Mutation in exon 7 of PTCH deregulates SHH/PTCH/SMO signaling: possible linkage to WNT

The novel PTCH mutation and clinical manifestations within Gorlin syndrome family links PTCH haploinsufficiency and aberrant activation of the Wnt pathway. We report a family case with Gorlin syndrome, characterized by the usual phenotype features such as widespread basocellular tumors and craniofacial and bone malformations, but also including a less common appearance of craniopharyngioma. These clinical manifestations might be associated with a novel constitutional mutation of the PTCH gene, 1047insAGAA, which we found in exon 7. It changes the normal amino acid sequence leading to termination of the PTCH protein at exon 9. The analyzed tumors of the family show extensive loss of heterozygosity in the PTCH region, both basocellular and in particular craniopharyngioma, and in the latter a high expression of beta-catenin was detected. Our findings suggest involvement of the SHH/PTCH/SMO pathway in pathogenesis of the analyzed disorders, including its possible contribution to aberrant activation of the Wnt pathway in craniopharyngioma.     

Identification of PATCHED mutations in medulloblastomas by direct sequencing

Medulloblastoma is the most common malignant embryonic tumors of the central nervous system. The nevoid basal cell carcinoma syndrome (NBCCS), which is caused by mutations of PTCH gene on chromosome 9q22, accounts for about 2% of all medulloblastomas. Previous studies of PTCH in sporadic medulloblastomas using single strand conformational polymorphism (SSCP) detected mutations in about 10% of the tumors. In this study, we directly sequenced the PTCH gene in 20 sporadic medulloblastoma DNA samples. A nonsense mutation (Q694X) and a splice site alteration (2875+1G>A) were identified in two of the samples. The mutations are predicted to result in a truncated PTCH protein and aberrant splicing, respectively. In both cases, only the mutant alleles were identified, indicating that the mutations were associated with loss of the wild-type PTCH allele in the tumor cells. Several novel variants, including 1653T>C, 1672C>T, and 2292C>T, were also found in these tumor samples. One of the two mutations detected in this study had been missed by SSCP, suggesting that the true rate of PTCH mutations in sporadic medulloblastomas may be underestimated by SSCP screening. Nevertheless, the frequency of mutations in this study did not differ from previous reports.     

PTCH mutations are not mainly involved in the pathogenesis of sporadic trichoblastomas

Trichoblastomas are rare, benign tumors of the appendix in human skin. The histopathology comprises elements of basal cell carcinoma and trichoepithelioma with a variable degree of follicular differentiation. Both basal cell carcinoma and trichoepithelioma reveal alterations of PTCH, the human homolog of the Drosophila segment polarity patched gene. Furthermore, heterozygous PTCH knockout mice develop trichoblastoma-like tumors. This suggests an involvement of the PTCH gene in the pathogenesis of human trichoblastomas. However, trichoblastomas arising in nevus sebaceus did not show loss of heterozygosity at the PTCH locus (9q22.3) in a previous study. Sequencing of the PTCH gene and analysis of sporadic human trichoblastomas have not been performed yet. We therefore screened 10 sporadic trichoblastomas and 1 trichoblastoma arising within a nevus sebaceus for PTCH mutations. After microdissection of the tumors, single-strand conformational polymorphism (SSCP)/heteroduplex analysis of exons 2 to 23 of PTCH was performed, and polymerase chain reaction products with aberrant band patterns were sequenced. One trichoblastoma revealed a silent mutation at codon 562 in exon 12. Another trichoblastoma showed a somatic C > T single nucleotide substitution at codon 1,315 (exon 23), which was not present in corresponding normal epidermis. This mutation at codon 1,315 represents an already described PTCH germline polymorphism and results in a heterozygous Pro to Leu substitution in the tumor. The Pro/Leu polymorphism in germline is associated with a higher risk for breast cancer, but a potential contribution to the tumorigenesis of trichoblastoma is unknown. We detected no classical PTCH mutations in the investigated trichoblastomas. Our results indicate that PTCH mutations are not mainly involved in the pathogenesis of sporadic trichoblastomas, in contrast to basal cell carcinomas and trichoepitheliomas. The genetic basis of this rare appendageal tumor remains elusive.     

Fine mapping of the PTGFR gene to 1p31 region and mutation analysis in human breast cancer

The 1p31 chromosomal region shows loss of heterozygosity (LOH) in up to 50% of human breast cancer, indicating the presence of a tumor suppressor gene at this location. Many efforts have been made to identify candidate genes responsible for breast cancer on the short arm of chromosome 1. It was shown that prostaglandins have been implicated in the tumorigenesis pathway, perhaps via interactions with their cell surface receptors. The prostaglandin F2 receptor gene (PTGFR) was tentatively mapped to 1p31 adjacent to the region undergoing LOH in human breast cancer. We undertook a mutation study in 34 sporadic human breast tumors using a variant of SSCP, incorporation PCR SSCP (IPS). Several nucleotide variants were detected in different tumors. Here we report the nature of these nucleotide changes and the possible involvement of the PTGFR gene in the etiology of human cancer.     

Analysis of the PTCH1 signaling pathway in ovarian dermoids

Dermoids belong to the group of developmental cysts and arise from germ cells. Studies on these tumors may therefore increase our understanding of normal germ cell development within different environments and cell lines derived from these lesions may also constitute an important vehicle for studying neoplasia and differentiation. Recently, we investigated the status of the PTCH1 locus in a large set of sporadic non-inflammatory, developmental cystic lesions. Our data showed allelic loss of microsatellite markers in close vicinity to the PTCH1 locus in both odontogenic keratocysts and dentigerous cysts as well as in ovarian dermoid cysts (ODC). In this study, we closely examined the status of the PTCH1 gene in ODCs. Although about 25% of cysts demonstrated LOH at the PTCH1 locus, no nonsense or missense mutations in the coding region of PTCH1 were detected in genomic DNA isolated from any of the ODCs examined by direct sequencing. Staining with PTCH1 and GLI1 antibodies showed that proteins were present in virtually all epithelial linings, with variable staining intensity not correlated with LOH and generally weaker for GLI1. However, cDNA microarray analysis performed on cell lines derived from ODCs did not show any significant alteration in the expression of the analyzed target genes of PTCH1 signaling in any of the cell lines examined, except for CyclinD1 (and several other genes generally not associated with PTCH1 signaling).     

Is loss of heterozygosity at 9q22.3 (PTCH gene) and 19p13.3 (STK11 gene) involved in the pathogenesis of ovarian stromal tumors?

Some ovarian fibromas and rare fibrosarcomas are associated with Gorlin syndrome, which is caused by mutation in the human homologue of Drosophila patched gene (PTCH), localized on chromosome 9q22.3. The relationship between PTCH gene and sporadic ovarian tumors in the thecoma-fibroma group has not been well characterized. On the other hand, we have recently described loss of heterozygosity (LOH) at 19p13.3 in 2 sporadic fibromas with sex-cord elements. We have analyzed DNA from 8 fibromas, 6 cellular fibromas, 2 fibrothecomas, 9 luteinized thecomas, and 2 fibrosarcomas of the ovary for LOH at 9q22.3 and 19p13.3, using polymerase chain reaction amplification for 10 microsatellite markers. LOH at 9q22.3 was detected in 4 (67%) of 6 cellular fibromas, with the highest frequency at microsatellite marker D9S15, which localizes proximal to the PTCH gene. Of 9 luteinized thecomas, 2 (22%) also exhibited LOH at 9q22.3 with 3 microsatellite markers other than D9S15. Allelic losses were not detected in any fibroma, fibrothecoma, or fibrosarcoma. LOH at 19p13.3 was found in 2 (25%) of 8 fibromas, 3 (50%) of 6 cellular fibromas, and 1 (11%) of 9 luteinized thecomas. None of the 2 fibrothecomas or 2 fibrosarcomas showed LOH at 19p13.3. LOH at both 9p22.3 and 19p13.3 was observed in 3 (50%) of 6 cellular fibromas, but not in luteinized thecomas. The results indicate that (1) LOH at both PTCH gene and STK11 gene is relatively frequent in cellular fibromas; (2) approximately a quarter of luteinized thecomas exhibited LOH of the PTCH gene; in both neoplasms, cellular fibromas and luteinized thecomas, LOH may play a role in their pathogenesis; and (3) sporadic cellular fibromas may arise through similar genetic pathways as cases of Gorlin syndrome.     

The spectrum of patched mutations in a collection of Australian basal cell carcinomas

Inactivating mutations in the human patched (PTCH) gene have been identified in both familial and sporadic basal cell carcinomas (BCCs). In some tumors mutations have been detected in both alleles thereby supporting the role of PTCH as a tumor suppressor gene. We have analyzed 22/23 coding exons of PTCH for mutations in 44 sporadic BCCs, and detected 10 novel mutations in nine tumors. In two of the mutant tumors the remaining allele was inactivated by loss of heterozygosity. Five novel PTCH polymorphisms were also identified. Most of the variations found were C>T substitutions at dipyrimidine sites, supporting previous studies which indicate a role for ultraviolet-B in the genesis of sporadic BCCs.     

APC mutations in sporadic medulloblastomas

The cerebellar medulloblastoma (WHO Grade IV) is a highly malignant, invasive embryonal tumor with preferential manifestation in children. Several molecular alterations appear to be involved, including isochromosome 17q and the p53, PTCH, and beta-catenin gene mutations. In this study, 46 sporadic medulloblastomas were screened for the presence of mutations in genes of the Wnt signaling pathway (APC and beta-catenin). Single-strand conformational polymorphism (SSCP) analysis followed by direct DNA sequencing revealed 3 miscoding APC mutations in 2 (4.3%) medulloblastomas. One case contained a GCA-->GTA mutation at codon 1296 (Ala-->Val), and another case had double point mutations at codons 1472 (GTA-->ATA, Val-->Ile) and 1495 (AGT-->GGT, Ser-->Gly). Miscoding beta-catenin mutations were detected in 4 tumors (8.7%). Three of these were located at codon 33 (TCT -->TTT, Ser-->Phe) and another at codon 37 (TCT-->GCT, Ser-->Ala). Adenomatous polyposis coli (APC) gene and beta-catenin mutations were mutually exclusive and occurred in a total of 6 of 46 cases (13%). Although germline APC mutations are a well established cause of familial colon and brain tumors (Turcot syndrome), this study provides the first evidence that APC mutations are also operative in a subset of sporadic medulloblastomas.     

在乳腺癌干细胞中Hedgehog信号通路相关基因表达增强

目的 了解Hedgehog信号通路在乳腺癌发生发展中的作用.方法 用免疫磁珠法从无血清培养的乳腺癌悬浮细胞中分选CD44+CD24-细胞和非CD44+CD24-细胞,用real-time RT-PCR法检测Hedgehog信号通路主要分子$HH、PTCH1、SMO和GLI1 mRNA在细胞中的表达,用免疫组织化学法检测上述因子在乳腺癌组织中的表达.结果 分选出的CD44+CIDA-细胞约占乳腺癌悬浮细胞总数的8.25%,分选出的CD44+CD24-细胞表达干细胞标志蛋白ALDHA1和Oct-4;SHH、PTCH1、SMO和GLI1 mRNA在CD44+CD24-细胞中的表达均高于其在非CD44+CD24-细胞中的表达(P＜0.05);SMO和GLI1蛋白在三阴性乳腺癌的表达均高于非三阴性乳腺癌组织(P＜0.05).结论 在乳腺癌干细胞CD44+CD24-细胞中Hedgehog信号通路被激活,抑制癌症干细胞中Hedgehog通路的活化可能会降低或阻止乳腺癌的复发及化疗耐受.     

Alterations in the SMARCB1 (INI1) tumor suppressor gene in familial schwannomatosis

Schwannomatosis is a third major form of neurofibromatosis that has recently been linked to mutations in the SMARCB1 (hSnf5/INI1) tumor suppressor gene. We analyzed the coding region of SMARCB1 by direct sequencing and multiplex ligation-dependent probe amplification (MLPA) in genomic DNA from 19 schwannomatosis kindreds. Microsatellite markers in the SMARCB1 region were developed to determine loss of heterozygosity (LOH) in associated tumors. We detected four alterations in conserved splice acceptor or donor sequences of exons 3, 4 and 6. Two alterations that likely affect splicing were seen in introns 4 and 5. An additional four alterations of unclear pathogenicity were found to segregate on the affected allele in eight families including two non-conservative missense alterations in three families. No constitutional deletions or duplications were detected by MLPA. Nine of 13 tumors examined showed partial LOH of the SMARCB1 region consistent with 'second hits.' Alterations were detected in tumors both with and without somatic NF2 gene changes. These findings support the hypothesis that SMARCB1 is a tumor suppressor for schwannomas in the context of familial disease. Further work is needed to determine its role in other multiple and single tumor syndromes.     

Genomic structure of the human tetratricopeptide repeat-containing gene, TTC4, from chromosome region 1p31 and mutation analysis in breast cancers

Loss of heterozygosity (LOH) in 1p31 is a frequent genetic alteration in breast tumors indicating the site of a tumor suppressor gene. We recently isolated a new member of the human tetratricopeptide repeat-containing family of genes, TTC4, which maps to this region. Other members of this gene family have been implicated in tumorigenesis suggesting that TTC4 may represent a breast cancer tumor suppressor gene. We now report the exon/intron structure of TTC4 and single strand conformation polymorphism (SSCP) analysis of DNA from 20 sporadic breast tumors. Although polymorphic variations were identified no mutations affecting the open reading frame of TTC4 were detected. Since the overall region of chromosome 1p31 which undergoes LOH can be relatively large, excluding involvement of newly isolated genes from this region in breast cancer tumorigenesis is an important process for the successful identification of the critical gene. Understanding the structure of TTC4 now makes mutation analysis possible for other cancers and diseases that map to this region.     

Novel somatic mutations in the BRCA1 gene in sporadic breast tumors

Germline mutations in two major susceptibility genes BRCA1 and BRCA2 contribute to the majority of inherited breast and ovarian cancers. Besides the germline mutation, tumor progression depends on the loss of a wild-type allele. Allelic losses in the BRCA1 and BRCA2 loci have also been detected in a high proportion of sporadic breast tumors, suggesting the role of these genes in the development of non-inherited breast cancer. Forty unselected breast tumors were analyzed for the loss of heterozygosity (LOH) at BRCA1 and BRCA2 regions and tumors with allelic deletions were screened for the presence of acquired genetic alterations in respective genes. 21.1% of 38 informative tumor samples carried LOH at the BRCA1 locus whereas 33.3% of 39 informative samples showed LOH at the BRCA2 locus. Pathogenic truncating mutations in the BRCA1 gene were found in two tumor samples with allelic losses, whereas no mutations were identified in the BRCA2 gene. Mutations were not detected in non-tumor samples from the same individuals, which indicated that the BRCA1 allele was inactivated by somatic mutations in tumor tissue. The c.1116G>A (1235G>A) nonsense mutation (p.W372X) belongs to the genetic abnormalities detected infrequently in hereditary tumors; the c.3862delG (3981delG) frameshift mutation (p.E1288fsX1306) is a novel gene alteration. The occurrence of inactivating somatic mutations in sporadic breast tumors suggested the role of the BRCA1 gene in tumorigenesis in at least a minor group of patients with non-familial breast cancer.     

BCL10 is not a major target for frequent loss of 1p in testicular germ cell tumors

The deletion of chromosome 1p is one of the frequent genetic alterations found in testicular germ cell tumors (GCTs), suggesting the presence of a tumor suppressor gene. BCL10, which was identified as a gene altered in mucosa-associated lymphoid tissue lymphoma, has been mapped at 1p22. The gene has been reported to be mutated in a variety of human cancers. In this study, we investigated the allelic deletions on 1p and the mutation of BCL10 in 51 GCTs comprising 30 seminomas and 21 non-seminomatous germ cell tumors. Loss of heterozygosity (LOH) on 1p was tested using three microsatellite markers. The search for BCL10 mutations in each of the three exons was screened by a single-stranded conformation polymorphism (SSCP) analysis and samples with abnormal bandshifts were directly sequenced. LOH at at least one locus tested was found in 42% (21/49) of the tumors (43% of seminomas and 38% of NSGCTs). SSCP and direct sequence analyses revealed that there were single nucleotide polymorphisms at codon 5, 8, 162, and intron 1. However, there were no somatic mutations of BCL10 in the 51 tumors. In support of the previous studies, our results demonstrated that LOH on 1p is frequent in both seminomas and NSGCTs, indicating that there is an important tumor suppressor on 1p in GCT. However, the results indicate that BCL10 is not a candidate target gene of the 1p deletion.     

Allelic deletion and mutation of the von Hippel-Lindau (VHL) tumor suppressor gene in pancreatic microcystic adenomas.

An association between pancreatic microcystic (serous) adenomas (MCAs) and von Hippel-Lindau (VHL) disease has been suggested. However, genetic alterations of the VHL gene in MCAs of the pancreas have never been reported. In this study, we performed genetic analysis of 12 pancreatic MCAs. In 2 cases, VHL disease was documented clinically, and 10 cases were sporadic. For LOH analysis, tumor and normal pancreatic cells were procured from formalin-fixed, paraffin-embedded material using tissue microdissection. After DNA extraction, the samples were amplified by polymerase chain reaction using the polymorphic markers D3S2452, D3S1110, D3S192, and D3S656. In addition, the sporadic tumors were analyzed for VHL gene mutations using probes 3b/10b and K55/K56. Both MCAs associated with VHL disease showed LOH with at least one of the microsatellite markers tested. Among the 10 sporadic cases, 7 tumors showed LOH at the VHL gene locus. A somatic VHL gene mutation on exon 2 was documented in one sporadic case. The study provides the first direct genetic evidence for the role of the VHL gene in MCA tumorigenesis. Furthermore, VHL gene alterations may be detected in both VHL-associated and sporadic pancreatic MCAs.     

Loss of heterozygosity and DNA ploidy point to a diverging genetic mechanism in the origin of peripheral and central chondrosarcoma.

Chondrosarcomas are malignant cartilaginous tumors arising centrally in bone (central chondrosarcoma), or secondarily within the cartilaginous cap of a hereditary or sporadic exostosis (peripheral chondrosarcoma). Loss of heterozygosity (LOH) was studied by microsatellite analysis at the loci harboring the EXT genes (implicated in hereditary multiple exostoses), the EXT-like genes, and at 9p21, 13q14, 17p13, and chromosome 10. Nineteen of 20 peripheral chondrosarcomas showed LOH at all loci tested, while only 3 of 12 central chondrosarcomas exhibited LOH, restricted to 9p21, 10, 13q14, and 17p13. LOH at 9p21 did not appear to involve the CDKN2A gene, as assessed by SSCP analysis. DNA flow cytometry demonstrated a wide variation in the ploidy status in peripheral chondrosarcomas (DNA indexes, 0.56-2.01), whereas central chondrosarcomas were predominantly peridiploid. Near-haploidy found in peripheral chondrosarcomas could explain part of the high LOH percentages. Ki-67 immunohistochemistry suggested a higher proliferation rate in peripheral chondrosarcomas. Our results indicate that peripheral chondrosarcomas, arising secondarily to an exostosis, may obtain genetic alterations during malignant transformation, with subsequent genetic instability as demonstrated by a high percentage of LOH and a wide variation in ploidy status. In contrast, peridiploidy and a low percentage of LOH in central tumors suggest that a different oncogenic molecular mechanism may be operative.     

Analysis of the PTCH coding region in human rhabdomyosarcoma

Inherited mutations of the human tumor suppressor gene Patched (PTCH) lead to an autosomal dominant disorder known as Nevoid Basal Cell Carcinoma Syndrome (NBCCS). The syndrome is characterized by a combination of developmental abnormalities and a predisposition to tumor formation. Tumors in patients with NBCCS include basal cell carcinoma, medulloblastoma, fibroma and rhabdomyosarcoma (RMS). RMS are also present in 15 % of mice haplodeficient for Ptch. To investigate whether mutations in PTCH are a general feature in rhabdomyosarcomagenesis we sequenced the protein-coding region in sporadic human cases of these tumors. For this purpose we first determined the distribution and frequency of polymorphisms in 23 exons of PTCH in 48 healthy caucasians. Ten new polymorphisms were identified (IVS11 + 15-17del AAA; IVS14 + 25T>C; 2485G>A; IVS15 + 9G>C; IVS17 + 21A>G; 3033T>C; 3149T>C; 3387T>C; 3617G>A; 4080C>T). Next, the PTCH coding region in 14 RMS was sequenced. Whereas one case with LOH at the PTCH locus was detected, none of the cases showed nonsense or missense mutations in the coding region of PTCH. These data do not support the existence of frequent mutations in the protein-coding region of PTCH in RMS.