Identification of an Interstitial 18p11.32-p11.31 Duplication Including the EMILIN2 Gene in a Family with Porokeratosis of Mibelli

Porokeratosis is a rare disease of epidermal keratinization characterized by the histopathological feature of the cornoid lamella, a column of tightly fitted parakeratocytic cells, whose etiology is still unclear. Porokeratosis of Mibelli is a subtype of porokeratosis presenting a single plaque or a small number of plaques of variable size located unilaterally on limbs. It frequently appears in childhood and occurs with a higher incidence in males. Cytogenetic analyses were performed in all members of the family on lesioned and uninvolved skin. An array-CGH analysis was also performed utilizing the Human Genome CGH Microarray Kit G3 400 with 5.3 KB overall median probe spacing. Gene expression was performed on skin fibroblasts. In this study, we describe a Caucasian healthy 4-year-old child and his father showing features of porokeratosis of Mibelli. Array-CGH analysis revealed an interstitial 429.5 Kb duplication of chromosome 18p11.32-p11.3 containing four genes, namely: SMCHD1, EMILIN2, LPIN2, and MYOM1 both in patient and his father. EMILIN2 resulted overexpressed on skin fibroblasts. Also other members of this family, without evident signs of porokeratosis, carried the same duplication. Among these genes, we focused our attention on elastin microfibril interfacer 2 (EMILIN2) gene. Apoptosis plays a fundamental role in maintaining epidermal homeostasis, balancing keratinocytes proliferation, and forming the stratum corneum. EMILIN2 is known to trigger the apoptosis of different cell lines negatively affecting cell survival. It is expressed in the skin. We could speculate that the duplication and overexpression of EMILIN2 cause an abnormal apoptosis of epidermal keratinocytes and alter the process of keratinization, even if other epigenetic and genetic factors could also be involved. Our results could contribute to a better understanding of the pathogenesis of porokeratosis of Mibelli.     

Cytogenetically marked clones in human fibroblasts cultured from normal subjects.

Clones of cytogenetically abnormal cells have been recognized in fibroblasts cultured from normal human adult skin. No such clones have been observed in human embryo skin fibroblasts cultured in the same way. Although the culture conditions may have played some part in the emergence of these clones, it is possible that the abnormal cells from which the clones were derived were present in vivo.     

Investigation of the cytotoxic effects of DNA damaging agents on neurofibromatosis cells

Cells cultured from individuals with neurofibromatosis, a genetic syndrome associated with a predisposition to malignancy, were studied. We examined survival as measured by colony formation in skin fibroblasts from 5 patients with neurofibromatosis after exposure to X-rays, ultraviolet light and an alkylating agent. We did not observe mutagen hypersensitivity in neurofibromatosis cells as compared to normal controls.     

Cell size and growth characteristics of cultured fibroblasts isolated from normal and keloid tissue.

No differences in appearance or in cell size distribution were observed between cultured fibroblasts derived from normal skin, mature scars, or keloids. Artifactual differences in cell size distributions between strains can result when populations are compared at different cell densities. Keloid derived fibroblasts remain euploid in culture, and they have the same growth rate and same degree of density-dependent growth inhibition as cultured normal human fibroblasts.     

The ultraviolet sensitivity of Cockayne syndrome cells is not a consequence of reduced cellular NAD content

Cells from individuals with Cockayne syndrome (CS) are hypersensitive to the lethal effects of ultraviolet light (uv) and show a number of abnormal biochemical responses following uv-irradiation. Fujiwara et al. recently reported that the NAD contents of CS fibroblasts were lower than those of normal fibroblasts, and that addition of NAD to the cellular growth medium rectified most of the abnormal responses of CS cells to uv-irradiation. In our experiments, however, the cellular NAD contents of normal and CS fibroblasts were similar, and addition of NAD to the growth medium had no effect on the hypersensitivity of CS cells to uv-irradiation, nor did it restore the inability of CS cells to recover normal rates of DNA or RNA synthesis following uv-irradiation.     

A DNA double strand break repair defect in Fanconi anemia fibroblasts

Fanconi anemia (FA) is a heterogeneous autosomal recessive disease characterized by congenital abnormalities, pancytopenia, and an increased incidence of cancer. Cells cultured from FA patients display elevated spontaneous chromosomal breaks and deletions and are hypersensitive to bifunctional cross-linking agents. Thus, it has been hypothesized that FA is a DNA repair disorder. We analyzed plasmid end-joining in intact diploid fibroblast cells derived from FA patients. FA fibroblasts from complementation groups A, C, D2, and G rejoined linearized plasmids with a significantly decreased efficiency compared with non-FA fibroblasts. Retrovirus-mediated expression of the respective FA cDNAs in FA cells restored their end-joining efficiency to wild type levels. Human FA fibroblasts and fibroblasts from FA rodent models were also significantly more sensitive to restriction enzyme-induced chromosomal DNA double strand breaks than were their retrovirally corrected counterparts. Taken together, these data show that FA fibroblasts have a deficiency in both extra-chromosomal and chromosomal DNA double strand break repair, a defect that could provide an attractive explanation for some of the pathologies associated with FA.     

Fate of HL-A antigens in aging cultured human diploid cell strains : II. Quantitative absorption studies

The expression of histocompatibility antigens on cultured human fibroblasts was studied utilizing a quantitative microabsorption assay. Trypsin treatment of cultured human embryonic and adult fibroblasts did not change their capacity to absorb selected HL-A alloantisera as compared with cells harvested by scraping. The density of HL-A antigens was found to remain unchanged throughout the finite in vitro lifetime of two human embryonic diploid cell strains (WI-38 and WI-26) and ten adult skin fibroblast cultures. Cultured fibroblasts derived from skin, lung, heart, and liver of one donor showed similar quantitative expression of HL-A1, 9, W5 and W16. These experiments support the contention that the HL-A marker system is at present the only system by which human fibroblasts derived from different normal human donors can be distinguished in vitro.     

Differential genetic susceptibility of cultured human skin fibroblasts to transformation by Kirsten murine sarcoma virus.

Hereditary adenomatosis of the colon and rectum (ACR), an autosomal dominant trait, is associated with a predisposition to neoplasia. The present study describes the differential genetic susceptibility of cultured human skin fibroblasts to transformation by Kirsten murine sarcoma virus. Primary cutaneous outgrowths were derived from normal appearing subepidermal biopsies of ACR phenotypes and appropriate controls. Exponentially growing cell cultures from ACR subjects and a portion of the clinically asymptomatic ACR progeny subjected to the viral probe were 100-1000 fold more susceptible to transformation than were normal skin fibroblast cultures. The virally transformed human skin fibroblasts showed a loss of anchorage dependency in carboxymethylcellulose suspension and formed tumors in athymic mice. The results suggest that skin fibroblasts obtained from individuals gine sarcoma virus.     

G2 chromosomal radiosensitivity in Fanconi's anemia

Both the peripheral lymphocytes from 4 patients affected with the inherited disease Fanconi's anemia (FA), and tissue-culture fibroblasts from skin biopsies from 3 patients similarly affected were found to be about twice as sensitive to the induction of chromatid-type chromosomal aberrations by X-rays administratered in the G2 phase of the cell cycle as cells from normal controls. Using tritiated thymidine labelling of peripheral lymphocytes and of cultured fibroblasts, it was determined that 3 affected patients and 3 normal controls all had similar percent labeled mitoses (PLM) curves, so the increased induced aberration yields seen in the FA cells do not appear to be simply a consequences of a longer than normal G2 phase of the cell cycle.     

The in vitro photosensitivity of systemic lupus erythematosus skin fibroblasts

To investigate the role of DNA damage in the pathogenesis of systemic lupus erythematosus (SLE), we studied the ability of skin fibroblasts derived from SLE patients to recover from ultraviolet (UV) light radiation of varying wavelengths. Four of five SLE cell strains were more sensitive to UV-C (254 nm), sun lamp, and UV-A (320 to 400 nm) light than were normal cells. SLE cellular recovery was most sensitive to broad spectrum, long wavelength light. This hypersensitivity did not appear to result from the UV light activation of a clastogenic factor. Experiments which examined the DNA repair capacity of irradiated cells indicated that SLE fibroblasts may be able to excise certain DNA lesions as well as normal cells. The mechanisms responsible for the hypersensitivity of SLE cells remain under investigation.     

Abnormalities of human chromosome 13 and in vitro radiosensitivity A study of 19 fibroblast strains

The in vitro X-ray sensitivity of 19 fibroblast strains derived from patients bearing a deletion, trisomy, inversion, or translocation of all or part of chromosome 13 were determined with a clonogenic survival assay. The results were compared with data from similar experiments involving strains from normal controls and from individuals trisomic for 3 other autosomes. The results suggest the involvement of this chromosome with hypersensitivity to the cytotoxic effects of X-irradiation. Experiments involving the partial monosomies and trisomies seem to implicate a locus on 13q14.     

Cytogenetic investigations in families with ataxia-telangiectasia.

Chromosomal studies were performed on peripheral blood lymphocytes and cultured skin fibroblasts from five Israeli-Moroccan families with ataxia-telangiectasia. A total of 24 individuals, including seven propositi, was investigated. Among the probands, significantly elevated rates of chromosome damage were observed in both blood and skin. Skin fibroblasts of affected individuals showed several orders of magnitude more chromosome breakage than lymphocytes. Increased rates of chromosome damage were also observed in the fibroblasts of some phenotypically normal family members (obligate heterozygotes and sibs) when compared to normal controls. An apparent abnormal clone of cells, possessing a large acrocentric marker chromosome (14q+), was observed in varying proportions among cells of all the propositi (2-5% of lymphocytes; 1-9% of fibroblasts).     

Cellular characterization of cells from the Fanconi anemia complementation group, FA-D1/BRCA2

Fanconi anemia (FA) is an inherited cancer-susceptibility disorder, characterized by genomic instability and hypersensitivity to DNA cross-linking agents. The discovery of biallelic BRCA2 mutations in the FA-D1 complementation group allows for the first time to study the characteristics of primary BRCA2-deficient human cells. FANCD1/BRCA2-deficient fibroblasts appeared hypersensitive to mitomycin C (MMC), slightly sensitive to methyl methane sulfonate (MMS), and like cells derived from other FA complementation groups, not sensitive to X-ray irradiation. However, unlike other FA cells, FA-D1 cells were slightly sensitive to UV irradiation. Despite the observed lack of X-ray sensitivity in cell survival, significant radioresistant DNA synthesis (RDS) was observed in the BRCA2-deficient fibroblasts but also in the FANCA-deficient fibroblasts, suggesting an impaired S-phase checkpoint. FA-D1/BRCA2 cells displayed greatly enhanced levels of spontaneous as well as MMC-induced chromosomal aberrations (CA), similar to cells deficient in homologous recombination (HR) and non-D1 FA cells. In contrast to Brca2-deficient rodent cells, FA-D1/BRCA2 cells showed normal sister chromatid exchange (SCE) levels, both spontaneous as well as after MMC treatment. Hence, these data indicate that human cells with biallelic BRCA2 mutations display typical features of both FA- and HR-deficient cells, which suggests that FANCD1/BRCA2 is part of the integrated FA/BRCA DNA damage response pathway but also controls other functions outside the FA pathway.     

Utilization of electron microscopy in the prenatal diagnosis of genetic disease.

The use of electron microscopy as a further method of diagnosis of disease in cultured skin fibroblasts and cultured amniotic fluid fibroblasts is presented. It was demonstrated that Tay-Sachs disease, Fabry's disease, and metachromatic leukodystrophy had distinctive abnormalities in both cultured skin fibroblasts and cultured amniotic fluid fibroblasts. It was shown that control of culturing conditions made it possible to distinguish normal cell lines from certain cell lines carrying known genetic diseases.     

Near-ultraviolet sensitivity of skin fibroblasts of patients with bloom's syndrome

Near-ultraviolet survival of colony forming ability was compared between fibroblast strains from normal individuals and seven strains from Bloom's Syndrome patients using monochromatic light at 313 nm. Six BS strains possessed abnormal survival properties. Two strains lacked the low dose shoulder, which is characteristic for normal fibroblasts, but their overall sensitivity was in the normal range. Four BS strains were hypersensitive and possessed biphasic survival curves with a sharp initial drop. They mimick in culture the characteristic solar sensitivity of BS patients.     

Aromatase activity in human skin fibroblasts: characterization by an enzymatic method

Human skin fibroblasts were incubated for 24 h with 10(-6) M androstenedione and the estrone + estradiol released in the culture medium were measured by an enzymatic assay. Aromatase activity was expressed as pmol (estrone + estradiol) formed in the medium per mg cell protein per day. Using this method we were able to investigate the kinetic properties of aromatase in different cell strains and its stimulation by dexamethasone. Values of 92 nM and 9.1 pmol/mg protein/day were obtained respectively for Km and Vmax in cultured fibroblasts derived from genital skin of normal prepubertal subjects. In patients with complete androgen insensitivity syndrome CAIS, the Km was 156 nM and the Vmax 42 pmol/mg protein/day. Aromatase activity varied from 7.9 +/- 1.2 pmol/mg protein/day (mean +/- SD; n = 19) in normal prepubertal boys to 24.5 +/- 4.7 pmol/mg protein/day (mean +/- SD; n = 11) in those from normal postpubertal boys. The values were even higher in fibroblasts cultured from genital skin of prepubertal patients with CAIS. Cell concentrations did not modify the pattern of estrogen formation and aromatase activity did not vary with serial subcultures. The stimulatory effect of dexamethasone on aromatase activity in cultured fibroblasts was measured after preincubation of the cells for 48 h with dexamethasone, by determining estrogen formation after 24 h incubation of the cells with androstenedione 10(-6) M using this enzymatic method. This data suggest that aromatase activity measured in cultured fibroblasts could be a useful tool for studying extraglandular estrogen formation in physiological and pathological conditions.     

A defect in DNA double strand break processing in cells from unaffected parents of retinoblastoma patients and other apparently normal humans

Cells from unaffected parents of retinoblastoma (RB) patients were previously shown to be hypersensitive to radiation induced G(1) arrest and cell killing [1]. The hypersensitivity was similar to that reported for cells from ATM heterozygotes. The latter was consistent with a mild DNA DSB rejoining defect which we demonstrated using a gamma-H2AX focus assay after low dose-rate (LDR) irradiation of non-cycling G(0) cells [2,3]. Since neither parent carried the mutant RB allele of the RB heterozygous probands, these results suggested the possibility of an enhanced germline mutation rate, perhaps resulting from some mild defect in genome maintenance. We therefore examined levels of gamma-H2AX foci for cells from these RB parents in this G(0) LDR assay, which reflects the non-homologous end joining (NHEJ) capacity of cells and in a G(2)/M assay, which reflects additional contributions from other G(2)-related damage processing systems. For several of the cell strains parallel radiosensitivity comparisons were made for cell killing and for G(2) chromosomal radiosensitivities. G(0) cells from the RB parents were clearly hypersensitive both in the LDR gamma-H2AX assay, and for cell killing. In addition, cultured fibroblasts from 6 of 15 apparently normal individuals in this study (and one of six in a previous study) were also hypersensitive in the same assays. In the G(2)/M gamma-H2AX assay, the relative sensitivities were similar to those seen in the low dose-rate G(0) assay and tracked with chromosomal radiosensitivity, but some differences were observed.     

Regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and the esterification of cholesterol in human long term lymphoid cell lines.

The regulation of the rate-controlling enzyme in cholesterol biosynthesis and of the incorporation of [14C]oleate into cholesterol esters were studied in established lymphoid cell lines from normal subjects and compared with that of eight patients with genetic abnormalities of lipid metabolism. The activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase, the rate-controlling enzyme in cholesterol biosynthesis, increases in lymphoid cell lines derived from normal subjects after the culture medium is changed to a lipid deficient medium and reaches peak activity after 48 hr. The addition of whole serum and of low density lipoproteins to cell lines derived from normal subjects suppressed 3-hydroxy-3-methylglutaryl coenzyme A reductase activity by 50%, but failed (almost completely) to suppress the activity in the lymphoid cell lines derived from two patients with homozygous familial hypercholesterolemia. When 7-ketocholesterol was added, the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase was markedly suppressed in both normal and abnormal lymphoid cell lines. Lymphoid cell lines derived from patients presumably heterozygous for familial hypercholesterolemia were difficult to distinguish from normal cells in these studies. The incorporation of [14C]oleate into the fatty acid fraction of cholesteryl esters was stimulated by the addition of the low density lipoproteins to the culture media of the lymphoid cell lines derived from the normal human subjects. The lymphoid cell lines derived from the patients with homozygous familial hypercholesterolemia showed no increase in [14C]oleate incorporation into cholesteryl esters even when a fourfold amount of low density lipoprotein was added to the media; a modest increase in [14C]oleate incorporation was observed in lymphoid cell lines from patients with heterozygous familial hypercholesterolemia. The results of these studies in lymphocyte cell lines are compared with the findings in cultured human fibroblasts obtained from normal subjects and from patients with homozygous familial hypercholesterolemia. Studies of the regulation of cholesterol biosynthesis in the apparently permanent lymphoid cell line maintained in suspension culture offer certain advantages over cultured skin fibroblasts, and, in addition, provide a second tissue for the study of genetic abnormalities from the same patient.     

The pathophysiology of neurofibromatosis. I. Resistance in vitro to 3-nitrotyrosine as an expression of the mutation

The in vitro expression of the autosomal dominant mutation responsible for neurofibromatosis was probed using the amino acid analogue 3-nitrotyrosine as a cell culture selective agent. The presence of 3-nitrotyrosine in culture medium led to inhibition of growth and cell death among normal skin fibroblasts in log phase growth, whereas cell strains derived from six different patients' neurofibromas or skin cells, or both, exhibited a consistently enhanced ability to survive under the same conditions. At 0.8 mM 3-nitrotyrosine, four patient-derived skin fibroblast strains could be differentiated from five strains of control skin fibroblasts with a high level of confidence (P < 0.0000). In the same way four neurofibroma-derived fibroblast strains were differentiated from control skin fibroblasts (P < 0.0022). Neurofibroma-derived cells were not different from control cells when treated with 5-fluorotryptophan or p-fluorophenylalanine.     

Growth abnormalities of cultured human skin fibroblasts derived from individuals with hereditary adenomatosis of the colon and rectum

A heritable propensity to develop malignant lesions is found in individuals with familial adenomatosis of the colon and rectum (ACR) and the Gardner's syndrome variant, an autosomal dominant trait. In the present study, the growth characteristics of cultured skin fibroblasts (SF) derived from normal-appearing flat skin biopsies of ACR families, representing all phenotypes, and appropriate controls were investigated. SF were obtained from stocks between the second and fifth passages and grown to confluency in Eagle's Minimal Essential Medium (EMEM) supplemented with 15% fetal calf serum (FCS). Following trypsinization, cells were replanted in EMEM supplemented with either 1% or 15% FCS at an initial density of 4 × 10³ cells/cm² and counted daily for five days. Normal SF representing several age groups (both sexes) and those obtained from non-afflicted individuals of ACR families grew only in 15% FCS. In contrast, SF from ACR subjects and from embryonal skin grew both in 1% and 15% FCS. SF from several clinically asymptomatic adults, children of ACR patients, grew in 1% FCS as well. Cell cultures from ACR individuals showed regions of criss-crossed arrays and multilayered pattern. These growth properties were not observed in normal cell cultures. The SF from ACR individuals did not grow in methocel, nor did they form tumors in athymic mice. These results suggest the occurrence of previously undetected biochemical alterations in SF taken from ACR genotypes.