Missense NR2F1 variant in monozygotic twins affected with the Bosch-Boonstra-Schaaf optic atrophy syndrome.

BACKGROUND: The Bosch-Boonstra-Schaaf optic atrophy syndrome (BBSOAS) is an autosomal-dominant disorder (OMIM615722) mostly characterized by optic atrophy and/or hypoplasia, mild intellectual disability, hypotonia, seizures/infantile epilepsy. This disorder is caused by loss-of-function alterations of NR2F1 (i.e., either whole gene deletions or single nucleotide variants) and, to date, 40 patients have been identified with deletions or mutations in this gene. Here we describe two monozygotic twins harboring a de novo missense variant in the DNA-binding domain of NR2F1 (c.313G>A, p.Gly105Ser), with well-characterized features associated to BBSOAS. METHODS: Patients' DNA was analyzed by exome sequencing identifying the missense variant c.313G>A in NR2F1 (NM_005654.4). Furthermore, molecular modeling was performed to evaluate putative differences in DNA binding between wild-type and mutated NR2F1. RESULTS: The missense variant is predicted to be likely pathogenetic following the ACMG (American College of Medical Genetics and Genomics)/AMP (Association for Molecular Pathology) guidelines. Indeed, dynamic simulation experiments highlighted that the Gly105Ser substitution let the formation of a hydrogen bond between the S105 side chain and R142 and a base (G5) of the DNA sequence, allowing us to hypothesize that the G105 residue might be evolutionary conserved due to the absence of a side chain, besides glycine conformational features. Therefore, the G105S variation seems to cause a stiffening and a possible deformation in the protein-DNA complex due to the interaction of residues R142-S105 and G5 on the DNA, compared to the wild-type. CONCLUSION: In summary, we described two monozygotic twins harboring a novel Gly105Ser mutation in NR2F1 DNA binding domain, displaying the classical phenotype of BBSOAS-affected patients. Our computational data suggest a dominant negative effect of this newly characterized missense variant. To date, this is the first genetic report analyzing in silico structural consequences of NR2F1 Gly105Ser substitution.

A new germline VHL gene mutation in three patients with apparently sporadic pheochromocytoma.

CONTEXT: Germline mutations in four genes (RET, VHL, SDHB and SDHD) are detected in about 17% of patients with apparently sporadic pheochromocytoma. Thus, genetic screening of all patients with this disease is suggested for a rational diagnostic approach and management. OBJECTIVE: To report the clinical, biochemical and genetic analysis of three unrelated patients affected by pheochromocytoma. DESIGN AND PATIENTS: All the coding regions and exon-intron boundaries of RET, VHL, SDHB and SDHD genes were sequenced in three unrelated patients with intra-adrenal pheochromocytoma: a 17-year-old girl, a 15-year-old boy and a 73-year-old man. The family history of all three cases was negative for von Hippel-Lindau lesions or other types of endocrine tumours. Structural modelling of the VHL protein was then performed. RESULTS: We identified a novel germline VHL gene point mutation, a G to A nucleotide substitution in exon 3, leading to an aspartate to asparagine amino acid change in codon 197 (D197N). No mutations were found in RET, SDHB and SDHD genes. Structural modelling of the VHL protein suggests that the D197N mutation could have a functional role. CONCLUSIONS: Our study expands the number of VHL gene known mutations and indicates the usefulness of performing the genetic analysis in all patients with apparently sporadic pheochromocytoma.

Plasminogen activator inhibitor-1 gene polymorphisms in pre-eclampsia.

Pre-eclampsia (P-EC) is a multisystem disorder of pregnancy, characterized by new-onset hypertension and proteinuria. Deregulation of the coagulation cascade and hypofibrinolysis appear to play a central role in the development of this disease. After a brief review of the genetic basis of P-EC and the role of genes encoding proteins involved in coagulation, we focus on polymorphisms of the plasminogen activator inhibitor (PAI-1) gene. The most relevant association studies between PAI-1 gene polymorphisms and P-EC are reviewed. Results indicate that the 4G/4G genotype of the -675 4G/5G polymorphism represents a weak risk factor for P-EC.

Effects of histone acetylation on sodium iodide symporter promoter and expression of thyroid-specific transcription factors.

Inhibitors of histone deacetylases (HDACs) activate the sodium iodide symporter (NIS) expression in thyroid tumor cells. In this study, mechanisms accounting for these effects were investigated. Various human thyroid tumor cell lines (ARO, BCPAP, FRO, TPC-1) were treated with the HDAC inhibitors Na butyrate (NaB) and tricostatin A (TSA), and the effects on the expression of NIS and several thyroid-specific transcription factors together with the activity of NIS promoter were evaluated. TSA and NaB increased NIS mRNA levels in all cell lines. Among thyroid-specific transcription factors, only expression of PAX8 in ARO cells was increased. Down-regulation of thyroid-specific transcription factor-1 expression was observed in BCPAP and TPC-1 cell lines. Thyroid-specific transcription factor-2 mRNA was reduced in FRO, BCPAP, and TPC-1 cells. Histone acetylation had no significant effects on HEX expression. Altogether, these data indicate that the increase of NIS expression is not mediated by modification of expression of thyroid-specific transcription factors. Accordingly, in transfection experiments performed in the HeLa cell line (which does not express thyroid-specific transcription factors), treatment with TSA and NaB increased NIS promoter activity. Stimulation of NIS promoter activity was also obtained by overexpressing histone acetylating proteins pCAF and p300 in HeLa cells. Conversely, overexpression of the HDAC 1 enzyme inhibited basal activity of the NIS promoter. Effects of TSA and NaB on NIS expression were also evaluated in nonthyroid cell lines MCF-7, Hep-G2, and SAOS-2. In all cell lines TSA and NaB greatly increased NIS mRNA levels. We concluded that control of NIS expression by inhibition of HDAC appears not to be mediated by cell-specific mechanisms, suggesting it as a potential strategy to induce radioiodine sensitivity in different human tumors.

Functional interaction among thyroid-specific transcription factors: Pax8 regulates the activity of Hex promoter.

The transcription factor Hex is expressed in the thyroid follicular cells (TFC) and in several other cell types. In TFC, Hex contributes to the control of the tissue-specific gene expression. By means of RT-PCR assays we found a correlation between the Hex and Pax8 (a different tissue-specific transcription factor, expressed in TFC) mRNA levels in normal and neoplastic thyroid tissues. This finding suggested the presence of a functional correlation between the two transcription factors. Therefore, we tested whether Pax8 regulates the transcriptional activity of Hex promoter. Indeed, by using cotransfection experiments in non-thyroidal cells, we show that increasing doses of Pax8 expression vector elicited a dose-dependent increase of the transcriptional activity of Hex promoter. Accordingly, gel-retardation assays indicated that in the Hex promoter are present several Pax8 binding sites. The Pax8 activating effect on Hex promoter was further increased by the contemporary presence of Hex protein. In fact, cotransfection of both Hex and Pax8 expression vectors doubled the transcriptional activity of Hex promoter with respect to the condition in which the Pax8 expression vector only was transfected. In addition, we show that also the transcriptional cofactor APE/Ref-1 cooperated with Pax8 for upregulation of Hex promoter activity. These findings, together with other published data, suggest that a network of functional interactions between transcriptional regulators is present in TFC.

Association between plasminogen activator inhibitor 1 gene polymorphisms and preeclampsia.

It is known that the plasminogen activator inhibitor 1 (PAI-1) protein levels are increased in placentas of preeclamptic subjects. Therefore, we assessed whether polymorphisms related to the transcriptional control of the PAI-1 gene (-675 4G/5G and -844G/A) are associated with mild preeclampsia. We compared 52 women with preeclampsia to 80 women with a normal pregnancy. None of the preeclamptic women suffered from the severe form of preeclampsia. DNA was extracted from blood, and -675 4G/5G and -844G/A genotypes of the PAI-1 gene were determined. Since it has been shown that the presence of factor V Leiden, prothrombin G20210A, and MTHFR C677T gene variants may be associated with preeclampsia, their frequency was also evaluated in our study groups. The factor V Leiden, PT G20210A, and MTHFR C677T gene variants were not associated with preeclampsia. In the case of the -675 4G/5G polymorphism, genotypes 4G/4G and 5G/5G were more prevalent in the preeclamptic and in the control group, respectively. In the case of -844 G/A polymorphism, genotypes A/A and G/G were more prevalent in the preeclamptic and in the control group, respectively. By using the chi(2) test for trend, differences for both genotypes were significant (p = 0.0141 for the -675 genotypes and p = 0.0492 for the -844 genotypes). The frequency of the 4G and 5G alleles of the -675 gene polymorphism was significantly different between preeclamptic and normal women (p = 0.032). Differently, the allelic frequency of the -844 gene polymorphism did not show significant differences between preeclamptic and normal women (p = 0.083). In conclusion, the hypofibrinolytic genotypes 4G/4G and A/A at positions -675 and -844 of the PAI-1 gene are associated with the occurrence of mild preeclampsia independently of thrombophilic mutations of the factor V, prothrombin, and MTHFR genes.

Thyroid-specific transcription factors control Hex promoter activity.

The homeobox-containing gene Hex is expressed in several cell types, including thyroid follicular cells, in which it regulates the transcription of tissue- specific genes. In this study the regulation of Hex promoter activity was investigated. Using co- transfection experiments, we demonstrated that the transcriptional activity of the Hex gene promoter in rat thyroid FRTL-5 cells is approximately 10-fold greater than that observed in HeLa and NIH 3T3 cell lines (which do not normally express the Hex gene). To identify the molecular mechanisms underlying these differences, we evaluated the effect of the thyroid- specific transcription factor TTF-1 on the Hex promoter activity. TTF-1 produced 3-4-fold increases in the Hex promoter activity. Gel- retardation assays and mutagenesis experiments revealed the presence of functionally relevant TTF-1 binding sites in the Hex promoter region. These in vitro data may also have functional relevance in vivo, since a positive correlation between TTF-1 and Hex mRNAs was demonstrated in human thyroid tissues by means of RT-PCR analysis. The TTF-1 effect, however, is not sufficient to explain the difference in Hex promoter activity between FRTL-5 and cells that do not express the Hex gene. For this reason, we tested whether Hex protein is able to activate the Hex promoter. Indeed, co-transfection experiments indicate that Hex protein is able to increase the activity of its own promoter in HeLa cells approximately 4-fold. TTF-1 and Hex effects are additive: when transfected together in HeLa cells, the Hex promoter activity is increased 6-7-fold. Thus, the contemporary presence of both TTF-1 and Hex could be sufficient to explain the higher transcriptional activity of the Hex promoter in thyroid cells with respect to cell lines that do not express the Hex gene. These findings demonstrate the existence of direct cross-regulation between thyroid-specific transcription factors.

Frequency of factor V, prothrombin and methylenetetrahydrofolate reductase gene variants in preeclampsia.

BACKGROUND: The association between thrombophilic variants (Leiden mutation of the factor V gene, G20210A mutation of the prothrombin gene and C677T polymorphism of the methylenetetrahydrofolate reductase (MTHFR) gene) with preeclampsia was investigated in a north-eastern Italian population. METHODS: Fifty-eight preeclamptic (PE) women and 74 normal pregnancies were evaluated. Genotypes were determined by polymerase chain reaction. RESULTS: The frequency of heterozygous carriers of the factor V Leiden was similar between PE women (5.2%) compared to the control subjects (4.1%; p 0.76). Also the frequencies of G20210A and C677T mutations were similar between PE and control subjects. CONCLUSIONS: In this population, we found no difference in the prevalence of genetic risk factors for thrombosis in women with preeclampsia compared with control subjects.

Expression and localization of the homeodomain-containing protein HEX in human thyroid tumors.

Homeobox genes are involved in neoplastic transformation of both epithelial and hemopoietic tissues. The divergent homeobox gene HEX is expressed in the anterior visceral endoderm during early mouse development and in some adult tissues of endodermal origin, including liver and thyroid. Whereas a role in leukemyogenesis has been proposed already, few data are available on the involvement of HEX in human epithelial tumors. Herein, we analyzed HEX expression and subcellular localization in a series of 55 human thyroid tumors and in several tumoral cell lines. HEX mRNA was detected by RT-PCR either in normal tissues or in thyroid adenomas and differentiated (papillary and follicular) carcinomas. HEX mRNA was also expressed in most undifferentiated carcinomas. Subcellular localization of HEX protein was investigated by immunohistochemistry. In normal tissues and adenomas, HEX protein was present both in nucleus and cytoplasm. In contrast, both differentiated and undifferentiated carcinomas, as well as the tumoral cell lines investigated, showed HEX protein only in the cytoplasm. These findings suggest that regulation of HEX entry in the nucleus of thyrocytes may represent a critical step during human thyroid tumorigenesis.