3p was decreased. The mRNA expression of Tetraspanin3 and Serpin family A member 3, as miR-21-3p targets, and L1 Cell Adhesion Molecule, as miR-197-3p target, was also evaluate. CTNNB1 mutations associated to miRNA dysregulation could affect the genesis and the progression of this disease and help histological diagnosis of sporadic DTs.