High levels of oxysterol sulfates in serum of patients with steroid sulfatase deficiency.

Steroid sulfatase (STS) deficiency is the underlying cause of the skin condition known as recessive X-linked ichthyosis (RXLI). RXLI patients show scales on their skin caused by high concentrations of cholesterol sulfate (CS), as they are not capable of releasing the sulfate group from its structure to obtain free cholesterol. CS has been reported, so far, as the sole sulfated steroid with increased concentrations in the blood of RXLI patients. A non-targeted LC-MS approach in negative mode detection (LC-MS precursor ion scan mode) was applied to serum samples of 12 RXLI patients and 19 healthy males. We found that CS was not the only sulfated compound consistently elevated in RXLI patients, because a group of compounds with a m/z of 481 was found in high concentrations too. Further LC-MS/MS demonstrated that the main contributor to the m/z 481 signal in RXLI serum is 27-hydroxycholesterol-3-sulfate (27OHC3S). Accordingly, a new method for 27OHC3S quantification in the context of RXLI has been developed and validated. Other hydroxycholesterol sulfate compounds were elevated as well in RXLI patients.

Role of cholesterol sulfate in epidermal structure and function: lessons from X-linked ichthyosis.

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. Guest Editors: Kenneth R. Feingold and Peter Elias.

Genetic analysis of a 12-year-old boy with X-linked ichthyosis in association with sclerosing glomerulonephritis.

In this study, we report the case of a 12-year-old male with X-linked ichthyosis (XLI) in association with glomerular sclerosis, and our investigation into the deletion pattern of the STS gene and the flanking regions in DNA samples of family members. We observed no features typical of renal osteodystrophy or rickets, with the exception of short stature, in the three afffected male family members. Audiometry, visual acuity and olfactory sensation were normal. By performing PCR analysis of the steroid sulfatase (STS) gene and flanking regions on our patients, we discovered a complete deletion that involved the entire region from DXS1139 to DXF22S1. Further studies are required to determine whether the STS gene or the co-deleted flanking sequences are the cause of renal disease associated with XLI.

Exacerbation of X-linked ichthyosis phenotype in a female by inheritance of filaggrin and steroid sulfatase mutations.

BACKGROUND: X-linked ichthyosis (XLI) is a relatively common, recessive condition caused by mutations in the steroid sulfatase (STS) gene. Common loss-of-function mutations in the filaggrin gene (FLG) cause ichthyosis vulgaris and predispose individuals to atopic eczema. OBJECTIVE: To test the hypothesis that co-inheritance of FLG mutations can act as a genetic modifier in XLI. METHODS: An unusually severe XLI phenotype in addition to eczema and mild childhood asthma was investigated in a female Indian patient by fluorescent in situ hybridization (FISH) for the common STS gene deletion. Direct sequencing of the entire FLG gene was also performed. RESULTS: FISH analysis revealed that the proband was homozygous for the common STS genomic deletion mutation. Further investigation revealed a frame-shift mutation 3672del4 in the gene encoding filaggrin (FLG), leading to premature termination of profilaggrin translation. Interestingly, her father, who had a very typical mild presentation of XLI, did not carry this FLG mutation in addition to his STS deletion. Her mother was a heterozygous carrier of the FLG mutation and consistent with this, had mild symptoms of ichthyosis vulgaris; she was also a heterozygous carrier of the STS deletion. CONCLUSION: This is the second reported case of the modifying effects of FLG null alleles on XLI and strengthens the hypothesis that filaggrin defects can synergize with STS deficiency to exacerbate the ichthyosis phenotype.

HDHD1, which is often deleted in X-linked ichthyosis, encodes a pseudouridine-5'-phosphatase.

Pseudouridine, the fifth-most abundant nucleoside in RNA, is not metabolized in mammals, but is excreted intact in urine. The purpose of the present work was to search for an enzyme that would dephosphorylate pseudouridine 5'-phosphate, a potential intermediate in RNA degradation. We show that human erythrocytes contain a pseudouridine-5'-phosphatase displaying a Km ≤ 1 µM for its substrate. The activity of the partially purified enzyme was dependent on Mg2+, and was inhibited by Ca2+ and vanadate, suggesting that it belonged to the 'haloacid dehalogenase' family of phosphatases. Its low molecular mass (26 kDa) suggested that this phosphatase could correspond to the protein encoded by the HDHD1 (haloacid dehalogenase-like hydrolase domain-containing 1) gene, present next to the STS (steroid sulfatase) gene on human chromosome Xp22. Purified human recombinant HDHD1 dephosphorylated pseudouridine 5'-phosphate with a kcat of 1.6 s-1, a Km of 0.3 µM and a catalytic efficiency at least 1000-fold higher than that on which it acted on other phosphate esters, including 5'-UMP. The molecular identity of pseudouridine-5'-phosphatase was confirmed by the finding that its activity was negligible (<10% of controls) in extracts of B-cell lymphoblasts or erythrocytes from X-linked ichthyosis patients harbouring a combined deletion of the STS gene (the X-linked ichthyosis gene) and the HDHD1 gene. Furthermore, pseudouridine-5'-phosphatase activity was 1.5-fold higher in erythrocytes from women compared with men, in agreement with the HDHD1 gene undergoing only partial inactivation in females. In conclusion, HDHD1 is a phosphatase specifically involved in dephosphorylation of a modified nucleotide present in RNA.

IFAP syndrome is caused by deficiency in MBTPS2, an intramembrane zinc metalloprotease essential for cholesterol homeostasis and ER stress response.

Ichthyosis follicularis with atrichia and photophobia (IFAP syndrome) is a rare X-linked, oculocutaneous human disorder. Here, we assign the IFAP locus to the 5.4 Mb region between DXS989 and DXS8019 on Xp22.11-p22.13 and provide evidence that missense mutations exchanging highly conserved amino acids of membrane-bound transcription factor protease, site 2 (MBTPS2) are associated with this phenotype. MBTPS2, a membrane-embedded zinc metalloprotease, activates signaling proteins involved in sterol control of transcription and ER stress response. Wild-type MBTPS2 was able to complement the protease deficiency in Chinese hamster M19 cells as shown by induction of an SRE-regulated reporter gene in transient transfection experiments and by growth of stably transfected cells in media devoid of cholesterol and lipids. These functions were impaired in five mutations as detected in unrelated patients. The degree of diminished activity correlated with clinical severity as noted in male patients. Our findings indicate that the phenotypic expression of IFAP syndrome is quantitatively related to a reduced function of a key cellular regulatory system affecting cholesterol homeostasis and ability to cope with ER stress.

Analysis of the VCX3A, VCX2 and VCX3B genes shows that VCX3A gene deletion is not sufficient to result in mental retardation in X-linked ichthyosis.

BACKGROUND: X-linked ichthyosis (XLI), an inborn error of metabolism, is due to steroid sulphatase (STS) deficiency. Most patients with XLI harbour complete deletion of the STS gene and flanking sequences. The presence of low copy number repeats on either side of the STS gene seems to have a major role in the high frequency of these deletions. Some patients with XLI with terminal deletions of Xp22.3 involving marker DXS1139 and the STS gene show mental retardation (MR); VCX3A is the only gene located on this critical region. OBJECTIVES: To analyse the VCX3A, VCX, VCX2 and VCX3B genes in 80 unrelated Mexican patients with XLI with normal intelligence. METHODS: STS activity was measured in the leucocytes using 7-[3H]-dehydroepiandrosterone sulphate as a substrate. Amplification of the regions from telomeric DXS89 to centromeric DXS1134 including both extremes of the STS and the VCX3A, VCX, VCX2 and VCX3B genes was performed using polymerase chain reaction. RESULTS: No STS activity was detected in the patients with XLI (0.00 pmol mg(-1) protein h(-1)). We observed two different deletion patterns: the first group included 62 patients with deletion of VCX3A and VCX genes. The second group included 18 patients with breakpoints at several regions on either side of the STS gene not including the VCX3A gene. CONCLUSIONS: These data indicate that more complex mechanisms, apart from possible VCX3A gene participation, are occurring in the genesis of MR in XLI, at least in the sample of Mexican patients analysed.

Three-dimensional structures of sulfatases.

The sulfatase family of enzymes catalyzes the hydrolysis of sulfate ester bonds of a wide variety of substrates. Nine human sulfatase proteins and their genes have been identified, many of which are associated with genetic disorders leading to reduction or loss of function of the corresponding enzyme. A catalytic cysteine residue, strictly conserved in prokaryotic and eukaryotic sulfatases, is modified posttranslationally into a formylglycine. Hydroxylation of the formylglycine residue by a water molecule forming the activated hydroxylformylglycine (a formylglycine hydrate or a gem-diol) is a necessary step for sulfatase activity of the enzyme. Crystal structures of three human sulfatases, arylsulfatases A and B (ARSA and ARSB) and C, also known as steroid sulfatase or estrone/dehydroepiandrosterone sulfatase (ES), have been determined. In addition, the crystal structure of a homologous bacterial arylsulfatase from Pseudomonas aeruginosa (PAS) is also available. While ARSA, ARSB, and PAS are water-soluble enzymes, ES has a hydrophobic domain and is presumed to be bound to the endoplasmic reticulum membrane. This chapter compares and contrasts four sulfatase structures and revisits the proposed catalytic mechanism in light of available structural and functional data. Examination of the ES active site reveals substrate-specific interactions previously identified in another steroidogenic enzyme. Possible influence of the lipid bilayer in substrate capture and recognition by ES is described. Finally, mapping the genetic mutations into the ES structure provides an explanation for the loss of enzyme function in X-linked ichthyosis.

Prenatal diagnosis for placental steroid salfatase deficiency with fluorescence in situ hybridization: a case of X-linked ichthyosis.

X-linked ichthyosis (XLI) is a relatively common genetic disorder that occurs in about one in every 2000-6000 male births. Clinically, XLI is characterized by a generalized scaling of the skin, with large, polygonal, dark brown scale, and more prominent on the extensor aspects of the limbs. It is known that an undetectable maternal serum, unconjugated estriol, associated with placental steroid sulfatase (STS) deficiency, may be the cause of cause of XLI. In most case, STS deficiency is caused by a complete or partial deletion of the STS gene mapped on chromosome Xp22.3. We describe here the prenatal detection of a male fetus affected with STS deficiency as a result of an undetectable unconjugated estriol in the second-trimester maternal serum screening. Microdeletion of the STS gene was confirmed by fluorescence in situ hybridization analysis of cultured amniotic fluid.

Carrier identification by FISH analysis in isolated cases of X-linked ichthyosis.

X-linked ichthyosis (XLI) is an inborn error of metabolism due to steroid sulfatase (STS) deficiency. STS assay and FISH are useful in diagnosing carrier status of XLI. Biochemical analysis appears to indicate that most sporadic cases are inherited. Since this method does not seem to be completely reliable in recognizing XLI-carriers, the aim of the present study was to corroborate by FISH whether or not most sporadic cases of XLI had de novo mutations. XLI patients were classified through STS assay and PCR amplification of 5'-3' ends of the STS gene. XLI patients had undetectable levels of STS activity and complete deletion of the STS gene. Patients' mothers were studied through STS assay and FISH. Nine out of 12 mothers presented an STS activity compatible with XLI-carrier state. These mothers also had only one copy of the STS gene, indicating that they carry the primary gene defect. One mother had normal STS activity but only one copy of the STS gene. This data corroborated that most sporadic cases do not represent de novo mutations, and that FISH must be included in the analysis of mothers of sporadic cases when they present with normal STS activity, in order to correctly diagnose the XLI carrier state.

Deletion pattern of the STS gene in X-linked ichthyosis in a Mexican population.

BACKGROUND: X-linked ichthyosis (XLI) is an inherited disorder due to steroid sulfatase deficiency (STS). Most XLI patients (>90%) have complete deletion of the STS gene and flanking sequences. The presence of low copy number repeats (G1.3 and CRI-S232) on either side of the STS gene seems to play a role in the high frequency of these interstitial deletions. In the present study, we analyzed 80 Mexican patients with XLI and complete deletion of the STS gene. MATERIALS AND METHODS: STS activity was measured in the leukocytes using 7-[(3)H]-dehydroepiandrosterone sulfate as a substrate. Amplification of the regions telomeric-DXS89, DXS996, DXS1139, DXS1130, 5' STS, 3' STS, DXS1131, DXS1133, DXS237, DXS1132, DXF22S1, DXS278, DXS1134-centromeric was performed through PCR. RESULTS: No STS activity was detected in the XLI patients (0.00 pmoles/mg protein/h). We observed 3 different patterns of deletion. The first two groups included 25 and 32 patients, respectively, in which homologous sequences were involved. These subjects showed the 5' STS deletion at the sequence DXS1139, corresponding to the probe CRI-S232A2. The group of 32 patients presented the 3' STS rupture site at the sequence DXF22S1 (probe G1.3) and the remaining 25 patients had the 3' STS breakpoint at the sequence DXS278 (probe CRI-S232B2). The third group included 23 patients with the breakpoints at several regions on either side of the STS gene. No implication of the homologous sequences were observed in this group. CONCLUSION: These data indicate that more complex mechanisms, apart from homologous recombination, are occurring in the genesis of the breakpoints of the STS gene of XLI Mexican patients.

Corneal manifestations of X-linked ichthyosis in two brothers.

PURPOSE: To report the unusual manifestation of X-linked ichthyosis in two brothers. METHODS: Leukocyte separation and sterylsulfatase assay are performed to show the deficiency of sterylsulfatase. RESULTS: Two brothers presented in our clinic with cutaneous alterations consistent with X-linked ichthyosis. Ocular examination disclosed fine, flour-like, punctate, evenly, and diffusely distributed opacities of the posterior corneal stroma, close to Descemet membrane in both patients. In one patient, superficial, small, granular opacities were detected. They were gray in color and seemed to involve the epithelium and the subepithelial and anterior stromal layers. In both patients, the deficiency of sterylsulfatase could be shown and confirmed the diagnosis. CONCLUSIONS: Flour-like opacities in the posterior stroma have been shown to be a common manifestation of X-linked ichthyosis. Though the underlying biochemical defect in X-linked ichthyosis has been discovered, the question of how these opacities develop is still a subject of debate. Subepithelial stromal keratopathies or epithelial irregularities are uncommon and are rarely described in the literature. The superficial corneal changes seen in one of our patients are unusual and are not similar to those reported by other authors.

[Occurrence of X-linked ichthyosis along with atopy]. / X-kromoszómához kötött ichthyosis és atopiás betegségek együttes elófordulása.

The cases of children indicate suffering from X-linked ichthyosis accompanied by atopic diseases (bronchial asthma, allergic rhinitis, allergic conjunctivitis, atopic dermatitis) which developed in infancy. The examinations revealed microdeletion of the steroid sulfatase region (Xp22.3), increased total IgE value and polysensibilization. It seems that the steroid sulphatase deficiency in ichthyosis played a role in the development of the atopic diseases. The present study suggests the commencement of a strict diet and antihistamine therapy, already in its infancy, in order to prevent the development of the accompanying atopic diseases.

Deletion patterns of the STS gene and flanking sequences in Israeli X-linked ichthyosis patients and carriers: analysis by polymerase chain reaction and fluorescence in situ hybridization techniques.

BACKGROUND: Deletion of the entire steroid sulfatase (STS) gene is the most common molecular defect in X-linked ichthyosis (XLI) patients. Usually, additional flanking sequences are also missing. The aim of this study was to estimate the extent of deletions in an ethnically heterogeneous population of Israeli XLI patients. METHODS: Multiplex polymerase chain reaction (PCR) and fluorescence in situ hybridization (FISH) techniques were applied in the analysis of blood samples of 24 patients and amniotic cells of seven affected fetuses from 22 unrelated families. RESULTS: In 19 families, a large deletion of the 2-3 megabase was found. It included the whole STS gene and spanned adjacent areas up- and downstream between the loci DXS 1139 and DXS 1132. Two unrelated families of Iraqi ancestry had a partial deletion of the gene and its centromeric adjacent sequence. In another family, the telomeric end of the extragenic segment was only partially missing. Application of FISH on metaphase blood cells and interphase amniotic cells confirmed the diagnosis of XLI in all patients, except the three with partial intragenic deletion. In those cases, the remaining fraction of the gene was sufficient to provide a false negative result. Diagnosis of carriers and prenatal diagnosis in uncultured cells was applicable only by FISH. CONCLUSIONS: Our study revealed a remarkable heterogeneity in the deletion pattern among Israeli patients with XLI. This heterogeneity could not be attributed to specific ethnic groups because of the small size of the study group. More studies involving patients of various ancestries should be carried out. In addition, this study demonstrated the usefulness of the FISH technique in the prenatal diagnosis of fetuses with suspected XLI.

CHILD syndrome caused by deficiency of 3beta-hydroxysteroid-delta8, delta7-isomerase.

CHILD (congenital hemidysplasia, ichthyosis, and limb defects) syndrome is a rare, usually sporadic disorder associated with unilateral distribution of ichthyosiform skin lesions, limb defects, punctate calcifications of cartilaginous structures, and visceral anomalies. CHILD syndrome shares some manifestations with X-linked dominant Conradi-Hünermann syndrome (CDPX2), although the skeletal defects and skin lesions in CDPX2 are bilateral and asymmetric. Because CDPX2 patients have abnormal 8-dehydrosterol metabolism caused by mutations in 3beta-hydroxysteroid-delta8,delta7-isomerase, we measured plasma sterols in a patient with CHILD syndrome and found levels of 8-dehydrocholesterol and 8(9)-cholestenol increased to the same degree as in CDPX2 patients. Subsequently, we identified a nonsense mutation in exon 3 of the patient's 3beta-hydroxysteroid-delta8,delta7-isomerase gene. We speculate that at least some cases of CHILD syndrome are allelic with CDPX2 caused by 3beta-hydroxysteroid-delta8,delta7-isomerase deficiency.

Mutations in the NSDHL gene, encoding a 3beta-hydroxysteroid dehydrogenase, cause CHILD syndrome.

We report for the first time that CHILD syndrome (MIM 308050), an X-linked dominant, male-lethal trait characterized by an inflammatory nevus with striking lateralization and strict midline demarcation, as well as ipsilateral hypoplasia of the body is caused by mutations in the gene NSDHL located at Xq28 (NAD(P)H steroid dehydrogenase-like protein) encoding a 3beta-hydroxysteroid dehydrogenase functioning in the cholesterol biosynthetic pathway. SSCA and genomic sequence analysis of NSDHL identified in 6 patients with CHILD syndrome, including one boy as well as a mother and her daughter, mutations potentially impairing protein function. This phenotype is distinct from, but shares various clinical and biochemical findings with chondrodysplasia punctata (CDPX2, MIM 302960). CDPX2 is due to mutations affecting a delta8-delta7 sterol isomerase (EBP, emopamil binding protein, at Xp11.22-p11.23) that functions downstream of NSDHL in a later step of cholesterol biosynthesis. EBP was unaffected in the patients analyzed by us demonstrating that CHILD syndrome and CDPX2 are not caused by allelic mutations. Two mouse X-linked dominant male-lethal traits, bare patches (Bpa) and striated (Str) had previously been associated with mutations in Nsdhl. They provide animal models for the study of CHILD syndrome, a further human condition due to mutations in a gene of the cholesterol synthesis pathway.

Submicroscopic Xpter deletion in a boy with growth and mental retardation caused by a familial t(X;14).

In a 3-year-old boy with short stature, developmental delay, and dry skin, steroid sulphatase deficiency and a submicroscopic terminal deletion of Xp were found. Except for the short stature, no major clinical signs of X-linked recessive chondrodysplasia punctata could be observed. His mother had lowered steroid sulphatase activity compatible with carriership for X-linked ichthyosis and a submicroscopic translocation (X;14)(p22.31;p11.1). This finding combined with a normal amplification of exons 1, 5, and 10 of the STS gene from propositus' DNA suggested a breakpoint upstream of the STS gene. The submicroscopic maternal translocation had important implications for genetic counseling. This case report illustrates that contiguous gene syndrome related to the Xpter region may have an atypical clinical presentation and the usefulness of combined clinical, biochemical, molecular, and fluorescence in situ hybridization analysis.