Introduction

Cathepsin C (CatC), also known as dipeptidyl peptidase I, is a papain-like cysteine protease involved in a variety of immune and inflammatory pathways, including activation of granule-associated proinflammatory serine proteinases in T lymphocytes, natural killers, and neutrophils [1]. Besides activation of immune cells, the proteolytic activity of CatC has been proposed to play a role in epithelial differentiation and desquamation [2]. CatC is processed into a proteolytically mature enzyme composed of three subunits (exclusion domain, light, and heavy chain) and acts as tetramers [3]. The exclusion domain is non-covalently attached to the heavy and light chains forming a heterodimeric structure [4].

Homozygous or compound heterozygous loss-of-function variants in the CatC coding gene, i.e., CTSC, cause Papillon–Lefèvre syndrome (PLS; OMIM#245000), a rare condition characterized by the combination of diverse degrees of palmoplantar keratoderma and prepubertal aggressive periodontitis leading to premature loss of both deciduous and permanent dentition [2, 5]. To date, over 75 causal variants have been identified in ethnically diverse populations [6]. While the vast majority (~97%) of the described variants cause typical PLS, a broad phenotypic spectrum has been described [6, 7]. CTSC variants have indeed also been reported in patients with Haim–Munk syndrome (HMS; OMIM#245010) that is characterized by palmoplantar hyperkeratosis, periodontal inflammation, arachnodactly, acroosteolysis, pesplanus, and onychogryposis [8]. In addition, CTSC variants were associated with isolated aggressive periodontitis (AP1; OMIM#170650), a nonsyndromic form of periodontitis which also leads to tooth loss due to severe periodontal inflammation [7, 9]. While additional genetic or environmental factors probably impact the expression of the disease [10, 11], the observed phenotypic heterogeneity, sometimes even observed for the same variant, complicates differential clinical diagnosis [7, 8, 12]. Despite the important number of known variants, to date no CTSC variant has been reported to be causing only AP1 without being also associated with the syndromic form PLS in other families.

In the present study we report a novel homozygous missense mutation in the CTSC gene (NM_148170; c.G302C; p.Trp101Ser) identified by whole exome sequencing in a three generations consanguineous Turkish family with AP1.

Subjects and methods

Subjects and whole exome sequencing

The subjects reported in this study were members of a three generation consanguineous family of Turkish origin. To determine the genetic etiology of aggressive periodontitis within the family we performed whole exome sequencing of the affected son (III-c, proband), his affected mother (II-b), as well as of four non-affected relatives: paternal grandmother (I-a), father (II-a), and two sisters (III-a, b). All subjects gave written informed consent to participate in the study. Genomic DNA was isolated from peripheral blood using standard protocols. The TruSeq Exome Kit (Illumina, San Diego, CA, USA) was used for the preparation and enrichment of exonic DNA libraries. Paired-end (2 × 75 bp) sequencing was performed on a NextSeq500 sequencer (Illumina, San Diego, CA, USA).

Variant analysis strategy

Sequence data was mapped on the hg19 reference genome using Isaac Genome Alignment Software 2.1.0 (Illumina, San Diego, CA, USA). For each sample, median coverage was at least 190-fold, and over 97% of target sequences were covered at a minimum of 10× (Supp. Table 1). Sequence variants were called using the Isaac Variant Caller 2.1.0 (Illumina, San Diego, CA, USA). Annotation was performed with the KGGSeq software package based on dbSNP build 135 [13]. Consecutive filtering steps were applied to variants identified in the proband (III-c) in order to focus on: (i) low frequency variants (minor allele frequency < 0.005 in 1000 Genomes project (http://www.internationalgenome.org/1000-genomes-browsers/), the genome aggregation database (gnomAD; http://gnomad.broadinstitute.org/), and an internal exome database with ~600 exomes accessed in october 2018); (ii) protein-altering variants (noncoding and synonymous variants were excluded); (iii) variants compatible with a recessive mode of transmission.

Targeted sequencing

Conventional capillary Sanger sequencing was performed to analyze and validate the genotype of the identified candidate variant in all family members. A 254 bp fragment spanning the missense mutation in position chr11:88068121 was amplified using the Expand Long Template PCR System (Roche Diagnostics, Rotkreuz, Switzerland) according to the manufacturer’s recommendations and using the following PCR primers, forward: 5′-AGTGGTGTACCTTCAGAAGCTGGATACA-3′; reverse: 5′-GGGAAGAGTGGTGTCAATTCCGGT-3′. After purification with the Exostar Kit (GE Healthcare Life Sciences, Marlborough, MA, USA), PCR products were bidirectionally sequenced using the same primers and the Big Dye Terminator Kit v3.1 (Thermo Fisher Scientific, Waltham, MA, USA). All targeted sequencing reactions were run on an ABI PRISM 3730xl (Thermo Fisher Scientific, Waltham, MA, USA).

3D modeling of CatC variants

The crystal structure solved at 1.4 Å, PDB 4OEL [14], was used as the model structure for the analysis. Structure visualization, distance measurement, and mutagenesis analysis were performed with the PyMOL program (DeLano Scientific, San Carlos, CA, USA). The RASP program was used for side-chain analysis [15].

Results

Clinical report

The index patient is a 6-year-old boy from a consanguineous family of Turkish origin. His parents are first cousins and his mother was also born from related parents (Fig. 1a). Premature loss of deciduous teeth started at the age of 3 years and a first orthopantomogram was performed at the age of 4 (Fig. 1b). At that time, deciduous teeth, except canines were prematurely lost. Oral hygiene was progressively introduced and mechanical periodontal treatment (root scaling with ultrasonic devices) as well as intensive maintenance therapy was combined to prevent adverse periodontal evolution [16, 17]. At the time of last examination (6 years of age), the patient presented his four permanent first molars and central incisors without detectable mobility or probing depths >2 mm (Fig. 1c, d). The patient showed additional dental abnormalities: hypodontia (agenesis of the two mandibular lateral incisors), taurodontism on the first right maxillary molar and molar incisor hypomineralization (Fig. 1c, d). Except aggressive periodontitis, physical, and intellectual development was unremarkable. Pregnancy, delivery, and neonatal period were normal except that the umbilical cord fell off relatively late (1 month). Language delay with absence of sentence construction was observed at the age of 3 years, motor development was normal. Complete blood count was normal, and the patient had no history of infections at the time of presentation. Importantly, no signs of palmoplantar hyperkeratosis could be detected.

Fig. 1
figure 1

Pedigree and clinical findings. a Pedigree of the three generation consanguineous family with autosomal recessive inheritance of aggressive periodontitis. DNA from individuals indicated by numbers was analyzed by whole exome and Sanger sequencing. The arrow denotes the proband. Solid symbols refer to affected individuals, half-solid symbols indicate unaffected, heterozygote carriers of the variant. bd Clinical findings in proband (III-c). Orthopantomogram at the age of 4 (b) and 6 years (c). Photograph at the age of 6 years (d). Additional dental abnormalities are highlighted: hypodontia (*), taurodontism (→), and hypomineralization (↓)

The patient’s mother (II-b; 39 years old) presented, according to her statements, severe periodontitis in early childhood, requiring dental implants, bone graft, and dental prosthesis early in adolescence (Supp. Fig. 1a). Other relatives, paternal grandmother (I-a), father (II-a), and sisters (III-a, III-b) were clinically unaffected (Supp. Fig. 1b–d). The first sister (16 years old, III-a) however showed slight gingivitis and small restorations (Supp. Fig. 1c). The second sister (14 years old, III-b) showed slight gingivitis, small restorations, and microdontia on the right maxillary lateral incisor (Supp. Fig. 1d).

Whole exome sequencing and variant interpretation

We used whole exome sequencing to identify pathogenetic variants with autosomal recessive inheritance in a three generation consanguineous family of Turkish origin with isolated AP1. Appling a consecutive filtering strategy based on variant frequency, functional consequences on coding sequence and heredity, a single missense variant in the CTSC gene was uncovered (NM_148170; c.G302C; p.Trp101Ser). This suspected pathogenic variant was confirmed by Sanger sequencing. It was found homozygous in the two affected individuals (patients III-c and II-b), while clinically unaffected family members (I-a, II-a, III-a, b) were heterozygous carriers of the variant (Fig. 2). To our best knowledge the missense variant has not been reported so far and is absent in public databases including the 1000 Genomes project (http://www.internationalgenome.org/1000-genomes-browsers/), the genome aggregation database (gnomAD; http://gnomad.broadinstitute.org/) and our internal exome database with ~600 exomes. Moreover, the variant is highly conserved and predicted to be disease causing by several bioinformatics tools (Table 1).

Fig. 2
figure 2

Sanger validation of the disease associated c.G302C missense mutation. The electropherograms show single base pair substitutions G302C of the CTSC coding sequence (NM_148170). Affected individuals (II-a and III-c) are homozygous, all unaffected individual (I-a, II-a, III-a) are heterozygous carriers of the nucleotide substitution

Table 1 Details of the variant of interest

The substituted residue, Trp101 is localized at the interface between the exclusion domain and the light and heavy chains of the mature CatC enzyme (Fig. 3a). Trp101 forms π–π interactions with Phe78, Trp110, and Tyr270, where lies the hydrophobic core of the protein complex. It may form a water-mediated sulfhydryl bond with Cys30 (at a distance of 3.6 Å) that stabilizes the structure of the CatC monomer (Fig. 3b). The Trp101Ser substitution is predicted to disrupt the π–π interaction based hydrophobic core and destruct the intimate interaction with Cys30, destabilizing both the three-dimensional structure of the exclusion domain and its interaction with the light and heavy chains of the mature CatC enzyme, which suggest complete or at least partially reduced enzyme function.

Fig. 3
figure 3

Predicted impact of the Trp101Ser substitution on CatC protein structure. a The CatC structure forms a beta-barrel (shown in green) in complex with its substrates (shown in cyan), while the mutant residue Trp101 is highlighted in magenta. b In the wild-type structure, the Trp110 (magenta) is surrounded by the hydrophobic residues Phe78 and Trp110 from CatC and Tyr270 from the substrates, while Trp101 is also in intimate contacts with Cys30 at a contact distance of 3.6 Å. c When mutated to Ser101, which has a hydrophilic side-chain, not only all the hydrophobic interactions, but also the interaction with Cys30 was disabled

Discussion

In the present study, we identified by whole exome sequencing a novel homozygous missense variant in the CatC coding gene CTSC (NM_148170; c.G302C; p.Trp101Ser) causing isolated AP1 in a consanguineous family of Turkish origin.

Most homozygous or compound heterozygous CTSC variants reported in the literature are associated with PLS. To date, only three studies reported CTSC variants (p.Tyr347Cys, p.Arg272His, and p.Thr189fs*10) associated with the nonsyndromic form AP1 [7, 9, 12]. All of these variants have however also been reported by other groups in the context of PLS [2, 18,19,20], suggesting that these variants may rather be linked to PLS with variable penetrance.

Here we report the first variant that is associated with only AP1 and no other syndromic form. The absence of skin manifestations cannot be explained by a potentially weak CTSC mutation, since predictive bioinformatics tools and the predicted effect of the Trp101Ser substitution on protein structure suggest a dramatic impact on CatC function. This observation is in line with previous functional studies reporting significantly reduced CatC activity regardless of the phenotypic penetrance in PLS patients [7]. We can however not exclude that the Trp101Ser variant could potentially lead to PLS in other so far unknown cases.

In conclusion, our finding might contribute to decipher the complex genotype–phenotype correlation of CTSC pathogenicity. Furthermore, it highlights the importance to consider CTSC in genetic screening of subjects or families with aggressive periodontitis without other symptoms.