Detection of mosaicism in amniotic fluid cultures: a CYTO2000 collaborative study.

PURPOSE: To evaluate the assumptions on which the American College of Medical Genetics (ACMG) Standards and Guidelines for detecting mosaicism in amniotic fluid cultures are based. METHODS: Data from 653 cases of amniotic fluid mosaicism were collected from 26 laboratories. A chi-square goodness-of-fit test was used to compare the observed number of mosaic cases with the expected number based on binomial distribution theory. RESULTS: Comparison of observed data from the in situ colony cases with the expected distribution of cases detected based on the binomial distribution did not reveal a significant difference (P = 0.525). CONCLUSIONS: The empirical data fit the binomial distribution. Therefore, binomial theory can be used as an initial discussion point for determining whether ACMG Standards and Guidelines are adequate for detecting mosaicism.

Unique mosaicism of tetraploidy and trisomy 8: clinical, cytogenetic, and molecular findings in a live-born infant.

We report on a live-born infant with mosaicism of tetraploidy and trisomy 8 who had craniofacial abnormalities, cardiac and genitourinary defects, agenesis of the corpus callosum, and anomalies of limbs. The infant died at age 14 weeks. Molecular studies were done on peripheral blood lymphocytes and cultured amniocytes to determine the origin of the cytogenetic abnormalities. On the basis of the results, we describe a possible mechanism to explain these abnormalities. To our knowledge, this infant represents the first reported case of mosaic trisomy 8 with a tetraploid cell line.

Incidence and significance of chromosome mosaicism involving an autosomal structural abnormality diagnosed prenatally through amniocentesis: a collaborative study.

Among 179,663 prenatal diagnosis cases collected from ten institutions and two publications, 555 (0.3 per cent) were diagnosed as having chromosome mosaicism. Of these, 57 (10.3 per cent) were mosaic for an autosomal structural abnormality, 28 (5 per cent) for a sex chromosome structural abnormality, and 85 (15.3 per cent) were mosaic for a marker chromosome. Ninety-five cases of prenatally diagnosed mosaicism with a structural abnormality in an autosome and a normal cell line, and with a known phenotypic outcome, were collected for karyotype-phenotype correlations through our collaboration (40 cases), a prior survey (26 cases), and published reports (29 cases). They included 13 balanced reciprocal translocations, one unbalanced reciprocal translocation, four balanced Robertsonian translocations, four unbalanced Robertsonian translocations, four inversions, 17 deletions, three ring chromosomes, 19 i(20q), seven +i(12p), six other isochromosomes, and 17 partial trisomies resulting from a duplication or other rearrangement. All cases mosaic for a balanced structural rearrangement resulted in a normal phenotype. All cases of 46/46,i(20q) resulted in normal liveborns. Five of seven cases with 46/47,+i(12p) had an abnormal phenotype compatible with Killian-Pallister syndrome. The overall risk for an abnormal outcome for a mosaic case with an unbalanced structural abnormality, excluding 46/46,i(20q) and 46/47,+i(12p), is 40.4 per cent. In the same category, the study also suggested a correlation between the percentage of abnormal cells and an abnormal phenotype. For mosaicism involving a terminal deletion, the possibility of a familial fragile site should be considered.

Effect of rabbit antimouse brain serum on marrow cells capable of curing W/Wv mice.

The W/Wv mouse has a recessively inherited defect in hematopoietic stem cells (HSC) but can be cured of its hematopoietic abnormalities by infusion of marrow from a co-isogeneic, +/+ mouse. The "curative" cell for the W/Wv is thought to be a subcompartment of the HSC that is capable of forming hematopoietic spleen colonies (CFU-S) in irradiated mice. The curative HSC must have a very high proliferative potential and it is known that HSC with variable degrees of proliferative potential are found within the CFU-S compartment. Rabbit antimouse brain serum (RAMBS) was used to treat +/+ marrow and its effect upon CFU-S and upon curative cells was compared with the effect of normal rabbit serum (NRS) or of sham treatment. CFU-S were reduced to 70%-79% of control by NRS and to 8%-9% by RAMBS. Curative cells for the W/Wv were not detectably reduced by NRS; they were reduced by RAMBS, but to only approximately 20%-30% of control. Thus, it appeared to a certain degree that RAMBS spared HSC with a high proliferative potential when compared with its effect on the entire CFU-S compartment.

Localization of the human myelin basic protein gene (MBP) to region 18q22----qter by in situ hybridization.

A restriction endonuclease fragment derived from a cloned portion of human genomic DNA corresponding to the myelin basic protein gene has been used to map the position of this gene by in situ hybridization to human metaphase chromosomes. Ten percent of the radioactively labeled sites observed were on chromosome 18. Eighty-four percent of the grains on chromosome 18 were located within the region corresponding to 18q22----qter. This represents a greater than 10-fold increase in labeling at this position over the background grain distribution found along all of the other chromosomes.

Transplantation of chromosomally marked syngeneic marrow cells into mice not subjected to hematopoietic stem cell depletion.

The percentage of donor-host chimerism was determined 4-6 weeks or six months after injection of normal bone marrow cells into normal syngeneic or coisogeneic recipient mice. Donor-recipient pairs had chromosome markers that provided easy identification of metaphase cells. The percentage of donor cells in marrow or spleen ranged from 0 to 16% and this percentage was independent of the age of recipient or attempts to stimulate hematopoiesis in donor and/or host mice. In adult C57BL/6 mice there was a roughly linear dose-response relationship between cell dose and percentage of chimerism. There was no apparent dose-response relationship for AKR mice. The percentage of donor cells in the spleen was correlated to that seen in the marrow of recipients. Neonatal mice given the same intraperitoneal marrow cell dose as weanlings, but a larger number of cells relative to their own marrow mass, did not show a larger percentage of chimerism than weanlings. Similarly, weanlings given the same intravenous dose as adults showed no greater degree of chimerism than adults. Temporary anemia, induced by bleeding donors prior to cell collection, or more chronic hemopoietic stimulus (produced by injecting recipients with phenylhydrazine prior to cell injection with subsequent bleeding at intervals) did not result in an increased percentage of chimerism. These results indicate that there are "empty" sites in bone marrow of normal mice in which injected hematopoietic stem cells can lodge and grow.

Aging and hematopoiesis. II. The ability of bone marrow cells from young and aged mice to cure and maintain cure in W/Wv.

The proliferative ability of hematopoietic stem cells (HSC) was compared in young and aged mice. Infusion of coisogeneic marrow cures the hematopoietic defect of the W/Wv, a mouse with a recessively inherited defect in stem cells, including HSC. W/Wv were cured with equal frequency by relatively small doses of marrow (5 X 10(4) nucleated cells, an average of 2-3 "curative" HSC per aliquot) from 3-month-old or 27-month-old +/+ donors. After functioning in the originally cured W/Wv for 26 months, the marrow was used to cure other W/Wv. After functioning in secondary recipients for 16 months, it was, in turn, used to cure still other W/Wv. There was no difference between "old" and "young" bone marrow with respect to the frequency of cure in W/Wv, the duration of cure--or, in regard to marrow from cured W/Wv, the number of nucleated and peroxidase-positive cells per humerus and the number of cells capable of producing spleen colonies in irradiated recipients. Thus, these studies fail to disclose any evidence for a proliferative limitation for old as compared to young HSC.

Trisomy of chromosome 6.15 is not necessary for proliferation of AKR(Rb6.15)1Ald lymphoma cells.

By utilizing a unique AKR(Rb6.15)Ald (AKRb) mouse line we have been able to determine the incidence and frequency of cells with and without abnormalities of chromosome number (specifically trisomy 6.15) in spontaneous and transplanted lymphoma. The transplanted lymphoma cells were easily distinguished from the normal cells of AKR/J recipients by the presence in diploid cells of 2 metacentric and 36 acrocentric chromosomes. The majority of the cells from 17 normal control AKRb mice were diploid and trisomy 6.15 (3 metacentric and 36 acrocentric chromosomes) was not seen. In 73% of the 59 AKRb mice with spontaneous lymphoma, the majority of cells were diploid. In 54% of the mice none of the cells had trisomy 6.15, and in 22% of these mice a majority (greater than 50%) of cells had trisomy 6.15. In order to determine which of the cells from AKRb mice with spontaneous lymphoma were malignant, they were transplanted into normal young AKR/J recipient mice, and when lymphoma developed in recipients, their cells were analyzed. The majority of first passage lymphoma cells from 30 recipient mice were donor-type and the majority of these donor cells were diploid. Ten (17%) of these recipients had a majority of cells with trisomy 6.15. In order to further determine whether or not trisomic lymphoma cells had a different proliferative rate than diploid lymphoma cells, eight first passage recipient mice were used to start sequential passage lines. In two diploid lines and four trisomic lines, the same relative frequency of the trisomy was maintained in the sequential passages. However, in two trisomic lines, the frequency of lymphoma cells with three metacentric chromosomes diminished after passage. These studies suggest that aneuploidy is not necessary for proliferation of lymphoma and that diploid cells are a major part of the malignant cell population.

Hematopoietic stem cells with high proliferative potential. Assay of their concentration in marrow by the frequency and duration of cure of W/Wv mice.

THIS STUDY WAS DESIGNED TO APPROACH TWO PRIMARY QUESTIONS CONCERNING HEMATOPOIETIC STEM CELLS (HSC) IN MICE: what is the concentration of HSC with extensive proliferative potential in marrow, and how long can an HSC continue to function in an intact animal? The assay system was the W/W(v) mouse, a mouse with an inherited HSC defect, reflected in a reduction in all myeloid tissue and most particularly in a macrocytic anemia.A single chromosomally marked HSC will reconstitute the defective hematopoietic system of the W/W(v). The concentration of HSC in normal littermate (+/+) marrow was assayed by limiting dilution calculation using cure of W/W(v) as an end point (correction of anemia and erythrocytes' macrocytosis) and found to be approximately 10/10(5). This is significantly less than spleen colony forming cell (CFU-S) concentration: approximately 220/10(5) in +/+ and ranging from 50 to 270/10(5) in various other studies. Blood values were studied at selected intervals for as long as 26 mo. Of 24 initially cured mice, which were observed for at least 2 yr, 75% remained cured. However, of all cured mice, 17 lost the cure, returning to a macrocytic anemic state. Cured mice had normal numbers of nucleated and granulocytic cells per humerus and a normal concentration of CFU-S. However, cure of secondary W/W(v) recipients by this marrow was inefficient compared with the original +/+ marrow. These studies suggest the CFU-S assay over-estimates extensively proliferating HSC or perhaps does not assay such a cell. A single such HSC can not only cure a W/W(v), but can sustain the cure for 2 yr or more, despite a relative deficit of cells capable of curing other W/W(v). However, the duration of sustained cure may be finite.

In vitro clonal assay for human metastatic melanoma cells.

An in vitro assay for clonogenic tumor cells was applied to human metastatic melanomas. Melanoma colony formation was observed in 22 of 33 samples obtained from a variety of involved tissues. The melanocytic tumor origin of colonies was established by serial observations by inverted light microscopy, staining of fixed colonies for melanin, and cytological and karyotypic analysis of cells within colonies. Two morphologically distinct types of colonies were identified, one consisting of light large cells and the second of dark small cells. Investigations of factors which modulate growth and differentiation of clonogenic melanoma cells may provide a clearer understanding of this neoplasm.

Biological and biochemical properties of a human uveal melanocyte-derived cell line.

A human uveal melanocyte-derived cell line (U1I) is described. The cell line has a doubling time of 27.2 hr, a plating efficiency on plastic surfaces of 10%, and a cloning efficiency in soft agar of < 0.1%. U1I displays marked chromosomal aneuploidy and sensitivity to ultraviolet light. Biochemical studies indicate the presence of tyrosinase, which is stimulated by several compounds, including theophylline, progesterone, and nerve growth factor.