Localization of the gene for Wieacker-Wolff syndrome in the pericentromeric region of the X chromosome

The Wieacker-Wolff syndrome (WWS, MIM* 314580), first described clinically in 1985, is an X-linked recessive disorder. In earlier studies, linkage between the WWS gene and DXYS1 at Xq21.2 and DXS1 at Xq11 as well as AR at Xq12 was reported. Here we report on a linkage analysis using highly polymorphic, short terminal repeat markers located in the segment from Xp21 to Xq24. No recombination between the WWS locus and ALAS2 or with AR (z = 4.890 at theta = 0.0) was found. Therefore, the WWS locus was assigned to a segment of approximately 8 cM between PFC (Xp11.3-Xp 11.23) and DXS339 (Xq11.2-Xq13).     

A family with mental retardation, variable macrocephaly and macro-orchidism, and linkage to Xq12-q21

A family with X linked inheritance of mental retardation (XLMR) is presented. There are 10 mentally retarded males and two affected females in two generations. There are four obligatory carriers, one of whom is described as "slow". Most affected males show macrocephaly and macro-orchidism, which are typical signs of the fragile X syndrome, but have been tested cytogenetically and by analysis of the FMR1 gene and do not have this syndrome. However, some normal males in the family also exhibit macro-orchidism and macrocephaly. Linkage analysis using markers derived from the X chromosome indicates that the causative gene in this family is located in the proximal long arm of the X chromosome, in the interval Xp11-q21. Maximum lod scores of 2.96 with no recombination were found at three loci in Xq13-q21: DXS1111, DXS566, and DXS986. Recombination was observed with DXS1002 (Xq21.31) and DXS991 (Xp11.2), loci separated by about 30 Mb. Although isolation of the gene in this family will be difficult because of the size of the region involved, the localisation should be helpful in investigating other similar families with XLMR, macrocephaly, and macro-orchidism not attributable to FMR1.     

Synthesis and binding of stable bisubstrate ligands for phosphoglycerate kinase

Stable bisubstrate ligands of phosphoglycerate kinase (PGK) have been synthesized with AMP or ADP conjugated to hydrolytically-stable, symmetrical analogues of 1,3-bisphosphoglycerate and their binding to yeast PGK evaluated. Their Kds decrease with net negative charge, with a penta-anionic analogue 7 showing highest affinity-in accordance with its approximation to the transition state for the reaction catalysed by PGK.     

Kinetics of grayanotoxin evoked modification of sodium channels in squid giant axons

Clonality analysis using the polymorphism of X-linked genes, such as the phosphoglycerate kinase (PGK), hypoxanthine phosphoribosyl transferase (HPRT) genes and CAG repeat of the human androgen receptor (HU-MARA) gene and the hypervariable DXS255 gene have been widely used in the assessment of many hematologic diseases. Monoclonal hematopoiesis was clearly demonstrated in myelodysplastic syndromes (MDS), myeloproliferative disorders (MPD) and leukemia by the X-inactivation analysis. Previous studies also found a higher incidence of monoclonality in aplastic anemia and 'clonal remission' in acute leukemia. However, recent studies have shown that clonal hematopoiesis in aplastic anemia or remission of leukemia is rarer than previously thought when skewed X-inactivation was extensively ruled out by comparison with T-lymphocytes as an internal control. However, a polyclonal pattern obtained by X-inactivation cannot exclude the possibility of a small clonal cell population presenting in aplastic anemia. However, recent studies have demonstrated that residual polyclonal (possibly normal) hematopoietic progenitor cells can be detected in the bone marrow of MDS and MPD patients whose peripheral blood granulocytes showed a monoclonal pattern. This may suggest a novel approach to treatment of these diseases.     

Refining the genetic location of the gene for X linked hydrocephalus within Xq28

The most common inherited form of hydrocephalus, X linked hydrocephalus (HSAS), is characterised by mental retardation, adducted thumbs, and spastic paraplegia. Genetic analysis has mapped the locus for HSAS to subchromosomal band Xq28 within a region of approximately 2 megabases of DNA. In order to refine the location of the disease gene we have conducted genetic linkage analysis with Xq28 marker loci in four additional HSAS families. A lod score of 4.26 with polymorphic marker DXS52 (St14) confirms the linkage of HSAS to Xq28. Identification of a recombination event between the HSAS gene and Xq28 loci F8C and DXS605 (2-19) reduces the size of the interval likely to contain the disease locus to about 1.5 megabases, the distance between DXS605 and DXS52. The locus for neural cell adhesion molecule, L1CAM, maps within this interval and therefore represents a candidate gene for HSAS.     

The relation between immunoglobulin G antibodies to Chlamydia trachomatis and poor ovarian response to gonadotropin stimulation before in vitro fertilization

Although several genes for mental retardation and epilepsy, including double cortex/X-linked lissencephaly (DC/XLIS), have been localized to Xq21.3-q23, there has been no complete physical map of this region available. We constructed a YAC/STS contig map by initiating two yeast artificial chromosome (YAC) walks from the markers that flanked the DC/XLIS candidate gene region. We report an approximately 4-Mb contig extending from DXS287 to DXS8088, encompassing DXS1072 and DXS1059, and composed of 52 YACs identified with 15 previously published STSs and 19 novel YAC-end STSs. This contig also contains two brain-specific genes, doublecortin (HGMW-approved symbol DCX), responsible for DC/XLIS, and PAK3, which may be responsible for neurological diseases localized to this region. The new contig extends and incorporates several previously published contigs, providing a total overlapping contig extending approximately 34 Mb from DXS441 in Xq13.1 to DXS8088 in Xq23.     

Loss of heterozygosity at chromosome segment Xq25-26.1 in advanced human ovarian carcinomas

To determine whether a tumor suppressor gene of importance to epithelial ovarian cancer resides on the X chromosome, we examined loss of heterozygosity (LOH) in 123 epithelial ovarian cancer cases. In 54 such cases, we examined LOH at 26 loci on the human X chromosome. In eight cases, we examined LOH in 14 loci and in 61 cases we examined LOH in 13 loci. Matched DNA samples from tumors and corresponding normal tissues were analyzed by polymerase chain reaction (PCR) amplification of microsatellite markers. Frequent losses were found in epithelial carcinomas at the Xq25-26.l region, including DXS1206 (34.5% loss in informative cases), DXS1047 (27.7%), HPRT (24.1%), and DXS1062 (33.3%). The minimum overlapping region of LOH was approximately 5 megabases (Mb), flanked by DXS1206 (Xq25) and HPRT (Xq26.1). The methylation status of the remaining allele of the androgen receptor gene in the tumors exhibiting LOH at the Xq25-26.1 region suggested that the loss was exclusively in the inactive X chromosome. We next determined whether a significant relationship exists between Xq LOH and other parameters, including histologic grade and/or clinical stage of the tumors and LOH at TP53. The Xq LOH had a significant association with grade 2 to 3 tumors at stages II to IV. Sixteen of 18 cases that showed Xq LOH revealed LOH at the TP53 locus, and 45% of tumors exhibiting LOH at TP53 showed Xq LOH. These results suggest that there may be a tumor suppressor gene or genes which escape inactivation of the X chromosome at Xq25-26.1, and that the loss of the gene(s) at Xq25-26.1 is frequently accompanied by loss of the TP53 or loss of another gene on chromosome 17. These losses may contribute to the progression from a well-differentiated to a more poorly differentiated state or to metastatic aggressiveness.     

Contribution to the physically anchored linkage map of the pig

Thirty-three microsatellites have been mapped on the PiGMaP porcine genetic map. By comparison with the previously published PiGMaP maps, the maps of chromosome 2 (140 cM/70 cM) and chromosome 3 (180 cM/110 cM) were extended and new markers were mapped on the p-arm extremity of chromosome 7 and on the centromeric extremity of chromosome 15. New orders are proposed for markers on chromosomes 3 and 17. Six microsatellites isolated from cosmids were also localized on the cytogenetic map by fluorescent in situ hybridization. We tested the subcloning ligation mixture-polymerase chain reaction (SLiM-PCR) method for isolating microsatellites from cosmids. Subcloning is more effective when the cosmid harbours several microsatellites whereas SLiM-PCR is more straightforward when the cosmid contains a single microsatellite. Fifteen anonymous microsatellites were regionally assigned by using a hybrid cell panel. For map integration, the determination of a regional assignment of anonymous microsatellites by using a hybrid cell panel offers an alternative to microsatellite isolation from cosmids and their localizations by in situ hybridization.     

Platelet aggregability in vivo is attenuated by verapamil but not by metoprolol in patients with stable angina pectoris

One of the most widely studied simple sequences in the mammalian genome is the (TG)n dinucleotide sequence. Because these microsatellites are highly polymorphic, we chose to study microsatellites from cosmids to provide genetic markers for the porcine genome. After screening a porcine cosmid library with a (CA)10 probe, 20 cosmids containing microsatellites were subcloned and 17 microsatellites identified by sequencing. Oligonucleotide primers flanking the repeat were designed for seven (TG)n microsatellites with n > 14. These seven microsatellites revealed polymorphism and were regionally assigned to chromosomes by fluorescent in situ hybridization of initial cosmids. These seven loci will be useful for both the construction of the genetic map and as landmark loci on the physical map of the porcine genome.     

Highly potent bisphosphonate ligands for phosphoglycerate kinase

We have synthesized a series of novel analogs of 1, 3-bisphospho-D-glyceric acid, 1,3-BPG,3 and evaluated their binding to phosphoglycerate kinase, PGK (EC 2.7.2.3). Nonscissile methanephosphonic acids replace the two phosphate monoesters of 1, 3-BPG and lead to several stable, tight-binding mimics of this intermediate species in glycolysis. Multiple fluorine substitution for hydrogen in the alpha-methylene groups of the phosphonic acid 1, 3-BPG analogs markedly improves their binding to PGK as determined by NMR analysis. The best ligands bind some 50-100 times more strongly than does the substrate 3-phospho-D-glyceric acid and show a requirement for pKa3 to be generally below 6.0, while the presence of a beta-carbonyl group seems to be of secondary importance.     

Mechanical gastritis as cause of upper gastrointestinal hemorrhage

The gene responsible for X linked agammaglobulinaemia (XLA) lies in Xq22 and has recently been identified as atk. DXS101 is a polymorphic locus which is closely linked to the disease locus. In this report we describe the identification, by pulsed field gel electrophoresis, of a new polymorphism at the DXS101 locus with a predicted heterozygosity of 4.9%. Despite this low value, we show how this polymorphism has been important in carrier status determination in a family with XLA where assessment was not possible by other means.     

Denatured states of yeast phosphoglycerate kinase

Structures of proteins in unfolded states have important implications for the protein folding problem and for the translocation of polypeptide chains. Acid-denatured, cold-denatured, and 6 M guanidine hydrochloride (GuHCl) denatured yeast phosphoglycerate kinase (PGK) are ensembles of flexible unfolded molecules with rapidly interconverting structures of the individual polypeptide chains. They differ, however, in their physical properties, such as in coil size and in stiffness over a short distance along the chain. These properties of polypeptide chains can be described well by persistence statistics. A solution containing 0.7 M GuHCl at 4.5 degrees C is nearly a Theta-solvent for PGK. By contrast, 6 M GuHCl is a good solvent for PGK. Acid-denatured PGK at low ionic strength has the most expanded and stiffest chains. The conformation of heat-denatured PGK should be more compact than that of random walk chains at the Theta-point, as can be inferred from measurements on other proteins. Investigations of heat-denatured PGK by scattering methods are unfeasible due to aggregation of the protein. The persistence length as a measure of chain stiffness varies between a = 1.74 nm for cold-denatured PGK and a = 3.0 nm for acid-denatured PGK. The distribution functions of the gyration radii were calculated from the X-ray scattering data for all unfolded states and compared with the radius of gyration of the natively folded molecule.     

Fine mapping of the dyskeratosis congenita locus in Xq28

Dyskeratosis congenita (DC) is characterised by reticulate skin pigmentation, mucosal leucoplakia, and nail dystrophy. Bone marrow failure occurs in 50% of patients and is the principal cause of early mortality. In the majority of families the pattern of inheritance of DC is compatible with an X linked recessive trait. The locus for the X linked recessive form of DC has been linked to Xq28. We have now extended our earlier studies by investigating five families with additional Xq28 polymorphic markers; analysis of recombination events in these families has located the DC1 locus between GABRA3 and DXS1108, an interval of approximately 4 Mb.     

Continuous nebulization with terbutaline sulfate under tent inhalation. Evaluation of the efficacy in children 2 to 5 years of age in asthmatic crises

X-linked mental retardation (XLMR) includes distinct entities in which mental deficiency is either associated with specific abnormalities (syndromal) or not (nonsyndromal). We report on the clinical, neuropsychological, and laboratory findings and linkage analysis in one family with XLMR and isolated growth hormone deficiency (IGHD). Mental retardation was associated in 3 males and 5 females with short stature, microcephaly, and particular facial traits, i.e., high curved forehead, midface hypoplasia, and concave nasal bridge with nasal end of normal size and broad traits. Significant lod scores (Zmax >2) at a recombination fraction of theta = 0 were detected for 6 marker loci between DXS178 (Xq22.1) and DXS292 (Xq27.2). This mapping region overlaps that of XLMR with IGHD, recently reported by Hamel et al. [1996: Am J Med Genet 64:35-41] (Xq24-q27.3), and that of agammaglobulinemia with IGHD (Xq21.33-q22.2). This observation may confirm the suspicion of a gene involved in growth hormone regulation being localized in Xq.     

Clonal analysis in acute myeloid leukemia by polymerase chain reaction

We have used PCR to amplify a polymorphic portion of the X-chromosome linked phosphoglycerate kinase gene (PGK) combined with a methylation-sensitive restriction enzyme digestion of the active X chromosome to examine the frequency of heterozygosity in Taiwanese females and analyze clonality in 18 female patients with acute myeloid leukemia (AML). We used hair follicles as normal tissue control. We found that the incidence of heterozygosity of the PGK gene in 102 hematological normal females and 18 patients tested was 35% (42/120). In five AML patients, a monoclonal X-inactivation pattern of leukemic blasts was found at presentation, which then returned to polyclonal at remission. In two of these five cases, a monoclonal pattern recurred at relapse. We also found that the hair follicles were readily accessible normal tissue for control, were easy to obtain, and were non-invasive.     

Repair of DNA alkylation damage

Alkylation damage of DNA is one of the major types of insults which cells must repair to remain viable. One way alkylation damaged ring nitrogens are repaired is via the Base Excision Repair (BER) pathway. Examination of mutants in both BER and Nucleotide Excision Repair show that there is actually an overlap of repair by these two pathways for the removal of cytotoxic lesions in Escherichia coli. The enzymes removing damaged bases in the first step in the BER pathway are DNA glycosylases. The coding sequences for a number of methylpurine-DNA glycosylases (MPG proteins) were cloned, and a comparison of the amino-acid sequences shows that there are some similarities between these proteins, but nonetheless, compared to other DNA glycosylases, MPG proteins are more divergent. MPG proteins have been purified to homogeneity and used to identify their substrates ranging from methylating agents to deamination products to oxidatively damaged bases. The ligation-mediated polymerase chain reaction has been used to study the formation of alkylation damage, and its repair in mammalian cells. We have studied DNA damage in the PGK1 gene for a series of DNA alkylating agents including N-methyl-N'-nitro-N-nitrosoguanidine, Mechlorethamine, and Chlorambucil and shown that the damage observed in the PGK1 (phosphoglycerate kinase 1) gene depends on the alkylating agent used. This report reviews the literature on the MPG proteins, DNA glycosylases removing 3-methyladenine, and the use of these enzymes to detect DNA damage at the nucleotide level.     

Cytosine methylation enhances DNA damage produced by groove binding and intercalating enediynes: studies with esperamicins A1 and C

Methylation of the C5 position of cytosine in CG dinucleotides represents an important element in the control of gene expression in eukaryotic cells. This major groove modification of DNA causes changes in DNA conformation that are recognized by DNA-binding proteins and DNA-damaging chemicals. We have observed that CG methylation affects the DNA damage produced by the enediyne esperamicin A1 and its analog lacking the intercalating anthranilate, esperamicin C. Fragments of the human phosphoglycerate kinase gene (PGK1) and the plasmid pUC19 were methylated with SssI methylase and subjected to damage by esperamicins A1 and C. Damage produced by esperamicin A1 was enhanced 1.5-2-fold near a single CG sequence at position -101 in PGK1 and in a region containing several methylated CG dinucleotides between positions -120 and -131. Esperamicin C-induced damage was enhanced to a similar degree in PGK1 but only at the site that contained multiple CG dinucleotides. There was enhancement of damage for both drugs in the pUC19 fragment at several sites near CG sequences. Analysis of the chemistry of esperamicin-induced DNA damage suggests that cytosine methylation does not affect the identity of drug-abstracted hydrogen atoms. The damage chemistry was also used to identify the DNA binding orientation of the esperamicins. The anthranilate of esperamicin A1 is predicted to intercalate in a CT step four base pairs in a 3'-direction to the CG sequence at -101 in PGK1 and in a CG dinucleotide at the site containing multiple CGs (positions -120 to -131). These observations are consistent with other studies that indicate a long range effect of cytosine methylation on DNA conformation.     

Age of initiating selected health-risk behaviors among high school students in the United States

The XYL1 and XYL2 genes from Pichia stipitis encoding xylose reductase (XR) and xylilitol dehydrogenase (XDH), respectively, were transformed into Saccharomyces cerevisiae. These two genes were placed in different directions under the control of the alcohol dehydrogenase I (ADHI) and phosphoglycerate kinase (PGK) promoters and inserted into the E. coli-yeast shuttle plasmid YEp24. Different recombinant S. cerevisiae strains were constructed with different specific activities of XR and XDH. The highest XR or XDH activities were obtained when the expressed gene was controlled by the PGK promoter and located downstream after the ADHI promoter-gene-terminator sequence. The XR/XDH ratio (ratio of specific enzyme activities of XR and XDH) in these recombinant S. cerevisiae strains varied from 17.5 to 0.06. In order to enhance xylose utilization, in the XYL1, XYL2 containing S. cerevisiae strains, the native TKL1 gene encoding transketolase and the TALI gene encoding transaldolase were also overexpressed, which showed considerably good growth on the xylose plate. Fermentation of the recombinant S. cerevisiae strains containing XYL1, XYL2, TKL1, and TAL1 were studied with mixtures of glucose and xylose. The strain with XR/XDH ratio of 0.06 consumed 3.25 g/L xylose and formed no xylitol and less glycerol and acetic acid, but more ethanol compared with the strains with a higher XR/XDH ratio.