TY - JOUR
AU1 - Shultz, Leonard D.
AU2 - Lyons, Bonnie L.
AU3 - Burzenski, Lisa M.
AU4 - Gott, Bruce
AU5 - Samuels, Rebecca
AU6 - Schweitzer, Peter A.
AU7 - Dreger, Christine
AU8 - Herrmann, Harald
AU9 - Kalscheuer, Vera
AU1 - Olins, Ada L.
AU1 - Olins, Donald E.
AU1 - Sperling, Karl
AU1 - Hoffmann, Katrin
AB - Abstract The nature of the wild-type gene product at the mouse ichthyosis (ic) locus has been of great interest because mutations at this locus cause marked abnormalities in nuclear heterochromatin, similar to those observed in Pelger–Huët anomaly (PHA). We recently found that human PHA is caused by mutations in the gene (LBR) encoding lamin B receptor, an evolutionarily conserved inner nuclear membrane protein involved in nuclear assembly and chromatin binding. Mice homozygous for deleterious alleles at the ichthyosis (ic) locus present with a blood phenotype similar to PHA, and develop other phenotypic abnormalities, including alopecia, variable expression of syndactyly and hydrocephalus. The ic locus on mouse chromosome 1 shares conserved synteny with the chromosomal location of the human LBR locus on human chromosome 1. In this study, we identified one nonsense (815ins) and two frameshift mutations (1088insCC and 1884insGGAA) within the Lbr gene of mice homozygous for either of three independent mutations (ic, icJ and ic4J, respectively) at the ichthyosis locus. These allelic mutations are predicted to result in truncated or severely impaired LBR protein. Our studies of mice homozygous for the icJ mutation revealed a complete loss of LBR protein as shown by immunofluorescence microscopy and immunoblotting. The findings provide the molecular basis for the heterochromatin clumping and other distinct phenotypes caused by ic mutations. These spontaneous Lbr mutations confirm the molecular basis of human PHA and provide a small animal model for determination of the precise function of LBR in normal and pathological states. INTRODUCTION The first reported mutation at the ichthyosis (ic) locus arose spontaneously in 1948 in a sib-mated stock of mice (1). There have been five additional spontaneous mutations at the ic locus in mouse stocks maintained at The Jackson Laboratory (2). One of these mutations, icJ, occurred on the C57BL/6J strain background in 1974 (3) and was cryopreserved. This C57BL/6J-icJ stock was rederived from frozen embryos for phenotypic analyses. Mice carrying the original ic mutation as well as the four other mutations that occurred at The Jackson Laboratory (ic2J, ic3J, ic4J and ic5J) are extinct (4,5). These remutations were considered as possible alleles at the ichthyosis locus based solely on gross phenotype. Allelism tests between heterozygotes for each of the presumptive remutations crossed with +/ic mice confirmed that the new mutations were allelic with ic. However, phenotypic analyses of the new ichthyosis mutations were not carried out prior to cryopreserving or discarding these mutations. DNA samples from several of these mutations are available from The Jackson Laboratory DNA Resource and were utilized in the current study. All previous studies of phenotypic abnormalities in ichthyosis mutant mice have focused on mice homozygous for the original ic mutation. Homozygotes (ic/ic) can be recognized at 2 days of age by their shortened vibrissae. Adults have coats with short, fine hairs of variable density or they may develop a severe alopecia depending on genetic background (6). The skin is mildly thickened and often develops hard scales. Results of skin graft experimentation following dermal–epidermal recombination indicate that the ic mutation exerts its effects on the skin at the level of the epidermis (7). In 1975, we reported clumping of heterochromatin in the nuclei of granulocytes, lymphocytes, intestinal epithelial cells and other cell types in IC/Le-ic/ic mice and suggested that the phenotype resembled Pelger–Huët anomaly (PHA) in humans (8). PHA in heterozygotes is a benign disorder seen in 0.01–1% of individuals and is transmitted as an autosomal dominant trait (9). Neutrophil and eosinophil nuclei in PHA heterozygotes show incomplete segmentation resulting in a bean shaped or rod-like appearance, with condensation of nuclear chromatin in contrast with the multilobulated appearance of nuclei in normal granulocytes. Homozygous PHA individuals have an extreme reduction in lobulation of granulocyte nuclei and may express a number of additional clinical features (10–21). Recently, we reported that human PHA was caused by mutations in the gene (LBR) that encodes lamin B receptor (LBR) (17). This receptor is an inner nuclear membrane protein that targets heterochromatin and lamins to the nuclear membrane (22,23). In the present investigation, we show that deleterious mutations at the mouse ic locus disrupt the lamin B receptor gene (Lbr) and provide an animal model for the study of PHA and for the function of the LBR. RESULTS Gross phenotypic changes, histopathologic abnormalities and skeletal lesions in C57BL/6J-icJ/icJ mice Although allelism tests carried out in 1974 confirmed that the icJ mutation was a new allele at the ichthyosis locus (3), no further characterization of C57BL/6J-icJ/icJ mice was carried out before the stock was cryopreserved. Therefore, we initiated our investigation by characterizing the genetics and phenotype of these mice. We identified icJ/icJ mice at 2 weeks of age by sparseness of hair accompanied by decreased body size. By 3–4 weeks, icJ/icJ homozygotes also develop scales on the tail and to a lesser degree on the truncal skin (Fig. 1A). Three icJ/icJ mice had syndactyly affecting one or more paws (Fig. 1B–E). Histological examination of truncal skin from icJ/icJ mice showed mild epidermal hyperplasia with orthokeratotic hyperkeratosis and dilation of the piliary canals (Fig. 1F and G). Three icJ/icJ homozygotes had gross evidence of hydrocephaly. Histological evaluation of H&E-stained sections of brains from these mice confirmed marked dilation of the lateral ventricles (not shown). Lymphocytes in the spleen, lymph nodes, thymus and Peyer's patches from icJ/icJ mice showed clumping of the nuclear chromatin, usually into three to four discreet masses located at the edges of the nucleus. In contrast, +/+ littermates had normal appearing lymphocytes with perinuclear chromatin often extending into the interior of the nucleus (Fig. 2A and B). Lymphocytes in blood smears from icJ/icJ mice also showed heterochromatin clumping (Fig. 2C and D). Nuclei of neutrophils in icJ/icJ blood smears varied from a bilobed appearance to an individual clump of heterochromatin, while neutrophils in +/+ blood smears had a normal polymorphonuclear appearance (Fig. 2E and F). Nuclei of eosinophils in icJ/icJ blood smears were mostly bilobed, while +/+ smears contained mostly band eosinophils (Fig. 2G and H). In cytospin preparations of bone marrow from icJ/icJ mice, neutrophils and eosinophils had a single clump of heterochromatin while bone marrow preparations from +/+ control mice had normal-appearing immature neutrophils and eosinophils (Fig. 2I and J). Chromatin clumping was also observed in other cell populations of icJ/icJ mice, including intestinal epithelial cells and granule cells of the cerebellum (not shown). Analyses of blood smears from icJ/+ mice revealed that nuclei in a small number of granulocytes had decreased lobulation compared with granulocytes from +/+ control animals (data not shown). Analyses of the ultrastructure of lymphocytes in spleens from icJ/icJ mice showed discrete masses of heterochromatin at the edges of the nuclear membrane with no indentation of the membrane, while +/+ lymphocyte nuclei had normal perinuclear and perinucleolar heterochromatin (Fig. 3). Expression of LBR protein in lymphoid tissues from +/+ but not icJ/icJ mice Frozen sections of spleens from C57BL/6 +/+ and icJ/icJ mice were analyzed for expression of LBR using an anti-LBR antibody recognizing the first 217 amino acids of the LBR protein. Spleen cells from icJ/icJ mice were uniformly negative for LBR expression while the nuclear envelopes of lymphocytes from +/+ mice showed intense LBR staining (Fig. 4A and B). Western blot analyses of LBR expression in spleen cells from C57BL/6 +/+ and icJ/icJ mice were carried out with the same anti-LBR antibody used for immunofluorescence studies. Spleen and lymph node cells from +/+ mice showed a strong band at ∼65 kDa. In contrast, spleen and lymph node cells from icJ/icJ mice failed to express LBR protein (Fig. 4C). Identification of the molecular basis for the ic, icJ and ic4J mutations Recently, we reported that PHA is caused by mutations in the human LBR structural gene (17). This was an excellent candidate for the mouse ic locus since the map position for ic on mouse chromosome 1 is syntenic with the map position of the human LBR locus on chromosome lq 41–43 (www.informatics.jax.org/reports/homologymap/mouse_human.shtml). The genomic sequence for Lbr (MGI:213821) was located on the February 2002 version of the public Mouse Genome Sequence Consortium draft mouse assembly. The Lbr gene is 27 289 bp long with coordinates of 183 211 378–183 238 667. We examined DNA samples from three available mutant alleles (ic, icJ and ic4J). The following coordinates are based upon the RefSeq entry NM_133815 for Lbr, which is 3494 bp long. The original ic mutation (C523T) introduces a premature stop (TAG) codon at amino acid position 175 (Fig. 5A, B and G). The icJ mutation corresponds to a 2 bp insertion at position 1088 (1088insCC) that creates a frame shift predicted to change amino acids 365–385 and to create a stop codon at position 386 (Fig. 5C, D and G). The ic4J mutation is an insertion of four nucleotide residues (1815insGGAA). The resulting frameshift is open until codon 648 and is predicted to change amino acids 606–627, and add 21 amino acids to the carboxy end of the altered protein (Fig. 5E, F, and G). None of these mutations were present in the background strain wild-type controls. PCR genotyping of C57BL/6-icJ/icJ, icJ/+ and +/+ mice Identification of the icJ mutation as a CC insertion in the Lbr structural gene facilitated the design of primers for genotyping of all offspring from (icJ/+×icJ/+) matings. After we determined the molecular basis of the icJ mutation, a PCR assay was designed to distinguish among icJ/icJ, icJ/+ and +/+ mice. The primers amplify a 121 bp DNA fragment from both the wild-type and mutant alleles. Digestion with BglI results in 100 and 21 bp fragments for wild-type, a 121 bp fragment for icJ/icJ and three fragments (100, 21 and 121 bp) for icJ/+ (Fig. 6). Genotypes of progeny from matings of C57BL/6J-icJ/+ mice Sib matings of C57BL/6J-icJ/+ mice would be expected to yield ∼25% mutant homozygotes (icJ/icJ). However, of 17 litters from icJ/+ breeding pairs, only 14 out of 99 mice born were identified as icJ/icJ on the basis of alopecia at 2 weeks of age (14.1% born versus 25% expected). Thus, approximately one-half of the homozygous icJ/icJ offspring probably die in utero or shortly after birth. Chi-square analyses show a significant decrease in the number of homozygotes born versus the number expected (χ2=3.86, P<0.05). In order to determine whether the icJ/+ heterozygotes and +/+ wild-type mice were produced in the expected ratios, 53 mice in 10 litters born from matings of icJ/+ heterozygotes were genotyped. Seven of the offspring had sparse hair and a scaly appearance of the skin. Genotyping by PCR confirmed that these affected mice were indeed icJ/icJ homozygotes. Thirty-one of the remaining 46 offspring were genotyped as icJ/+ heterozygotes and 15 offspring were +/+. A ratio of 1:2:1 of icJ/icJ, icJ/+ and +/+ mice would be expected from matings of heterozygotes. The observed ratio was 7:31:15 icJ/icJ, icJ/+ and +/+ mice, respectively. These data show that the icJ/+ and +/+ mice are born at the expected ratio (2:1) and confirm the loss of icJ/icJ homozygotes. DISCUSSION Homozygosity for deleterious alleles at the ichthyosis locus in mice results in pleiotropic changes that include clumping of heterochromatin in the nuclei of granulocytes, lymphocytes and other cell populations, skeletal defects, hydrocephalus, abnormalities of the skin and hair and increased prenatal mortality. The nuclear morphology seen in icJ/icJ mice and characteristic of PHA is consistent in several mammalian species with LBR mutations that include humans (9,17,24), rabbits (25–27), dogs (28–31) and cats (32). The present study focused on LBR-deficient mice and our earlier publication on LBR-deficient humans (17) clearly demonstrate that adequate levels of LBR are required for neutrophil nuclear lobulation and normal heterochromatin distribution. However, changes in nuclear lobulation are not an inevitable consequence of elevated LBR expression levels (33), suggesting that other factors modulate LBR activity. Although PHA results in abnormal granulocyte nuclear morphology in human and all known animal models, the mouse is the only known species in which LBR deficiency results in marked pathological changes in the skin. The pronounced skin abnormality in mice with deleterious alleles at the ic locus compared with the normal skin of homozygous PHA individuals might result from the complete loss of LBR caused by the known deleterious alleles at the ic locus. In contrast, human PHA homozygotes express detectable LBR protein. It is also possible that the development and function of mouse skin are more vulnerable to altered LBR function than are those of other species. Skeletal defects, such as the syndactyly that was observed in icJ/icJ mice, are frequently associated with homozygous PHA individuals. Of the few Pelger–Huët homozygotes described (10–21), at least two presented with syndactyly or brachydactyly (10,17). Homozygotes from an extinct strain of Pelger–Huët rabbits, referred to as ‘Super-Pelgers’, had severe skeletal deformities characterized as chondrodystrophy. In addition, they had low birth weight, failure to grow, and increased prenatal and perinatal death (25–27). A reduced prenatal and early postnatal mortality of homozygous PHA individuals is suggested by a report that a family with one confirmed homozygote had two miscarriages and three children who died within the first year of life (16). Increased prenatal mortality in icJ/icJ mice was suggested by a significant loss in numbers of icJ/icJ mice born from icJ/+ heterozygotes. Future studies will include genotyping of embryos conceived from icJ/+ parents to evaluate loss of icJ/icJ mice in utero. LBR is a multifunctional protein, capable of structurally integrating nuclear lamins, the nuclear membrane and heterochromatin. Pathological changes caused by mutations at the Lbr locus might be a direct consequence of the failure of appropriate binding of lamin B to the LBR. A number of human diseases, termed laminopathies, are associated with defects in proteins of the nuclear envelope (34). Mutations in the human LMNA gene that encodes lamins A and C by alternative splicing cause at least five distinct diseases, including skeletal defects. One of these diseases, mandibuloacral dysplasia, is a rare autosomal-recessive disorder that is characterized by postnatal growth retardation, craniofacial abnormalities, skeletal malformations and abnormalities in skin pigmentation (35). Although PHA is inherited as a dominant trait, phenotypic changes caused by the known ichthyosis mutations are all recessive. The absence of detectable LBR protein in icJ/icJ mice could be due to nonsense-mediated RNA decay, impaired translation of mutant RNA into protein or enhanced posttranslational degradation of the LBR protein. By comparison, the LBR mutation in the homozygous PHA individual whom we recently studied resulted in decreased mRNA splicing efficiency; the small amount of remaining LBR protein might be sufficient to reduce the severity of the phenotype (17). The null mutation at the Lbr locus in icJ/icJ mice results in morphological alterations in the nuclei of a number of cell types, increased prenatal mortality, and developmental abnormalities that include changes in the skin, alopecia, fusions of bone and soft tissue in the paws and hydrocephalus. The mechanisms by which LBR deficiency results in the diverse phenotypic abnormalities in icJ/icJ mice are not well understood. The pathogenesis of diseases associated with lamin A/C mutations is under investigation by a number of laboratories (34,36–38). Possible mechanisms by which abnormalities in lamin interactions with their receptors might cause pathological changes include increased nuclear fragility, alterations in nuclear positioning, changes in regulation of gene expression, or secondary perturbations in the endoplasmic reticulum (34,39). We hypothesize that the primary defect in the LBR and consequent lamina disruption may have downstream effects on chromatin structure or gene expression. Indeed, several transcription factors involved in tissue differentiation are associated with the nuclear matrix, and it has been demonstrated that mutations that interfere with this process can severely affect the function of transcription factors (40). LBR has potential recognition sites for a number of transcription factors, including SP-1, AP-1, AP-2 and NFκB (22). The diverse nature of phenotypic changes in icJ/icJ mice might be related to the interaction of LBR with specific transcription factors since these are expressed in a tissue-specific manner. The normal LBR protein consists of a hydrophilic nucleoplasmic domain and a hydrophobic domain with eight transmembrane segments (22). The nucleoplasmic domain contains regions critical for interactions with lamin B, double stranded DNA, and HP-1 type heterochromatin proteins (23). The hydrophobic domain contains 8 transmembrane segments and belongs to the sterol reductase family. This domain has C-14 sterol reductase activity when expressed in Neurospora crassa or Saccharomyces cervisiae (41,42). Both the ic and icJ mutations are predicted to eliminate parts of the transmembrane domain of the lamin B receptor. The ic4J mutation is predicted to result in severe alteration of LBR structure or it might cause premature RNA or protein decay. Although the transmembrane carboxyterminal domain of LBR has homology with C14 sterol reductases (43), it is not yet known whether LBR has any metabolic, functional or regulatory steroid reductase activity in humans or mice. The possibility of such functional activity is supported by recent investigation of the FACKEL gene in plants. This gene, studied in Arabidopsis dwarf mutants, encodes a protein with C14 sterol reductase activity and plays a critical role in embryonic patterning (44). Thus, the developmental abnormalities found in human and rabbit Pelger–Huët homozygotes and in icJ/icJ mice might also be related to changes in sterol metabolism. Indeed, defects in sterol metabolism underlie the human skin disease, X-linked ichthyosis. In this disease, steroid sulfatase deficiency severely affects cholesterol metabolism, leading to increased cholesterol sulfate in serum and stratum corneum (45). Defects in sterol metabolism also cause a number of skeletal malformations, reviewed in (46). The transmembrane domain of LBR is conserved among vertebrates (22). Moreover, at least two related genes, SR1 (TM7SF2) and SR2 (7-dehydrocholesterol reductase, DHCR7) are members of the sterol reductase family (43). Deficiency of DHCR7 causes Smith–Opitz syndrome, an autosomal recessive disease of cholesterol metabolism presenting with multiple developmental errors (48,49). It has also been suggested that the sterol reductase domain acts as a receptor for sterol molecules that function in signaling pathways involved in regulation of cell cycling and that LBR might be involved in the inactivation of the signaling pathway (42). In summary, mice homozygous for the ichthyosis mutation provide a single gene model for human PHA and for determination of the role of LBR in normal and pathologic states. The functional consequences of perturbations in LBR—a pivotal architectural protein in the nuclear envelope—to chromosomal functions and cell cycle dynamics remain to be fully elucidated. Ongoing experimentation is focused on determination of the biochemical and cellular mechanisms of LBR function. MATERIALS AND METHODS Mice The original ic mutation (1) was previously mapped to mouse chromosome 1 and was backcrossed from the IC/Le incipient inbred strain background onto the AKR/J strain at The Jackson Laboratory (www.informatics.jax.org/searches/linkmap.cgi) (8,50). Five additional spontaneous mutations at the ichthyosis locus occurred from 1975 to 1995 at The Jackson Laboratory. The mouse strains and allele designations are: C57BL/6-icJ (3); A/J-ic2J and A/J-ic3J (Hope Sweet, personal communication); C3H/HeJ-ic4J (51); and SK/Rk-ic5J (5,51). We sequenced the Lbr gene in: (a) homozygous AKR-ic/ic, the corresponding AKR/J+/+, and the IC/Le +/+ controls; (b) C57BL/6J-icJ/icJ and the corresponding C57BL/6 +/+ control; and (c) C3H/HeJ-ic4J/ic4J and the C3H/HeJ +/+ control. The C57BL/6-icJ mouse stock used in the current study was originally obtained from The Jackson Laboratory Cryopreservation Service. The colony was maintained by matings of C57BL/6J-icJ/+ breeders. Initial breeding required test matings to identify icJ/+ heterozygotes because these heterozygotes appear phenotypically normal. The icJ/icJ homozygotes were readily identifiable prior to weaning by sparseness of hair. Additional C57BL/6J +/+ mice were produced by sib-matings of wild-type mice. All mice were reared on NIH 31M diet and acidified water ad libitum in a research mouseroom under modified barrier conditions at The Jackson Laboratory. Histopathology and hematology Mice were euthanized by CO2 asphyxiation. Tissues were fixed in Bouin's solution, embedded in paraffin and sectioned at 5 µm. Slides were stained with Mayer's hematoxylin and eosin (H&E). Blood was collected from the retro-orbital sinus using heparinized capillary tubes. Blood smears were fixed in methanol and stained with Wright-Giemsa (Sigma Chemical Co., St Louis, MO, USA). Bone marrow was obtained by flushing the medullary cavities of femurs with cold HBSS (Sigma). Marrow plugs were disrupted by passage through a 25 g needle. The resulting single cell suspensions were counted with a model Z1 Coulter counter (Beckman Coulter, Miami, FL, USA) and resuspended in HBSS containing 50% FBS. Cell suspensions were spun at 600 rpm for 6 min in a Shandon Cytospin 2 (Shandon Southern Instruments, Inc., Sewickley, PA, USA). The cytospin slides were air-dried, fixed in absolute methanol and stained with Wright–Giemsa. Immunofluorescence microscopy Samples of spleens from icJ/icJ and +/+ mice were collected, frozen in Tissue-Tek OCT compound (Sakura Finetek, Torrance, CA, USA) and sectioned at 6 µm. Slides were air dried for 30 min, fixed in absolute methanol for 3 min at room temperature and rinsed once in PBS. Sections were blocked with 20% normal goat serum for 1 h at room temperature. Guinea pig anti-LBR antibody (1:4000 dilution) or control PBS was applied and incubated overnight at 4°C. The guinea pig anti-LBR antibody was generated against a recombinant fragment representing the 217 amino-terminal amino acids of LBR (17). Sections were then washed three times in PBS containing 0.05% Tween-20 and then incubated in the dark for 1 h at room temperature with goat anti-guinea pig Alexa Fluor 546 (Molecular Probes, Eugene, OR, USA) diluted 1:500 in PBS. Sections were counterstained with DAPI and were rinsed three times in PBS before mounting with PBS. Digital images were collected with a Leica DMRE microscope equipped for epifluorescence. DAPI was imaged in green. Transmission electron microscopy Small pieces of spleen from icJ/icJ and +/+ littermate control mice were minced with a razor blade in cold 1.5% glutaraldehyde in 0.l M cacodylate buffer, pH 7.2. After overnight fixation at 4°C, samples were washed three times in 0.1 M cacodylate buffer, and then postfixed in 1.5% osmium tetroxide–0.1 M cacodylate buffer, pH 7.4. The samples were stained en bloc with 1% uranyl acetate–70% ethanol, dehydrated in a graded series of ethanol and embedded in Spurr's resin. Ultrathin sections were post-stained with uranyl acetate followed by lead citrate. Samples were imaged on a JOEL JEM 100 CX II electron microscope. Radiography C57BL/6J-icJ/icJ and littermate control mice were anesthetized with an intraperitoneal injection of tribromoethanol (0.2 ml per 10 g body weight of a 1.2% solution). The mice were radiographed at 25 kV for 2 s at a source-to-object distance of 11.4 cm with a cabinet X-ray system (Faxitron, Wheeling, IL, USA). Images were collected on Kodak MIN-R mammography film. Western blotting Mouse spleen and lymph nodes were collected and frozen in liquid nitrogen. Total cellular protein lysates were prepared in buffer consisting of 50 mM Tris base (pH 8.2), 150 mM NaCl, 1% Igepal, and Complete protease inhibitor cocktail tablets (Roche, Chicago, IL, USA) for 20 min at 4°C, followed by centrifuging at 10 000g for 20 min at 4°C. Total protein levels were measured using a detergent compatible protein assay (BioRad, Hercules, CA, USA). Lysates were loaded on 10% SDS–polyacrylamide gels using 10µg total protein/lane and transferred to PVDF membranes (Millipore, Bedford, MA, USA). Membranes were blocked with 2% non-fat dry milk (BioRad), then incubated with guinea pig anti-LBR antibody diluted 1:5000 followed by HRP-conjugated rabbit anti-guinea pig antibody at 1:10 000 (Roche). Signal was detected using an enhanced chemiluminescence kit (Amersham, Piscataway, NJ, USA). PCR amplification and sequencing of PCR products Genomic DNA samples from C57BL/6J-icJ/icJ, AKR-ic/ic, and C3H/HeJ-ic4J/ic4J mice and their respective wild-type controls were obtained from The Jackson Laboratory DNA Resource. The lamin B receptor gene in both mouse and human has an untranslated first exon. All 13 translated exons of the mouse Lbr gene were amplified from genomic DNA with intronic primers. The entire mouse Lbr gene spans about 27.3 kb (from the 5′ UTR to the 3′ UTR) and contains one untranslated and 13 translated exons. Standard procedures were used for bi-directional automatic sequencing with fluorescent dye terminators on the MegaBACE 1000 DNA-Sequencer. The following intronic primer sequences were used: Me1F 5′-cacactccttccttggctttct-3′, Me1R 5′-ttgtgtaagagcacctcacagg-3′, Me2F 5′-tagaatgcacggcaacagagag-3′, Me2R 5′-tctgaacacgcatttgtataggg-3′, Me3F 5′-gcctttactccagacggtttag-3′, Me3R 5′-tcacatgcactcttcaggacag-3′, Me4F 5′-tgctgagttagtagtgtttgcac-3′, Me4R 5′-aagttcttccctagtcctccac-3′, Me6F 5′-gctgttgcacagaaaacctgtc-3′, Me6R 5′-agataaccttccgttcaccacc-3′, Me7F 5′-gcaacctagtgtgacttgtctc-3′, Me7R 5′-ctctatgagtagtcatggctgc-3′, Me8F 5′-aggtgaagtgagctctcaagatc-3′, Me8R 5′-ctccaagcctgaaactgggtca-3′, Me9F 5′-gctgaggtgtcttgttctctgg-3′, Me9R 5′-acattcctctaatcccgtgtgg-3′, Me10F 5′-acaggacttaagccagagcaac-3′, Me10R 5′-cgacagacaccaaccagaaaac-3′, Me11/12F 5′-caagatggatgatgacaagtgag-3′, Me11/12R 5′-tgttagacaccaagagagtagag-3′, Me13F 5′-tgaacgtttggtttgcgtttgg-3′, Me13R 5′-tcctgtcagtgcatcacattag-3′. Mouse exon 6 was analyzed by nested primary PCR/sequencing reactions with PCR primers Me5F 5′-ccttacaaattggtttcagacagc-3′ and Me5R 5′-cctggtctacagagtgagttcc-3′, and sequencing primers Me5F2 5′-agttacaracagatggttgtgagc-3′ and Me5R2 5′-gggcacacgtttataatccaagc-3′. Genotyping +/+, icJ/+ and icJ/icJ mice Tail DNA was prepared using hot sodium hydroxide and Tris–HCl (52). Genotyping at the Lbr locus used primers flanking the icJ mutation (F: 5′GTACCTGTATCACTTCC-3′, R: 5′TGGCTGGCGACAGCTCCGCC-3′). Before agarose electrophoresis, the amplification products were digested with the BglI restriction enzyme to distinguish among +/+, icJ/+ and icJ/icJ mice. Database analysis The genomic and cDNA sequences of human LBR have been published (22). The mouse genomic and cDNA sequences were reconstructed by combining information from both the public Mouse Genome Sequence Consortium draft mouse assembly (MGI:213821) and CELERA database (previous contig GA_x5J8B7W6MNY, updated contig GA_x6K02T2P20D). The coordinates for the Lbr mutations in ichthyosis mice are based on Refseq entry NM_133815. Homology search and exon assembly were done with BLAST programs at the National Center for Biotechnology Information (NCBI). Interspecies comparison was done by MegAlign in the DNAStar software package. Protein sequences for LBR of human, chick and frog were obtained from the public database. Mouse protein sequence was determined from cDNA sequence based on Refseq entry NM_133815. In addition, BLAST search revealed a rat protein called nbp60 that is highly homologous to LBR in the other species (53). ACKNOWLEDGEMENTS This manuscript is dedicated to Dr Margaret Green with great appreciation for introducing me (LDS) to ichthyosis mice and to the wonders of spontaneous mutations. We thank Heike Fischer and Barbara Lucke for sequence analyses of the Lbr mutations in ichthyosis mice. We are grateful to Ken Johnson and Greg Cox for helpful discussions and critical review of the manuscript. We thank Carol Bult for expertise in DNA sequence analyses and annotation. This work was supported in part by National Institutes of Health grants CA20408 (L.D.S.), CA34196 to The Jackson Laboratory, and grant Sp 144/18-1 from the Deutsche Forschungsgemeinschaft (K.H.). A.L.O. and D.E.O. supported by the Davis Family Foundation while at the Foundation for Blood Research, Scarborough, ME, and by the German Cancer Research Center while they were guests at the laboratory of Peter Lichter. * To whom correspondence should be addressed at: The Jackson Laboratory, 600 Main Street, Bar Harbor, ME 04609, USA. Tel: +1 2072886405; Fax: +1 2072886079; Email: lds@jax.org Open in new tabDownload slide Figure 1. Gross, radiographic and histologic specimens from C57BL/6-icJ/icJ and littermate +/+ control mice at 5 weeks of age. (A) icJ/icJ and littermate +/+ control mice. The icJ/icJ mouse shows sparseness of hair, presence of scales, most predominantly on the tail, and decreased body size. (B–E) Radiographs of front and hind paws of 5-week-old mice. (B) icJ/icJ mouse hind paw showing bony fusion of digits four and five at the level of the proximal interphalangeal joint (arrow). (C) +/+ mouse hind paw showing normal anatomy. (D) icJ/icJ mouse front paw showing syndactyly characterized by soft tissue fusion between digits two and three and between digits four and five (arrows). (E) +/+ front paw showing normal anatomy. (F, G) Truncal skin sections (H&E). (F) Epidermal hyperplasia with orthokeratotic hyperkeratosis and abnormal piliary canals in an icJ/icJ mouse. (G) Normal skin in +/+ control. Open in new tabDownload slide Figure 2. Leukocytes in spleen, peripheral blood, and bone marrow of 4–5-week-old C57BL/6J-icJ/icJ and +/+ control mice. (A, B) Lymphoid follicles in spleen sections (H+E). (A) Lymphocytes showing clumping of heterochromatin in an icJ/icJ mouse. (B) Normal lymphocytes showing dispersed heterochromatin in +/+ control. (C–H) Blood smears (Wright–Giemsa). (C) Lymphocytes with heterochromatin clumping in an icJ/icJ mouse. (D) Normal lymphocyte in +/+ control with dispersed heterochromatin. (E) Neutrophil showing bilobed nucleus with clumped heterochromatin in an icJ/icJ mouse. (F) Normal multilobulated neutrophil in +/+ control. (G) Eosinophil showing bilobed nucleus with clumped heterochromatin in an icJ/icJ mouse. (H) Normal band eosinophil in +/+ control. (I, J)Bone marrow cytospins (Wright–Giemsa). (I) Immature neutrophils and eosinophils showing clumped heterochromatin in an icJ/icJ mouse. (J) Normal immature neutrophils and eosinophils in a +/+ control. Open in new tabDownload slide Figure 3. Transmission electron photomicrographs of splenic lymphocytes from 5-week-old C57BL/6J-icJ/icJ and +/+ control mice. (A) Heterochromatin in discrete clumps at periphery of nucleus in an icJ/icJ mouse. (B) Heterochromatin is dispersed throughout nucleus of normal +/+ lymphocytes. Open in new tabDownload slide Figure 4. Expression of immunoreactive LBR in spleens from C57BL/6J-icJ/icJ mice. (A, B) Frozen sections of spleen. Immuofluorescence for lamin B receptor, red; DAPI imaged in green. (A) Absence of lamin B receptor expression in icJ/icJ mouse. (B) Nuclear membrane localization of lamin B receptor in +/+ mouse. (C) Immunoblot analysis of lamin B receptor expression in spleen and lymph node (LN) from icJ/icJ and control +/+ mice. Tissue lysates from icJ/icJ mice lack lamin B receptor expression. Tissue lysates from +/+ control mice show a strong single band in lymph node and a moderately intense band in spleen at ∼65 kDa. Spleen cells from +/+ spleen cells show an additional faint band at ∼70 kDa. Open in new tabDownload slide Figure 5. Mutations in the Lbr gene in DNA from AKR/J-ic/ic, C57BL/6J-icJ/icJ and C3H/HeJ-ic4J/ic4J mice. Sequences from the ic, icJ and ic4J mutated regions are shown. (A, B) A substitution from wild-type C to T in AKR/J-ic/ic at position 523 (C523T) creates a premature stop codon at position 175. (C, D) A CC insertion (1088insCC) in C57BL/6J-icJ/icJ causes a frameshift with predicted amino acid substitutions and a subsequent stop codon at position 386. (E, F) A frameshift caused by a 4 bp insertion (1815insGGAA) in C3H/HeJ-ic4J/ic4J is predicted to cause substitution of amino acids 606–627, addition of 21 novel amino acids, and a postponed stop codon at position 648 (instead of 627). None of these three mutations were detected in the AKR/J +/+, C57BL/6J +/+ or C3H/HeJ +/+ control animals. Base positions are counted from the start of the ATG of the mouse Lbr gene. (G) Lamin B receptor protein structure. Organization of the lamin B receptor protein including mutation sites and structural effects. Domains are given with their approximate amino acid positions, known binding sites and functions. The dashed lines represent the putative missing protein domains caused by the mutations C523T (ic), 1088insCC (icJ), and 1815insGGAA (ic4J), respectively. Structure and function are presented according to previous studies (22,23,42). Domain sizes and distances are approximate. Open in new tabDownload slide Figure 6. PCR amplification of DNA from C57BL/6J +/+, icJ/+ and icJ/icJ mice. The primers amplify a 121 bp DNA fragment from both alleles. The R primer differs from the Lbr genomic sequence by containing a T to G substitution at nucleotide 18, which in conjunction with the sequence at the mutation site, introduces a BglI restriction enzyme cleavage site into the PCR products from the wild-type allele. PCR products from the mutant allele do not contain the restriction enzyme site. Digestion with BglI results in a 100 bp and 21 bp fragment for wild-type; 100, 21 and 121 bp fragments for icJ/+, and a 121 bp fragment for ic/ic. The 21 bp fragment is not seen. 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TI - Mutations at the mouse ichthyosis locus are within the lamin B receptor gene: a single gene model for human Pelger–Huët anomaly
JF - Human Molecular Genetics
DO - 10.1093/hmg/ddg003
DA - 2003-01-01
UR - https://www.deepdyve.com/lp/oxford-university-press/mutations-at-the-mouse-ichthyosis-locus-are-within-the-lamin-b-2PvpAkCChb
SP - 61
EP - 69
VL - 12
IS - 1
DP - DeepDyve
ER -