Response to genetic counseling and testing for the APC I1307K mutation.

The APC I1307K gene mutation is associated with increased colorectal cancer (CRC) risk in Ashkenazi Jews. Factors predicting acceptance of this and other hereditary colon cancer mutation tests in a clinical setting are unknown. We analyzed sex, age, family history, personal history, and gene test results of patients at increased risk for cancer who sought cancer risk counseling at the Johns Hopkins (JH) CRC Risk Assessment Clinic (n = 91), and those submitting samples to the JH Pathology Molecular Diagnostic Laboratory (n = 256) for APC I1307K testing. Of patients seen at the JH Clinic, 77/91 (84.6%) elected APC I1307K testing after pretest counseling (acceptors). There were no statistically significant differences in demographic characteristics between acceptors and decliners. In comparison, only 8 of 57 (14.0%) patients offered HNPCC testing proceeded with testing (P < 0.001). Of 256 individuals tested at the JH laboratory, most were male (61.3%) and most had a personal or family history of colorectal cancer or polyps. Test positivity correlated with increasing risk of colorectal cancer. Acceptance of testing for the APC I1307K mutation is high, with more men than women pursuing counseling and testing. The reported association between the APC I1307K mutation and colon cancer risk was supported by a correlation in these data between personal or family history of CRC or polyps and a gene mutation.

A human-murine chimera model for in utero human hematopoietic stem cell transplantation.

To date, 16 in utero hematopoietic stem cell (HSC) transplants for diseases other than immunodeficiency disorders have been reported. No therapeutic level of engraftment was detected in 15 of these transplants. To overcome engraftment failure, we transplanted a very large number (5 billion paternal CD34+ cells/kg) of HSCs to a fetus with leukodystrophy during the first trimester of gestation. As reported previously, the fetus died in utero 7 weeks after the procedure and the cause of death appeared to be overwhelming donor engraftment. In the present investigation, we developed a human-murine chimera model to test for the optimal donor cell dose for human in utero transplantation. We found a strong correlation between the level of donor engraftment in three human fetuses transplanted for leukodystrophy during the first trimester of gestation and the results of parallel xenotransplants of the same human donor cells using the NOD/SCID mouse model. This small animal model appears to predict both extremes of hyperengraftment (seen in the first human fetus transplanted) and engraftment failure (seen in the second and third human fetuses transplanted in utero). These and future correlated clinical and laboratory assay results may be useful for the development of in utero transplants for a variety of congenital disorders.

Mosaic trisomy 16 ascertained through amniocentesis: evaluation of 11 new cases.

Trisomy 16, once thought to result uniformly in early pregnancy loss, has been detected in chorionic villus samples (CVS) from on-going pregnancies and was initially ascribed to a second, nonviable pregnancy. Prenatally detected trisomy 16 in CVS and its resolution to disomy has led to the reexamination of the viability of trisomy 16. This study evaluates 11 cases of mosaic trisomy 16 detected through second trimester amniocentesis. In 9 of the 11 cases, amniocenteses were performed in women under the age of 35 because of abnormal levels of maternal serum alpha-fetoprotein (MSAFP) or maternal serum human chorionic gonadotropin (MShCG). The other two amniocenteses were performed for advanced maternal age. Five of the 11 pregnancies resulted in liveborn infants, and six pregnancies were electively terminated. The liveborn infants all had some combination of intrauterine growth retardation (IUGR), congenital heart defects (CHD), or minor anomalies. Two of them died neonatally because of complications of severe congenital heart defects. The three surviving children have variable growth retardation, developmental delay, congenital anomalies, and/or minor anomalies. In the terminated pregnancies, the four fetuses evaluated by ultrasound or autopsy demonstrated various congenital anomalies and/or IUGR. Cytogenetic and fluorescent in situ hybridization studies identified true mosaicism in 5 of 10 cases examined, although the abnormal cell line was never seen in more than 1% of cultured lymphocytes. Placental mosaicism was seen in all placentas examined and was associated with IUGR in four of seven cases. Maternal uniparental disomy was identified in three cases. Mosaic trisomy 16 detected through amniocentesis is not a benign finding but associated with a high risk of abnormal outcome, most commonly IUGR, CHD, developmental delay, and minor anomalies. The various outcomes may reflect the diversity of mechanisms involved in the resolution of this abnormality. As 80% of these patients were ascertained because of the presence of abnormal levels of MSAFP or MShCG, the increased use of maternal serum screening should bring more such cases to clinical attention.

Esophagin cDNA cloning and characterization: a tissue-specific member of the small proline-rich protein family that is not expressed in esophageal tumors.

Cancer may be understood as the net effect of differences in gene expression between normal and transformed cells. In a novel direct approach applying this principle, complete genes expressed at altered mRNA levels in malignant versus normal esophageal epithelium were identified and isolated from cDNA libraries. One clone was expressed in normal esophageal mucosae but absent in esophageal carcinomas. By in situ hybridization, Northern blotting, and immunohistochemistry, expression of this gene was restricted to normal esophageal mucosa; it is designated esophagin. Esophagin expression was greatest in the superficial, most mature layers of esophageal squamous mucosa and was restricted to this organ, being undetectable in other squamous epithelia. A genomic clone localized esophagin to chromosomal region 1q21-q22. The expressed protein contains multiple direct repeats of an 8-amino acid motif rich in proline, with significant homology to the cornifin, pig 20K, monkey MT5, and human small proline-rich genes spri and spril. Esophagin constitutes the newest and largest member of this small proline-rich gene family and is associated with differentiation and the benign phenotype of the human esophageal epithelial cell.

Complex chromosomal rearrangements: some breakpoints may have cellular adaptive significance.

Cytogenetic study of a 3-year-old girl with developmental delay and some minor abnormalities revealed a complex chromosome rearrangement (CCR) involving seven chromosomes with eight breakpoints, leading to monosomy of segment 5q15-q22. According to breakpoint distribution, CCRs may be classified as those with primary intrachromosomal abnormalities (including inversions, insertions, duplications, etc.) and those without them. Only the latter group of CCRs was used in this analysis. Comparison of theoretical and observed breakpoint distributions in 33 cases demonstrated that recurrent involvement of some chromosome(s) ("re-entry") occurs more frequently than expected. One possible explanation for this observation suggests that the initial event leads to an unstable provisional rearrangement, and subsequent breaks are necessary to stabilize the karyotype.

Phenotypic variability of del(2) (q22-q23): report of a case with a review of the literature.

An interstitial deletion of 2q22-q23 was found in a 2.5 year old boy with multiple congenital abnormalities (including Hirschsprung's disease) and severe mental retardation. Comparison with seven additional cases of 2q deletions from the literature does not allow the delineation of a clinically recognizable syndrome. Some of the previously reported patients had features rarely observed in chromosomal syndromes (i.e., occipital encephalocele and tracheo-esophageal fistula). Possible reasons for such phenotypic variability are briefly discussed.

Human cytogenetics. A current overview.

Chromosomal abnormalities are the basis for a substantial proportion of human morbidity and mortality. During the past 35 years, the field of human cytogenetics has helped to elucidate the etiology of many congenital malformation/mental retardation syndromes. Through adaptation of technological advances and integration of molecular biological techniques, cytogenetics continues to contribute significantly to our knowledge of clinical genetics, chromosomal fine structure and function, gene mapping, and prenatal diagnosis. This review outlines the basic concepts, recent findings, and current laboratory approaches to cytogenetic diagnosis.

Cytogenetic studies of primary cultures of esophageal squamous cell carcinoma.

Cytogenetic analysis was performed on primary cultures of 21 squamous cell carcinomas of the esophagus (SCCE). Seven cases exhibited mosaic clonal chromosome abnormalities distributed as follows: two contained tetraploid cell populations, one with t(3;7)(p21;q11); two showed loss of the Y chromosome, one with double minutes; single cases demonstrated der(11)t(4;11)(q?27;q23); add(1)(p35) and del(4)(p12); and del(7)(p13), del(7)(q22q34), and der(11)t(7;11)(p?15;p?13). The remaining 14 cases had apparently normal karyotypes, possibly derived from stromal elements. These results demonstrate numerical abnormalities and the multiple occurrence of rearrangements involving chromosomes 7 and 11 in SCCE.

Structure and organization of amplified DNA on double minutes containing the mdm2 oncogene.

We have been studying a transformed derivative of a mouse fibroblast line (3T3DM) that stably maintains double minute chromosomes (DMs). In this report we describe a comprehensive analysis of the structure of the DMs within this cell line, utilizing a combination of long-range mapping via pulsed-field gel electrophoresis, screening of DM-enriched genomic libraries, and DM sizing using contour-clamped homogeneous electric field (CHEF) gel electrophoresis. Our data indicate that the minute particles in these cells exist as a homogeneous population of circular molecules, roughly 4 Mb in size, upon which three genes are amplified. One of these is the mdm2 oncogene, which has also been found to be amplified in a number of human sarcomas. Further, we present evidence that these three genes are arranged as two identical inverted repeat units linked by spacer regions of heterogeneous size. This work has led to the first model for the structure of an entire double minute particle containing an amplified oncogene; this model provides clues to later events occurring in the gene amplification process in tumor cells.

The ROX3 gene encodes an essential nuclear protein involved in CYC7 gene expression in Saccharomyces cerevisiae.

The ROX3 gene was identified during a hunt for mutants with increased expression of the heme-regulated CYC7 gene, which encodes the minor species of cytochrome c in the yeast Saccharomyces cerevisiae. The rox3 mutants caused a 10-fold increase in CYC7 expression both in the presence and absence of heme, had slightly increased anaerobic expression of the heme-activated CYC1 gene, and caused decreases in the anaerobic expression of the heme-repressed ANB1 gene and the aerobic expression of its heme-induced homolog. The wild-type ROX3 gene was cloned, and the sequence indicated that it encodes a 220-amino-acid protein. This protein is essential; deletion of the coding sequence was lethal. The coding sequence for beta-galactosidase was fused to the 3' end of the ROX3 coding sequence, and the fusion product was found to be localized in the nucleus, strongly suggesting that the wild-type protein carries out a nuclear function. Mutations in the rox3 gene showed an interesting pattern of intragenic complementation. A deletion of the 5' coding region complemented a nonsense mutation at codon 128 but could not prevent the lethality of the null mutation. These results suggest that the amino-terminal domain is required for an essential function, while the carboxy-terminal domain can be supplied in trans to achieve the wild-type expression of CYC7. Finally, RNA blots demonstrated that the ROX3 mRNA was expressed at higher levels anaerobically but was not subject to heme repression. The nuclear localization and the lack of viability of null mutants suggest that the ROX3 protein is a general regulatory factor.

A yeast protein with homology to the beta-subunit of G proteins is involved in control of heme-regulated and catabolite-repressed genes.

The product of the Saccharomyces cerevisiae AER2 gene is responsible for maintaining repression of at least two distinct regulatory pathways: heme activation/repression and catabolite repression. Mutations in the gene caused an eightfold increase in the expression of the heme-activated CYC1 gene in the absence of heme, a substantial increase in the expression of the heme-repressed ANB1 gene in the presence of heme, and a 13-fold increase in the expression of the catabolite-repressed GAL1 gene in the presence of glucose. Lesser or no increases in the expression of these genes were observed under derepressed or activation conditions. The aer2 mutations also caused a large increase in CYC7 gene expression under all conditions; this gene is subject to heme activation/repression, as well as catabolite repression. The AER2 gene was cloned and the sequence determined. The large open reading frame contiguous with the transcript from the complementing region encoded a 713-amino acid polypeptide chain with extensive homology to the beta-subunit of G proteins. The sequence revealed that AER2 is the TUP1 gene. A deletion mutation was constructed and the null phenotype was the same as the original mutants. The aer2 null mutant was shown to have increased aerobic and anaerobic levels of RNA encoding the ROX1 repressor, normally expressed only aerobically and responsible for the aerobic repression of ANB1 expression. The increase in both ROX1 and ANB1 RNAs aerobically in this mutant suggests that the repressor is nonfunctional in the mutant.