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Abstract
Gingival fibromatosis (GF) occurs in several genetic forms as a simple Mendelian trait, in malformation syndromes, and in some chromosomal disorders. Specific genes responsible for GF have not been identified. An autosomal dominant form of hereditary gingival fibromatosis (HGF, MIM 135300) was recently mapped to chromosome 2p21 in a large Brazilian family and there was an earlier report of GF in a boy with a cytogenetic duplication involving 2p13→p21. We thus hypothesised that a common gene locus may be responsible for GF in both the Brazilian family and the boy with the chromosome 2p duplication. We performed additional genetic linkage studies on the Brazilian family and molecular cytogenetic studies on the patient with the cytogenetic duplication to correlate more precisely the genetic interval of the HGF phenotype with the duplicated 2p interval. Additional linkage analysis of new family members resulted in refinement of the candidate region for HGF to an 8 Mb region. Molecular cytogenetic analysis of the 2p13→p21 duplication associated with GF showed that the duplicated region was proximal to the candidate interval for HGF. Thus, our results support the presence of two different gene loci on chromosome 2p that are involved in GF.
- gingival fibromatosis
- chromosome duplication
- chromosome 2
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Gingival fibromatosis (GF) is characterised by overgrowth of the oral epithelium (gingiva) that surrounds the teeth, resulting in functional and aesthetic problems. Heritable forms of GF may occur as an isolated condition, as part of a syndrome, or a chromosomal abnormality.1-3 Gingival fibromatosis also occurs secondary to exposure to certain pharmacological agents, including calcium channel blockers, phenytoin, and cyclosporin.4There may be a genetic susceptibility in the drug induced forms, since not everyone exposed to these drugs develops gingival fibromatosis.5
While GF is seen as a part of some well described genetic conditions, specific genes aetiologically important in isolated GF have not been identified. Elucidation of the genetic basis for GF may permit understanding of the molecular determinants of the condition. A major gene locus for autosomal dominant hereditary gingival fibromatosis (HGF) (MIM 135300) has recently been mapped on chromosome 2p21-p22 in a Brazilian family.6 Fryns et al 7 initially reported a 3 year old boy with a partial duplication of chromosome 2, involving bands 2p13→p21, resulting in mental retardation and an Aarskog-like phenotype. At the age of 4 years, he developed massive gingival overgrowth.3This patient had no medical history of exposure to drugs associated with GF or a family history of GF. Fryns3 hypothesised that there may be a causal relationship between the cytogenetic duplication and GF. Since the chromosome duplication in this boy potentially included the candidate region of HGF identified in the Brazilian family, it raised the possibility that a common gene locus within the 2p region was aetiologically responsible for GF in both the Brazilian family and in the boy with 2p duplication. A review of published reports did not find any report of GF in the other six cases of chromosome 2p duplication.8 However, it is notable that the duplication reported by Fryns3 7 is more proximal than the others (table 1). Although mental retardation was reported in all cases, other features were variable, possibly owing to gene dosage differences or specific breakpoint differences between the patients.
Findings in patients with a de novo dup(2p)1-150
To correlate the region of cytogenetic duplication reported by Fryns3 7 with the linked interval reported by Hartet al,6 further molecular studies were pursued. We performed linkage analysis to refine the HGF interval in the Brazilian family and FISH analysis on Fryns’s patient with 20 yeast artificial chromosomes (YACs) spanning the region 2p13→23 to define more precisely the 2p duplicated region associated with GF.
Methods
REFINEMENT OF THE HGF CANDIDATE GENE INTERVAL
Five additional family members were ascertained (III.15, 16, 17, 18, 19, 20, fig 1) and genotyped for 12 STRP markers that spanned the HGF chromosome 2p candidate interval (table 2). Peripheral venous blood was obtained by standard venepuncture, and genomic DNA was extracted using the QIAamp blood kit (QIAMP). Subjects were genotyped for the STRP markers using standard techniques for PCR amplification with radioactively labelled γ-[32P] primers as previously described.9 PCR amplification products were separated on a 6% PAGE 7 mol/l urea gel (30 W, 1500 V). Following electrophoresis, gels were exposed in a phosphorimaging cassette for 15 minutes and scanned (Molecular Dynamics). Two point linkage analysis was performed by use of the MLINK program version 5.1 from the LINKAGE computer program.10 Assumptions of the analysis included autosomal dominant transmission of HGF with complete penetrance and an affected allele frequency of 0.0001. Marker allele frequencies were assumed to be uniformly distributed. At any given locus, results for the pedigree were used to generate a final lod score for each marker tested (table2). Precise values for maximum lod scores (Zmax) were calculated with the ILINK program from the same computer package.
Pedigree of Brazilian family with haplotype data showing subjects and site of genetic recombinants.
Two point lod scores at standard recombination rates and at the maximum likelihood estimate of the recombination fraction (Zmax and θmax), equal recombination rates in both sexes, for markers from chromosome 2p
MOLECULAR CYTOGENETIC ANALYSIS
Twenty YACs (Whitehead Institute for Biomedical Research, Cambridge, MA) spanning the 2p13→p23 interval were obtained from Research Genetics (Huntsville, AL, table 3, fig 2). The YACs were cultured for 48 hours in an enriched medium (YPD). Protein precipitation was performed with sodium dodecyl sulphate (SDS) and potassium acetate. Whole YAC DNA extraction was performed according to a standard protocol.11 Human sequences from the YAC clones were generated by polymerase chain reaction (PCR) with primers directed against Alu sequences.12 The PCR products suitable for in situ hybridisation ranged from 300-500 base pairs in size. The amplified DNA was nick translated with biotin-14-dATP (Gibco-BRL, Gaithersburg, MD) or directly labelled with spectrum orange or green (Vysis, Downer’s Grove, IL). Fluorescence in situ hybridisation (FISH) was performed as described in Pettenati et al. 13Metaphases were captured with a Zeiss Axioskop microscope. Images were captured with a CCD camera (Photometrics) and viewed using the VYSIS Quips mFISH imaging software system. To localise the YACs, a Giemsa-like banding pattern was obtained by reversing the DAPI counterstain. The YACs were mapped onto normal chromosomes to determine their precise cytogenetic location and then to the patient’s metaphases to characterise the duplication. Dual colour hybridisation onto normal lymphocytes was performed with YACs that were close to one another (798-F-2 and 930-A-1, 972-C-5 and 929-F-5, 894-F-8 and 873-G-3, 873-G-3 and 915-F-7) to determine their precise order on 2p.
Summary of molecular cytogenetic analysis
Summation of HGF genetic linkage interval, relation to cytogenetic map, and correlation with the refined duplicated region reported by Fryns.3
Results
LINKAGE ANALYSIS OF BRAZILIAN FAMILY
Fig 1 shows a pedigree of family members including the haplotype segregating with the HGF phenotype. Two point lod scores for the HGF trait with the 12 STRP loci tested are shown in table 2. As a result of linkage analysis, a meiotic recombinant event was detected for III.20, permitting us significantly to reduce the candidate interval containing the HGF locus. The genetic interval containing the HGF locus is flanked by D2S1788 and D2S2298. This genetic interval is 11 cM and corresponds to a physical map distance of approximately 8 Mb.
MOLECULAR CYTOGENETIC ANALYSIS OF CHROMOSOME 2p DUPLICATION
FISH analysis on Fryns’s patient using 20 YACs spanning the 2p13→p23 region determined the 2p duplication more precisely as 2p13→p16. YACs in the region (n=10) were duplicated (fig 2, fig 3B, table 3). The genetic interval containing the HGF locus was determined to be 2p21→p22 (fig 2, fig 3A). YACs in this region (n=8) were not duplicated. Two YACs located proximal to 2p13 were not duplicated in the patient (data not shown). These results clearly indicate that the two intervals are distinct. YACs spanning the HGF interval (fig 2, table 3) contain several candidates genes for HGF including sodium calcium exhanger protein (NCX 1), calmodulin 2 (CALM 2), and the cytochrome p450 geneCYP1B1. The genetic loci D2S1788 (CHLC > GATA > 86E02) and D2S2298, flanking the HGF interval, were localised to 2p22 and 2p21 respectively (table3).
(A) FISH with YAC 809_B_6 on Fryns’s patient3 shows no duplication of signals indicating that the duplication is outside this region. The yellow arrow indicates the duplicated chromosome 2. (B) FISH with YAC 873_G_3 on Fryns’s patient shows duplicated signals on the abnormal chromosome 2, indicating that this YAC is within the duplicated region. The yellow arrow indicates the duplicated chromosome 2.
Discussion
Gingival fibromatosis may occur as an isolated heritable condition, as part of a syndrome, or secondary to exposure to certain pharmacological agents. While there appears to be a genetic basis for each of these forms of gingival fibromatosis, the genes responsible are unknown. Most non-syndromic forms of hereditary gingival fibromatosis are transmitted as autosomal dominant traits.2 Hartet al 6 recently identified a major gene locus for autosomal dominant hereditary gingival fibromatosis on chromosome 2p. This was the first report to localise a major gene locus for HGF within a 37 cM genetic interval flanked by the STRP loci D2S1788 and D2S441. Initial analysis suggested possible repression of meiotic recombination in this interval, although cytogenetic abnormalities were not detected in the Brazilian family.6 Although this region of chromosome 2p is not well characterised, this genetic interval corresponds to a physical map of approximately 43 Mb and is estimated to contain at least 250 genes and expressed sequences (ESTs). This genetic interval containing the HGF gene locus appeared to overlap the chromosome 2p duplicated region initially reported by Fryns3 7 and raised the possibility of a common gene aetiologically important in both Fryns’s patient and in the Brazilian family. The report by Fryns et al 7 is significant because the affected subject did not develop gingival fibromatosis until 4 years of age and gingival fibromatosis in the Brazilian family with HGF does not appear clinically until the same age.
Results of the linkage studies presented here permit us to refine the genetic interval containing the HGF locus to an 11 cM genetic interval flanked by D2S1788 (CHLC.GATA.86E02) and D2S2298. These genetic markers have been localised to chromosome 2, on p22 and p21 respectively. Based on our FISH analysis, we have refined the duplicated region of the chromosome 2p duplication in Fryns’s patient to bands 2p13→p16. Thus, the duplicated region in Fryns’s patient is proximal to the HGF locus identified in the Brazilian family (fig 3). These data appear to exclude a common locus as the cause of GF in the boy and the Brazilian family.
These findings indicate that there are at least two gene loci on the short arm of chromosome 2 that are responsible for GF. One locus is located in 2p21→2p22 and the other is located more proximally in the region of 2p13→p16. These findings are consistent with the existence of two or more syntenic loci that are important in the growth and development of the oral gingival tissues. Further analysis of the breakpoints of this duplication will help determine if the breakpoints of the duplication disrupt one or more genes important in gingival development or if the condition results from gene dosage.
Acknowledgments
We would like to thank Ruby Griffin for manuscript preparation. This work was supported by NIDCR grant DE 10990 (T C Hart, M J Pettenati, and V Shashi).