Germline mutations of the PTEN gene in Cowden disease, an inherited breast and thyroid cancer syndrome.

Cowden disease (CD) is an autosomal dominant cancer predisposition syndrome associated with an elevated risk for tumours of the breast, thyroid and skin. Lhermitte-Duclos disease (LDD) cosegregates with a subset of CD families and is associated with macrocephaly, ataxia and dysplastic cerebellar gangliocytomatosis. The common feature of these diseases is a predisposition to hamartomas, benign tumours containing differentiated but disorganized cells indigenous to the tissue of origin. Linkage analysis has determined that a single locus within chromosome 10q23 is likely to be responsible for both of these diseases. A candidate tumour suppressor gene (PTEN) within this region is mutated in sporadic brain, breast and prostate cancer. Another group has independently isolated the same gene, termed MMAC1, and also found somatic mutations throughout the gene in advanced sporadic cancers. Mutational analysis of PTEN in CD kindreds has identified germline mutations in four of five families. We found nonsense and missense mutations that are predicted to disrupt the protein tyrosine/dual-specificity phosphatase domain of this gene. Thus, PTEN appears to behave as a tumour suppressor gene in the germline. Our data also imply that PTEN may play a role in organizing the relationship of different cell types within an organ during development.

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.

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.

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.

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.

Cultured human melanocytes express the intermediate filament vimentin.

Human melanocytes are neural crest-derived cells that synthesize the pigment melanin. These cells migrate from a central location to the dermal-epidermal junction early in gestation and situate themselves between keratinocytes of the basal layer of the epidermis, extending thin dendritic processes upwards into the epidermis. In vitro, neonatal melanocytes can assume a variety of morphologies, depending on the culture conditions. Using standard immunofluorescent, immunoblotting, and Northern blotting techniques, we investigated the expression of intermediate filament proteins and demonstrate here that cultured human melanocytes express vimentin gene and protein under a variety of culture conditions. Vimentin is a 57-kD intermediate filament protein synthesized primarily by cells of mesenchymal origin. It is transcribed as a single-messenger RNA species of 2.0 kb and the human gene is located on chromosome 10. As a member of the intermediate filament family of proteins, we suggest that vimentin is an important component of the cytoskeleton of neonatal, human melanocytes.

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.

Erythropoietic protoporphyria: four novel frameshift mutations in the ferrochelatase gene.

Human erythropoietic protoporphyria is an inherited disorder of the heme metabolic pathway caused by defects in the gene for ferrochelatase, the terminal enzyme of the pathway that catalyzes chelation of ferrous iron into protoporphyrin IX to form heme. Mutation analysis was performed for families with erythropoietic protoporphyria and four novel frameshift mutations were identified. Two of the mutations, 205insA and 215insT in exon 3 of the ferrochelatase gene, are single bp insertions. The other two, 400delA in exon 4 and 678delG in exon 6, are single bp deletions. All of the mutations result in premature termination codons downstream shortly after the mutation sites, and in one case the premature termination codon caused by 400delA was also shown to reduce mRNA level via nonsense-mediated mRNA decay.

Clonality of basal cell carcinoma--molecular analysis of an interesting case.

Tumor cells represent a single clone of cells that have undergone a series of mutations in genomic DNA. This process, known as clonal evolution, is a distinguishing feature of cancer. The human androgen receptor gene (HUMARA; GenBank) contains a highly polymorphic cytosine-adenine-guanine trinucleotide repeat that can be used to determine clonality by depicting X chromosome inactivation patterns. Random X chromosome inactivation is consistent with polyclonality; nonrandom X chromosome inactivation indicates a clonal population of cells. Basal cell carcinoma (BCC) demonstrates an atypical growth pattern in that it grows slowly, rarely metastasizes, and is rarely lethal. Whether this tumor results from the accumulation of mutations in a single cell with subsequent clonal expansion or reflects a polyclonal response by a group of cells to a growth stimulus is unknown. To provide further insight into the molecular events characterizing BCCs, we determined the clonal origin of five modular BCCs from a female patient by analyzing X chromosome inactivation patterns at the HUMARA locus. All tumors demonstrated a nonrandom pattern of X chromosome inactivation, consistent with monoclonal proliferation. These findings provide strong genetic evidence that sporadic BCCs develop by clonal evolution and support the contention that a series of mutations in a single cell is responsible for the altered growth state seen in these transformed epithelial cells.

Clonal origin of dermatofibrosarcoma protuberans.

Dermatofibrosarcoma protuberans (DFSP) is a malignant tumor originating in the dermis. Although it is known to be locally aggressive, it only rarely metastasizes and will recur unless completely excised. The exact cell responsible for the development of a DFSP has been a matter of controversy for several decades; however, most histochemical and electron microscopic studies support a fibroblastic origin, with the tumor cells staining uniformly for vimentin and containing active endoplasmic reticulum synthesizing collagen. Cytogenetic analysis of some of these tumors has demonstrated at least two specific chromosomal abnormalities in DFSP and suggested that this tumor may be polyclonal in origin. To further address the clonal origin of this locally invasive, mesenchymal tumor, we analyzed DNA from two female patients by restriction fragment length polymorphisms and methylation analysis. Our data strongly support the concept that DFSP is monoclonal in origin and that this tumor mass reflects the clonal expansion of a single cell.

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.

Interferon and the epidermis: implications for cellular senescence.

Human keratinocytes are epithelial cells that cover the external surface of the body and function primarily to protect against physical, chemical and biological injury. Since 1975, serial cultivation of disaggregated human keratinocytes in vitro has been possible and these cultures have been utilized to assess in vitro physiological age. As has been demonstrated in human dermal fibroblasts harvested from donors of different ages, human epidermal keratinocytes from older donors manifest a decreased proliferative response to serum and to selected mitogens when compared to younger donors. Recent work suggests an increased sensitivity to negative growth modulators as well. The epidermis constantly renews itself. In normal skin, the majority of the cells in the germinative, basal cell layer are blocked in Go and do not cycle unless stimulated. Interferons (IFN) are a family of glycoproteins well known for their antiviral activity and their ability to inhibit growth and alter the behavior of various normal and transformed cell types, both in vitro and in vivo. As such, we examined intact skin for the presence of negative modulators of growth and demonstrated the presence, by immunofluorescence and Western blotting, of a protein of approximate molecular weight 40 kilodaltons with polyclonal antiserum directed against recombinant IFN-alpha. Extracts from healthy, non-virally infected keratinocyte cultures contained IFN activity as determined by viral plaque inhibition assays. Further, we have demonstrated that IFN inhibits the growth of the human keratinocyte in a non-cytotoxic, reversible manner and that the keratinocytes harvested from older adult donors are significantly more sensitive to the growth inhibitory effects of IFN than are the keratinocytes of young donors.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Clinical and pathological features of breast disease in Cowden's syndrome: an underrecognized syndrome with an increased risk of breast cancer.

Cowden's syndrome (CS), or multiple hamartoma syndrome, is an autosomal dominant disorder associated with benign skin tumors and an increased risk of breast cancer. In an effort to understand the basic mechanisms regulating the development of breast cancer in this patient population, as well as to define diagnostic aspects of the disorder, we describe for the first time the clinical and pathological spectrum of breast disease in CS. We obtained the clinical histories and examined the histopathology of 59 cases from 19 women with CS sent to us from a variety of institutions. The 19 women showed a spectrum of benign histopathological findings, including ductal hyperplasia, intraductal papillomatosis, adenosis, lobular atrophy, fibroadenomas, and fibrocystic change. Seventeen (89%) showed features suggestive of a breast hamartoma. Fourteen women (74%) showed malignant disease, most of which was ductal carcinoma. Twelve patients (86%) showed ductal carcinoma in situ (DCIS), and 12 (86%) showed infiltrating ductal carcinoma. One patient had only DCIS and another patient showed both infiltrating tubular carcinoma and lobular carcinoma in situ. Ten patients (71%) actually showed foci of tumor involving densely fibrotic, hamartomatous areas. In summary, we show that women with CS have a spectrum of exuberant benign and malignant breast pathology. A common benign breast lesion in CS is a densely fibrotic hyalinized nodule, whereas the most frequent breast malignancy is ductal carcinoma.

Induction of c-fos but not c-myc in S-91 cells by melanization signals.

Synthesis of the pigment melanin is a complex differentiated function performed by pigment cells in response to a variety of stimuli. The possible roles of the proto-oncogenes c-fos and c-myc in the control of pigmentation were studied using subconfluent, actively proliferating Cloudman S-91 murine melanoma cells stimulated to synthesize melanin by melanocyte stimulating hormone (MSH) or forskolin. Stimulation caused a significant increase in melanin synthesis when compared to control cells, but had no effect on cell growth. Northern analysis of total cellular RNA demonstrated rapid, transient induction of c-fos mRNA as early as 30 min after stimulation with MSH or forskolin. In contrast, there was no effect on the high constitutive expression of c-myc in these actively proliferating cells. These data strongly suggest that the induction of c-fos mRNA is an early genetic event in stimulation of melanin synthesis and thus this proto-oncogene may play a major role in the regulation of this differentiated function, as reported for other forms of cellular differentiation. In contrast, c-myc expression is unaffected and instead correlated with cellular proliferative capacity. These results are consistent with the hypothesis that the down-regulation of c-myc frequently observed during cell differentiation is not a necessary event, but rather reflects an associated decrease in cell growth rate. The S-91 melanoma system appears to provide a convenient model for study of the regulation for a single, well defined differentiated function that is independent of growth rate.

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.

A diagnostic assay for the Wiskott-Aldrich syndrome and its variant forms.

BACKGROUND: The Wiskott-Aldrich syndrome (WAS) is an X-linked recessive disease characterized by severe thrombocytopenia, eczema, and impaired immunity. While the diagnosis is usually straightforward, the syndrome may be expressed in an attenuated form, a phenotype which is difficult to distinguish from other types of congenital thrombocytopenia. Although a molecular-based assay for diagnosis of the spectrum of WAS patients has not been available, recent data indicate that WAS is associated with a specific profile of impaired mitogen responsiveness and suggest that detection of this abnormality may provide a diagnostic marker for all forms of the disease. To address this issue, we have studied patients with classical and atypical WAS for their lymphocyte proliferative responses to four T cell mitogenic stimuli and compared their response patterns to those detected in unaffected children. METHODS: Clinical histories and informed consent were obtained from 23 patients with either classical or putative (ie, atypical) WAS, 16 subjects with other disorders, and 12 healthy children. Peripheral blood mononuclear cells (PBMCs) collected from patients and controls were resuspended in culture medium, stimulated with the T cell mitogens phytohemagglutinin (PHA), concanavalin A (Con A), neuraminidase/galactose oxidase (NAGO), or periodate, and cultured for 60 h in 0.2 mL aliquots. Following a 20 h pulse with 3H-thymidine, cultures were harvested and the 3H-thymidine uptake was evaluated by liquid scintillation counting. RESULTS: The most striking observation involved response to periodate. While lymphocytes from all healthy control children proliferated in response to periodate treatment, cells from both classical as well as atypical WAS patients consistently failed to proliferate in response to this mitogen. By contrast, lymphocyte proliferative responses to PHA, Con A, and NAGO were detected in all patients and controls, although responses generally were lower in cells from classical WAS patients compared to other children. In two WAS patients, bone marrow transplantation and clinical improvement were associated with a change from no periodate response (pre-transplant) to periodate responsiveness (post-transplant). In contrast to the WAS patients, cells from patients with other hematologic and primary immune deficiency diseases responded uniformly to all four mitogens, including periodate. CONCLUSIONS: The data presented here indicate that T cells from patients with either classical or attenuated WAS fail to undergo proliferation in response to periodate, an agent that induced extensive T cell mitogenesis of cells from all healthy controls as well as patients with diseases other than WAS. As the WAS patients' cells did proliferate in response to treatment with other T cell mitogens, it appears that periodate induced T cell proliferation is selectively impaired in WAS and that detection of this defect may be of value in the distinction of both classical and attenuated WAS from other thrombocytopenic conditions.

Cellular senescence: a reflection of normal growth control, differentiation, or aging?

Normal cells, with few exceptions, cannot proliferate indefinitely. Cell populations--in vivo and in culture--generally undergo only a limited number of doublings before proliferation invariably and irreversibly ceases. This process has been termed the finite lifespan phenotype or cellular senescence. There is long-standing, albeit indirect, evidence that cellular senescence plays an important role in complex biological processes as diverse as normal growth control, differentiation, development, aging, and tumorigenesis. In recent years, it has been possible to develop a molecular framework for understanding some of the fundamental features of cellular senescence. This framework derives primarily from the physiology, genetics, and molecular biology of cells undergoing senescence in culture. Our understanding of senescence, and the mechanisms that control it, is still in its infancy. Nonetheless, recent data raise some intriguing possibilities regarding potential molecular bases for the links between senescence in culture and normal and abnormal growth control, differentiation, and aging.