Luke's Tuberous Sclerosis Page
Genetic Research Part One |
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TUBEROUS SCLEROSIS - A GENETIC OVERVIEW Professor Sue Povey, University College London Genetics is the study of the inheritance of characteristics either from parent to child or from one cell in the body to the two cells which form when it divides. Both of these have very important consequences for the understanding of Tuberous Sclerosis and for its impact on the lives of patients. At the family level considerable progress has been made. It seems likely that all cases of TS are caused by alteration within the TSC2 gene on chromosome l6pl3 or TSCl on 9q34. From the relatively small number of families where there are enough affected members of a family to be sure which gene is involved, it seems that each gene accounts for half the cases. However most cases do not have a family history, and it is by no means certain that the proportion of TSCl and TSC2 is the same in these sporadic patients. The tremendous achievements in identifying the TSC2 gene, and now an increasing number of mutations, are enabling us to make a new estimate of the proportion of cases which are TSC2 and some figure on this should emerge by the end of this meeting. Technical advances have speeded up ways of finding these mutations but it remains a large task. It is hoped but not proved that all patients who do not have a defective TSC2 gene will ultimately be found to have a defect in the elusive TSC 1 gene on chromosome 9. Although it is increasingly embarrassing that (at the time of writing) this gene has not been identified, a small reduction in the region of chromosome to be searched has been achieved in the past year by the finding of new genetic markers. Although the generally favored region is now quite a reasonable target of about 230 kilobases between D9Sl22 and A6 (the result of work by Dr Pitiot and Dr H Northrup respectively), the much larger region between D9S 149 and D9SI 14 cannot be entirely excluded. The search for TSCl, much more than the search for TSC2, has produced puzzling data which do not all tell the same story. As always the accurate clinical diagnosis of a few critical cases carrying recombinant chromosomes remains of paramount importance. One piece of encouragement is that by combined efforts of several groups virtually the whole 1.7Megabases of the largest candidate region have now been isolated in cosmid clones. From this point there are several ways forward. The clones can be employed in systematic searching for small chromosome re-arrangements in a large panel of unrelated patients, in the hope of narrowing the region further. The identification of all genes in the region is being approached by diverse methods, ranging from isolation of equivalent genes in puffer fish to experiments carried out entirely on the Internet, which take advantage of the tremendous amount of random DNA sequencing of genes of unknown function already done. The availability of these clones would also allow sequencing of the whole region (an approach recently used in the identification of a gene giving susceptibility to breast cancer). Although TS is a dominantly inherited disease at the level of the person, work done over the past two years particularly by groups in Cambridge, Italy and in Boston has shown that at a cellular level it is probably recessive. That is, most of the cells in the body of a person with TS are quite healthy. Trouble is caused only when a second event occurs which presumably causes the loss of function of the relevant TSC gene in that cell and in all its progeny. Genetics has been able to tell us the perhaps rather surprising result that each TS lesion tested so far arises from a single cell. This information also suggests that the products of the TSC genes so not diffuse through the body but are used in the cell in which they are made. Looking at genetic changes in several lesions in the same person may be able to tell us in which cells and in what stage of development the second hits occur. The number and distribution of these events is probably the main determinant of the severity of the disease in a particular patient. Once again, the story from the TSCl region is less clear than that from TSC2.Genetics has already told us a lot about TS, but we have much more to learn! TSC2 AND TUBERIN: MUTATIONAL ANDFUNCTIONAL ANALYSIS J R Sampson, on behalf of the TSC research group, University of Wales College of Medicine, Cardiff, Wales, UK. The TSC2 gene was isolated by positional cloning, enabling commencement of studies on the mutational and cellular basis of tuberous sclerosis. We have characterized the gene in detail, showing that it is organized as 41 codingexons (3 of which are subject to alternative splicing) and a noncoding leader exon. Comparative analysis with the Fugu rubripes homologue of TSC2 showed conservation of gene structure and of alternative splice forms and highlighted several areas of high evolutionary conservation within the gene. These included the rap 1 GAP homologous domain. We have shown that this domain carries frequent missed mutations in patients with TSC(see poster Maheshwar et al. at this meeting), confirming the functional importance of the domain. We have also characterized a large number of deletion mutations at the TSC2 locus, including contiguous mutations involving TSC2 and the immediately adjacent PKD 1 gene in patients with tuberous sclerosis and renal cystic disease, establishing the first relationship between genotype and phenotype in TSC. In total we have now identified and characterized TSC2 mutations in over 40 unrelated families. Recently we have initiated studies to investigate the cellular role of tuberin, theTSC2 gene product. Preliminary data support a role in activation of the GTPase activity of rap 1. In order to establish other roles of this multidomain protein we are investigating protein-protein interactions using the yeast based two hybrid system. TSC2 MUTATIONAL ANALYSIS IN COHORT OF 90 PROBANDS Authors: Hope Northrup1, Kit-Sing Au, E. Steve Roach2, Mauricio R. Delgado2, Estanislado Rodriguez, Jr.', and Joseph A. Rodriguez' Division of Medical Genetics, Department of Pediatrics, The University of Texas Medical School -Houston, Houston Texas, USA Division of Pediatric Neurology, The University of Texas Southwestern Medical School and The Scottish Rite Hospital, Dallas, Texas, USA Tuberous sclerosis complex (TSC) is an autosomal dominant disease of benign tumors (hamartomas) with a population prevalence of 1/6,000 to 1/10,000. The tumors in TUBEROUS SCLEROSIS CLINICAL POLYMORPHISMS IN INFANCY AND CHILDHOOD A.S.Petrukhin, M.I.Medvedev ,L.O.Karpukhina. Child Neurology Department of the Pediatric Faculty, Russian State Medical University, Moscow, Russia We investigated 38 infants and children with Tuberous Sclerosis (TSC) diagnosed by the Gomez criteria. The period of observation varied from 2 to S years and the earliest onset of TSC was at the age of 3 months. Almost all cases were sporadic (27patients). In S family cases the patient's parents had TSC monosymptomatic forms (depigmentations and cafe au 1ait spots) without any neurological and intellectual damage. In 12 sporadic patients TSC started with epileptic seizures (infantile spasms or complex partial seizures) which were resistant to monotherapy. There were no other neurological signs, and the CNS morphological damage as a cause of the seizures was suspected because of the presence of automatic and stereotypic movements and by monotonous emotional and behavioral activity. 8 patients of this group had subependymal nodular calcifications on CT scans, mostly localized in the areas of the lateral ventricles anterior horns and bodies. There were no skin signs at the time of seizure onset, but they appeared in the following 1 to 2 years. It is interesting that after the skin signs appeared, the seizures stopped in 8of these patients. Among all 38 patients, only 1 girl of7 years of age had a normal intellectual level. She also had typical skin signs, subependymal nodular calcifications and seizures which were stopped by a combination of valproate and lamictal. Her neuropsychological examination revealed mild intellectual problems. The molecular genetic analysis of TSC family cases revealed both the TSC1 and TSC2 phenotypes. mild (minor skin manifestations) to severe(seizures and mental retardation). TSC exhibits genetic heterogeneity with one causative gene mapping to chromosome 9q34 (TSC1) and the other mapping to chromosome 16p13.3(TSC2). The TSC2 gene is cloned. Reported TSC2 mutations include gross deletions in 3-4% of patients studied and a dozen subtle mutations identified to date. We have studied a cohort of 90 probands (58 sporadic and 32 familial TSC patients) for mutations in the TSC2 gene by Southern blotting and single strand conformational analysis (SSCA). The SSCA is being done on each of the 41 exons of TSC2 using intronic primers and genomic DN SSCA has been completed for 36 TSC2exons. A total of 32 SSCP variants have been detected, 9 representing normal polymorphic variation and 23 representing unique potentially disease causing variants. Of the unique variants seven mutations have been characterized: exon 5 del. 2nt.(505-506), exon 9 dup. 15nt. (916-929), eon 9 sub. T893-C, exon 18 del. int. C (2088-2089), exon 21 ins. AT at nt.2509, exon 32 sub. A3905-T, and exon 34 del. 4 nt. (4493-4496). Efforts are underway to sequence the remaining 16 unique SSCP variants and screen the remaining 5 exons of the TSC2 gene. conditionally, two probands, one familial and one sporadic, were found to have gross deletions. genotype/phenotype correlations will be presented. MITOCHONDRIAL INSUFHCIENCY SIGNS IN TUBEROUS SCLEROSIS V.S.Soukhoroukov, A.I.Klembovski,P.A.Temin, M.J.Dorofeeva, V.V.Nevstrueva, A. F.Nazarenko, Kh.M.Makkaev. Institute of Paediatrics & Paediatric Surgery, Dept. of Pathomorphology, Moscow, Russia At present the problem of mitochondrial pathology attracts great attention. One of the reasons for mitochondrial dysfunction may be a calcium metabolism disorder. High amounts of calcium in mitochondria is a reflection of one of the special functions of mitochondria connected with superfluous cytoplasmic calcium. The calcium transport protein of the inner mitochondrial membrane competes for energy with ATP synthesis. If the calcium concentration in a cytosol significantly increases, the ATP synthesis may cut off. So the mitochondrial insufficiency and the calcium distribution disorder are two factors which may accompany one another. Indices of mitochondrial dysfunction have not been examined in tuberous sclerosis, the disease closely associated with calcium metabolism disorder. Morphological analysis of skeletal muscle is one of the leading methods of mitochondrial insufficiency diagnostics. Rather homogeneous and rich in mitochondria, skeletal muscle tissue represents an ideal model for morphological investigation of these organelles' pathology. The " mitochondrial distress" signal is the formation of "ragged-red" fibres (RRF). The RRF phenomenon is characterized by mitochondrial proliferation under a sarcolemma and among myofibrils. Apart from this, histochemical analysis of myobioptates permits easy detection of changes of mitochondrial enzymes’ activity, and electron microscopy permits analysis of ultrastructural changes of mitochondria. We examined skeletal muscle bioptates from 11 children and two of their mothers with tuberous sclerosis. The morphological analysis was performed with hematoxylin and eosin staining of cryostat and paraffin sections, modified Gomori trichrome stain, Mallori and Van Gison stains, staining with phosphotungstic acid-hemtoxylin; histochemical exposure of succinct dehydrogenase (SDH), cytochrome oxydase (COX) (two mitochondrial enzymes), glycogen, calcium, lipids; and electron microscopy. The degrees of pathomorphological changes were estimated with rann: criterion. Quantitative examination was performed with correlation (simple and ranr:), regression, factor and cluster analyses. Nine children had RRF in their muscles, 1 had COX-negative RRF, the others had COX-positive RRF. RRF in all the children was SDH-positive. Two children had moderate decreases of COX activity in muscle. In other respects activity of SDH and COX were not changed. Electron microscopy showed mitochondrial conglomerates that were typical for RRF. Mitochondria were polymorphous and mainly small in size. There were accumulations of the organelles with clarified matrix and disorganized cristae. RRF were found also in the mothers' muscle. 1 was SDH positive and COX negative, the other SDH and COX positive. The activity of both enzymes was unchanged. The average amount of RRF in muscle for all the patients was12.1% +3.4% (p=O.006). (Norm <5 ) Correlation analysis revealed that expression of mitochondrial insufficiency signs positively correlates with the amount of calcium conglomerates (Correlation coefficient 0.63, p=0.05). This was confirmed by regression and factor analyses. Among other pathomorphological changes, the degree of connective tissue coat changes was the one most connected with the degree of mitochondrial insufficiency and the amount of calcium conglomerates. The cluster analyses revealed the following clusters: 1) Older children and mothers (20%) Skeletal muscle tissue was characterized by relatively large numbers of RRF and the expression of other signs of mitochondrial insufficiency, increased moderate signs of regeneration, the absence or low degree of lipid accumulation. Other signs were heterogeneous. 2) The basic group of children(60%) with heterogeneous signs of fibres and coat damage and mitochondrial insufficiency. 3) Children under age2(20%) with an absence of obvious myons and coat damage symptoms. There were no calcium and lipid pathological accumulation. The signs of mitochondrial insufficiency were minimal. These facts testify to the presence of the mitochondrial insufficiency signs in patients with tuberous sclerosis. The cause of this insufficiency may be the calcium metabolism disorder. It is significant that both calcium conglomerates in skeletal muscle and the signs of mitochondrial insufficiency are hardly found in infancy and accumulate with age. MUTATION DETECTION IN TUBEROUS SCLEROSIS RESTRICTION ENDONUCLEASE FINGERPRINTING (REF) AND PROTEIN TRUNCATION TEST (PTT) Van Bakel, Inge1;Yates, JRwl,2 ; Green, AJl,2,3 1Department of Pathology, University of Carnbridge, UK 2Department of Medical Genetics, Addenbrooke's NHS Trust,Cambridge, UK 3Department of Medical Genetics, Universityof Cambridge, UK The TSC2 gene on chromosome l6pl3.3,responsible for about 300_ of tuberous sclerosis, has recently been cloned. The gene has 41 snill esons covering 50 lrb encoding a 5.5kb cDNA Large germline deletions of TSC2 occur in nearly 5% of cases. We analyzed TSC2 mRNA in lymphoblastoid cells from 21 cases of TSC, representing 18 different TSC mutations. Three of the cases were linked to the TSC2 gene, either by linkage analysis, or because a hamartoma from the patient showed loss of heterzygosity for 16p13.3. markers. TSC2 cDNA was generated in six overlapping RT-PCR fragments. For restriction endonuclease fingerprinting (REF), each fragment was digested by restriction enzymes, end-labeled, and analyzed on a non-denaturing polyacrylamide gel. interpreuion of these results was difficult due to high background in several fragments. We analyzed 67% of the TSC2 gene by REF, and have to date found several REF alterations, but no confirmed mutations. As TSC2 mutations are likely to be inactivating, we analyzed TSC2 RiRNA for mutations by the protein truncation test (PTT). The same 6 RT-PCR fragments were generated, using a primer for each which allowed transcription/translation of the PCR product in a reticulocyte lysate. The protein product was 3S5 labeled, and analyzed by SDS/PAGE. All of the TSC2 cDNA has been analyzed by PIT in these cases, and 6 PIT shifts have been detected to date. These Pat products are being sequenced. PIT may offer more convenient detection of inactivating TSC2 mutations than SSCP based techniques. ANALYSIS OF TSC2 GENE AND ITS PROTEIN PRODUCT. M. Nellist, R. Willemsen, A.L.W.Hesseling-Janssen, Q. Wang, A.M.P. Tempelaars, B. Eussen, S. Verhoef, D.Lindhout, A.J.J. Reuser, A.M.W. van den Ouweland and D.J.J. Halley. MGC Department of Clinical Genetics, Erasmus University, Dr. Molewaterplein 50, 3Ol5GE Rotterdam, The Netherlands. The TSC2 gene on chromosome 16p13.3contains 41 coding exons and produces a 5.Skb itiRNA transcript. Homologous transcripts have been identified in organisms as diverse as the rat, pufferfish (F. rip ides) and nematode worm (C. Elegans). An insertion in theTsc2 gene is responsible for the predisposition to renal cell carcinoma in the Eker rat. The function of tuberin, the protein product of the TSC2 gene, is not known, However, a small region of homology to rap 1 GIPase activating protein suggests that part of its function may be to help mediateras-related GTPase activity. Members of the ras protein fatally are implicated in many aspects of cell proliferation and differentiation, processes which are perturbed in lesions from tuberous sclerosis (TSC) patients. We have raised polyclonal antisera against portions of the tuberin molecule expressed as either glutathione - S - transferase or histidine tagged fusions in E. coli, and generated several full-length TSC2 cDNA constructs, Because exons 25 and 30b of the TSC2 gene are alternatively spliced we have made one construct containing the sequences of both exons, and one lacking the 69 base pairs encoded by exon30b. We have expressed our full-length constructs in a coupled in vin’o transcription-translation assay, and detected the predicted 200kDa protein product. This was specifically immunoprecipitated by your antisera. In Immunocytochemicai analysis of COS cells transfected with the same constructs revealed dense granular staining, predominantly in the perinuclear region. Electron microscopy confirmed the cytoplasmic localisation. No specific association with any cell 'anelles was observed. The presence or absence of exon 30b in the plasmid construct -de no detectable difference to the subcellular localization or the level of expression. We have used our antisera in Western blotting experiments to investigate tuberin expression in human cultured cells and frozen tissue samples. Normal fibroblast, transformed lymphoblastoid, HeLa cervical carcinoma andHepG2 hepatoma cell lines all express the protein, consistent with the pattern of TSC2 mDNA expression. In all samples tested the estimated size of the protein was approximately200kDa, indicating that the extent of post translational modification was not extensive. Tuberin was also detected in normal fibroblasts derived from patients carrying a known TSC2 mutation. No mutant Protein species could be detected because our antisera are directed against the tuberin C-terminal domain and most of the tested mutations were predicted to lead to premature tuncation of the peptide chain, Immunocytochemical analysis of normal cultured fibroblasts indicated that tuberin was predominantly cytoplasmic, consistent with the results of the transfection experiments, In frozen tissues, tuberin could be detected in normal adult cerebellum, but not in rain stem, correlating with the frequency of TSC-associated lesions found in these two regions of the brain, A low level of tuberin was detected in adult kidney. To determine the spectrum of TSC2mutations in a large group of tuberous sclerosis patients we have performed FISH, Southern blot analysis, and SSCP, To date we have identified 16mutations including 8 deletions,two splice site mutations, 3 point mutations leading to' premature stop codons and TUBERIN IS HIGHLY EXPRESSED IN MANY HUMAN FETAL AND MOUSE TISSUES AND IN CULTURED HUMAN CELLS, BUT IS DETECTABLE ONLY IN ADULT HUMAN BRAIN. H.Onda and Dj.Kwintkowskl, Bright and Women’s Hospital, Harvard Medical School, Boston, USA We have raised monoclonal antibodies against bacterially expressed tuberin cDNA fragments. Several hybridoma produced monoclonal antibody that react specifically with a 200 IrDa band in human fetal tissues which comigrates with a 200 IrDaprotein product from a CHO cell line transfected with full4ength tuberin cDNA on Western blots. The specificity of the antibodies were further confirmed by immunohistochemical staining of COS and CHO cells expressing exogenous human tuberin. We have used these monoclonal antibodies to study the pattern and level of expression of tuberin in normal tissues of fetal and adult humans by Western blot. Tuberin is expressed in human fetal brain, lung, kidney, heart, skin, skeletal muscle, small intestine, thymus, spleen, liver and spinal cord. The brain expressed the highest level of tuberin uniformly including cerebral cortex, germinal matrix, ventricular surface, cerebellum and brain stem.. No detectable tuberin expression was seen in adult human lung, kidney, heart, skin, skeletal muscle, small intestine, thyroid, spleen, liver, esophagus, pancreas, adrenal gland and peripheral WBC (i.e. less than in tuberin/lOOug of total protein), but some expression was present in adult human brain. One monoclonal antibody cross reacted with mouse tuberin. In the adult mouse, the brain expressed the highest amount of tuberin, and smaller amounts were seen in kidney, heart and lung, but not in liver or spleen. In both humans and mice, tuberin expression decreases about five-fold during brain development from fetal to adult. Tuberin was expressed in several cultured cells including endothelial cells, smooth muscle cells, skin fibroblasts (from skin of all ages), lymphoblastoid cells and bladder carcinoma. Overexpression of tuberin in CHO cells at relatively high levels(approximately 0.2% of total cellular protein) appears to have growth retarding effects. We have examined one subependymal giant cell astrocytoma (SEGA) from a TS patient and one glioblastomamultiforme (GEM) from a non-TS patient. Tuberin was expressed by the GEM but not in the SEGA. We have successfully cultured this SEGA for further characterization of these tumor cells. LOSS OF HETEROZYGOSITY IN TUBEROUS SCLEROSIS HAMARTOMAS. Green, Andrew J1'2'3; Sepp, T2 ; Yates, JRWy2,3 1Department of Medical Genetics, University of Cambridge, UK 2Department of Pathology, University of Cambridge, UK 3Department of Medical Genetics, Addeebrooke's NHS Trust, Cambridge, UK We have previously described in tuberous sclerosis (TSC) basartomas the phenomenon of loss of heterozygosity (LOH) for DNA markers in the region of both the TSC2 gene on chromosome l6p13.3, and the TSCl gene on 9q34. We now describe the specttusn of LOH in 51 TSC hamartomas from 34 cases of TSC. DNA was extracted from leucocytes or normal parailtin-embedded tissue, and from frozen paraitin-embedded hamartoma tissue from the same patient. The samples were analyzed for 10 markers spanning the TSC 1 locus, and S markers spanning the TSC2 locus. The markers on chromosome 9 were ASS, D9564, D95149, D95150, ABO, DEH,D9S122, D9S298, D9Sl 14, D9S67, and on chromosome 16 were D165291, D16S665,KG8, a coding EcoRV polymorphism in the TSC2 gene, D165525, D165309, D16585and HBAP1. Twenty one of 51 hatomas showed LOH (41%), 16 for markers around TSC2, andS for markers in the vicinity of TSC1. No hamartoma showed LOH for markers around both loci. Seven of 17 renal angiomyolipomas showed LOH and S of 9giant cell astrocytomas showed LOH. Three nail fibromas, 3 cortical tubers, a shagreen patch, a cardiac rhabdomyoma, and a renal cell carcinoma showed LOH. All the areas of LOH on chromosome 9 were large, but the smallest region of overlap lay between the markers D95149 andD951 14, providing independent evidence for the localization of the TSC1gene. These data show that LOH is a common finding in a wide range of harrartomas, affecting the same TSC locus in different lesions from the same patient and never affecting both loci. These data support the hypothesis that both the TSC genes act as tumour uppressors, and that the manifestations of TSC in patients with gerinline TSC mutations is from 'second hit' somatic mutations inactivating the remaining normal copy of the TSC -gene. FREQUENT CHROMOSOME 16P13 LOSS OF HETEROZYGOSITY OCCURS IN TUBEROUS SCLEROSIS KIDNEY LESIONS BUT NOT IN BRAIN LESIONS. Elizabeth Petri Henske1, Bernd W. Scheithauer2, M. Priscilla Shor@,Robert Wollrnann4, Joseph Nahshias51 Nick Hornigold61 Marion van Slegtenhorst7, Cynthia T.Welsh8, and David J. Kwiatkowskil Divisions of Experimental Medicine andHematology-Oncology, Brigham and Women's Hospital, Boston NjA1; Department of Pathology, Mayo Clinic, Rochester MN2; Department of Neurology3 and Section of Neuropathology4, University of Chicago Medical Center, Chicago IL; Medical Research Council, Human Biochemical Genetics Unit, London, UK5 Department of Genetics and Biometry, University College London, London, UK6; Erasmus University, Rotterdam, the Netherlands7; and Department of Pathology, University of Utah, Salt Lake City UT8 Tuberous sclerosis (TSC) is an autosomal dominant disorder characterized by seizures, mental retardation, and multi-organ hamartomatous lesions. Loss of heterozygosity (LOH) in TSC lesions has been reported on chromosomes 16p13and 9q34, the locations of the TSC2 and TSC1 genes, respectively. This suggests that the TSC genes are tumor suppressor genes. In this study, 82 TSC lesions were analyzed for LOH in the TSCl andTSC2 chromosomal regions. Three findings resulted from this analysis. First, based on a renal angiomyolipoma with D95114 LOU and retained heterozygosity at D9S149, we confirm that the TSCl critical region is distal to D9S149. Second, we found LOU more frequently on chromosome 16p13 than on 9q34. Of the 27 patients with angiomyolipomas or rhabdomyomas, 16p13 LOU was detected in lesions from 13 patients (48%) while 9q34 LOU was detected in lesions from only 1 (4%). This finding could indicate that important differences ex1st between TSC1 and TSC2 disease. For example,TSC2-associated angiomyolipomas may tend to be larger or more symptomatic than TSC1 lesions, and thus more often surgically removed. Most lesions in this study were surgical specimens. It is also possible that TSC2 disease is more frequent than TSC1 disease in the sporadic TSC population, despite the fact that the familial incidence of TSC2 andTSC1 appears equal. This would occur if new TSC2 mutations are more likely to decrease reproductive fitness than TSC1 new mutations. Lastly, if the chromosomal regions with 9q34 LOU are small, they could have been missed. However, large regions with 9q34LOU were detected in four angiomyolipomas. The third finding of this study is that LOU is rare in TSC brain lesions. LOU was detected in 30 of 54 (56%) renal angiomyolipomas and cardiac rhabdomyomas but in only 1 of 25 TSC brain lesions (4%). This raises the possibility that brain lesions result from a different pathogenic mechanism than kidney and heart lesions. The second hits could more often be point mutations or small deletions, or the brain lesions could result from haplo insufficiency of TSC1 or TSC2. This home page is intended to be a family resource for families affected by Tuberous Sclerosis. It does not intend to constitute medical advise. Viewers are warned not to take any action with regard to medical treatment relying on the information provided on this page without first consulting the patient's physician.
Deanna Runyan-Wall E-mail address: deannadawn@lukets.org Last updated: April 5, 2008 Created: December 5, 1996 |
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