Regional assignment of the gene locus for steroid sulfatase

Summary The gene locus for steroid sulfatase, deficiency of which causes X-linked ichthyosis, is assigned to Xp11Xpter by analysis of 24 man-Chinese hamster somatic cell hybrids. High steroid sulfatase,activity in a hybrid clone having retained only part of Xq is explained by demonstration of an additional late-replicating human X chromosome. This observation confirms previous evidence for noninactivation of the STS locus.     

Height discordance in monozygotic females is not attributable to discordant inactivation of X-linked stature determining genes.

We tested the hypothesis that X-linked genes determining stature which are subject to skewed or non-random X-inactivation can account for discordance in height in monozygotic female twins. Height discordant female monozygotic adult twins (20 pairs) were identified from the Australian Twin Registry, employing the selection criteria of proven monozygosity and a measured height discordance of at least 5 cm. Differential X-inactivation was examined in genomic DNA extracted from peripheral lymphocytes by estimating differential methylation of alleles at the polymorphic CAG triplet repeat of the Androgen receptor gene (XAR). There were 17/20 MZ pairs heterozygous at this locus and informative for analysis. Of these, 10/17 both had random X-inactivation, 5/17 showed identical X-inactivation patterns of non random inactivation and 2/17 (12%) showed discordant X-inactivation. There was no relationship between inactivation patterns and self-report chorionicity. We conclude that non-random X-inactivation does not appear to be a major contributor to intra-pair height discordance in female MZ twins.     

Long-range physical mapping around the human steroid sulfatase locus

The region of the human X chromosome containing the steroid sulfatase locus was analyzed by pulsed-field gel electrophoresis. Restriction site maps were generated for the X chromosome in the blood of a normal male individual and that in the mouse-human hybrid cell line ThyB-X; these maps extend over approximately 4.3 Mb of DNA of the former, and 3.2 Mb of the latter. Physical linkage was defined between the STS locus and sequences detected by the probes GMGX9 (DXS237), GMGXY19 (DYS74), CRI-S232 (DXS278), and dic56 (DXS143), and the order telomere--(STS, DYS74)--DXS237--DXS278--DXS143--centromere was deduced. The pulsed-field maps were used to demonstrate a deletion of 180 kb of DNA from the X chromosome of an individual with X-linked ichthyosis. Also, possible locations for the Kallmann syndrome gene were revealed, and the distance between the steroid sulfatase locus and the pseudoautosomal region was estimated to be at least 4 Mb.     

Activity of steroid sulfatase in fibroblasts with numerical and structural X chromosome aberrations

Summary In cultured fibroblasts of patients with numerical and structural X chromosome aberrations the activity of steroid sulfatase (STS) is correlated with the number of functional STS gene copies. While normally, this X-linked gene is not inactivated, our data suggest that it may be subject to inactivation when carried on a structurally altered X-chromosome. Similar inactivation patterns have been reported earlier for the Xg locus which, like STS, is located on the distal protion of Xp.     

Assignment by deletion mapping of the steroid sulfatase X-linked ichthyosis locus to Xp223

Summary A male child and his mother who are nullisomic and monosomic, respectively, for the distal portion of Xp because of an unbalanced X-Y translocation were tested for steroid sulfatase activity after clinical examination had yielded evidence for ichthyosis in the boy. Deficiency of steroid sulfatase was found in the male patient, while in his mother enzyme levels were in the heterozygous range. These results, based on cytogenetic evidence obtained with an elongation technique, indicate that the STS locus is at Xp223.     

Biochemical evidence for the non-inactivation of the steroid sulfatase locus in human placenta and fibroblasts

Summary Steroid sulfatase activities are significantly higher in placentas obtained after the birth of girls than after the birth of boys, and also in female fibroblasts compared to male strains. This constitutes biochemical evidence for the non-inactivation of the X-linked sulfatase locus. No hydrolytic activity is found in the fibroblasts of ichthyotic boys. Heterozygosity is demonstrated in the fibroblasts of the four mothers studied, as they have steroid sulfatase activity of less or equivalent to the normal male value.     

Steroid sulfatase. Biosynthesis and processing in normal and mutant fibroblasts

Antibodies raised against steroid sulfatase purified from human placenta were used to follow the biosynthesis of this enzyme in human skin fibroblasts. Steroid sulfatase is synthesized as a membrane-bound Mr-63 500 polypeptide with asparagine-linked oligosaccharide chains. Within 2 days, newly synthesized steroid sulfatase is processed to a mature Mr-61 000 form. The decrease in size is due to processing of the oligosaccharide chains, which are cleavable by endoglucosaminidase H in both the early and the mature form of steroid sulfatase. The processing involves mannosidase(s) sensitive to 1-deoxy-manno-nojirimycin. The half-life of the steroid sulfatase polypeptides is 4 days. Synthesis of steroid-sulfatase-related polypeptides and steroid sulfatase activity were not detectable in fibroblasts from four patients with X-linked ichthyosis.     

Localization of Y chromosome sequences and X chromosomal replication studies in XX males

Summary By in situ hybridization, Y-specific DNA sequences were localized on Xp22.3-Xpter of one of the two X chromosomes in all of eleven XX males studied. In nine of the cases the presence of the Y-specific DNA did not affect random X inactivation in fibroblasts. Fibroblasts of the other two cases showed a preferential inactivation of the Y DNA-carrying X chromosome. In only one of these two exceptions blood lymphocytes could also be studied, and here, random inactivation of the Y DNA-carrying X chromosome occurred. Furthermore, the gene dosage of steroid sulfatase (STS) was examined by Southern blot analysis. In ten of the cases including the one showing random X-inactivation in lymphocytes but not in fibroblasts, a double dosage of the STS gene is present. The remaining case with non-random inactivation shows a single STS gene dosage. This case was reported previously to have STS enzyme activity in the male range. It is assumed that, as a consequence of an unequal X-Y interchange, a deletion of X-specific DNA sequences may result in the preferential inactivation of the Y DNA-carrying X chromosome.     

Genetic background modifies inner ear and eye phenotypes of jag1 heterozygous mice

Mice heterozygous for missense mutations of the Notch ligand Jagged1 (Jag1) exhibit head-shaking behavior indicative of an inner ear vestibular defect. In contrast, mice heterozygous for a targeted deletion of the Jag1 gene (Jag1del1) do not demonstrate obvious head-shaking behavior. To determine whether the differences in inner ear phenotypes were due to the types of Jag1 mutations or to differences in genetic background, we crossed Jag1del1 heterozygous mice onto the same genetic background as the missense mutants. This analysis revealed that variation of the Jag1 mutant inner ear phenotype is caused by genetic background differences and is not due to the type of Jag1 mutation. Genome scans of N2 backcross mice identified a significant modifier locus on chromosome 7, as well as a suggestive locus on chromosome 14. We also analyzed modifiers of an eye defect in Jag1del1 heterozygous mice from this same cross.     

A comparative study on steroid sulfatase and arylsulfatase C in fibroblast clones from 45,X/47,XXX and 69,XXY

Summary Steroid sulfatase (STS) and arylsulfatase C (ARSC) were studied in fibroblast clones from a 45,X/47,XXX mosaic and from a 69,XXY triploidy with one or two active X chromosomes. The comparison of the 47,XXX with 45,X clones showed an incomplete gene dosage effect (1.8 for STS and 2.0 for ARSC). This was not the case for the triploid clones with different X-inactivation patterns. These results confirm previous reports on the non-inactivation of the STS gene, and establish X linkage and non-inactivation for the ARSC gene as well.     

Comparison of arylsulfatase C and steroid sulfatase from human placenta and liver

Human placental and hepatic arylsulfatase C (ASC) were purified to homogeneity and about 1,000-fold, respectively. Placental ASC hydrolyzed sterol sulfates at the same active site, whereas the major hepatic ASC did not. This major hepatic ASC isozyme was more thermolabile than placental ASC and steroid sulfatase from both placenta and liver. It was not precipitated by anti-bovine ASC IgG which quantitatively precipitated both placental ASC and steroid sulfatase activities from placenta and liver. A minor hepatic ASC isozyme with similar electrophoretic mobility to the placental enzyme copurified with the major hepatic ASC and is likely responsible for the steroid sulfatase activity in this organ. Hence, placental ASC and steroid sulfatase are biochemically and antigenically identical to hepatic steroid sulfatase. In contrast, the major hepatic ASC is a distinct protein whose catalytic and structural properties differ from all the above enzymes.     

Association of steroid sulfatase with one of the arylsulfatase C isozymes in human fibroblasts

When arylsulfatase C, a microsomal membrane-bound enzyme, is assayed with its natural substrates, the 3-beta-hydroxysteroid sulfates, it is also known as steroid sulfatase. Whether arylsulfatase C and steroid sulfatase are identical enzymes or not, however, has long been disputed. We now report that two electrophoretic variants of arylsulfatase C occur in normal human fibroblasts: one has a single anodic band of activity, "s," and the other has an additional faster migrating band, "f". The two types, s and "f + s", occur in cells from either sex. When fibroblast strains with the f + s forms of arylsulfatase C were cloned, two types of primary clones were always obtained: s and f + s. A single f band was never seen. When these primary clones were subcloned, however, the arylsulfatase C phenotype remained unchanged: primary s clones gave rise to s subclones and f + s clones to f + s subclones only. Therefore, these forms were clonal in origin and demonstrated a novel inheritance pattern in human cultured cells. The appearance of increasing amounts of the f band was correlated with up to 4-fold increase of arylsulfatase C activity, whereas the steroid sulfatase activity remained constant, thus demonstrating that arylsulfatase C was not identical with steroid sulfatase activity. Polyclonal antibodies raised against the s form immunoprecipitated activities of the s form of arylsulfatase C and steroid sulfatase but not the f form of arylsulfatase C. Therefore, we conclude that only the s form of arylsulfatase C is immunologically related to steroid sulfatase so that arylsulfatase C per se is not necessarily identical with steroid sulfatase. In addition, a novel form of genetic heterogeneity of isozymes in human fibroblasts is demonstrated.     

Lack of effect of insulin or insulin-like growth factor I on the steroid sulfatase activity of human placental cytotrophoblasts

Hyperinsulinemia is known to reduce serum dehydroepiandrosterone sulfate (DHEA-S) levels in normal females. A possible mechanism for this phenomenon would be an insulin-mediated increase in steroid sulfatase activity, with insulin acting either via activation of the insulin receptor or via cross-reaction with the insulin-like growth factor I (IGF-I) receptor. Using a well characterized human cytotrophoblast system, the presence of steroid sulfatase activity in isolated cytotrophoblasts was documented. Half maximal cellular hydrolysis of DHEA-S was observed at a substrate concentration of 9.6-14.5 microM, and maximal hydrolysis at a concentration of 75-100 microM. The hypothesis that insulin increases steroid sulfatase activity was examined by exposing cytotrophoblasts to supraphysiological concentrations of either insulin (2 micrograms/ml) or IGF-I (20 ng/ml) for 24 h and then measuring the rate of DHEA-S hydrolysis. Insulin failed to affect cytotrophoblastic steroid sulfatase activity, irrespective of whether the substrate concentration was 20 microM or 100 microM. IGF-I also exerted no effect on steroid sulfatase activity. These data indicate that neither insulin nor IGF-I affect the steroid sulfatase activity of human cytotrophoblasts. An effect of insulin or IGF-I on the steroid-sulfatase activity of other tissues has not been excluded. These observations suggest that the decline in serum DHEA-S levels during hyperinsulinemia is not mediated via an insulin-induced increase in steroid sulfatase activity.     

Linkage analysis in X-linked ichthyosis (steroid sulfatase deficiency)

Summary Linkage analysis has been carried out in nine unrelated families segregating for X-linked ichthyosis (steroid sulfatase deficiency) using seven polymorphic DNA markers from the distal Xp. Close linkage was found between the disease locus and the loci DXS16, DXS89, and DXS143. In all families except one, Southern hybridization with the human steroid sulfatase cDNA and GMGX9 probes showed a deletion of corresponding loci in affected males. Three patients belonging to the same family had no evident deletion with either of the two above-mentioned probes. None of the other six DNA loci included in the linkage analysis were found to be deleted.     

Fabry disease: molecular diagnosis of hemizygotes and heterozygotes

Fabry disease, an X-linked inborn error of glycosphingolipid catabolism, results from the deficient activity of the lysosomal hydrolase, alpha-galactosidase A. Previously, the diagnosis of affected hemizygous males and heterozygous females was based on clinical findings and the levels of alpha-galactosidase A activity in easily obtained sources such as plasma and isolated lymphocytes or granulocytes. Since the gene encoding alpha-galactosidase A undergoes random X-inactivation, the expressed level of enzymatic activity in females heterozygous for the disease gene may vary significantly, thereby making accurate carrier detection difficult. The recent cloning and characterization of the full-length cDNA encoding human alpha-galactosidase A now permits the accurate diagnosis of affected hemizygotes and heterozygous females. In families with gene rearrangements or an altered restriction endonuclease cleavage site, precise diagnosis can be accomplished by Southern hybridization analysis using the alpha-galactosidase A cDNA as probe. In families with normal restriction patterns, two restriction fragment length polymorphisms have been identified in and adjacent to the alpha-galactosidase A gene which also allow precise hemizygote and heterozygote diagnosis. In addition, the recent identification of polymorphic, random DNA sequences (DXS17 and DXS87) located near the alpha-galactosidase A locus permits molecular diagnosis in informative families. Further evaluation of DXS17, DXS87 and other closely linked random DNA probes is required in order to determine their informativeness, proximity to the alpha-galactosidase A locus and, hence, accuracy for molecular diagnosis.     

An estimate of unique DNA sequence heterozygosity in the human genome

Summary Patients with recessive X-linked ichthyosis Patients with recessive X-linked ichthyosis (RXLI), one hereditary form of scaly skin, lack activity of the enzyme steroid sulfatase in all tissues studied. To investigate the molecular defect underlying the lack of enzyme activity, we prepared antisera against normal enzyme by injecting normal placental microsomal suspensions or partially purified steroid sulfatase into rabbits. Antibody activity was assessed by immunoprecipitation of detergent solubilized steroid sulfatase. In addition, we prepared rabbit antisera against RXLI placental microsomal suspensions. To detect immunologically cross-reactive material in patients' placentas, extracts were studied by immunoblot techniques and by competition with normal enzyme for antibody binding. Patients' extracts did not contain immunoreactive material co-migrating on electrophoresis with purified enzyme nor did they inhibit immunoprecipitation of normal enzyme. Sera from rabbits immunized with RXLI placental microsomes contain no antibodies to normal steroid sulfatase, as judged by their failure to immunoprecipitate normal enzyme or to react with normal steroid sulfatase on immunoblot. Thus the mutation in RXLI appears to reduce steroid sulfatase enzyme protein as well as enzyme activity. Portions of this material have appeared in abstract form in Clinical Research 31:564A, 1983 and 32:138A, 1984     

Retinoid-mediated stimulation of steroid sulfatase activity in myeloid leukemic cell lines requires RARalpha and RXR and involves the phosphoinositide 3-kinase and ERK-MAP kinase pathways

All-trans retinoic acid and 9-cis-retinoic acid stimulate the activity of steroid sulfatase in HL60 acute myeloid leukemia cells in a concentration- and time-dependent manner. Neither of these 'natural retinoids' augmented steroid sulfatase activity in a HL60 sub-line that expresses a dominant-negative retinoic acid receptor alpha (RARalpha). Experiments with synthetic RAR and RXR agonists and antagonists suggest that RARalpha/RXR heterodimers play a role in the retinoid-stimulated increase in steroid sulfatase activity. The retinoid-driven increase in steroid sulfatase activity was attenuated by inhibition of phospholipase D (PLD), but not by inhibitors of phospholipase C. Experiments with inhibitors of protein kinase C (PKC) show that PKCalpha and PKCdelta play an important role in modulating the retinoid-stimulation of steroid sulfatase activity in HL60 cells. Furthermore, we show that pharmacological inhibition of the RAF-1 and ERK MAP kinases blocked the retinoid-stimulated increase in steroid sulfatase activity in HL60 cells and, by contrast, inhibition of the p38-MAP kinase or JNK-MAP kinase had no effect. Pharmacological inhibitors of the phosphatidylinositol 3-kinase, Akt, and PDK-1 also abrogated the retinoid-stimulated increase in steroid sulfatase activity in HL60 cells. These results show that crosstalk between the retinoid-stimulated genomic and non-genomic pathways is necessary to increase steroid sulfatase activity in HL60 cells.