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The Journal of Clinical Endocrinology & Metabolism Vol. 86, No. 7 3410-3420
Copyright © 2001 by The Endocrine Society

Original Articles

Familial Neurohypophysial Diabetes Insipidus in a Large Dutch Kindred: Effect of the Onset of Diabetes on Growth in Children and Cell Biological Defects of the Mutant Vasopressin Prohormone1

Marga Nijenhuis2, Erica L. T. van den Akker2,3, Robbert Zalm, Anton A. M. Franken, Andre P. Abbes, Henk Engel, David de Wied and J. Peter H. Burbach

Isala Klinieken Zwolle, Departments of Pediatrics (E.L.T.v.d.A.), Internal Medicine (A.A.M.F.), Clinical Chemistry (A.P.A., H.E.), 8025 AB Zwolle, The Netherlands; and Rudolf Magnus Institute for Neurosciences, Department of Medical Pharmacology, Utrecht University (M.N., R.Z., D.d.W., J.P.H.B.), 3508 TA Utrecht, The Netherlands

Address all correspondence and requests for reprints to: Dr. J. Peter H. Burbach, Rudolf Magnus Institute for Neurosciences, Utrecht University, P.O. Box 80040, 3508 TA Utrecht, The Netherlands. E-mail: j.p.h.burbach{at}med.uu.nl

Abstract

Familial neurohypophysial diabetes insipidus (FNDI) is an autosomal dominant trait in which expression of a mutant vasopressin prohormone reduces vasopressin production. We investigated the NP85 Cys->Gly mutant vasopressin prohormone in a large kindred in The Netherlands. We demonstrate that growth retardation is an important early sign in two children from this kindred, which recuperates by substitution therapy with 1-desamino-8-D-arginine vasopressin. To obtain clues about the basis for the dominant inheritance of FNDI, we analyzed the trafficking and processing of the mutant vasopressin prohormone in cell lines by metabolic labeling and immunoprecipitation. The mutant vasopressin prohormone was retained in the endoplasmic reticulum and thus was not processed to vasopressin. This defect was not caused by dimerization of the vasopressin prohormone via its unpaired cysteine residue. High level expression of the mutant vasopressin prohormone in cell lines resulted in strong accumulation in the endoplasmic reticulum and an altered morphology of this organelle. We hypothesize that disturbance of the endoplasmic reticulum results in dysfunction and ultimately cell death of the cells expressing the mutant prohormone. Our data support the hypothesis that FNDI is a progressive neurodegenerative disease with delayed onset of symptoms. Its treatment requires early detection of symptoms for which growth parameters are useful.

FAMILIAL NEUROHYPOPHYSIAL diabetes insipidus (FNDI) is characterized by excessive thirst, excessive urine production, and a lack of the day-night rhythm of urine production (1, 2, 3). The disease is caused by a mutation in the vasopressin (VP) prohormone gene, resulting in a defective preprohormone and a deficiency of arginine VP. FNDI reduces not only the plasma levels of VP, but also the amount of VP stored in the posterior pituitary, as evidenced by the absence of the characteristic high signal intensity of the posterior pituitary in T1-weighted magnetic resonance images of most FNDI patients (4, 5, 6, 7, 8 ; see also 9). At present, 36 different mutations have been identified in FNDI families (8, 10, 11, 12, 13, 14, 15, 16, 17). All except 1 (14) are inherited in an autosomal dominant mode. Moreover, a delayed onset of the disease is characteristic (1, 2, 3, 10). The disease is not manifest during the first months or years of life and progressively develops thereafter. The delayed onset and the autosomal dominant inheritance indicate that the mutant VP preprohormone gradually induces the lack of VP secretion despite the presence of the normal allele. It has been speculated that accumulation of the mutant prohormone may block the secretory pathway or can result in degeneration of VP neurons (11, 18). The latter is supported by postmortem studies showing, respectively, a strong reduction in the amount of magnocellular neurons in the supraoptic and paraventricular nuclei of the hypothalamus of FNDI patients (19, 20, 21, 22).

FNDI mutations have been found within the signal sequence, VP, and neurophysin (NP) moieties of the VP preprohormone (Fig. 1Go) (8, 10, 11, 12, 13, 14, 15, 16, 17). The mutations within the signal sequence reduce signal sequence cleavage and, correspondingly, endoplasmic reticulum (ER) exit of the mutant prohormone (18, 23, 24). Most of the FNDI mutations (30 of 36) have been identified within the NP domain of the precursor. NP is an intracellular binding protein for VP. It has been postulated that this binding is required for appropriate folding and targeting of the prohormone to the regulated secretory pathway (25, 26). NP is tightly folded. Although it is only 93 amino acids long, it contains 7 disulfide bridges. In addition, the central core (amino acids 10–85) consists of 2 domains of 4 antiparallel ß-strands, separated by an {alpha}-helix and a loop (25, 26, 27, 28). Except for 1, all FNDI mutations within NP are present within this highly folded central core, suggesting that they affect the folding of the NP domain. The same hypothesis can be made for the VP2Tyr->His mutation within the VP moiety. Tyrosine at position 2 of VP is essential for binding of VP to NP, which, in turn, enhances the stability of NP (25, 26, 27, 28). In agreement with this, the only mutation causing recessive FNDI (VP7Pro->Leu within VP) changes a residue within VP that is not required for NP binding (25, 26, 27).



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  		Figure 1. Schematic representation of the VP preprohormone and the established diabetes insipidus mutations. The four moieties of the VP prohormone, the signal peptide (SP), VP (VP), NP II (NP), and the glycopeptide (GP) are indicated, as are the sequences of the cleavage sites (GKR and R) and the position of the glycan (lollipop). The established diabetes insipidus mutations are indicated underneath the bar representing the VP preprohormone. According to conventions in the nomenclature of the FNDI mutations (10 ), amino acids of each moiety of the VP preprohormone were numbered separately. Thus, the abbreviation G14R underneath the NP moiety indicates substitution of the G at position 14 of NP by an R and is indicated as NP14G->R in the rest of this manuscript. {Delta}, Deletion of the indicated residue(s). The only mutation resulting in a recessively inherited phenotype, VP-P7L, is indicated in italics and marked with an asterisk. The mutation NP85C->G investigated in this study is depicted in gray. The disulfide bridges and secondary elements present in the VP preprohormone are depicted on top of the bar. ->, ß-Strand; , {alpha}-helix; , loop.

 
In this report we describe a Dutch family with a recently identified FNDI mutation (17). In two children within this kindred we analyzed the relationship between diabetes insipidus symptoms (i.e. excessive fluid intake) and growth rate. In addition, to obtain clues about the cause of the dominant nature of the disease, we analyze the intracellular transport of the mutant VP prohormone in stably transfected cell lines that contain a regulated secretory pathway.

Subjects and Methods

Subjects

A family with FNDI was studied. We recently described the genetic screening of 1 branch of the family (17). The pedigree of this branch (branch A) is shown in Fig. 2AGo. In this manuscript 3 more branches of this family were studied. The family includes 56 individuals symptomatic with polyuria and polydipsia from 4 generations (22 in branch A, 14 in branch B, 15 in branch C, and 5 in branch D). All family members reported an onset in early infancy. The pedigree shows an autosomal dominant inheritance pattern of clinical overt diabetes insipidus.



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  		Figure 2. Pedigree of a Dutch kindred with FNDI caused by the NP85C->G mutation. A, Branch A. The founders of branches B, C, and D are shown next to the founder of this branch (subject A-I-1). B, Branch B. C, Branch C. D, Branch D. The legend is depicted next to branch D.

 
Subjects A-IV-21 and A-III-18 (referred to, respectively, as subject IV-21 and III-18 in Ref. 17) were the first patients of the family who visited the out-patient clinics. A-IV-21 was a 2-yr-old boy who presented with failure to thrive. He drank 4 L/day. The father of subject A-IV-21 (A-III-18), who was 34 yr old, reported life-long symptoms of polydipsia and polyuria. He complained of nocturia and drank up to 10 L/day. We previously described the genetic screening for the two described members of the family (17). Briefly, genomic DNA was extracted from the two subjects. Each of the three exons of the VP prohormone gene was PCR amplified, and the nucleotide sequences of these PCR products were determined by primer dye cycle sequencing (17). The identified mutation introduces an additional Sau96I restriction site in the PCR fragment, and mutations were confirmed by restriction enzyme analysis (17).

Subject B-IV-9 was diagnosed with FNDI at the age of 7 yr. The presence of the nucleotide (nt) 2110 T->G mutation in one of the VP prohormone alleles of this subject was confirmed by PCR and restriction analysis with Sau96I (Fig. 3Go). The details of this method were described previously (17). The same method was employed to analyze the presence of the nt 2110 T->G mutation in the VP prohormone genes of the other members of branches B, C, and D of the kindred.



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  		Figure 3. Screening of members of branches B and C by restriction endonuclease digestion. Genomic DNA from two unaffected (C-III-48 and C-II-9) and five affected (C-III-60, C-II-18, B-II-16, B-IV-9, and C-II-7) members of branches B and C of the family was isolated, and a 257-bp PCR fragment encompassing exon III of the VP prohormone gene was generated and digested with restriction endonuclease Sau96I. Exon 3 of the wild-type VP prohormone gene has two Sau96I restriction sites, yielding three fragments of 160, 87, and 10 bp. The mutation creates an extra Sau96I site and produces four fragments of 160, 64, 23, and 10 bp. The 10-bp fragment is not shown. Pos.con., Positive control heterozygous for the mutation; Neg.con., negative control. A molecular weight marker (in base pairs) is indicated at the left. The migration position, length, and origin (wild-type or mutant gene) of the Sau96I fragments are indicated on the right. Note that only affected members of the family possess the mutation. Note in addition that all affected members are heterozygous for the mutation. They display both the 87-bp fragment originating from the wild-type VP prohormone gene and the 64- and 23-bp fragments originating from the mutated gene.

 
Construction of expression plasmids encoding wild-type and mutant human VP genes

The human VP gene (GenBank accession no. M11166) was a gift from Dr. Jim Battey (Bethesda, MD) (29). The gene was cloned into the hygromycin B resistance-conferring expression plasmid pRSVhyg (Dr. Van Tol, University of Toronto, Toronto, Canada) as previously described (11). The resulting plasmid was named phygHVP.

A PCR fragment encompassing the last part of the second intron and first part of the third exon of the NP85C->G mutant VP prohormone gene was obtained by PCR of patient material with the following primers: NAR1 M13FW, 5'-GTTGTAAAACGACGGCCAGCCGGCAGGGAGGGTGTGGG-3'; and AVP3-M13BK, 5'-GAAACAGCTATGACCATGCCTCTCTCCCCTTCCCTCTT-3' (17). These primers amplify a 409-bp PCR fragment extending from nt 2008 in intron 2 to nt 2379 in intron 3 of the VP prohormone gene. Due to a polymorphism in the second intron, the 5'-primer is specific for the mutant gene (17), resulting in amplification of the mutant gene fragment only. The underlined primer sequences are M13 sequences, which were introduced because the primers were previously employed for primer-dye cycle sequencing with M13 primers (17). The PCR fragment was digested with NarI, and the resulting 160-kb NarI fragment (nt 2059–2219) containing the nt 2110 T->G mutation, was substituted for the wild-type NarI fragment in the phygHVP plasmid via a series of three cloning steps. The cloned NarI fragment was completely sequenced to verify the presence of the nt 2110 T->G mutation and the absence of mutations induced by the PCR.

Culture and transfection of cells

The rat adrenal pheochromocytoma cell line PC12 transfected with mouse prohormone convertase 2 (PC12/PC2) was a gift from Dr. Sharon Tooze (30) and was cultured in poly-L-lysine-coated flasks in DMEM (Life Technologies, Inc., Gaithersburg, MD) supplemented with 4 mmol/L glutamine, nonessential amino acids (Life Technologies, Inc.), 200 IU/mL penicillin, 200 µg/mL streptomycin, 5% FCS, and 10% horse serum (Life Technologies, Inc.) in an atmosphere of 10% CO2.

The mouse pituitary tumor cell line AtT-20 (ATCC CCL89, American Type Culture Collection, Manassas, VA) and the mouse neuroblastoma cell line Neuro-2A (ATCC CCL 131) were cultured in DMEM (Life Technologies, Inc.) supplemented with 4 mmol/L glutamine, nonessential amino acids (Life Technologies, Inc.), 200 IU/mL penicillin, 200 µg/mL streptomycin, and 10% FCS (Life Technologies, Inc.) in an atmosphere of 5% CO2.

PC12/PC2 and AtT-20 cells were transfected with gene constructs cloned into the pRSVhyg vector by incubating cells plated in 6-cm dishes overnight in 2 mL serum-free medium with 15 µg plasmid DNA and 25 µL Lipofectamine (Life Technologies, Inc.) as previously described (30). Stable PC12/PC2 clones were selected in medium supplemented with 100 µg/mL hygromycin B (Roche Molecular Biochemicals, Indianapolis, IN), and maintained in medium with 50 µg/mL hygromycin B. Stable AtT-20 clones were selected on and maintained in medium with 200 µg/mL hygromycin B. After selection, hygromycin-resistant clones were pooled and employed for the experiments described here. Analysis of (pooled) clones from independent PC12/PC2 transfections gave similar results. In addition, similar results were obtained for clones expressing different levels of wild-type or mutant prohormone.

Neuro-2A cells were transiently transfected with gene constructs cloned into the pRSVhyg vector with the calcium phosphate method (31).

Antisera

Rabbit antiserum D7 raised against swine NP and cross-reacting with human NP was a gift from Dr. Iain Robinson (32). Rabbit antiserum HenryK was raised against rat NP and displays cross-reactivity with human NP (33). Monoclonal antibody 1D3 was raised against the C-terminus of the ER protein protein-disulfide-isomerase and recognizes both protein-disulfide-isomerase and calreticulin (34).

Labeling of cells, immunoprecipitation, and gel electrophoresis

Labeling of cells with 25 µCi [35S]cysteine (ICN Biomedicals, Inc., Costa Mesa, CA) for the times indicated and immunoprecipitation with the D7 antiserum were performed as previously described (11), except for the supplementation of medium with 10% FCS instead of 5% FCS and 10% horse serum in the case of AtT-20 cells. Where indicated, endoglycosidase H digestions of the immunoprecipitates were performed with 4 U endoglycosidase H (Roche Molecular Biochemicals) for 32 h at 37 C in digestion buffer (50 mmol/L sodium citrate, pH 5.5, and 0.2% SDS). Immunoprecipitated proteins were analyzed by 10% Tricine-SDS-PAGE (35). Depending on the experiment, nonreducing or reducing gel electrophoresis was employed. Nonreducing gel electrophoresis gave sharper bands, thus improving the separation of proteins. However, because the separation between the VP prohormone and the slightly slower migrating background band from the medium was better on reducing gels, we used reducing SDS-PAGE when we wanted to focus at the VP prohormone (in the case of endoglycosidase H digestions). The MW-SDS-17S marker (Sigma, St. Louis, MO) was used as a molecular weight marker. Protein gels were dried, and radioactive bands were detected by analysis on a BAS1000 phosphorimager (Fujix, Fuji, Tokyo, Japan).

Immunofluorescence

Cells were grown on coverslips and transiently transfected for 48 h. Fixation and immunofluorescence were performed as previously described (36, 37). Immunofluorescence was performed with a 1:1000 dilution of the HenryK antiserum followed by a 1:300 dilution of fluorescein-conjugated donkey antirabbit Igs (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA). In the case of double labeling, the second labeling was performed with a 1:50 dilution of the 1D3 monoclonal antibody (hybridoma supernatant) followed by a 1:2400 dilution of Cy3-conjugated donkey antimouse Igs (Jackson ImmunoResearch Laboratories, Inc.). The coverslips were mounted with DABCO/Mowiol and examined with a x63 Planapo objective on a Leitz DMIRB fluorescence microscope (Leica Corp., Voorburg, The Netherlands) interfaced with a Leica Corp. TCS4D confocal scanning laser microscope (Leica Corp., Heidelberg, Germany).

Results

Clinical characterization of individuals affected with FNDI

Recently, a novel diabetes insipidus mutation was identified in a Dutch kindred (Fig. 2AGo) (17). Subjects A-IV-21 and A-III-18 were the first patients in this family who visited the out-patient clinics. A-IV-21 was a 2-yr-old boy, who presented with failure to thrive. In this subject, formal assessment of the diagnosis of diabetes insipidus was carried out in the hospital. The patient was subjected to a standard water deprivation test with hourly measurement of plasma and urinary osmolality (38, 39). The test was concluded when plasma osmolality rose to 315 mosmol/kg while urinary osmolality as well as the plasma VP concentration remained relatively low (respectively, 400 mosmol/kg and 1.3 pg/mL). Normally the plasma VP concentration is higher than 8 pg/mL at this plasma osmolality. The boy had more than a 50% increase in urinary osmolality after the administration of exogenous VP, confirming the diagnosis of neurohypophysial diabetes insipidus.

In response to the water deprivation test, subject A-III-18, the father of the boy, had a rise in plasma osmolality to 307 mosmol/kg with a maximum urinary osmolality of 305 mosmol/kg and plasma VP of 0.33 pg/mL (normally >5 pg/mL). His symptoms were relieved by the use of 1-desamino-8-D-arginine vasopressin.

Identification of the VP prohormone gene mutation in affected individuals

We previously identified a mutation in the VP prohormone gene by sequencing all three exons of this gene for the two affected individuals, A-III-18 and A-IV-21 (17). The DNA sequence indicated heterozygosity for the normal sequence and a T->G transition mutation at nucleotide 2110 (codon 116) of the VP prohormone gene (17). This mutation encodes a cysteine->glycine substitution at amino acid position 85 of the NP domain of the preprohormone (NP85C->G; Fig. 1Go). Samples from two unaffected individuals from the family demonstrated homozygosity for the normal DNA sequence for all three exons of the VP prohormone gene (17).

The mutation detected by direct sequencing of the PCR-amplified VP prohormone gene was confirmed by restriction endonuclease analysis. PCR fragments of 257 bp containing exon 3 of the normal VP prohormone allele have two Sau96I restriction sites and yield three restriction fragments of, respectively, 160, 87, and 10 bp. The T->G mutation introduces an extra Sau96I restriction site and produces four fragments of 160, 64, 23, and 10 bp (17). Thirty-one members of branch A of the family were tested by restriction analysis (17) (Fig. 2AGo).

In this study 3 additional branches of this family were screened, encompassing 61 individuals (23 of branch B, 30 of branch C, and 8 of branch D; Fig. 2Go, B–D). For 7 subjects, the resulting restriction fragment patterns are displayed in Fig. 3Go. All affected subjects were heterozygous for the mutation, whereas none of the healthy family members displayed the mutation (Fig. 2Go). The results are consistent with autosomal dominant inheritance of FNDI in this family.

Diabetes insipidus and growth in children

Subject A-IV-21 was a 2-yr-old boy who presented with failure to thrive. Retrospectively, this subject showed decelerated weight gain as an early sign from the age of 9 months and a retarded length from the age of 1.5 yr (Fig. 4AGo). Closer study of his diet revealed a shortage of calorie intake due to excessive fluid intake. He drank 4 L/day. He was successfully treated with DDAVP. Within 3 months after starting treatment his weight rose dramatically to a normal level. His length curve normalized slowly (Fig. 4AGo). Thus, the growth-related symptoms appear as early, indirect signs of diabetes insipidus.



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  		Figure 4. Effects of diabetes insipidus and treatment on growth in children. A, Growth chart of subject IV-21, identified with FNDI at the age of 2 yr, with growth retardation as an early sign. The weight preceded length retardation. Growth normalized with treatment. B, Growth chart of subject IV-9, identified with FNDI at the age of 7 yr, with growth retardation from the age of 3 yr. Weight gain accelerated with treatment.

 
Subject B-IV-9 was identified with FNDI by mutation screening at the age of 7 yr (Fig. 3Go, lane 7). He had not presented in the clinic because there were no complaints, although he had been drinking progressively more for a few years. At the age of 7 yr he drank 2.5–5 L/day. His growth curve already demonstrated growth retardation from the age of 3 yr (Fig. 4BGo). Treatment of diabetes insipidus with DDAVP normalized polydipsia and improved his appetite and his weight gain (Fig. 4BGo).

The NP85C->G mutation abolishes processing and secretion of the VP prohormone

To prove that the NP85C->G mutation results in a defective VP prohormone and to investigate the dominant nature of FNDI caused by this mutation, we examined intracellular transport and processing of the mutant VP prohormone in cell lines. PC12/PC2 cells, stably transfected with wild-type or NP85C->G mutant VP prohormone gene, were metabolically labeled, and NP-containing proteins were immunoprecipitated and analyzed. PC12/PC2 cells possess a regulated secretory pathway and are able to process the wild-type VP prohormone (11) (Fig. 5Go). Analysis of NP-containing proteins in cells stably expressing the wild-type prohormone demonstrates that most of the prohormone has been processed to the processing intermediate VP-NP or to the final product, NP (Fig. 5Go, lane 3). Of these proteins, NP is selectively retained intracellularly, indicating sorting into and storage in the regulated secretory pathway (Fig. 5Go, lane 4). In contrast, in cells expressing the NP85C->G mutant VP prohormone, the only NP-containing protein present is the intact prohormone (Fig. 5Go, lane 5). No processing could be detected. In addition, whereas part of the wild-type VP prohormone was secreted via the constitutive secretory pathway, this secretion was abolished by the NP85C->G mutation (Fig. 5Go, lanes 4 and 6). Although the partial missorting of regulated secretory proteins to the constitutive pathway is an imperfection of cell lines (11, 18, 40, 41, 42), the absence of secretion in the case of the NP85C->G mutant VP prohormone suggests an intracellular transport defect for this prohormone.



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  		Figure 5. The NP85C->G mutation abolishes processing and secretion of the VP prohormone in PC12/PC2 cells. PC12/PC2 cells, stably transfected with either empty vector or the gene encoding the wild-type or NP85C->G mutant VP prohormone, were metabolically labeled for 18 h. NP-containing proteins were immunoprecipitated from the cell lysate (C) or the medium (M), and aliquots were analyzed by reducing or nonreducing Tricine SDS-PAGE. Molecular weight markers are indicated on the left. The migration positions of the VP prohormone, the VP-NP-processing intermediate, and NP are indicated on the right. The bands marked with asterisks are background bands that were also present in the medium of cells transfected with only the empty vector. Note the absence of any specific bands with a molecular weight higher than the prohormone in lanes 11 and 12, indicating that the NP85C->G mutant prohormone does not form disulfide-linked homo- or heterodimers.

 
Formation of disulfide-linked dimers is not involved in the processing and secretion defect of the NP85C->G VP prohormone

Recently, defective processing and intracellular transport of an FNDI VP prohormone with a mutation in the signal sequence were demonstrated to coincide with the formation of disulfide-linked multimers by this mutant prohormone (24). Because the NP85C->G mutant VP prohormone possesses an uneven number of cysteines and thus at least one unpaired cysteine, we analyzed whether this mutant forms disulfide-linked multimers. Analysis of NP-containing proteins by nonreducing gel electrophoresis gave similar results as that by reducing gel electrophoresis, indicating the absence of disulfide-linked dimers or multimers (Fig. 5Go). The only higher molecular weight bands observed after nonreducing gel electrophoresis were also obtained from the medium of PC12/PC2 cells that had been transfected with the empty vector, indicating that these are background bands.

Comparison of the processing and secretion defect of the NP85C->G VP prohormone and other FNDI VP prohormones

Recently, we demonstrated different FNDI mutant VP prohormones to vary in the degree of processing deficiency in PC12/PC2 cells (11). Whereas no processing was observed for NP14G->R and NP65G->V prohormones, which both have their mutations within the ß-pleated sheet domains of the NP moiety, some processing was observed for the NP47{Delta}E and NP47E->G mutant prohormones, which have their mutations within the {alpha}-helix. The processing defect was the least strong for the NP57G->S mutation, which is located within the loop connecting the {alpha}-helix and the second ß-pleated domain. Thus, the processing defect of the NP85C->G mutant is among the most severe that we have observed to date. To examine whether this secretion and processing defect is as severe in a cell line that expresses higher levels of proprotein convertases (43) and thus processes the VP prohormone more efficiently, we expressed wild-type and mutant prohormones in the cell line AtT-20. Whereas in this cell line the majority of the wild-type VP prohormone had been processed to VP-NP and NP during the metabolic labeling, no processing was observed for the NP85C->G mutant (Fig. 6Go, lanes 3–6). In addition, the NP85C->G mutation abolished secretion of the VP prohormone. Similar results were obtained for the NP14G->R mutant VP prohormone (Fig. 6Go, lanes 7 and 8). The NP47E->G mutant prohormone demonstrated some processing and secretion, albeit at much reduced amounts compared with the wild type. These data indicate that the processing and secretion defect observed for the NP85C->G mutant prohormones is among the most severe observed for FNDI mutant prohormones.



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  		Figure 6. Comparison of the severity of reduction in processing and secretion of the VP prohormone by different diabetes insipidus mutations. AtT-20 cells, stably transfected with empty vector or wild-type, NP85C->G, NP14G->R, or NP47E->G VP prohormones, were analyzed as described in Fig. 5Go. The Tricine SDS-PAGE was nonreducing. The bands marked with asterisks are background bands that were also obtained from cells transfected with only the empty vector. Note that the deficiency of processing and secretion of the NP85C->G VP prohormone is comparable to that of the NP14G->R prohormone.

 
The NP85C->G VP prohormone is retained in the ER

Because other FNDI VP prohormones have been demonstrated to be retained in the ER (11, 18, 24, 42, 44), we examined whether this was also the case for the NP85C->G mutant. NP-containing proteins were immunoprecipitated from the cell lysates of PC12/PC2 transfectants and either mock-treated or treated with endoglycosidase H. Whereas most of the wild-type prohormone was resistant to endoglycosidase H digestion, all of the NP85C->G mutant prohormone was deglycosylated (Fig. 7Go). This indicates that the NP85C->G mutant prohormone still carries the high mannose-type sugar that is present on proteins within the ER. In contrast, most of the wild-type prohormone has left the ER, and its glycan has been converted to a mature type glycan in the Golgi apparatus. These data indicate that the NP85C->G mutant VP prohormone is retained in the ER.



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  		Figure 7. The NP85C->G VP prohormone is retained in the ER. PC12/PC2 cells stably transfected with the wild-type or NP85C->G VP prohormone were metabolically labeled for 24 h. NP-containing proteins were immunoprecipitated from the cell lysates and either mock-treated (-) or treated with endoglycosidase H (+). Immunoprecipitates were analyzed by reducing Tricine SDS-PAGE. Note that all of the NP85C->G VP prohormone was deglycosylated by endoglycosidase H, indicating its retention in the ER. In contrast, only a minor portion of the wild-type VP prohormone was endoglycosidase H sensitive.

 
High expression of the NP85C->G VP prohormone induces large accumulates in the ER and a changed morphology of this organelle

Because the magnocellular neurons that synthesize VP in the supraoptic nucleus and paraventricular nucleus of the hypothalamus express very high amounts of the VP prohormone, we examined the effect of very high level expression of the NP85C->G mutant VP prohormone. We did so by immunocytochemistry of Neuro-2A neuroblastoma cells transiently transfected with plasmids encoding either wild-type or mutant prohormone. The NP-containing proteins in cells transiently expressing wild-type prohormone were homogeneously stained throughout the entire cytoplasm of the cells. In contrast, in approximately 25% of cells transiently expressing the NP85C->G mutant prohormone, staining concentrated at distinct sites within the cytoplasm (Fig. 8AGo). Costaining with an antibody against the ER marker protein-disulfide-isomerase demonstrated that the large sites of accumulation of the NP85C->G prohormone were located within the ER (Fig. 8BGo). In addition, in cells displaying these large accretions of mutant prohormone, a changed morphology of the ER was observed. Whereas the ER marker demonstrated the normal reticulate staining in nontransfected cells, in transfected cells the marker concentrated in the same large accretions as the mutant prohormone (Fig. 8BGo, middle panel). We hypothesize that these large accretions might also form in magnocellular neurons expressing the NP85C->G mutant VP prohormone and that this will result in severe dysfunction of the cells or even cell death due to the strong disturbance of the ER in these cells.



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  		Figure 8. The NP85C->G VP prohormone demonstrates an enhanced tendency to form large accumulates in the ER. A, Neuro-2A cells were transiently transfected with wild-type or NP85C->G VP prohormone and labeled with an antiserum against NP. Note the homogenous staining in cells transfected with the wild-type VP prohormone compared with the formation of large accumulates in cells expressing the NP85C->G VP prohormone. B, Neuro-2A cells, transiently transfected with the NP85C->G VP prohormone gene, were double stained with antiserum against NP (anti-NP, in green) and a monoclonal antibody against the ER marker protein-disulfide-isomerase (anti-PDI, in red). The right panel shows the overlap of both stainings. The top and bottom panels demonstrate different cell groups obtained from the same experiment. Note that the large NP-containing aggregates colocalize with the ER marker, indicating that they are present within the ER. Note in addition the severely changed distribution of the ER marker in cells expressing high levels of the NP85C->G VP prohormone compared with that in nontransfected cells (middle panel).

 
Discussion

In this study we investigated the mutant NP85C->G VP prohormone from individuals of a Dutch kindred with FNDI. We recently identified one branch of this family (17). We now extend the data to three other branches and prove the cell biological deficit of this mutant precursor. The kindred we identified is the largest kindred with FNDI reported to date. Heterozygosity for a mutation in the VP prohormone gene was identified, and was confirmed in all affected individuals by restriction endonuclease digestion.

The mutation cosegregated with clinical symptoms of the disease. Although some family members believed they had no complaints, personal history revealed polydipsia, and in the children signs of growth retardation were found. The mutation affects males and females and was transmitted to approximately 50% of those at risk. These findings are consistent with an autosomal dominant trait.

This family demonstrates a typical clinical presentation of FNDI, with onset of symptoms of polyuria and polydipsia in early infancy. It is well known that the excessive drinking associated with diabetes insipidus can decrease food and thus caloric intake and so influence growth and development (45, 46, 47). However, this effect is most severe when the diabetes insipidus starts at birth and is usually unnoticed in patients with FNDI. Of the four generations of FNDI patients we describe, only one patient presented in the clinic with a failure to thrive. Nonetheless, we demonstrate that a deceleration in growth can be an early sign in childhood of FNDI and that this deceleration can also be measured in a patient in whom growth retardation was not problematic. Symptoms and growth recover during treatment with DDAVP. In the clinical management of children with an inherited FNDI mutation, this deceleration in growth can be employed as an early symptom. It may mark the moment that substitution therapy with DDAVP is required.

To proof that the NP85C->G mutation results in a defective VP prohormone and thus underlies the symptoms of FNDI in this kindred, the cell biological properties of the mutant prohormone were evaluated. Analysis of the intracellular transport of the mutant VP prohormone in cell lines demonstrated that the mutation abolished ER exit and thus processing of the VP prohormone. The ER retention caused by the NP85C->G mutation was almost absolute and stronger than that caused by FNDI mutations in the {alpha}-helix (NP47E->G and NP47{Delta}E) or in the loop connecting the {alpha}-helix with the second ß-pleated sheet domain (NP57G->S) that were examined previously (11). It was comparable in severity with the retention caused by two mutations within a ß-pleated sheet domain (NP14G->R and NP65G->V) (11). This suggests that the folding defect caused by the NP85C->G mutation is more severe than that caused by the NP47E->G, NP47{Delta}E, and NP57G->S mutations.

Although the NP85C->G mutant VP prohormone contains 15 cysteine residues and at least 1 unpaired cysteine, it did not form disulfide-linked homo- or heterodimers. This excludes dimer formation as the cause of ER retention of the NP85C->G VP prohormone. This is remarkable, because formation of disulfide-linked oligomers was proven for and implicated as the cause of ER retention of the SP{Delta}(M1P2D3T4) FNDI VP prohormone (24). In the gene encoding this mutant, the G of the ATG start codon has been deleted. Protein translation now starts at Met5 of the SP moiety, resulting in a VP preprohormone with a truncated signal peptide that is not cleaved. Due to the presence of a Cys residue at position 8 of the SP moiety, the SP{Delta}(M1P2D3T4) VP preprohormone contains an uneven number of cysteine residues, like the NP85C->G VP prohormone we investigated. However, whereas the majority of the SP{Delta}(M1P2D3T4) VP preprohormone was present as disulfide-linked oligomers or aggregates, no disulfide-linked NP85C->G VP prohormone was detected. We conclude the existence of at least two different causes of ER retention of FNDI VP (pre)prohormones with at least one unpaired cysteine residue. Beuret et al. (24) elegantly demonstrated that disulfide-linked aggregate formation by the SP{Delta}(M1P2D3T4) VP preprohormone was probably due to a failure in formation of the disulfide bridge within the VP moiety. NP folding seemed to be intact in this mutant. In our mutant (and in other prohormones with mutations within the NP moiety) it is probable that the folding problem is located within the NP moiety.

The mutant VP prohormone demonstrates an enhanced tendency to form large accumulates within the ER when it is expressed at very high levels. Similar accretions have been observed in vivo in the magnocellular neurons of rat expressing a mutant prohormone resulting from the nonhomologous crossing over of the VP and oxytocin genes (48, 49). They have been demonstrated to consist of accumulations of globular aggregates in dilated saccules of the rough ER (48, 49). A similar change in ER morphology has recently been observed in the magnocellular neurons of transgenic rats expressing a human diabetes insipidus prohormone (50). Because accumulation of mutant VP prohormone causes an aberrant ER morphology (11, 48, 49, 50), it is likely to disturb the function and possibly even the viability of the cell. A disturbed function or viability of magnocellular neurons caused by FNDI mutant VP prohormones would explain the autosomal dominant inheritance of FNDI. In this light, FNDI may even be considered a neurodegenerative disease. Moreover, the delayed onset of the disease at only a few months or years of age could be explained by the time required for the mutant prohormone to accumulate in high enough amounts to change ER morphology and block cell function. Several observations support this possible mechanism for FNDI. First, a strong reduction in the amount of magnocellular neurons in the paraventricular and supraoptic nuclei of the hypothalamus was observed in autopsies of FNDI patients (19, 20, 21, 22). Second, human FNDI VP prohormones were demonstrated to cause cytotoxicity in differentiated Neuro-2A cells and to decrease the secretion of the wild-type VP prohormone (18, 51). Third, cytotoxicity caused by ER accumulation of a mutant protein has been proven for another disease, {alpha}1-antitrypsin deficiency (52, 53). The proposed mechanism for the dominance of FNDI would predict that the only recessive FNDI mutation identified (VP7P->L), would not result in ER retention of the mutant VP prohormone. Instead, the prohormone would be normally transported, and the symptoms in homozygotes would be due to the reduced biological activity of the mutant VP peptide (14). In agreement with this, the mutation changes a residue within VP that is not required for NP binding (25, 26, 27) and, in addition, is oriented toward the solvent in the x-ray crystals of NP II and oxytocin (27), suggesting a minor influence of this residue on folding of the oxytocin and VP prohormones. It would be interesting to determine whether the VP7P->L mutant VP prohormone is indeed transported efficiently out of the ER.

As described above, the severity of ER retention of the mutant VP prohormone varies between different FNDI mutations. However, there is probably no linear correlation between the severity of ER retention and the severity of the disease (i.e. the time of onset of FNDI). The time needed for formation of large accretions of mutant VP prohormone in the ER, resulting in an aberrant ER morphology and proposed dysfunction of the cell, will not only be dependent on the extent of ER retention, but also on the expression level and the extent of intracellular degradation of the mutant prohormone. This also predicts that the onset of the disease will display individual variations. Drinking habits and thus VP prohormone production might vary between individuals. In addition, individual differences in expression levels of proteins involved in degradation of mutant proteins have been observed (52). Indeed, even for a certain FNDI mutation, large differences were found in the onset of the disease (54). This concerns both differences between two kindred expressing the same FNDI mutation and differences between the affected members of one kindred. In the clinical management of persons afflicted with the FNDI mutation it is essential to note early symptoms and to provide adequate substitution therapy. This study shows that observation of growth may provide early parameters.

Acknowledgments

We express our gratitude to Dr. Iain Robinson for providing us with the last batches of the D7 antiserum, to Dr. Sharon Tooze for her kind gift of the PC12/PC2 cells, to Dr. (Ph.D.) Bill North for the Henry-K antiserum, to Dr. Peter van der Sluijs for providing us with the 1D3 monoclonal, and Dr. Fuller for the permission to use it, to Dr. Jim Battey for his kind gift of the cloned human VP prohormone gene, and to Dr. Oscar Schoots for the pRSVhyg vector and AtT-20 cells.

Footnotes

1 This work was supported by Research Grant NWO-MW-903-46-150 from the Council for Medical and Health Research of the Netherlands Organization for Scientific Research (to M.N.) and Research Grant GRN-94002 from the Glaxo Research Foundation Netherlands (to R.Z.). Part of this work was supported by a grant from the Hersenstichting Nederland to this consortium (Grant 8F00.09). Back

2 M.N. and E.L.T.v.d.A. share first authorship. Back

3 Present address: Sophia Children’s Hospital, Department of Pediatric Endocrinology, Rotterdam, The Netherlands. Back

Received November 22, 2000.

Revised February 27, 2001.

Accepted March 19, 2001.

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