Katherine A. Siminovitch

X-Chromosome inactivation in the Wiskott-Aldrich syndrome: A marker for detection of the carrier state and identfication of cell lineages expressing the gene defect

Recently developed techniques for the direct analysis of DNA have made possible the determination of patterns of cellular X-chromosome inactivation. These techniques provide a potential method for carrier detection for several X-linked human disorders in which obligate carriers show nonrandom X inactivation. By using restriction fragment length polymorphic (RFLP) gene-specific probes in conjunction with methylation-sensitive enzymes, we have characterized the patterns of X-chromosome inactivation in cell subsets from females belonging to 10 kindreds segregating for the X-linked immune deficiency disorder Wiskott-Aldrich syndrome (WAS). We show that selective inactivation of the X chromosome distinguishes obligate WAS carriers from noncarrier females and constitutes a valuable marker of the WAS carrier state. Selective inactivation phenomena were observed in the monocytes and T and B lymphocytes of obligate carriers, implying that the WAS gene defect is expressed in each of these cellular lineages. In conjunction with the use of linked DNA markers, RFLP-methylation analysis should render carrier detection feasible for the majority of females from WAS families. The results of such analyses also provide an initial step toward identifying the cellular level and molecular basis for WAS.

Linkage studies of the Wiskott-Aldrich syndrome: polymorphisms at TIMP and the X chromosome centromere are informative markers for genetic prediction

SummaryEleven families segregating for the X-linked recessive immune deficiency disorder, Wiskott-Aldrich syndrome (WAS), were studied by linkage analysis with an alpha satellite DNA probe, pBamX-7, which detects polymorphism at the X chromosome centromere, locus DXZ1, as well as three other polymorphic markers defining loci on the proximal short arm of the X chromosome. Linkage has been established between WAS and DXZ1 (ž (θ)=7.08 at θ=0.03) and WAS and the TIMP gene locus (ž (θ)=5.09 at θ=0.0). We have also confirmed close linkage between DXZ1 and two marker loci, DXS14 and DXS7, previously shown to be linked to the WAS locus. The probe pBamX-7 detected allelic variation in all females tested, reflecting the high frequency of polymorphism at the centromere. One WAS carrier revealed a recombination between WAS and both marker loci DXZ1 and DXS14, indicating that WAS does not map between these loci. In conjunction with previous data from genetic mapping studies of WAS, these results confirm the pericentromerix Xp localization of WAS and demonstrate the usefulness of alpha satelite DNA probes as tools for genetic prediction in WAS as well as other pericentric X-linked diseases.

Toward the molecular dissection of primary biliary cirrhosis

Primary biliary cirrhosis (PBC) is the archetypal autoimmune disease of the liver, characteristically associated with highly specific antimitochondrial antibodies directed against the E2 subunits of mitochondrial 2-oxo-acid dehydrogenase enzymes. Primarily affecting women, PBC occurs in about 1 in 1000 women over the age of 40, with other autoimmune diseases frequently developing in these individuals and their families.1 Until recently, relatively little has been known about the genetic and environmental factors underpinning the inflammatory nonsuppurative granulomatous cholangitis and bile duct–infiltrating, autoreactive, T lymphocytes that define PBC.2 However, as for other autoimmune diseases, molecular dissection of PBC is now well underway, with recent data from animal3 and human4 genetic studies identifying the interleukin-12 (IL-12) signaling pathway as a central player in the etiopathogenesis of PBC. Appreciation of the integral link between IL-12 signaling and emergence of PBC has arisen in the context of major improvements in technologies for animal modeling of human disease and disease gene mapping. PBC-like phenotypes have been reported, for example, in the NODc3.c4 mouse, a recently-derived congenic variant of the diabetes-prone NOD (nonobese diabetic) strain,5 in scurfy mice expressing a mutant form of the Foxp3 (forkhead box p3) transcription factor,6 and in mice deficient for the Cl /HCO3 anion exchanger 2 transporter.7 Xenobiotic immunization has also been shown to recreate facets of the disease in mice.8 Although they facilitate characterization of PBC pathophysiology, these models have not been extensively investigated as of yet, and do not faithfully replicate the human phenotype, the animals lacking the precise histologic lesions, the female predominance, and/or the late onset and slow progression of disease typical of human PBC. PBC-like disease has also been reported in mice manipulated for T cell–restricted expression of a dominantnegative (dn) form of transforming growth factor-beta receptor II (TGF RII).9 While again not exactly mimicking the human disease, models such as this one are particularly exciting in view of the integral relationship of TGF signaling with development of T helper 17 (TH17) cells, a T helper subset with key roles in driving autoimmune, inflammatory responses.10,11 Animal model studies have also confirmed a significant influence of the IL-12 cytokine, a heterodimer comprising the p35 and p40 subunits, in expression of autoimmune disease, raising the potential that the IL-12 and TGF signaling pathway coordinately contribute to the autoimmune cholangitis seen in dnTGF RII mice, and the emergence of human PBC. Yoshida and colleagues3 have delved further into this pathway through the analysis of dnTGF RII mice rendered deficient for either IL-12p40 (IL-12p40 / ) or for IFN(IFN/ ), an IL-12–induced cytokine effector of proinflammatory TH1 responses, as well as a suppressor of TH17 cells. IL-12p40 (encoded by the IL12B gene) covalently associates with the smaller p35 subunit encoded by IL12A to form the heterodimeric cytokine IL-12. It also comprises one of the subunits that form the immunoregulatory and TH17-cell–triggering cytokine IL-23 (p40p19). In this issue of HEPATOLOGY, this group reports that the elevation in intrahepatic proinflammatory cytokine levels and the histopathologic signs of inflammatory cholangitis typically manifested by dnTGF RII mice are dramatically decreased in the context of concomitant IL-12p40 deficiency, but relatively unchanged by concomitant interferon-gamma (IFN) deficiency. Based on these findings, the authors conclude that IFNmay be dispensable for development of autoimmunity, but that IL-12p40 is an essential requirement for development of autoimmune cholangitis. The link between IL-12 signaling and the PBC-like disease manifested by dnTGF RII mice is particularly exciting in view of our group’s recent genetic data implicating the IL-12 pathway in genesis of human PBC.4 Through a genomewide association screen of more than 500 PBC cases and more than 1000 healthy controls, we identified IL12A, IL12 receptor (IL12R)B2, as well as HLA (human leukocyte antigen) gene variants as the loci most strongly associated with risk for PBC in our study cohort (Fig. 1). Not only were the population-attributable risks for all three of these loci very high, but another of the Abbreviations: IFN, interferon; IL, interleukin; PBC, primary biliary cirrhosis; TGF RII, transforming growth factor-beta receptor II; TH17, T helper 17 cell. Address reprint requests to: Dr. G. M. Hirschfield, Liver Centre, Toronto Western Hospital, 6B Fell, Room 160, 399 Bathurst Street, Toronto, Ontario, M5T 2S8 Canada. E-mail: gideon.hirschfield@uhn.on.ca; fax: 647-438-4730 Copyright

T cell receptor genes in rheumatoid arthritis

ConclusionRheumatoid arthritis is an oft debilitating chronic disease of an autoimmune nature. Although the putative antigen remains unknown, the recent elucidation of the structure and functional relationships of the trimolecular complex governing the immune response to antigen, facilitates the development of novel approaches to the treatment of RA and enhances our understanding of the etiopathogenesis of this disorder and autoimmunity in general. The characterization of immune active T cell populations, particularly at inflammatory sites such as the synovium, with respect to their expressed and genomic TCR repetoire, may permit the identification of crucial genetic components which contribute either directly or indirectly to the susceptibility and/or the development of RA.