Iris Scala

Analysis of seven maternal polymorphisms of genes involved in homocysteine/folate metabolism and risk of Down syndrome offspring

PURPOSE: We present a case-control study of seven polymorphisms of six genes involved in homocysteine/folate pathway as risk factors for Down syndrome. Gene-gene/allele-allele interactions, haplotype analysis and the association with age at conception were also evaluated.METHODS: We investigated 94 Down syndrome-mothers and 264 control-women from Campania, Italy.RESULTS: Increased risk of Down syndrome was associated with the methylenetetrahydrofolate reductase (MTHFR) 1298C allele (OR 1.46; 95% CI 1.02–2.10), the MTHFR 1298CC genotype (OR 2.29; 95% CI 1.06–4.96), the reduced-folate-carrier1 (RFC1) 80G allele (1.48; 95% CI 1.05–2.10) and the RFC1 80 GG genotype (OR 2.05; 95% CI 1.03–4.07). Significant associations were found between maternal age at conception ≥34 years and either the MTHFR 1298C or the RFC 180G alleles. Positive interactions were found for the following genotype-pairs: MTHFR 677TT and 1298CC/CA, 1298CC/CA and RFC1 80 GG/GA, RFC1 80 GG and methylenetetrahydrofolate-dehydrogenase 1958 AA. The 677–1298 T-C haplotype at the MTHFR locus was also a risk factor for Down syndrome (P = 0.0022). The methionine-synthase-reductase A66G, the methionine-synthase A2756G and the cystathionine-beta-synthase 844ins68 polymorphisms were not associated with increased risk of Down syndrome.CONCLUSION: These results point to a role of maternal polymorphisms of homocysteine/folate pathway as risk factors for Down syndrome.

Impairment of methyl cycle affects mitochondrial methyl availability and glutathione level in Down's syndrome

In Downs syndrome there is evidence that increased gene expression coding for specific cystathionine beta-synthase translates directly into biochemical aberrations, which result in a biochemical and metabolic imbalance of the methyl status. This event is destined to impact mitochondrial function since methylation is a necessary event in mitochondria and relies on the availability and uptake of the methyl donor S-adenosylmethionine. Indeed mitochondrial dysfunctions have been widely described in Downs syndrome, but they have never been correlated to a possible mitochondrial methyl unbalance. In the present study we find that the mitochondrial levels of S-adenosylmethionine are reduced in Downs syndrome compared to control cells demonstrating the effect of the methyl unbalance on mitochondria. The possible role of methylation in mitochondria is discussed and some preliminary results on a possible methylation target are presented.

Expanding the clinical and neuroradiologic phenotype of primary microcephaly due to ASPM mutations

Objective: To determine the spectrum of clinical, neuropsychological, and neuroradiologic features in patients with autosomal recessive primary microcephaly (MCPH) due to ASPM gene mutations. Methods: ASPM was sequenced in 52 unrelated MCPH probands. In patients with ASPM mutations, we evaluated the clinical phenotype, cognition, behavior, brain MRI, and family. Results: We found homozygous or compound heterozygous ASPM loss-of-function mutations in 11 (22%) probands and 5 siblings. The probands harbored 18 different mutations, of which 16 were new. Microcephaly was severe after 1 year of age in all 16 patients, although in 4 patients the occipital-frontal circumference (OFC) at birth was decreased by only 2 SD. The OFC Z score consistently decreased after birth. Late-onset seizures occurred in 3 patients and significant pyramidal tract involvement in 1 patient. Intellectual quotients ranged from borderline-normal to severe mental retardation. Mild motor delay was noted in 7/16 patients. Language development was delayed in all patients older than 3 years. Brain MRI (n = 12) showed a simplified gyral pattern in 9 patients and several malformations including ventricle enlargement (n = 7), partial corpus callosum agenesis (n = 3), mild cerebellar hypoplasia (n = 1), focal cortical dysplasia (n = 1), and unilateral polymicrogyria (n = 1). Non-neurologic abnormalities consisted of short stature (n = 1), idiopathic premature puberty (n = 1), and renal dysplasia (n = 1). Conclusions: We provide a detailed description of features associated with ASPM mutations. Borderline microcephaly at birth, borderline-normal intellectual efficiency, and brain malformations can occur in ASPM-related primary hereditary microcephaly.

Epigallocatechin-3-gallate prevents oxidative phosphorylation deficit and promotes mitochondrial biogenesis in human cells from subjects with Down's syndrome

A critical role for mitochondrial dysfunction has been proposed in the pathogenesis of Downs syndrome (DS), a human multifactorial disorder caused by trisomy of chromosome 21, associated with mental retardation and early neurodegeneration. Previous studies from our group demonstrated in DS cells a decreased capacity of the mitochondrial ATP production system and overproduction of reactive oxygen species (ROS) in mitochondria. In this study we have tested the potential of epigallocatechin-3-gallate (EGCG) - a natural polyphenol component of green tea - to counteract the mitochondrial energy deficit found in DS cells. We found that EGCG, incubated with cultured lymphoblasts and fibroblasts from DS subjects, rescued mitochondrial complex I and ATP synthase catalytic activities, restored oxidative phosphorylation efficiency and counteracted oxidative stress. These effects were associated with EGCG-induced promotion of PKA activity, related to increased cellular levels of cAMP and PKA-dependent phosphorylation of the NDUFS4 subunit of complex I. In addition, EGCG strongly promoted mitochondrial biogenesis in DS cells, as associated with increase in Sirt1-dependent PGC-1α deacetylation, NRF-1 and T-FAM protein levels and mitochondrial DNA content. In conclusion, this study shows that EGCG is a promoting effector of oxidative phosphorylation and mitochondrial biogenesis in DS cells, acting through modulation of the cAMP/PKA- and sirtuin-dependent pathways. EGCG treatment promises thus to be a therapeutic approach to counteract mitochondrial energy deficit and oxidative stress in DS.

Aberrant Glycosylation as Biomarker for Cancer: Focus on CD43

Glycosylation is a posttranslational modification of proteins playing a major role in cell signalling, immune recognition, and cell-cell interaction because of their glycan branches conferring structure variability and binding specificity to lectin ligands. Aberrant expression of glycan structures as well as occurrence of truncated structures, precursors, or novel structures of glycan may affect ligand-receptor interactions and thus interfere with regulation of cell adhesion, migration, and proliferation. Indeed, aberrant glycosylation represents a hallmark of cancer, reflecting cancer-specific changes in glycan biosynthesis pathways such as the altered expression of glycosyltransferases and glycosidases. Most studies have been carried out to identify changes in serum glycan structures. In most cancers, fucosylation and sialylation are significantly modified. Thus, aberrations in glycan structures can be used as targets to improve existing serum cancer biomarkers. The ability to distinguish differences in the glycosylation of proteins between cancer and control patients emphasizes glycobiology as a promising field for potential biomarker identification. In this review, we discuss the aberrant protein glycosylation associated with human cancer and the identification of protein glycoforms as cancer biomarkers. In particular, we will focus on the aberrant CD43 glycosylation as cancer biomarker and the potential to exploit the UN1 monoclonal antibody (UN1 mAb) to identify aberrant CD43 glycoforms.

Homocysteine Lowering by Folate-Rich Diet or Pharmacological Supplementations in Subjects with Moderate Hyperhomocysteinemia

Background/Objectives: To compare the efficacy of a diet rich in natural folate and of two different folic acid supplementation protocols in subjects with “moderate” hyperhomocysteinemia, also taking into account C677T polymorphism of 5,10-methylenetetrahydrofolate reductase (MTHFR) gene. Subjects/Methods: We performed a 13 week open, randomized, double blind clinical trial on 149 free living persons with mild hyperhomocyteinemia, with daily 200 μg from a natural folate-rich diet, 200 μg [6S]5-methyltetrahydrofolate (5-MTHF), 200 μg folic acid or placebo. Participants were stratified according to their MTHFR genotype. Results: Homocysteine (Hcy) levels were reduced after folate enriched diet, 5-MTHF or folic acid supplementation respectively by 20.1% (p < 0.002), 19.4% (p < 0.001) and 21.9% (p < 0.001), as compared to baseline levels and significantly as compared to placebo (p < 0.001, p < 0.002 and p < 0.001, respectively for enriched diet, 5-MTHF and folic acid). After this enriched diet and the folic acid supplementation, Hcy in both genotype groups decreased approximately to the same level, with higher percentage decreases observed for the TT group because of their higher pre-treatment value. Similar results were not seen by genotype for 5-MTHF. A significant increase in RBC folate concentration was observed after folic acid and natural folate-rich food supplementations, as compared to placebo. Conclusions: Supplementation with natural folate-rich foods, folic acid and 5-MTHF reached a similar reduction in Hcy concentrations.

Functional and structural characterization of novel mutations and genotype-phenotype correlation in 51 phenylalanine hydroxylase deficient families from Southern Italy.

Hyperphenylalaninemia (Online Mendelian Inheritance in Man ®database: 261600) is an autosomal recessive disorder mainly due to mutations in the gene for phenylalanine hydroxylase; the most severe form of hyperphenylalaninemia is classic phenylketonuria. We sequenced the entire gene for phenylalanine hydroxylase in 51 unrelated hyperphenylalaninemia patients from Southern Italy. The entire locus was genotyped in 46 out of 51 hyperphenylalaninemia patients, and 32 different disease‐causing mutations were identified. The pathologic nature of two novel gene variants, namely, c.707‐2delA and p.Q301P, was demonstrated by in vitro studies. c.707‐2delA is a splicing mutation that involves the accepting site of exon 7; it causes the complete skipping of exon 7 and results in the truncated p.T236MfsX60 protein. The second gene variant, p.Q301P, has very low residual enzymatic activity (∼ 4.4%), which may be ascribed, in part, to a low expression level (8–10%). Both the decreased enzyme activity and the low expression level are supported by analysis of the 3D structure of the molecule. The putative structural alterations induced by p.Q301P are compatible with protein instability and perturbance of monomer interactions within dimers and tetramers, although they do not affect the catalytic site. In vivo studies showed tetrahydrobiopterin responsiveness in the p.Q301P carrier but not in the c.707‐2delA carrier. We next investigated genotype–phenotype correlations and found that genotype was a good predictor of phenotype in 76% of patients. However, genotype–phenotype discordance occurred in approximately 25% of our patients, mainly those bearing mutations p.L48S, p.R158Q, p.R261Q and p.P281L.

Accumulation of altered aspartyl residues in erythrocyte proteins from patients with Down's syndrome

Spontaneous protein deamidation of labile Asn residues, generating l‐isoaspartates and d‐aspartates, is associated with cell aging and is enhanced by an oxidative microenvironment; to minimize the damage, the isoaspartate residues can be ‘repaired’ by a specific l‐isoaspartate (d‐aspartate) protein O‐methyltransferase (PIMT). As both premature aging and chronic oxidative stress are typical features of Downs syndrome (DS), we tested the hypothesis that deamidated proteins may build up in trisomic patients. Blood samples were obtained from children with karyotypically confirmed full trisomy 21 and from age‐matched healthy controls. Using recombinant PIMT as a probe, we demonstrated a dramatic rise of l‐isoaspartates in erythrocyte membrane proteins from DS patients. The content of d‐aspartate was also significantly increased. The integrity of the repair system was checked by evaluating methionine transport, PIMT specific activity, and intracellular concentrations of adenosylmethionine and adenosylhomocysteine. The overall methylation pathway was directly monitored by incubating fresh red blood cells with methyl‐labeled methionine; a three‐fold increase of protein methyl esters was detected in trisomic children. Deamidated species include ankyrin, band 4.1, band 4.2 and the integral membrane protein band 3; ankyrin and band 4.1 were significantly hypermethylated in DS. When DS red blood cells were subjected to oxidative treatment in vitro, the increase of protein deamidation paralleled lipid peroxidation and free radical generation. We observed a similar pattern in Epstein–Barr virus B‐lymphocytes from trisomic patients. In conclusion, our findings support the hypothesis that protein instability at asparagine sites is a biochemical feature of DS, presumably depending upon the oxidative microenvironment. The possible pathophysiological implications are discussed.

Cancer-Associated CD43 Glycoforms as Target of Immunotherapy

CD43 is a sialoglycosylated membrane protein that is involved in cell proliferation and differentiation. CD43 glycoforms that are recognized by the UN1 monoclonal antibody (mAb) were expressed in lymphoblastoid T-cell lines and solid tumors, such as breast, colon, gastric, and squamous cell lung carcinomas, while unexpressed in the normal counterparts. The cancer association of UN1/CD43 epitope suggested the possibility to use the UN1 mAb for tumor diagnosis and therapy. In this study, we show that the UN1 mAb was endowed with antitumor activity in vivo because its passive transfer inhibited the growth of UN1-positive HPB-ALL lymphoblastoid T cells in mice. Furthermore, we demonstrate that tumor inhibition was due to UN1 mAb-dependent natural killer–mediated cytotoxicity. By screening a phage-displayed random peptide library, we identified the phagotope 2/165 as a mimotope of the UN1 antigen, as it harbored a peptide sequence that was specifically recognized by the UN1 mAb and inhibited the binding of the UN1 mAb to UN1-positive tumor cells. On the basis of sequence homology with the extracellular region of CD43 (amino acids 64 to 83), the 2/165 peptide sequence was likely mimicking the protein core of the UN1/CD43 epitope. When used as vaccine in mice, the 2/165 phagotope raised antibodies against the UN1/CD43 antigen, indicating that the 2/165 phagotope mimicked the UN1 antigen structure, and could represent a novel immunogen for cancer immunotherapy. These findings support the feasibility of using monoclonal antibodies to identify cancer-associated mimotopes for immunotherapy. Mol Cancer Ther; 13(3); 752–62. ©2013 AACR.

Eukaryotic Initiation Factor 4H Is under Transcriptional Control of p65/NF-κB.

Protein synthesis is mainly regulated at the initiation step, allowing the fast, reversible and spatial control of gene expression. Initiation of protein synthesis requires at least 13 translation initiation factors to assemble the 80S ribosomal initiation complex. Loss of translation control may result in cell malignant transformation. Here, we asked whether translational initiation factors could be regulated by NF-κB transcription factor, a major regulator of genes involved in cell proliferation, survival, and inflammatory response. We show that the p65 subunit of NF-κB activates the transcription of eIF4H gene, which is the regulatory subunit of eIF4A, the most relevant RNA helicase in translation initiation. The p65-dependent transcriptional activation of eIF4H increased the eIF4H protein content augmenting the rate of global protein synthesis. In this context, our results provide novel insights into protein synthesis regulation in response to NF-κB activation signalling, suggesting a transcription-translation coupled mechanism of control.