Antonella Izzo

Chronic Pro-oxidative State and Mitochondrial Dysfunctions are more Pronounced in Fibroblasts from Down Syndrome Foeti with Congenital Heart Defects

Trisomy of chromosome 21 is associated to congenital heart defects in ∼50% of affected newborns. Transcriptome analysis of hearts from trisomic human foeti demonstrated that genes involved in mitochondrial function are globally downregulated with respect to controls, suggesting an impairment of mitochondrial function. We investigated here the properties of mitochondria in fibroblasts from trisomic foeti with and without cardiac defects. Together with the upregulation of Hsa21 genes and the downregulation of nuclear encoded mitochondrial genes, an abnormal mitochondrial cristae morphology was observed in trisomic samples. Furthermore, impairment of mitochondrial respiratory activity, specific inhibition of complex I, enhanced reactive oxygen species production and increased levels of intra-mitochondrial calcium were demonstrated. Seemingly, mitochondrial dysfunction was more severe in fibroblasts from cardiopathic trisomic foeti that presented a more pronounced pro-oxidative state. The data suggest that an altered bioenergetic background in trisomy 21 foeti might be among the factors responsible for a more severe phenotype. Since the mitochondrial functional alterations might be rescued following pharmacological treatments, these results are of interest in the light of potential therapeutic interventions.

A novel CISD2 intragenic deletion, optic neuropathy and platelet aggregation defect in Wolfram syndrome type 2.

BackgroundWolfram Syndrome type 2 (WFS2) is considered a phenotypic and genotypic variant of WFS, whose minimal criteria for diagnosis are diabetes mellitus and optic atrophy. The disease gene for WFS2 is CISD2. The clinical phenotype of WFS2 differs from WFS1 for the absence of diabetes insipidus and psychiatric disorders, and for the presence of bleeding upper intestinal ulcers and defective platelet aggregation. After the first report of consanguineous Jordanian patients, no further cases of WFS2 have been reported worldwide. We describe the first Caucasian patient affected by WFS2.Case presentationThe proband was a 17 year-old girl. She presented diabetes mellitus, optic neuropathy, intestinal ulcers, sensorineural hearing loss, and defective platelet aggregation to ADP. Genetic testing showed a novel homozygous intragenic deletion of CISD2 in the proband. Her brother and parents carried the heterozygous mutation and were apparently healthy, although they showed subclinical defective platelet aggregation. Long runs of homozygosity analysis from SNP-array data did not show any degree of parental relationship, but the microsatellite analysis confirmed the hypothesis of a common ancestor.ConclusionOur patient does not show optic atrophy, one of the main diagnostic criteria for WFS, but optic neuropathy. Since the “asymptomatic” optic atrophy described in Jordanian patients is not completely supported, we could suppose that the ocular pathology in Jordanian patients was probably optic neuropathy and not optic atrophy. Therefore, as optic atrophy is required as main diagnostic criteria of WFS, it might be that the so-called WFS2 could not be a subtype of WFS. In addition, we found an impaired aggregation to ADP and not to collagen as previously reported, thus it is possible that different experimental conditions or inter-patient variability can explain different results in platelet aggregation. Further clinical reports are necessary to better define the clinical spectrum of this syndrome and to re-evaluate its classification.

Mental Retardation, Congenital Heart Malformation, and Myelodysplasia in a Patient With a Complex Chromosomal Rearrangement Involving the Critical Region 21q22

The region 21q22 is considered crucial for the pathogenesis of both Down syndrome (DS) and the partial monosomy 21q syndrome. Haploinsufficiency of the RUNX‐1 gene, mapping at 21q22 is responsible for a platelet disorder and causes predisposition to myelodysplastic syndrome (MDS). We describe a 3‐year‐old girl with mental retardation, congenital heart malformation, and subtle dysmorphic facial features. The patient developed thrombocytopenia when she was 2 years old. Bone marrow smear led to the diagnosis of myelodysplasia. Prenatal karyotyping had shown chromosome 21 pericentric inversion. Postnatally the array‐CGH revealed duplication at bands 21q11.2–21q21.1 and a simultaneous deletion involving the region 21q22.13–21q22.3. RUNX‐1 mRNA levels analyzed in patients skin fibroblasts were reduced. In this child the monosomy of the region 21q22 likely had the main role in determining the phenotype. Although the RUNX‐1 gene is localized outside the deleted region, we speculate that RUNX‐1 reduced expression, is probably due to the deletion of regulatory factors and caused the hematologic disorder in the patient. The present report underlines also the importance of array‐CGH in characterizing patients with a complex phenotype.

Metformin restores the mitochondrial network and reverses mitochondrial dysfunction in Down syndrome cells.

Alterations in mitochondrial activity and morphology have been demonstrated in human cells and tissues from individuals with Down syndrome (DS), as well as in DS mouse models. An impaired activity of the transcriptional coactivator PGC-1α/PPARGC1A due to the overexpression of chromosome 21 genes, such as NRIP1/RIP140, has emerged as an underlying cause of mitochondrial dysfunction in DS. We tested the hypothesis that the activation of the PGC-1α pathway might indeed reverse this mitochondrial dysfunction. To this end, we investigated the effects of metformin, a PGC-1α-activating drug, on mitochondrial morphology and function in DS foetal fibroblasts. Metformin induced both the expression of PGC-1α and an augmentation of its activity, as demonstrated by the increased expression of target genes, strongly promoting mitochondrial biogenesis. Furthermore, metformin enhanced oxygen consumption, ATP production, and overall mitochondrial activity. Most interestingly, this treatment reversed the fragmentation of mitochondria observed in DS and induced the formation of a mitochondrial network with a branched and elongated tubular morphology. Concomitantly, cristae remodelling occurred and the alterations observed by electron microscopy were significantly reduced. We finally demonstrated that the expression of genes of the fission/fusion machinery, namely OPA1 and MFN2, was reduced in trisomic cells and increased by metformin treatment. These results indicate that metformin promotes the formation of a mitochondrial network and corrects the mitochondrial dysfunction in DS cells. We speculate that alterations in the mitochondrial dynamics can be relevant in the pathogenesis of DS and that metformin can efficiently counteract these alterations, thus exerting protective effects against DS-associated pathologies.

Variegated silencing through epigenetic modifications of a large Xq region in a case of balanced X;2 translocation with Incontinentia Pigmenti-like Phenotype

Molecular mechanisms underlying aberrant phenotypes in balanced X;autosome translocations are scarcely understood. We report the case of a de novo reciprocal balanced translocation X;2(q23;q33) presenting phenotypic alterations highly suggestive of Incontinentia Pigmenti (IP) syndrome, a genodermatosis with abnormal skin pigmentation and neurological failure, segregating as X-linked dominant disorder. Through molecular studies, we demonstrated that the altered phenotype could not be ascribed to chromosome microdeletions or to XIST-mediated inactivation of Xq24-qter. Interestingly, we found that the Xq24-qter region, which translocated downstream of the heterochromatic band 2q34, undergoes epigenetic silencing mediated by DNA methylation and histone alterations. Among the downregulated genes, we found the inhibitor of kappa light polypeptide gene enhancer in B cells, kinase gamma (IKBKG/NEMO), the causative gene of IP. We hypothesize that a mosaic functional nullisomy of the translocated genes, through a Position Effect Variegation-like heterochromatization, might be responsible for the proband’s phenotypic anomalies. Partial silencing of IKBKG may be responsible for the skin anomalies observed, thereby mimicking the IP pathological condition. In addition to its clinical relevance, this paper addresses fundamental issues related to the chromatin status and nuclear localization of a human euchromatic region translocated proximally to heterochromatin. In conclusion, the study provides new insight into long-range gene silencing mechanisms and their direct impact in human disease.

Rab7 Regulates CDH1 Endocytosis, Circular Dorsal Ruffles Genesis, and Thyroglobulin Internalization in a Thyroid Cell Line

Rab7 regulates the biogenesis of late endosomes, lysosomes, and autophagosomes. It has been proposed that a functional and physical interaction exists between Rab7 and Rac1 GTPases in CDH1 endocytosis and ruffled border formation. In FRT cells over‐expressing Rab7, increased expression and activity of Rac1 was observed, whereas a reduction of Rab7 expression by RNAi resulted in reduced Rac1 activity, as measured by PAK1 phosphorylation. We found that CDH1 endocytosis was extremely reduced only in Rab7 over‐expressing cells but was unchanged in Rab7 silenced cells. In Rab7 under or over‐expressing cells, Rab7 and LC3B‐II co‐localized and co‐localization in large circular structures occurred only in Rab7 over‐expressing cells. These large circular structures occurred in about 10% of the cell population; some of them (61%) showed co‐localization of Rab7 with cortactin and f‐actin and were identified as circular dorsal ruffles (CDRs), the others as mature autophagosomes. We propose that the over‐expression of Rab7 is sufficient to induce CDRs. Furthermore, in FRT cells, we found that the expression of the insoluble/active form of Rab7, rather than Rab5, or Rab8, was inducible by cAMP and that cAMP‐stimulated FRT cells showed increased PAK1 phosphorylation and were no longer able to endocytose CDH1. Finally, we demonstrated that Rab7 over‐expressing cells are able to endocytose exogenous thyroglobulin via pinocytosis/CDRs more efficiently than control cells. We propose that the major thyroglobulin endocytosis described in thyroid autonomous adenomas due to Rab7 increased expression, occurs via CDRs. J. Cell. Physiol. 231: 1695–1708, 2016.

Complex chromosomal rearrangements causing Langer-Giedion syndrome atypical phenotype: genotype-phenotype correlation and literature review.

Langer–Giedion syndrome (LGS) is caused by a deletion of chromosome 8q23.3–q24.11. The LGS clinical spectrum includes intellectual disability (ID), short stature, microcephaly, facial dysmorphisms, exostoses. We describe a 4‐year‐old girl with ID, short stature, microcephaly, distinctive facial phenotype, skeletal signs (exostoses on the left fibula, coccyx agenesis, stubby and dysmorphic sphenoid bone, osteoporosis), central nervous system malformations (hypoplastic and dysmorphic corpus callosum and septum pellucidum), pituitary gland hypoplasia and hyperreninemia. Array‐CGH revealed complex chromosomal rearrangements. A diagnosis of LGS was confirmed by the detection of a 8q23.3–q24.1 deletion. Associated chromosomal abnormalities were a 21q22.1 deletion and a balanced reciprocal translocation t(2;11)(p24;p15) de novo, confirmed by FISH analysis. We document the patients atypical findings, never described in LGS patients, in order to update the genotype–phenotype correlation. We speculate that the disruption of regulatory elements mapping upstream CYP11B2 involved in the deleted region could cause hyperreninemia.

40 Mb duplication in chromosome band 5p13.1p15.33 with 800 kb terminal deletion in a foetus with mild phenotypic features

Large duplication of the short arm of chromosome 5 is a rare condition normally associated to severe phenotype anomalies including heart and brain malformations. We report a prenatal case of a large 5p duplication with sub-telomeric deletion in a foetus with very mild phenotypic abnormalities. Foetal ultrasonographic examination at 22 weeks of gestation showed short femur, clubfeet, pielectasy, and facial dysmorphisms. Chromosome investigations revealed an inverted duplication of the short arm of chromosome 5 from 5p13.1 to 5p15.33 and a 800 kb deletion at 5pter. The absence of severe anomalies such as cardiac and cerebral defects, observed so far in all large 5p duplications, and the comparison to previous cases described both in literature and in DECIPHER database suggest that the critical region for the severe phenotype in 5p duplication syndrome might be smaller than that previously described, excluding half of the 5p13 band. This might help in prenatal genetic counselling.

Gene Expression Profile in Liver Transplantation and the Influence of Gene Dysregulation Occurring in Deceased Donor Grafts

Background: Brain dead patients are the main source of organs for transplants. Brain death causes changes in peripheral organs. We define modifications of gene expression in specific pathways occurring in donor livers and their influence on gene expression profile of livers after transplant. Methods: We compared gene expression profile of both deceased donor livers and transplanted livers to gene expression data of liver tissue, retrieved from Array Express database, used as control. All expression data were obtained by microarray analysis. Results: The expression of about 33,000 genes has been compared in liver samples from three groups: deceased donor livers, transplanted livers two hours after reperfusion, and control livers. We found that about 900 genes are dysregulated in deceased donor versus control livers. Up-regulated genes are mainly involved in apoptosis, immune response and inflammation. Down-regulated genes are mostly involved in metabolism and electron transport. We also re-evaluated a group of genes that in a previous study were found dysregulated in transplanted livers when compared to donor livers. Most of these genes, but not all, were dysregulated also when compared to control livers. Moreover 317 additional genes, dysregulated after liver transplant, were identified in this study; they were undetectable in the previous study because they had the same dysregulation both in donor and in transplanted livers. Conclusions: Understanding molecular mechanisms that in the donor compromise graft function is crucial in order to discriminate between basal graft damages and ischemia-reperfusion injuries and therefore to identify therapeutic targets aiming to improve liver transplantation performances.

Overexpression of Chromosome 21 miRNAs May Affect Mitochondrial Function in the Hearts of Down Syndrome Fetuses

Dosage-dependent upregulation of most of chromosome 21 (Hsa21) genes has been demonstrated in heart tissues of fetuses with Down syndrome (DS). Also miRNAs might play important roles in the cardiac phenotype as they are highly expressed in the heart and regulate cardiac development. Five Hsa21 miRNAs have been well studied in the past: miR-99a-5p, miR-125b-2-5p, let-7c-5p, miR-155-5p, and miR-802-5p but few information is available about their expression in trisomic tissues. In this study, we evaluated the expression of these miRNAs in heart tissues from DS fetuses, showing that miR-99a-5p, miR-155-5p, and let-7c-5p were overexpressed in trisomic hearts. To investigate their role, predicted targets were obtained from different databases and cross-validated using the gene expression profiling dataset we previously generated for fetal hearts. Eighty-five targets of let-7c-5p, 33 of miR-155-5p, and 10 of miR-99a-5p were expressed in fetal heart and downregulated in trisomic hearts. As nuclear encoded mitochondrial genes were found downregulated in trisomic hearts and mitochondrial dysfunction is a hallmark of DS phenotypes, we put special attention to let-7c-5p and miR-155-5p targets downregulated in DS fetal hearts and involved in mitochondrial function. The let-7c-5p predicted target SLC25A4/ANT1 was identified as a possible candidate for both mitochondrial and cardiac anomalies.