Toshiyuki Miyashita

Patched1 haploinsufficiency increases adult bone mass and modulates Gli3 repressor activity.

Hedgehog (Hh)-Patched1 (Ptch1) signaling plays essential roles in various developmental processes, but little is known about its role in postnatal homeostasis. Here, we demonstrate regulation of postnatal bone homeostasis by Hh-Ptch1 signaling. Ptch1-deficient (Ptch1+/-) mice and patients with nevoid basal cell carcinoma syndrome showed high bone mass in adults. In culture, Ptch1+/- cells showed accelerated osteoblast differentiation, enhanced responsiveness to the runt-related transcription factor 2 (Runx2), and reduced generation of the repressor form of Gli3 (Gli3rep). Gli3rep inhibited DNA binding by Runx2 in vitro, suggesting a mechanism that could contribute to the bone phenotypes seen in the Ptch1 heterozygotes. Moreover, systemic administration of the Hh signaling inhibitor cyclopamine decreased bone mass in adult mice. These data provide evidence that Hh-Ptch1 signaling plays a crucial role in postnatal bone homeostasis and point to Hh-Ptch1 signaling as a potential molecular target for the treatment of osteoporosis.

Two cases of nevoid basal cell carcinoma syndrome associated with meningioma caused by a PTCH1 or SUFU germline mutation

Nevoid basal cell carcinoma syndrome (NBCCS) is an autosomal dominant disorder characterized by developmental defects and tumorigenesis. The gene responsible for NBCCS is PTCH1. The development of meningioma in NBCCS patients is a rare event. Here, we report two cases of NBCCS in which meningiomas did develop. The first patient carried a germline mutation in one allele of PTCH1, c.290dupA (p.N97KfsX43). In addition, the meningioma sample carried a somatic mutation, c.307delG (p.Val103LeufsX15), in the other allele of the same gene, suggesting a second hit. This is the first case of NBCCS-associated meningioma explained by the standard two-hit hypothesis. The second patient had a germline nonsense mutation in the SUFU gene, c.550C>T (p.Q184X). SUFU is located downstream of PTCH1 in the sonic hedgehog signaling pathway. This is the second time a germline mutation in SUFU has been found to cause NBCCS. Together with the previous report describing three cases of non-NBCCS medulloblastoma carrying a germline mutation in this gene, individuals with a SUFU germline mutation are expected to have a markedly high risk of developing medulloblastoma and probably meningioma.

Nationwide survey of nevoid basal cell carcinoma syndrome in Japan revealing the low frequency of basal cell carcinoma.

Nevoid basal cell carcinoma syndrome (NBCCS) is characterized by developmental defects and tumorigenesis. The clinical manifestations of NBCCS have been reported in large epidemiological studies from the United States, the United Kingdom, and Australia, but not from an Asian country. We conducted a nationwide survey and identified 311 NBCCS patients in Japan. We investigated the detailed clinical manifestations of 157 patients ranging in age from 9 months to 77 years old (mean: 33.1 years). We then compared the frequency and age of onset for various tumors developed in Japanese NBCCS patients with patients from the three countries listed above in which NBCCS studies were previously conducted. Our most significant finding was the low frequency of basal cell carcinoma (BCC) in Japanese patients. Frequency of BCC in patients over 20 years of age was 51.4%, a much lower rate compared to the United States, Australia, and the United Kingdom (91%, 85%, and 73%, respectively). The mean age of BCC onset was 37.4 years of age, a much older age compared to the above‐mentioned countries. These findings suggest that differences in ethnicity and/or environmental factors affect the incidence of BCC. Because the age of BCC onset is generally higher in Japanese NBCCS patients, careful skin examination over a prolonged period of time is warranted.

Gorlin syndrome (nevoid basal cell carcinoma syndrome): Update and literature review

Gorlin syndrome, also called nevoid basal cell carcinoma syndrome, is an autosomal dominant neurocutaneous disease characterized by developmental anomalies such as palmar pits and rib anomaly, and tumorigenesis such as medulloblastoma and basal cell carcinoma. This syndrome is mainly caused by a mutation of PTCH1, a human homologue of Drosophila patched, including frameshift, missense, or nonsense mutations. Genotype–phenotype correlation has not been established. PTCH1 is a member of hedgehog signaling, which is a highly conserved pathway in vertebrates, composed of hedgehog, SMO, and GLI proteins as well as PTCH1. Given that hedgehog signaling regulates cell growth and development, disorder of this pathway gives rise to not only developmental anomalies but also diverse tumors such as those seen in Gorlin syndrome. We recently reported, for the first time, a nationwide survey of Gorlin syndrome in Japan, noting that the frequency was 1/235u2009800 in the Japanese population, and that the frequency of basal cell carcinomas was significantly lower in Japan than in the USA and Europe, suggesting that ethnicity and genetic background contribute to these differences. Given that many clinical trials using newly discovered molecular inhibitors are still ongoing, these agents should become the new therapeutic options for hedgehog pathway‐dependent tumors in patients with or without Gorlin syndrome.

Frameshift mutation in the PTCH2 gene can cause nevoid basal cell carcinoma syndrome

Nevoid basal cell carcinoma syndrome (NBCCS) is an autosomal dominant disorder characterized by developmental defects and tumorigenesis. The gene responsible for NBCCS is PTCH1, encoding a receptor for the secreted protein, sonic hedgehog. Recently, a Chinese family with NBCCS carrying a missense mutation in PTCH2, a close homolog of PTCH1, was reported. However, the pathological significance of missense mutations should be discussed cautiously. Here, we report a 13-year-old girl diagnosed with NBCCS based on multiple keratocystic odontogenic tumors and rib anomalies carrying a frameshift mutation in the PTCH2 gene (c.1172_1173delCT). Considering the deleterious nature of the frameshift mutation, our study further confirmed a causative role for the PTCH2 mutation in NBCCS. The absence of typical phenotypes in this case such as palmar/plantar pits, macrocephaly, falx calcification, hypertelorism and coarse face, together with previously reported cases, suggested that individuals with NBCCS carrying a PTCH2 mutation may have a milder phenotype than those with a PTCH1 mutation.

Downregulation of microRNA-431 by human interferon-β inhibits viability of medulloblastoma and glioblastoma cells via upregulation of SOCS6

miRNAs are small non-coding RNAs that inhibit gene expression by cleaving or hindering the translation of target mRNAs. In this study, we focused on miR-431, which mediated inhibition of cell viability by human interferon-β (HuIFN-β). We aimed to demonstrate an antineoplastic effect of HuIFN-β via miR-431 expression against medulloblastoma and glioblastoma, because HuIFN-β is frequently used in adjuvant therapy of these tumors. Addition of HuIFN-β to medulloblastoma and glioblastoma cells reduced viability, significantly decreased miR-431 expression, upregulated expression of SOCS6 (putative miR-431 target genes) and inhibited Janus kinase (JAK) 1 and signal transducer and activator of transcription (STAT) 2. The mitogen-activated protein kinase (MAPK) pathway, but not the phosphoinositide 3-kinase (PI3K)-Akt pathway, was downregulated in medulloblastoma cells, whereas the PI3K-Akt pathway, but not the MAPK pathway, was downregulated in glioblastoma cells. Addition of HuIFN-β and transient transfection with miR-431 to medulloblastoma and glioblastoma cells did not reduce viability, downregulated expression of SOCS6, and concomitantly activated the JAK1 and STAT2. We propose that, in medulloblastoma and glioblastoma cells, HuIFN-β decreases miR-431 expression and upregulates SOCS6 expression, and consequently inhibit cell proliferation by suppressing the JAK-STAT signaling pathway.

Entire PTCH1 deletion is a common event in point mutation-negative cases with nevoid basal cell carcinoma syndrome in Japan.

To the Editor : Nevoid basal cell carcinoma syndrome [NBCCS (OMIM 109400)], also called Gorlin syndrome, is an autosomal dominant neurocutaneous disorder characterized by large body size, developmental and skeletal abnormalities, sensitivity to radiation, and an increased incidence of cancers such as basal cell carcinoma (BCC) and medulloblastoma (1). NBCCS is caused by inactivating mutations in the Patched-1 (PTCH1 ) gene (2, 3). Heterozygous loss of PTCH1 found in certain sporadic and familial cases of BCC indicates that PTCH1 is also a tumor suppressor gene (4, 5). Despite extensive efforts to detect mutations, they are still unidentified in 25–60% of patients (6–8). To date, we have analyzed 38 patients with NBCCS from 32 families. Eight of the families did not harbor any PTCH1 mutations detectable by polymerase chain reaction (PCR)based direct sequencing of the exons. To investigate the possibility of large deletions involving the PTCH1 gene, we employed a high-resolution array-based comparative genomic hybridization technology. Consequently, we identified genomic deletions involving PTCH1 in seven individuals from five of the eight point mutation-negative families (Fig. S1). These patients are listed in Table 1. Some of them have been reported previously by us (9) and one patient reported by others (NS6) (10) is also included in the table, all of which are of Japanese origin. To our knowledge, this table includes all the patients with PTCH1 deletions in which the breakpoints have been identified at the nucleotide level. A schematic representation of each deletion’s size together with the deleted genes is shown in Fig. 1a. Unlike in cases of Sotos syndrome and neurofibromatosis type 1, no recurrent breakpoints were observed in these patients (11, 12). Whereas deletions larger than 2.4 Mb were generated by non-homologous end joining, smaller ones (less than 1.2 Mb) were produced by Alu-mediated nonallelic homologous recombination (Fig. S2). G19 and G36 inherited the deletion from their mothers (G27 and G43, respectively), whereas the deletion in NS6 is of paternal origin. The breakpoint sequences in these cases were completely conserved through generations. Other patients (G35, G10 and G5) did not have a family history of NBCCS and, therefore, the deletions seemed to be de novo. Patients harboring deletions of less than 2.4 Mb did not exhibit any phenotypes atypical for NBCCS despite that up to 22 RefSeq genes (four disease genes) were included in the deleted region. This implies that hemizygous loss of these genes, except for PTCH1, might not have an influence on any observable phenotypes. In contrast, deletions larger than 5.3 Mb led to phenotypes unusual for NBCCS including severe mental and motor retardation, epilepsy, and hypotonia (Table 1). Interestingly, each Alu-mediated deletion was mediated by a distinct path of rearrangement (Fig. 1b). G36/43 had a crossing over point within the Alu elements generating a hybrid Alu element. In G19/27, however, the crossing over occurred near the poly-A tail of the proximal Alu element (9). Therefore, the proximal Alu remained intact while the distal Alu was deleted. In the third case, NS6, crossing over occurred at the 5′ end of the Alu elements and removed both Alu sequences leaving two short direct repeats flanking an Alu element on both sides called target-site duplications (10). To date, we have analyzed 32 NBCCS families and identified entire deletions of PTCH1 in 5 families. This implies that 16% of NBCCS families (five of the eight point mutation-negative families) can be explained by the entire loss of PTCH1. Mutations are not observed in the PTCH1 -coding sequences in considerable numbers of NBCCS cases not only in Japanese but also in other ethnicities and, apart from PTCH1, only one PTCH2 and one SUFU mutation in NBCCS have been

Multiple keratocystic odontogenic tumors associated with nevoid basal cell carcinoma syndrome having distinct PTCH1 mutations: a case report

Nevoid basal cell carcinoma syndrome (NBCCS) is a rare autosomal dominant disorder characterized by developmental abnormalities and a predisposition to cancers. Although multiple jaw tumors, such as keratocystic odontogenic tumors (KCOTs), are one of the most frequent complications in NBCCS, the molecular mechanism for how KCOTs develop in NBCCS is poorly understood. A 15-year-old girl with 2 jaw tumors was diagnosed as NBCCS according to the clinical criteria. The pathologic findings indicated that the 2 tumors were consistent with KCOTs. A PTCH1 mutation, c.1472delT, was detected in her peripheral blood as well as in the 2 tumors. Interestingly, an additional PTCH1 mutation, c.264_265insAATA, that was not present in the peripheral blood, was found in the maxillary tumor but not the mandibular tumor. The Ki-67 labeling index was significantly higher in the maxillary KCOT (17.7%) than in the mandibular KCOT (14.3%). These findings indicate distinct molecular mechanisms of tumorigenesis in these KCOTs.

Molecular pathogenesis of keratocystic odontogenic tumors developing in nevoid basal cell carcinoma syndrome.

OBJECTIVEnThe aim of this study was to investigate the molecular pathogenesis of keratocystic odontogenic tumors (KCOTs) that developed in nevoid basal cell carcinoma syndrome (NBCCS) patients.nnnSTUDY DESIGNnWe analyzed germline and somatic mutations of the PTCH1 and its related genes, SMO and SUFU in 10 KCOTs that developed in 8 unrelated NBCCS patients. Methylation status of the PTCH1 promoter was also investigated by bisulfite sequencing.nnnRESULTSnSomatic mutations of PTCH1 were detected in 3 KCOTs. Two out of 3 somatic mutations were either identified as a polymorphism or located on the same allele as the germline mutation. Neither abnormal methylation of the PTCH1 promoter, loss of PTCH1, nor somatic mutation of SMO or SUFU was detected in any of the samples.nnnCONCLUSIONSnOur results suggest that the tumorigenesis of most KCOTs associated with NBCCS cannot be explained by the classical 2-hit theory.

Selective haploinsufficiency of longer isoforms of PTCH1 protein can cause nevoid basal cell carcinoma syndrome

Nevoid basal cell carcinoma syndrome (NBCCS) is an autosomal dominant disorder characterized by developmental defects and tumorigenesis. The gene responsible for NBCCS is PTCH1. The PTCH1 gene has five alternatively used first exons resulting in the translation of three isoforms of the PTCH1 protein; that is, PTCHL, PTCHM and PTCHS. However, the biological significance of each isoform is unclear. Here we show an individual with NBCCS carrying a nonsense mutation in PTCH1 exon2, c.387G>A (p.W129X). As the mutation lay upstream of the ATG codon used for PTCHS translation, the mutant allele still expressed RNA isoforms that encode PTCHS. These results clearly demonstrate that a selective haploinsufficiency of longer isoforms of the PTCH1 protein, PTCHL and PTCHM, but not PTCHS is sufficient to cause NBCCS. Although mice selectively deficient in PTCHS isoforms are currently unavailable, this study sheds light on the complex in vivo roles of PTCH1 isoforms.