A loss-of-function mutation in natriuretic peptide receptor 2 (Npr2) gene is responsible for disproportionate dwarfism in cn/cn mouse

The achondroplastic mouse is a spontaneous mutant characterized by disproportionate dwarfism with short limbs and tail due to disturbed chondrogenesis during endochondral ossification. These abnormal phenotypes are controlled by an autosomal recessive gene (cn). In this study, linkage analysis using 115 affected mice of F2 progeny mapped the cn locus on an approximately 0.8-cM region of chromosome 4, and natriuretic peptide receptor 2 (Npr2) gene was identified as the most potent candidate for the cn mutant in this region. This gene encodes a receptor for C-type natriuretic peptide (CNP) that positively regulates longitudinal bone growth by producing cGMP in response to CNP binding to the extracellular domain. Sequence analyses of the Npr2 gene in cn/cn mice revealed a T to G transversion leading to the amino acid substitution of highly conserved Leu with Arg in the guanylyl cyclase domain. In cultured chondrocytes of cn/cn mice, stimulus with CNP did not significantly increase intracellular cGMP concentration, whereas it increased in +/+ mice. Transfection of the mutant Npr2 gene into COS-7 cells also showed similar results, indicating that the missense mutation of the Npr2 gene in cn/cn mice resulted in disruption of the guanylyl cyclase activity of the receptor. We therefore concluded that the dwarf phenotype of cn/cn mouse is caused by a loss-of-function mutation of the Npr2 gene, and cn/cn mouse will be a useful model to further study the molecular mechanism regulating endochondral ossification by CNP/natriuretic peptide receptor B signal.     

A novel hypothyroid dwarfism due to the missense mutation Arg479Cys of the thyroid peroxidase gene in the mouse

Recently, we found a novel dwarf mutation in an ICR closed colony. This mutation was governed by a single autosomal recessive gene. In novel dwarf mice, plasma levels of the thyroid hormones, T3 and T4, were reduced; however, TSH was elevated. Their thyroid glands showed a diffuse goiter exhibiting colloid deficiency and abnormal follicle epithelium. The dwarfism was improved by adding thyroid hormone in the diet. Gene mapping revealed that the dwarf mutation was closely linked to the thyroid peroxidase (Tpo) gene on chromosome 12. Sequencing of the Tpo gene of the dwarf mice demonstrated a C to T substitution at position 1508 causing an amino acid change from arginine (Arg) to cysteine (Cys) at codon 479 (Arg479Cys). Western blotting revealed that TPO protein of the dwarf mice was detected in a microsomal fraction of thyroid tissue, but peroxidase activity was not detected. These findings suggested that the dwarf mutation caused a primary congenital hypothyroidism by TPO deficiency, resulting in a defect of thyroid hormone synthesis.     

Identification and characterization of a novel mouse prion gene allele

The major determinant of prion disease incubation time in mice is thought to be the amino acid sequence of the prion protein. Two alleles of the mouse prion gene (Prnp) have been described, where Prnp^a (Leu-108, Thr-189) and Prnp^b (Phe-108, Val-189) are associated with short and long incubation times, to defined prion strains, respectively. As part of a survey of inbred mouse lines, the prion gene open reading frame was sequenced and revealed a new allele, Prnp^c (Phe-108, Thr-189), in the strain MAI/Pas. To study the influence of Prnp^c independently of the MAI/Pas genetic background, we generated a congenic line in which Prnp^c was bred onto the C57BL/6JOlaHsd background. Following intracerebral inoculation with Chandler/RML scrapie prions, the congenic mice showed an increased mean incubation time relative to C57BL/6JOlaHsd, of over 100 days. However, no differences were observed in the intensity and pattern of PrP immunoreactivity deposition or spongiosis. We conclude that the new allele, Prnp^c, modulates incubation time but not neuropathology and that the previous classification of mice into two distinct groups based on incubation time and Prnp genotype should now be revised.     

Regulation of collagen gene expression in the Tsk2 mouse

The tight skin 2 (Tsk2) mutation is an ENU induced dominant mutation localized on mouse chromosome 1. While the molecular defect is unknown, Tsk2/+ mice display cutaneous thickening associated with excessive matrix production and are used as a model of scleroderma. The purpose of this study was to examine the cellular mechanisms associated with the excessive synthesis of matrix macromolecules using a collagen promoter GFP reporter transgene (pOBCol3.6GFP) as a marker of Col1a1 expression. This analysis of pOBCol3.6GFP expression in Tsk2/+ skin showed an increase in transgene activity compared to wild-type (+/+) samples. In addition, an increased area of "high" GFP fluorescence in Tsk2/+ dermis in both 1- and 4-month-old mice was observed that was also associated with an increased number of dermal fibroblasts per unit area of dermis. These data collectively suggest an important mechanism of Tsk2/+ skin fibrosis; an increased number of collagen expressing cells as well as elevated collagen expression on a per cell basis. During this study it was noted that Tsk2/+ mice appeared consistently smaller than wild-type (+/+) siblings and measurements of body length revealed a decrease (5-10%) in 1- and 2-month-old Tsk2/+ mice as well as a decrease in body weight in both age groups as compared to wild-type (+/+) control mice. Femur length was also decreased (2-9%) in Tsk2/+ mice. Finally, in contrast to Tsk/+ mice that display an emphysema-like lung pathology, histological sections of lungs from Tsk2/+ mice were normal and indistinguishable from wild-type (+/+) controls.     

Generation of a floxed allele of the mouse BMP type II receptor gene

Bone morphogenetic proteins (BMPs) regulate a wide range of cellular functions that contribute to embryonic development from mesoderm formation to organogenesis. BMP type II receptor (BMPR-II) transduces BMP signals by forming heteromeric complexes with and phosphorylating BMP type I receptors. Heterozygous germline mutations of BMPR-II gene have been identified in patients with familial and sporadic primary pulmonary hypertension, indicating that BMPR-II may contribute to the maintenance of normal pulmonary vascular structure and function. Since embryos homozygous for a null BMPR-II allele died during gastrulation, precluding further studies of BMPR-II function in organ formation and in adult tissues, we generated mice carrying a conditional mutant BMPR-II allele in which exons 4 and 5 were flanked by loxP sequences. We anticipate that studies of mice carrying a floxed BMPR-II allele and a Cre transgene (under the control of a tissue-specific promoter) will enable characterization of the role of BMPR-II in specific cell types during development and in the pathogenesis of cardiovascular diseases.     

TaqIB allele polymorphism of the dopamine receptor D2 gene in patients with endogenous psychoses

The genotype at theTaqIB locus of the dopamine receptor D2 (DRD2) gene was established by PCR in 113 healthy donors and 340 patients with endogenous psychoses. The allele and genotype frequencies in patients with affective psychoses significantly differed from those in controls and in patients with schizophrenia. The proportion of B2/B2 homozygotes decreased with increasing contribution of the affective component to the clinical manifestation of schizo-affective and affective disorders. TheTaqIB polymorphism was assumed to play a role in mood aberrations in patients with mental disorders.     

Congenital hypothyroidism, dwarfism, and hearing impairment caused by a missense mutation in the mouse dual oxidase 2 gene, Duox2

Dual oxidases generate the hydrogen peroxide needed by thyroid peroxidase for the incorporation of iodine into thyroglobulin, an essential step in thyroid hormone synthesis. Mutations in the human dual oxidase 2 gene, DUOX2, have been shown to underlie several cases of congenital hypothyroidism. We report here the first mouse Duox2 mutation, which provides a new genetic model for studying the specific function of DUOX2 in the thyroid gland and in other organ systems where it is hypothesized to play a role. We mapped the new spontaneous mouse mutation to chromosome 2 and identified it as a T>G base pair change in exon 16 of Duox2. The mutation changes a highly conserved valine to glycine at amino acid position 674 (V674G) and was named "thyroid dyshormonogenesis" (symbol thyd) to signify a defect in thyroid hormone synthesis. Thyroid glands of mutant mice are goitrous and contain few normal follicles, and anterior pituitaries are dysplastic. Serum T(4) in homozygotes is about one-tenth the level of controls and is accompanied by a more than 100-fold increase in TSH. The weight of adult mutant mice is approximately half that of littermate controls, and serum IGF-I is reduced. The cochleae of mutant mice exhibit abnormalities characteristic of hypothyroidism, including a delayed formation of the inner sulcus and tunnel of Corti and an abnormally thickened tectorial membrane. Hearing thresholds of adult mutant mice are on average 50-60 decibels (dB) above those of controls.     

A novel HLA-DRB1-DR2 allele associated with HLA mistyping

This work was supported by a research grant from the Federal Ministry of Research and Technology (BMFT/DFVLR) 01 VM 8608/9.     

Autosomal recessive disorder otospondylomegaepiphyseal dysplasia is associated with loss-of-function mutations in the COL11A2 gene

Otospondylomegaepiphyseal dysplasia (OSMED) is an autosomal recessive skeletal dysplasia accompanied by severe hearing loss. The phenotype overlaps that of the autosomal dominant disorders-Stickler and Marshall syndromes-but can be distinguished by disproportionately short limbs, severe hearing loss, and lack of ocular involvement. In one family with OSMED, a homozygous Gly-->Arg substitution has been described in COL11A2, which codes for the alpha2 chain of type XI collagen. We report seven further families with OSMED. All affected individuals had a remarkably similar phenotype: profound sensorineural hearing loss, skeletal dysplasia with limb shortening and large epiphyses, cleft palate, an extremely flat face, hypoplasia of the mandible, a short nose with anteverted nares, and a flat nasal bridge. We screened affected individuals for mutations in COL11A2 and found different mutations in each family. Individuals from four families, including three with consanguineous parents, were homozygous for mutations. Individuals from three other families, in whom parents were nonconsanguineous, were compound heterozygous. Of the 10 identified mutations, 9 are predicted to cause premature termination of translation, and 1 is predicted to cause an in-frame deletion. We conclude that the OSMED phenotype is highly homogenous and results from homozygosity or compound heterozygosity for COL11A2 mutations, most of which are predicted to cause complete absence of alpha2(XI) chains.     

A loss-of-function mutation in the rice KNOX type homeobox gene, OSH3

KNOX homeodomain (HD) proteins encoded by KNOTTED1-like homeobox genes (KNOX genes) are thought to work as switches for cells to change from an indeterminate to a determinate state, although their direct functions are not clear. In the process of isolating KNOX genes from rice, we found that one gene, named OSH3, has two amino acid substitutions in three of the invariant amino acid residues in the HD of KNOX proteins. These amino acid substitutions are not universal in rice: two of the cultivars from the Indica variety of rice do not carry those substitutions but two of the cultivars from Japonica variety do. We tested the effect of these amino acid substitutions on their ability to form dimers and to induce abnormal morophologies when overexpressed in transgenic plants. We found that OSH3 without those substitutions can form dimers and can induce an abnormal phenotype in overexpression studies, and that OSH3 with those amino acid substitutions is defective in both. Based on these observations, we concluded that OSH3 from two of the cultivars from the Japonica variety could have lost its original function, or could have acquired a novel function by modifying the action of HD, or both.