High-resolution melting analysis (HRM) for mutational screening of Dnajc17 gene in patients affected by thyroid dysgenesis.

BACKGROUND: Congenital hypothyroidism is a frequent disease occurring with an incidence of about 1/1500 newborns/year. In about 75% of the cases, CH is caused by alterations in thyroid morphogenesis, defined "thyroid dysgenesis" (TD). TD is generally a sporadic disease but in about 5% of the cases a genetic origin has been demonstrated. Previous studies indicate that Dnajc17 as a candidate modifier gene for hypothyroidism, since it is expressed in the thyroid bud, interacts with NKX2.1 and PAX8 and it has been associated to the hypothyroid phenotype in mice carrying a single Nkx2.1 and Pax8 genes (double heterozygous knock-out). PURPOSE: The work evaluates the possible involvement of DNAJC17 in the pathogenesis of TD. METHODS: High-resolution DNA melting analysis (HRM) and direct sequencing have been used to screen for mutations in the DNAJC17 coding sequence in 89 patients with TD. RESULTS: Two mutations have been identified in the coding sequence of DNAJC17 gene, one in exon 5 (c.350A>C; rs79709714) and one in exon 9 (c.610G>C; rs117485355). The last one is a rare variant, while the rs79709714 is a polymorphism. Both are present in databases and the frequency of the alleles is not different between TD patients and controls. CONCLUSIONS: DNAJC17 mutations are not frequently present in patients with TD.

Hepatocyte nuclear factor (HNF)-4alpha-driven epigenetic silencing of the human PED gene.

AIMS/HYPOTHESIS: Overexpression of PED (also known as PEA15) determines insulin resistance and impaired insulin secretion and may contribute to progression toward type 2 diabetes. Recently, we found that the transcription factor hepatocyte nuclear factor (HNF)-4alpha binds to PED promoter and represses its transcription. However, the molecular details responsible for regulation of PED gene remain unclear. METHODS: Here we used gain and loss of function approaches to investigate the hypothesis that HNF-4alpha controls chromatin remodelling at the PED promoter in human cell lines. RESULTS: HNF-4alpha production and binding induce chromatin remodelling at the -250 to 50 region of PED, indicating that remodelling is limited to two nucleosomes located at the proximal promoter. Chromatin immunoprecipitation assays also revealed concomitant HNF-4alpha-induced deacetylation of histone H3 at Lys9 and Lys14, and increased dimethylation of histone H3 at Lys9. The latter was followed by reduction of histone H3 Lys4 dimethylation. HNF-4alpha was also shown to target the histone deacetylase complex associated with silencing mediator of retinoic acid and thyroid hormone receptor, both at the PED promoter, and at GRB14 and USP21 regulatory regions, leading to a reduction of mRNA levels. Moreover, HNF-4alpha silencing and PED overexpression were accompanied by a significant reduction of hepatic glycogen content. CONCLUSIONS/INTERPRETATION: These results show that HNF-4alpha serves as a scaffold protein for histone deacetylase activities, thereby inhibiting liver expression of genes including PED. Dysregulation of these mechanisms may lead to upregulation of the PED gene in type 2 diabetes.

Glucosamine-induced endoplasmic reticulum stress affects GLUT4 expression via activating transcription factor 6 in rat and human skeletal muscle cells.

AIMS/HYPOTHESIS: Glucosamine, generated during hyperglycaemia, causes insulin resistance in different cells. Here we sought to evaluate the possible role of endoplasmic reticulum (ER) stress in the induction of insulin resistance by glucosamine in skeletal muscle cells. METHODS: Real-time RT-PCR analysis, 2-deoxy-D: -glucose (2-DG) uptake and western blot analysis were carried out in rat and human muscle cell lines. RESULTS: In both rat and human myotubes, glucosamine treatment caused a significant increase in the expression of the ER stress markers immunoglobulin heavy chain-binding protein/glucose-regulated protein 78 kDa (BIP/GRP78 [also known as HSPA5]), X-box binding protein-1 (XBP1) and activating transcription factor 6 (ATF6). In addition, glucosamine impaired insulin-stimulated 2-DG uptake in both rat and human myotubes. Interestingly, pretreatment of both rat and human myotubes with the chemical chaperones 4-phenylbutyric acid (PBA) or tauroursodeoxycholic acid (TUDCA), completely prevented the effect of glucosamine on both ER stress induction and insulin-induced glucose uptake. In both rat and human myotubes, glucosamine treatment reduced mRNA and protein levels of the gene encoding GLUT4 and mRNA levels of the main regulators of the gene encoding GLUT4 (myocyte enhancer factor 2 a [MEF2A] and peroxisome proliferator-activated receptor-gamma coactivator 1alpha [PGC1alpha]). Again, PBA or TUDCA pretreatment prevented glucosamine-induced inhibition of GLUT4 (also known as SLC2A4), MEF2A and PGC1alpha (also known as PPARGC1A). Finally, we showed that overproduction of ATF6 is sufficient to inhibit the expression of genes GLUT4, MEF2A and PGC1alpha and that ATF6 silencing with a specific small interfering RNA is sufficient to completely prevent glucosamine-induced inhibition of GLUT4, MEF2A and PGC1alpha in skeletal muscle cells. CONCLUSIONS/INTERPRETATION: In this work we show that glucosamine-induced ER stress causes insulin resistance in both human and rat myotubes and impairs GLUT4 production and insulin-induced glucose uptake via an ATF6-dependent decrease of the GLUT4 regulators MEF2A and PGC1alpha.

Toxic cure: Hyperfractionated radiotherapy with concurrent cisplatin and fluorouracil for Stage III and IVA head-and-neck cancer in the community.

PURPOSE: To evaluate efficacy and toxicity of the Duke University chemoirradiation regimen for locally advanced head-and-neck cancer in a regional community cancer center. METHODS AND MATERIALS: Between June 1998 and June 2002, 50 patients with Stage III or IVA squamous cell carcinoma of the head and neck were treated definitively with concurrent combined modality therapy (CMT). Patients received accelerated, hyperfractionated radiotherapy (AFRT), 1.2-1.25 Gy b.i.d., to a median prescribed dose of 70 Gy. Chemotherapy consisted of cisplatin 12 mg and fluorouracil 600 mg/m(2) daily for 5 consecutive days during Weeks 1 and 6, followed by two cycles after AFRT. Patients with N2-N3 neck disease (n = 21; 42%) were considered for neck dissection depending on their response to AFRT and chemotherapy. Twenty-nine patients with Stage III and IVA disease treated between 1991 and 1997 with definitive RT alone served as historical controls. RESULTS: Forty-nine patients (98%) in the CMT group completed the prescribed AFRT and 38 (76%) completed four cycles of chemotherapy. Three of 8 patients who underwent neck dissection had a pathologically complete response. The median follow-up for all patients was 23 months. The actuarial progression-free survival rate at 2 years was 75% for the CMT group vs. 40% (p <0.01) for the RT group. The overall survival rate was 80% and 43% (p <0.01), respectively, for the CMT and RT groups. Acute Radiation Therapy Oncology Group Grade 3 toxicities for the CMT group were mucosal (n = 50; 100%), skin (n = 9; 18%), and hematologic (n = 3; 6%). Late Grade 3-4 toxicities consisted of pharyngeal stricture (n = 7; 14%), laryngeal chondritis (n = 3; 6%), osteoradionecrosis (n = 2; 4%), and peripheral neuropathy (n = 1; 2%). CONCLUSION: This aggressive regimen of AFRT with concurrent cisplatin and fluorouracil with or without neck dissection is feasible in the community setting for patients with Stage III and IVA head-and-neck cancer. Early results indicated excellent survival, albeit with universal acute mucosal, and considerable, although acceptable, late toxicity.

Molecular characterisation of four cases of intrachromosomal triplication of chromosome 15q11-q14.

CONTEXT: Chromosomal abnormalities that involve the proximal region of chromosome 15q occur relatively frequently in the human population. However, interstitial triplications involving one 15 homologue are very rare with three cases reported to date. OBJECTIVE: To provide a detailed molecular characterisation of four additional patients with interstitial triplications of chromosome 15q11-q14. DESIGN: Molecular analyses were performed using DNA markers and probes specific for the 15q11-q14 region. SETTING: Molecular cytogenetics laboratory at the University of Chicago. SUBJECTS: Four patients with mild to severe mental retardation and features of Prader-Willi syndrome (PWS) or Angelman syndrome (AS) were referred for molecular cytogenetic analysis following identification of a suspected duplication/triplication of chromosome 15q11-q14 by routine cytogenetic analysis. MAIN OUTCOME MEASURES: Fluorescence in situ hybridisation (FISH) was performed to determine the type of chromosomal abnormality present, the extent of the abnormal region, and the orientation of the extra chromosomal segments. Molecular polymorphism analysis was performed to determine the parental origin of the abnormality. Methylation and northern blot analyses of the SNRPN gene were performed to determine the effect of extra copies of the SNRPN gene on its methylation pattern and expression. RESULTS: Fluorescence in situ hybridisation (FISH) using probes within and flanking the Prader-Willi/Angelman syndrome critical region indicated that all patients carried an intrachromosomal triplication of proximal 15q11-q14 in one of the two chromosome 15 homologues (trip(15)). In all patients the orientation of the triplicated segments was normal-inverted-normal, suggesting that a common mechanism of rearrangement may have been involved. Microsatellite analysis showed the parental origin of the trip(15) to be maternal in three cases and paternal in one case. The paternal triplication patient had features similar to PWS, one maternal triplication patient had features similar to AS, and the other two maternal triplication patients had non-specific findings including hypotonia and mental retardation. Methylation analysis at exon 1 of the SNRPN locus showed increased dosage of either the paternal or maternal bands in the paternal or maternal triplication patients, respectively, suggesting that the methylation pattern shows a dose dependent increase that correlates with the parental origin of the triplication. In addition, the expression of SNRPN was analysed by northern blotting and expression levels were consistent with dosage and parental origin of the triplication. CONCLUSIONS: These four additional cases of trip(15) will provide additional information towards understanding the phenotypic effects of this abnormality and aid in understanding the mechanism of formation of other chromosome 15 rearrangements.

Relaxation of insulin-like growth factor 2 imprinting and discordant methylation at KvDMR1 in two first cousins affected by Beckwith-Wiedemann and Klippel-Trenaunay-Weber syndromes.

Beckwith-Wiedeman syndrome (BWS) and Klippel-Trenaunay-Weber syndrome (KTWS) are different human disorders characterized, among other features, by tissue overgrowth. Deregulation of one or more imprinted genes located at chromosome 11p15.5, of which insulin-like growth factor 2 (IGF2) is the most likely candidate, is believed to cause BWS, whereas the etiology of KTWS is completely obscure. We report a case of BWS and a case of KTWS in a single family. The probands, sons of two sisters, showed relaxation of the maternal IGF2 imprinting, although they inherited different 11p15.5 alleles from their mothers and did not show any chromosome rearrangement. The patient with BWS also displayed hypomethylation at KvDMR1, a maternally methylated CpG island within an intron of the KvLQT1 gene. The unaffected brother of the BWS proband shared the same maternal and paternal 11p15.5 haplotype with his brother, but the KvDMR1 locus was normally methylated. Methylation of the H19 gene was normal in both the BWS and KTWS probands. Linkage between the insulin-like growth factor 2 receptor (IGF2R) gene and the tissue overgrowth was also excluded. These results raise the possibility that a defective modifier or regulatory gene unlinked to 11p15.5 caused a spectrum of epigenetic alterations in the germ line or early development of both cousins, ranging from the relaxation of IGF2 imprinting in the KTWS proband to disruption of both the imprinted expression of IGF2 and the imprinted methylation of KvDMR1 in the BWS proband. Analysis of these data also indicates that loss of IGF2 imprinting is not necessarily linked to alteration of methylation at the KvDMR1 or H19 loci and supports the notion that IGF2 overexpression is involved in the etiology of the tissue hypertrophy observed in different overgrowth disorders, including KTWS.

Role of histone acetylation and DNA methylation in the maintenance of the imprinted expression of the H19 and Igf2 genes.

H19 and Igf2 are linked and reciprocally imprinted genes. We demonstrate that the histones associated with the paternally inherited and unexpressed H19 allele are less acetylated than those associated with the maternal expressed allele. Cell growth in the presence of inhibitors of either histone deacetylase or DNA methylation activated the silent Igf2 allele, whereas derepression of the silent H19 allele required combined inhibition of DNA methylation and histone deacetylation. Our results indicate that histone acetylation as well as DNA methylation contribute to the somatic maintenance of H19 and Igf2 imprinting and that silencing of the imprinted alleles of these two genes is maintained via distinct mechanisms.

Relaxation of insulin-like growth factor-2 imprinting in rat cultured cells.

The parental-specific expression of the insulin-like growth factor-2 (Igf-2) and H19 genes was studied in rat fibroblast cells derived from a 3 day-old first-generation hybrid animal obtained by crossing Fisher and Wistar strains (F x W cells). Results showed that the reciprocal imprinting of the Igf-2 and H19 genes was conserved in the rat tissues and in the derived F x W cells when cultured with frequent transfer. Igf-2 and H19 gene expression was coordinately up-regulated upon reaching confluence, but Igf-2 RNA levels were further increased in a time-dependent manner and the repressed state of the maternal Igf-2 allele was progressively relaxed in cultures held in the confluent state and in the presence of low serum for more than 3 days. The active expression and relaxed imprinting status of the Igf-2 gene persisted over cell generations when the growth-constraining conditions were released by trypsinization and dilution. On the contrary, the imprinting of the H19 gene appeared to be unaffected by changes in growth conditions and its expression was down-regulated when the confluent cells were passaged. Methylation of the H19 promoter and Igf-2 coding regions was increased in the F x W cells extensively held under confluence and in the derived 'post-confluent' cultures. The heritable changes in the expression, and imprinting status of the Igf-2 and H19 genes observed in the F x W cells closely resembles events described in human embryonal cancers and cancer-predisposing syndromes. The occurrence of imprinting relaxation under strong growth-inhibitory conditions supports the hypothesis that it is an epigenetic change.

Loss of heterozygosity of imprinted genes in SV40 t/T antigen-induced hepatocellular carcinomas.

Expression of the chromosomally linked Insulin-like Growth Factor II (IGF-II) and H19 genes is regulated by parental imprinting during development, since the maternally inherited IGF-II and the paternally inherited H19 alleles are inactive in fetal tissues. Here we show that expression of IGF-II and H19 genes is activated in transgenic mice during SV40 Tag-induced hepatocarcinogenesis and that imprinting of both genes is conserved in the liver tumors. Allelic imbalances of IGF-II and H19 genes and other chromosome 7 markers were detected in one third (13/39) of the hepatocellular carcinomas analysed. A strong bias on the allele retained in the neoplasms was observed, since underrepresentation or complete loss of maternal chromosome 7 was recognised in 12/13 cases. High levels of IGF-II mRNA were expressed by all carcinomas with relative excess of paternal chromosome 7 alleles and suppressed H19 expression was found in the neoplasms lacking the maternal alleles. Overall the results indicate that expression of imprinted genes is involved in progression of experimental liver tumors and suggest that the murine chromosome 7, whose loss may possibly cause the inactivation of a growth-inhibitory gene, is preferentially retained as paternal copy in the liver tumors because of parental imprinting of IGF-II gene.

Parental imprinting of rat insulin-like growth factor II gene promoters is coordinately regulated.

The insulin-like growth factor II (IGF-II) gene is parentally imprinted in the mouse and human species. By following the inheritance of natural polymorphisms of IGF-II mRNA, we demonstrated that the tissue-specific parental imprinting of the IGF-II gene is conserved in the rat. The expression of the paternal IGF-II allele exceeded by more than 3 orders of magnitude that of the maternal allele in livers of 3-day-old Wistar x Fisher interstrain rat crosses. In contrast, the two alleles were both expressed in the rat central nervous system, which is also the only district of the organism where this gene is active in adult rodents. We also analyzed the allelic usage of the three IGF-II promoters, which generate alternatively spliced transcripts, and showed that parental imprinting of all transcription starts sites is coordinately regulated since P1, P2, and P3 are all repressed on the maternal allele in neonatal rat liver, and all of them are activated on both alleles in the choroid plexus of the central nervous system. RNase protection assays demonstrated that the activity ratio of the three IGF-II promoters can be different in tissues that show the same imprinting mode.

Mono- and bi-allelic expression of insulin-like growth factor II gene in human muscle tumors.

Insulin-like growth factor II (IGF-II) is a mitogen for many cell types and an important modulator of muscle growth and differentiation. IGF-II gene is prevalently expressed during prenatal development and its gene activity is regulated by genomic imprinting, in that the allele inherited from the father is active and the allele inherited from the mother is inactive in most normal tissues. IGF-II expression is activated in several types of human neoplasms and an alteration of IGF-II imprinting has been described in Beckwith-Wiedemann syndrome and Wilms' tumor. Here we show that monoallelic expression of IGF-II gene is conserved in normal adult muscle tissue whereas two or more copies of active IGF-II alleles, arising by either relaxation of imprinting or duplication of the active allele, are found in 9 out of 11 (82%) rhabdomyosarcomas retaining heterozygosity at 11p15, regardless of the histological subtype. Since IGF-II has been indicated as an autocrine growth factor for rhabdomyosarcoma cells, these findings strongly suggest that acquisition of a double dosage of active IGF-II gene is an important step for the initiation or progression of rhabdomyosarcoma tumorigenesis. Among different types of muscle tumors, relaxation of imprinting seems to arise prevalently in rhabdomyosarcomas, since we have detected only one case of partial reactivation of the maternal IGF-II allele out of 7 leiomyosarcomas tested.

A common response element mediates differential effects of phorbol esters and forskolin on type-1 plasminogen activator inhibitor gene expression in human breast carcinoma cells.

We have characterized regulation of type-1 plasminogen activator inhibitor (PAI-1) gene expression by phorbol 12-myristate 13-acetate (PMA) and the cAMP-inducing agent forskolin in the human breast carcinoma cell line MCF-7. PMA caused a strong induction of PAI-1, while forskolin suppressed the PMA response. Transfection experiments with fusion genes showed that sequences mediating PMA induction as well as forskolin suppression were present between base pairs -100 and -30 of the 5'-flanking region of the PAI-1 gene. The region was found to contain two Sp1 binding sites. A proximal sequence in the region, TGAGTTCA (P box), with sequence similarity to phorbol ester response elements (TRE) as well as to cAMP response elements (CRE), bound a low-abundance, as yet unidentified nuclear protein in MCF-7 cells. This sequence had a higher affinity to purified c-jun homodimer than to c-jun/c-fos heterodimer in MCF-7 nuclear extracts; it had no affinity to the proteins binding to CRE consensus sequences in these extracts. A distal TRE-like sequence, TGAGTGG (D box), had a weak affinity to c-jun/c-fos heterodimer and c-jun homodimer; binding of proteins to this sequence was facilitated by binding of proteins to the P box. Both the P box and the D box were necessary for PMA responsiveness, suggesting a cooperativity between the two binding sites. A mutation of the P box removing the CRE similarity abolished the forskolin suppression of the PMA response. We propose that the protein kinase C and the protein kinase A signal-transduction pathways, with opposite effects on PAI-1 gene expression converge by modulating differently P-box-binding proteins.

[Vaccination against hepatitis B: 3 years' experience at the Latium Operative Unit]. / Vaccinazione contro l'epatite B: tre anni di esperienza presso l'Unità Operativa di Latina.

A campaign against hepatitis B was launched in 1985 in Latium Region, Italy, aimed at hospital workers, newborns of HBsAg positive mothers, hemodialysis patients, thalassemics and hemophiliacs. Subsequently, since the beginning of 1987 other at risk categories were included, namely households of HBsAg positive carriers, subjects with accidental exposure to Hepatitis B virus (HBV) (i.e exposure to street syringes), health care personnel working outside the hospital setting such as dentists, private clinics and laboratory workers, etc. A protocol was defined by the Regional Epidemiologic Unit (Osservatorio Epidemiologico Regionale) in order to evaluate the immunogenicity and safety af the two plasma-derived (pd) vaccines registered in Italy, MSD and Pasteur, in field conditions. Subjects belonging to these at risk categories were distributed among 21 hospital based vaccination units, to which the two vaccines were randomly allocated. Subjects were considered eligible for vaccination if they were HBsAg negative and Anti-HBs negative or Anti-HBs positive at low titer i.e. less than 20 milli-International units per milliliter. Subjects with insulin dependent diabetes, chronic liver disease or known hypersensitivity to vaccine components were also excluded. Antibody response was checked at six months since the beginning of the vaccination, i.e. after two doses of the MSD and three doses of Pasteur vaccine and expressed in miU/ml by use of Hollinger formula. Pre-vaccination screening, vaccination and post-vaccination anti-HBs testing were offered free of charge.(ABSTRACT TRUNCATED AT 250 WORDS)

Large cell lymphoma presenting as a coronal penile lesion.

Although the majority of penile malignancies are primary epithelial carcinomas, consideration of secondary involvement, especially from lymphoma, is important, since there are alternatives to the standard therapeutic approach of penile amputation.