Description of Two New Cathepsin CGene Mutations in Patients WithPapillon-Lefevre SyndromeAijaz Ahmad Wani,* Nihal Devkar,† Milind S. Patole,* and Yogesh S. Shouche*

Background: Papillon-Lefevre syndrome (PLS) is a rare au-tosomal disorder characterized by severe periodontitis andpalmar plantar hyperkeratosis (PPK). PLS is caused by muta-tions in the cathepsin C (CTSC) gene. Dipeptidyl peptidase I(DPPI) encoded by the CTSC locus removes dipeptides fromthe amino terminus of the protein substrate and mainly playsan important role in immune and inflammatory processes.Several mutations have been reported in this gene in patientswith PLS. This study reports two novel deletion mutation of theCTSC gene in two Indian families with PLS.

Methods: Peripheral blood samples were obtained for ge-nomic DNA isolation from individuals belonging to two Indianfamilies. Exon-specific intronic primers were used to amplifyDNA from all individuals, and the PCR products were subse-quently sequenced to detect the mutations. Heteroduplexanalysis (HDA) was used to confirm heterozygosity and to de-termine the presence of mutations in control individuals.

Results: All patients from both families had a classic PLSphenotype, which included PPK and severe periodontitis. Se-quence analysis of the CTSC gene revealed two novel deletionmutations, one (1213-1215delCAT) in exon 7 and the other(629-630delGA) in exon 4 of the CTSC gene. For both muta-tions, the patients were homozygous, whereas the parentswere heterozygous.

Conclusions: This study reports two novel deletion muta-tions in two Indian families with PLS. One of the mutationsintroduces a premature stop codon, thereby producing atruncated protein. In the other case, the mutation observedleads to the loss of a highly conserved histidine moleculethat is present in the active site of the enzyme. In both cases,mutations may result in a conformation change, causingloss of the enzymatic activity. J Periodontol 2006;77:233-237.

KEY WORDS

Cathepsin C; hyperkeratosis; mutation; Papillon-Lefevresyndrome.

Papillon-Lefevre syndrome (PLS;Mendelian Inheritance in Man[MIM] #245000) is an autosomal

recessive disorder, which occurs with anincidence of one to four per million.1

Males and females are equally affected,and approximately one-third of familiesshow consanguinity.1,2 Clinically, it ischaracterized by severe periodontitis,leading to premature shedding of bothdeciduous and permanent teeth, andpalmar plantar hyperkeratosis (PPK). Inaddition, PPK affects not only the palmsand soles but also other areas such asthe knees and elbows. Several additionalclinical features have been reported tooccur with PLS, including hearing loss,calcification of the dura, follicular hyper-keratosis, nail abnormalities, recurrent in-fections, and mental retardation.3 Theseabnormalities have been suggested tooccur with the slow and effective loss ofimmunity in PLS patients. Mutations inthe cathepsin C (CTSC) gene are im-plicated in PLS.4,5 The CTSC geneconsists of seven exons and encodes alysosomal cysteine protease or dipep-tidyl aminopeptidase I (DPPI), whichfunctions by removing dipeptides fromthe amino terminus of the protein sub-strate. It also has endopeptidase activ-ity. DPPI is a high molecular weight(200 kDa) oligomeric enzyme found inmost tissues of the body and is highlyexpressed in cells of the immune system(polymorphic nuclear leukocytes, alveo-lar macrophages, and their precursors).

* Molecular Biology Unit, National Center for Cell Science, Pune, Maharashtra, India.† Jodhpur Dental College General Hospital, Jodhpur, Rajasthan, India.

doi: 10.1902/jop.2006.050124

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These cells are implicated in a wide variety of immuneand inflammatory processes, including cell-mediatedcytotoxicity, phagocytic destruction of bacteria, acti-vation and deactivation of cytokines and other in-flammatory mediators, and extracellular matrixdegradation. Additionally, DPPI is thought to play animportant role in protein degradation and proenzymeactivation.4 Therefore, it is likely that any effectivechange in the coding sequences of the cathepsin Cgene or its allele may result in a defective or truncatedprotein with improper function, which ultimately resultsin abolished activity in the homozygous patients com-pared to those with heterozygous genetic makeup.

Here, we report the results of sequence analysisand identification of two novel mutations in the CTSCgene from two unrelated Indian families.

CASE DESCRIPTION AND METHODS

PatientsTwo families from central India were included in thestudy. Although consanguineous marriages were re-ported in family 1, no consanguinity was reported infamily 2 for as long as three previous generations.The study was conducted in accordance with theHelsinki Declaration of 1975, as revised in 2000,approved by a local institutional ethics committee,and conducted from 15 July 2004 to 25 May 2005.Consent was obtained from all available familymembers, and a total of 20 individuals includingfour patients were selected for mutation analysis. Af-fected individuals received diagnoses of PLS, and theyshowed severe periodontitis and the clinical appear-ance of hyperkeratosis on palmar and plantar sur-faces. Patients also showed hyperkeratosis on theknees.

PCR Amplification and Mutation AnalysisPeripheral venous blood was obtained by vein punc-ture, and genomic DNA was extracted.‡ GenomicDNAfrommembersofboth familieswasPCR-amplifiedfor all seven exons spanning the CTSC gene by use ofexon-specific intronic primers as described by Toomeset al.5 Following DNA amplification, unincorporatedpolymerase chain reaction (PCR) primers and free de-oxynucleoside triphosphates (dNTPs) were inacti-vated prior to sequencing by enzymatic treatment.Briefly, this was accomplished by mixing 6 ml PCRproduct with 0.5 ml exonuclease I (10 U/1 ml) and 2ml shrimp alkaline phosphatase§ (1 U/1 ml) and incu-bation at 37�C for 30 minutes followed by inactivationof enzyme at 80�C for 15 minutes. Sequencing reac-tions were performedi and samples were sequencedon a DNA analyzer.¶ Sequence data were analyzedfor mutations by the use of a sequencing program#

and were also confirmed by a computer program.**DNA sequences were compared to published CTSC

gene sequences (GenBank accession no. NM-001814).

Heteroduplex Analysis (HDA)Segregation of mutations within families and the pres-ence of mutations in the parents and other clinicallyunaffected siblings were confirmed by PCR amplifica-tion of the mutated exons followed by HDA. For HDA,the PCR products were denatured for 5 minutes andallowed to renature at room temperature for 1 hour.Thesamples were subjected toelectrophoresis through8% polyacrylamide gel electrophoresis (PAGE) at 250V for 6 hours and were visualized by ethidium bromidestaining.6

RESULTS

In family 1, all three patients (two brothers, aged 11and 7 years, and their younger sister, aged 4 years)showed severe periodontitis leading to shedding ofmost of their teeth. They also had severe palmarplantar keratosis resulting in hyperkeratosis of theknuckles, knees, and elbows. However, only one pa-tient (female, aged 17 years) from family 2 had a his-tory of onset of the disorder by the age of 5 years. Themedical examination report indicated that the patienthad lost all of her permanent teeth by the age of 14.Extraoral examination revealed hyperkeratosis of thepalms, soles, and knees. The parents and other sib-lings from both families were clinically unaffected.Figure 1 illustrates the pedigree analysis of the twofamilies included in the study.

We sequenced all seven exons of the CTSC genefrom all four patients of both families and detectedtwo mutations specific to them. Once the particularmutation was identified, other family members werescreened by HDA. The patients were homozygousfor these two mutations. In all cases, both parentswere heterozygous, as indicated by multiple bandson HDA gel (Fig. 2A). The three patients from family1 showed a 3-basepair (bp) deletion (nucleotideposition: 1213-1215delCAT) in exon 7 of the CTSCgene (Fig. 2B). Amino acid analysis revealed thatthis mutation resulted in complete loss of a highlyconserved amino acid (histidine) at position 405(H405del) of the amino acid sequence of CTSC(Fig. 3A).

In family 2, sequence analysis of all seven exonsfrom the single patient with clinical phenotype forPLS showed a 2-bp deletion at nucleotide position629-630delGA in exon 4 of the CTSC gene (Fig. 2B).This mutation introduced a frameshift, resulting in a

‡ DNeasy tissue kit, Qiagen, Hilden, Germany.§ Amersham Life Sciences, Arlington Heights, IL.i Big-Dye terminator mix, Applied Biosystems, Foster City, CA.¶ DNA analyzer (3730), Applied Biosystems.# Seqscape version 2.1, Applied Biosystems.** nBLAST, National Institutes of Health, Bethesda, MD.

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premature termination codon, 21 bases downstream ofthe mutation site. This mutation is predicted to result ina truncated protein of only 221 amino acids instead ofthe wild-type protein of 463 amino acids. Sequencingof 30 ethnically matched normal control individualsshowed that these two nucleotide deletions were notpresent in any of the control individuals (data notshown).

DISCUSSION

DPPI is a lysosomal protein and is necessary for theactivation of serine proteinases in polymorphonuclearleukocytes.7 It has also been suggested that DPPI is in-volved in a wide variety of immune responses, such asthe activation of phagocytes and T lymphocytes. Ifthe protein is truncated, it may not be transported tothe organelle and may not be able to activate proteinkinases. Inaddition, itwill not be able toactivatephago-cytes and T lymphocytes, thereby leading to diseasephenotype.5 Therefore, any typical mutation mayeither result in truncation or alteration in the confor-

mation of CTSC encoded enzyme DPPI. The 629-630delGA mutation would reduce any residual activityof DPPI through frameshift-mediated decay of thetranscript, which might result in very low steady-statelevels of the CTSC messages, leading to the diseasephenotype as identified in the patient from family 2.Mutation 1213-1215delCAT would produce a proteinlacking one histidine residue at position 405 of theCTSC amino acid sequence. This amino acid positionis evolutionarily conserved in various species fromhumans to Schistosoma mansoni (Fig. 3B), and itsdeletion may cause a conformational alteration thatmay decrease or abolish DPPI activity. Recently, aC > A substitution has been found at nucleotide posi-tion 1213 of the CTSC gene that changes amino acidhistidine to arginine.7 This mutation was reported asthe first mutation in the active site of the enzyme(DPPI) that results in the complete loss of enzyme ac-tivity. Because H405 residue is crucial for the enzy-matic action of CTSC/DPPI, it is likely that the H405Amutation causes the absence of cathepsin C activityin PLS patients. The 3-bp deletion (1213-1215del-CAT), which we report, also falls within the active site

Figure 1.Pedigree diagram of families 1 and 2 included in the study. DNAsamples from individuals 1 through 14 were analyzed from family 1,and from individuals 1 through 6 from family 2. A horizontal doubleline indicates consanguineous marriage. Filled symbols indicateaffected individuals. Asterisks indicate those who were unavailablefor genetic analysis. Squares indicate males, ovals indicate females,and half-filled symbols indicate heterozygous unaffected.

Figure 2.A) Segregation of two cathepsin C deletion mutations in families 1and 2 as shown by HDA. Three patients (III, IV, and V) from family 1were homozygous for this mutation, and other family members, i.e.,father (II), mother (VII), and grandfather (VIII), were heterozygous.The grandmother (IX) and two maternal uncles (I and VI) werehomozygous normal. The deletion mutation (629-630delGA) wasfound to be homozygous in the affected patient (I) from family 2, andother family members, i.e., patient’s sister (II), father (III), mother (IV),and grandfather (V) were heterozygous carriers with both thewild-type and mutant alleles. Lane V is the wild-type control. B) DirectDNA sequence of exon 7 and 4 showing the wild-type andhomozygous deletion of 3 bp in exon 7 and 2-bp deletion in exon 4.In family 1, the parents and grandfather carry the heterozygousmutation, whereas in family 2, the parents and patient’s sister carrythe heterozygous mutation. The mutations are numbered according toreference cDNA sequence (GenBank accession no. NM-001814.2).

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of the enzyme and might also result in the complete lossof enzyme activity. This mutation produced a very se-vere PLS phenotype in family 1 patients compared tothe 2-bp deletion mutation found in the family 2 patient.

According to Online Mendelian Inheritance inMan (OMIM) and currently available literature, thetotal number of mutations in the CTSC gene to dateis 44.8-11 Of these, the most common ones are mis-sense/nonsense mutations (present in 85% of pa-tients), deletions (18%), splice site mutations (2%),and insertions (4.5%). Mutations reported in the pre-sent paper do not match any of the known mutationsand thus appear to be novel, bringing the total numberof mutations in the CTSC gene to 46.

Other than PLS, mutations in the cathepsin C genehave been shown to result in two closely related con-ditions: Haim-Munk syndrome (HMS)12 and prepu-bertal periodontitis (PPP).13 A common mutation,1040A >G, has been shown to cause two distinct phe-notypes, PLS and PPP, suggesting that other geneticand environmental factors play an important role inthe ultimate phenotype.14 Therefore, it is very impor-tant to study these other factors at the molecular andclinical levels in all patients. A missense mutation,857A > G (p. Q286R), in the CTSC gene has beenfound to cause HMS.12 This mutation has also beenfound in a homozygous state in a Spanish PLS patient,suggesting that HMS and PLS are clinical variants ofthe same homozygous cathepsin C gene mutation.15

It is possible that some of the clinical manifestations in

HMS patients (i.e., hyper-keratosis and periodontitis)are caused by mutations inthe CTSC gene, whereasother features of HMS(i.e., onychogryposis, pesplanus, arachnodactyly, andacroosteolysis) are causedby mutations in another hith-ertoundescribed locus.How-ever, this possibility remainsto be proven.

CONCLUSIONS

We report two novel deletionmutations responsible for avery severe PLS phenotypein two Indian families. Withthis study, the total numberof mutations in the CTSCgene reaches 46.

ACKNOWLEDGMENTS

The authors thank SachinSurve (National Center forCell Science [NCCS], Pune)

for his help in collecting samples from one of the fam-ilies. The authors also thank K.M. Dayananda (NCCS)and Raamesh Gowri Raghavan (National ChemicalLaboratory, Pune) for their helpful suggestions.

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Figure 3.A) Alignments of the parts of CTSC protein sequence harboring the mutations identified in PLS family 1,demonstrating the high conservation of amino acid H405 (exon 7). B) Alignments of the parts of CTSCprotein sequence harboring the mutations identified in PLS family 2, demonstrating the moderateconservation of amino acid 205S (exon 4). The aligned sequences are from human (GenBank accessionno. ·87212), rat (D90404), mouse (CTSC, U74683), dog (AF060171), Schistosoma japonicum(SchJp, U77932), and Schistosoma mansoni (SchMa, Z32531).

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8. Online Mendelian Inheritance in Man (OMIM). MIM#245000. Baltimore, MD: Johns Hopkins University.Available at: http://www.ncbi.nlm.nih.gov/omim.Accessed March 28, 2005.

9. Selvaraju V, Markandaya M, Prasad PV, et al. Mutationanalysis of the cathepsin C gene in Indian familieswith Papillon-Lefevre syndrome. BMC Med Genet2003;4:5.

10. Noack B, Gorgens H, Hoffmann T, et al. Novel muta-tions in the cathepsin C gene in patients with pre-pubertal aggressive periodontitis and Papillon-Lefevresyndrome. J Dent Res 2004;83:368-370.

11. Hewitt C, McCormick D, Linden G, et al. The role ofcathepsin C in Papillon-Lefevre syndrome, prepubertalperiodontitis, and aggressive periodontitis. Hum Mutat2004;23:222-228.

12. Hart TC, Hart PS, Michalec MD, et al. Haim-Munksyndrome and Papillon Lefevre syndrome are allelicmutations in cathepsin C. J Med Genet 2000;37:88-94.

13. Hart TC, Hart PS, Michalec MD, et al. Localization of agene for prepubertal periodontitis to chromosome11q14 and identification of cathepsin C gene muta-tion. J Med Genet 2000;37:95-101.

14. Hart PS, Zhang Y, Firatli E, et al. Identification ofcathepsin C mutations in ethnically diverse Papillon-Lefevre syndrome patients. J Med Genet 2000;37:927-932.

15. Allende LM, Garcia-Perez MA, Moreno A, et al.Cathepsin C gene: First compound heterozygous pa-tient with Papillon-Lefevre syndrome and a novel symp-tomless mutation. Hum Mutat 2001;17:152-153.

Correspondence: Dr. Yogesh S. Shouche, MolecularBiology Unit, National Center for Cell Science, Pune4110077, Maharashtra, India. Fax: 91-20-25692259;e-mail: yogesh@nccs.res.in.

Accepted for publication July 21, 2005.

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