PTCH mutations in squamous cell carcinoma of the skin.

Ultraviolet light exposure is the major risk factor for the development of squamous cell carcinoma in Caucasians. Mutations in the tumor suppressor gene p53 have been identified in both squamous cell carcinomas and basal cell carcinomas. The human homolog of the Drosophila patched gene, has been shown to be mutated in sporadic basal cell carcinomas; however, mutations in the patched gene have not been found in squamous cell carcinoma. In this study, we screened a total of 20 squamous cell carcinoma samples for mutations in the patched gene. Using polymerase chain reaction-single strand conformation polymorphism as an initial screening method, we identified one non-sense mutation, two mis-sense mutations and three silent mutations in five squamous cell carcinoma samples. In one squamous cell carcinoma sample, we identified a tandem GG-->AA transitional change at nucleotide 3152 in exon 18 of the patched gene that resulted in a premature stop codon at codon 1051. The three squamous cell carcinoma samples containing non-sense and mis-sense mutations were isolated from individuals with histories of multiple basal cell carcinoma. Sequence analysis of the p53 gene in these five squamous cell carcinoma samples identified one CC-->TT and three C-->T ultraviolet-specific nucleotide changes. Our study provides evidence that the patched gene is mutated in squamous cell carcinoma from individuals with a history of multiple basal cell carcinoma. The identification of ultraviolet-specific nucleotide changes in both tumor suppressor genes supports the notion that ultraviolet exposure plays an important part in the development of squamous cell carcinoma.

Identification of a PTEN mutation in a family with Cowden syndrome and Bannayan-Zonana syndrome.

Cowden syndrome (CS) and Bannayan-Zonana syndrome (BZS) are two inherited hamartoma syndromes characterized by distinct phenotypic features. Mutations in the PTEN gene have been identified in patients with CS and BZS, suggesting the presence of a common genetic basis. We describe a single kindred with individuals manifesting both CS and BZS phenotypes (CS/BZS overlap family) in which we have identified a novel mutation in PTEN by DNA sequencing. We have confirmed these results by means of restriction enzyme analysis. The presence of individuals with CS and BZS within the same family, and moreover the identification of identical PTEN gene mutations in these individuals, suggest that these two syndromes represent different phenotypic expressions of one disease. Furthermore, these findings imply that, like patients with CS, individuals with BZS should be monitored for the onset of malignancy.

UV-specific p53 and PTCH mutations in sporadic basal cell carcinoma of sun-exposed skin.

UVB irradiation is known to produce DNA damage at mutation hotspots in the p53 tumor suppressor gene, leading to the development of skin cancers. Mutations in the PTCH tumor suppressor gene, which is known to be responsible for the development of nevoid basal cell carcinoma syndrome, have also been identified in sporadic basal cell carcinomas (BCCs). We describe the case of an 80-year-old welder in whom 3 novel p53 mutations, as well as UV-specific PTCH mutations, were detected in two BCC samples from sun-exposed skin. The simultaneous presence of UV-specific p53 and PTCH mutations in the same BCC sample has not previously been reported.

Role of PTCH and p53 genes in early-onset basal cell carcinoma.

Basal cell carcinoma (BCC) is the most common skin cancer in the Western world. Ultraviolet (UV) exposure, race, age, gender, and decreased DNA repair capacity are known risk factors for the development of BCC. Of these, UVB irradiation from sunlight is the most significant risk factor. The incidence of sporadic BCC increases in individuals older than age 55, with the greatest incidence reported in individuals who are older than 70, and is rare in individuals who are younger than 30. In this study, we analyzed 24 BCC samples from individuals who had BCC diagnosed by the age of 30. Fifteen single-stranded conformation polymorphism variants in the PTCH gene were identified in 13 BCC samples. Sequence analysis of these single-stranded conformation polymorphism variants revealed 13 single nucleotide changes, one AT insertion, and one 15-bp deletion. Most of these nucleotide changes (nine of 15) were predicted to result in truncated PTCH proteins. Fifteen p53 mutations were also found in 11 of the 24 BCC samples. Thirty-three percent (five of 15) and 60% (nine of 15) of the nucleotide changes in the PTCH and p53 genes, respectively, were UV-specific C-->T and CC-->TT nucleotide changes. Our data demonstrate that the p53 and PTCH genes are both implicated in the development of early-onset BCC. The identification of UV-specific nucleotide changes in both tumor suppressor genes suggests that UV exposure is an important risk factor in early onset of BCC.

Association of splicing defects in PTEN leading to exon skipping or partial intron retention in Cowden syndrome.

Cowden syndrome (CS) and Bannayan Zonana syndrome (BZS) are two autosomal dominantly inherited conditions characterized by hamartomas. Mutations in PTEN, a tumor suppressor gene located on chromosome 10q23, have been identified in patients with phenotypic findings of both CS and BZS. These mutations are found throughout the entire gene, with exon 5 being the most common site, and include point mutations, insertions and deletions. To date, 11 point mutations at the splice junctions of the PTEN gene have been reported, however, data on the alterations in the transcripts have been lacking. In this study, we have identified three novel splice site mutations in PTEN, in two families with CS and in one individual with BZS. One mutation affected the splice-acceptor site, which resulted in out-of-frame skipping of an entire exon. By contrast, the other two mutations affected the splice-donor sites, and both showed inclusion of partial intronic sequences in the transcript due to activation of cryptic splice sites. These data demonstrate mRNA alterations as a consequence of splice site mutations in the PTEN gene.

Identification of PTEN mutations in metastatic melanoma specimens.

CONTEXT: PTEN, a tumour suppressor gene located on chromosome 10q23, develops somatic mutations in various tumours and tumour cell lines including brain, endometrium, prostate, breast, kidney, thyroid, liver, and melanoma. OBJECTIVES: To investigate the mutational profile of this gene further, as well as its role in tumour progression in melanoma. DESIGN, SETTINGS: We examined 21 metastatic melanoma samples for 10q23 allelic losses and PTEN sequence alterations. Additionally, we screened these samples for mutations in CDKN2A, a gene in which alterations are well documented in primary melanoma as well as in the germline of familial melanoma. RESULTS: Loss of heterozygosity (LOH) at 10q23 was observed in 33% (7/21) of the samples tested. We identified four sequence alterations in PTEN (19%) and two in CDKN2A (9.5%). Of interest, only one case showed mutations in both genes. CONCLUSIONS: These data support the notion that PTEN alterations occur in some metastatic melanomas, and that mutation of this gene plays a role in the progression of some forms of melanoma.

Expression of senescence-associated beta-galactosidase in enlarged prostates from men with benign prostatic hyperplasia.

OBJECTIVES: Cellular senescence is a unique cellular response pathway thought to be closely associated with the aging process. The senescent phenotype is characterized by the loss of a cell's ability to respond to proliferative and apoptotic stimuli even while normal metabolic activity and vitality is maintained. Recently, a novel biomarker, senescent-associated beta-galactosidase (SA-beta-gal), was found to identify cells with the senescent phenotype. In the present study, we examined whether human prostatic epithelial cells adopt a senescence-associated phenotype after prolonged culture and analyzed a series of human benign prostatic hyperplasia (BPH) specimens to determine whether the cellular senescence process might be a factor in the development of BPH. METHODS: A primary culture of epithelial cells was established from the normal tissue of the peripheral zone of a radical prostatectomy specimen and was serially passaged until senescence. Forty-three human prostate specimens were obtained subsequent to radical prostatectomy or transrectal ultrasound-guided biopsy. The cultured cells and tissue specimens were histochemically stained to reveal the expression of SA-beta-gal, the cellular senescence biomarker. RESULTS: As has been reported for other types of cultured cells, human prostatic epithelial cells demonstrated widespread expression of the cellular senescence marker, SA-beta-gal, on prolonged culture. In our survey of hypertrophied human prostate tissues, 17 specimens (40%) of the 43 analyzed demonstrated positive staining for SA-beta-gal. In these tissues, SA-beta-gal expression was noted only in the epithelial cells. No statistical correlation (P = 0.42) between the chronologic age of the patient donor and SA-beta-gal expression was found. However, a high prostate weight (greater than 55 g) was found to correlate strongly with the expression of the SA-beta-gal biomarker (P = 0. 0001). CONCLUSIONS: Cultured prostatic epithelial cells expressed SA-beta-gal on reaching replicative senescence in vitro. The survey of human BPH specimens for the senescent marker showed that prostatic epithelial cells in patients with BPH with more advanced enlargement of the prostate (greater than 55 g prostate weight) expressed SA-beta-gal, and the prostates from patients with BPH that weighed less than 55 g tended to lack senescent epithelial cells. On the basis of these results, we propose that the accumulation of senescent epithelial cells may play a role in the development of the prostatic enlargement associated with BPH.

Germline PTEN mutations in three families with Cowden syndrome.

Cowden syndrome (CS) is an autosomal dominant inherited disorder characterized by hamartomas in a variety of tissues including the skin, thyroid, breast, endometrium, and the brain. Individuals with CS are predisposed to development of malignancy in these organs, especially the breast and the thyroid. We describe 3 unrelated individuals with CS associated with germline PTEN mutations. While the frameshift (375insTTTA) and the missense (Gly69Arg) mutations reported herein are novel in CS, the nonsense (Arg130stop) mutation has been described in 2 families with CS and in a single family exhibiting both CS and Bannayan Zonana phenotype.

Mutation report: identification of a germline mutation in keratin 17 in a family with pachyonychia congenita type 2.

Pachyonychia congenita type 2 (PC-2), also known as Jackson-Lawler type PC, is an autosomal dominant disorder characterized by hypertrophic nail dystrophy associated with focal keratoderma and multiple pilosebaceous cysts. It has been demonstrated that PC-2 is associated with germline mutations in the keratin 17 (K17) gene and in its expression partner keratin 6b. In this report, we describe a novel germline mutation in K17, M88T, in a family with PC-2.

Absence of PTEN germ-line mutations in men with a potential inherited predisposition to prostate cancer.

Epidemiological studies have demonstrated that men with a family history of prostate cancer are at an increased risk for this disease. This important observation has led a number of research teams, including our own, to collect DNA samples and clinical data from prostate cancer families, with the goal of localizing and characterizing prostate cancer susceptibility genes. The candidate tumor suppressor gene PTEN (also called MMAC1) has recently been shown to be somatically altered in several common malignancies, including cancers of the brain, kidney, skin, thyroid, endometrium, breast, and prostate. Germ-line mutations in this gene, which maps to chromosome 10q23, have been associated with Cowden disease, an autosomal dominant cancer predisposition syndrome that is characterized by multiple hamartomas. Although prostate cancer is not typically associated with Cowden disease, previous studies of sporadic prostate cancers demonstrate loss of heterozygosity at 10q23 loci in approximately 25% of cases. We, therefore, hypothesized that germ-line mutations in the PTEN gene may predispose to prostate cancer in a subset of families, particularly those in which cancers of the breast, kidney, and/or thyroid also segregate. To test this hypothesis, DNA was isolated from whole blood of 11 prostate cancer patients from 10 unrelated families. Four of the 10 families met the previously established clinical criteria for hereditary prostate cancer. Eight of the II men had at least one second primary malignancy, including cases of neuroendocrine cancer, glioblastoma multiforme, melanoma, kidney, and thyroid cancer. Although we identified some common as well as some unique polymorphisms, no nonsense or missense mutations were identified in any of the 11 samples. To further examine the possibility that PTEN mutations contribute to prostate cancer predisposition, we also studied the probands from each of 10 families with early-onset and/or multiple individuals with prostate cancer. Sequence analysis of the PTEN gene in these 10 men also revealed no mutations or novel polymorphisms. We conclude that germ-line mutations in the PTEN are unlikely to contribute in a significant way to the inherited predisposition to prostate cancer.

Phenotypic findings of Cowden syndrome and Bannayan-Zonana syndrome in a family associated with a single germline mutation in PTEN.

Cowden syndrome (CS) and Bannayan-Zonana syndrome (BZS) are two hamartoma syndromes with distinct phenotypic features. Although partial clinical overlap exists between CS and BZS, they are considered to be separate entities. PTEN has been identified as the susceptibility gene for both disorders, suggesting allelism. We have identified a germline mutation, R335X, in PTEN in a family consisting of two female members with the phenotypic findings of CS and two male members with the phenotypic findings of BZS. To our knowledge, this is the first report that shows the presence of separate subjects with CS and with BZS in a single family associated with a single germline PTEN mutation.

Identification of PTEN mutations in five families with Bannayan-Zonana syndrome.

Germline mutations in PTEN, a putative tumor suppressor gene, has been identified in 2 autosomal dominant inherited hamartoma syndromes, Cowden syndrome (CS) and Bannayan-Zonana syndrome (BZS). While both diseases exhibit distinct phenotypic features, there seems to be a partial clinical overlap between the 2 diseases. To date, 9 families with BZS have been screened for PTEN mutations, of which 5 were found to exhibit mutations in this gene. We report 5 novel germline mutations in the PTEN coding sequence from 5 unrelated families with the BZS phenotype. While all the mutations we identified are novel in BZS, 1003C-->T (nonsense mutation) and 209+5G-->A (putative splice site mutation) have been previously reported in unrelated families with CS and Lhermitte Duclos disease. Interestingly, 1 of the families has an individual with BZS and 1 with CS phenotype, associated with a single PTEN mutation, 885insA. These data support the notion that CS and BZS may be within the spectrum of the same primary disorder.

PTEN/MMAC1 mutations in hepatocellular carcinomas.

Mutations in the PTEN/MMAC1 gene have been identified in several types of human cancers and cancer cell lines, including brain, endometrial, prostate, breast, thyroid, and melanoma. In this study, we screened a total of 96 hepatocellular carcinoma (HCC) samples from Taiwan, where HCC is the leading cancer in males and third leading cancer in females, for mutations in the PTEN/MMAC1 gene. Complete sequence analysis of these samples demonstrated a missense mutation in exon 5 (K144I) and exon 7 (V255A) from HCC samples B6-21 and B6-2, respectively. A putative splice site mutation was also detected in intron 3 from sample B6-2. Both B6-21 and B6-2 were previously shown to contain missense mutations in the coding sequences of the p53 gene. Functional studies with the two missense mutations demonstrated that while mutation V255A in exon 7 resulted in a loss of phosphatase activity, mutation K144I in exon 5 retained its phosphatase activity. Additionally, we identified a silent mutation (P96P) in exon 5 of the PTEN/MMAC1 gene from HCC sample B6-22. These data provide the first evidence that the PTEN/MMAC1 gene is mutated in a subset of HCC samples.

Severe Lhermitte-Duclos disease with unique germline mutation of PTEN.

Germline mutations in the PTEN gene have recently been identified in some individuals with Cowden disease (CD), Lhermitte-Duclos disease (LDD), and Bannayan-Zonana syndrome. We report on a patient with CD and LDD in whom a unique de novo germline missense mutation is present in the PTEN gene. Direct sequence analysis detected a transitional change (T-->C) at nucleotide 335, resulting in substitution of the amino acid proline for leucine. The mutation is in exon 5, which has been proposed as a "hot-spot" for germline mutations. Comparison of this patient's clinical course with the previously reported cases of CD and LDD shows more extensive and more severe clinical findings than reported previously. Findings in this patient contribute to the current understanding of germline PTEN mutations and clinical outcome.

Similarities of cutaneous and breast pathology in Cowden's Syndrome.

Cowden's Syndrome (CS), or multiple hamartoma syndrome, is an autosomal dominant disorder characterized by mucocutaneous lesions, multiple benign tumors of internal organs and an increased risk of breast cancer. Here, we describe and illustrate in detail the benign breast pathology of 59 breast cases from 19 women with CS. Fibrosis is a significant characteristic of the breasts of patients with CS. Fibroadenomas appear to hyalinize at an early age and are frequently complex. The specimens demonstrate a spectrum of dense hyalinization of both the lobule and the stroma, and hyaline nodules appear to be the most characteristic lesion. This hyalinization process shares striking similarities with keloids, as well as the sclerotic nodules seen in the skin of CS individuals. Ductal carcinoma in CS was common, and it appeared to be associated with stromal hyalinization. Other frequently found benign features are adenosis and cysts. Of interest, the features of the benign breast disease in CS show certain similarities with senescent lobules, fibrous mastopathy of diabetes mellitus, and mammary hamartomas. These observations provide a framework for pathologists to identify this underrecognized syndrome.

Repression of transactivation of the retinoic acid receptor beta2 promoter in human breast cancer cells.

In previous studies, we have shown that the expression of retinoic acid receptor beta2 (RARbeta2) is altered in certain breast cancer cell lines. To investigate the mechanism responsible for this change, we studied in detail the RARbeta2 promoter in cell lines which demonstrated altered expression and compared these results to cell lines in which RARbeta2 was expressed normally. Direct DNA sequencing failed to identify alterations in the sequences of the known response elements in the cell lines manifesting altered expression patterns. By contrast, electrophoretic mobility shift studies of the proteins binding to these response elements demonstrated striking differences in the cells in which expression was altered, when compared to patterns seen in normal cells. Moreover, transient transfection studies using constructs of the RARbeta2 promoter demonstrated an absence of transactivation in the lines in which the expression of this gene was altered. These data suggest that the mechanism responsible for loss of induction of RARbeta2 in breast tumor cells is, at least in part, transcriptional repression.

The genetic basis of Cowden's syndrome: three novel mutations in PTEN/MMAC1/TEP1.

Cowden's syndrome (CS) is an autosomal dominant disorder associated with an increased risk of developing benign and malignant tumors in a variety of tissues, including the skin, thyroid, breast and brain. Women with CS are felt to have an increased risk of developing breast cancer, and virtually all women with CS develop bilateral fibrocystic disease of the breast. Recently, a series of germline mutations have been identified from CS families in a gene known as PTEN/MMAC1/TEP1. In this study, we used heteroduplex analysis and direct sequencing analysis and identified three novel germline mutations in the PTEN/MMAC1/TEP1 coding sequence from unrelated individuals with CS. We report a de novo transition (T-->C) at nucleotide 335 in exon 5. This missense mutation resulted in a leucine to proline (CTA to CCA) change at codon 112. We also describe a novel splice site mutation (801+2T-->G) in intron 7 that caused exon skipping in PTEN/MMAC1/TEP1 mRNA. The third mutation we report is a missense mutation, consisting of a transition (T-->C) at nucleotide 202 in exon 3, resulting in a tyrosine to histidine (TAC to CAC) change at codon 68. Finally, we also detected a rare polymorphism in exon 7 of the PTEN/MMAC1/TEP1 coding sequence. These data confirm the observation that mutations of the PTEN/MMAC1/TEP1 coding sequence are responsible for at least some cases of CS, and further define the spectrum of mutations in this autosomal dominant disorder.

Alopecia universalis associated with a mutation in the human hairless gene.

There are several forms of hereditary human hair loss, known collectively as alopecias, the molecular bases of which are entirely unknown. A kindred with a rare, recessively inherited type of alopecia universalis was used to search for a locus by homozygosity mapping, and linkage was established in a 6-centimorgan interval on chromosome 8p12 (the logarithm of the odds favoring linkage score was 6.19). The human homolog of a murine gene, hairless, was localized in this interval by radiation hybrid mapping, and a missense mutation was found in affected individuals. Human hairless encodes a putative single zinc finger transcription factor protein with restricted expression in the brain and skin.