Lucia Giordani

Association of Breast Cancer and Polymorphisms of Interleukin-10 and Tumor Necrosis Factor-α Genes

We investigated the potential relationship between breast cancer and polymorphisms of the interleukin 10 (IL-10) and tumor necrosis factor-α (TNF-α) genes. High plasma TNF-α concentrations predict poor cancer outcome (1), weight loss, cachexia, poor immune response, and anemia (2)(3). We studied the −308(G/A) polymorphism because it is the most frequently investigated, it is included in an AP-2 transcription-factor binding site of the TNF-α gene (4)(5)(6), and its importance in TNF-α gene expression has been demonstrated by reporter gene assays (7). The TNF-α −308 polymorphism affects TNF-α gene transcription in both a cell-type and stimulus-specific manner, with a remarkable effect in macrophage-like cells (8). IL-10, an important immunoregulatory cytokine, induces T-cell anergy (9) and prevents tumor antigen presentation to CD8+ cytotoxic T lymphocytes by suppressing expression of MHC class I and II antigens (10). This effect may contribute to the lack of immune response toward transformed cells (11)(12)(13). Three polymorphisms [−1082(G/A), −819(C/T), and −592(C/A)] have been described in the IL-10 promoter region, but data are unclear regarding their influence, alone or in reciprocal linkage, on transcription (14). Higher IL-10 production seems to be associated with −1082 G/G. The positive effect on IL-10 gene expression was substantiated by reporter gene assays (15)(16), although, in some cases, differing results have been described in cells undergoing distinct stimuli (17). There appears to be conflicting evidence on the interplay between IL-10 and cancer. Indeed, it has been proposed that IL-10 might contribute to tumor escape from the immune response, but it may also exert an antitumor effect. In one study, IL-10-deficient mice developed colitis and colorectal cancer, mirroring the increased colorectal cancer incidence observed in patients with inflammatory bowel disease (18). In another study, the results indicated that the G/G genotype, …

Homozygosis for (12) CA repeats in the first intron of the human IFN‐γ gene is significantly associated with the risk of aplastic anaemia in Caucasian population

Interferon‐γ (IFN‐γ) mediates the final damage of the stem cell compartment in Aplastic Anaemia (AA). Normal subjects homozygous for 12 (CA) repeats of polymorphism variable number of dinucleotide (CA) repeat (VNDR) in position 1349 of the IFN‐γ gene (IFNG) were shown to overproduce IFN‐γin vitro. We studied the distribution of polymorphism VNDR 1349 of IFNG in 67 Caucasian AA patients and in normal controls. Genotype (CA)12‐12, (homozygosis for allele 2) and the single allele 12 were significantly more frequent (P = 0·005 and 0·004 respectively) in patients versus controls. The polymorphism was equally distributed in AA patients regardless of their response to immunosuppression. Homozygosity for 12 (CA) repeats of polymorphism VNDR 1349 of IFNG is strongly associated with the risk of AA in Caucasian subjects.

Dyserythropoietic Anemia and Thrombocytopenia due to a Novel Mutation in GATA-1

Hematopoiesis is a complex process regulated by nuclear proteins that coordinate lineage-specific patterns of gene expression. Targeted mutagenesis has revealed critical roles for the X-linked transcription factor GATA-1 in erythrocyte and megakaryocyte differentiation. GATA-1 has two zinc fingers essential for normal function. The C-terminal finger is necessary for DNA binding. The N-terminal finger mediates interaction with FOG-1, a cofactor for GATA-1. Mutations in the N-terminal zinc finger of GATA-1 result in abnormal hematopoiesis. Here we report a family with a novel single base mutation that results in an amino acid substitution (Gly208Arg) within the highly conserved portion of the GATA-1 N-terminal finger domain, leading to dyserythropoietic anemia and macrothrombocytopenia. Another mutation described at the same codon (208) has been found to be associated with thrombocytopenia only. Our data support and extend the effect of the amino acid substitution at codon 208 on GATA-1 function not only regarding megakaryocyte but also regarding erythroid development.

Caspase 3 and 8 deficiency in human neuroblastoma.

An altered apoptotic response represents a pivotal feature of cancer and is involved in cancerogenesis and resistance to chemotherapy. So far, however, only a few studies have been devoted to survey caspase content in malignant cell lines and primary tumor specimens. In this report, we investigated the expression of two pivotal caspases, 3 and 8, in 63 neuroblastoma specimens by three complementary techniques (i.e., reverse transcriptase polymerase chain reaction, immunoblotting, and immunohistochemistry). We confirmed the frequent absence of caspase 8 expression. Moreover and most important, we demonstrated, for the first time to our knowledge, that a significant percentage of neuroblastomas lack caspase 3 mRNA and protein. Both caspase alterations do not show any correlation with tumor stage and MYCN status. Immunohistochemistry showed a large number of caspase-negative cell islets also present in positive samples. Our findings suggest that the absence of caspases might play an important role in neuroblastoma development and resistance to apoptosis-based treatments.

Reduced expression of transforming growth factor-beta receptor type III in high stage neuroblastomas

Transforming growth factor beta (TGF-β) is a powerful inhibitor of cell proliferation and a potent inducer of differentiation. Resistance to TGF-β action is a characteristic of many malignancies and has been attributed to alterations of TGF-β receptors as well as disturbance of downstream transduction pathways. To analyse the TGF-β response in neuroblastoma, the expression of TGF-β1 and TGF-β type I, II and III receptor genes was investigated in 61 cancer samples by means of reverse transcription polymerase chain reaction. The specimens analysed belong to different stages, namely nine samples of stage 1, ten of stage 2, nine of stage 3 and 28 of stage 4. Moreover, five samples were of stage 4S, which represents a tumour form undergoing spontaneous regression. The results obtained show that TGF-β1 and TGF-β type I and II receptor genes appear to be almost equally expressed in neuroblastomas of all stages. Conversely, TGF-β type III receptor gene expression, which is required for an efficacious TGF-β binding and function, is strongly reduced exclusively in neuroblastomas of stages 3 and 4. These findings were directly confirmed by immunohistochemical analyses of ten neuroblastoma specimens. Our results suggest the occurrence of an altered TGF-β response in advanced neuroblastomas which might be an important mechanism for escaping growth control and for developing invasiveness. Moreover, our findings allow the proposal of a novel mechanism, namely down-regulation of TGF-β type III receptor gene expression, to avoid TGF-β inhibitory activity.

IGF2 Gene Variants and Risk of Hypertension in Obese Children and Adolescents

Obese children have a great risk of hypertension and cardiovascular morbidity in adults. The insulin-like growth factor type II (IGF-II) regulates glucose homeostasis, cardiovascular functions, and lipid metabolism. IGF2 gene variants have shown a strong association with weight, body mass index (BMI), and metabolic profile in adults. We performed the molecular screening of two IGF2 polymorphisms (6815 A/T, 820 G/A), in 227 obese children to evaluate the potential association between IGF2 variants with either obesity or high blood pressure (assessed with a 24-h holter system) or both. A second cohort of age-, sex-, and BMI-matched children were enrolled to confirm any eventual association. We observed a significant association between the 6815 A/T IGF2 gene variant and high systolic blood pressure in obese children. Homozygote subjects for the T6815 allele showed, even in 24-h measurements, a higher risk to develop hypertension than those carrying the A6815 allele (OR = 3.7, 95% CI: 1.59–8.66). This result was confirmed in the second cohort (OR = 4.1, 95% CI: 1.41–6.50). Any statistically significant difference in terms of BMI between the genotype groups was observed. Our results suggest that IGF2 gene variants are involved in the blood pressure regulation in obese children.

Bilirubin levels in the acute hemolytic crisis of G6PD deficiency are related to Gilbert's syndrome

Abstract:  In this study we analyzed the effect of the (TA)7 polymorphism of the UGT1A gene associated with Gilberts syndrome in G6PD‐deficient subjects during an acute hemolytic crisis (tabic crisis). DNA from 44 subjects originating from the same geographic area in Sardinia was analyzed for the UGT1A promoter polymorphism. The increase of unconjugated bilirubin level during fabic crisis and its relationship with UGT1A polymorphism was evaluated. The UGT1A (TA)7 TATA box variant was found in 9/44 (21%) of the G6PD deficient subjects examined. The median value for unit of increase of bilirubin (mg/dl)/unit of decrease of hemoglobin (g/dl) was higher in variant homozygous than in heterozygous and normal subjects. These findings imply a contribution of the UGT1A polymorphism associated to Gilberts syndrome to development of the hyperbilirubinemia in G6PD deficient subjects during acute hemolytic anemia.

Interstitial and large chromosome 1p deletion occurs in localized and disseminated neuroblastomas and predicts an unfavourable outcome

We studied chromosome 1p loss of heterozygosity (1p-LOH) in 53 neuroblastomas (NBs) using 15 (CA)n repeat loci, which covered a region of 90 cM. We also assessed chromosome 1p36 deletion by fluorescence in situ hybridization (FISH) on interphase nuclei. 1p-LOH was found in 19 (36%, 95% confidence interval (CI) 23-50%) NBs. We detected interstitial and large deletion in both localized and disseminated tumours and in one tumour of a patient at stage 4S. Allelic loss was frequently observed in 1p36 and 1p32 regions. In patients older than 1 year of age (53 versus 13%, P < 0.002) we detected significant chromosome 1p deletion and it was associated with MYCN amplification (P = 0.001). Overall survival (OS) analysis showed that 1p-LOH is predictive of a poor outcome (odds ratio 16.5, 95% CI 5.4-50.9%); therefore, 1p-LOH should be regarded as an additional tumour progression marker in neuroblastoma.

Structural and Functional Analysis of Cyclin-Dependent Kinase Inhibitor Genes(CDKN2A, CDKN2B, and CDKN2C) in Neuroblastoma

The status of the CDKN2A gene family, including CDKN2A, CDKN2B, and CDKN2C, was investigated in 24 cases of neuroblastoma. These genes were selected on the basis of 1) high incidence of their inactivation in several human cancers and 2) their localization on chromosomal regions (9p and 1p) frequently rearranged in neuroblastomas. Detailed molecular analyses indicated the absence of homozygous deletions and point mutations involving these genes in all investigated tumor samples. However, when loss of heterozygostity for chromosome 9p21 (the region where CDKN2A and CDKN2B are localized) was investigated, 16% of cases showed abnormalities in an area telomeric to the CDKN2A locus. To study transcriptional silencing of the CDKN2A gene, the methylation status of exon 1 was examined. In about 35% of cases, a partial methylation was evidenced. Analysis of the CDKN2A mRNA expression, however, did not show any relationship between methylation status and gene transcription. Finally, expression of the CDKN2B gene was demonstrated in all stage IV neuroblastomas, whereas none of stage I tumors expressed this gene. This finding suggests the occurrence of a correlation between CDKN2B transcription and tumor phenotype.

PTPN11 Gene Mutation and Severe Neonatal Hypertrophic Cardiomyopathy: What Is the Link?

Noonan syndrome (NS) is an autosomal dominant disorder characterized by multiple dysmorphic features and a broad spectrum of congenital heart defects. Specific mutations of the PTPN11 gene are associated with 50% of the NS cases and 90% of the multiple lentigines/LEOPARD syndrome (ML/LS) cases. These two allelic conditions have several overlapping clinical features. This study describes the association between the Gln510Glu mutation of the PTPN11 gene and lethal progressive hypertrophic cardiomyopathy (HCM) in a newborn with the NS phenotype. The findings confirm the intriguing relationship between site-specific mutations of the PTPN11 gene and rapidly progressive HCM.