The role of the sex-determining region Y gene in the etiology of 46,XX maleness.

The condition of 46,XX maleness is characterized by testicular development in subjects who have two X chromosomes but who lack a normal Y chromosome. Several etiologies have been proposed to explain 46,XX maleness: 1) translocation of the testis-determining factor from the Y to the X chromosome, 2) mutation in an autosomal or X chromosome gene which permits testicular determination in the absence of TDF, and 3) undetected mosaicism with a Y-bearing cell line. We evaluated 10 affected subjects who were ascertained for different reasons and who had several distinct phenotypes. Six subjects had inherited sequences from the short arm of the Y chromosome including the sex-determining region Y gene (SRY). Five of the subjects were pubertal at the time of evaluation and had a phenotype similar to that of Klinefelter syndrome with evidence of Sertoli cell and Leydig cell dysfunction. One subject had evidence from Southern blot analysis and in situ hybridization for the presence of an intact Y chromosome in approximately 1% of cells. Three subjects lacked Y sequences by Southern blot analysis and by polymerase chain reaction amplification of SRY. These subjects were ascertained in the newborn period because of congenital anomalies. One had multiple anomalies including cardiac abnormalities; one had cardiac anomalies alone; and one had ambiguous genitalia. Our data confirm the genetic heterogeneity of 46,XX maleness, in which some subjects have SRY while other subjects lack it. In addition, there is phenotypic heterogeneity among subjects who lack SRY suggesting that there is also genetic heterogeneity within this subgroup.

Mosaic trisomy 22: a case presentation and literature review of trisomy 22 phenotypes.

In a case of mosaic trisomy 22 the trisomic cells were detected primarily in fibroblasts. Results of initial lymphocyte chromosome analysis were normal. However, mosaicism was suspected because the patient had hypomelanosis of Ito, hemiatrophy, failure to thrive, and mental retardation. Mosaicism was confirmed in cultured fibroblasts. Repeat cytogenetic analysis of peripheral blood demonstrated a low level of trisomic metaphase cells, which was confirmed by interphase fluorescent in situ hybridization (FISH) analysis. Molecular studies supported maternal disomy in the child's disomic cells. The phenotype of this condition overlaps that of non-mosaic trisomy 22 chromosome mosaicism in general and to some extent the Ullrich-Turner syndrome phenotype. Improved cytogenetic and molecular techniques now allow better delineation of aneuploidy syndromes. Molecular and FISH studies added information about this case (mosaicism and uniparental disomy) not appreciated by routine cytogenetic analysis of lymphocytes. The detection of low-level mosaicism and/or uniparental disomy in such cases may change the clinical classification and our understanding of pathogenesis and recurrence risk of these disorders.

Reaction of monoclonal H-Y antibody in the ELISA.

Monoclonal H-Y antibody of demonstrated specificity was reacted with soluble H-Y antigen in a newly-developed enzyme-linked immunosorbent assay (ELISA). Typing of cell lines was accomplished by qualitative and quantitative absorption. In one case, the antibody was subdivided into equal portions, each of which was absorbed with 1, 3, 6 or 12 X 10(6) cells from male or female; after absorption, the cells were discarded and the antibody was titrated for residual activity in the ELISA. With increasing cell numbers, optical density scores for male-absorbed antibody approached baseline levels. Although male-female differences were pronounced and highly significant, there was also a fall in reactivity after absorption with female cells.

Sex selection with monoclonal H-Y antibody.

H-Y antigen defined by antibody from male-sensitized female mice has been reported in male embryos of mouse, rat, cattle, goat, pig, and sheep. We now describe the use of monoclonal H-Y antibodies in identification of male and female during embryo transfer in cattle. Monoclonal H-Y antibodies were applied with fluorescein-isothiocyanate-conjugated goat antimouse immunoglobulin (Ig), and the embryos were scored for fluorescence under ultraviolet light. In alternative trials with 149 embryos, the method could be applied with 73% to 82% efficiency. Pregnancy rates for treated embryos were at least as high as those expected for untreated embryos. The calves that developed from the treated embryos are normal and growing.

Lens protein phylogeny: immunocrossreactivity of vertebrate lens antigens to anti-shark crystallin antibody.

Antisera prepared against total water-soluble lens proteins of the shark, Scoliodon sorrakowah were reacted with homologous antigen and analysed reaction products by immunoelectrophoresis (IE) and two dimensional crossed antigen-antibody electrophoresis (2D-CE). On IE, shark antigens formed 5 precipitin lines including 1 alpha, 3 beta and 1 gamma crystallins and on 2D-CE 3 alpha, 6 beta and 6 gamma peaks accounting for 8%, 27% and 65% antigen in the respective group were obtained from the total crystallins. Using anti-shark antisera, the immunocrossreactivity of lens proteins from 6 Chondropterygii, 23 teleosts and 16 higher vertebrates was examined by IE. It is found that beta crystallins are the most conserved and crossreact with all vertebrate classes, whereas gamma crystallin crossreactivity is specific to the class Pisces and alpha crystallins are least conserved and their crossreactivity is confined to subclass Chondropterygii. Based on IE patterns, a phylogenetic tree is constructed which demonstrates the intrafamily closeness except in case of adaptive radiation.

Differential expression of H-Y antigen on lymphocyte subsets: analysis by flow cytometry.

Expression of H-Y antigen in human white blood cells was measured using flow cytometry with monoclonal antibodies. In this system, lymphocytes were stained preferentially in the male, and to a lesser extent in the female. Analysis of the lymphocyte subsets with biotinylated H-Y antibody conjugated with streptavidin-fluorescein isothiocyanate (FITC) and subset-specific antibody conjugated with phycoerythrin derivative (RD1) revealed differential expression of H-Y among the subsets of the male. In samples from eight men, 41.1% +/- 21.7% of B cells (B1) were stained, compared with 20.7% +/- 12.8% of cytotoxic-suppressor T cells (T8) and 5.4% +/- 3.0% of helper-inducer T cells (T4). In samples from seven women, 12.4% +/- 10.9% of B cells were stained, but staining of T cells was negligible.

Studies on cell-mediated immunity in women using different fertility regulating methods.

Cell-mediated immunity (CMI) was assessed in women using fertility regulating methods for 1-5 months, 6-11 months or 12-18 months. The CMI as assessed by phytohaemagglutin in (PHA)-induced lymphocyte transformation of treated groups were compared with that of normal subjects who were not using any contraceptive methods and women on conventional methods of contraception. The data obtained indicates that there is no significant alteration of CMI in women fitted with IUCD or women on estrogen progestogen combination. However, a significant suppression of CMI is observed in women in progestogen pills for 12-18 months. The short term therapy did not affect the CMI. In a prospective study it was found that the CMI in women before and after the use of combination therapy for 1-5 and 6-11 months revealed no change. Estradiol and progesterone at concentration on 1 microgram/ml in culture medium suppressed PHA-induced lymphocyte transformation.

Inability to detect fetal metaphases in flow-sorted lymphocyte cultures based on maternal-fetal HLA differences.

Separation of fetal cells from maternal blood could provide a means for prenatal diagnosis that would not endanger the fetus. In this pursuit, we attempted cytogenetic analysis of candidate fetal cells flow sorted on the basis of parental HLA disparity. Metaphases showing 46,XY or aneuploidy and concordant with prenatal diagnostic studies (i.e., amniocentesis, chorionic villus sampling) would presumably be fetal in origin. Blood samples were obtained from 78 pregnant women and their partners. Among 18 HLA informative cases in which metaphases were recovered, 15 involved fetuses that were 46,XY or aneuploid. From these 15 cases, 2,483 metaphases were analyzed. All metaphases were 46,XX. Cytogenetic analysis of flow-sorted fetal cells thus probably will need to emphasize not metaphase analysis but in situ hybridization with chromosome-specific probes.