European Journal of Medical Genetics 57 (2014) 630e635

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Clinical report

Root dentin anomaly and a PLG mutation Napaporn Tananuvat a, Pimlak Charoenkwan b, Atsushi Ohazama c, James R. Ketuda Cairns d, e, Massupa Kaewgahya f, g, Piranit Nik Kantaputra f, g, h, * a

Department of Ophthalmology, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand Department of Pediatrics, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand c Division of Oral Anatomy, Department of Oral Biological Science, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan d School of Biochemistry, Institute of Science, and Center for Biomolecular Structure, Function and Application, Suranaree University of Technology, Nakhon Ratchasima, Thailand e Laboratory of Biochemistry, Chulabhorn Research Institute, Bangkok, Thailand f Center of Excellence in Medical Genetics Research, Chiang Mai University, Chiang Mai, Thailand g Craniofacial Genetics Laboratory, Division of Pediatric Dentistry, Department of Orthodontics and Pediatric Dentistry, Faculty of Dentistry, Chiang Mai University, Chiang Mai, Thailand h Dentaland Clinic, Chiang Mai, Thailand b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 17 June 2014 Accepted 21 September 2014 Available online 30 September 2014

We report a Thai girl affected with plasminogen deficiency, Type I. Ligneous conjunctivitis was first observed when she was one-month-old. The newly recognized findings include tapered incisor roots as a result of thin root dentin, generalized short tooth roots, and mandibular prognathism. Mutation analysis of PLG demonstrated homozygous c.1193G>A missense mutation. The parents were heterozygous for c.1193G>A mutation. The c.1193G>A mutation is novel and predicted to cause amino acid substitution p.Cys398Tyr. Thin root dentin in the patient who was affected with PLG mutation and immunolocalization of Plg during early root development in mice imply the role of plasminogen in root dentin formation. Ó 2014 Elsevier Masson SAS. All rights reserved.

Keywords: Plasminogen Tooth development Root development Dentin formation Dentinogenesis Dental morphology

1. Introduction Plasminogen deficiency, Type I (OMIM#217090) or inherited severe hypoplasminogenaemia is a rare autosomal recessive disorder of the plasminogen gene (PLG) characterized by severely impaired extravascular fibrinolysis leading to ligneous fibrin-rich pseudomembrane formation on mucosa. The most common clinical manifestation is ligneous conjunctivitis (80%), a rare form of chronic conjunctivitis characterized by the formation of pseudomembranes on the palpebral surfaces of the eyes mainly on the tarsal conjunctivae and progress to thick, white or yellow-white masses that replace the normal mucosa. Ligneous gingivitis or periodontitis (destructive membranous periodontitis) (34%), ligneous laryngitis, ligneous vaginitis (8%), and congenital occlusive hydrocephalus have also been reported in patients affected with

* Corresponding author. Center of Excellence in Medical Genetics Research, Division of Pediatric Dentistry, Department of Orthodontics and Pediatric Dentistry, Faculty of Dentistry, Chiang Mai University, Chiang Mai, 50200, Thailand. E-mail address: [email protected] (P.N. Kantaputra). http://dx.doi.org/10.1016/j.ejmg.2014.09.006 1769-7212/Ó 2014 Elsevier Masson SAS. All rights reserved.

plasminogen deficiency. No genotypeephenotype correlation has yet been described. Intrafamilial clinical variability has been reported [Klammt et al., 2011; Tefs et al., 2006]. Here, we report a Thai girl affected with type I plasminogen deficiency, ligneous conjunctivitis, thin root dentin, tapered incisor roots, generalized short tooth roots, and a novel PLG mutation. 2. Case report We report a 10-year-old Thai girl who came to the Departments of Ophthalmology and Pediatrics, Faculty of Medicine, Chiang Mai University for the treatment of ligneous conjunctivitis (Fig. 2A). She was the third child of Karen tribe parents in northern Thailand. There was no history of consanguinity. However, the parents’ families have lived in the same village for many years. Both parents, the older brother, and sister were healthy. The patient’s birth weight was 2600 g (A missense mutation. The parents were heterozygous for the c.1193G>A mutation. The c.1193G>A mutation is predicted to cause the amino acid substitution p.Cys398Tyr in the plasminogen precursor protein (Supplemental Fig. 3S). This mutation is novel and classified as a disease-causing mutation based on its absence in the dbSNP and the Human Gene Mutation Database (HGMD), absence in our 200 normal chromosome samples, evolutionary conservation of the amino acid across species, and its prediction as a disease-causing variant by the SIFT, Polyphen2, Mutation Taster, and HANSA programs. As shown in Fig. 4, the Cys398Tyr mutation found in our patient affects a disulfide bridge in kringle domain 4 of plasminogen

[Petersen et al., 1990]. The kringle domains in plasminogen have been described to control plasminogen activation, plasmincatalyzed fibrinolysis, and the binding of plasmin to fibrin [Patthy et al., 1984]. Kringle 4 domain interacts with the Pan-apple (P-Ap) domain and forms an interface with the activation loop and the serine protease (SP) domain [Law et al., 2012]. These interactions create a closed, activation-resistant conformation that protects the proenzyme from undesirable activation. The closed plasminogen contains a high-affinity lysine-binding site that is important for interaction with C-terminal lysine residues present in fibrin clots and cell-surface plasminogen receptors [Law et al., 2012; Ponting et al., 1992]. Once plasminogen is recruited to targets, additional lysine kringle interactions trigger a major conformational change by opening up the closed state of the plasminogen molecule in order to allow cleavage and conversion to plasmin [Law et al., 2012]. Disulfide bonds are linked between sulfur atoms of two cysteine residues and generally increase the stability of extracellular proteins. Most disulfide bonds remain unchanged for the life of the protein [Schmidt et al., 2006]. The loss of amino acid Cys398 disrupts the disulfide bond with Cys437, which is likely to cause the kringle 4 domain to be less stable [Betz, 1993]. Moreover, the remaining Cys437 sulfhydryl is 3.8 Å from the disulfide bond between Cys426 and Cys449 (Fig. 5B,C), which could allow it to cleave that bond by disulfide exchange or form an improper disulfide link during the folding of this domain, depending on the folding pathway of the protein [Hwa et al., 1999]. The presence of a free sulfhydryl may also allow a disulfide bond to be formed with a free Cys bonding to plasminogen monomer or other proteins. The occurrence of ligneous conjunctivitis and the low Plg activity indicate that this mutation leads to a deficiency in Plg, rather than constituently active Plg which might occur if the protein were in an open conformation due to this mutation. If the mutation leads to misfolding, the protein is likely to be degraded in the endoplasmic reticulum [Ciechanover and Schwartz, 2002], but deficiency in functions of the kringle 4 domain other than blocking the activation peptide might also lead to these symptoms. 4. Immunohistochemical study of Plg expression during root development In order to investigate if PLG has role in root dentin formation, we did immunolocalization of Plg during root development in mice. CD1 mouse heads were fixed in 4% buffered paraformaldehyde at post-natal day 10 (P10). Heads were embedded in wax and serially sectioned at 7 mm. After deparaffinization of sections, specimens were treated by proteinase K and then incubated with primary antibody to plasminogen (Proteintech, Chicago, IL, USA). Biotinylated antibody (Jackson ImmunoResearch, West Grove, PA, USA), Avidin Biotin Complex conjugated with peroxidase (Vector Lab, Burlingame, CA, USA) and DAB (Nichirei, Tokyo, Japan) were used for detecting the primary antibody. At P10, mouse crowns of the 1st and 2nd molars are almost fully developed, and molar tooth root formation has been initiated. At the apical end of the developing root, epithelial root sheath is observed and odontoblasts are present on the pulpal surface of root dentin. Plasminogen immunolocalization was observed in periodontal bone, newly formed predentin matrix, and odontoblasts facing the apical end of the developing root, whereas it could not be detected in matured dentin (Fig. 5AeC). 5. Discussion

Fig. 3. Panoramic radiograph at age 10 years. Note thin root dentin and tapered incisor tooth roots. Generalized short tooth roots.

Dental anomalies found in our patient have not been reported in patients affected with plasminogen deficiency. Tapered incisor

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Fig. 4. The context of the Cys398Tyr mutation within the human plasminogen structure. A) The overall structure of human plasminogen [Law et al., 2012] in cartoon representation, with the Pan-apple domain in dark blue, the kringle 1 domain in sky blue, the kringle 2 domain in cyan, the kringle 3 domain in aquamarine, the kringle 4 domain in green, the kringle 5 domain in yellow, the linker/activation peptide in orange, and the serine protease in red. B) A close-up of the kringle 4 domain with the cysteines involved in disulfide bonds labeled. The amino acid residue numbering is according to the position in the precursor protein, as used in this paper, rather than in the mature protein numbering used by Law et al. [2012]. C) The kringle 4 domain with Cys398 converted to Tyr. The Cys437 sulfhydryl is within range to disulfide exchange with the Cys426-Cys449 disulfide (3.8 Å) and the introduction of the large Tyr398 side chain will clash with several residues (not shown), which will disrupt the domain structure.

roots and generalized short tooth roots, which are uncommon in general population, might imply the role of plasminogen in tooth root formation. The strong Plg expression in newly formed predentin matrix and odontoblasts facing the apical end of the developing root found in our study supports the role of plasminogen in root dentin formation. The coexpression of plasminogen activator activity and mineral accretion has previously been identified in predentin [Robinson et al., 1984]. It has also been shown that the plasminogen/plasmin pathway is involved in degradation of extracellular matrix including proteoglycans [de Bart et al., 1995; Daci et al., 1999; Mignatti and Rifkin, 1993; Ronday et al., 1997]. Proteoglycans are known to be present in predentin, supporting the role of plasmin-dependent proteolysis in extracellular regulation of dentin matrix mineralization [Moriguchi et al., 2004]. It is interesting to note that crown dentin in our patient appears clinically and radiographically normal. This implies that the molecular mechanisms of crown and root dentin formations are different and this is supported by the difference in noncollagenous matrix composition of crown and root dentin of bovine incisors [Takagi et al., 1988]. However, the thin root dentin and generalized short tooth roots need to be confirmed by findings in other patients affected with this condition. The strong expression of Plg in periodontal bone of the mouse tooth suggests the role of plasminogen in periodontal tissue formation. This might be the reason why a number of patients (34%) with PLG mutations have destructive membranous periodontal disease (ligneous gingivitis) [Sivolella et al., 2012]. Our patient and many patients with PLG mutations did not have destructive membranous periodontal disease. There might be effects of other

genes and there may be genotypeephenotype correlation. It has been shown that plasminogen activator deficiency leads to increased bone formation in bone remodeling, whereas less dentin formation was observed in this study. Plasminogen activators have also been shown to be involved in the degradation of bone by osteoclasts [Everts et al., 2008]. Plasminogen immunostaining pattern in bone tissue was significantly different from those in developing dentin, indicating that the role of plasminogen system is likely to be different between dentin and bone formation [Daci et al., 2003]. Although there was no reported consanguinity, the parents came from the Karen tribe and were from the same small village, therefore homozygosity for a second recessive gene is possible. The prevalence of plasminogen deficiency in Thai population has not been reported. However, the prevalence in the Japanese population has been reported to be 4.3% [Okamoto et al., 2003]. Plasminogen deficiency results in spontaneous fibrin deposition as a result of impaired thrombolysis. It is interesting to note that patients with plasminogen deficiency do not have venous thrombosis [Demarmels Biasiutti et al., 1998; Okamoto et al., 2003; Shigekiyo et al., 2000]. It is hypothesized that there may be other plasminogen-independent mechanisms that compensate for intravascular fibrinolysis in humans with plasminogen deficiency. Plasminogen, a proenzyme precursor of the primary fibrinolytic protease plasmin produced mainly in the liver, is cleaved and activated to be the protease plasmin by tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator, upon binding to the neuronal surface through specific binding molecules

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Unfortunately the tooth morphology of the patients with plasminogen deficiency and Plg / mice have not been reported. 6. Conclusion We report a Thai girl with plasminogen deficiency, type I who carried a novel homozygous c.1193G>A mutation. Ligneous conjunctivitis was the first clinical manifestation, observed when she was one month old. The thin root dentin and short tooth roots found at age 10 years appears to be newly recognized findings of this rare genetic disorder. Immunolocalization of PLG during early root development suggests the role of plasminogen in tooth root development. Conflict of interest All authors declare no conflict of interest. Acknowledgments We are grateful to the patient and her family for their kind cooperation and allowing us to use their medical and dental information for publication. This research was supported by Center of Excellence in Medical Genetics Research, Chiang Mai University, The Thailand Research Fund (TRF; Grant number BRG5580019), the Dental Association of Thailand, and Suranaree University of Technology (J.R.K.C.). The authors thank Dr. Neelaya Sukhamwang for providing the histopathology figures. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.ejmg.2014.09.006. References

Fig. 5. Plasminogen immunolocalization in murine molar root development. Sagittal sections showing plasminogen immunolocalization in murine lower first molar tooth at P10. A) Apical region of developing molar. Plasminogen immunoreactivity in odontoblasts (red arrow). B) Crown region of developing molar. Blue arrow indicating plasminogen deposition in newly formed predentin. C) Developing alveolar bone. Blue arrowheads indicating plasminogen deposition in newly formed bone. Enamel matrix (E), dentin (D) and pulp (P).

on the Raft membrane domains [Dotti et al., 2004]. Plasmin has important roles in hemostasis via a controlled dissolution of fibrin blood clots. Partial or complete absence of PLG is characterized by low or undetectable PLG antigen levels (normal range 6e25 mg/dl) and activity (normal range 75%e140%) [Tefs et al., 2006]. This leads to the inability to clear the fibrin deposit mainly at mucosal surfaces, causing the formation of an amorphous, fibrin-rich, amyloid-like substance that accumulates in the lamina plasminogen deficiency [Sivolella et al., 2012]. Ligneous conjunctivitis found in our patient is the most common clinical manifestation found in patients with type I plasminogen deficiency, with female predilection (1.4:1) [Schuster and Seregard, 2003; Tefs et al., 2006]. Ligneous conjunctivitis has also been reported in mice with plasminogen deficiency [Drew et al., 1998] and the wound healing in female mice has been reported to be better than that of the males [Rønø et al., 2013].

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Root dentin anomaly and a PLG mutation.

We report a Thai girl affected with plasminogen deficiency, Type I. Ligneous conjunctivitis was first observed when she was one-month-old. The newly r...
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