Functional Analysis of PTH1R Variants Found in Primary Failure of Eruption.

Although many variants of the parathyroid hormone 1 receptor (PTH1R) gene are known to be associated with primary failure of eruption (PFE), the mechanisms underlying the link remains poorly understood. We here performed functional analyses of PTH1R variants reported in PFE patients-namely, 356C>T (P119L), 395C>T (P132L), 439C>T (R147C), and 1148G>A (R383Q)-using HeLa cells with a lentiviral vector-mediated genetic modification. Two particular variants, P119L and P132L, had severe reduction in a level of N-linked glycosylation when compared with wild-type PTH1R, whereas the other 2 showed modest alteration. PTH1R having P119L or P132L showed marked decrease in the affinity to PTH1-34, which likely led to severely impaired cAMP accumulation upon stimulation in cells expressing these mutants, highlighting the importance of these 2 amino acid residues for ligand-mediated proper functioning of PTH1R. To further gain insights into PTH1R functions, we established the induced pluripotent stem cell (iPSC) lines from a patient with PFE and the heterozygous P132L mutation. When differentiated into osteoblastic-lineage cells, PFE-iPSCs showed no abnormality in mineralization. The mRNA expression of RUNX2, SP7, and BGLAP, the osteoblastic differentiation-related genes, and that of PTH1R were augmented in both PFE-iPSC-derived cells and control iPSC-derived cells in the presence of bone morphogenetic protein 2. Also, active vitamin D3 induced the expression of RANKL, a major key factor for osteoclastogenesis, equally in osteoblastic cells derived from control and PFE-iPSCs. In sharp contrast, exposure to PTH1-34 resulted in no induction of RANKL mRNA expression in the cells expressing P132L variant PTH1R, consistent with the idea that a type of heterozygous PTH1R gene mutation would spoil PTH-dependent response in osteoblasts. Collectively, this study demonstrates a link between PFE-associated genetic alteration and causative functional impairment of PTH1R, as well as a utility of iPSC-based disease modeling for future elucidation of pathogenesis in genetic disorders, including PFE.

Haploinsufficiency of Sf3b1 leads to compromised stem cell function but not to myelodysplasia.

SF3B1 is a core component of the mRNA splicing machinery and frequently mutated in myeloid neoplasms with myelodysplasia, particularly in those characterized by the presence of increased ring sideroblasts. Deregulated RNA splicing is implicated in the pathogenesis of SF3B1-mutated neoplasms, but the exact mechanism by which the SF3B1 mutation is associated with myelodysplasia and the increased ring sideroblasts formation is still unknown. We investigated the functional role of SF3B1 in normal hematopoiesis utilizing Sf3b1 heterozygous-deficient mice. Sf3b1(+/-) mice had a significantly reduced number of hematopoietic stem cells (CD34(-)cKit(+)ScaI(+)Lin(-) cells or CD34(-)KSL cells) compared with Sf3b1(+/+) mice, but hematopoiesis was grossly normal in Sf3b1(+/-) mice. When transplanted competitively with Sf3b1(+/+) bone marrow cells, Sf3b1(+/-) stem cells showed compromised reconstitution capacity in lethally irradiated mice. There was no increase in the number of ring sideroblasts or evidence of myeloid dysplasia in Sf3b1(+/-) mice. These data suggest that SF3B1 plays an important role in the regulation of hematopoietic stem cells, whereas SF3B1 haploinsufficiency itself is not associated with the myelodysplastic syndrome phenotype with ring sideroblasts.

Molecular analysis of non-syndromic preaxial polydactyly: preaxial polydactyly type-IV and preaxial polydactyly type-I.

Human GLI3 gene mutations have been identified in several phenotypes of digital abnormality such as Greig cephalopolysyndactyly syndrome, Pallister-Hall syndrome, preaxial polydactyly type-IV (PPD-IV) and postaxial polydactyly. However, the different phenotypes resulting from GLI3 mutations have not yet been properly defined. We have experienced two types of digital abnormality without other complicating developmental defects; a family with foot PPD-IV with syndactyly of the third and fourth fingers, and four sporadic cases with biphalangeal thumb polydactyly (PPD-I). The genes responsible for syndactyly of the third and fourth fingers (syndactyly type-I) and PPD-I have not yet been identified; we therefore examined the involvement of the GLI3 gene in these subtypes of digital abnormality. We found a non-sense mutation in the GLI3 gene in the family with foot PPD-IV accompanied with hand syndactyly of the third and fourth fingers, but no mutations were detected in the GLI3 gene in the four other cases with PPD-I alone. Thus, the phenotype of foot PPD-IV accompanied with hand syndactyly of the third and fourth fingers may result from a GLI3 mutation, whereas the PPD-I phenotype alone is not caused by GLI3 gene defect. These results will help to define the phenotypic spectrum of GLI3 morphopathies, which have been recently proposed.

Somatic mosaicism in Wiskott--Aldrich syndrome suggests in vivo reversion by a DNA slippage mechanism.

Somatic mosaicism caused by in vivo reversion of inherited mutations has been described in several human genetic disorders. Back mutations resulting in restoration of wild-type sequences and second-site mutations leading to compensatory changes have been shown in mosaic individuals. In most cases, however, the precise genetic mechanisms underlying the reversion events have remained unclear, except for the few instances where crossing over or gene conversion have been demonstrated. Here, we report a patient affected with Wiskott--Aldrich syndrome (WAS) caused by a 6-bp insertion (ACGAGG) in the WAS protein gene, which abrogates protein expression. Somatic mosaicism was documented in this patient whose majority of T lymphocytes expressed nearly normal levels of WAS protein. These lymphocytes were found to lack the deleterious mutation and showed a selective growth advantage in vivo. Analysis of the sequence surrounding the mutation site showed that the 6-bp insertion followed a tandem repeat of the same six nucleotides. These findings strongly suggest that DNA polymerase slippage was the cause of the original germ-line insertion mutation in this family and that the same mechanism was responsible for its deletion in one of the propositus T cell progenitors, thus leading to reversion mosaicism.

Comparison of five retrovirus vectors containing the human IL-2 receptor gamma chain gene for their ability to restore T and B lymphocytes in the X-linked severe combined immunodeficiency mouse model.

X-linked severe combined immunodeficiency (XSCID) is caused by mutations in the IL-2 receptor gamma chain (IL2RG) gene, resulting in absent T lymphocytes and nonfunctional B lymphocytes. Recently T lymphocyte production and B lymphocyte function were restored in XSCID patients infused with autologous stem cells transduced with a retrovirus containing the human IL2RG cDNA. To optimize the expression of human IL2RG for future clinical trials, we compared five retroviral vectors expressing human IL2RG from different LTR enhancer-promoter elements in a mouse model. Northern and Southern blot analysis of hematopoietic tissues from repopulated mice revealed that the retroviral vector with the highest expression per copy number was MFG-S-hIL2RG, followed by MND-hIL2RG. All five vectors were capable of restoring lymphopoiesis in irradiated XSCID mice transplanted with transduced IL2RG-deficient hematopoietic stem cells. Transduction of IL2RG-deficient hematopoietic stem cells with all five vectors restored T lymphopoiesis in transplanted stem cell-deficient W/W(v) mouse recipients. However, only XSCID stem cells transduced with the MFG-S-hIL2RG vector generated B lymphocytes in W/W(v) mice. We conclude that the MFG-S-hIL2RG vector provides the best opportunity for in vivo selection and development of B and T lymphocytes for human XSCID gene therapy.

Lack of dominant-negative effects of a truncated gamma(c) on retroviral-mediated gene correction of immunodeficient mice.

A recent clinical trial of gene therapy for X-linked severe combined immunodeficiency (XSCID) has shown that retroviral-mediated gene correction of bone marrow stem cells can lead to the development of normal immune function. These exciting results have been preceded by successful immune reconstitution in several XSCID mouse models, all carrying null mutations of the common gamma chain (gamma(c)). One question not formally addressed by these previous studies is that of possible dominant-negative effects of the endogenous mutant gamma(c) protein on the activity of the wild-type transferred gene product. The present work was therefore undertaken to study whether corrective gene transfer was applicable to an XSCID murine model with preserved expression of a truncated gammac molecule (Deltagamma(c+)-XSCID). Gene correction of Deltagamma(c+)-XSCID mice resulted in the reconstitution of lymphoid development, and preferential repopulation of lymphoid organs by gene-corrected cells demonstrated the selective advantage of gamma(c)-expressing cells in vivo. Newly developed B cells showed normalization of lipopolysaccharide-mediated proliferation and interleukin-4 (IL-4)-induced immunoglobulin G1 isotype switching. Splenic T cells and thymocytes of treated animals proliferated normally to mitogens and responded to the addition of IL-2, IL-4, and IL-7, indicating functional reconstitution of gammac-sharing receptors. Repopulated thymi showed a clear increase of CD4-/CD8- and CD8+ fractions, both dramatically reduced in untreated Deltagamma(c+)-XSCID mice. These improvements were associated with the restoration of Bcl-2 expression levels and enhanced cell survival. These data indicate that residual expression of the endogenous truncated gamma(c) did not lead to dominant-negative effects in this murine model and suggest that patient selection may not be strictly necessary for gene therapy of XSCID.

Gene therapy for primary immune deficiencies.

Primary immunodeficiency diseases have been important targets of corrective gene transfer approaches since the very early days of gene therapy. The potential for selective survival advantage of gene-corrected cells over populations carrying the mutated, causative gene translates into the possibility of obtaining clinical meaningful results in patients with primary immunodeficiency diseases even if levels of gene transfer are low. This critical prospect has fueled the interest of researchers since the mid-1980s and has recently determined the success of a clinical trial of gene therapy for X-linked severe combined immunodeficiency.

In vivo competitive studies between normal and common gamma chain-defective bone marrow cells: implications for gene therapy.

Corrective gene transfer into hematopoietic stem cells (HSCs) is being investigated as therapy for X-linked severe combined immunodeficiency (XSCID) and it is hoped that selective advantage of gene-corrected HSCs will help in achieving full immune reconstitution after treatment. Lines of evidence from the results of allogeneic bone marrow transplantation in patients with XSCID support this hypothesis that, however, has not been rigorously tested in an experimental system. We studied the competition kinetics between normal and XSCID bone marrow (BM) cells using a murine bone marrow transplantation (BMT) model. For easy chimerism determination, we used genetic marking with retrovirus-mediated expression of the enhanced green fluorescent protein (EGFP). We found that XSCID BM cells were able to compete with normal BM cells for engraftment of myeloid lineages in a dose-dependent manner, whereas we observed selective repopulation of T, B, and NK cells deriving from normal BM cells. This was true despite the evidence of competitive engraftment of XSCID lineage marker-negative/c-Kit-positive (Lin-/c-Kit+) cells in the bone marrow of treated animals. From these results we extrapolate that genetic correction of XSCID HSCs will result in selective advantage of gene-corrected lymphoid lineages with consequent restoration of lymphocyte populations and high probability of clinical benefit.

Lentiviral-mediated gene transfer into human lymphocytes: role of HIV-1 accessory proteins.

Resting lymphocytes are refractory to gene transfer using Moloney murine leukemia virus (MMLV)-based retroviral vectors because of their quiescent status. Recently, it has been shown that lentiviral vectors are capable of transferring genes into nondividing and terminally differentiated cells. We used human immunodeficiency virus type-1 (HIV-1)-based vectors expressing enhanced green fluorescent protein (EGFP) driven by different promoters (CMV, MPSV, or PGK) and investigated their ability to transduce human T- and B-cell lines, as well as resting or activated primary peripheral and umbilical cord blood lymphocytes. The effects of the presence or the absence of HIV-1 accessory proteins (Vif, Vpr, Vpu, and Nef) in the vector system were also assessed. Flow cytometry analysis showed no differences in the ability of these vectors of transferring the reporter gene into lymphocytic lines and mitogen-stimulated primary lymphocytes in the presence or the absence of HIV-1 accessory proteins (APs). Similarly, viral supernatants generated in the presence of accessory genes could efficiently transduce various subsets of resting lymphocytes and provide long-term expression of the transgene. No significant transduction-induced changes in cell activation or cycling status were observed and Alu-HIV-1 long terminal repeat polymerase chain reaction (LTR PCR) analysis demonstrated integration of the vector sequences at the molecular level. In contrast, in the absence of HIV-1 APs, lentiviral vectors failed to integrate and express the transgene in resting lymphocytes. These results show that transduction of primary resting lymphocytes with HIV-1-based vectors requires the presence of viral accessory proteins. (Blood. 2000;96:1309-1316)

Lymphoid development and function in X-linked severe combined immunodeficiency mice after stem cell gene therapy.

Mutations of the common gamma chain (gammac) of cytokine receptors cause X-linked severe combined immunodeficiency (XSCID), a candidate disease for gene therapy. Using an XSCID murine model, we have tested the feasibility of stem cell gene correction. XSCID bone marrow (BM) cells were transduced with a retroviral vector expressing the murine gammac (mgammac) and engrafted in irradiated XSCID animals. Transplanted mice developed mature B cells, naive T cells, and mature natural killer (NK) cells, all of which were virtually absent in untreated mice. The mgammac transgene was detected in all treated mice, and we could demonstrate mgammac expression in newly developed lymphocytes at both the RNA and protein level. In addition, treated mice showed T cell proliferation responses to mitogens and production of antigen-specific antibodies upon immunization. Four of seven treated animals showed a clear increase of the transgene positive cells, suggesting in vivo selective advantage for gene-corrected cells. Altogether, these results show that retroviral-mediated gene transfer can improve murine XSCID and suggest that similar strategies may prove beneficial in human clinical trials.

Expression and purification of the extracellular ligand binding region of metabotropic glutamate receptor subtype 1.

Each metabotropic glutamate receptor possesses a large extracellular domain that consists of a sequence homologous to the bacterial periplasmic binding proteins and a cysteine-rich region. Previous experiments have proposed that the extracellular domain is responsible for ligand binding. However, it is currently unknown whether the extracellular ligand binding site can bind ligands without other domains of the receptor. We began by obtaining a sufficient amount of receptor protein on a baculovirus expression system. In addition to the transfer vector that encodes the entire coding region, transfer vectors that encode portions of the extracellular domain were designed. Here, we report a soluble metabotropic glutamate receptor that encodes only the extracellular domain and retains a ligand binding characteristic similar to that of the full-length receptor. The soluble receptor secreted into culture medium showed a dimerized form. Furthermore, we have succeeded in purifying it to homogeneity. Dose-response curves of agonists for the purified soluble receptor were examined. The effective concentration for half-maximal inhibition (IC50) of quisqualate for the soluble receptor was 3.8 x 10(-8) M, which was comparable to that for the full-length receptor. The rank order of inhibition of the agonists was quisqualate >> ibotenate >/= L-glutamate approximately (1S,3R)-1-aminocyclopentane-1, 3-dicarboxylic acid. These data demonstrate that a ligand binding event in metabotropic glutamate receptors can be dissociated from the membrane domain.

AIM-1: a mammalian midbody-associated protein required for cytokinesis.

Mitosis is a highly coordinated process that assures the fidelity of chromosome segregation. Errors in this process result in aneuploidy which can lead to cell death or oncogenesis. In this paper we describe a putative mammalian protein kinase, AIM-1 (Aurora and Ipl1-like midbody-associated protein), related to Drosophila Aurora and Saccharomyces cerevisiae Ipl1, both of which are required for chromosome segregation. AIM-1 message and protein accumulate at G2/M phase. The protein localizes at the equator of central spindles during late anaphase and at the midbody during telophase and cytokinesis. Overexpression of kinase-inactive AIM-1 disrupts cleavage furrow formation without affecting nuclear division. Furthermore, cytokinesis frequently fails, resulting in cell polyploidy and subsequent cell death. These results strongly suggest that AIM-1 is required for proper progression of cytokinesis in mammalian cells.

A possible role of ER-60 protease in the degradation of misfolded proteins in the endoplasmic reticulum.

Wild-type human lysozyme (hLZM) is secreted when expressed in mouse L cells, whereas misfolded mutant hLZMs are retained and eventually degraded in a pre-Golgi compartment (Omura, F., Otsu, M., Yoshimori, T., Tashiro, Y., and Kikuchi, M. (1992) Eur. J. Biochem. 210, 591-599). These misfolded mutant hLZMs are associated with protein disulfide isomerase (Otsu, M., Omura, F., Yoshimori, T., and Kikuchi, M. (1994) J. Biol. Chem. 269, 6874-6877). From the observation that this degradation is sensitive to cysteine protease inhibitors, such as N-acetyl-leucyl-leucyl-norleucinal and N-acetyl-leucyl-leucyl-methioninal, but not to the serine protease inhibitors, 1-chloro-3-tosylamido-7-amino-2-heptanone and (p-amidinophenyl)methanesulfonyl fluoride, it was suggested that some cysteine proteases are likely responsible for the degradation of abnormal proteins in the endoplasmic reticulum (ER). ER-60 protease (ER-60), an ER resident protein with cysteine protease activity (Urade, R., Nasu, M., Moriyama, T., Wada, K., and Kito, M. (1992) J. Biol. Chem. 267, 15152-15159), was found to associate with misfolded hLZMs, but not with the wild-type protein, in mouse L cells. Furthermore, denatured hLZM is degraded by ER-60 in vitro, whereas native hLZM is not. These results suggest that ER-60 could be a component of the proteolytic machinery for the degradation of misfolded mutant hLZMs in the ER.

Immunohistochemical study of p53, EGF, EGF-receptor, v-erb B and ras p21 in squamous cell carcinoma of hypopharynx.

We have characterized 24 hypopharyngeal squamous cell carcinomas and 5 normal hypopharyngeal tissues by immunostaining with antibodies against epidermal growth factor (EGF), EGF-Receptor (EGFR), p53, v-erb B, and ras p21. The Avidin-Biotin Complex (ABC) technique was employed. Overexpression of p53 appeared in 17 of 24 cases of squamous cell carcinoma of the hypopharynx (normal mucosa: none, well differentiated: 60%, moderately differentiated: 71.4%, poorly differentiated: 71.4%). Some dysplastic mucosae surrounding cancer lesions showed overexpression of p53. EGF and EGFR tended to be expressed more strongly in carcinoma [EGF: 29.1% (well differentiated: 30%, moderately differentiated: 28.6%, poorly differentiated: 28.6%); EGFR: 50% (well differentiated: 60%, moderately differentiated: 42.9%, poorly differentiated: 42.9%)] than in normal mucosa (EGF: 0%, EGFR: 20%). The v-erb B stained positively in carcinoma [62.5% (well differentiated: 70%, moderately differentiated: 71.4%, poorly differentiated: 42.9%)] but negatively in normal mucosa. These data suggest that genetic mutations of p53 probably play an important role at an early stage of tumorigenesis, and that the networks of EGF, EGFR and v-erb B probably are involved in the development of hypopharyngeal squamous cell carcinoma.

Site-specific O-glycosylation of cell adhesive lysozyme in yeast.

The cell adhesive protein RGD8 has been constructed using a yeast expression system by inserting eight amino acid residues (TGRGDSPA) between Val74 and Asn75 of human lysozyme [Yamada et al. (1993) J. Biol. Chem. 268, 10588-10592]. Purified RGD8 from yeast culture supernatant was found to contain glycosylated variants, in addition to the unglycosylated form. Peptide mapping analyses suggested that the glycosylation occurred at the inserted Thr residue in the RGD8 molecule. Electrospray ionization mass spectrometric analysis demonstrated the presence of four or five hexose residues in the glycosylated variants. Only mannose was detected in the sugar analysis of the oligosaccharide mixture obtained by mild alkaline treatment of the variants, and the structures of these carbohydrate chains were identified as Man alpha 1-3Man alpha 1-2Man alpha 1-2Man alpha and Man alpha 1-3Man alpha 1-3Man alpha 1-2Man alpha 1-2Man alpha by 1H-NMR spectroscopy. No other glycosylation was found, although the RGD8 molecule possesses a total of 13 Thr and Ser residues. In addition, no O-glycosylation was observed when the RGD8 protein was expressed in mouse L-cells. Thus, this O-glycosylation looks specific for yeast and the site of the Thr residue. The O-glycosylated variants of RGD8 exhibited a high level of adhesion activity to baby hamster kidney cells, which was almost comparable to that of the unglycosylated form.

Protein disulfide isomerase associates with misfolded human lysozyme in vivo.

Wild-type human lysozyme (hLZM) is quantitatively secreted into the media when expressed in mouse fibroblast cells, but some misfolded hLZMs are retained and rapidly degraded in a pre-Golgi compartment (Omura, F., Otsu, M., Yoshimori, T., Tashiro, Y., and Kikuchi, M. (1992) Eur. J. Biochem. 210, 591-599). To detect the association with misfolded hLZMs of cellular proteins involved in their folding, retention, and pre-Golgi degradation, a co-precipitation experiment was carried out using anti-hLZM antibody and metabolically labeled cell lysates, which were treated with a membrane-permeable cross-linking reagent. Here we report that protein disulfide isomerase associated in vivo with misfolded hLZMs, but not with the wild-type protein, and discuss the possible role of protein disulfide isomerase in the quality control of newly synthesized proteins in the endoplasmic reticulum.

Successful intravenous immunoglobulin therapy for recurrent pneumococcal otitis media in young children.

Serum immunoglobulin levels and naturally occurring antibody titres against Streptococcus pneumoniae were measured in seven children aged 1-1.9 years with recurrent pneumococcal acute otitis media (AOM). Three of them had low IgG2 levels. Mean antibody levels of anti-pneumococcal IgG1 and anti-pneumococcal IgG2 were significantly lower in patients when compared to those of healthy controls and children who had less frequent episodes of AOM. Following treatment with intravenous immunoglobulin (IVIG) for 6 months, anti-pneumococcal IgG1 and IgG2 antibody levels increased and the number of episodes of AOM decreased in all patients. Following the discontinuation of IVIG therapy, no AOM episode occurred. Serum levels of anti-pneumococcal IgG1 and IgG2 were normal, which were measured in three subjects at 5, 6, and 12 months after the cessation of IVIG therapy. These results suggested that delayed maturation of anti-pneumococcal antibody production caused recurrent AOM and this condition was corrected by IVIG therapy.