Novel PTCH1 mutations in Japanese familial nevoid basal cell carcinoma syndrome

Nevoid basal cell carcinoma syndrome (NBCCS), also known as Gorlin syndrome, is inherited in an autosomal dominant manner and is characterized by a combination of developmental abnormalities and a predisposition to tumor formation. Hedgehog receptor Patched 1 (PTCH1) has been identified as the mutated gene in NBCCS. We identified the PTCH1_c.3298_3299insAAG_p.1099_1100insE mutation in the transmembrane region, which comprises a sterol transporter whose abnormal function is reportedly related to pathogenicity.

Nevoid basal cell carcinoma syndrome (NBCCS, OMIM: 109400), also known as Gorlin syndrome, was first reported by Gorlin and Goltz in 1960 1 . NBCCS is an autosomal dominant inherited disease characterized by bifid ribs and palmar pits, as well as a predisposition to various tumors, including basal cell carcinoma (BCC), medulloblastoma, ovarioma, cardiac fibroma, odontogenic keratocyst, and skin patch [2][3][4] . At birth, patients with NBCCS typically exhibit macrocephaly or rib anomalies. As the patients' age, palmar and plantar pits become evident. At the age of approximately 10 years, a odontogenic keratocyst, formerly known as a jaw cyst, is observed as the first notable symptom in the diagnosis of NBCCS. At the age of approximately 20 years, BCC can develop at any location on the body, particularly on the eyelid. However, in Japanese patients with NBCCS, the incidence of BCC is significantly low, whereas the frequency of odontogenic keratocysts is relatively high 3,5 .
The gene responsible for causing NBCCS is the human homologue of the Drosophila patched gene Patched 1 (PTCH1) 6 . PTCH1, a Hedgehog (HH) receptor, is located on chromosome 9q22.3, consists of 23 exons, and encodes a 1447-amino-acid integral membrane protein with 12 transmembrane (TM) regions, five of which form a sterolsensing domain and two extracellular loops at which the N-terminal domain of the HH ligand binds 7,8 . The PTCH1 protein functions to inhibit the transmembrane protein Smoothened (SMO). Once extracellular HH ligands bind to the PTCH1 receptor, PTCH1 releases SMO inhibition, allowing SMO to participate in downstream signaling and activate GLI transcription factors. HH signaling plays an essential role during embryogenesis and maintains stem cell populations in certain adult tissues 9,10 . Unliganded PTCH1 inhibits HH signaling; this repression is released when HH ligands bind to PTCH1 7,8 . In this study, we investigated PTCH1 germline mutations in Japanese familial NBCCS.
Patients were diagnosed with NBCCS in the Department of Oral and Maxillofacial Surgery, Kawasaki Medical University Hospital, and Hiroshima University Hospital based on both clinical and genetic analyses. Each patient in the family with NBCCS was designated F1, F2, F3, F4, F5, F6, and F7. The pedigree is shown in Fig. 1a. F1-F5 had odontogenic keratocysts. F4 and F5 had undergone surgery for removal of odontogenic keratocysts several times at the Kawasaki Medical University Hospital. F1-F7 exhibited palmar and plantar pits, F3 and F4 showed kyphoscoliosis and calcification of the falx cerebri, and F4 had BCC, skin patch, and hairy skin patch. F6 and F7 had a congenital deficiency of the second premolar. Based on interviews with the patients, the husband of F5 also had a deficiency of the second premolar, and his mother had congenital deficiency of several incisors. The main phenotypes of F1-F7 are shown in Fig. 1b.
The detected mutation, PTCH1_c.3298_3299in-sAAG_p.1099_1100insE, was located in the 10 th transmembrane region and mapped closely to sterol-binding sites. Recently, through structure-guided mutational analysis, Gong et al. revealed that the interaction between Shh-N and PTCH1 is steroid-dependent 11 . Moreover, PTCH1_c.3298_3299insAAG was not found in Togo Var, Exome Variant Server, dbSNP, dbVar, or ClinVar, confirming that the mutation is novel. In addition, PTCH1_c.3298_3299insAAG was predicted as disease causing by Mutation Taster2 and as deleterious by PROVEAN software. Thus, we predicted that the specifically shared mutation, PTCH1_c.3298_3299insAAG_p.1099_1100insE, would be responsible for the pathogenesis of NBCCS 11,12 .
Of note, c.1665T>C (rs1805155), located in a sterolsensing domain, is synonymous and the most common SNP in PTCH1. Clinical significance is benign in ClinVar. To our knowledge, there is no prior report about the pathogenicity of this SNP. c.2560+9G>C and c.2887 +21G>A are intronic SNPs. Although c.2887+21G>A is not reported, c.2560+9G>C (rs2066829) is registered as an intronic variant and benign in ClinVar. It is unknown whether the PTCH1 polymorphisms located in introns cause a functional change. However, intronic polymorphisms have been demonstrated in association with other complex diseases, including the association of IRF6 with cleft lip/palate 13,14 . c.3944C>T (rs357564) results in an amino acid change in the topological domain of the Cterminus of PTCH1. Though the interpretation is benign in ClinVar, some researchers have concluded that, in combination with oral contraceptive use, c.3944C>Tcarrier in PTCH1 is associated with an increased risk of breast cancer 15 . Because PTCH1 has a sterol-sensing domain, long-term exogenous hormone use is reportedly related to breast cancer risk. Although the above three SNPs were also detected in wild-type PTCH1 in this family, it was recently suggested that SNPs in key genes involved in the HH signaling pathway are associated with susceptibility to odontogenic cystic lesions 16 .
Here, we reported the detection of a novel mutation, PTCH1_c.3298_3299insAAG_p.1099_1100insE, in Japanese familial NBCCS. Therefore, we conclude that PTCH1_ c.3298_3299insAAG is the "likely pathogenic" mutation of NBCCS.

HGV Database
The relevant data from this Data Report are hosted at the Human Genome Variation Database at https://doi.org/10.6084/m9.figshare.hgv.2927.