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Identification of a Novel Nonsense Mutation and a Missense Substitution in the Vasopressin-Neurophysin II Gene in Two Spanish Kindreds with Familial Neurohypophyseal Diabetes Insipidus

Endocrinology and Diabetes Research Group, Department of Endocrinology, Hospital de Cruces, Barakaldo-Basque Country E-48903; and the Department of Pediatrics, Hospital del Bidasoa (J.E.), Irun-Basque Country E-20280, Spain

Address all correspondence and requests for reprints to: Dr. Luis Castaño, Pediatric Endocrinology, Endocrinology and Diabetes Research Unit, Hospital de Cruces, Barakaldo-Bizkaia, E-48903 Spain.

	Abstract

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Familial neurohypophyseal diabetes insipidus (FNDI) is an autosomal dominant disease caused by deficiency in the antidiuretic hormone arginine vasopressin (AVP) encoded by the AVP-neurophysin II (AVP-NPII) gene on chromosome 20p13. In this study, we analyzed two families with FNDI using direct automated fluorescent, solid phase, single-stranded DNA sequencing of PCR-amplified AVP-NPII DNA. In one of the families, affected individuals presented a novel nonsense mutation in exon 3 of the gene, consisting in a G to T transition at nucleotide 2101, which produces a stop signal in codon 82 (Glu) of NPII. The premature termination eliminates part of the C-terminal domain of NPII, including a cysteine residue in position 85, which could be involved in the correct folding of the prohormone. In the second family, a G279A substitution at position -1 of the signal peptide was observed in all affected individuals. This missense mutation, which replaces Ala with Thr, is frequent among FNDI patients and is thought to reduce the efficiency of cleavage by signal peptidases.

	Introduction

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CENTRAL diabetes insipidus is a polyuric syndrome caused by insufficient circulating active arginine vasopressin (AVP), the antidiuretic hormone involved in the conservation of water through urine concentration (1, 2). The hereditary form of this disorder, termed familial neurohypophyseal diabetes insipidus (FNDI), is transmitted as an autosomal dominant trait (1, 2, 3, 4, 5, 6).

The AVP-neurophysin II gene (AVP-NPII) has been assigned to chromosome 20p13 (7, 8) and consists of three exons (9, 10). The gene product is synthesized as a precursor polypeptide (prepro-AVP-NPII), which undergoes posttranslational processing to yield its three functional peptides: AVP, NPII, and the C-terminal glycopeptide (copeptin) (11).

To date, at least 21 different mutations associated with FNDI have been located in the AVP-NPII gene (12, 13, 14). The heterogeneity in type and location of the mutations contrasts with the minimal variation in the clinical phenotype of patients. However, it has been postulated that mutations tend to cluster in discrete areas of the gene coding for nonhydrophobic regions of the signal peptide or NPII (12).

In the present study, we analyzed the AVP-NPII gene in two independent FNDI pedigrees using automated fluorescent, solid phase, single-stranded DNA sequencing and identified a novel nonsense mutation in exon 3 in one of the families. In a second pedigree, we found a missense substitution (alanine to threonine) in the last amino acid of the signal peptide, the most common mutation associated with FNDI reported to date.

	Subjects and Methods

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Studies were performed on nine (family A) and six (family B) members of two Spanish kindreds in which FNDI is segregating. In pedigree A, a 3-yr-old boy was diagnosed as FNDI based on clinical features of polyuria (>5 L/day) and polydipsia. Basal urinary osmolality was 83 mosmol/kg, rising to 341 mosmol/kg after a 10-h dehydration test; at this point, plasma osmolality was 318 mosmol/kg. After intranasal administration of DDAVP (10 µg), urinary osmolality rose to 818 mosmol/kg. The proband’s mother and an aunt were diagnosed as having FNDI at 27 and 34 yr of age, respectively, and are currently controlled with intranasal desmopressin.

The proband of family B was a 15-yr-old boy with polyuria (up to 20 L/day) and polydipsia since approximately 3–4 months of age, and enuresis until 13 yr of age. Basal urinary osmolality was 74 mosmol/kg and rose to 147 mosmol/kg after a 7-h dehydration test, with a weight loss of 3.4% and a coincidental plasma osmolality of 299 mosmol/kg. Intravenous DDAVP (1-desamino-8-D-arginine vasopressin; 5 U) was able to rise urinary osmolality to 321 mosmol/kg. The proband’s mother, his grandmother, and one brother had a history of FNDI (data not available).

Molecular studies

Amplification of the AVP-NPII gene. Genomic DNA was extracted from peripheral whole blood following standard methods (15). Two independent 100-µL PCR reactions and four primers (A, B, C, and D) were used to amplify the entire AVP-NPII-coding region (16). Thermocycling profiles were 30 cycles of 95 C for 1 min and 72 C for 2 min (exon 1) (17) or 3 min (exons 2 and 3). For subsequent purification of single-stranded DNA for solid phase sequencing, one of the amplification primers in each reaction (corresponding to the strand to be sequenced) was bio-tinylated in its 5'-end.

Solid-phase sequencing. Biotinylated strands were purified using streptavidin-coated Dynabeads M280 magnetic particles (Dynal, Oslo, Norway), according to the manufacturer’s instructions. Sequencing was performed in a microplate format using T7 DNA polymerase and internal Cy5-labeled primers: E and H (16) and the antisense primer K [5'-CGCAGGCCCGCGTCCCCCCCACCCAAGCGT-3', corresponding to nucleotides 1984–1955 (intron 2) numbered according to the published AVP-NPII sequence (10)]. Electrophoresis of the sequencing reactions was carried out in an ALFexpress (Pharmacia, Uppsala-Sweden) automated fluorescent DNA sequencing apparatus.

Restriction endonuclease analysis. Purified amplification products were digested with either MvnI (exon 1) or MaeI (exons 2 and 3), according to the manufacturer’s instructions. Restriction fragments were separated by agarose gel electrophoresis and were visualized by ethidium bromide staining.

	Results

Top Abstract Introduction Subjects and Methods Results Discussion References

 
In pedigree A, sequencing of the three exons revealed the presence of a heterozygous G to T transition in nucleotide 2101 (exon 3) in the affected patient (Fig. 1). No other alteration was observed in the remaining coding region. This G2101T mutation produces a stop signal in codon 82 (Glu) and thus a truncated NPII peptide. The mutation also creates a restriction site for endonuclease MaeI, which is useful to verify the sequencing data and to check for the presence of the same mutation in affected and asymptomatic members of the family (Fig. 2).

View larger version (33K): [in this window] [in a new window]   Figure 1. Partial sequence data of exon 3 of the AVP-NPII gene in A) a patient with FNDI from pedigree A, harboring a heterozygous nonsense mutation (G to T) in codon 82 of the NPII protein, resulting in a stop codon, compared to B) normal sequence from a healthy individual. K, G/T heterozygote.

View larger version (33K): [in this window] [in a new window]   Figure 2. Agarose gel electrophoresis of MaeI-digested (d) and undigested (u) amplified exons 2 and 3 of the AVP-NPII gene, showing the presence of the mutation among FNDI-affected members in pedigree A. The G2101T transition in the mutant allele introduces a MaeI site in exon 3 of the gene yielding 453- and 279-bp fragments. All affected individuals (filled symbols) show the two fragments of the mutant allele and the 732-bp fragment corresponding to the normal allele.

View larger version (44K): [in this window] [in a new window]   Figure 3. Restriction fragment analysis of pedigree B members. Agarose gel electrophoresis of MvnI digestion of the 345-bp amplification of exon 1 results in 164-, 103-, and 78-bp fragments in normal alleles. The G279A substitution eliminates one of the MvnI restriction sites, and digestion of the mutant alleles produces only two fragments of 267 and 78 bp. Four affected subjects of this family (filled symbols) have both the mutant and normal alleles, whereas unaffected individuals lack the 267-bp fragment. u, Undigested; d, MvnI digested.

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

The mutation present in family A creates a premature stop signal in codon 82 of neurophysin, eliminating the cysteine residue in position 85 that is involved in the formation of disulfide bridges in the molecule. This novel mutation adds genetic evidence to support the misfolding/toxicity hypothesis, because, like other mutations described previously in FNDI patients, it could affect the correct folding of the prohormone (12, 18). This G2101T transition also causes loss of the carboxyl-terminal copeptin in the mutated allele. Nevertheless, the importance of this glycopeptide has not been defined, and no mutation in this region has been found in patients with FNDI. In addition, oxytocin neurophysin is produced and processed efficiently by neurosecretory neurons even though this prohormone normally lacks the copeptin moiety.

On the other hand, the mutation in family B affects the last amino acid of the signal peptide (Ala^-1Thr) and is one of the most common substitutions reported in FNDI (6, 12, 22, 23, 24). Amino acids at position -1 are essential for correct cleavage by signal peptidases, and alanine is, by far, the most preferred residue at this site; nevertheless, although threonine has similar physical characteristics, it does not compete well with Ala for that position and has been shown to impair correct cleavage of the signal peptide (25, 26, 27).

Although it has been shown that mutations in the region coding for NPII are responsible for FNDI, the molecular and cellular pathways involved in the development of the disease are not completely clear. Little is known about the role of the C-terminus glycoprotein encoded by the AVP-NPII gene, and further studies are necessary to clarify the maturation processes involved in functional AVP production and to understand the mechanisms by which a varying array of genetic mutations can induce a constant and dominant pathological phenotype.

	Acknowledgments

 
We would like to express our gratitude to Dr. M. Oyarzabal for her collaboration and thoughtful scientific advice, and to Dr. A. Rodriguez and M. A. Antón for providing affected family samples for this project.

Received March 20, 1997.

Revised July 22, 1997.

Revised November 17, 1997.

Accepted December 4, 1997.

	References

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