Davut Pehlivan et al.: SOS1 Mutation in Hereditary Gingival Fibromatosis Journal of Hard Tissue Biology 18[3] (2009) p131-134 © 2009 The Hard Tissue Biology Network Association Printed in Japan, All rights reserved. CODEN-JHTBFF, ISSN 1341-7649 Original Cytogenetic Analysis and Examination of SOS1 Mutation in a Turkish Family with Hereditary Gingival Fibromatosis

Davut Pehlivan1, 3), Shinichi Abe2), Sukru Ozturk3), Kivanc Bektas Kayhan4), Esra Gunduz5), Kivanc Cefle3), Aysegul Bayrak3), Nebil Ark6), Mehmet Gunduz6, 7) and Sukru Palanduz3)

1)Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA 2)Oral Health Science Center hrc7 and Department of Anatomy, Tokyo DentalCollege 1-2-2 Masago, Mihama-ku, Chiba-City, Chiba 261-8502, Japan 3)Department of Internal Medicine, Division of Medical Genetics, Istanbul University, Istanbul, Turkey 4)Department of Oral Surgery and Medicine, Faculty of Dentistry, Istanbul University, Istanbul, Turkey 5)Department of Medical Genetics, Faculty of Medicine, Fatih University, Ankara, Turkey 6)Department of Otolaryngology Head and Neck Surgery, Faculty of Medicine, Fatih University, Ankara, Turkey 7)Department of Oral Pathology and Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Shikatacho 2-5-1, Okayama, Japan. (Accepted for publication, December 5, 2009)

Abstract: Hereditary Gingival Fibromatosis (HGF) is a rare, benign disorder characterized by slowly progressive fibrous overgrowth of the gingiva. HGF occurs in several forms as a Mendelian trait (usually as an autosomal dominant condition), in malformation syndromes, in chromosomal abnormality syndromes and side effect of several pharmacological agents. Except -1 (SOS1) gene mutation, molecular basis of HGF is unclear. Here, we reported the cytogenetic and SOS1 gene mutation analysis in a Turkish family with 7 affected members through three generations, whose features are consistent with the diagnosis of autosomal dominant, isolated hereditary gingival fibromatosis. To the best of our knowledge this is the first large Turkish family with hereditary gingival fibromatosis. In this study, we excluded the chromosomal abnormalities and the mutation in SOS1 gene at this family.

Key Words: Hereditary gingival fibromatosis, SOS1, Mutation

Introduction abnormalities and mental retardation. Although the recessive cases Hereditary Gingival Fibromatosis (HGF) is a benign, have been described in some literatures, HGF is mainly inherited genetically heterogeneous overgrowth disease, which is in autosomal dominant manner6, 7). Chromosomal duplication and characterized by slowly progressive fibrous enlargement of the deletion anomalies have been reported for syndromic forms of keratinized gingival tissues (MIM 135300, 605544, 609955). The HGF8, 9). In addition to Mendelian and chromosomal forms, HGF most common manifestations are diastemata due to gingival may also seen as a side effect of several pharmacological agents, overgrowth are diastemata, malposition of the teeth and prolonged including calcium channel blockers, phenytoin and cyclosporine10- retention of primary teeth. More severe lesions result in both 12). Although understanding the molecular basis of HGF has been aesthetic and functional problems by leading to improper tooth expanded in the last decade, genetic etiology still remains unclear13- eruption and malocclusions. Treatment by surgical resection is 17). Hart et al reported a mutation in son of sevenless-1 (SOS1) generally followed by re-growth of the gingival tissues. Its gene on 2p21 and this is the only article explaining estimated incidence is 1/750.0001). It usually develops as an the molecular basis of the HGF so far15). isolated condition or as part of an uncommon genetic syndrome; In this study, we present the clinical features of 7 patients with syndromic HGF is usually associated with mental retardation2-5). HGF in a Turkish family. We performed molecular and cytogenetic However, familial form is rare and is not associated with congenital analysis and excluded the chromosomal abnormalities as well as the previously defined mutation in codon 1083 of the SOS1 gene. Correspondencce to : Prof. Dr. Sukru Ozturk, Istanbul Tip Fakultesi Ic Hastaliklari Anabilim Dali, Tibbi Genetik Bilim Dali, Sehremini-Fatih Materials and Methods Istanbul- Turkey 34390, Tel: 0090 212 414200 x 32919,Fax: 0090 212 5324208.E-mail: [email protected] Clinical data and samples collection 131 J.Hard Tissue Biology Vol. 18(3):131-134, 2009

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Figure 1. Oral region of the index patient, which shows gingival overgrowth and misalignment of teeth 19 20 21 22 X Y

Figure 3. Karyotype of the index patient, which reveals normal 46,XY male karyotype

analyses were performed in two affected individuals (proband and II-4) and one unaffected individual (II-3) at the Istanbul University, Istanbul Medical Faculty, Department of Internal Medicine, Division of Medical Genetics, Cytogenetic Laboratory. Peripheral blood cultures were prepared and stimulated by phytohemagglutinin at the 72-nd hour. Routine cultures were established in RPMI 1640 (Sigma-Aldrich Co.LTD, United Kingdom) supplemented with 15 % FCS (Biochrom LTD, Cambridge, United Kingdom), 1 %

Figure 2. Pedigree of the family. Arrow shows index patient. Black PHA-M (Biochrom LTD, Cambridge, United Kingdom), Pen- labeled members are affected from the same disease Strep (Biological Industries Beit Haemek LTD, Israel), 1 % L- glutamin (Biochrom AG, Berlin, Germany). After routine A 9 year-old child was consulted from Faculty of Dentistry, harvesting procedures, at least 30 GTG-banded metaphases Department of Periodontology to the Outpatient Clinics of were analyzed at 550 band level for each patient Genetical Counceling due to familial HGF. Then 8 family according to ISCN (International System of Cytogenetic members were examined and 6 of them were diagnosed as affected. Nomenclature). Clinical examination revealed generalized, severe gingival hyperplasia involving both the mandibular and maxillary arches. Molecular analysis The gingiva was pink with a firm and dense consistency (Fig.1). Molecular analysis has been performed at Okayama University, No acute inflammatory signs were observed. There were no other Graduate School of Medicine, Dentistry and Pharmaceutical symptoms such as pain, bleeding or halitosis. His weight and Sciences, Department of Oral Pathology and Medicine. Peripheral height were within the normal limits and he had normal venous samples (10 ml) were obtained from examined affected intelligence. The onset of symptoms was 3 years. Exposure to and unaffected family members. Genomic DNA were extracted any medications and exposure to any drug associated with gingival using Qiagen blood kit following manufacturer‘s recommended overgrowth, including phenytoin, cyclosporine and calcium- protocol. Designed primer sequences and expected size of the channel blockers were excluded. All of the affected individuals PCR products were summarized in Table 1. of the family received an oral/dental examination on the basis of Reactions were performed in a 20 µl volume of the LightCycler- presence or absence of gingival enlargement (Fig.2). To be DNA Master SYBR Green I master mix (Roche Diagnostics Ltd., classified as affected, an individual had to have keratinized Applied Science, Penzberg, Germany) with a 10 pmol of each gingival tissues covering at least one-third of the clinical crowns. primer, MgCl2 concentration optimized between 2 and 5 mM, 200 Informed written consent form was provided by all participants µM of each dNTP, Taq DNA polymerase and 10x buffer. Initial prior to their inclusion in this study. denaturation at 94°C for 3 min was followed by 35 cycles of a denaturation step at 94°C for 30 s, an annealing step at 58 °C for Cytogenetic analysis 1 min and an extension step at 72°C for 1 min. A final extension In order to exclude chromosomal abnormalities, cytogenetic step at 72°C for 7 min was added. The amplified PCR products

132 Davut Pehlivan et al.: SOS1 Mutation in Hereditary Gingival Fibromatosis

Figure 4: Upstream and downstream of the previously mutation detected region in exon 21 of SOS1 gene

Table 1. Primer sequences for SOS1 gene mutation in exon 21

Primer Sequence Product Size Annealing (bp) Temperature SOS.S1 CCTAAAATCTCCTGGTGTTCG SOS.AS1 CTGCCAATTTTACCACTAGGC 497 62

were separated by 1.5% agarose gel electrophoresis and visualized dominant, isolated hereditary gingival fibromatosis (Fig.2). by exposure to ultraviolet light after ethidium bromide staining. Mutation analysis was performed by direct sequencing. The Cytogenetic analysis resultant PCR products reamplified with BigDye terminator Cytogenetic analysis on stimulated peripheral blood sequencing kit (Applied Biosystems, Foster City, CA), ethanol lymphocytes of the three affected members of the family revealed precipitated and direct sequenced on an automated sequencer using normal karyotypes (proband, II-3 and II-4) (Fig.3). the 5'-CCTAAAATCTCCTGGTGTTCG-3' sense primer and 5'- CTGCCAATTTTACCACTAGGC-3' antisense primer (ABI Molecular analysis Prisme 3100P Genetic Analyzer). Approximately 250 bp upstream and downstream of the previously mutation detected region was directly sequenced in Results one affected and one unaffected family members and neither Clinical findings mutation nor variation was detected in those two family members An extended multigenerational kindred segregating HGF was (Fig.4). studied. Maternal grandfather, who was dead, was reported to have similar signs. Totally 8 family members were examined and Discussion 6 of them were diagnosed as affected. In affected individuals, Hereditary Gingival Fibromatosis is a rare disorder intraoral examination revealed generalized, severe gingival characterized by overgrowth of the gingival tissue, resulting in overgrowth involving both the mandibular and maxillary arches. functional and aesthetic problems. HGF may occur as an isolated The severity of gingival enlargement was not varied too much condition, as part of a syndrome or a chromosomal abnormality, between affected individuals. The age of onset was between 6-8 and secondary to certain pharmacological agents. The genetic basis years old in all affected individuals. None of the affected family for HGF is heterogeneous. Hart et al. identified a major gene locus members reported a history of taking medications associated with for autosomal dominant HGF in a Brazilian family on chromosome gingival overgrowth. None of the family members were 2p2113). This was the first report to localize a major gene locus for intellectually abnormal and had no associated hypertrichosis, HGF within a 37 cM genetic interval flanked by the STRP loci hearing deficit or other abnormalities in extraoral examination. D2S1788 and D2S441. 4 years after detecting this localization, HGF was present in all three generations and male-to-male Hart et al. found the first mutation in the SOS1 gene for isolated transmission was observed. Due to normal intelligence, lack of HGF in the same family. Shashi et al. showed at least two loci on congenital malformations, analysis of pedigree and absence of the short arm of that were responsible for gingival drug history, our diagnosis was consistent with autosomal fibromatosis18). One of the regions was located on 2p21 and the 133 J.Hard Tissue Biology Vol. 18(3):131-134, 2009 other was located in the region of 2p13-p16. The second region Oral Pathol Med 20(9): 457-459, 1991 was the result of partial duplication of the chromosome 2p which 6. Jorgensen RJ and Cocker ME. Variation in the inheritance was published by Fryn et al8). Xiao et al. identified a new locus and expression of gingival fibromatosis. J Periodontol 45: on 5q13-q22 region after excluding 2p21 and 2p13-p16 loci14). In 472-477, 1974 the Xiao‘s family, all affected individuals were exhibiting clinical 7. Goldblatt J and Singer SL. Autosomal recessive gingival symptoms within the first year of life without the combined fibromatosis with distinctive facies. Clin Genet 42(6): 306- phenotypes such as mental retardation, deafness and hypertrichosis 308, 1992 as a striking feature. This resulted in to a new classification in 8. Fryns JP. Gingival fibromatosis and partial duplication of the HGF and called as HGF2 (MIM 605544). Ye et al. described short arm of chromosome 2 (dup(2)(p13Æp21)) Ann Genet another region on chromosome 2p, which is telomeric to the SOS1 39(1):54-55, 1996 gene, in a five generation affected Chinese family 16). This was 9. Rivera HM, Ramirez-Duenas L, Figuera LE, Gonzales-Montes the third region in HGF and called as HGF3 (MIM 609955). RM and Vasquez AI. Opposite imbalances of distal 14q in Recently, Zhu et al. identified a major gene locus in two Chinese two unrelated patients. Ann Genet 35:97-100, 1992 family on chromosome 11p15 with a maximum two point LOD 10. Dongari A, McConnell HT and Langlais RP. Drug-induced score of 8,70 at D11S4046 for the first family and of 6,02 at gingival overgrowth. Oral Surg Oral Med Oral Pathol 76:543- D11S1318 for the second family17). This region is well known as 548, 1993 an imprinted region and mutations occurring at the non-imprinted 11. Hassell TM and Hefti AF. Drug-induced gingival overgrowth: gene that cause a maternally transmitted HGF. The clinical findings old problem, new problem. Crit Rev Oral Biol Med 2:103- of Turkish family are consistent with Hart and Ye‘s families as an 137, 1991 autosomal dominant, non-syndromic hereditary gingival 12. Pernu HE, Knuuttila MLE, Huttunen KRH and Tiilikainen fibromatosis. Presence of male to male transmission on the ASK. Drug-induced gingival overgrowth and class II major pedigree, we rule out the imprinting mutations. histocompatibility antigens. Transplantation 57: 1811-1823, In conclusion, findings in the Turkish family are consistent with 1994 the other families in the literature. Identification of the genetic 13. Hart TC, Pallos D, Bowden DW, Bolyard J, Pettenati MJ and pathways and mechanisms will be the most important factor in Cortelli JR. Genetic linkage of hereditary gingival classifying these heterogeneous disorders with the minor role of fibromatosis to chromosome 2p21. Am J Hum Genet 62(4): phenotype. In this stage, we excluded the chromosomal 876-883, 1998 abnormalities and mutation in the same region previously reported 14. Xiao S, Bu L, Zhu L, Zheng G, Yang M, Qian M, Hu L, by Hart. Future planned studies are excluding the mutation in the Liu J, Zhao G and Kong X. A new locus for hereditary SOS1 gene, four known HGF loci and if yet no mutation has been gingival fibromatosis (GINGF2) maps to 5q13-q22. detected, genome-wide linkage analysis study. Genomics 74(2): 180-185, 2001 15. Hart TC, Zhang Y, Gorry MC, Hart PS, Cooper M, Marazita References ML, Marks JM, Cortelli JR and Pallos D. A mutation in the 1. Gorlin RJ, Cohen MM and Hennekam RCM. Klinefelter SOS1 gene causes hereditary gingival fibromatosis type 1. syndrome. In: Syndromes of the Head and Neck 4th Ed. Am J Hum Genet 70(4): 943-954, 2002 2001, pp62-65 16. Ye X, Shi L, Cheng Y, Peng Q, Huang S, Liu J, Huang M, 2. Kasaboglu O, Tumer C and Balci S. Hereditary gingival Peng B and Bian Z. A novel locus for autosomal dominant fibromatosis and sensorineural hearing loss in a 42-year-old hereditary gingival fibromatosis, GINGF3, maps to man with Jones syndrome. Genet Couns 15(2): 213-218, 2004 chromosome 2p22.3-p23.3. Clin Genet 68(3): 239-244, 2005 3. Katz J, Guelmann M and Barak S. Hereditary gingival 17. Zhu Y, Zhang W, Huo Z, Zhang Y, Xia Y, Li B, Kong X and fibromatosis with distinct dental, skeletal and developmental Hu L. A novel locus for maternally inherited human gingival abnormalities. Pediatr Dent May-Jun; 24(3): 253-256, 2002 fibromatosis at chromosome 11p15. Hum Genet 121(1): 113- 4. Wynne SE, Aldred MJ and Bartold PM. 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