Jonathan M.J. Derry

Isolation of a novel gene mutated in Wiskott-Aldrich syndrome

Wiskott-Aldrich syndrome (WAS) is an X-linked recessive immunodeficiency characterized by eczema, thrombocytopenia, and recurrent infections. Linkage studies have placed the gene at Xp11.22-p11.23. We have isolated from this interval a novel gene, WASP, which is expressed in lymphocytes, spleen, and thymus. The gene is not expressed in two unrelated WAS patients, one of whom has a single base deletion that produces a frame shift and premature termination of translation. Two additional patients have been identified with point mutations that change the same arginine residue to either a histidine or a leucine. WASP encodes a 501 amino acid proline-rich protein that is likely to be a key regulator of lymphocyte and platelet function.

Wiskott–Aldrich Syndrome Protein, a Novel Effector for the GTPase CDC42Hs, Is Implicated in Actin Polymerization

The Rho family of GTPases control diverse biological processes, including cell morphology and mitogenesis. We have identified WASP, the protein that is defective in Wiskott-Aldrich syndrome (WAS), as a novel effector for CDC42Hs, but not for the other Rho family members, Rac and Rho. This interaction is dependent on the presence of the G protein-binding domain. Cellular expression of epitope-tagged WASP produces clusters of WASP that are highly enriched in polymerized actin. This clustering is not observed with a C-terminally deleted WASP and is inhibited by coexpression with dominant negative CDC42Hs-N17, but not with dominant negative forms of Rac or Rho. Thus, WASP provides a novel link between CDC42Hs and the actin cytoskeleton, which suggests a molecular mechanism for many of the cellular abnormalities in WAS. The WASP sequence contains two novel domains that are homologous to other proteins involved in action organization.

Impaired regulation of NF-κB and increased susceptibility to colitis-associated tumorigenesis in CYLD-deficient mice

Cylindromatosis (CYLD) is a deubiquitinating enzyme that is altered in patients with familial cylindromatosis, a condition characterized by numerous benign adnexal tumors. However, the regulatory function of CYLD remains unsettled. Here we show that the development of B cells, T cells, and myeloid cells was unaffected in CYLD-deficient mice, but that the activation of these cells with mediators of innate and adaptive immunity resulted in enhanced NF-kappaB and JNK activity associated with increased TNF receptor-associated factor 2 (TRAF2) and NF-kappaB essential modulator (NEMO) ubiquitination. CYLD-deficient mice were more susceptible to induced colonic inflammation and showed a dramatic increase in the incidence of tumors compared with controls in a colitis-associated cancer model. These results suggest that CYLD limits inflammation and tumorigenesis by regulating ubiquitination in vivo.

Chromosomal localization of GABAA receptor subunit genes: relationship to human genetic disease

Hybridization of GABAA receptor probes to human chromosomes in situ and to DNA from sorted human chromosomes has localized the genes encoding a beta subunit and three isoforms of the alpha subunit. The alpha 2 and beta genes are both located on chromosome 4 in bands p12-p13 and may be adjacent. The alpha 1 gene is on chromosome 5 (bands q34-q35) and the alpha 3 gene is on the X chromosome. The alpha 3 locus was mapped also on the mouse X chromosome using genetic break-point analysis in an interspecies pedigree. The combined results locate the human alpha 3 gene within band Xq28, in a location that makes it a candidate gene for the X-linked form of manic depression.

Specific NEMO mutations impair CD40-mediated c-Rel activation and B cell terminal differentiation

Hypomorphic mutations in the zinc finger domain of NF-kappaB essential modulator (NEMO) cause X-linked hyper-IgM syndrome with ectodermal dysplasia (XHM-ED). Here we report that patient B cells are characterized by an absence of Ig somatic hypermutation (SHM) and defective class switch recombination (CSR) despite normal induction of activation-induced cytidine deaminase (AID) and Iepsilon-Cepsilon transcripts. This indicates that AID expression alone is insufficient to support neutralizing antibody responses. Furthermore, we show that patient B cells stimulated with CD40 ligand are impaired in both p65 and c-Rel activation, and whereas addition of IL-4 can enhance p65 activity, c-Rel activity remains deficient. This suggests that these NF-kappaB components have different activation requirements and that IL-4 can augment some but not all NEMO-dependent NF-kappaB signaling. Finally, using microarray analysis of patient B cells we identified downstream effects of impaired NF-kappaB activation and candidate factors that may be necessary for CSR and SHM in B cells.

RIP4 Is an Ankyrin Repeat-Containing Kinase Essential for Keratinocyte Differentiation

The epidermis is a stratified, continually renewing epithelium dependent on a balance among cell proliferation, differentiation, and death for homeostasis. In normal epidermis, a mitotically active basal layer gives rise to terminally differentiating keratinocytes that migrate outward and are ultimately sloughed from the skin surface as enucleated squames. Although many proteins are known to function in maintaining epidermal homeostasis, the molecular coordination of these events is poorly understood. RIP4 is a novel RIP (receptor-interacting protein) family kinase with ankyrin repeats cloned from a keratinocyte cDNA library. RIP4 deficiency in mice results in perinatal lethality associated with abnormal epidermal differentiation. The phenotype of RIP4(-/-) mice in part resembles that of mice lacking IKKalpha, a component of a complex that regulates NF-kappaB. Despite the similar keratinocyte defects in RIP4- and IKKalpha-deficient mice, these kinases function in distinct pathways. RIP4 functions cell autonomously within the keratinocyte lineage. Unlike IKKalpha, RIP4-deficient skin fails to fully differentiate when grafted onto a normal host. Instead, abnormal hair follicle development and epidermal dysplasia, indicative of progression into a more pathologic state, are observed. Thus, RIP4 is a critical component of a novel pathway that controls keratinocyte differentiation.

Expression of human Wiskott-Aldrich syndrome protein in patients' cells leads to partial correction of a phenotypic abnormality of cell surface glycoproteins.

The Wiskott–Aldrich syndrome (WAS) is an uncommon X-linked recessive disease characterized by thrombocytopenia, eczema and immunodeficiency. The biochemical defect of this disorder primarily affects cells derived from bone marrow. To understand better the molecular mechanisms underlying this disease and to evaluate the possibility of correcting the genetic defects in hematopoietic cells, a Moloney murine leukemia virus (MoMLV)- based retroviral vector carrying a functional Wiskott–Aldrich syndrome protein (WASp) cDNA driven by an SV40 promoter (LNS-WASp) was constructed. A packaging cell line containing this vector produced a stable level of WAS protein and maintained a high titer of viral output. Epstein–Barr virus (EBV)-transformed B lymphoblastoid cell lines (B-LCL) from WAS patients, which lack expression of the WAS protein, were transduced by the LNS-WASp retroviral vector and showed expression of WASp by Western blot. Analysis of the O-glycan pattern on cell surface glycoproteins from WAS patients’ B-LCL showed an altered glycosylation pattern, due to increased activity of β-1,6-N-acetylglucosaminyltransferase (C2GnT). Transduction by the retroviral vector carrying the functional WASp cDNA partially restored the abnormal glycosylation pattern, and was accompanied by a decreasing C2GnT activity. These findings imply a functional linkage between the WAS protein and the expression of the glycosyltransferase involved in the O-glycosylation, and also suggest a potential gene therapy via transferring a functional WASp cDNA into hematopoietic cells for Wiskott–Aldrich syndrome.

Mapping of the glycine receptor α2-subunit gene and the GABAA α3-subunit gene on the mouse X chromosome

We have mapped the gene for the α2-subunit of the inhibitory glycine receptor (Glra2) to the telomeric end of the mouse X chromosome by backcross analysis of a Mus musculus/Mus spretus interspecific cross. In addition, we have extended the mapping of the GABAA α3-subunit receptor gene (Gabra3). A deduced gene order of cen-Cybb-Hprt-DXPas6-Gabra3-Rsvp-Gdx/Cf-8-Dmd-Pgk-1-DXPas2-Plp-DXPas1-Glra2-tel places Gabra3 proximal to the visual pigment gene Rsvp and Glra2 in the region of loci for hypophosphatemia (Hyp), steroid sulfatase (Sts), and the E1α-subunit of pyruvate dehydrogenase (Pdha1). This establishes the XF region of the mouse X chromosome as homologous with the Xp22.1–p22.3 region of the human X chromosome and indicates the presence of an evolutionary breakpoint in the region of Xp21.3.

Transgenic rescue of the tattered phenotype by using a BAC encoding Ebp

Tattered ( Td) is an X-linked semi-dominant mouse mutation in which affected males have severe skeletal defects, lack intestines, and die before birth (Uwechue et al. 1996; Derry et al. 1999). Heterozygous females are small and at 4–5 days post partum develop patches of hyperkeratotic skin where hair fails to grow (Uwechue et al. 1996). Td/+ females also exhibit mild skeletal defects including craniofacial dysmorphia and asymmetric microophthalmia. The phenotype of Td is similar to that seen in patients with X-linked dominant chondrodysplasia punctata (CDPX2), where females, heterozygous at the CDPX2 locus, display skin, hair, craniofacial, and skeletal abnormalities and cataracts (MIM 302960). Affected males are not observed, suggestive that CDPX2, like Td, is a prenatal lethal in the hemizygous state. Recently the molecular basis for Td and CDPX2 has been established to be a consequence of mutations in the X-linked gene emopamil-binding protein gene EBP (Derry et al. 1999; Braverman et al. 1999). EBP is an integral endoplasmic reticulum membrane protein that functions as a sterol isomerase, catalyzing the conversion of cholest-8(9)-en-3 b-ol to lathosterol (a late step in cholesterol biosynthesis). EBP is also a high-affinity binding protein for the antiischemic phenylalkylamine Ca ++ antagonist emopamil and additionally binds a number of structurally diverse molecules including the immunosuppressant SR31747A and the chemotherapeutic agent tamoxifen. Td mice harbor a single nucleotide substitution, G454A, resulting in an amino acid substitution of arginine for glycine at amino acid position 107 (Derry et al. 1999). Analysis of CDPX2 patients identified a diverse set of mutations including missense mutations, in-frame deletions, and splice site, frameshifts, and nonsense mutations (Derry et al. 1999; Braverman et al. 1999). All of the missense mutations in human CDPX2, and the R107G substitution in Td, affect amino acids conserved between human, mouse, and guinea pig EBP. Heterologous expression of alanine-scanning mutants of huamn EBP in a D-D isomerase-deficient yeast strain has demonstrated that missense mutations in the area that encompasses the Td mutation substantially reduce the isomerase activity of the protein (Moebius et al. 1999). This supports a mechanism in which a loss of function of the mutant allele coupled with X-inactivation generates a mosaic of cells, some expressing normal EBP and some the mutant protein. However, the alternative mechanism of a dominant negative effect of the mutant protein in cells owing to a failure of complete X-inactivation cannot be excluded, particularly as EBP functions as a homodimer. We wished to establish which, if any, aspects of theTd phenotype could be rescued by introducing a wild-type copy ofEbp into all cells by transgenesis, and to distinguish a loss of function from dominant negative mechanisms. Transgenic mice were generated from an Ebp-encoding BAC (Research Genetics, ; 446O24: BAC75) by pronuclear injection.