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Steroid Sulfatase Deficiency and the Genetics of the Short Arm of the Human X Chromosome
Abstract
It is considered axiomatic in human genetics that the study of relatively rare disorders may yield far more in dividends than might be anticipated based on the incidence of the condition in question. This has clearly been demonstrated in studies of human steroid sulfatase deficiency and the steroid sulfatase system during the past few years. Investigations of this infrequent human variation have led to expanded studies of sulfated steroid metabolism, the physiological control of epidermal keratinization, estrogen biosynthesis in pregnancy, testosterone biosynthesis, and the molecular mechanism of X-chromosome inactivation and the escape of inactivation of certain portions of the human X. In addition, the availability of a readily scoreable marker for the distal human short arm provides the potential basis for a number of observations regarding X/Y interchange involving this portion of the X and has raised a number of evolutionary issues as well. Further studies may help clarify several of these questions and substantially add to our understanding of a variety of human X-chromosome disorders, such as X aneuploid states, XX males, and true hermaphrodites.
... Serum lipoprotein electrophoresis is also diagnostic, demonstrating more rapid mobility of the LDL (beta) and pre-LDL (pre-beta) fractions due to an increase in sulfated sterol content [15,17]; however, this assay is no longer widely available. Because most XLI cases arise from deletion of the STS gene [18][19][20][21][22][23][24][25][26], fluorescence in situ hybridization (FISH) analysis is commonly employed for diagnosis of XLI and its carrier state [27], but FISH testing provides false negatives in XLI patients who have point mutations (≈10% of affected XLI subjects). ...
... Placental sulfatase deficiency syndrome (PSD), which occurs in pregnancies of XLI fetuses, can manifest as failure of labor either to initiate or to progress, defective cervical softening, and a poor response to exogenous pitocin. PSD syndrome can be detected prior to the development of these complications by low maternal urinary and blood estriol levels due to the placenta's (a largely fetal structure) failure to desulfate estrone sulfate [19,29,30]. Since maternal estriol levels are part of the so-called 'triple screen' employed to detect pregnancies at risk for trisomies 18/21, Smith-Lemli-Opitz syndrome, and neural tube defects, many XLI fetuses are now detected in this manner [31][32][33][34]. ...
X-linked ichthyosis is a relatively common syndromic form of ichthyosis most often due to deletions in the gene encoding the microsomal enzyme, steroid sulfatase, located on the short area of the X chromosome. Syndromic features are mild or unapparent unless contiguous genes are affected. In normal epidermis, cholesterol sulfate is generated by cholesterol sulfotransferase (SULT2B1b), but desulfated in the outer epidermis, together forming a 'cholesterol sulfate cycle' that potently regulates epidermal differentiation, barrier function and desquamation. In XLI, cholesterol sulfate levels my exceed 10% of total lipid mass (≈1% of total weight). Multiple cellular and biochemical processes contribute to the pathogenesis of the barrier abnormality and scaling phenotype in XLI. This article is part of a Special Issue entitled The Important Role of Lipids in the Epidermis and their Role in the Formation and Maintenance of the Cutaneous Barrier.
... Thus, it is likely that some partial dosage compensation occurs at the STS locus in man and that this is due to down regulation of one of the two STS loci in females. (For a comprehensive review of STS in man, we refer the reader to Shapiro 1985.) Steroid sulphatase activity has also been observed in other mammals and in birds, amphibians, and fish. ...
... Lam et al. (1983) In contrast to the similar levels of the reference enzymes found in male and female fibroblasts of the root vole, STS levels were found to be significantly higher in root vole male cells (ratio 1.6:1, male to female). In the root vole, thus, there is a negative correlation between the number of X chromosomes and the STS level, which is just the reversed situation to that found in man (reviewed by Shapiro 1985) and in the wood lemming (Ropers and Wiberg 1982). The root vole ratio 1.6 : 1, male : female, also differs from the 1 : 1 ratio found in various tissues of the mouse (Crocker and Craig 1983;Gartler and Rivest 1983;Keitges et al. 1985). ...
Steroid sulfatase (STS; EC 3.1.6.2) levels were assayed in cultured fibroblasts of root voles captured in the wild. Four independent experiments were performed using two different substrates (DHEAS and E1S). Evidence is presented that in this species, STS levels are significantly higher in males than in females (ratio 1.6:1). We discuss our findings on a comparative basis and suggest that in the root vole the STS gene(s) is X- and Y-linked (as in the mouse) and that it is subject to X-inactivation, or partially so, on one of the X chromosomes in the female.
... Evaluation of Xg expression in XX males has shown that XX males do not always express their father's XG allele, indicating that the aberrant X-Y interchange leading to the transfer of TDF to the paternal X may result in the loss of XG from the X chromosome (de la Chapelle 1986; Petit et al. 1987). The functional locus for the steroid sulfatase (STS) gene (STS) also maps to the distal short arm of the human X at an estimated genetic distance of 13 cM proximal to XG (reviewed in Shapiro 1985; Yates et al. 1987). It is therefore of interest to determine whether STS is lost from the TDF-bearing X chromosome in XX males. ...
... The results showed that, of a total of 20 XX males evaluated , 17 had STS levels in the female range, indicating that they had two copies of STS, while three had activities in the male range, suggesting that only a single copy of STS was present in their genome. Results of dosage studies are sometimes difficult to interpret in the case of STS, as this gene does not appear to escape X inactivation completely and as the ratio of female-to malespecific activities is less than 2 and averages about 1.6 (reviewed in Shapiro 1985). Recently, cDNA and genomic probes for STS have been isolated (Ballabio et al. 1987; Bonifas et al. 1987; Conary et al. 1987; Yen et al. 1987 Yen et al. , 1988). ...
The human X and Y chromosomes pair and recombine at their distal short arms during male meiosis. Recent studies indicate that the majority of XX males arise as a result of an aberrant exchange between X and Y chromosomes such that the testis-determining factor gene (TDF) is transferred from a Y chromatid to an X chromatid. It has been shown that X-specific loci such as that coding for the red cell surface antigen, Xg, are sometimes lost from the X chromosome in this aberrant exchange. The steroid sulfatase functional gene (STS) maps to the distal short arm of the X chromosome proximal to XG. We have asked whether STS is affected in the aberrant X-Y interchange leading to XX males. DNA extracted from fibroblasts of seven XX males known to contain Y-specific sequences in their genomic DNA was tested for dosage of the STS gene by using a specific genomic probe. Densitometry of the autoradiograms showed that these XX males have two copies of the STS gene, suggesting that the breakpoint on the X chromosome in the aberrant X-Y interchange is distal to STS. To obtain more definitive evidence, cell hybrids were derived from the fusion of mouse cells, deficient in hypoxanthine phosphoribosyltransferase, and fibroblasts of the seven XX males. The X chromosomes in these patients could be distinguished from each other when one of three X-linked restriction-fragment-length polymorphisms was used. Hybrid clones retaining a human X chromosome containing Y-specific sequences in the absence of the normal X chromosome could be identified in six of the seven cases of XX males.(ABSTRACT TRUNCATED AT 250 WORDS)
... The STS locus occupies a unique position bordering the pseudoautosomal region of distal Xp. Although its inheritance is strictly sex linked, the locus shows incomplete inactivation, with STS levels in normal females approximately 1.6 times higher than enzyme levels in normal males (for review, see Shapiro 1985). This contrasts with what appears, on the basis of the ophthalmoscopic findings in OA1 carriers, to be potentially complete and random inactivation at the OA1 locus. ...
... Second, both diseases are relatively rare. The incidence of XI in males is approximately 1/6,000 (Shapiro 1985), and that of OA1 has recently been estimated to be about 1/150,000 in the Netherlands (van Dorp 1987). Our patients first sought medical attention because of only one of their problems (their poor visual acuity). ...
Ocular albinism of the Nettleship-Falls type (OA1) and X-linked ichthyosis (XI) due to steroid sulfatase (STS) deficiency are cosegregating in three cytogenetically normal half-brothers. The mother has patchy fundal hypopigmentation consistent with random X inactivation in an OA1 carrier. Additional phenotypic abnormalities that have been observed in other STS "deletion syndromes" are not present in this family. STS is entirely deleted on Southern blot in the affected males, but the loci MIC2X, DXS31, DXS143, DXS85, DXS43, DXS9, and DXS41 are not deleted. At least part of DXS278 is retained. Flow cytometric analysis of cultured lymphoblasts from one of the XI/OA1 males and his mother detected a deletion of about 3.5 million bp or about 2% of the X chromosome. Southern blot and RFLP analysis in the XI/OA1 family support the order tel-[STS-OA1-DXS278]-DXS9-DXS41-cen. An unrelated patient with the karyotype 46,X,t(X;Y) (p22;q11) retains the DXS143 locus on the derivative X chromosome but loses DXS278, suggesting that DXS278 is the more distal locus and is close to an XI/OA1 deletion boundary. If a contiguous gene deletion is responsible for the observed XI/OA1 phenotype, it localizes OA1 to the Xp22.3 region.
... However, the proba-ble recent loss of ADMLY function suggests that ADMLX still escapes X-inactivation. This is further supported by the fact that ADMLX is flanked on its distal side by the STS gene, which at least partially escapes inactivation (see Shapiro, 1985), and on its proximal side by a CpG island undermethylated on the inactive X (Petit et al., 1990a). ...
... STS has been detected in prostate tissues by a number of investigators, and it can be inhibited by aryl sulfamates including 667COUMATE (STX64, Irosustat) and other compounds (Purohit et al. 1995(Purohit et al. , 1996. However, it should not be forgotten that STS deficiency is associated with X-linked ichthyosis, a scalyskin disease affecting roughly 1 in every 2000-6000 males (Shapiro 1979(Shapiro , 1985, and would be an expected phenotype of chronic sulfatase inhibition in which cholesterol can no longer be mobilized from cholesterol sulfate. ...
Prostate cancer is the second leading cause of cancer death in adult males in the United States. Recent advances have found that the fatal form of this cancer known as castration resistant prostate cancer (CRPC) remains hormonally driven despite castrate levels of circulating androgens. CRPC arises since the tumor undergoes adaptation to low levels of androgens by either synthesizing its own androgens (intratumoral androgens) or by alterations in the androgen receptor (AR). This article reviews the major routes to testosterone and dihydrotestosterone in CRPC, examines the enzyme targets and progress in the development of isoform specific inhibitors that could block intratumoral androgen biosynthesis. Because redundancy exists in these pathways it is likely that inhibition of a single pathway will lead to up-regulation of another so that drug resistance would be anticipated. Drugs that target multiple pathways or agents that block intratumoral androgen biosynthesis and antagonize the AR offer the most promise. Optimal use of enzyme inhibitors or AR antagonists to ensure maximal benefit to CRPC patients will also require application of precision molecular medicine to determine that a tumor in a particular patient will be responsive to these treatments either alone or in combination.
... La parte distal del brazo corto del cromosoma X es una región del genoma humano que muestra varias características muy peculiares: se escapa a la inactivación de cromosoma X [18]; se ha demostrado que la actividad de STS es mayor en las mujeres normales que en los hombres normales, un hallazgo compatible con la 'no lyonización' [19]; y comparte homología tanto con el brazo corto [20][21][22][23] como con el brazo largo [24][25][26][27] del cromosoma Y. Por otra parte, en esta región se ha demostrado una alta frecuencia de deleciones [28], que pueden deberse a los efectos fenotípicos no letales de estas supresiones en el estado hemicigoto masculino, proponiéndose un posible papel de recombinación aberrante entre las regiones homólogas de los cromosomas sexuales [26,27]. ...
X-chromosome-linked ichthyosis is caused by mutation or deletion of the STS gene associated with a deficiency of the enzyme steroid sulphatase, located in the distal part of the short arm of the X chromosome (Xp22.3-pter), close to the pseudo-autosomal region. Depending on its size, it can present as an isolated entity or combined with a syndrome caused by neighbouring genes, thus associating itself with other monogenic diseases as well as other mental disorders. The most relevant findings from the literature review are the importance of the Xp22.3-pter region and the higher incidence of neurological disorders among males (attention deficit hyperactivity disorder, autism and X-linked mental retardation). The role and implication of these genes in the disease are discussed and the authors suggest a possible contribution of the gene PNPLA4, which was originally described as GS2 and codes for calcium-independent phospholipase A2 beta, involved in lipoprotein metabolism, as one of the causes of autism. Improvements have been observed following treatment with citicoline, thanks to the role this nootropic plays in the biosynthesis of structural phospholipids involved in the formation and repair of the neuronal membrane.
... However, the proba-ble recent loss of ADMLY function suggests that ADMLX still escapes X-inactivation. This is further supported by the fact that ADMLX is flanked on its distal side by the STS gene, which at least partially escapes inactivation (see Shapiro, 1985), and on its proximal side by a CpG island undermethylated on the inactive X (Petit et al., 1990a). ...
Kallmann syndrome associates hypogonadotropic hypogonadism and anosmia and is probably due to a defect in the embryonic migration of olfactory and GnRH-synthesizing neurons. The Kallmann gene had been localized to Xp22.3. In this study 67 kb of genomic DNA, corresponding to a deletion interval containing at least part of the Kallmann gene, were sequenced. Two candidate exons, identified by multiparameter computer programs, were found in a cDNA encoding a protein of 679 amino acids. This candidate gene (ADMLX) is interrupted in its 3' coding region in the Kallmann patient, in which the proximal end of the KAL deletion interval was previously defined. A 5' end deletion was detected in another Kallmann patient. The predicted protein sequence shows homologies with the fibronectin type III repeat. ADMLX thus encodes a putative adhesion molecule, consistent with the defect of embryonic neuronal migration.
... STS deficiency might cause other deleterious effects to the affected patients. Thus a high occurrence of testicular maldescent has been found in RXLI males ( 10, I I), and an increased risk of testis cancer cannot be excluded (11,12,13). The link between the STS deficiency and the development of gonadal diseases might be found in the recent uncovering of an abnorma! ...
Steroid sulphatase (STS) activity was measured with tritiated dehydroepiandrosterone sulphate (DHEAS) and oestrone sulphate (OES) in leucocytes as well as in skin fibroblasts from 31 women who were presumed to be carriers of STS deficiency and recessive X-linked ichthyosis. Overall, 30 of the 31 women (96.8%) could be identified as heterozygotes in at least one of the four assay systems used, i.e. on the basis of having an STS activity below the 2.5 percentile calculated for normal control females. In the individual assay systems, the highest carrier detection rate was achieved with OES in leucocytes (96.2%), followed by DHEAS in leucocytes (80.8%), whereas a more pronounced overlap was present in the fibroblast systems. In leucocytes as well as in fibroblasts, the STS activity determined with DHEAS was positively correlated with the STS activity determined with OES (p less than 0.001) suggesting that a single sulphatase is responsible for the hydrolysis of both steroid sulphates.
... X-linked ichthyosis (XLI) has been shown to be associated with deficiency of steroid sulfatase (STS) [16]. The STS gene is localized to the distal short arm of the X chromosome in band Xp22.3 [15,17]. ...
Three families segregating for X-linked ichthyosis (XLI) were analysed using the full-length STS cDNA probe and an anonymous polymorphic DNA sequence closely linked to the STS gene. In patients from two of the families, submicroscopic chromosomal deletions could be detected using both the STS and the GMGX9 (DXS237 locus) probes. Patients in the third family showed the same hybridization pattern as healthy males following molecular hybridization with either of the probes. The results of DNA analysis (indirect genotype diagnosis) agree well with those based on the arysulfatase C/beta-gal determination and prove the reliability of the biochemical test. Both methods are discussed for carrier detection, prenatal diagnosis, and genetic counseling.
... Indeed, the human MIC2 gene and the murine STS locus seem to escape X chromosome inactivation (Shapiro, 1985). The STS gene in man maps close to, but not within, the pseudoautosomal region. ...
The mammalian X and Y chromosomes are thought to have evolved from a common, nearly homologous chromosome pair. Although there is little sequence similarity between the mouse or the human X and Y, there are several regions in which moderate to extensive sequence homologies have been found, including, but not limited to, the so-called pseudoautosomal segment, in which X-Y pairing and recombination take place. The steroid sulfatase gene is in the pseudoautosomal region of the mouse, but not in man. We have cloned and characterized the human STS X-encoded locus and a pseudogene that is present on the long arm of the Y chromosome. Our data in humans and other primates suggest that there has been a pericentric inversion of the Y chromosome during primate evolution that has disrupted the former pseudoautosomal arrangement of these genes. These results provide additional insight into the evolution of the sex chromosomes and into the nature of this interesting portion of the human genome.
The molecular genetics of keratinizing disordersIchthyosisCongenital ichthyosesIsolated genetic syndromes with ichthyosisAcquired ichthyosesPityriasis rotundaPeeling skin syndromes (MIM 270300)ErythrokeratodermaFolliculocentric keratotic disordersPityriasis rubra pilarisDarier's disease and related disordersOther focal disordersPalmoplantar keratodermasKeratodermas and associated disordersAcanthosis nigricansConfluent and reticulate papillomatosis
The human X chromosome constitutes one of the most intensively examined stretches of genetic material in a higher eukaryote. This is probably the result of certain technical features that facilitate the study and recognition of X-linked genes, the relative frequency of X-linked disorders as a group, and the quantitative importance of several specific conditions, such as Duchenne muscular dystrophy and hemophilia. It has been relatively easy to recognize X-encoded mutant genes by virtue of their typical pattern of inheritance, and several X-linked diseases have been known since antiquity. Clear descriptions of X-linked pedigrees of hemophilia, color blindness, ichthyosis, muscular dystrophy, nystagmus, and ectodermal dysplasia antedate the postulation of Mendel’s laws and the recognition of sex chromosomes (McKusick, 1983). More than 120 X-linked single-gene Mendelian human conditions have been catalogued. The vertical transmission of traits through a number of generations affecting only males, but involving carrier females, and the absence of male-to-male transmission have come to be appreciated as the hallmark of this form of inheritance. Since a single copy of a mutant gene in males is sufficient to produce disease, it is not uncommon to find many affected individuals in a single pedigree in addition to many at-risk women, creating significant complexities in genetic counseling.
Some disorders of sulfur metabolism affect thousands of people. Many, however, are rare. Interest in these conditions reaches beyond their clinical significance because genetically determined metabolic alterations, defects and deficiencies are biochemical windows giving insight into mechanisms that is normally denied the investigator. Apropos of steroid sulfate sulfatase deficiency, Shapiro (1985) has elegantly written that, “it is considered axiomatic in human genetics that the study of relatively rare disorders may yield far more in dividends than might be anticipated based on the incidence of the condition in question”. To understand the normal, one must study the abnormal. Study of the disorders discussed in this chapter have permitted the testing and refining of our concepts of sulfur metabolism, its quantitation and regulation. Thus, much of our understanding of Met biochemistry has been triumphantly borne out by the study of the inherited defects discussed in Section 8.2.
Many birth defects, although rare individually, are encountered in clinical practice and have now become treatable if properly diagnosed. In the Atlas of Genetic Diagnosis and Counseling, Harold Chen, MD, shares his almost 40 years of clinical genetics practice in a comprehensive pictorial atlas of 203 genetic disorders, malformations, and malformation syndromes. The author provides a detailed outline for each disorder, describing its genetics, basic defects, clinical features, diagnostic tests, and counseling issues, including recurrence risk, prenatal diagnosis, and management. Numerous color photographs of prenatal ultrasounds, imagings, cytogenetics, and postmortem findings illustrate the clinical features of patients at different ages, patients with varying degrees of severity, and the optimal diagnostic strategies. The disorders cited are supplemented by case histories and diagnostic confirmation by cytogenetics, biochemical, and molecular techniques, when available. Also available in a CD-ROM edition (ISBN: 1-58829-974-5).
Authoritative and up-to-date, the Atlas of Genetic Diagnosis and Counseling will help all physicians to understand and recognize genetic diseases and malformation syndromes, and consequently better evaluate, counsel, and manage affected patients.
The various manifestations of ichthyoses are classified either by their appearance or their molecular genetics. This volume focuses on generalized, inherited disorders of cornification, which constitute an ever-enlarging group of monogenic diseases caused by a large number of genes that affect a broad array of cellular functions. The authors' overview reflects their unique perspective that the clinical phenotype in the inherited ichthyoses mirrors a 'best attempt' by a metabolically compromised epidermis to maintain a barrier sufficiently impermeable for survival in a desiccating external environment. The basis for threats to survival is illuminated, and the systemic problems, including growth failure, also reflect a compromised barrier. A new consensus classification of these disorders is provided, and the distinguishing clinical features of each disorder are described. Further, the latest molecular genetic information is succinctly reviewed with up-to-date and comprehensive references. Yet, the major emphases of this volume are on disease pathogenesis and on the identification of key ultrastructural features. This publication will prove an invaluable aid to dermatologists, pediatric dermatologists and pediatricians dealing with patients with inherited ichthyoses. In addition, clinical geneticists and dermatopathologists will find it interesting reading.
The genetic functions carried by the sex chromosomes have become the subject of investigation because of the opportunity afforded by the functional monosomy of the X and Y chromosomes in males. The view of the mammalian sex chromosomes perceives their evolution from an ancestral pair of homologs. Morphological differentiation has accompanied the emergence of a sex-determining role on the Y and a mechanism of dosage compensation for X-linked genes in females, mediated by random X inactivation. It might be expected that sequence homology from ancestral times might have been retained in functionally important domains of the sex chromosomes. Despite their disparity in size and morphology, the X and Y chromosomes pair at meiosis, ensuring correct segregation during gametogenesis. The failure of the sex chromosomes to participate in recombination beyond the pairing region has precluded a conventional genetic analysis for most of the Y. Suppression of recombination is likely to be an essential requirement for preserving the sex-determining role of the differential part of the Y. Models of the Y have relied on phenotype-karyotype correlations through detailed cytogenetic analysis of abnormal Y chromosomes. Models of the Y derived from such studies reflect the level of resolution that cytogenetics can provide.
The success of pregnancy depends on the appropriate process and timing of parturition. For each viviparous species, natural selection has affected extant physiology such that birth processes and timing complement overall reproductive strategy to maximize reproductive efficiency. This chapter synthesizes the current understanding of the physiology and pathophysiology of human parturition from the perspective of the evolutionary biology and comparative physiology of birth timing. We examine selective pressures that may have influenced parturition physiology and pathophysiology; the principal organ for parturition, the uterus, and its functional tissue types: the myometrium, cervix, and decidua; and the hormonal interactions that control their function to either sustain pregnancy or facilitate labor and delivery. Conserved uterotropic (causing uterine growth) and uterotonic (causing uterine contraction) hormonal pathways are present across viviparous species, although their regulatory patterns and sites of production differ. An emerging theme is that parturition involves positive-feedback loops leading to intrauterine inflammation, enhanced uterine contractility, membrane rupture, and cervical dilation, functioning at a paracrine level within each of the gestational tissues and in an endocrine fashion between the uterus and extrauterine sites.
El mosaicismo genético designa la presencia en un paciente de dos o varios patrimonios genéticos diferentes derivados del mismo cigoto. En el ser humano se ha demostrado la existencia de un mosaicismo genético en múltiples enfermedades. El objetivo de este artículo es explicar el concepto y la patogenia del mosaicismo genético y describir las manifestaciones cutáneas que permitan sospecharlo.
X-linked recessive ichthyosis (XRI) is caused by a deficiency of steroid sulfatase. Because this enzyme plays an important role in androgen metabolism, we advance the hypothesis that men with XRI do not show androgenetic alopecia or develop only mild forms of this common type of hair loss. Clinical studies should show whether this hypothesis can be supported.
A total of 30 cases of 46,XX true hermaphroditism was analysed for Y-DNA sequences including the recently cloned gene for male testis-determination SRY. In 3 cases, a portion of the Y chromosome including SRY was present and, in 2 cases, was localised, to Xp22 by in situ hybridisation. Since previous studies have shown that the majority of XX males are generated by an X-Y chromosomal interchange, the Xp22 position of the Yp material suggests that certain cases of hermaphroditism can arise by the same meiotic event. The phenotype in the 3 SRY-positive cases may be caused by X-inactivation resulting in somatic mosaicism of testis-determining factor expression giving rise to both testicular and ovarian tissues. Autosomal or X-linked mutation(s) elsewhere in the sex-determining pathway may explain the phenotype observed in the remaining 27 SRY-negative cases.
X chromosome rearrangements usually convey clinical manifestations in the hemizygous males and are, thus, readily ascertained.
They are found in all parts of the X chromosome and are associated with more than 20 disorders. Some of the rearrangements
are the results of homologous recombination between low-copy repeats (LCRs) on the X chromosome or between large homologous
regions on the X and Y chromosome, whereas others are caused by nonhomologous end-joining (NHEJ). For most large deletions
associated with contiguous gene syndromes, the deletion breakpoints remain uncharacterized. The deletions, as well as inversions
and duplications on the X chromosome, occur mainly in male germ cells, indicating intrachromatid or sister chromatid exchange
as the underlying mechanism.
Steroid sulfatase (STS) deficiency is the biochemical defect of X-linked ichthyosis (XLI), one of the most common X-linked disorders. We studied 57 European unrelated patients affected by STS deficiency. Twentyeight patients were from Italy, 24 from the United Kingdom, 4 from The Netherlands, and 1 from Denmark. In two families XLI was associated with Kallmann syndrome (hypogonadotropic hypogonadism and anosmia). STS enzymatic activity was profoundly deficient in all cases. Direct DNA analysis, using cDNA and genomic probes from the STS gene and linked regions, demonstrated heterogeneity of the molecular defect. Forty-eight patients (84%) showed a deletion of the STS gene. In 44 cases the deletion also involved the STS flanking locus DXS237. In 1 patient a partial deletion of the STS gene was detected and in 9 patients no evidnce of deletion was found. Locus DXS31 (probe M1A), previously mapped to Xp22.3-pter, was not deleted either in 24 patients with X-linked ichthyosis or in two families with X-linked ichthyosis associated with Kallmann syndrome. Consequently, the following loci order could be suggested: telomere-DXS31-(DXS237, STS)-Kallmann-centromere. Immunoblotting experiments, performed using anti-STS polyclonal antibodies, revealed the absence of cross-reacting material to STS in all cases tested, including 4 patients without evidence of deletions.
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.
Cholesterol 3-sulphate (CS) is a component of the intercellular lipid found in the uppermost layer of human epidermis (the 'stratum corneum') and is thought to play an important role in tissue cohesion. In this investigation we have compared the influence of cholesterol (CH) and CS on the gel-to-liquid crystalline phase behaviour, the polymorphic phase behaviour, and the hydrocarbon order profile in selected model membranes. It is shown that in sphingomyelin (SPM) systems, the presence of equimolar amounts of either CH or CS eliminates the gel-to-liquid crystalline transition as detected by calorimetry. Similarly, in 1-palmitoyl,2-oleoyl-phosphatidylethanolamine (POPE) dispersions containing a perdeuterated palmitoyl chain (POPE-d31), it is shown that both CH and CS exert an ordering effect as determined by 2H-NMR techniques, however, CS is less potent at temperatures both above and below that of the main transition for the native phospholipid. Alternatively, in mixed systems containing dioleoylphosphatidylethanolamine (DOPE) and SPM (DOPE/SPM, 6:1 mol/mol) CH promotes thermotropic L alpha-->HII phase transitions, whereas CS stabilizes the bilayer organization. These bilayer stabilization effects can be diminished by addition of Ca2+. These effects are consistent with a larger area per molecule of CS as compared to CH, presumably related to the presence of the negatively charged sulphate moiety of CS.
There are several copies of related sequences on the distal short arm of the human X chromosome and the proximal long arm of the Y chromosome which were originally detected by cross hybridization with a genomic DNA clone, CRI-S232. Recombination between two S232-like sequences flanking the steroid sulfatase locus has been shown to cause frequent deletions in the X chromosome short arm, resulting in steroid sulfatase deficiency. We now report the characterization of several S232-like sequences. Restriction mapping and sequence analysis show that each S232 unit contains 5 kb of unique sequence in addition to two elements, RU1 and RU2, composed of a variable number of tandem repeats. RU1 consists of 30 bp repeating units and its length shows minimal variation between individuals. The RU2 elements in the hypervariable S232 loci on the X chromosome consist of repeating sequences which are highly asymmetric, with about 90% purines and no C's on one strand. The X-derived RU2 elements range from 0.6 kb to over 23 kb among different individuals, accounting entirely for the observed polymorphism at the S232 loci. Although the repeating units of the RU2 elements in the nonpolymorphic S232 loci on the Y chromosome share high sequence homology with those on the X chromosome, they exhibit much higher intrarepeat sequence variation. S232 homologous sequences are found in great apes, old world and new world monkeys. In chimpanzees and gorillas the S232-like sequences are polymorphic in length.
Parallel to the inactivation of the X chromosome in somatic cells of female, the male X in mammals is rendered inactive during spermatogenesis. Pseudoautosomal genes, those present on the X-Y meiotically pairable region of male, escape inactivation in female soma. It is suggested, but not demonstrated, that they may also be refractory to the inactivation signal in male germ cells. We have assayed activity of the enzyme steroid sulfatase, product of a pseudoautosomal gene, in testicular cells of the mouse and shown its presence in premeiotic, meiotic (pachytene), and postmeiotic (spermatid) cell types. It appears that, as in females, pseudoautosomal genes may escape inactivation in male germ cells also.
Testis cancer and ichthyosis are both relatively rare diseases. Hence the finding of six individuals with both these conditions in a small population with testicular cancer is highly conspicuous and indicates some kind of connection among such persons. Despite the identical clinical appearances of their ichthyoses, three of the ichthyotic subjects had no measurable activity of the enzyme, steroid sulfatase (STS) in leucocytes, a distinct characteristic of recessive X-linked ichthyosis (RXLI). However, the remaining three subjects had normal STS activity, a strong indicator of autosomal dominant ichthyosis (ADI). The STS activity in patients with testicular cancer who do not have ichthyosis (N = 30) was also within the normal range. The patients with testicular cancer with no skin disease had elevated serum levels of 4-androstenedione (4-AD), follicle stimulating hormone (FSH), and luteinizing hormone (LH) but had reduced levels of estrone and estrone sulfate. The other serum parameters measured did not significantly differ from normal levels. In essence, the hormone levels obtained for the patients with ichthyotic testicular cancer followed the same pattern, although their dehydroepiandrosterone sulfate (DHEAS) and estrone sulfate levels tended to be slightly higher than normal. However, no conspicuous aberrations in any of the parameters examined were observed, and why men with ichthyosis are at high risk for testicular cancer remains an unresolved issue.
Scaling in patients with recessive X-linked ichthyosis is caused by lack of activity of the enzyme steroid sulfatase. In approximately 90% of kindreds, this lack is the result of a DNA deletion large enough to eliminate the coding region completely. We have used Southern blot hybridization of DNA isolated from peripheral blood leukocytes to measure gene dosage of the steroid sulfatase gene. This readily detects a half-normal dosage in women who are carriers and therefore can be used to diagnose the carrier status of female relatives of 90% of patients with the disease. We have found one family in whom the deletion arose on an allele inherited from the proband's clinically normal maternal grandfather.
Mendelian inherited disorders due to deletions of adjacent genes on a chromosome have been described as "contiguous gene syndromes." Short stature, chondrodysplasia punctata, mental retardation, steroid sulfatase deficiency, and Kallmann syndrome have been found as isolated entities or associated in various combinations in 27 patients with interstitial and terminal deletions involving the distal short arm of the X chromosome. The use of cDNA and genomic probes from the Xp22-pter region allowed us to identify 12 different deletion intervals and to confirm, and further refine, the chromosomal assignment of X-linked recessive chondrodysplasia punctata and Kallmann syndrome genes. A putative pseudoautosomal gene affecting height and an X-linked non-specific mental retardation gene have been tentatively assigned to specific intervals. The deletion panel described is a useful tool for mapping new sequences and orienting chromosome walks in the region.
Several DNA sequences from two homologous regions, localized on the distal part of the human X chromosome short arm and on the long arm of the Y chromosome, have been hybridized to DNAs from seven human-rodent hybrids containing human X; Y translocation chromosomes. Molecular characterization of the translocated chromosomes has revealed, in all but one case, transfer of the Y cluster of sequences and complete deletion of the corresponding X-chromosomal sequences. The possible role of X/Y homology in the aetiology of X; Y translocations is proposed.
We report on a male infant with X-linked ichthyosis, X-linked Kallmann syndrome, and X-linked recessive chondrodysplasia punctata (CPXR). Chromosome analysis showed a terminal deletion with a breakpoint at Xp22.31, inherited maternally. This patient confirms the localization of XLI, XLK, and CPXR to this region of the X chromosome and represents an example of a “contiguous gene syndrome.”
A comparison of the manifestations of patients with CPXR, warfarin embryopathy, and vitamin K epoxide reductase deficiency shows a remarkable similarity. However, vitamin K epoxide reductase deficiency does not appear to be the cause of CPXR. We propose that CPXR may be due to a defect in a vitamin K-dependent bone protein such as vitamin K-dependent bone carboxylase, osteocalcin, or matrix Gla protein.
The human steroid sulfatase gene (STS) is located on the distal X chromosome short arm close to the pseudoautosomal region but in a segment of DNA that is unique to the X chromosome. In contrast to most X chromosome-encoded genes, STS expression is not extinguished during the process of X chromosome inactivation. Deficiency of STS (steryl-sulfatase; steryl-sulfate sulfohydrolase, EC 3.1.6.2) activity produces the syndrome of X chromosome-linked ichthyosis, which is one of the most common inborn errors of metabolism in man. Approximately 90% of STS- individuals have large deletions at the STS locus. We and others have found that the end points of such deletions are heterogeneous in their location. One recently ascertained subject was observed to have a 40-kilobase deletion that is entirely intragenic, permitting the cloning and sequencing of the deletion junction. Studies of this patient and of other X chromosome sequences in other subjects permit some insight into the mechanism(s) responsible for generating frequent deletions on the short arm of the X chromosome.
Gonadal androgens are known to regulate the rate of growth in height during adolescence, particularly in males, but little is known of the role of adrenal androgens in this process. In a prospective multiple regression model we show that both adrenal and gonadal androgens contribute to the increasing rate of growth in height prior to peak height velocity and the decreasing rate of height growth in later adolescence. Since adrenal androgen secretion begins in mid-childhood, long before gonadal maturation and the secretion of testicular androgens, these findings may prove helpful in explaining population variation in adolescent growth.
Patients with steroid sulphatase deficiency develop ichthyosis with accumulation of cholesterol sulphate in plasma and in the stratum corneum. The present study was undertaken to determine whether desulphation of the C19 steroid DHEAS is also impaired. The mean plasma concentrations of DHEA and androstenedione were significantly lower for patients than for controls (p less than 0.02 and 0.001) while the mean concentration of DHEAS was higher (p less than 0.002). Following intravenous administration of 3H-DHEAS, one patient failed to desulphate 3H-DHEAS as evidenced by an absence of urinary 3H-glucuronides. A second produced normal amounts of urinary 3H-glucuronides (indicative of desulphation capacity) in a baseline study but did not desulphate 3H-DHEAS following ampicillin treatment to alter gut microflora. A third patient had consistent sulphatase activity with and without ampicillin.
We report a large Italian pedigree in which five out of six males are affected by a syndrome, following an X-linked inheritance pattern, characterized by ichthyosis, hypogonadotropic hypogonadism, and anosmia. The concurrence of features of X-linked ichthyosis (XLI) with those of Kallmann syndrome, another disease often inherited as an X-linked trait, prompted us to perform biochemical, cytogenetic, and molecular studies in relation to the short arm of the X chromosome (Xp). Steroid sulphatase (STS) activity was found to be completely deficient in all affected members of the family. Prometaphase chromosome analyses of two obligate heterozygous women and one affected male showed normal karyotypes. Xg blood group antigen analysis and molecular studies employing cloned DNA sequences from the distal segment of the Xp (probes RC8, 782, dic56, and M1A), did not provide evidence for deletions or rearrangements of the X chromosome. The linkage analysis showed no crossovers between the disease, Xg, and DXS143, the locus defined by probe dic56, thus suggesting the possibility of a linkage between these two markers of the distal segment of Xp and the X-linked ichthyosis, hypogonadism, and anosmia syndrome.
Human STS is a microsomal enzyme important in steroid metabolism. The gene encoding STS is pseudoautosomal in the mouse but not in humans, and escapes X inactivation in both species. We have prepared monoclonal and polyclonal antibodies to the protein which has been purified and from which partial amino acid sequence data have been obtained. cDNA clones containing the entire coding sequence were isolated, sequenced, and expressed in heterologous cells. Variable length transcripts have been shown to be present and due to usage of alternative poly(A) addition sites. The functional gene maps to Xp22.3-Xpter and there is a pseudogene on Yq suggesting a recent pericentric inversion. Absence of STS enzymatic activity occurs frequently in human populations and produces a visible phenotype of scaly skin or ichthyosis. Ten patients with inherited STS deficiency were studied and eight had complete gene deletions. The possibility that STS deficiency results from aberrant X-Y interchange is discussed.
Three cDNA clones with inserts of 1.2-1.6 kb that reacted both with antibodies and oligonucleotides specific for steroid sulfatase were isolated from a human placental library in lambda gt11. The 5'-end of one of the inserts, STS-3, was sequenced and colinearity with the amino acid sequence of 3 peptides of steroid sulfatase encompassing 64 amino acids was demonstrated. STS-3 hybridized with 2.5, 4.6 and 6.3 kb species in poly(A)+RNA and with 2.5, 4 and 9 kb fragments of EcoRI digested human DNA. The frequency of the EcoRI fragments in DNA from females was approximately twice that in DNA from males. DNA from two patients with steroid sulfatase deficiency and X-linked ichthyosis did not hybridize with STS-3. DNA from a third patient showed a normal hybridization pattern. It is concluded that steroid sulfatase deficiency is a genetically heterogenous disorder.
In Triton X-100 solubilized leukocytes of 17 patients and 8 obligate carriers of X-linked recessive ichthyosis (XLI) the activity of arylsulphatase C (ASC) was determined and expressed as the ratio to beta-galactosidase activity. The ASC/beta-gal ratio of XLI patients is markedly decreased (range 0.07-0.48) in comparison to the corresponding control group of males (range 1.3-2.7). The enzyme ratios of 8 obligate carriers of XLI are decreased (range 0.90-1.9) in comparison to the normal females (2.13-5.52). These results indicate that the determination of the enzyme ratio of ASC/beta-gal in Triton X-100 solubilized leukocytes is a sensitive test for biochemical identification of patients and probably of carriers of XLI.
Immunologically cross-reacting material to antibodies against steroid sulphatase has not been found in fibroblasts from patients with steroid sulphatase deficiency.
We describe a family with two male members showing an X/Y translocation (karyotype: 46,Y,der(X)t(X;Y)(p22;q11]. At physical examination both patients showed ichthyosis, mental retardation and dysmorphic features. Chondrodysplasia punctata and short stature were present in one case. Direct DNA analysis, using a steroid sulphatase cDNA probe, was performed in one patient, his mother and sister, both carriers of the translocation. We found that the translocated region of the Y chromosome includes the steroid sulphatase pseudogene. These results suggest that in our patients the X/Y translocation may be derived from a recombinational event between homologous regions located on the short arm of the X chromosome and the long arm of the Y chromosome. Clinical and molecular studies on the present family add further information for the construction of a tentative physical map of the distal Xp.
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.
The control of X inactivation during development can be considered under four main aspects as follows: (1) the initiation of inactivation or differentiation of the X chromosomes in the early embryo, (2) the spreading of the inactivation process along the chromosome, (3) the maintenance of the active or inactive state throughout the further life of the animal, and (4) the reactivation of the inactive X in the oocyte or early embryo.
X-linked genes are conserved among all mammalian species, but the organization of genes on the X chromosome varies from one species to another. This review summarizes the evidence for established gene homologies between mice and human beings. It also describes genes that are possible homologies because of their locations in the human and murine X chromosomes and similarities in the phenotypes they produce. Based on current knowledge of homologous gene location, the human and murine X chromosomes appear to contain four highly conserved segments and differ in organization by only three to four simple chromosomal rearrangements.
A pericentric inversion of a human X chromosome and a recombinant X chromosome [rec(X)] derived from crossing-over within the inversion was identified in a family. The rec(X) had a duplication of the segment Xq26.3----Xqter and a deletion of Xp22.3----Xpter and was interpreted to be Xqter----Xq26.3::Xp22.3----Xqter. To characterize the rec(X) chromosome, dosage blots were done on genomic DNA from carriers of this rearranged X chromosome using a number of X chromosome probes. Results showed that anonymous sequences from the distal end of the long arm to which probes 4D8, Hx120A, DX13, and St14 bind as well as the locus for glucose-6-phosphate dehydrogenase (G6PD) were duplicated on the rec(X). Mouse-human cell hybrids were constructed that retained the rec(X) in the active or inactive state. Analyses of these hybrid clones for markers from the distal short arm of the X chromosome showed that the rec(X) retained the loci for steroid sulfatase (STS) and the cell surface antigen 12E7 (MIC2); but not the pseudoautosomal sequence 113D. These molecular studies confirm that the rec(X) is a duplication-deficiency chromosome as expected. In the inactive state in cell hybrids, STS and MIC2 (which usually escape X chromosome inactivation) were expressed from the rec(X), whereas G6PD was not. Therefore, in the rec(X) X chromosome inactivation has spread through STS and MIC2 leaving these loci unaffected and has inactivated G6PD in the absence of an inactivation center in the q26.3----qter region of the human X chromosome. The mechanism of spreading of inactivation appears to operate in a sequence-specific fashion. Alternatively, STS and MIC2 may have undergone inactivation initially but could not be maintained in an inactive state.
In an elaborate paper upon this subject in the last two numbers of the ‘British and Foreign Review’ (April and July, 1863), Mr. Sedgwick has recorded numerous facts, which he has been at the pains to collect from various English and French sources; so numerous, indeed, are his observations, that his paper will supply a valuable storehouse of references to those who may wish specially to study the subject. The more general reader, overwhelmed by the multitude of unconnected details which seem to point to no conclusion, may, perhaps, find the ancient adage involuntarily rise to his lips— Non numerandæ sed perpendendæ observationes . It is an adage, however, which is more often the refuge of idleness unwilling to labour at the tedious collection of facts, than it is the legitimate expression of a just censure.
Three cytidine analogs containing modifications in the 5-position of the cytosine ring (5-azacytidine, 5-aza-2'-deoxycytidine and pseudoisocytidine) induced the expression of human hypoxanthine/guanine phosphoribosyltransferase (IMP; pyrophosphate phosphoribosyltransferase, EC 2.4.2.8) gene (HPRT) from a structurally normal inactive human X chromosome retained in a mouse-human somatic cell hybrid. Between 0.1% and 8% of the cells surviving treatment with these analogs were able to form colonies in selective medium (hypoxanthine/aminopterin/thymidine/glycine medium), but two other analogs, 5-fluoro-2'-deoxycrytidine and 5.6-dihydro-5-azacytidine, did not induce HPRT expression. The inactive X chromosome present in the hybrid, was found to be late replicating, and experiments with synchronized cells showed that the induction of HPRT expression by 5-aza-2'-deoxycytidine occurred maximally in cells treated in the latter half of the S phase. Two division cycles were required after analog treatment for the highest frequency of expression of the induced gene. Because these analogs are powerful inhibitors of the methylation of cytosine residues in DNA, the results imply that demethylation of specific DNA sequences may be required for the reexpression of human HPRT.
Genetics of human lysosomal arylsulfatases A and B (aryl-sulfate sulfohydrolase, EC 3.1.6.1), associated with childhood disease, has been studied with human-rodent somatic cell hybrids. Deficiency of arylsulfatase A (ARS(A)) in humans results in a progressive neurodegenerative disease, metachromatic leukodystrophy. Deficiency of arylsulfatase B (ARS(B)) is associated with skeletal and growth malformations, termed the Maroteaux-Lamy syndrome. Simultaneous deficiency of both enzymes is associated with the multiple sulfatase deficiency disease, suggesting a common relationship for ARS(A) and ARS(B). The genetic and structural relationships of human ARS(A) and ARS(B) have been determined by the use of human-Chinese hamster somatic cell hybrids. Independent enzyme segregation in cell hybrids demonstrated different chromosome assignments for the structural genes, ARS(A) and ARS(B), coding for the two lysosomal enzymes. ARS(A) activity showed concordant segregation with mitochondrial aconitase encoded by a gene assigned to chromosome 22. ARS(B) segregated with beta-hexosaminidase B encoded by a gene assigned to chromosome 5. These assignments were confirmed by chromosome analyses. The subunit structures of ARS(A) and ARS(B) were determined by their electrophoretic patterns in cell hybrids; a dimeric structure was demonstrated for ARS(A) and a monomeric structure for ARS(B). Although the multiple sulfatase deficiency disorder suggests a shared relationship between ARS(A) and ARS(B), independent segregation of these enzymes in cell hybrids did not support a common polypeptide subunit or structural gene assignment. The evidence demonstrates the assignment of ARS(A) to chromosome 22 and ARS(B) to chromosome 5. A third gene that affects ARS(A) and ARS(B) activity is suggested by the multiple sulfatase deficiency disorder.
As a result of routine screening of ante-natal patients by urinary total oestrogens and plasma human placental lactogen (HPL) from 35 weeks, a rare placental sulphatase deficiency was indicated which was later confirmed by in vitro studies of placental enzyme activities.
An animal model using dexamethosone-suppressed, castrated dogs was developed to test the hypothesis that a pituitary hormone other than ACTH modulates adrenal androgen (AA) secretion. Plasma samples were obtained every 15 min during infusions of saline, synthetic alpha 1-24 corticotropin, porcine 1-39 corticotropin (ACTH), or bovine pituitary gland extract (PE) in a wide range of doses. Androstenedione (A), dehydroepiandrosterone (DHA), and cortisol (F) were quantified by radioimmunoassay. When the ratio of AA levels was related to those of F, in order to correct for ACTH content in the PE, the slopes of the dose-response curves for corticotropin and PE were different at the 0.01 level. For A the dose-response slope for the PE was 0.18 +/- 0.5 SE, whereas that of ACTH was 0.02 +/- 0.01. For the DHA response the slopes were 0.17 +/- 0.04 for the PE and 0.04 +/- 0.03 for ACTH. Related studies showed no increase in AA levels in response to luteinizing hormone-releasing hormone, bovine growth hormone (GH), bovine prolactin, ovine thyroid-stimulating hormone (TSH), or synthetic aqueous arginine vasopressin (AVP). We conclude that a pituitary factor other than ACTH, prolactin, GH, luteinizing hormone, follicle-stimulating hormone, TSH, or AVP may be responsible for the observed increase in AA concentrations.
Natural selection operates among individual organisms which differ in their genetic constitution. The degree of hereditary variability within a species is greatly enhanced by cross-fertilization. Indeed, the mechanism of sexual reproduction occurred very early in evolution, for it is seen today even in bacteria. In Escherichia coli, fertilization occurs by passage of the single chromosome from the male into the female bacterium (LEDERBERG, 1959). In multicellular organisms, the separation of germ from soma, and the production of haploid gametes became mandatory. The gametes were of two types. One, extremely mobile, was designed to seek out and penetrate the other, which loaded with nutrients, received the mobile gamete and intiated the development of a new individual. The foundation for true bisexuality was thus laid. In the primitive state of bisexuality, whether an individual is to be a sperm-producing male or an egg-producing female appears to be decided rather haphazardly. In the worm, Banelia viridis, the minute males are parasites in the female. Larvae that become attached to the proboscis of an adult female become males, while unattached larvae sink to the bottom and become females (BALTZER, 1935). The more sophisticated state of bisexuality was initiated by setting aside a particular pair of chromosomes for specialization and making either the male or the female a heterogametic sex. Sex chromosomes as we know them were thus born.
The subject matter of this chapter could, if taken in its widest sense, encompass a very large part of modern enzymology because of the very close relationships between the enzymes of steroid conjugation and those of the general metabolic pathways of an animal. Here, however, attention will be restricted to those enzymes directly related to the metabolism of steroid conjugates and, furthermore, of only those steroid conjugates whose metabolism has been investigated in detail; that is, of the steroid glucuronides and the steroid sulfates.
1. The properties of enzyme activities hydrolysing the sulphate esters of dehydroepiandrosterone, oestrone and p-nitrophenol are reported. The preparation studied was obtained from the microsomal fraction of human placenta by ultrasonic treatment and addition of Triton X-100. 2. The behaviour of the preparation during sedimentation at 105000g and attempts at purification indicated that the activities were particulate. Electron microscopy demonstrated the rupture of vesicular structures approx. 0·5μ in diameter concurrent with the release of activity. 3. The three activities were always associated throughout repeated attempts at separation by sucrose-density-gradient centrifugation and Sephadex-gel filtration. On the basis of kinetic studies, stability studies and treatment with butanol and ribonuclease it was concluded that a separate enzyme is responsible for each of the three activities. Widely varying plots of activity as a function of pH were consistent with this conclusion. 4. On the basis of sensitivity of the enzymes hydrolysing dehydroepiandrosterone sulphate and oestrone sulphate to ribonuclease and sensitivity of all three enzymes to lipase, it was concluded that the three enzymes are bound to a particle containing lipid and RNA. Enzymic activity is dependent on structural integrity of the particle. 5. A spectrophotometric method for the assay of oestrone sulphate hydrolysis is described. 6. Hydrolysis of nitrocatechol sulphate by human placenta under conditions described for arylsulphatases A and B is reported.
The very bulk of our knowledge of the biochemistry of the sulfates of the 3β—hydroxy—∆
5—steroids would appear to be ample justification for including in this volume a separate chapter devoted especially to this kind of steroid conjugate. However, it seems to us that there are other, more trenchant, reasons for this exceptional treatment. Although scores of steroid sulfates have been isolated or detected in body tissues and fluids, only those of the 3β—hydroxy—∆
5—series seem to possess a uniquely different metabolic role. While the evidence existing at this time does not establish unequivocally the exact nature and importance of their function, there is available now sufficient information about their unusual character to warrant a distinct chapter.
A new inhibitor of cholesterol synthesis, 20,25 diazachlestenol, produced myotonia in 31 persons; and, in six of these, a keratoderma which resembles the natural disease, keratoderma climactericum. These observations further emphasize the importance of normal lipid synthesis in orderly cornification.
The process of epidermal cell renewal, differentiation, and shedding is an exquisitely well-balanced, smoothly functioning system in normal persons. Modern interest in the epidermis has focused primarily on rates and control of cell renewal and on the composition of keratin and keratohyalin polypeptides. Until recently, except for pharmacologic studies of percutaneous penetration, comparatively little consideration has been directed toward the stratum corneum as an organized tissue with cohesive forces that initially hold together its cells and later permit their orderly shedding as small, clinically inapparent bits of material. Yet excessive accumulation and retention of stratum corneum are clinically of great importance in patients with both generalized scaling dermatoses (eg, psoriasis and ichthyosis) and localized hyperplasias (eg, warts and calluses).RECESSIVE X-LINKED ICHTHYOSIS (RXLI) AND STEROID SULFATASE
Although many pharmacologic agents are available for the treatment of hyperkeratosis, usually these are only modestly effective, and little is known about their mechanism
We are accustomed to viewing drug reacactions as representing toxic, allergic, or idiosyncratic responses to the chemical administered; yet a drug may perform its normal action in an undesired tissue site and so give notable side-effects. Triparanol (MER 29) can produce such effects. It blocks synthesis of plasma cholesterol in the liver, but in certain individuals it also causes a slowly progressive, noninflammatory degeneration of the skin provisionally attributed to depression of cutaneous cholesterol synthesis.
A few years ago Parsons and Flinn1 reported that dryness of the skin and a picture of pseudoacanthosis nigricans were produced by nicotinic acid administered in large quantities to patients for hypercholesterolemia. Ruiter and Meyler2 confirmed this observation. Consequently, it was logical to look for such changes in patients taking a different medication but for a similar purpose, namely, to decrease plasma cholesterol. Six months after we started administration of triparanol to patients
Estrogen biosynthesis during human pregnancy has been studied by examination of the radioactive urinary metabolites excreted following the intravenous administration of isotope-labelled steroids to pregnant women under a variety of conditions. Dehydroisoandrosterone sulfate has been shown to be efficiently converted to estrogen in women pregnant with a hydatidiform mole, an anencephalic fetus, and a normal fetus. The fraction of dehydroisoandrosterone sulfate converted to ‘estradiol’ ranged from 4 to 55 per cent. Under certain conditions a major portion of the total endogenous estrogen production may arise by this means. In addition, several lines of evidence indicate that estriol may be derived from dehydroisoandrosterone sulfate by a biochemical pathway which does not involve estrone or estradiol. It is concluded that the placenta does not exclusively utilize mechanisms for estrogen production and may, in fact, rely entirely upon externally supplied steroidal precursors.
Histological features of 12 biopsy specimens from patients with ichthyosis inherited as a dominant trait were compared with 15 specimens from patients with ichthyosis inherited as a sex-linked recessive trait and with 12 control sections from normal skin. Characteristic features that distinguished each inherited variety were described. The histology of sex-linked ichthyosis is very similar to that of non-bullous ichthyosiform erythrodermia.
In an attempt to obtain information not otherwise available about the complex problem of androgen secretion in the human subject, various experiments involving the administration of isotopically labeled steroids have been carried out. The experiments consisted of injecting various steroids labeled with 14C or 3H, singly or in combination, into various subjects and isolating appropriate metabolites from their urine. The C19 steroids used as tracers were dehydroisoandrosterone (D), dehydroisoandrosterone sulfate (DS), androstenedione (Δ) and testosterone (T). After the administration of labeled D it was observed that the specific activities of the urinary glucuronoside of D (DG) were higher than the specific activities of urinary DS, androsterone (A) and etiocholanolone (E). The comparison of these specific activities indicated the existence of secreted precursors, other than D, for DS, A and E (DS itself for DS and T or Δ for A and E). When tracers of D and DS labeled with 3H and 14C were simultaneously ad...
A translocation of the Y to the X chromosome was found in a woman who had habitual abortion. Endocrinologic studies disclosed only an abnormal elevation of her prolactin and thyroid-stimulating hormone (TSH) levels. Phenotypically, she has a rare form of dwarfism and abnormal dermatoglyphic patterns. This new chromosomal rearrangement in the human has several implications.
Inactivation of the single X chromosome in the primary spermatocytes of species with heterogametic males is postulated as
a basic control mechanism on the chromosomal level that is required for normal spermatogenesis. This view is supported by
(a) cytological observations of X-chromosome allocycly in the primary spermatocytes of all male-heterogametic organisms that
were adequately examined, (b) autoradiographic evidence of early cessation of transcription by the X chromosome in the mouse and three species of grasshopper,
and (c) the male sterility of animals with certain X-chromosome rearrangements that cannot be attributed to misfunction of specific
genes. X-chromosome inactivation during spermatogenesis is proposed as the ideal system for studies of genetic control at
the chromosomal level.
The endogenous neutral steroids in human, boar and rat testis tissue were investigated by gas-liquid chromatography and gas chromatography-mass spectrometry. In addition, the secretion of these steroids by human testis was investigated under basal conditions as well as during human chorionic gonadotrophin (HCG) administration. In man and rat testis, testosterone was the main steroid hormone present, whereas in boar, 16-androstenes quantitatively occupied the most important position. In man and boar, a large number of 3β-hydroxy-5-ene steroids, to a large extent sulphate-conjugated, were detected. Some of these compounds were secreted by human testis tissue, and their secretion was increased during HCG administration. When considered together with the findings of other authors, these results stress the importance of the 3β-hydroxy-5-ene pathway of testosterone biosynthesis in testicular tissue. Further, they give support to the view that testicular steroidogenesis might be regulated by modulation of the activity of steroid sulphatase in testis. In the evaluation of the regulation of steroidogenesis in testis, species differences must be given serious consideration, as in rat testes no steroid sulphates could be detected.
The oestrogen excretion of a pregnant woman, subsequently shown to have placental sulphatase deficiency, was only 1-2 mg/24 hr, whereas pregnanediol and 17-oxogenic steroid excretion were within the normal ranges. Plasma concentrations of oestradiol-17β, progesterone, 17α-hydroxyprogesterone, 11β-hydroxycortico-steroids and corticosteroid-binding globulin were subnormal. Placental sulphatase activity in vitro towards the sulphates of DHA, pregnenolone and oestrone was negligible, although extracts of a normal placenta showed high activity under identical conditions. The activities of placental enzymes concerned with metabolism of non-conjugated steroids (3β-hydroxysteroid dehydrogenase-isomerase for DHA and for pregnenolone, aromatase complex and 17β-hydroxysteroid for oestradiol-17β) were less than those of a normal placenta but were not diminished as severely as sulphatase activity.
Satisfactory uterine contractions were achieved during infusion of prostaglandin E2 or Syntocinon but the cervix failed to dilate. A healthy male infant (3330 g) was delivered at term by Caesarean section.
From the evidence obtained during the study of this patient it appears that (i) the presence of appropriate non-conjugated substrates may be required for the induction of placental enzymes concerned with the metabolism of free steroids; (ii) measurement of maternal 17-oxogenic steroid excretion may help to distinguish patients with placental sulphatase deficiency from those whose fetus has adrenal hypoplasia; (iii) further investigations, possibly involving steroid replacement therapy, are required to identify which, if any, of the alterations in plasma steroid concentrations are associated with the failure to achieve cervical dilatation.
A method has been developed for the assay of arylsulfatase C in tissue extracts containing arylsulfatases A and B. Significant variation of enzyme activity was observed among 26 inbred murine strains. Activity differences were apparent at all stages evaluated between 1 and 70 days postnatal age. Arylsulfatase C from representative high- and low-activity strains exhibited similar Michaelis constants, temperature optima, pH optima, thermostabilities and inhibitor profiles.
1. The determination of the relationship of crossing-over to chiasmata and of chiasmata to segregation has made it possible
to define the conditions of variation of pairing chromosomes in complex-heterozygotes and sex-heterozygotes and to analyse
their behaviour in genetic terms. The object of the present studies is, first, to test the predictions made with regard to
sex-chromosomes deducing their genetic structure from cytological observations and, secondly, to note those respects in which
the behaviour of the sex-chromosomes throws light on the problems of chromosome mechanics.
2. The sex-chromosomes of the Norway rat each consist of a pairing segment and a differential non-pairing segment. The pairing
segment includes the spindle attachment and chiasmata may be formed on one or both sides of it so that the first division
is either reductional or equational for the differential segments (Text-fig. 17).
3. The differential segments will have complete sex-linkage, the pairing segments will have partial sex-linkage diminishing
in proportion to the crossing-over distance from the differential segments.
4. The shape, movements, and staining capacity of the sex-chromosomes in the rat and elsewhere agree in suggesting that they
have a lower surface charge than the autosomes, and this is held to be responsible for the special mechanism ofX-chromosome segregation in organisms lacking theY.
In the course of studies on the incorporation of sodium [35S]sulfate into sulfatide in patients with neurological disorders, a metabolically active substance was found which resembles sulfatide both in solubility and behavior on column chromatography but is separable from it on thin-layer chromatography, A method was devised for the isolation of larger quantities of this material. It was found to have the same melting point and Chromatographie properties as authentic cholesterol sulfate and strongly resembles it in its infrared spectrum, Mild acid hydrolysis yielded a substance identical to cholesterol in its melting point, and infrared and nuclear magnetic spectra. A survey of human tissues revealed the presence of small amounts of cholesterol sulfate in plasma, bile, urine, liver, kidney, and larger quantities in feces. It was also found in the brain of a 4-day-old infant. Intravenously administered sodium [35S]sulfate was rapidly incorporated into cholesterol sulfate. Until recently, cholesterol sulfate had not been isolated from any biological material. This study demonstrates that it is metabolically active and that it is present in a variety of human tissues and body fluids.
Cholesterol sulfate is a normal constituent of human erythrocytes at a concentration which is approximately 2-fold higher than plasma cholesterol sulfate. In these cells, the major fraction of the cholesterol sulfate is firmly bound to the membrane.Cholesterol sulfate as well as certain analogs can project the red blood cell against hypotonic hemolysis. This effect is produced in vitro at physiological concentrations of the sterol sulfate and both the sulfate moiety as well as the side chain of the molecule are necessary for biological activity.
In spite of the rather intense interest in the biology of X inactivation, little experimental data have been obtained that provide insight into the molecular basis for the initiation or the maintenance of X inactivation. Indeed, although the preponderance of data would indicate that supernumerary X chromosomes are inactivated, some investigators still consider it possible that the differentiative event is activation of a single X chromosome with the other X chromosomes remaining 'passively' inactive. Furthermore, it is clear that active X-encoded genes are not constitutively expressed in all cell types, and it is therefore probable that further levels of control of gene expression are superimposed on inactivation. In addition, it is not unlikely that the initiation of X inactivation will turn out to have a mechanism different from the process responsible for the maintenance of an inactivation pattern once it is established. At the time of inactivation, a chromosomal event clearly appears to be implicated. However, as discussed, individual segments and, perhaps, even individual genes can be independently 'reactivated' in vitro. Finally, it is possible that there may be several types of X inactivation, as perhaps is exemplified by the preferential and partial inactivation of the parental X chromosome in marsupials as opposed to the random and complete inactivation of the X chromosome seen in eutherian mammals. Preferential paternal X inactivation may also exist in mouse extraembryonic tissues, and a reversible form of X inactivation seems to occur in female primordial germ cells. Basically, three general types of models have been put forth in attempts to encompass and explain numerous biological observations regarding mammalian X-chromosome inactivation. These include specific DNA-protein interaction, physical rearrangement of the inactive X genes, and DNA modification.
A family is reported in which the mother and two sons are carriers of a Y-to-X translocation, der (X)t(X;Y) (p22;q11). All the the three carriers have short stature and disproportion of extremities, but otherwise normal phenotype. One of the sons, the propositus, has been affected with schizophrenia. Evidence was obtained that male carriers are probable sterile; both sons aged 26 and 30 years had azoospermia and the biopsied specimens of the testis had histologic pictures showing spermatogenetic arrest. The mother was H-Y weakly positive, and the normal X chromosome was inactivated in the majority of the cells analyzed. Dermatoglyphics of the three carriers were unusual and dissimilar to the features of Turner's syndrome. The clinical and cytogenetic findings in the present study are compared with those of the previously reported familial cases, and the genetic background causing phenotypic abnormalities in the male and female carriers is discussed.
Steroid sulfatase, a membrane-bound enzyme present in many mammalian tissues, was extracted from rat liver microsomes by treatment with Miranol H2M, a zwitterion detergent, and sonication. It has been purified approximately 33-fold. All steps of the purification, which included salt and solvent fractionation, hydroxylapatite treatment, ion-exchange chromatography, and gel filtration were performed in the presence of Miranol H2M, most of which was removed from the final preparation by gel filtration. The final preparation did not contain any detectable NADPH-cytochrome c reductase or glucose-6-phosphate phophatase activities. According to the elution volume on a Sephadex G-200 column, steroid sulfatase has a molecular weight of approximately 130,000. Polyacrylamide-gel electrophoresis in the presence of Miranol H2M revealed one major protein band which was enzymatically active. Purified steroid sulfatase hydrolyzes all the sulfate esters of estrone, dehydroepiandrosterone, pregnenolone, testosterone, and cholesterol as well as p-nitrophenyl sulfate, the substrate for arylsulfatase C, during the purification. However, estrone sulfatase and arylsulfatase C activities were enriched more than the others. Analysis of kinetic data and the effects of different buffers and of Miranol H2M also suggested that estrone sulfatase and arylsulfatase C are identical but that they are distinct from the other sulfatases. Competitive inhibition studies suggest that estrone sulfatase also catalyzes the hydrolysis of the sulfate esters of other estrogens.
Multiple deficiency disorder fibroblasts cultured in MEM-CO2 showed deficiencies of arylsulfatase A(ARS A) comparable to the deficiency in metachromatic leukodystrophy fibroblasts. However, the MSDD fibroblasts cultured in MEM-HEPES contained near normal levels of ARS A. Moreover, the enzyme from the latter fibroblasts was indistinguishable from ARS A of control fibroblasts on DEAE-cellulose chromatography, ratio of activity with several substrates, thermal inactivation, sensitivity to inhibitors, and precipitation by antiserum to human ARS A. These data support the conclusion that the ARS A genome is intact in MSDD fibroblasts and, by extension, in MSDD patients. Other sulfatases were present at levels ranging from mildly deficient to near normal but never as low as seen in the corresponding specific sulfatase deficient disorders.
It has been observed that multiple sulfatase deficiency disorder (MSDD) fibroblasts contained from profoundly deficient to near normal amounts of arylsulfatase (ARS) A depending on the medium in which they were cultured. Our present findings show that the major factor determining the enzyme level is the pH of the medium during growth. In media which became acidic or was maintained at low pH (less than 7), the cells expressed the enzymopathy, while in high pH media (7.4), the cells produced enzyme. The high and low enzyme states were reversible. The ARS A deficiency in MSDD must, therefore, be a secondary manifestation of a mutation in another system.
An assay of cultured skin fibroblasts identified several individuals with 3 beta-hydroxysteroid-sulphate sulphatase deficiency. All patients with this inborn error of metabolism had clinically apparent ichthyosis and a family history of this skin disorder compatible with X-linked inheritance. It is concluded that steroid-sulphatase deficiency is the bio-chemical basis of at least some cases of X-linked ichthyosis.
Steroid sulphatase activity was determined in cultured fibroblasts from 25 individuals with X-linked ichthyosis from four countries. All those with X-linked disease had markedly reduced enzyme levels compared with controls and patients with other types of ichthyosis. X-linked ichthyosis seems to be the result of a common mutation affecting the expression of steroid-sulphatase activity.
Multiple sulphatase deficiency disease is an unusual autosomal recessive disorder characterised biochemically by a deficiency of several sulphohydrolase activities. The laboratory diagnosis of this combined neurological connective tissue disorder is made on the basis of decreased activities of the lysosomal enzymes, arylsulphatase A and arylsulphatase B and the microsomal enzyme, arylsulphatase C. The primary defect in this multi-enzyme deficiency has not been identified. Using immunological techniques to characterise further the residual activities of arylsulphatases A and B in the multiple sulphatase deficiency disease, we have examined the levels of cross-reaching material (CRM) to arylsulphatases A and B in cultured skin fibroblasts from controls and patients with multiple sulphatase deficiency, metachromatic leukodystrophy (deficiency of only arylsulphatase A activity) and Maroteaux-Lamy syndrome (deficiency of only arylsulphatase B activity). We report here results indicating that arylsulphatases A and B in multiple sulphatase deficiency are reduced in their levels of CRM while retaining a normal activity/CRM ratio. Because the two enzymes are apparently structurally unrelated, these data are consistent with the possibility that their combined deficiencies in this disorder may result from a defect in the coordinated expression of sulphohydrolases.
Our studies on the steroid composition and steroid secretion by human testis are summarized. Using gas-liquid chromatography and gas chromatography-mass spectrometry 24 neutral steroids were identified and measured in human testis tissue obtained from cadavers or in connexion with orchiectomy due to prostatic cancer. The compounds were partly present in unconjugated form and partly as mono- and disulphates. The main compounds present were unconjugated testosterone, pregnenolone and its sulphate, dehydroepiandrosterone sulphate and 5-androstene-3β,17β-diol monosulphate. C19 steroid diol monosulphates with a 17β-hydroxyl carried their sulphate group at carbon 3, whereas those with a 17α-hydroxyl had the sulphate group at carbon 17. The main unconjugated compound secreted by human testis was testosterone followed by 5-androstene-3β, 17β-diol, 17α-hydroxyprogesterone, pregnenolone, 5-androstene-3β,17α-diol, 17α-hydroxypregnenolone, androstenedione and dehydro-epiandrosterone. The monosulphates of pregnenolone, 5-androstene-3β,17β-diol and testosterone were also secreted. The secretion of all these compounds was increased during the administration of human chorionic gonadotropin (HCG) and dehydroepiandrosterone sulphate secretion also became evident. These results, when considered in the light of the findings of other authors strongly suggest that the 5-ene pathway is the quantitatively more important one in the formation of testosterone in human testis. Further, it is possible that sulphated steroids also act as precursors and that testosterone biosynthesis could be regulated through regulation of testicular steroid sulphatase activity.
The 5-ene-steroid-3β-monosulfates (SMS) are accepted as potential intermediates in androgen biosynthesis in vivo. Evidence from this laboratory and others are reviewed to support the premise that despite low conversions, the physiological significance of the regulation of SMS cleavage is important for androgen biosynthesis primarily at the target site of responsive tissue. Steroid sulfatase enzyme, regardless of tissue source in mammals, appears to be a monomeric unit having a steroid sulfate binding-site and a second regulatory steroid binding-site. Monomer or its aggregates intimately associated with particulate cell membrane implicate both transport and metabolism of steroid hormones. A model is presented in which SMS (substrates) contribute to androgen biosynthesis through hormonally (steroid allosteric modifiers and inhibitors) regulated cleavages at the cell membrane by steroid sulfatase enzyme at the site of hormone (steroid product) utilization. This is suggested to occur at the blood-organ barrier in target tissues sensitive to androgens, and in particular as a means of regulating the level of androgens within the seminiferous tubule.
Placental sulfatase deficiency has been found in four pregnancies (cases 1 to 4) with inappropriately low levels of urinary estriol excretion (less than 1.3 mg. per day near term gestation) associated with healthy neonates. The basis of the diagnosis in these cases was the greatly limited capacities for hydrolysis of 14C-dehydroepiandrosterone sulfate (DHA-S) and 3H-estrone sulfate (0.2 mugCi each) to the free steroids during incubation of placental homogenates. Placental aromatase activities in vitro for free DHA and the concentrations of appropriate estrogen precursors in cord blood were normal or elevated. The defect was diagnosed prenatally in two of these cases on the basis of failure to increase the maternal excretion of urinary estriol (0.6 to 0.7 and 1.3 to 1.3 mg. per day, respectively) following acute instillation of DHA-S (250 mg.) into the amniotic fluid and on normal levels of estrogen precursors in cord blood. In comparison, a twofold increase in maternal estriol excretion was observed after infusing DHA-S into the amniotic cavity of a "high-risk" pregnancy having normal sulfatase and aromatase activities in vitro (case 5). These enzyme activities were also found to be similarly normal in another placenta from an undergrown fetus (case 6) and in six normal placentas. The clinical features of these pregnancies, the first ones described from the western hemisphere, are similar to reported cases: the newborn progeny are healthy males who appear to be developing normally. The prenatal diagnosis of the sex-specific placental enzyme defect has been made possible by the use of an intra-amniotic DHA-S loading test.
A pregnancy with placental sulfatase deficiency was suspected when a 36-year-old patient at 41 weeks of gestation was found to have extremely low urinary estriol excretion and an otherwise normal prenatal course. The maternal plasma levels of estriol and estradiol 17-beta (E2) were extremely low and estetrol (E4) was undetectable (less than 40 pg/ml), whereas dehydroepiandrosterone sulfate (DS) was normal. THE AMNIOTIC Fluid DS concentration was 22-fold higher than the mean of normal pregnancy, while that of dehydroepiandrosterone (D) and androstenedione (A) was normal. Following intravenous infusion of 50 mg DS, no rise of plasma E2 was noted and plasma E4 levels remained undetectable. At 42 weeks of pregnancy, after induction of labor failed, a healthy male infant was delivered by cesarean section. The umbilical vein (UV) and umbilical artery (UA) levels of DS were extremely high, and those of E2 and E4 were subnormal. The UA level of A was normal and the levels of D and testosterone were slightly elevated. In vitro studies of placental microsomes and the 10,000 x g supernantant confirmed the diagnosis of placental sulfatase deficiency. The infant at 6 months of age had normal growth and development and normal peripheral plasma DS concentration.