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Pachyonychia congenita (PC) is a rare, autosomal dominant keratin disorder caused by mutations in four genes (KRT6A, KRT6B, KRT16, or KRT17). The International PC Research Registry is a database with information on patients' symptoms as well as genotypes. We sought to describe the heterogeneity of clinical symptoms and to investigate possible genotype–phenotype correlations in patients with two types of K16 mutations, p.Asn125 and p.Arg127, causing the PC-16 subtype of PC. We found that clinical symptoms depended on the type of amino-acid substitution. Patients with p.Asn125Asp and p.Arg127Pro mutations exhibited more severe disease than patients carrying p.Asn125Ser and p.Arg127Cys mutations in terms of age of onset of symptoms, extent of nail involvement, and impact on daily quality of life. We speculate that amino-acid substitutions causing larger, more disruptive changes to the K16 protein structure, such as a change in amino-acid charge in the p.Asn125Asp mutation or a bulky proline substitution in the p.Arg127Pro mutation, may also lead to more severe disease phenotypes. The variation in phenotypes seen with different substitutions at the same mutation site suggests a genotype–phenotype correlation. Knowledge of the exact gene defect is likely to assist in predicting disease prognosis and clinical management.
Pachyonychia congenita (PC; OMIM (Online Mendelian Inheritance in Man) #167200 and #167210) is a rare, autosomal dominant genetic disorder caused by mutations in any one of four keratin (KRT) genes (KRT6A, KRT6B, KRT16, and KRT17) encoding the keratin cytoskeletal proteins K6a, K6b, K16, and K17, respectively (Smith et al., 2005). Historically, K6a and K16 mutations have been associated with PC-1 (Jadassohn-Lewandowski type) and K6b and K17 mutations with PC-2 (Jackson-Lawler type) (Smith et al., 2005). Recently, data from the International PC Research Registry (IPCRR) have revealed considerable phenotypic overlap between these subtypes; therefore, a new molecular classification linking the mutant gene and clinical subtype has been adopted (PC-6a, PC-16, and so on, see Wilson et al., 2010).
In this study, we sought to characterize the clinical characteristics of PC-16 patients with amino-acid substitutions at two sites on the K16 protein, asparagine 125 (p.Asn125) and arginine 127 (p.Arg127), which lie at the helix initiation motif within the helix 1A domain (Steinert et al., 1993). Previous studies have shown that mutations in this critical area for keratin filament assembly can produce variable clinical manifestations, suggesting a correlation between the type of amino-acid substitution and severity of disease (Shamsher et al., 1995; Covello et al., 1998; Smith et al., 1999; Munro, 2001). However, these studies lacked the numbers required to draw conclusions based on statistically robust data.
Larger disruptions at the helix boundary motif sequences have been shown to abrogate the normal assembly and function of intermediate filaments (Smith et al., 1997, 1999). Particularly disruptive changes may include bulky proline substitutions such as p.Arg127Pro (Serrano et al., 1992) or charge-associated changes from asparagine to aspartic acid, as in p.Asn125Asp. We hypothesized that PC-16 patients with more disruptive missense mutations, p.Asn125Asp and p.Arg127Pro, would manifest more severe symptoms than patients carrying their more conservative counterparts, p.Asn125Ser and p.Arg127Cys.
As of June 2010, 72 individuals in the IPCRR had confirmed K16 mutations; 34 had complete survey data. Of the 34 patients in this study, 100% reported focal plantar keratoderma, 29/34 (85%) reported some form of nail dystrophy, and 16/34 (47%) reported oral leukokeratosis (Table 1). Almost all patients (94%) reported some amount of plantar pain; 74% had pain that was “very painful” or “painful enough to require medication.” Only two patients reported steatocystomas, one reported pilosebaceous cysts, three reported follicular hyperkeratosis, and none reported a history of prenatal/natal teeth, similar to previous reports (Leachman et al., 2005). In all, 20 individuals had mutations at the Asn125 site and 14 had mutations at the Arg127 site. Patients in each mutation group were compared against those with different amino-acid substitutions at the same site; i.e., p.Asn125Asp versus p.Asn125Ser, and p.Arg127Pro versus p.Arg127Cys.
Within the Asn125 group of patients, 4 patients had p.Asn125Asp mutations, a potentially more disruptive change, whereas 16 patients had the more conservative change p.Asn125Ser. Interestingly, p.Asn125Asp patients had more severe disease compared with p.Asn125Ser patients (Figure 1). Patients with p.Asn125Asp mutations were more likely to have 8–10 thickened toenails (P=0.006) and fingernails (P=0.048) beginning at an earlier age (1.3 vs. 13 years, P=0.01 and 1.8 vs. 23 years, P=0.06, respectively). All Asn125 patients reported plantar keratoderma and most (70%) reported severe plantar pain. Patients with p.Asn125Asp mutations had more palmar keratoderma (P=0.09) at an earlier age of onset (4.6 vs. 11 years, P=0.06). There was more variability in palmar pain and fingernail involvement (Table 1).
Of the 14 patients with mutations at the Arg127 site, 3 had p.Arg127Pro mutations, the more disruptive substitution, and 11 patients had p.Arg127Cys mutations. Patients with p.Arg127Pro mutations were more likely to have 8–10 thickened toenails (P=0.002) and fingernails (P=0.002). Although all patients reported persistent plantar keratoderma and most (79%) reported plantar pain, p.Arg127Pro patients were much more likely to report painful palmar keratoderma (P=0.001) and a significant impact on their quality of life (P=0.002). Interestingly, all p.Arg127Pro patients reported oral leukokeratosis compared with 18% of p.Arg127Cys patients (P=0.01).
Most clinicians consider hypertrophic nail dystrophy the dominant feature of PC. In our analysis, we found that most, but not all, patients with K16 mutations exhibit nail symptoms. Five patients reported no nail disease and all had the milder p.Asn125Ser or p.Arg127Cys mutations, consistent with one previous report (Shamsher et al., 1995). All of our subjects reported persistent plantar keratoderma, suggesting that this is not a distinguishing feature. A majority (67%) also reported persistent palmar keratoderma, but several patients with the milder mutations did not.
Although our sample size was limited, we found that PC clinical symptoms depended on the type of amino-acid substitution. p.Asn125Asp and p.Arg127Pro patients exhibited more severe disease than their counterparts p.Asn125Ser and p.Arg127Cys in terms of age of onset of symptoms, extent of nail involvement, and impact on daily quality of life. In the p.Asn125Asp mutation, asparagine, an uncharged amino acid, is changed to aspartic acid, a negatively charged molecule. In the p.Arg127Pro mutation, the change from arginine to proline introduces a bulky proline side chain into the protein structure. These changes in charge and protein structure may affect the folding and function of the K16 filament. We speculate that mutations causing these larger structural changes may lead to more severe disease phenotypes and earlier age of onset. Correspondingly, mutations involving relatively minor structural changes at the same sites are associated with milder disease; e.g., at the Asn125 site, patients with mutations from asparagine to serine exhibited different, less severe disease characteristics than those with asparagine to aspartic acid mutations. That different mutations at the same site lead to different disease phenotypes suggests a genotype–phenotype correlation. Interestingly, we also found that symptoms varied within families with identical mutations. In one family with four adult members carrying the p.Asn125Asp mutation, two patients reported oral leukokeratosis whereas two did not, suggesting that other modifier effects may also be at play. Nevertheless, this study emphasizes the importance of genotyping for clinical prognosis and patient counseling. In the future, patients with mutations causing severe disease may be ideal candidates for trials of new, gene-targeted therapies for PC.
In 2004, the IPCRR established an international registry for PC with the goal of gathering patient data to improve understanding of the disease process and support investigation into potential therapies. The registry was approved by the Western Institutional Review Board (no. 20040468); more details on the survey and IPCRR can be found online at www.pachyonychia.org. All subjects in the IPCRR database underwent genetic testing, as previously described (Smith et al., 2005). Sequencing of the KRT16 gene was performed on genomic DNA from blood samples and confirmed using an independent buccal DNA sample by GeneDx (Gaithersburg, MD). Each subject received a standardized survey with questions on the presence/absence and severity of PC clinical features, including persistent plantar and palmar keratoderma, plantar and palmar pain, thickened toenails and fingernails, and oral leukokeratosis (Table 1). Subjects also answered questions regarding age of onset of each PC symptom. For questions on severity of symptoms, answer choices ranged from “never affected” to “always affected.” For questions addressing presence of pain, answer choices ranged from “no pain” to “often require medication for pain.” Severity of nail disease was assessed using absolute number of affected fingernails or toenails. Impact of disease on quality of life was assessed by asking how often symptoms affected daily life. Symptom severity, pain, and impact on quality of life were assessed using a four-category Likert scale (Table 1). Differences in proportions were analyzed with χ2 tests and continuous variables with two-sided t-tests. Statistical significance was defined as P<0.05.
We thank Roger Kaspar, Transderm, and Irwin McLean, University of Dundee, for their help in reviewing and editing this manuscript, and Holly Evans, Pachyonychia Congenita Project, for administrative assistance. NJW and FJDS are funded by grants from Pachyonychia Congenita Project. Finally, we are grateful to the PC patients who have contributed to the IPCRR and who made this study possible.
CONFLICT OF INTEREST The authors state no conflict of interest.