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Novel Mutations of Cathepsin C Gene in Two Chinese Patients with Papillon-Lefèvre Syndrome
1 Department of Pedodontics and Correspondence: * corresponding author, gelh{at}public.bta.net.cn
Papillon-Lefèvre syndrome (PLS) is an inherited human disease characterized by early-onset periodontitis and palmoplantar hyperkeratosis. Mutations of the lysosomal protease cathepsin C (CTSC) gene have been shown to be the genetic cause of Papillon-Lefèvre syndrome. There are several case reports in China, while there has been no study on the genetic analysis of PLS. We studied two Chinese patients carrying Papillon-Lefèvre syndrome and showing premature tooth loss and palmoplantar hyperkeratosis. Mutation screening and sequence analysis of the CTSC gene revealed a compound heterozygous mutation (c.415 G>A and c.778 T>C) in one patient, and two novel compound heterozygous mutations (c.851G>A and c.112delCCTG) in the other patient. Our novel discovery indicates that the phenotypes observed in these two patients are due to the CTSC gene mutation.
Key Words: Cathepsin C mutation • Papillon-Lefèvre syndrome • pre-pubertal periodontitis
Papillon-Lefèvre syndrome (PLS, MIM no. 245000) is a rare autosomal-recessive disorder characterized by early-onset periodontitis and palmoplantar hyperkeratosis. Two Frenchmen, Papillon and Lefèvre, first described it (Papillon et al., 1924). Periodontitis begins early in life, with gum swelling, abscess formation, alveolar bone loss, and eventual tooth loss in both the primary and permanent dentition. The palmoplantar lesions are erythematous, keratotic, and sharply demarcated, and knees and elbows may also be involved (Gorlin et al., 1964). Other clinical features not often reported include: increased susceptibility to infections, especially furunculosis and pyoderma; pyogenic liver abscess; and calcification of the dura (Cury et al., 2002). The incidence of the disease is estimated at 1–4 per million (Gorlin et al., 1964), with males and females equally affected. At the end of 1999, two groups identified the candidate gene cathepsin C (CTSC), whose mutations result in Papillon-Lefèvre syndrome (Hart et al., 1999; Toomes et al., 1999). The CTSC gene encodes a lysosomal protease (cathepsin C) that plays an important role in the activation of many serine proteinases of immune and inflammatory cells (Toomes et al., 1999; De Haar et al., 2004). Most mutations in the CTSC gene have been associated with dramatically decreased enzyme activity, which may result in a susceptibility to bacterial infection (Toomes et al., 1999; Hart et al., 2000; Zhang et al., 2002). To date, more than 40 mutations in the CTSC gene have been identified worldwide (Hart et al., 2000; Selvaraju et al., 2003). Although there have been several case reports in China, there has been no genetic analysis of PLS. In the present study, we report compound heterozygous mutations of the CTSC gene in two Chinese patients with Papillon-Lefèvre syndrome, each from a non-consanguineous nuclear pedigree.
Patients The present study was conducted under a protocol approved by Peking University Health Science Center’s Ethical Committee. The two probands with Papillon-Lefèvre syndrome were diagnosed at the Peking University School of Stomatology based on clinical examinations. Each person was from a non-consanguineous nuclear pedigree. The parents of the patients and 50 normal individuals with no evidence of aggressive periodontitis and palmoplantar hyperkeratosis were also included in this study after providing informed consent, but the other relatives of the patients were not available for examination and genetic research.
PCR Analysis PCR reactions were carried out with the Perkin-Elmer 9700 GeneAmp System (Perkin-Elmer, Waltham, MA, USA). Each amplification reaction was performed in 50-µL reaction volume containing 1.25 U Taq DNA polymerase (MBI); 10x buffer (MBI: 50 mM KCl, 10 mM Tris-HCl [pH 8.8]); 1.5 mM MgCl2; 50 ng template DNA; 200 µM of each dNTP; and 500 nM of each primer. After initial denaturation at 95°C for 5 min, 35 cycles of denaturation at 95°C for 30 sec, annealing for 30 sec, and extension at 72°C for 30 sec, final extensions were performed at 72°C for 7 min.
Sequencing To determine the exact site of mutation, we cloned PCR fragments of exon 1 from Patient II and his father into pGEM®-T Easy Vectors (Promega Corporation, Madison, WI, USA), then transformed them into competent E. coli strain TOP 10. Sequencing analysis was performed with the clonies carrying recombinant plasmid.
Clinical Diagnosis Both patients had a history of early-onset periodontitis at the age of 2–3 yrs. Intra-oral examination indicated red, swollen gums, with most teeth missing. Patient I was a three-year-old boy, his palmoplantar hyperkeratosis, first noted at 3 mos of age, was moderate, and the hyperkeratotic lesions worsened in winter, with deep fissures on the soles of his feet. He had recurrent respiratory infections, indicating an increased susceptibility to bacterial infection. Patient II was a four-year-old boy, and oral examination showed the loss of his 16 primary teeth. He had severe, extensive hyperkeratotic lesions, involving knuckles, knees, buttocks, and rump, in addition to palmoplantar hyperkeratosis. The typical clinical manifestations of Patient II with PLS are shown in Fig. 1  .
Neither patient had siblings. The non-consanguineous parents were also examined, but no sign of aggressive periodontitis and palmoplantar hyperkeratosis could be found, nor did they know any relative or antecedent with the disease in their family.
Mutation Analysis
Sequence analysis of Patient II also showed compound heterozygous mutations in c.851G>A (Fig. 3A ) and a frameshift mutation after nucleotide 112 of the cDNA (Fig. 3B). Sequencing of the clones carrying recombinant plasmid found a four-base-pair CCTG deletion of exon 1 (c.112delCCTG). His unaffected parents were responsible for each mutation: His mother was heterozygous for mutation c.851G>A, and his father carried the c.112delCCTG mutation. Neither the c.851G>A mutation nor the c.112delCCTG mutation was found in the 50 healthy individuals.
Previous studies have revealed the gene of the Papillon-Lefèvre syndrome locus, being mapped to chromosome 11q14-21 (Fischer et al., 1997; Laass et al., 1997; Hart et al., 1998). At the end of 1999, two groups identified Papillon-Lefèvre syndrome-related CTSC gene mutations (Hart et al., 1999; Toomes et al., 1999). Here we report novel mutations of the CTSC gene, which were responsible for Papillon-Lefèvre syndrome in two Chinese patients. Sequencing the CTSC gene in Patient I revealed 2 heterozygous mutations. The mutations include 2 missense changes: the maternal mutation c.415 G>A and the paternal mutation c.778 T>C. The first mutation resulted in the substitution of a hydrophobic glycine at codon 139 by a hydrophilic arginine (p.G139R), which has been reported previously (Zhang et al., 2002), and the second mutation is a novel mutation of the CTSC gene, changing a highly conserved serine to proline at codon 260 (p.S260P). Examination of CTSC in Patient II also revealed two novel compound heterozygous mutations. The maternal mutation c.851 G>A changed a highly evolutionarily conserved serine to asparagine at codon 284 (p.S284N). The paternal mutation showed overlapping peaks after nucleotide 112. After subcloning the PCR products and sequencing the clones carrying recombinant plasmid, we identified a deletion of four-base-pair CCTG in exon 1, which created a premature stop codon and resulted in a truncated short enzyme of 62 amino acids, in contrast to a normal enzyme of 463 amino acids. CTSC is a lysosomal protease playing an important role in the activation of many serine proteinases of immune and inflammatory cells. CTSC gene mutations have also been found to result in Haim-Munk syndrome (Hart et al., 2000a) and pre-pubertal periodontitis (Hart et al., 2000b; Hewitt et al., 2004; Noack et al., 2004). A common clinical manifestation of the 3 diseases is severe early-onset periodontal destruction, which indicates the important role of cathepsin C in the immune and inflammatory processes. The phenotype of Papillon-Lefèvre syndrome also indicates the possible role of CTSC in keratin processing or epithelial differentiation (Nuckolls and Slavkin., 1999; Toomes et al., 1999). The c.112del CCTG mutation in Patient II deleted all the functional parts of the protein, which seriously affected the function of CTSC. This mutation would be related to the phenotype of Patient II, with prominent and widespread hyperkeratosis involving both palms, soles, heels, knees, the rump, and hips. Here we describe, for the first time, compound heterozygous mutations in the CTSC gene in two Chinese patients. Five compound heterozygous mutations of the CTSC gene in Papillon-Lefèvre syndrome have been reported in other ethnic groups (Hart et al., 2000; Allende et al., 2001; Zhang et al., 2002; Noack et al., 2004).
Our study is the first to analyze the CTSC gene mutations of Papillon-Lefèvre syndrome patients in China (Table
This work was supported by a grant from the National Natural Science Foundation, P.R. China (30371536), to Lihong Ge. We appreciate the patients and their family members for participating in this study, Chang Yu for his help in collecting the pedigree information, Dr. Yuming Zhao for her review of the manuscript, and members of the Peking University Stem Cell Research Center for their technical assistance. Received for publication March 21, 2006. Revision received January 8, 2007. Accepted for publication February 28, 2007.
Journal of Dental Research, Vol. 86, No. 8,
735-738 (2007)
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ABSTRACT
INTRODUCTION
