High frequency de novo alterations in the long-range genomic structure of the mouse pseudoautosomal region.

The pseudoautosomal region (PAR) is a segment of shared homology between the X and Y chromosomes. Here we report physical linkage of three mouse PAR probes: DXYHgu1, DXYMov15 and (TTAGGG)n. Steroid sulphatase (Sts) maps distal to these probes, indicating that there is an internal array of the telomere sequence (TTAGGG)n in the PAR. Pseudoautosomal PacI restriction fragments, up to 2 Mb in size, are unstable in C57BL/6 x C57BL/6 crosses. New alleles, often several hundred kilobases different in size, occur at a sex-averaged rate of approximately 30% per allele. Such frequent large-scale germline genome arrangements are without precedent in mammals.

Expression of the X-inactivation-associated gene XIST during spermatogenesis.

Mammalian X-chromosome inactivation is thought to be controlled by the X inactivation centre (XIC, X-controlling element -Xce-in mice). A human gene, XIST and its mouse counterpart, Xist, which map to the XIC/Xce, are expressed exclusively from inactive X chromosomes, suggesting their involvement in the process of X-inactivation. We now report the presence of Xist/XIST transcripts in newborn and adult mouse testes, and in human testicular tissue with normal spermatogenesis, but not in the testes of patients who lack germ cells. Our results indicate that while the X chromosome in males is active in somatic cells, it undergoes inactivation during spermatogenesis.

Cloning and expression of the mouse pseudoautosomal steroid sulphatase gene (Sts).

Steroid sulphatase (STS) is an important enzyme in steroid metabolism. The human STS gene has been cloned and mapped to Xp22.3, proximal to the pseudoautosomal region (PAR). Using quantitative differences in STS activity among various mouse strains, a segregation pattern consistent with autosomal linkage was first reported, but more recent studies have linked Sts to the mouse PAR. Failed attempts to clone the mouse Sts gene using human reagants (STS cDNA and anti-STS antibodies) suggest a substantial divergence between these genes. However, partial amino-terminal sequence from purified rat liver Sts is very similar to its human counterpart, and several domains are conserved among all the sulphatases. We followed a degenerate-primer reverse transcriptase-PCR (RT-PCR) approach to amplify a conserved fragment of the rat Sts cDNA that was then used to clone the mouse Sts cDNA. This 2.3-kb cDNA revealed 75% similarity with rat Sts cDNA, while it was only 63% similar to human STS cDNA. Transfection of STS(-) A9 cells with the mouse Sts cDNA restored STS enzymatic activity. Sts was also mapped physically to the distal end of the mouse sex chromosomes, and our backcross studies placed Sts distal to the 'obligatory' cross-over in male meiosis.

The murine Xe169 gene escapes X-inactivation like its human homologue.

Among a number of genes that escape X-chromosome inactivation in humans, three have been evaluated in mice and unexpectedly all three are subject to X-inactivation. We report here the cloning and expression studies of a novel mouse gene, Xe169, and show that it escapes X-inactivation like its human homologue. Xe169 was assigned to band F2/F3 on the mouse X chromosome by fluorescent in situ hybridization and Southern analysis indicates that the gene is located outside the pseudoautosomal region. Homologous, but divergent, sequences exist on the Y chromosome. In vitro and in vivo studies show that Xe169 is expressed from both the active and the inactive X chromosomes. Xe169 is the first cloned non-pseudoautosomal gene that escapes X-inactivation in mice.

Reactivation of an inactive human X chromosome: evidence for X inactivation by DNA methylation.

A mouse-human somatic cell hybrid clone, deficient in hypoxanthine-guanine phosphoribosyltransferase (HPRT) and containing a structurally normal inactive human X chromosome, was isolated. The hybrid cells were treated with 5-azacytidine and tested for the reactivation and expression of human X-linked genes. The frequency of HPRT-positives clones after 5-azacytidine treatment was 1000-fold greater than that observed in untreated hybrid cells. Fourteen independent HPRT-positive clones were isolated and analyzed for the expression of human X markers. Isoelectric focusing showed that the HPRT expressed in these clones is human. One of the 14 clones expressed human glucose-6-phosphate dehydrogenase and another expressed human phosphoglycerate kinase. Since 5-azacytidine treatment results in hypomethylation of DNA, DNA methylation may be a mechanism of human X chromosome inactivation.

Non-inactivation of an x-chromosome locus in man.

Cloned fibroblasts from women heterozygous for X-linked ichthyosis (steroid sulfatase deficiency) were examined to see whether or not this locus is subject to X-inactivation. Of 103 clones examined, all had normal levels of steroid sulfatase activity. Two of the women studied were also heterozygous for glucose-6-phosphate dehydrogenase deficiency. This allowed the demonstration that both X chromosomes were represented as the active X in various clones and that selection did not account for these findings. Thus, the steroid sulfatase locus, like the Xga locus to which it is linked, appears to escape X-inactivation in man.

Expression of steroid sulfatase during embryogenesis.

Neurosteroids are steroids that are synthesized de novo in the brain from cholesterol and, in general, mediate their effects through ion-gated channel receptors such as gamma-aminobutyric acidA (GABA[A]) and N-methyl-D-aspartate receptors rather than through classical nuclear steroid hormone receptors. Steroid hormones are known to exist not only as free compounds, but also as sulfated derivatives. Pharmacological studies indicate that unconjugated and sulfated steroids, such as pregnenolone and pregnenolone sulfate, may have opposite effects on GABA(A) receptors. Thus, pregnenolone acts as a potent positive allosteric modulator of gamma-aminobutyric acid action at GABA(A )receptors, whereas pregnenolone sulfate acts as a potent negative modulator. Recent experiments also suggest that dehydroepiandrosterone and dehydroepiandrosterone sulfate may have distinct effects on growth of neurites from embryonic neocortical neurons in vitro. Thus, regulation of steroid sulfation may have profound behavioral and morphological effects on the nervous system. We, therefore, studied the developmental expression of the enzyme steroid sulfatase (STS), which converts sulfated steroids to free steroids. By in situ hybridization, STS messenger RNA was expressed in the embryonic mouse cortex, hindbrain, and thalamus during the last third of gestation. The sites of expression of STS were similar to those of P450c17, suggesting that these two enzymes may have concerted actions in similar functional processes.

Increased cholesterol sulfate in plasma and red blood cell membranes of steroid sulfatase deficient patients.

Steroid sulfatase deficiency is an inborn error of metabolism characterized during fetal life by decreased estriol production and postnatally by x-=linked ichthyosis. No consistent substrate abnormalities have been found beyond the perinatal period. Utilizing gas chromatography, we found that the cholesterol sulfate concentration was less than 350 micrograms/100 ml plasma in 9 normal adults, 2 subjects with ichthyosis vulgaris and 2 subjects with lamellar ichthyosis. Control red cell membranes had less than 300 micrograms/100 ml erythrocytes. Eight subjects (age 3 months-74 years) with steroid sulfatase deficiency had strikingly elevated cholesterol sulfate levels with means and ranges as follows: plasma - 3,300 micrograms/100 ml (2,700-4,000), red cell membranes- 7,500 (5,200-9,800) Cholesterol sulfate is known to effect membrane stability and the present observations may help to explain the pathogenesis of STS deficiency and x-linked ichthyosis.

Steroid sulfatase expression in human placenta: immunocytochemistry and in situ hybridization study.

Steroid sulfatase (STS), an important enzyme in the pathway of estrogen synthesis from sulfated steroid precursors, was localized to the syncytial trophoblast of human placentas during different periods of pregnancy by using a mouse monoclonal antibody and immunocytochemical techniques. Preembedding immunoelectron microscopy revealed STS immunoreactivity associated with the rough endoplasmic reticulum of the syncytial trophoblast. STS mRNA was also localized to this outermost layer of the human trophoblast. At a cellular level, both STS message and immunoreactivity seemed to be more abundant during the late first and early second trimesters than in term placentas. Although the syncytial trophoblast is known to originate from the cytotrophoblast, neither STS immunoreactivity nor STS mRNA was detected in the cytotrophoblast at any stage of placental development studied.

Prenatal in situ hybridization test for deleted steroid sulfatase gene.

X-linked ichthyosis results from steroid sulfatase (STS) deficiency; 90% of affected patients have a complete deletion of the entire 146 kb STS gene on the distal X chromosome short arm (Xp22.3). In these families prenatal diagnosis and carrier testing can be completed in 2 days by hybridizing simultaneously 2 different cosmid probes labeled with fluorescein or Texas red and counterstaining interphase nuclear DNA with DAPI. An STS gene probe labeled with Texas red hybridizes specifically to the steroid sulfatase gene on the X chromosome. A second flanking probe labeled with fluorescein hybridizes to both the normal Y chromosome and normal and STS deleted X chromosomes. In this fashion the interphase nuclei of normal males, affected males, normal females, and carrier females can be distinguished unambiguously. Because normal males and carrier females each show two yellow-green fluorescein spots and one Texas red STS spot, use of this test prenatally requires determining fetal sex independently with repetitive X and Y chromosome-specific probes. This procedure can be used with lymphocytes, direct and cultured chorionic villus cells, direct and cultured amniocytes, and fibroblasts. Similar methods are anticipated to be useful for rapid diagnostic assessment of other aneuploid gene disorders.

Prenatal diagnosis of Maroteaux-Lamy syndrome.

Maroteaux-Lamy syndrome exhibits deficient activity of the enzyme arylsulfatase-B in cultured skin fibroblasts. Prenatal diagnosis was successfully attempted in two pregnancies of a consanguineous Chaldean couple whose first child is affected with Maroteaux-Lamy syndrome. In both instances, deficient arylsulfatase-B activity was observed in amniotic fluid cell cultures, and the diagnosis was confirmed by 35S-sulfate studies and postmortem enzymology and electron microscopy. The prenatal diagnosis of Maroteaux-Lamy syndrome remains problematic. Residual activity of arylsulfatase-B in the affected homozygote can make interpretation difficult, and the behavior of many lysosomal enzymes varies greatly in response to tissue culture conditions and enzyme extraction processes.

Familial X-linked ichthyosis, steroid sulfatase deficiency, mental retardation, and nullisomy for Xp223-pter.

Steroid sulfatase (STS)-deficient X-linked ichthyosis was diagnosed in a man with short stature and mental retardation. His generation includes five similarly affected male members. A translocation chromosome is segregating in this Newfoundland kindred. The proband's mother and grandmother have normal skin and are of normal intelligence. From his carrier mother, the proband inherited an X short arm (Xp) to Y long arm (Yq) translocation chromosome, with the entire Y short arm and the X short arm terminal segment deleted (Xp223-pter). His cells are completely deficient in STS activity, confirming assignment of the STS locus to Xp223-pter. Effective management of his ichthyosis included treatment with 6% salicylic acid gel under plastic occlusion and removal of the scales by scrubbing.

Serum pyruvate-kinase (PK) and creatine-phosphokinase (CPK) in female relatives and patients with X-linked muscular dystrophies (Duchenne and Becker).

Determination of serum creatine phosphokinase (CPK) activity is often used in efforts to detect carriers of X-linked muscular dystrophies. We have recently demonstrated that another serum enzyme, pyruvate-kinase (PK) may also be of use in the diagnosis of patients affected with a variety of neuromuscular disorders. To evaluate the usefulness of this assay for carrier detection, a comparative study of serum PK and CPK activity was performed in 74 female relatives of patients affected with Duchenne (DMD) and Becker (BMD) muscular dystrophies. For obligate carriers of the DMD gene, 10 of 14 had elevated CPK's, 11 of 14 had elevated PK's and 12 of 14 had abnormal results for either of the two enzymes. Three of 16 mothers of isolated cases had increased serum CPK activity and 6 of 16 had increased PK activity (7 had elevation of at least one enzyme). These preliminary data suggest that the use of PK may enhance the capability to discriminate carriers for these X-linked recessive genes.

Serum pyruvate-kinase (PK) and creatine-phosphokinase (CPK) in progressive muscular dystrophies.

PK and CPK have been determined in the serum from 208 individuals including 70 normal controls (61 adults and 9 children) and 138 patients with a variety of neuromuscular disorders. In adult controls the mean activity (+/- SE) for PK is 1.2 +/- 0.05 mumol/ml/h. In normal children PK activity was about twice as high as in normal adults and decreases with increasing age. In 26 patients with Duchenne dystrophy the range of serum PK was 4.0-150.4 and in 17 individuals with the Becker type, 3.0 to 148.7. All had elevated PK and CPK levels. Eighteen of 20 patients with the facio-scapulo-humeral (FSH) from of muscular dystrophy had increased PK while only 9 had elevated CPK. Regression analyses have shown an inverse correlation between PK levels and age (or degree of disability in DMD). Kinetic and electrophoretic studies indicate that the PK isozyme found in the serum from affected patients and from heterozygotes for the DMD gene is mainly the M1 type PK, which is the only PK isozyme found in skeletal muscle and brain and the major component from myocardium.

The ADA at three years: a statute in flux.

In the three years since the Americans with Disabilities Act (ADA) was enacted, significant developments have occurred in the form of new administrative and judicial interpretations of the Act. The new guidelines and decisions will assist employers in complying with the ambiguous, and sometimes confusing, provisions of the ADA. Recent developments in areas such as the definition of a disability and the permissibility of medical inquiries, along with continuing developments in the areas of mental disabilities and defining reasonable accommodations, give employers insight into the obligations created by the ADA. These developments are a good starting point to understanding the ADA, but there is still a long way to go. This article surveys the recent developments in the law and examines the considerations that have become important to the Equal Employment Opportunity Commission and the courts in interpreting the ADA's provisions.