Irene M. Leigh

Germline mutations in FH predispose to dominantly inherited uterine fibroids, skin leiomyomata and papillary renal cell cancer.

Uterine leiomyomata (fibroids) are common and clinically important tumors, but little is known about their etiology and pathogenesis1,2,3. We previously mapped a gene that predisposes to multiple fibroids, cutaneous leiomyomata and renal cell carcinoma to chromosome 1q42.3–q43 (refs 4–6). Here we show, through a combination of mapping critical recombinants, identifying individuals with germline mutations and screening known and predicted transcripts, that this gene encodes fumarate hydratase, an enzyme of the tricarboxylic acid cycle. Leiomyomatosis-associated mutations are predicted to result in absent or truncated protein, or substitutions or deletions of highly conserved amino acids. Activity of fumarate hydratase is reduced in lymphoblastoid cells from individuals with leiomyomatosis. This enzyme acts as a tumor suppressor in familial leiomyomata, and its measured activity is very low or absent in tumors from individuals with leiomyomatosis. Mutations in FH also occur in the recessive condition fumarate hydratase deficiency7,8,9,10,11, and some parents of people with this condition are susceptible to leiomyomata. Thus, heterozygous and homozygous or compound heterozygous mutants have very different clinical phenotypes. Our results provide clues to the pathogenesis of fibroids and emphasize the importance of mutations of housekeeping and mitochondrial proteins in the pathogenesis of common types of tumor12,13,14.Uterine leiomyomata (fibroids) are common and clinically important tumors, but little is known about their etiology and pathogenesis. We previously mapped a gene that predisposes to multiple fibroids, cutaneous leiomyomata and renal cell carcinoma to chromosome 1q42.3–q43 (refs 4–6). Here we show, through a combination of mapping critical recombinants, identifying individuals with germline mutations and screening known and predicted transcripts, that this gene encodes fumarate hydratase, an enzyme of the tricarboxylic acid cycle. Leiomyomatosis-associated mutations are predicted to result in absent or truncated protein, or substitutions or deletions of highly conserved amino acids. Activity of fumarate hydratase is reduced in lymphoblastoid cells from individuals with leiomyomatosis. This enzyme acts as a tumor suppressor in familial leiomyomata, and its measured activity is very low or absent in tumors from individuals with leiomyomatosis. Mutations in FH also occur in the recessive condition fumarate hydratase deficiency, and some parents of people with this condition are susceptible to leiomyomata. Thus, heterozygous and homozygous or compound heterozygous mutants have very different clinical phenotypes. Our results provide clues to the pathogenesis of fibroids and emphasize the importance of mutations of housekeeping and mitochondrial proteins in the pathogenesis of common types of tumor.

Role of a p53 polymorphism in the development of human papillomavirus-associated cancer

The E6 oncoprotein derived from tumour-associated human papillomaviruses (HPVs) binds to and induces the degradation of the cellular tumour-suppressor protein p53. A common polymorphism that occurs in the p53 amino-acid sequence results in the presence of either a proline or an arginine at position 72. The effect of this polymorphism on the susceptibility of p53 to E6-mediated degradation has been investigated and the arginine form of p53 was found to be significantly more susceptible than the proline form. Moreover, allelic analysis of patients with HPV-associated tumours revealed a striking overrepresentation of homozygous arginine-72 p53 compared with the normal population, which indicated that individuals homozygous for arginine 72 are about seven times more susceptible to HPV-associated tumorigenesis than heterozygotes. The arginine-encoding allele therefore represents a significant risk factor in the development of HPV-associated cancers.

Identification of the familial cylindromatosis tumour-suppressor gene

Familial cylindromatosis is an autosomal dominant genetic predisposition to multiple tumours of the skin appendages. The susceptibility gene (CYLD) has previously been localized to chromosome 16q and has the genetic attributes of a tumour-suppressor gene (recessive oncogene). Here we have identified CYLD by detecting germline mutations in 21 cylindromatosis families and somatic mutations in 1 sporadic and 5 familial cylindromas. All mutations predict truncation or absence of the encoded protein. CYLD encodes three cytoskeletal-associated-protein–glycine-conserved (CAP–GLY) domains, which are found in proteins that coordinate the attachment of organelles to microtubules. CYLD also has sequence homology to the catalytic domain of ubiquitin carboxy-terminal hydrolases (UCH).

Human papillomavirus infection and non‐melanoma skin cancer in immunosuppressed and immunocompetent individuals

The role of human papillomavirus (HPV) in anogenital carcinogenesis is established firmly, but a similar role in non‐melanoma skin cancer remains speculative. Certain immunosuppressed individuals have an increased incidence of both viral warts and non‐melanoma skin cancer, that has prompted the suggestion that HPV may play a pathogenic role. Differences in the techniques used to detect HPV DNA in skin, however, have led to discrepancies in the prevalence and spectrum of HPV types reported in these malignancies. This study describes the use of a comprehensive degenerate PCR technique to compare the HPV status of 148 Non‐melanoma skin cancers from immunosuppressed and immunocompetent individuals. HPV DNA was detected in 37/44 (84.1%) squamous cell carcinomas, 18/24 (75%) basal cell carcinomas and 15/17 (88.2%) premalignant skin lesions from the immunosuppressed group compared with 6/22 (27.2%) squamous cell carcinomas, 11/30 (36.7%) basal cell carcinomas and 6/11 (54.4%) premalignancies in the immunocompetent group. Epidermodysplasia verruciformis HPV types prevailed in all lesion types from both groups of patients. In immunosuppressed individuals, cutaneous HPV types were also identified at high frequency, and co‐detection of multiple HPV types within single tumours was commonly observed. This study represents the largest and most comprehensive analysis of the HPV status of non‐melanoma skin cancers yet undertaken; whereas there are clearly significant differences in non‐melanoma skin cancers from immunosuppressed and immunocompetent populations, we provide evidence that the prevalence and spectrum of HPV types does not differ in squamous cell carcinomas, basal cell carcinomas or premalignancies within the two populations. These data have important implications for future investigation of the role of HPV in cutaneous carcinogenesis at a functional level. J. Med. Virol. 61:289–297, 2000.

Plectin deficiency results in muscular dystrophy with epidermolysis bullosa.

We report that mutation in the gene for plectin, a cytoskeleton–membrane anchorage protein, is a cause of autosomal recessive muscular dystrophy associated with skin blistering (epidermolysis bullosa simplex). The evidence comes from absence of plectin by antibody staining in affected individuals from four families, supportive genetic analysis (localization of the human plectin gene to chromosome 8q24.13–qter and evidence for disease segregation with markers in this region) and finally the identification of a homozygous frameshift mutation detected in plectin cDNA. Absence of the large multifunctional cytoskeleton protein plectin can simultaneously account for structural failure in both muscle and skin.

Mutations in the plakophilin 1 gene result in ectodermal dysplasia/skin fragility syndrome

Members of the armadillo protein gene family, which includes plakoglobin and β-catenin, have important functions in cytoskeleton/cell membrane interactions1,2. These proteins may act as linker molecules at adherens junctions and desmosomes at the plasma membrane3; in addition, they may have pivotal roles in signal transduction pathways and significant effects on cell behaviour during development4–7. Here, we describe the first human mutations in one of these dual function proteins, plakophilin 1 (band-6 protein; refs 8–10). The affected individual has a complete absence of immunostaining for plakophilin 1 in the skin and is a compound heterozygote for autosomal-recessively inherited premature termination codons of translation on both alleles of the plakophilin 1 gene (PKP1). Clinically, there are features of both cutaneous fragility and congenital ectodermal dysplasia affecting skin, hair and nails. There is no evidence of significant abnormalities in other epithelia or tissues. Desmosomes in the skin are small and poorly formed with widening of keratinocyte intercellular spaces and perturbed desmosome/keratin intermediate filament interactions. The molecular findings and clinical observations in this patient attest to the dual importance of plakophilin 1 in both cutaneous cell–cell adhesion and epidermal morphogenesis.

Aberrant integrin expression during epidermal wound healing and in psoriatic epidermis.

We have examined integrin expression during the remodeling of the epidermis that takes place during wound healing, using a suction blister model in which the epidermis is detached from the dermis, leaving the basement membrane intact. By immunofluorescence microscopy, we found that the same integrin subunits were expressed during wound healing as in normal epidermis with very little change in the relative intensity or distribution of staining at the leading edge of the migrating epidermis. However, at the time of wound closure, when the epidermis is still hyperproliferative, alpha 2, alpha 3, alpha 6, and beta 1 were no longer confined to the basal layer, as in normal epidermis, but were also found in all the living suprabasal cell layers, coexpressed with the terminal differentiation markers involucrin, keratin 10, and keratin 16. Strong suprabasal staining for alpha v was also found in one specimen. beta 4, which normally forms a heterodimer with alpha 6, and alpha 5 remained predominantly basal. Three of the integrin ligands, fibronectin, type IV collagen, and laminin, remained largely confined to the basement membrane zone and dermis. By 14 d after wounding, the integrins were once more restricted to the basal layer. Suprabasal integrin expression was also observed in involved psoriatic lesions. Thus, in two situations in which the epidermis is hyperproliferative, there is a failure to downregulate integrin expression on initiation of terminal differentiation. The functional consequences of this aberrant integrin expression remain to be explored.

Treatment of chronic venous ulcers with sheets of cultured allogenic keratinocytes

Cultured keratinocytes were used as allografts to treat 51 patients with chronic venous ulccration or rheumatoid ulcers unresponsive to all previous conventional treatments including split skin grafts. Although early epithelialization could be seen in the centre of some ulcers, a major effect appeared to be healing from the previously indolent edge. This treatment appears to provide some clinical benefit in healing of chronic ulceration.

Abnormal expression of wild type p53 protein in normal cells of a cancer family patient

Mutations in the p53 gene are the commonest specific genetic change in human cancer. In normal tissues, p53 protein is present in such low quantities that it is not readily detectable by immunochemical techniques. However, in many tumour cells large amounts of p53 protein accumulate and can be seen by simple immunohistochemical staining; this is generally attributed to the accumulation of stabilised, mutant protein. We have found a mother and daughter, who both have a history of breast cancer, who show strong immunohistochemical staining of p53 in most of their normal epithelial and mesenchymal cells. Their family has a history of multiple cancers developing at an early age. Detailed protein analysis and gene sequencing of material obtained from cultured cells, grown from a skin biopsy taken from the daughter, suggest that her cells contained large quantities of normal (unmutated) p53. We suggest that this phenotype defines a new inherited cancer susceptibility syndrome that is distinct from the germ-line mutations in p53 found in some Li-Fraumeni families. This new syndrome affects p53 tumour suppressor function through an indirect mechanism that stabilises normal p53. It remains to be established whether this mechanism also contributes to the accumulation of p53 in sporadic cancers.

Identification of an epidermal basement membrane defect in recessive forms of dystrophic epidermolysis bullosa by LH 7:2 monoclonal antibody: use in diagnosis

LH 7:2 is a monoclonal antibody that was raised against an extract of human epidermal cells and identifies an epitope within the lamina densa of the basement membrane of stratified squamous epithelia. Using indirect immunofluorescence we found intense labelling with LH 7:2 at the epidermal basement membrane (EBM) of normal skin, and in skin samples from patients with simplex, junctional, dominantly inherited dystrophic and acquired forms of epidermolysis bullosa (EB), as well as bullous pemphigoid. Staining was absent or only very faint in generalized recessive dystrophic EB (RDEB), and patchily reduced in the localized form of RDEB.