Cathepsin-C (CTSC) Gene Mutations in Papillon-Lefevre Syndrome in India.

Background: Papillion Lefevre Syndrome (PLS) is a rare autosomal recessive disorder characterized by severe early onset periodontopathia and palmoplantar hyperkeratosis leading to premature loss of both primary and permanent dentition. PLS is caused by mutations in cathepsin C (CTSC) gene. The prevalence being 1-4 per million individuals with a carrier rate of 2 to 4 per million with no sex prediction and no racial predominance, onethird of all reported cases having consanguineous parents. A major gene locus for PLS has been mapped on chromosome 11q14. Correlation of physical and genetic maps of this interval indicate it includes at least 40 ESTs and six known genes including the lysosomal protease CTSC gene. Methods: Peripheral blood samples were obtained from three families with a total of 14 members of which 7 with PLS and others as ethnically matched controls were studied. DNA was extracted and all the 7 exons of the CTSC gene amplified by the polymerase chain reaction (PCR) using specific primers. All the PCR amplicons were subjected to automated DNA sequencing for mutations analysis. Results: The mutation spectrum shows that, in exon 2, 5 and 6 only missense mutations and in exon 3 only frame shift insertion were observed. Hence the total number of mutations observed in this study are 6. Out of this 5 are missense mutations and 1 is a frame shift insertion. Conclusions: This study describes a novel mutation of the cathepsin C gene in an Indian population with Papillon-Lefévre syndrome.


Introduction
The Papillon-Lefevre syndrome (PLS) is a rare genodermatosis of autosomal recessive inheritance manifesting as palmar plantar hyperkeratosis with early onset periodontitis[1]. It was first described by two French physicians, Papillon and Lefevre, in 1924. It has a prevalence of 1-4 cases per million persons and both males and females are equally affected with no racial predominance [2], [3]. The disorder is characterized by diffuse palmoplantar keratoderma and premature loss of both deciduous and permanent teeth. The palmoplantar keratoderma typically has its onset between the ages one and four years [4]. The sharply demarcated erythematous keratotic plaques may occur focally, but usually involve the entire surface of palms and soles resulting in foul-smelling odor [5]. Well-demarcated psoriasiform plaques occur on elbows and knees [6]. This may worsen in winter and be associated with painful fissures.
The second major feature of PLS is severe periodontitis, which starts at the age of three or four years [7]. The development and eruption of deciduous teeth proceeds normally, but their eruption is associated with gingival inflammation and subsequent rapid destruction of the periodontium. The resulting periodontitis characteristically is unresponsive to traditional periodontal treatment modalities and the primary dentition is usually exfoliated prematurely by the age four years. After exfoliation, the inflammation subsides and gingiva appears healthy. However, with eruption of the permanent dentition the process of gingivitis and periodontitis is usually repeated and there is subsequent premature exfoliation of the permanent teeth, although the third molars are sometimes spared [8], [9] .
Although the cause of PLS is not well understood, it has been reported that loss of the functional mutation effecting both the alleles of cathepsin C gene, located on chromosome 11q14.1-q14.3 is associated with PLS. [10], [11] The cathepsin C (CTSC) gene encodes a cystine lysosomal protease, also known as dipeptidyl peptidase I, which functions to remove dipeptides from the amino terminus of protein substrate. It also has endopeptidase activity. CTSC gene is expressed in the epithelial region commonly affected by PLS such as palms, soles, knees, and keratinized oral gingiva. It is also expressed at high levels in various immune cells including polymorphonuclear leukocytes, macrophages, and their precursors. Several mutations have been reported in the cathepsin C gene in individuals from diverse ethnic groups.
An increased prevalence of parental consanguinity has been reported in PLS patients [2]. All PLS patients are homozygous for the same CTSC gene mutation inherited from a common ancestor. It would be pertinent to mention that there are reports of at least six cases of late onset variation of PLS without underlying CTSC gene mutation [12].

Materials And Methods Patient selection and clinical sample collection
In this study three families, total 14 (PLS1 to PLS14) individuals were recruited and analyzed for mutations in CTSC gene. Out of 14 the individuals PLS1, PLS2, PLS3, PLS7, PLS8, PLS12 and PLS13 were exibiting extreme clinical manifestation of PLS. Rest of the PLS individuals (4, 5, 6, 9, 10, 11 and 14) were family members not exhibiting any clinical features of PLS. PLS1 -PLS5 belong to family 1 which was reported from SDM College of Dental Sciences and hospital, Dharwad, India. PLS6-PLS8 belong to family 2 and PLS 10-14 belong to family 3, both the families were reported in S. S Dental College, Davangere.
The pedigree investigations of the affected families showed similar clinical features in the siblings.

Primer design and PCR
The nucleotide sequence for CTSC gene was retrieved from NCBI (NG_007952.1), which is 51182 bp linear DNA. The primers were designed for all the 7 exons, which encode for Dipeptidyl -peptidase I.
The primers were designing was done consedering that no exonic region was left unread during automated sequencing.. Exon 1 to 6 were covered by a single set of primers whereas exon 7 was amplified with three overlapping primer sets (Table 1). Final extension was carried out at 72 °C for 5 minutes (Eppendorf Mastercycler Gradient-Germany).

Sequencing of the PCR amplicons
The amplified PCR products were subjected to automated sequencing ABI-3130E at Center for Cellular and Molecular Biology, Hyderabad, India. All the amplicons including the affected and normal were sequenced. However, we could not rule out a mutation in the intronic sequence that would lead to abnormal splicing of the transcript, or a mutation in nearby regulatory sequences that could reduce gene expression.

Discussion
The results of the present study clearly indicate that, among the seven PLS patients studied, mutations in the CTSC gene are observed only in exons 2, 3, 5 and 6. The exons 1, 4 and 7 do not show any mutations in this population.
The mutation spectrum shows that, in exon 2, 5 and 6 only missense mutations and in exon 3 only frame shift insertion are observed. Hence the total numbers of mutations observed in this study are 6.
Out of these 5 are missense mutations and 1 is a frame shift insertion.
All the six nucleotide changes reported in this study fulfilled the criteria of a mutation, as these changes were not present in the reference-sequence of the CTSC which encodes for the enzyme PCR products of Exon 3 were analyzed for all the 14 individuals of PLS families g.30126-30127 insA was found in all the affected as well as non-affected members of the PLS families. The insertion was found to cause the frame shift insertion and not leading to any amino acid change as reading frame change. The said frame shift insertion is also never been reported in past published studies. However, this insertion was not found in any family members of PLS 2 family.
The mutation g.42281T > C was found in exon 5 in all the 14 members of the all the PLS families. This is a missense mutation leading to the amino acid change L14094S. This is the novel mutation found in our study in all the family members and never been reported elsewhere. Comparing with the reference sequence it was found that the mutation g.42281T > C is restrict to the Indian PLS family members. The change in amino acid is expected to change the structure of the protein dipeptidyl-pemptidase1. But when the mutation is present in all the affected and non affected PLS individuals it is also suspected that only these mutations are not responsible for causing the phenotypic changes but also some other mutations or expressions of genes are responsible for causing PLS.
In exon 6 of PLS1, mutation g.46325A > G and G46357A > G were found. Both the mutations are missense mutation and leading to the amino acid change E15442G and M15453V respectively. The mutation g.4625A > G is novel mutation found in exon 6 of specimen 1, whereas the mutation g.46357A > G has been already reported in Indian families [13] and in Spanish families [14].
The mutation g.46325A > G, g.46357A > G and g.46663C > T were found in specimen 4. Among the 3 mutations in specimen 4. The mutations g.46357A > G and g.46663C > T were already reported in the earlier studies [13] in Indian families as well as in Turkish families.
In exon 6 of specimen 5, mutation g.46325A > G, g.46357A > G, g.46519G > T and g.46663C > T has been reported. All the 4 mutation were missense mutations and leading to the amino acid change.
The mutations g.46357A > G and g.46663C > T were already reported and rest of the two mutations are novel found in our study.
Among the 3 mutations, mutation g.46357A > G has been already reported and the rest of the two mutations are novel. All the mutations are missense and leading to the aminoacid change E15442G, M15453V and A15507S respectively.
The mutation g.46325A > G and g.46357A > G were found in the specimen PLS1, PLS4, PLS5, PLS6 and PLS7 An interesting feature of the CTSC gene is that mutations in this gene also result in two other closely  [14]. No correlation was found as affected subjects with transgressions of dermal lesions onto knees or elbows or both had mutations in both the pro-and mature regions of the enzyme. Our analysis also did not find any correlation between the types of mutations (missense, nonsense, insertion, deletion and splice site) and the presence or absence of the lesions on the knees or elbows or both as observed previously [14].      PLS13 showing Palmar hyperkeratosis.