Rapid identification of female carriers of DMD/BMD by quantitative real-time PCR

Recently developed PCR systems offer online-monitoring of amplification and allow simple and reliable DNA quantification. We have used the LightCycler system to develop a simple and rapid method for direct identification of female carriers of deletions and duplications in the dystrophin gene. The challenge resides in the ability to identify the presence of a deleted or duplicated allele over the background contributed by the normal allele. Quantification is based on the determination of the ratio between potentially deleted/duplicated dystrophin exons and non-deleted/-duplicated reference exons using the unspecific dsDNA-dye SYBRgreen I. In a retrospective study, we evaluated our method in female relatives of DMD/BMD patients with known carrier status by comparative analysis of deleted or duplicated versus non-deleted/-duplicated exons. Carrier status was accurately attributed in 100% of cases, the mean ratios being 0.52+/-0.12 for deletion carriers (expected value: 0.5) and 1.56+/-0.18 for duplication carriers (expected value: 1.5) vs. 1.022+/-0.17 for non-carriers (expected value: 1.0). The method proved to be simple, rapid, reliable, and cost-effective. It may be used for direct determination of deletions/duplications in potential DMD/BMD carriers and may easily be adapted for other genetic conditions involving deletions and duplications.     

Two Cases of Peritoneal Serous Papillary Adenocarcinoma

Two cases of peritoneal papillary carcinoma are reported. The patient in the first case was a 71-year-old woman with symptoms of obstructive ileus. Laparotomy revealed a tumor in the omentum involving the transverse colon, and several small tumors in the peritoneum and pelvic wall. However, no primary site of the tumor was seen in the ovary, pancreas, or gastrointestinal tract. The patient in the second case was a 44-year-old woman with carcinomatous peritonitis. Postmortem examination revealed multiple tumors in the peritoneum, omentum, and pelvic wall. Tumors were also found in the cortex with mild invasion of the underlying parenchyma of the bilateral ovaries, although these lesions were thought to be metastatic. The histologic features of the tumor in both cases were those of tubulopapillary adenocarcinoma containing scattered psammoma bodies. The cells were positive with the PAS D technique, but negative with alcian blue staining. In both cases, the serum levels of CA-125 were considerably elevated, and the tumor cells showed positivity for CA-125, S 100 protein, cytokeratin and EMA by im-munohistochemistry. The present cases were most likely peritoneal serous papillary adenocarcinoma derived from extraovarian peritoneal mesothelium with miillerian potential, being different from the usual type of diffuse malignant mesothelioma. Acta Pathol Jpn 41: 642-646, 1991.     

Charcot-Marie-Tooth disease: molecular characterization of patients from Central and Southern Italy

The syndrome of peroneal muscular atrophy, or Charcot-Marie-Tooth (CMT), disease represents the most common inherited peripheral neuropathy, with a prevalence of about 1 per 2500. The disease is usually transmitted in an autosomal dominant fashion, although it can display all the mendelian patterns of inheritance. The chromosome 17-linked form (CMT1a) appears to be the most common form of the disease in all the ethnic groups studied so far, Italians included, and is due to a tandem duplication in 17p11.2. In order to study the distribution of CMT types and to establish a genotype-phenotype correlation in patients from Central and Southern Italy, we collected 19 CMT pedigrees diagnosed in the years 1992–1993. Simple tandem repeats (STR) polymorphism analysis with the marker RM11-GT and Southern blotting with the probes pVAW409R3 and pVAW412 were performed, demonstrating a high prevalence (about 60%) or 17p duplication in the families studied. No clinical or electrophysiological differences were noted between CMT1 patients with or without 17p duplication, respectively. Two families affected by CMT2 showed no evidence of rearrangement at the D17S122 locus. These data are consistent with the hypothesis of a different molecular basis for CMT2.     

The CA 125 tumour-associated antigen: a review of the literature

CA 125 is an antigenic determinant on a high-molecular-weight glycoprotein recognized by a monoclonal antibody which was raised using an ovarian cancer cell line as an immunogen. During the last 5 years the studies reviewed in this paper have provided information concerning the nature, distribution and clinical significance of CA 125. The CA 125 determinant is expressed by epithelial ovarian tumours and various other pathological and normal tissues of Müllerian origin. The function of the glycoprotein expressing CA 125 remains unclear but the distribution of the antigen suggests that it may have a physiological role. The highest serum levels of CA 125 are found in ovarian cancer patients, but elevation of serum CA 125 may also be associated with other malignancies and benign and physiological states, including pregnancy, endometriosis and menstruation. Despite limitations of sensitivity and specificity serum CA 125 estimation is of clinical value in the pre-operative diagnosis and monitoring of ovarian malignancy and may be a prognostic indicator for this disease. The role of CA 125 in screening for early-stage ovarian cancer is currently under investigation. Recent reports suggest that serum CA 125 measurement may also be of value as a prognostic indicator in endometrial cancer and as a reflection of disease status in advanced endometriosis.     

CA125-producing adenocarcinoma of the seminal vesicle

A case of primary seminal vesicle carcinoma is reported. The tumor was a CA125-producing adenocarcinoma consisting of fine papillary-tubular, intricate branching or anastomosing glandular structures and was composed of small cuboidal, but occasionally hobnailed, cells with mostly clear, but occasionally granular, cytoplasm. Some tumor cells showed evidence of secretion of seromucinous materials into the interpapillary and cystic space. lmmunohistochemically, almost half of the tumor cells expressed a positive reaction with anti-CAl25, a common serological marker for ovarian epithelial carcinomas; however, no tumor cells expressed any other serological tumor markers such as carcinoem-bryonic antigen, α-fetoprotein, human chorionic gonadotropin, prostatic specific acid phosphatase, or prostatic specific antigen. The patient showed a high level of serological CA125, which fluctuated parallel with the growth, removal and recurrence of the tumor. The morphological and immunohistochemical findings suggested a close relationship between the present tumor and clear cell carcinoma of the ovary, which is thought to be of a Müllerian-Wolfian duct origin.     

Lattices of Type I Collagen Select Invasive Variants of K-ras Oncogene-Transfected NIH3T3 with Less Cellular fibronectin

A clone of NIH3T3 transformant (H3) can yield subcutaneous tumors and experimental pulmonary metastasis in nude mice. Compared to H3 in culture, the cells after in vivo tumor growth (H3-N) acquired enhanced tumorigenicity and metastatic ability. Also, indirect immunofluorescence revealed that cellular fibronectin (c-FN) of H3-N was decreased remarkably. We have studied the interactions between H3 and extracellular matrices to elucidate these phenomena. In the present study, we observed the effect of NIH3T3, H3, and H3-N cultured in type I collagen gel. Morphologically in the collagen gel, NIH3T3 assumed an extensive elongated fiber-like shape, H3 assumed a moderately elongated shape, and H3-N assumed a round or spindle shape with short pseudopodia. Compared to conventional cultures on dishes, cell proliferation of all three types was suppressed in collagen gel, but the degree of the suppression was least in H3-N. As a result, H3-N grew fastest in collagen gel. The variants which acquired growth advantage in the subcutaneum of mice also kept it in collagen gel. H3 cells were cultured in type I collagen gel for 4 weeks, a period comparable to that of tumor formation in nude mice. The cells after this long-term culture (H3-C) acquired enhanced tumorigenicity and metastatic ability nearly equal to that of H3-N. FACS analysis revealed that the c-FN of H3-C had decreased to a value comparable to that of H3-N. This means that type I collagen gel as well as subcutaneous tissues could select variants of H3 with less c-FN through proliferation. Moreover, it is suspected that lattices of type I collagen regulate cell proliferation of fibroblast via c-FN. This revised version was published online in August 2006 with corrections to the Cover Date.     

Cat shedding of Fel d I is not reduced by washings, Allerpet-C spray, or acepromazine

Background: No published studies have compared the effectiveness of several treatments proposed to reduce cat allergenicity. Cat washing studies demonstrating efficacy involved very small sample sizes or infrequent washings. Allerpet-C (Allerpet, Inc., New York, N.Y.), a widely advertised topical spray, and acepromazine, a tranquilizer advocated as efficacious in subsedating doses, have never been scientifically studied. Objective: We compared the effects of cat washing, Allerpet-C spray, and acepromazine with that of no treatment on the shedding of the primary cat allergen, Felis domesticus I by cats. Methods: In a blinded, comparative, controlled study, we measured the amounts of Fel d I shed during an 8-week treatment period with a sample of 24 female mongrel cats randomly assigned to four groups; one group received weekly distilled water washings, one received weekly Allerpet-C spray applications, one received daily oral acepromazine, and one had no treatment (control). Thirty-minute, twice-weekly air samples were collected from each cat with a laminated plastic–acrylic chamber and air sampler. Results: One-sample, two-sided t tests comparing baseline to final-week measurements revealed no significant change in Fel d I within each group (mean change ±SD: washing; 487.6 ± 1896.4 mU per 30 minutes, p = 0.63; Allerpet-C spray, 429.2 ± 871.6 mU per 30 minutes, p = 0.46 acepromazine; −620.6 ± 1031.2, p = 0.52 per 30 minutes). Furthermore, analysis of covariance revealed no significant change in Fel d I levels between groups (p = 0.72). Conclusions: Our data do not show significant reductions in Fel d I shedding as a result of any of these treatments. Therefore we cannot recommend them to patients allergic to cats. (J ALLERGY CLIN IMMUNOL 1995;95:1164-71.)     

Ras-transfection up-regulated HaCaT cell migration: Inhibition by Marimastat

Cell migration is an essential process in physiological and pathological conditions such as wound healing and tumor invasion. This phenomenon involves cell adhesion on the extracellular matrix mediated by integrins, and cell detachment promoted in part by metalloproteinases (MMPs). In the present study, the migration of two HaCaT-ras clones (metastatic or not), was compared with HaCaT cells, and normal human primary cultured keratinocytes. Using colloidal gold migration assay, the migration index on type I and type IV collagen was similar for primary cultured keratinocytes and HaCaT, whereas it was markedly higher for the HaCaT-ras clones. High motility of ras-transfected cells was confirmed from an in vitro wound healing assay. It was not correlated with changes in integrin expression or related to a different adhesion on extracellular matrix. The Marismastat (BB-2516), a MMP inhibitor, inhibited in a dose-dependent effect the migration in both assays, demonstrating the important role of MMPs in the migration process. Under our experimental conditions, MMP-1 activity was not detected in HaCaT and MMP-9 activity was secreted by these cells only after their stimulation by EGF. Here, MMP-2 was the major gelatinolytic activity secreted by all the cells and its secretion was markedly higher for HaCaT-ras clones compared with HaCaT. In addition, Western blotting results confirmed a higher expression of MMP-2 associated with a lower expression of TIMP-2 in HaCaT-ras compared with HaCaT. These results suggest that Ha-ras oncogene could be a stimulating factor of migration and might modified the balance between MMP-2 and TIMP-2 in keratinocyte cell lines. This revised version was published online in July 2006 with corrections to the Cover Date.     

Pathogenesis of antiphospholipid syndrome: recent insights and emerging concepts

Introduction: Even though our understanding of the antiphospholipid syndrome (APS) has improved tremendously over the last decades, we are still not in a position to replace symptomatic anticoagulation by pathogenesis based causal treatments.

Areas covered: Recent years have provided further insights into pathogenetically relevant mechanisms. These include a differentiation of pathogenic subtypes of antiphospholipid antibodies (aPL), novel mechanisms modulating disease activity, for example, extracellular vesicles and microRNA, and novel players in pathogenesis, for example, neutrophils and neutrophil extracellular traps (NETs).

Expert commentary: It is evident that aPL induce a proinflammatory and procoagulant state and recent data suggest that different aPL species activate different signaling pathways which sometimes converge into a common cellular response. This implies that presence of more than one aPL species may disproportionally increase the risk for the major manifestations of APS, that is, thrombosis and fetal loss. Further delineation of the pathogenic mechanisms will hopefully provide clues to causal rather than symptomatic treatments of APS.     

V180I mutation of the prion protein gene associated with atypical PrP glycosylation

A valine to isoleucine mutation at residue 180 was identified in a French patient with Creutzfeldt-Jakob disease (CJD). The mutation is located in the close vicinity of one of the two N-glycosylation sites of the cellular prion protein (PrP^C). Western blot analysis revealed accumulation in the brain of the pathogenic proteinase K-resistant PrP (PrP^Sc) isoform with the notable absence of the diglycosylated band. The mutant protein expressed in CHO cells was correctly glycosylated, suggesting that the atypical glycosylation pattern of PrP^Sc was not due to the mutation at position 180. These results suggest that the diglycosylated form of the mutant PrP^180I prevents its conversion into the pathogenic mutant form PrP^Sc180I, supporting a central role of N-linked glycan chains in the PrP conversion process.