Showing papers by "Carolyn J. Brown published in 1993"

The interleukin-2 receptor γ chain maps to Xq13.1 and is mutated in X-linked severe combined immunodeficiency, SCIDX1

Abstract: The gene encoding the gamma chain of the lymphocyte interleukin-2 receptor has been cloned and shown to be required to associate with the beta chain in order for IL-2 internalization and cell activation to occur (1). We considered this gene, IL2RG, a candidate for the X-linked form of severe combined immunodeficiency at the SCIDX1 locus, in which affected males have impaired lymphocyte development. Using fluorescence in situ hybridization and PCR amplification of somatic cell hybrid DNAs, we mapped IL2RG to human Xq13.1, a location within the SCIDX1 critical region established by linkage analysis. The 4.2 kb IL2RG gene was sequenced, and its genomic organization was elucidated. Seven of 19 transformed B-lymphocyte cell lines with independent SCIDX1 mutations had absent or minimal IL2RG mRNA. Unique point mutations were documented to be specifically associated with the disease and the carrier state in four unrelated affected males and their family members: one in a boy with no detectable IL2RG mRNA, in which the mutation ablated a splice donor site; one causing premature chain termination; and two causing distinct amino acid changes. The demonstration of impaired IL2RG mRNA expression in males with X-linked SCID and of unique point mutations in SCIDX1 pedigrees constitutes powerful evidence that the SCIDX1 gene is IL2RG. Noguchi et al. (2) have independently published IL2RG mapping to Xq13 and discovery of mutations in three affected males. The specific pathogenesis of IL2RG mutations and approaches to gene therapy can now be addressed in the X-linked form of SCID.

Evolutionary conservation of possible functional domains of the human and murine XIST genes

Abstract: The human XIST gene, a candidate for a role in X chromosome inactivation, has recently been cloned and sequenced, yielding a 17 kb cDNA with no apparent significant, conserved open reading frame. In addition, the XIST transcript has been localized within the nucleus to the Barr body by RNA in situ hybridization. This subnuclear localization and lack of any significant protein-coding potential suggest that XIST may act as a functional RNA within the nucleus. In the absence of a conserved open reading frame, we have turned to evolutionary studies as a first step toward elucidating a function for XIST in the process of X inactivation. While probes for XIST detect homologues in numerous eutherians, sequence comparisons require significant gapping and reveal identity levels intermediate between those seen for coding and non-coding regions in other genes. Further, sequence comparison of the most likely candidate open reading frame among several primate species reveals sequence changes not normally associated with protein-coding regions. Other features of XIST are conserved in different species, however, including the position of a major transcription start site and active X chromosome-specific DNA methylation patterns at the gene's 5' end. Finally, a possible molecular basis for differing propensity toward X inactivation between Xce alleles in mouse is investigated by comparing the sequence of the Xist conserved 5' repeats in mouse strains carrying different Xce alleles.

2.6 Mb YAC contig of the human X inactivation center region in Xq13: physical linkage of the RPS4X, PHKA1, XIST and DXS128E genes

Abstract: X chromosome inactivation is a mechanism of dosage compensation that regulates the expression of mammalian X-linked genes between XY males and XX females. This phenomenon is cis-acting, clonally heritable, and requires the presence of an X inactivation center (XIC). In our attempts to characterize this phenomenon, we have focused on the physical organization of the human XIC localized to Xq13. From previous studies, we had determined that the candidate XIC interval contained two loci (DXS128 and XIST) and was bound by the breakpoints of two structurally abnormal inactivated X chromosomes, a t(X;14) and an idic(Xp). Here we present a refined mapping of the XIC-containing region using the breakpoint of a late replicating rearranged X (rea(X)), and the initial characterization of a set of 40 yeast artificial chromosomes (YACs) derived from the XIC-containing region. These YACs form a 2.6 Mb contig which completely covers the XIC, and physically links the RPS4X, PHKA1, XIST, and DXS128E genes, as well as a laminin receptor pseudogene (LAMRP4). Furthermore, we have determined the relative orientations of these four genes, and have derived a restriction map of the region using the rare cutter enzymes BssHII, EagI, MluI, NruI, SalI, SfiI, SstII (or SacII), and NotI. We have identified at least 9 CpG-rich islands within this region, and have discovered a large (approximately 125 kb) inverted duplication proximal to the XIC based on symmetrical restriction patterns and homologous probes. We estimate the maximum size of the XIC-containing interval to be between 680 kb and 1200 kb, based on the localization of the breakpoints of the rearranged X chromosomes mentioned above. This lays the groundwork for the further characterization of the XIC region and the isolation of other expressed sequences therefrom.

Mapping of the distal boundary of the X-inactivation center in a rearranged X chromosome from a female expressing XIST

Abstract: A female patient with primary amenorrhea, immature secondary sexual characteristics, and tall stature was found to have a normal X chromosome and a rearranged X [rea(X)] chromosome that resembled an 'isochromosome' Xp, but retained the proximal portion of Xq. The rea(X) was interpreted as rec(X)dup p,inv(X)(p11.4q13). Replication studies demonstrated that the rea(X) was always the late-replicating and, therefore, presumably inactive X chromosome, which must contain the X-inactivation center. Consistent with this interpretation, fluorescence in situ hybridization demonstrated that the rea(X) retained the XIST gene, and reverse transcription polymerase chain reaction analysis showed that XIST was expressed in the patient's cells. By fluorescence in situ hybridization with previously mapped probes, the breakpoint of the rea(X) was located within an approximately 500-kb region located approximately 200 to 700 kb distal to the XIST locus. This is the closest breakpoint distal to XIST in an inactivated X chromosome and, therefore, defines a new distal boundary for the X-inactivation center in humans.

Characterization of a small supernumerary ring X chromosome by fluorescence in situ hybridization.

Abstract: We report on a male with mild learning disabilities who has a supernumerary marker chromosome. The marker chromosome was defined by fluorescence in situ hybridization as a ring X chromosome with breakpoints in the juxacentromeric region. Replication studies suggest that the ring X is late-replicating. However XIST, a gene in the X inactivation centre interval which is expressed exclusively from the inactive X chromosome, is not present on the marker, nor is it expressed in the patient's cells. These results are discussed with respect to karyotype-phenotype correlations and X inactivation. © 1993 Wiley-Liss, Inc.

Molecular and Genetic Studies of Human X Chromosome Inactivation

Abstract: Publisher Summary The chapter discusses the molecular and genetic studies of human X chromosome inactivation X chromosome inactivation is a unique developmental event that results in the cis -limited inactivation of most genes on one of the pair of X chromosomes in mammalian females The inactive X also becomes heterochromatic, asynchronously replicating, and differentially methylated Analysis of the evidence for inactivation of human X-linked genes suggests that inactivation is transcriptionally based and does not affect all X-linked genes The genes known to escape X inactivation are found in multiple locations along the human X chromosome X inactivation requires the presence, in cis , of a region of the proximal long arm called the “X inactivation center” (XIC) Both in humans and in mice, the XZST gene is expressed from the XIC region, only from the inactive X chromosome, not the active X chromosome This suggests that XZST is either involved in or directly affected by the process of X inactivation The inactivation process is described in the chapter in three stages—initiation, promulgation, and maintenance—and models are discussed for these stages