Deletion of a DNA sequence in eight of nine families with X-linked ichthyosis (steroid sulphatase deficiency).

Deficiency of steroid sulphatase (STS) is associated with ichthyosis, with failure of the placental production of oestriol in late pregnancy and with difficulties in childbirth. The STS gene has been localised by deletion mapping to the distal tip of the snort arm of the X chromosome, and is of interest in that it appears to escape X-inactivation. We have constructed an X-specific DNA library and screened it for single copy DNA sequences which lie at the distal end of Xp. The sequence GMGX9 was found to map in the interval Xp22.3-pter and to detect a frequent HindIII polymorphism. We have used GMGX9 in linkage studies in families with classical X-linked ichthyosis and this has not only shown tight linkage with STS deficiency but has also revealed that the sequence is deleted in affected males in eight of nine families. GMGX9 is present in all of 26 normal male individuals so far examined. Our findings suggest that a high proportion of the mutations at the STS locus leading to enzyme deficiency are deletions, presumably generated by unequal cross-over events in female meiosis or by illegitimate X-Y interchange in male meiosis.     

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.     

核基质结合区对转爬山虎芪合酶基因烟草中产生白藜芦醇的作用

采用核基质结合区(MARs)来提高转芪合酶基因(STS)烟草(Nicotianatabacum L.)中白藜芦醇产物的含量.MARs是细胞中能与核基质特异紧密结合的DNA片段,体外结合实验表明克隆自酵母的MARs序列能特异地与烟草核基质结合.芪合酶是白藜芦醇生物合成中的关键酶,用RT-PCR方法从川鄂爬山虎(Parthenocissus henryana(Hemsl.)Diels et Gilg)中克隆了与葡萄芪合酶基因有较高同源性的芪合酶编码区,将其置于CaMV35SΩ强启动子下,分别构建两侧带有MARs及不含MARs序列的表达载体,通过农杆菌介导转化烟草.Northern blot及HPLC等分析表明STS基因已整合至烟草染色体中并正常转录,且表达的外源芪合酶在烟草中可催化其底物合成白藜芦醇产物.与对照相比,MARs的存在使转芪合酶基因烟草中白藜芦醇的含量平均提高了约一倍.MARs在转芪合酶基因植物中的应用也为获得抗病性更强、白藜芦醇含量更高、更保健的转基因果蔬的研究奠定了基础.     

Deletions of the steroid sulphatase gene in “classical” X-linked ichthyosis and in X-linked ichthyosis associated with Kallmann syndrome

Summary We have studied 16 men, from 10 unrelated Italian families, affected by steroid suphatase (STS) deficiency, which is the basic defect of X-linked ichthyosis (XLI). The patients' clinical diagnoses were of either isolated ichthyosis or ichthyosis associated with Kallmann syndrome (KS) (hypogonadotropic hypogonadism and anosmia). DNA from patients and their relatives was analysed by Southern blotting followed by hydridization with an STS cDNA probe. None of the patients affected by either XLI or XLI/KS showed any hybridization signal, thus revealing a deletion in the STS gene. We suggest that a gene deletion may be the most common molecular defect involved in XLI and that the syndrome XLI/KS may be due to a deletion of both the STS and the KS loci.     

Molecular heterogeneity of steroid sulfatase deficiency: a multicenter study on 57 unrelated patients, at DNA and protein levels

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. Twenty-eight 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 evidence 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.     

Cloning of the rat steroid sulfatase gene (Sts), a non-pseudoautosomal X-linked gene that undergoes X inactivation

Although the human steroid sulfatase (STS) gene has been cloned and characterized in detail, several attempts to clone its mouse homologue, with either anti-human STS antibodies or human STS cDNA probes, have failed, suggesting a substantial divergence between these genes. However, partial amino-terminal sequence from purified rat liver STS is very similar to its human counterpart, and sequence comparisons have revealed several domains that are conserved among all the sulfatases characterized to date. Thus, we used a degenerate-primer RT-PCR approach to amplify a 321-bp fragment from rat liver cDNA, which was used as a probe to clone and characterize the complete cDNA. Comparison of the protein coding region between the rat and human genes showed 66% homology both at the DNA and the protein levels. STS activity was conferred to STS(−) A9 cells upon transfection with a rat Sts expression construct, indicating the authenticity of the cloned cDNA. While Sts has been shown to be located in the mouse pseudoautosomal region, both physical and genetic mapping demonstrate that Sts is not pseudoautosomal in the rat. The overall genomic organization of rat Sts and human STS is very similar, except that the insertion site for intron 1 in the rat is 26 bp upstream from that in the human. Rat Sts is only 8.2 kb long, while the human STS spans over 146 kb. Received: 24 October 1995 / Accepted: 5 March 1996     

Multipoint linkage analysis of steroid sulfatase (X-linked ichthyosis) and distal Xp markers

Six families with steroid sulfatase deficiency (STS; X-linked ichthyosis) have been studied with the Xg blood group (XG) and the DNA markers dic56 (DXS143), 782 (DXS85), pD2 (DXS43), and GMGX9. Carrier status of females was determined by assay of STS in hair roots. GMGX9 detects a frequent restriction fragment length polymorphism and also identifies a deletion in the majority of families with STS deficiency, including five of the six reported here. The linkage relationship of this marker to the others was studied in normal three-generation families yielding 32 phase-known meioses informative for two or more markers. No recombinants were observed between STS and GMGX9, giving a maximum lod score of 8.73 at zero recombination. Multipoint linkage analysis taking STS and GMGX9 as a single locus and incorporating two-point marker data and STS-XG data from published studies gave the map (Sequence: see text). This order was 2.4 times more likely than with (STS,GMGX9) and dic56 reversed and is supported by our findings in a male with steroid sulfatase deficiency due to a deletion of Xp22.3 which encompasses the XG locus. He is deleted for GMGX9 but shows normal hybridization to dic56 and 782.     

Molecular analysis of X-linked ichthyosis in Japan

BACKGROUND: X-linked ichthyosis (XLI) is an inherited skin disorder caused by a deficiency of steroid sulfatase (STS). The gene and protein of STS were examined in 19 Japanese patients with XLI. RESULTS: In Western blotting analysis, no cross-reacting peptide was detected in the patients' placenta, although a single band (63 kD) corresponding to STS in a normal subject was observed. Southern blotting was performed using EcoRI digests of cellular DNA from 13 XLI patients and full-length human STS cDNA as a probe. Normal males had bands of 20, 15, 10, 9.0, 6.1, 4.2, 2.6, and 1.5 kb. Twelve of the 19 patients had only 20- and 1.5-kb bands. Only one patient had the same band pattern as that of normal males. The STS gene was analyzed by PCR in 6 of the 19 patients. PCR amplification products were sequenced to analyze the STS gene. Two cases with one-base change in the STS gene and variation in amino acids H444R and E560P were found. Mutant STS cDNA was transfected into COS-1 cells and the STS enzyme activity was assayed. The enzyme activities were less than the minimum detection value of the detection system. CONCLUSIONS: These results suggest that XLI is mainly caused by an extensive deletion of the STS gene and that the PCR method is useful for detection of STS point mutations.     

Steroid sulphatase in man: A non inactivated X-locus with partial gene dosage compensation

Summary Steroid sulphatase (STS) activity was measured with two different steroid substrates in leucocytes from normal human males and females, from females heterozygous for STS deficiency and recessive X-linked ichthyosis, and from individuals with numerical X chromosome aberrations. The results indicate non-inactivation with a partial gene dosage compensation at the STS locus. It is estimated that STS loci on inactive X chromosomes express approximately 45% of the STS activity originating from STS loci on active X chromosomes. It is also demonstrated that 45.XO (Turner syndrome) and 47,XXY (Klinefelter syndrome) individuals have abnormal STS enzyme levels compared with normal women and men, respectively.Supported by the Danish Cancer Society     

Frequent deletions of the human X chromosome distal short arm result from recombination between low copy repetitive elements

Substantial DNA deletions appear to be the molecular basis of several human genetic disorders but rarely account for the majority of observed mutations at any given locus. Exceptions in which deletions do account for the majority of observed abnormalities include the alpha-thalassemias, Duchenne muscular dystrophy, and steroid sulfatase deficiency. Variable deletion breakpoints have been recognized at the alpha-globin and dystrophin loci, but no information is available regarding STS deletions. We have found that these STS alterations usually involve breakpoints within highly similar sequence elements situated approximately 1.9 megabases apart on the X chromosome. It is surprising that these very large deletions produce such mild clinical abnormalities. These results may provide insight into the molecular mechanism of a number of human genetic defects.     

Correction of Steroid Sulfatase Deficiency by Gene Transfer into Basal Cells of Tissue-Cultured Epidermis from Patients with Recessive X-Linked Ichthyosis

To develop an experimental model for somatic gene therapy we have tried to correct the steroid sulfatase (STS) deficiency in tissue-cultured primary epidermal keratinocytes from patients suffering from recessive X-linked ichthyosis. An efficient Epstein-Barr virus-based vector was constructed, in which full-length steroid sulfatase cDNA is located between an SV40 early promotor and processing signals. After STS gene transfer into cultured basal cells from ichthyotic skin, the cells produce large amounts of enzymatically active steroid sulfatase protein. The subpopulation of transfected cells can be made to produce approximately 100 times more STS activity than normal keratinocytes. Keratinocytes from patients suffering from recessive X-linked ichthyosis display an abnormal phenotype when developing a multilayered tissue in culture: Initially an extensive burst of keratinization is observed, followed by rapid, premature shedding and degradation of most suprabasal cell layers, leaving a culture with hyperproliferative relatively immature keratinocytes. Transfection of these immature ichthyotic cells with the functional STS construct led to an increase in the amount of retained cell material in the culture medium, indicating an increased cell maturation. It is possible to genetically label individual transfected epidermal cells with a reporter gene. Cotransfection experiments with STS and reporter gene vectors show that the cohort of transfected cells had a tendency to develop less rapidly since they became overrepresented in the smaller size classes at the same time the total population was somewhat shifted toward higher cell sizes. We interpret these results as an indication that restoration of the enzymatic activity induces a more normal maturation of the transfected keratinocytes.     

Genetic variation of petaloid male-sterile cytoplasm of carrots revealed by sequence-tagged sites (STSs)

The mitochondrial DNA of various carrot lines was characterized by random amplified polymorphic DNA (RAPD) analysis, and six sequence-tagged sites (STSs) led to identification of the petaloid type of cytoplasmic male sterility (CMS). Using six STS primer combinations, we were able to classify five CMS lines into two groups and eight fertile carrots into six groups. Both the STS1 and the STS4 primer combinations differentiated CMS cytoplasms from the fertile cytoplasms, and the STS2 primer combination revealed two different types of CMS cytoplasms – of Wisconsin Wild and Cornell origins. Cybrid carrot lines with petaloid flowers which had been obtained by asymmetric cell fusion could also be separated from fertile cybrids by the STS1 primer combination. The STS1 fragment contained a homologous sequence with the orfB gene. DNA gel blot analysis indicated that homologous regions to the STS1 fragment existed in fertile types as well as the CMS types, although the restriction fragment size patterns differed. These observations demonstrate that rearrangements involving this region occurred in the mitochondrial genome. The STS4 fragment had a more complicated gene structure, including retrotransposon-like sequences and small segments of chloroplast genome. Received: 10 September 1998 / Accepted: 24 February 1999     

A long range restriction map of the distal human X chromosome short arm around the steroid sulfatase locus.

The distal short arm of the human X chromosome is of interest because it contains genes which escape X chromosome inactivation and because it is subject to frequent deletions in human patients. The steroid sulfatase gene has been particularly well studied as an example of a gene which escapes X inactivation and which is included in a number of these deletion events. For these reasons a physical map of the region around the STS gene would be of interest. We have constructed a rare cutting enzyme map of this area and have determined the position of several nearby markers with respect to STS. We have also oriented the 5' and 3' ends of the STS gene on this map and have determined the centromeric and telomeric portions of the region. Finally, we have shown that this map can be used to locate deletion breakpoints in STS deficient patients.     

Cloning and characterization of human FTCD on 21q22.3, a candidate gene for glutamate formiminotransferase deficiency

We have identified a new human gene, FTCD, which maps to chromosome 21q22.3 and encodes the enzyme formiminotransferase cyclodeaminase, an intermediate metabolism enzyme that links histidine catabolism to folate metabolism. The major cDNA encodes a protein containing 541 amino acid residues and shows 84% identity with porcine FTCD. Several other cDNAs have been isolated, which may result from alternative splicing events and have the potential to code for three different protein isoforms. The gene is highly expressed in human fetal and adult liver. The two FTCD protein domains show high sequence similarity to two distinct open reading frames from eubacterial genomes, suggesting that eukaryotic FTCD appeared through a gene fusion event. Defects in the glutamate formiminotransferase pathway have been documented, and the deficiency is presumed to be inherited as an autosomal recessive trait. The sequence reported here may be helpful in identifying the primary defect in glutamate formiminotransferase deficiency and establishing a molecular diagnosis.     

一个新的水稻叶形突变体(tll1)的遗传分析与精细定位

利用甲基磺酸乙酯(ethylmethane sulphonate, EMS)诱变粳稻品种日本晴获得了一个遗传稳定的叶形突变体 thread-like leaf 1 (tll1)。该突变体在杭州表现为矮化、窄叶, 极端时仅剩主脉, 呈细丝状。将该突变体分别与籼稻品种南京6号、浙辐802和9311进行正反交配组, 遗传分析表明该突变体性状由1对隐性单基因控制。通过SSR和STS分子标记对F₂代分离群体进行遗传定位, 将该基因初步定位在第12染色体SSR标记RM247和RM101之间。随后利用已公布的粳稻品种日本晴和籼稻品种9311的基因组序列, 发展了7对有多态的STS标记, 最终将该基因定位在FL13和FL14之间约94.3 kb的区间内, 为进一步克隆TLL1基因奠定了基础。     

New STS molecular markers for assessment of genetic diversity and DNA fingerprinting in hop (Humulus lupulus L.).

Molecular markers have been increasingly used in genetic studies of crop species for their applicability in breeding programs. In this work, we report on the development of new sequence-tagged site (STS) markers based on sequence information from several identified hop (Humulus lupulus L.) genes. We demonstrate the usefulness of these STS markers and compare them to SSRs for identifying hop genotypes and estimating genetic diversity in a collection of 68 hop cultivars from around the world. We found 3 individual gene variants (A, B, C) of the chs_H1 gene in this collection. The most frequent gene variant, B (AJ304877), was not detected in Mt. Hood, Glacier, and Horizon (US) cultivars. Gene variant A came from an American germplasm through wild hops. We found length polymorphism in intron 1 of the chs2 gene, and 4 different amplified markers were detected in PCRs. The chs3 gene was found in only one third of the cultivars. None of the variants of the studied CHS genes were found in Humulus japonicus. We detected 5 major gene variants of DNA-binding protein in the collection of H. lupulus cultivars and 2 others in H. japonicus. We also found 3 individual gene variants of an endochitinase gene. The distribution of gene variants did not correlate with any resistance. We proved that developed STS markers can be successfully used for the analysis of genetic diversity and can substitute and supplement SSR markers in hop.     

Genomic structure and chromosomal localization of the mouse gene Punc

The mouse gene Punc encodes a member of the immunoglobulin superfamily of cell surface proteins. It is highly expressed in the developing embryo in nervous system and limb buds. At mid-gestation, however, expression levels of Punc decrease sharply. To allow investigation of such a regulatory mechanism, the genomic locus encompassing the Punc gene was cloned, characterized, and mapped. Fluorescent in situ hybridization was used to determine the chromosomal location of the Punc gene of mouse and human. Mouse Punc maps to Chromosome (Chr) 9 in the region D-E1, whereas the human PUNC gene is localized to Chr 15 at 15q22.3-23, a region known to be syntenic to mouse 9D-E1. The human PUNC gene therefore maps close to a genetic locus that is linked to Bardet-Biedl Syndrome, an autosomal recessive human disorder. Confirmation for the location of human PUNC was obtained through sequence relationships between mouse Punc cDNA, human PUNC cDNA, genomic sequence upstream of the murine Punc gene, and human STS markers that had been previously mapped on Chr 15. The STS sequence WI-14920 is in fact derived from the 3′-untranslated region of the human PUNC gene. WI-14920 had been placed at 228cR from the top of the Chr 15 linkage group, which provided positional information for the human PUNC gene at high resolution. Thus, this study identifies PUNC as the gene corresponding to a previously anonymous marker and serves as a basis to investigate its role in genetic disorders. Received: 8 July 1998 / Accepted: 14 October 1998     

The human X-linked steroid sulfatase gene and a Y-encoded pseudogene: evidence for an inversion of the Y chromosome during primate evolution

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.     

Fine mapping of the distal short arm of the human X chromosome using X/Y translocations.

The loci for steroid sulfatase (STS), the deficiency of which causes X-linked ichthyosis, the cell surface antigen 12E7 (MIC2X), and the blood group antigen Xg (Xg) have been mapped to Xp22.3. These loci are of particular interest since they do not appear to undergo X-chromosome inactivation. In an attempt to establish the relative order of STS and MIC2X, fibroblasts from carriers of four different X/Y translocations and an X/10 translocation were obtained and fused with mouse cell lines deficient in hypoxanthine phosphoribosyltransferase. The breakpoints on the X chromosome in these five translocations are in Xp22. Several independent clones from each fusion were isolated in HAT medium. The clones were examined cytogenetically, and in each case at least two independent clones were identified that have an active X/Y or X/10 translocation chromosome in the absence of other X or Y material. These clones were then tested for STS and 12E7 expression. In two of the X/Y translocations, the markers, STS and 12E7, were both absent. In the X/10 and a third X/Y translocation, both markers were retained. In each of three clones containing the fourth X/Y translocation, STS activity was retained but 12E7 antigenicity was lost. Assuming that this is a simple translocation and does not represent a more complex rearrangement, these results suggest that MIC2X is distal to STS.