Rescue of human RET gene expression by sodium butyrate: a novel powerful tool for molecular studies in Hirschsprung disease.

BACKGROUND: The RET gene encodes a tyrosine kinase receptor involved in different human neurocristopathies, such as specific neuroendocrine tumours and Hirschsprung disease (HSCR). Gene expression is developmentally regulated and the RET transcript is undetectable in most adult cells, including lymphocytes. The impossibility of performing functional studies on RET mRNA has to date limited the detection and characterisation of an indefinite proportion of gene anomalies that cannot be identified by conventional DNA genomic screening in HSCR cases. AIMS: Development of a protocol suitable to activate RET expression in RET negative cell lines and therefore to investigate directly RET mRNA, extending the conventional gene mutation analysis to detection of splicing anomalies and impaired expression of the RET gene. METHODS: The effect of sodium butyrate (NaB), a histone deacetylase inhibitor, on rescuing RET expression was tested by one round of reverse transcription- polymerase chain reaction from total RNA of treated lymphoblasts from both HSCR patients and control individuals. RESULTS: Analysis of RET expression was possible by NaB treatment of RET negative cells, such as lymphoblasts. This treatment allowed us to detect impaired RET expression as well as a splicing defect in two HSCR patients previously believed to be devoid of any gene abnormality. CONCLUSIONS: The full application of the proposed protocol in most of the unexplained HSCR cases will allow us to establish the precise role of RET not only in causing but also in predisposing to HSCR pathogenesis.

Echographic study of extraocular muscle thickness in children and adults.

BACKGROUND: Echobiometric evaluation of extraocular muscles in normal subjects has been performed previously, but only in adults. We determined extraocular muscle thickness in normal subjects in three age groups. METHODS: Extraocular muscle thickness was studied in 75 normal subjects divided into three age groups (5-10, 11-15 and 28-37 years) using a Biovision B-scan-S instrument in standardized A-mode (frequency, 10 MHz; biometry resolution, 0.15 mm; depth, 40-60 mm; points on X axis, 512; levels on Y axis, 256). All measurements were performed by the same operator and repeated five times. The reproducibility of the technique was determined using the coefficient of variation. The one-way ANOVA test was used to compare the three groups, and the two-tailed unpaired t-test was used to compare subjects aged 5-10 years and those aged 11-15 years, and subjects aged 11-15 years with those aged 28-37 years. RESULTS: The technique showed good reproducibility. In subjects 5-10 years old, the coefficient of variation was 8%; in subjects 11-15 years and 28-37 years old, it was 5%. Increased muscle thickness was observed with age (p < 0.001). A statistically significant difference between the medial and inferior recti muscles in subjects 11-15 years and 28-37 years old was found (p < 0.001). CONCLUSIONS: The increased thickness of all recti muscles may be influenced by growth (primarily during puberty), and the variations in thickness of the extraocular muscles may be attributable to near-vision stimulus of the inferior and medial recti muscles.

Nuclear run-on assay using biotin labeling, magnetic bead capture and analysis by fluorescence-based RT-PCR.

In this report, we present a fluorescence-based approach to the assessment of cellular gene expression and transcription rates. Nuclear run-on was performed by supplying biotin-16-UTP to nuclei, and labeled transcripts were bound to streptavidin-coated magnetic beads. Total cDNA was then synthesized by means of random hexamer primed reverse transcription of captured molecules. To monitor transcript abundance in cDNA, both from nuclear run-on and total RNA, we propose a semiquantitative PCR approach based on the use of fluorescent primers.

A single-nucleotide polymorphic variant of the RET proto-oncogene is underrepresented in sporadic Hirschsprung disease.

Hirschsprung disease (HSCR) is an inherited disorder characterised by absence of intrinsic ganglion cells in the distal gastrointestinal tract. Different susceptibility genes, involved in either the Ret-tyrosine kinase or the endothelin signalling pathways, contribute to HSCR phenotype. Interestingly, alterations of these genes are detected in only 30-50% of all HSCR patients, suggesting the involvement of modifier genes and/or additional genetic or environmental risk factors. In complex disorders common polymorphic variants can be associated with the disease phenotype, thus modifying the risk of recurrence. To investigate whether sequence variants of the RET proto-oncogene may be associated with the development of the HSCR phenotype, we analysed 92 Italian patients for the 2508C > T synonymous substitution in exon 14 (S836S) finding that the T allele is clearly less frequent than in control individuals (Fisher exact test P = 0.0002). On the other hand, this RET variant allele is overrepresented in patients affected with medullary thyroid carcinoma. Assuming a direct effect of this single-nucleotide polymorphism in predisposing to RET associated pathologies, we have performed functional tests which excluded any possible involvement of the C and T alleles in DNA-protein binding, transcript stability and RNA splicing and editing.

Evaluation of the analgesic effect of 0.1% indomethacin solution on corneal abrasions.

PURPOSE: The authors wished to verify the analgesic action of 0.1% indomethacin in a water-based solution on patients affected by traumatic corneal abrasions. METHODS: 347 patients affected by traumatic corneal abrasions, having been randomly divided into 2 groups on the basis of the administration of indomethacin, were evaluated at 30 min, 12 h and 24 h after the initial treatment of the abrasion. The level of pain experienced was evaluated on a verbal pain scale and the healing time was evaluated relative to the dimension of the abrasion. RESULTS: The pain level was initially overwhelming for both groups: p = 0.737; at successive check-ups it was possible to verify a reduction of the symptomatology, with a more pronounced decrease in pain in the group treated with indomethacin (p < 0.0001), which also demonstrated a lower sensitivity to pain in the case of larger lesions (p < 0.0001). There was no difference in the healing time between groups, and the reduction of pain is not correlated with corneal anesthesia and healing time. CONCLUSIONS: Our study highlighted the efficacy of indomethacin as a pain reducer for acute corneal pathology and suggested that the medication may act on the corneal nociceptors in a qualitative way.

Small incision nucleus capture: results of 200 cases.

PURPOSE: To compare the learning curve in a series of 200 cataract surgeries performed using small incision nucleus capture with that of phacoemulsification as reported in the literature. SETTING: Department of Ophthalmology, University of Genoa, Genoa, Italy. METHODS: Two hundred eyes of 163 consecutive patients with cataract had small incision nucleus capture, a relatively new cataract surgery technique that allows small incisions and in-the-bag intraocular lens implantation. Patients were divided into 4 groups of 50 each according to when they had surgery between August 1996 and October 1997. The incidence of intraoperative complications (capsule break with or without vitreous loss, capsulorhexis tears, Descemet's detachment, transient iris damage) and postoperative complications (raised intraocular pressure, corneal epithelial edema, Descemet's folds, and permanent iris damage) were evaluated at the different time points. Also recorded was final visual acuity. These results were compared with those obtained with phacoemulsification. RESULTS: The study comprised 92 women and 71 men with an age range of 41 to 93 years. Overall final results showed that the learning curve of nucleus capture is comparable to that of phacoemulsification. CONCLUSION: Nucleus capture cataract extraction resulted in a low incidence of complications and good visual recovery that was comparable to that obtained with phacoemulsification.

A 5'-CG-3'-rich region in the promoter of the transcriptionally frequently silenced RET protooncogene lacks methylated cytidine residues.

In a large proportion of familial and sporadic cases of Hirschsprung disease (HSCR) mutations in the RET (rearranged during transfection) protooncogene have been described. We have investigated the structure of the RET gene promoter and have analysed a region of approximately 1000 nucleotides in its promoter and 5'-upstream segments for the occurrence of 5-methyldeoxycytidine (5-mC) residues by using the bisulfite protocol of the genomic sequencing method. With an estimated sensitivity of about 93% of this technique, not a single 5-mC residue could be detected in the control region of a gene that seems to be silenced or exhibit low activity in many adult tissues. In these experiments, the DNAs of peripheral white blood cells (PWBC) from four healthy individuals, from seven patients with familial HSCR, as well as DNAs from different human tissues and from a human embryonic kidney (HEK) cell line have been included. In a DNA segment starting 790 nucleotides upstream of the transcriptional start site of the RET gene, a few 5-mC residues have been identified. This region possibly constitutes the transition site from an unmethylated promoter to a more extensively methylated region in the human genome. The data presented are remarkable in that a highly 5'-CG-3'-enriched segment of the human genome with 49 5'-CG-3' dinucleotide pairs in 400 bp within the putative promoter region is completely devoid of 5-mC residues, although this control region is not actively transcribed in most adult human tissues. By hybridization of a PCR-amplified RET protooncogene cDNA probe harboring exons 9-15 to a membrane (Clontech) containing poly-A selected RNAs from 50 different human tissues, weak RET protooncogene expression in many of the neural cell derived tissues has been detected. RNAs extracted from many other human tissues do not share sequence homologies to this 32P-labeled probe. Mechanisms other than DNA methylation obviously play the crucial role in the inactivation of the RET gene promoter in these tissues. We have also demonstrated by the in vitro premethylation of a RET promoter-chloramphenicol acetyltransferase (CAT) gene construct and transient transfection experiments into neuroblastoma cells that the transcriptional activity of the RET promoter is decreased by HpaII (5'-CCGG-3') methylation and abolished by SssI (5'-CG-3') methylation. Hence, the RET promoter region is sensitive to this regulatory signal. However in vivo, DNA methylation of the promoter region seems not to be the predominant regulatory mechanism for the RET protooncogene. Possibly, in adults the RET gene can be occasionally activated.

A repeated element in the human lamin B2 gene covers most of an intron and reiterates the exon/intron junction.

Nuclear lamins are intermediate filament-type proteins forming a fibrillar meshwork that underlies the inner nuclear membrane. We have previously reported the identification of the human lamin B2 gene that maps to the subtelomeric band p13.3 of chromosome 19 in close proximity of a human DNA replication origin. Here we report the identification within the human lamin B2 gene of a novel repeated element (variable number of tandem repeats: VNTR) that appears to have a very recent origin, being absent in the genome of mouse and primates such as cercopitheques, lemurs and macaques. The VNTR is adjacent to exon 8 of the lamin B2 gene which, albeit encoding the nuclear localization signal of the protein, is highly divergent both at amino acid and nucleotide level among species. Moreover the VNTR, characterized by a repeated unit of about 100 bp, covers most of intron 8 of the gene and reiterates both the last 7 bp of the upstream exon and the exon/intron junction. RT-PCR experiments carried out on HeLa cell RNA suggest that none of the downstream junctions is used during the processing of the lamin B2 pre-mRNA.

Gorlin's syndrome. Case report.

Gorlin's syndrome (GS) is a hereditary dominant autosomal disease linked to a gene which has a strong discerning and an extremely variable expressiveness. It is an ecto-mesodermic polydysplasia generally characterized by multiple nevoid basal-cell carcinomas, palmo-plantar pits, cysts of cutis and long bones, intracranial ectopic calcification; less frequently it is accompanied by tumors of other organs such as the ovaries, and by brain, neurological and ocular defects. Chalazions and squinting are the most typical ocular complications. We describe a patient with GS detected at an ophthalmology consultancy during which by chance we found multiple peripheral retinal breaks with a partial retinal detachment and retinoschisis.

Sequence and characterisation of the RET proto-oncogene 5' flanking region: analysis of retinoic acid responsiveness at the transcriptional level.

The RET proto-oncogene encodes a receptor tyrosine kinase expressed during neural crest development. RET expression is enhanced in vitro by several differentiating agents, including retinoic acid (RA), which up-regulates RET expression in neuroblastoma cell lines. In the present work we sequenced and analysed a 5 kbp genomic fragment 5' to RET. Three deletion fragments of this region were tested for their RA inducibility in transient transfection assays and failed to support the hypothesis of a direct transcriptional activation. Finally, our functional analysis of a candidate RA response element present in the RET promoter provides new hints for the understanding of the interaction between nuclear receptors and their specific recognition sites.

The physical map of the human RET proto-oncogene.

The RET proto-oncogene, a transmembrane tyrosine kinase receptor, is involved in the development of at least five different disease phenotypes. RET is activated through somatic rearrangements in a number of cases of papillary thyroid carcinoma while germ-line point mutations are associated with three inherited cancer syndromes MEN 2A, MEN 2B and FMTC. Moreover, point mutations or heterozygous deletions of RET are found in the dominant form of Hirschsprung disease or congenital colonic aganglionosis. We cloned the entire RET genomic sequence in a contig of cosmids encompassing 150 kb, from the CA repeat sTCL-2 to the region upstream the RET promoter, and established the position of the 20 exons of the RET gene with respect to a detailed restriction map based on eight endonucleases. A new highly polymorphic CA repeat sequence was identified within intron 5 of RET (RET-INT5). Finally the orientation of RET on chromosome 10q11.2 made it possible to orientate three other genes rearranged with RET in papillary thyroid carcinomas, namely H4/D10S170 on 10q21, R1 alpha on 17q23 and RFG2/Ele1 on 10q11.2.