Christine Ambrose

A novel gene containing a trinucleotide repeat that is expanded and unstable on Huntington's disease chromosomes

The Huntingtons disease (HD) gene has been mapped in 4p16.3 but has eluded identification. We have used haplotype analysis of linkage disequilibrium to spotlight a small segment of 4p16.3 as the likely location of the defect. A new gene, IT15, isolated using cloned trapped exons from the target area contains a polymorphic trinucleotide repeat that is expanded and unstable on HD chromosomes. A (CAG)n repeat longer than the normal range was observed on HD chromosomes from all 75 disease families examined, comprising a variety of ethnic backgrounds and 4p16.3 haplotypes. The (CAG)n repeat appears to be located within the coding sequence of a predicted approximately 348 kd protein that is widely expressed but unrelated to any known gene. Thus, the HD mutation involves an unstable DNA segment, similar to those described in fragile X syndrome, spino-bulbar muscular atrophy, and myotonic dystrophy, acting in the context of a novel 4p16.3 gene to produce a dominant phenotype.

A novel moesin-, ezrin-, radixin-like gene is a candidate for the neurofibromatosis 2 tumor suppressor

Neurofibromatosis 2 (NF2) is a dominantly inherited disorder characterized by the occurrence of bilateral vestibular schwannomas and other central nervous system tumors including multiple meningiomas. Genetic linkage studies and investigations of both sporadic and familial tumors suggest that NF2 is caused by inactivation of a tumor suppressor gene in chromosome 22q12. We have identified a candidate gene for the NF2 tumor suppressor that has suffered nonoverlapping deletions in DNA from two independent NF2 families and alterations in meningiomas from two unrelated NF2 patients. The candidate gene encodes a 587 amino acid protein with striking similarity to several members of a family of proteins proposed to link cytoskeletal components with proteins in the cell membrane. The NF2 gene may therefore constitute a novel class of tumor suppressor gene.

Identification of a Palmitic Acid-modified Form of Human Sonic hedgehog

During hedgehog biosynthesis, autocatalytic processing produces a lipid-modified amino-terminal fragment (residues 24–197 in the human Sonic hedgehog sequence) that is responsible for all known hedgehog signaling activity and that is highly conserved evolutionarily. Published in vitro biochemical studies using Drosophila hedgehog identified the membrane anchor as a cholesterol, and localized the site of attachment to the COOH terminus of the fragment. We have expressed full-length human Sonic hedgehog in insect and in mammalian cells and determined by mass spectrometry that, in addition to cholesterol, the human hedgehog protein is palmitoylated. Peptide mapping and sequencing data indicate that the palmitoyl group is attached to the NH2 terminus of the protein on the α-amino group of Cys-24. Cell-free palmitoylation studies demonstrate that radioactive palmitic acid is readily incorporated into wild type Sonic hedgehog, but not into variant forms lacking the Cys-24 attachment site. The lipid-tethered forms of hedgehog showed about a 30-fold increase in potency over unmodified soluble hedgehog in a cell- based (C3H10T1/2 alkaline phosphatase induction) assay, suggesting that the lipid tether plays an important role in hedgehog function. The observation that an extracellular protein such as Shh is palmitoylated is highly unusual and further adds to the complex nature of this protein.

BAFF selectively enhances the survival of plasmablasts generated from human memory B cells

The generation of Ig-secreting cells (ISCs) from memory B cells requires interactions between antigen-specific (Ag-specific) B cells, T cells, and dendritic cells. This process must be strictly regulated to ensure sufficient humoral immunity while avoiding production of pathogenic autoantibodies. BAFF, a member of the TNF family, is a key regulator of B cell homeostasis. BAFF exerts its effect by binding to three receptors - transmembrane activator of and CAML interactor (TACI), B cell maturation antigen (BCMA), and BAFF receptor (BAFF-R). To elucidate the contribution of BAFF to the differentiation of B cells into ISCs, we tracked the fate of human memory B cells stimulated with BAFF or CD40L. BAFF and CD40L significantly increased the overall number of surviving B cells. This was achieved via distinct mechanisms. CD40L induced proliferation of nondifferentiated blasts, while BAFF prevented apoptosis of ISCs without enhancing proliferation. The altered responsiveness of activated memory B cells to CD40L and BAFF correlated with changes in surface phenotype such that expression of CD40 and BAFF-R were reduced on ISCs while BCMA was induced. These results suggest BAFF may enhance humoral immunity in vivo by promoting survival of ISCs via a BCMA-dependent mechanism. These findings have wide-ranging implications for the treatment of human immunodeficiencies as well as autoimmune diseases.

Interactions of tumor necrosis factor (TNF) and TNF receptor family members in the mouse and human

Ligands of the tumor necrosis factor superfamily (TNFSF) (4–1BBL, APRIL, BAFF, CD27L, CD30L, CD40L, EDA1, EDA2, FasL, GITRL, LIGHT, lymphotoxin α, lymphotoxin αβ, OX40L, RANKL, TL1A, TNF, TWEAK, and TRAIL) bind members of the TNF receptor superfamily (TNFRSF). A comprehensive survey of ligand-receptor interactions was performed using a flow cytometry-based assay. All ligands engaged between one and five receptors, whereas most receptors only bound one to three ligands. The receptors DR6, RELT, TROY, NGFR, and mouse TNFRH3 did not interact with any of the known TNFSF ligands, suggesting that they either bind other types of ligands, function in a ligand-independent manner, or bind ligands that remain to be identified. The study revealed that ligand-receptor pairs are either cross-reactive between human and mouse (e.g. Tweak/Fn14, RANK/RANKL), strictly species-specific (GITR/GITRL), or partially species-specific (e.g. OX40/OX40L, CD40/CD40L). Interestingly, the receptor binding patterns of lymphotoxinα andαβ are redundant in the human but not in the mouse system. Ligand oligomerization allowed detection of weak interactions, such as that of human TNF with mouse TNFR2. In addition, mouse APRIL exists as two different splice variants differing by a single amino acid. Although human APRIL does not interact with BAFF-R, the shorter variant of mouse APRIL exhibits weak but detectable binding to mouse BAFF-R.

Structure and expression of the Huntington's disease gene: Evidence against simple inactivation due to an expanded CAG repeat

Huntingtons disease, a neurodegenerative disorder characterized by loss of striatal neurons, is caused by an expanded, unstable trinucleotide repeat in a novel 4p16.3 gene. To lay the foundation for exploring the pathogenic mechanism in HD, we have determined the structure of the disease gene and examined its expression. TheHD locus spans 180 kb and consists of 67 exons ranging in size from 48 bp to 341 bp with an average of 138 bp. Scanning of theHD transcript failed to reveal any additional sequence alterations characteristic of HD chromosomes. A codon loss polymorphism in linkage disequilibrium with the disorder revealed that both normal and HD alleles are represented in the mRNA population in HD heterozygotes, indicating that the defect does not eliminate transcription. The gene is ubiquitously expressed as two alternatively polyadenylated forms displaying different relative abundance in various fetal and adult tissues, suggesting the operation of interacting factors in determining specificity of cell loss. TheHD gene was disrupted in a female carrying a balanced translocation with a breakpoint between exons 40 and 41. The absence of any abnormal phenotype in this individual argues against simple inactivation of the gene as the mechanism by which the expanded trinucleotide repeat causes HD. Taken together, these observations suggest that the dominant HD mutation either confers a new property on the mRNA or, more likely, alters an interaction at the protein level.

Cutting Edge: BAFF Regulates CD21/35 and CD23 Expression Independent of Its B Cell Survival Function

Herein we demonstrate that B cell-activating factor of the TNF family (BAFF), a B cell survival factor, also regulates CD21/35 and CD23 expression. BAFF blockade in wild-type mice down-modulates CD21/35 and CD23 on B cells while survival remains intact, and BAFF exposure causes elevated CD21/35 and CD23 expression. Similar down-modulation is observed in bcl-2-transgenic mice treated with a BAFF inhibitor. This is the first evidence that BAFF has a function independent of B cell survival. Reports using CD21/35 and CD23 expression to assess splenic B cell subsets in BAFF-null mice concluded a lack of B cells beyond the immature stage. Since CD21/35 and CD23 are inadequate for delineating B cell subpopulations in BAFF-null mice, we used expression of BAFF-R and several B cell markers to identify more mature splenic B cells in these mice. These data broaden our understanding of BAFF function and correct the view that BAFF-null mice lack mature B cells.

Gametic but not somatic instability of CAG repeat length in Huntington's disease.

Instability of a CAG repeat in 4p16.3 has been found in Huntingtons disease (HD) chromosomes. Unlike a similar repeat in the fragile X syndrome, the expanded HD repeat showed no evidence of somatic instability in a comparison of blood, lymphoblast, and brain DNA from the same persons. Four pairs of monozygotic HD twins displayed identical CAG repeat lengths suggesting that repeat size is determined in gametogenesis. In contrast with the fragile X syndrome and with HD somatic tissue, mosaicism was readily detected as a diffuse spread of repeat lengths in DNA from HD sperm samples. Typically, the modal repeat size was larger in the sperm DNA than in corresponding lymphoblast DNA, with the greatest degree of gametic mosaicism coinciding with the longest somatic CAG repeats. These data indicate that the developmental timing of repeat instability appears to differ between HD and fragile X syndrome, and that the fundamental mechanisms leading to repeat expansion may therefore be distinct.

The TNF family members BAFF and APRIL: the growing complexity

B cell activating factor belonging to the TNF family (BAFF) and apoptosis-inducing ligand (APRIL) are two related members of the TNF ligand superfamily. Although they share two receptors, TACI and BCMA, transgenic and knockout mice in this system reveal that their functions are not redundant. BAFF is a critical survival/maturation factor for peripheral B cells and this activity is mediated through a BAFF-specific receptor, BAFF-R. Overexpression of BAFF has been linked to autoimmune disease and aspects of B cell neoplasia. APRIL appears to play a role in T-independent type II antigen responses and T cell survival, but can also induce proliferation/survival of non-lymphoid cells. Elevated expression of APRIL has been found in some tumor cell lines and in tumor tissue libraries. Therapies designed to inhibit the BAFF and APRIL pathways holds great promise for the future.

Comparison of Soluble Decoy IgG Fusion Proteins of BAFF-R and BCMA as Antagonists for BAFF

BAFF is considered a therapeutic target because dysregulated production of BAFF can induce systemic lupus erythematosus-like phenotype in mice, and elevated levels of BAFF are associated with disease severity in systemic lupus erythematosus and rheumatoid arthritis patients. Fc fusion decoy receptors, BCMA-Fc and BAFF-R-Fc, are therapeutic candidates for blocking BAFF. While studying their interactions with BAFF, we found that BAFF-R-Fc is more effective than BCMA-Fc for blocking BAFF binding to its receptors. We also found that a trimeric BAFF can bind more than one BAFF-R-Fc but only one BCMA-Fc. Moreover, we show that, in contrast to monovalent BAFF-R-Fc, monovalent BCMA does not form stable complexes with BAFF. Differences in their interaction with BAFF predict BAFF-R-Fc would be a better inhibitor. Indeed, we show BAFF-R-Fc is 10-fold more efficacious than BCMA-Fc for blocking BAFF-induced B cell proliferation in vitro and for blocking BAFF-mediated survival of mouse splenic B lymphocytes in vivo.