Bernadette Keitz

Detection of epithelial ovarian cancer using 1H-NMR-based metabonomics

Currently available serum biomarkers are insufficiently reliable to distinguish patients with epithelial ovarian cancer (EOC) from healthy individuals. Metabonomics, the study of metabolic processes in biologic systems, is based on the use of 1H‐NMR spectroscopy and multivariate statistics for biochemical data generation and interpretation and may provide a characteristic fingerprint in disease. In an effort to examine the utility of the metabonomic approach for discriminating sera from women with EOC from healthy controls, we performed 1H‐NMR spectroscopic analysis on preoperative serum specimens obtained from 38 patients with EOC, 12 patients with benign ovarian cysts and 53 healthy women. After data reduction, we applied both unsupervised Principal Component Analysis (PCA) and supervised Soft Independent Modeling of Class Analogy (SIMCA) for pattern recognition. The sensitivity and specificity tradeoffs were summarized for each variable using the area under the receiver‐operating characteristic (ROC) curve. In addition, we analyzed the regions of NMR spectra that most strongly influence separation of sera of EOC patients from healthy controls. PCA analysis allowed correct separation of all serum specimens from 38 patients with EOC (100%) from all of the 21 premenopausal normal samples (100%) and from all the sera from patients with benign ovarian disease (100%). In addition, it was possible to correctly separate 37 of 38 (97.4%) cancer specimens from 31 of 32 (97%) postmenopausal control sera. SIMCA analysis using the Coomans plot demonstrated that sera classes from patients with EOC, benign ovarian cysts and the postmenopausal healthy controls did not share multivariate space, providing validation for the class separation. ROC analysis indicated that the sera from patients with and without disease could be identified with 100% sensitivity and specificity at the 1H‐NMR regions 2.77 parts per million (ppm) and 2.04 ppm from the origin (AUC of ROC curve = 1.0). In addition, the regression coefficients most influential for the EOC samples compared to postmenopausal controls lie around δ3.7 ppm (due mainly to sugar hydrogens). Other loadings most influential for the EOC samples lie around δ2.25 ppm and δ1.18 ppm. These findings indicate that 1H‐NMR metabonomic analysis of serum achieves complete separation of EOC patients from healthy controls. The metabonomic approach deserves further evaluation as a potential novel strategy for the early detection of epithelial ovarian cancer.

Linkage of amelogenin (Amel) to the distal portion of the mouse X chromosome

Amelogenins are hydrophobic, proline-rich proteins that are the primary biosynthetic products of ameloblasts. These cells are responsible for the formation of tooth enamel, and amelogenins play an important role in the process of biomineralization. A cDNA, corresponding to the mouse 26-kDa amelogenin, has been molecularly cloned and sequenced. Southern blot analysis of genomic DNA from the mouse using this cDNA as a probe indicates that there is only one mouse amelogenin (Amel) gene. This paper describes restriction site variation for the Amel gene that we have identified between C57BL/6 and M. spretus and the segregation of that variation as an X-chromosome gene. The position of the amelogenin locus (Amel) relative to the loci for alpha-galactosidase (Ags), proteolipoprotein (Plp), and the random genomic probe DXWas31 has been determined. Amel is established as: (1) the most distal locus in the genetic map of the mouse X chromosome, (2) lying proximal to the X:Y pairing region, and (3) being restricted to the mouse X chromosome.

Mapping and expression of the ubiquitin-activating enzyme E1 (Ube1) gene in the mouse

The nucleotide sequence of the human cDNA encoding ubiquitin-activating enzyme E1 is more than 99% identical with the human A1S9T cDNA, a gene that has been shown to complement the temperature-sensitive mutant mouse cell line, tsA1S9. The amino acid sequences of the proteins encoded by these two cDNA sequences are identical, and both cDNAs were previously shown to be located in the same region of the human X chromosome; thus, ubiquitin-activating enzyme E1 and A1S9T appear to be the same gene, designated UBE1. By in situ hybridization to metaphase chromosomes from male mice and by Southern blot analysis of male and female mouse DNA, we show that, in the mouse, a human UBE1 cDNA probe identified both X- and Y-linked loci. Ube1 is located at band A2 of the mouse X Chromosome (Chr) and Ube2 on the short arm of the Y Chr. This is in contrast to the situation in the human, where there is no evidence for Y-linked sequences related to UBE1. Mapping of the Ube1 gene in interspecific backcrosses between Mus spretus and C57BL/6 shows that the Ube1 locus maps close to Timp, in a conserved region of the mouse and human X Chrs that include Otc, Cybb, Syn1, Timp, and Araf. Expression of Ube1 on the inactive X Chr was examined to determine whether this gene is subject to X-Chr inactivation in the mouse, as there is previous evidence that the human UBE1 gene escapes, at least partially, X inactivation. Sequencing of reverse transcriptase polymerase chain reaction (RT-PCR) products from M. spretus, C57BL/6J, and T(X;16)16H x M. spretus F1 female mice indicates that the mouse Ube1 gene is subject to X-Chr inactivation in vivo. This represents a new example of differences between the sex chromosomes of mouse and human.

Linkage of a gene for neural cell adhesion molecule, L1 (CamL1) to the Rsvp region of the mouse X chromosome

L1 is a glycoprotein with an apparent molecular weight of 200 kDa in the developing fetus and adult central nervous system. In the peripheral nervous system, it has a molecular weight of 230 kDa. The L1 protein appears to be encoded by a single gene that has been located on the human X chromosome by in situ hybridization. In this paper we describe restriction variation in genomic DNA Southern analysis between Mus species for the K13 cDNA probe for the L1 neural cell adhesion molecule. We have designated the locus described by this variation as cell adhesion molecule L1, CamL1. The X chromosome linkage and the relative position on the X chromosome coincident with the genes Rsvp/G6pd/Cf-8 were defined in backcross matings involving M. spretus and M. musculus.

Linkage of the erythroid transcription factor gene (Gf-1) to the proximal region of the X chromosome of mice

We have used a cDNA probe for mouse Gf-1 gene that encodes the erythroid cell transcription factor to identify genetic variation in genomic DNA between Mus species. The segregation of Gf-1 DNA variation was analyzed in Mus species crosses that have been previously typed for the segregation of more than 30 genes spanning 80 cM of the mouse X chromosome from the centromere to the border of the X-Y pairing region. We identified a single X chromosome locus in the mouse, Gf-1, and an analysis of recombinants from 203 backcross progeny mapped Gf-1 to the proximal portion of the chromosome, coincident with the Cybb locus and proximal to Otc gene locus. A gene order of centromere, DXWas70, Cybb/Gf-1, Otc, Timp was established for the mouse X chromosome, which is in agreement with the map position observed on the human X chromosome.

Microsatellite instability in large cell neuroendocrine carcinoma of the female genital tract

which has a probable mesonephric origin, and retiform ovarian Sertoli–Leydig tumours, which in their turn have a sex-cord stromal origin. Retiform UTROSCTs are unusual, since they mimic the morphology of retiform ovarian Sertoli–Leydig cell tumours with the formation of epithelial structures akin to the early rete ovarii. Indeed, we demonstrated that these retiform epithelia found in UTROCSTs have an immunophenotype not substantially different from rete ovarii, Wolffian adnexal tumours or retiform areas of ovarian Sertoli–Leydig cell tumours. Indeed, a myoid differentiation was not found in any of these. Thus, we believe that the absence of myoid phenotype in retiform areas of UTROCSTs is a common feature of the areas that morphologically reproduce epithelial retiform structures. However, in our cases we found focal caldesmonpositive myoid differentiation in the minor, non-retiform stromal sex-cord-like areas UTROSCTs.

Linkage of phosphoribosylpyrophosphate synthetases 1 and 2, Prps1 and Prps2, on the mouse X Chromosome

The X Chromosome (Chr) genes for phosphoribosylpyrophosphate synthetases 1 and 2, Prps1 and Prps2, were mapped on the mouse X Chr with interspecific backcrosses between C57BL/6 (B6) and M. spretus (S). Southern analysis showed that Prps1 mapped between Plp and DXWas31, a mouse X Chr region that is homologous to Xq21-24 on the human X Chr while Prps2 mapped between DXWas31 and Amg, a region that is homologous to the map position of PRPS2 on Xp22 of the human X Chr. Additionally, other restriction fragments highlighted by PRS II showed autosomal segregation. In situ hybridization and FISH analysis of metaphase chromosome spreads prepared from lymphocytes of B6 or S male mice confirmed that there were in fact two different locations on the X Chr, X F1-2 and X F2-3 for Prps1 and 2 respectively, as well as two autosomal sites for Prps-like genes.