A missense mutation in the vasopressin-neurophysin precursor gene cosegregates with human autosomal dominant neurohypophyseal diabetes insipidus.

Familial neurohypophyseal diabetes insipidus in humans is a rare disease transmitted as an autosomal dominant trait. Affected individuals have very low or undetectable levels of circulating vasopressin and suffer from polydipsia and polyuria. An obvious candidate gene for the disease is the vasopressin-neurophysin (AVP-NP) precursor gene on human chromosome 20. The 2 kb gene with three exons encodes a composite precursor protein consisting of the neuropeptide vasopressin and two associated proteins, neurophysin and a glycopeptide. Cloning and nucleotide sequence analysis of both alleles of the AVP-NP gene present in a Dutch ADNDI family reveals a point mutation in one allele of the affected family members. Comparison of the nucleotide sequences shows a G----T transversion within the neurophysin-encoding exon B. This missense mutation converts a highly conserved glycine (Gly17 of neurophysin) to a valine residue. RFLP analysis of six related family members indicates cosegregation of the mutant allele with the DI phenotype. The mutation is not present in 96 chromosomes of an unrelated control group. These data suggest that a single amino acid exchange within a highly conserved domain of the human vasopressin-associated neurophysin is the primary cause of one form of ADNDI.

Expression of a mutant vasopressin gene: differential polyadenylation and read-through of the mRNA 3' end in a frame-shift mutant.

Sequence analysis of cDNA clones derived from hypothalamic mRNA of diabetes insipidus (Brattleboro) rats shows that the vasopressin gene transcript also includes the single base deletion demonstrated in the gene. This causes a frame-shift in the C terminus of the vasopressin precursor with a reading frame open through the 3' end of the mRNA including the poly(A) sequence. Antibodies raised against a synthetic tetradecapeptide (CP-14) corresponding to the frame-shifted C terminus identified a product of mol. wt approximately 26 000 in a reticulocyte lysate system programmed with Brattleboro hypothalamic mRNA. Immunohistochemical analysis indicated that a similar precursor is also present in vivo in neurones of the Brattleboro hypothalamus. Electrophoretic analysis of vasopressin mRNA from wild-type and mutant rat tissues revealed that (i) the hypothalamic mRNA from Brattleboro rats contains a longer stretch of poly(A) sequence than the wild-type strains; (ii) vasopressin mRNA is also present in the adrenal, ovary, testis and cerebellum, at very low levels; however, (iii) the extra-hypothalamic mRNA is considerably shorter than that in the hypothalamus because of a curtailed poly(A) sequence. Thus similar vasopressin gene transcripts are subject to a tissue-specific differential polyadenylation.

Immunocytochemical evidence for the presence of a mutant vasopressin precursor in the supraoptic nucleus of the homozygous Brattleboro rat.

CP-14, a tetradecapeptide from the predicted mutant vasopressin precursor in the homozygous Brattleboro rat was detected immunocytochemically in the supraoptic nucleus of homozygous Brattleboro but not normal rats. The staining was localized to the periphery of the perikarya. CP-14 immunoreactivity was not found in the neural lobes, paraventricular nuclei, accessory nuclei or suprachiasmatic nuclei of either homozygous Brattleboro or normal rats. Vasopressin immunoreactivity was found in the neural lobe and in the perinuclear region of neurons of the supraoptic, paraventricular, suprachiasmatic and accessory nuclei of normal rats. Vasopressin immunoreactivity was also found in homozygous Brattleboro rats, mainly in the ventral part of the supraoptic nucleus: densely stained solitary cells were found amongst other faintly stained perikarya. In both cell-types the staining was mainly in the periphery of the perikarya. No vasopressin immunoreactivity was detected in the paraventricular nuclei, suprachiasmatic nuclei, accessory nuclei or neural lobe of homozygous Brattleboro rats. CP-14 and vasopressin immunoreactivities were found to be co-localized; both were present in the periphery of the same perikarya of the supraoptic nuclei of homozygous Brattleboro rats. Differential staining was found with antioxytocin serum in both normal rats and homozygous Brattleboro rats: separate neurons were stained for either oxytocin or vasopressin and CP-14. Immunoreactive oxytocin was found mainly in the perinuclear region of the neurons from the supraoptic, paraventricular and accessory nuclei.

Immunocytochemical staining of supraoptic neurons from homozygous Brattleboro rats by use of antibodies against two domains of the mutated vasopressin precursor.

By use of antibody against the 14 amino acids in the mutated vasopressin precursor (CP-14) characteristic of the homozygous Brattleboro rat, an immunohisto- and -cytochemical study was performed on the supraoptic nuclei of homozygous Brattleboro rats. At the light-microscopic level, varying numbers of perikarya per section exhibited a positive reaction. The most intense staining was observed in a patchy manner on the peripheral portions of the cytoplasm, its central portion being stained less intensely. The antiserum did not react with the supraoptic perikarya of the Wistar rat. In the homozygous Brattleboro rat, antibodies against normal vasopressin only rarely resulted in a positive immunoreaction. However, when it was observed, incubation of the subsequent section with CP-14-antiserum suggested a co-localization of both peptides in the same perikaryon. At the ultrastructural level, CP-14 immunoreactivity was demonstrated on the secretory cisternae of the Golgi apparatus, on lysosome-like bodies and on parts of the rough endoplasmic reticulum. With the use of an antibody against normal vasopressin, immunoreactivity was confined to very limited areas of the rough endoplasmic reticulum. The oxytocin immunoreactivity in supraoptic perikarya of Brattleboro rats did not differ from that in the Wistar rat, either at the light- or at the electron-microscopic levels.