Rapid aneuploid diagnosis of high-risk fetuses by fluorescence in situ hybridization.

OBJECTIVE: Our purpose was to develop fluorescence in situ hybridization to repetitive chromosome-specific sequences to detect chromosome aneuploidy faster than hybridization to unique targets or karyotyping. STUDY DESIGN: Aneuploidy involving chromosomes 13, 18, 21, X, and Y comprises 70% of chromosome abnormalities in 10- to 12-week fetuses, 95% of the phenotypically significant newborn chromosome abnormalities. Our improved 8-hour protocol used repetitive probes to label and count the number of these centromeric chromosome domains. RESULTS: This protocol correctly determined chromosome 13, 18, and 21 status in 50 of 50 unselected direct amniocyte samples and found abnormal patterns in 27 of 27 archived trisomy 21 cases. Altogether karyotyping confirmed 744 of 745 chromosome-specific repetitive sequence test results. CONCLUSION: This protocol rapidly tests abnormal fetuses and newborn infants in whom diagnosis is made at the initiation of labor or before urgent surgery when a cytogenetic result cannot be completed.

Testicular amplification and impaired transmission of human butyrylcholinesterase cDNA in transgenic mice.

Gene amplification occurs frequently in tumour tissues yet is, in general, non-inheritable. To study the molecular mechanisms conferring this restraint, we created transgenic mice carrying a human butyrylcholinesterase (BCHE) coding sequence, previously found to be amplified in a father and son. Blot hybridization of tail DNA samples revealed somatic transgene amplifications with variable restriction patterns and intensities, suggesting the occurrence of independent amplification events, in 31% (11/35) of mice from the FII generation but in only 3.5% (2/58) of the FIII and FIV generations. In contrast, > 10-fold amplifications of the BCHE transgene and the endogenous acetylcholinesterase and c-raf genes appeared in both testis and epididymis DNA from > 80% of FIII mice. Drastic, selective reductions in testis BCHEmRNA but not in actin mRNA were detected by the PCR amplification of testis cDNA from the transgenic mice, and apparently resulted in the limited transmission of amplified genes. The testicular amplification of the BCHE transgene may potentially represent a general phenomenon with clinical implications in human infertility.

In vivo gene amplification in non-cancerous cells: cholinesterase genes and oncogenes amplify in thrombocytopenia associated with lupus erythematosus.

The ACHE and BCHE genes, encoding the acetylcholine hydrolysing enzymes acetylcholinesterase (ACHE) and butyrylcholinesterase (BCHE), co-amplify with several oncogenes in leukemic patients with platelet deficiency (thrombocytopenia). This and other experiments implicated ACHE and BCHE in the development of bone marrow megakaryocytes, the progenitors of platelets. Therefore, we wished to find out whether cholinesterase gene amplification would also occur in non-cancerous platelet disorders and, if so, whether oncogenes would amplify in such cases as well. The autoimmune disease systemic lupus erythematosus (SLE) presents an appropriate model system for this issue, since patients with SLE may suffer from thrombocytopenia resistant to most treatment modalities. Here, we report a 40-80-fold amplification of genomic sequences from the ACHE and BCHE genes as well as the C-raf, V-sis and C-fes/fps oncogenes in peripheral blood cells from an SLE patient with severe thrombocytopenia. PvuII restriction analysis and DNA blot hybridization of the amplified ACHE and BCHE sequences demonstrated apparent aberrations in both genes, suggesting that malfunctioning of modified, partially amplified cholinesterase genes may be involved in the etiology of thrombocytopenia associated with SLE. These observations imply that cholinergic mechanisms regulate megakaryocytopoiesis, shed new light on the diverse hematologic findings characteristic of SLE, and may become valuable as diagnostic, treatment and prognostic tools in the follow-up of patients suffering from thrombocytopenia associated with SLE. Furthermore, these findings reinforce the notion that cholinesterase gene amplifications are causally related with platelet abnormalities in multiple hemopoietic disorders.

Cloning and antisense oligodeoxynucleotide inhibition of a human homolog of cdc2 required in hematopoiesis.

Mechanisms triggering the commitment of pluripotent bone marrow stem cells to differentiated lineages such as mononuclear macrophages or multinucleated megakaryocytes are still unknown, although several lines of evidence suggested correlation between cholinergic signaling and hematopoietic differentiation. We now present cloning of a cDNA coding for CHED (cholinesterase-related cell division controller), a human homolog of the Schizosaccharomyces pombe cell division cycle 2 (cdc2)-like kinases, universal controllers of the mitotic cell cycle. Library screening, RNA blot hybridization, and direct PCR amplification of cDNA reverse-transcribed from cellular mRNA revealed that CHED mRNA is expressed in multiple tissues, including bone marrow. The CHED protein includes the consensus ATP binding and phosphorylation domains characteristic of kinases, displays 34-42% identically aligned amino acid residues with other cdc2-related kinases, and is considerably longer at its amino and carboxyl termini. An antisense oligodeoxynucleotide designed to interrupt CHED's expression (AS-CHED) significantly reduced the ratio between CHED mRNA and actin mRNA within 1 hr of its addition to cultures, a reduction that persisted for 4 days. AS-CHED treatment selectively inhibited megakaryocyte development in murine bone marrow cultures but did not prevent other hematopoietic pathways, as evidenced by increasing numbers of mononuclear cells. An oligodeoxynucleotide blocking production of the acetylcholine-hydrolyzing enzyme, butyrylcholinesterase, displayed a similar inhibition of megakaryocytopoiesis. In contrast, an oligodeoxynucleotide blocking production of the human 2Hs cdc2 homolog interfered with production of the human 2Hs cdc2 homolog interfered with cellular proliferation without altering the cell-type composition of these cultures. Therefore, these findings strengthen the link between cholinergic signaling and cell division control in hematopoiesis and implicate both CHED and cholinesterases in this differentiation process.

A role for cholinesterases in tumorigenesis?

Hydrolysis of the neurotransmitter acetylcholine by acetylcholinesterase (ACHE) and butyrylcholinesterase (BCHE) is the rate-limiting step in the termination of cholinergic signaling at neuromuscular junctions. A growing body of evidence suggests that these enzymes also play a role in tumorigenesis. The ACHE and BCHE genes are amplified, mutated, and/or aberrantly expressed in a variety of human tumor types. These changes could be the result of chromosome breakage, since there is an unusually high frequency of chromosomal abnormalities near the map positions of these genes (3q26-ter and 11p-ter, respectively) in such tumors, particularly hemopoietic malignancies. Both ACHE and BCHE contain the consensus peptide motif S/T-P-X-Z, which is found in many substrates of cdc2-related protein kinases. Here we consider the intriguing possibility that phosphorylation by cdc2-related kinases may be the molecular mechanism linking cholinesterases with tumor cell proliferation. We also discuss the notion that inhibition of these enzymes by commonly used organophosphorous poisons may be tumorigenic in humans.

Coamplification of human acetylcholinesterase and butyrylcholinesterase genes in blood cells: correlation with various leukemias and abnormal megakaryocytopoiesis.

To study the yet unknown role of the ubiquitous family of cholinesterases (ChoEases) in developing blood cells, the recently isolated cDNAs encoding human acetylcholinesterase (AcChoEase; acetylcholine acetylhydrolase, EC 3.1.1.7) and butyrylcholinesterase (BtChoEase; cholinesterase; acylcholine acylhydrolase, EC 3.1.1.8) were used in blot hybridization with peripheral blood DNA from various leukemic patients. Hybridization signals (10- to 200-fold intensified) and modified restriction patterns were observed with both cDNA probes in 4 of the 16 leukemia DNA preparations examined. These reflected the amplification of the corresponding AcChoEase and BtChoEase genes (ACHE and CHE) and alteration in their structure. Parallel analysis of 30 control samples revealed nonpolymorphic, much weaker hybridization signals for each of the probes. In view of previous reports on the effect of acetylcholine analogs and ChoEase inhibitors in the induction of megakaryocytopoiesis and production of platelets in the mouse, we further searched for such phenomena in nonleukemic patients with platelet production disorders. Amplifications of both ACHE and CHE genes were found in 2 of the 4 patients so far examined. Pronounced coamplification of these two related but distinct genes in correlation with pathological production of blood cells suggests a functional role for members of the ChoEase family in megakaryocytopoiesis and raises the question whether the coamplification of these genes could be causally involved in the etiology of hemocytopoietic disorders.