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Cathepsin C Gene Variants in Aggressive Periodontitis
1 Department of Conservative Dentistry, Correspondence: * corresponding author, Barbara.Noack{at}uniklinikum-dresden.de
Cathepsin C (CTSC) mutations are known to cause Papillon-Lefèvre syndrome. The aim of this study was to examine the association of CTSC genotype with susceptibility to non-syndromic aggressive periodontitis. The CTSC gene was analyzed in 110 persons with generalized aggressive periodontitis in comparison with 78 control individuals, after identifying different variants in a cohort of 100 persons. Five out of 19 discovered variants were included in this association study, representing 5 single-nucleotide polymorphism groups in tight linkage disequilibrium. The relevance of genotypes on enzyme function was examined. The carrier frequency of the missense variant p.I453V was significantly increased in persons with disease compared with healthy control individuals (17.3% vs. 6.4%, p < 0.05). CTSC activity in leukocytes from individuals harboring this variant was significantly reduced (119.8  OD/min*105 cells, 95% confidence interval 17.4–174.9, p = 0.018). No influence of promoter variants was found on mRNA expression. The results support the hypothesis that CTSC gene variants contribute to increased susceptibility in generalized aggressive periodontitis.
Key Words: aggressive periodontitis • Papillon-Lefèvre syndrome • genetic susceptibility • cathepsin C gene variants
The quality and quantity of local inflammatory and immune reactions of the periodontium to the bacterial challenge differ widely among individuals (Page et al., 1997). Genetic variations contribute to the multi-factorial background, particularly in aggressive periodontitis (Hodge and Michalowicz, 2001; Kinane and Hart, 2003; Loos et al., 2005). Numerous candidate genes that are involved in immunity and inflammation are under consideration, including the Cathepsin C (CTSC) gene. More than 50 mutations have already been published since the initial "loss of function" CTSC mutations causing Papillon-Lefèvre syndrome (PLS) were reported (Hart et al., 1999; Toomes et al., 1999). PLS is a rare autosomal-recessive disorder characterized by palmoplantar keratosis and severe periodontitis. Mutations in the CTSC gene have also been reported in otherwise healthy families with a severe, prepubertal-onset form of aggressive periodontitis (TC Hart et al., 2000; Hewitt et al., 2004; Noack et al., 2004). These investigations confirmed that at least a subset of prepubertal aggressive periodontitis constitutes an allelic variant of PLS. CTSC plays an essential role in the host defense against bacteria (Nuckolls and Slavkin, 1999). It has been implicated in the processing of serine proteases for activity of natural killer cells (Pham and Ley, 1999; Pham et al., 2004). Impaired killer cell cytotoxicity might contribute to the pathogenesis of PLS-associated periodontitis (Lundgren et al., 2005; Meade et al., 2006). Since there is an association of CTSC mutations with an impaired host response to bacterial pathogens, variants in the CTSC gene may also be associated with non-syndromic generalized aggressive periodontitis. Therefore, we searched initially for CTSC sequence variants in the whole coding and regulatory regions of the gene. Subsequently, we examined the CTSC genotype in persons with generalized aggressive periodontitis compared with healthy control individuals. Finally, functional analyses in persons harboring periodontitis-associated haplotypes were performed.
Participants In total, 188 Caucasian German individuals volunteered to participate in the study. They received a description of the study and signed a consent form according to the Helsinki Declaration, which was approved by the local ethics committees. The assignment of 110 persons (mean age, 34.2 ± 5.9 yrs) to the generalized aggressive periodontitis group (case group) was based on the recommendations of the 1999 international classification workshop (American Academy of Periodontology, 1999). The control group was comprised of 78 unrelated individuals. As recommended (Morton and Collins, 1998), only controls who were older than the oldest patient (mean age, 52.9 ± 9.9 yrs; age of youngest control individual, 40.25 yrs; age of oldest patient, 39.75 yrs, respectively) were included. All controls were periodontally healthy or showed only minimal signs of periodontal disease. Thirty-one and 79 cases were identified in the University Hospitals of Greifswald and Dresden, respectively. In a previous study (Steffens et al., 2006), we have extensively studied stratification in the German population, and observed a low degree of population substructure. Regarding the present study, both case groups were identified in Eastern Germany, which renders the population substructure negligible. Detailed group descriptions are provided in the Appendix (www.dentalresearch.org).
Identification of CTSC Gene Variants and Association Study To study the association between aggressive periodontitis and the identified CTSC gene variants, we enrolled 60 case individuals as well as 28 control persons in the case-control study, in addition to the individuals already included in screening for DNA variants. They were genotyped by direct sequencing for 5 identified CTSC variations, representing 5 variant groups in tight linkage disequilibrium.
Functional Analysis In a second experiment, we analyzed mRNA expression in six Caucasian individuals to evaluate whether an impaired CTSC enzyme function could be due to an impaired expression of an allele harboring variants in the promoter region. Four persons were heterozygous carriers of variants in this region. The periodontal status was not relevant for that experiment. Reverse-transcription PCR (RT-PCR) was performed with 25 to 35 amplification cycles, and the resulting PCR products were sequenced. The RT-PCR segment contains 2 heterozygous variants in exon 7 which are in close linkage disequilibrium with promoter variants. Functional analyses are described in detail in the Appendix (www.dentalresearch.org).
Statistical Analysis
Prior to association analysis, sample size calculations were performed for the two-sided allelic
Deviation from Hardy-Weinberg equilibrium was investigated by a
Search for CTSC Gene Variants Nineteen sequence variants have been identified in 50 persons with generalized aggressive periodontitis and 50 healthy control individuals (Table 1  ). Thirteen variants occurred in non-coding regions. Three out of the 6 variants in the coding region resulted in an amino acid exchange.
In the promoter region, linkage disequilibrium was complete for single-nucleotide polymorphisms c.-1209 _-1219del, c.-816C>T, and c.-659del. We found only 2 different haplotypes of these 3 single-nucleotide polymorphisms in the studied population by sequencing allele-specific PCR products. In haplotype 1, all 3 loci were wild-type, while in haplotype 2, all 3 loci were variants. The corresponding promoter region was sequenced in a second population of African origin. In this population, a third haplotype with a 23% frequency, comprising 1 variant (c.-1209 _-1219del) and 2 wild-type alleles, was found.
Four additional single-nucleotide polymorphism subgroups showed a tight linkage disequilibrium (r2
Association Study
Enzyme Activity Enzyme function of CTSC was analyzed in 20 homozygous carriers of the wild-type allele and nine heterozygous carriers of the c.1357A>G variant. Additional pathogenic CTSC-sequence variants were excluded by complete sequence analysis of these persons. CTSC activity was significantly lower in leukocytes from individuals harboring the c.1357G allele compared with individuals harboring wild-type A/A (median difference, 119.8 OD/min*105 cells; 95% CI, 17.4–174.9; p = 0.018; Fig. 1).
Relative Quantification of mRNA Expression The c.1357A>G variation in exon 7 was found in tight linkage disequilibrium with 3 promoter variants (c.-260del, c.-259del, and c.-232T>C). Thus, reduced enzyme activity may be caused by a decreased expression level of the variant allele. RT-PCR products spanning the heterozygous variants in exon 7 were sequenced simultaneously with PCR fragments derived from genomic DNA of the same affected persons. An almost equal abundance of the wild-type and variant cDNA, as well as genomic DNA, sequence was observed in all four heterozygous carriers examined (Fig. 2 ).
In previous studies, only 3 CTSC variants have been identified: c.458C>T, c.1173T>G, and c.1357A>G. The allele frequencies of the known variants detected in the present study were similar to the allele frequencies from these studies in different populations (Allende et al., 2001; Nakano et al., 2001; Zhang et al., 2002). No comparable data are available for the other detected variants. In the promoter, 3 variants occurred in complete linkage disequilibrium, and only 2 different haplotypes were observed by sequencing of allele-specific PCR products in the study population. Three simultaneous mutation events seemed rather unlikely in a sequence comprised of 550 bases. However, a third haplotype was found in an African population, suggesting non-simultaneous mutations. In our association study, we could not detect a significant relation between generalized aggressive periodontitis and four of five analyzed single-nucleotide polymorphisms, which represent different haplotypes in the CTSC gene. However, the carrier frequency of the missense variant c.1357A>G (p.I453V) was significantly increased in case individuals compared with healthy control individuals. Even after adjustments for established prognostic factors (smoking and gender), there was a clear tendency for an increased frequency of the G-allele in persons with generalized aggressive periodontitis.
The amino acid exchange p.I453V has been previously reported as a polymorphism (Nakano et al., 2001), and was found in a heterozygous carrier of a PLS family (Hewitt et al., 2004). Although an isoleucine at position 453 constitutes a part of the S2-substrate-binding site (Turk et al., 2001), the latter authors dispute a significant effect on protein function by considering structural changes resulting from the deletion of a methyl group when replacing isoleucine with valine. In contrast, analysis of our experimental data clearly showed a significant effect on CTSC activity. A significantly reduced enzyme activity was found in leukocytes from persons harboring the p.453V allele compared with those harboring the wild-type, which may also be caused by promoter variants in strong linkage disequilibrium with p.I453V. However, the results from mRNA analysis suggest a normal mRNA expression of the variant allele, rendering it unlikely that the diminished CTSC activity is due to reduced CTSC expression. Nevertheless, analysis of our data does not show that p.453V is a total null allele. A wide range of activity reduction has to be considered (95% confidence interval, 17.4–174.9; Recent studies have analyzed the pathogenic role of CTSC deficiency in periodontitis. It has been shown that reduced CTSC activity results in diminished activity and stability of human leukocyte-derived serine proteases (Pham and Ley, 1999; de Haar et al., 2004; Pham et al., 2004). As a result, leukocyte capacity to neutralize leukotoxin and to eliminate Aggregatibacter actinomycetemcomitans seems to be reduced (de Haar et al., 2006). Further studies have highlighted the importance of CTSC in human killer cell function to control infection, and an impaired killer cell cytotoxicity in PLS-associated periodontitis is under discussion (Lundgren et al., 2005; Meade et al., 2006). The real role of reduced CTSC activity in more common types of aggressive periodontitis is still controversial, despite the plausible underlying mechanisms. It is known from most mutation studies in PLS families that heterozygous family members showing a reduced activity are not affected by periodontitis. Likewise, Hewitt et al. failed to show an impaired CTSC activity in 30 persons with aggressive periodontitis compared with 30 control individuals (Hewitt et al., 2004). Yet, a recent study reported a decreased CTSC activity in gingival tissue extract as well as in crevicular fluid in persons with chronic periodontitis, which supports a pathogenic role at least for reduced local CTSC activity in periodontitis (Soell et al., 2002). We have identified CTSC alleles associated with decreased enzyme activity in approximately 17% of cases, more than twice as much as in control individuals (6.4%). Thus, our results suggest the existence of a subgroup of persons with generalized aggressive periodontitis carrying a haplotype which confers a diminished CTSC activity that could contribute to periodontitis susceptibility. Nonetheless, aggressive periodontitis is a multifactorial disease, and it is likely that individuals carrying c.1357A>G do not develop aggressive periodontitis. Similarly to other complex diseases, it is estimated that several genes and environmental factors might be involved in disease susceptibility and create a high-susceptibility profile (Kinane and Hart, 2003). This high-risk profile seems to exist in only a very small part of a population, since aggressive periodontitis is a very rare periodontal disorder. Thus, despite numerous analyzed unaffected family members of PLS patients with decreased CTSC activity, their number could be still too low for persons with aggressive periodontitis to be detected in that population. The impact magnitude of functionally relevant CTSC gene variants in combination with other risk factors on generalized aggressive periodontitis susceptibility, as well as the pathogenic role of impaired function of Cathepsin C in periodontitis, remains to be seen.
The study was supported by the ARPA Research Foundation of the German Society of Periodontology as well as by the German Society of Dentistry.
A supplemental appendix to this article is published electronically only at http://jdr.iadrjournals.org/cgi/content/full/87/10/958/DC1. Received for publication June 12, 2007. Revision received June 18, 2008. Accepted for publication July 10, 2008.
Journal of Dental Research, Vol. 87, No. 10,
958-963 (2008)
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ABSTRACT
INTRODUCTION
0.8 (
2 test, a 5% test level, and power of 80%. When assuming a minimum carrier frequency of the risk allele of 0.06 in the control group and a relative risk of 2.5, a minimum of 110 case individuals and 78 control individuals was required if 2% of genotypes were missing non-differentially. 
