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Identification of Mutations of the Arginine Vasopressin-Neurophysin II Gene in Two Kindreds with Familial Central Diabetes Insipidus

Klinik II und Poliklinik für Innere Medizin der Universität zu Köln, Cologne, Germany

Address all correspondence and requests for reprints to: Dirk Müller-Wieland, M.D., Klinik II und Poliklinik für Innere Medizin der Universität zu Köln, 50924 Koln, Germany.

	Abstract

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Familial central diabetes insipidus is transmitted as an autosomal dominant trait with almost complete penetrance. Twenty-three different mutations of the arginine vasopressin-neurophysin II gene have been reported to date, located within the signal peptide-, the arginine vasopressin-, or the neurophysin II-coding region. In the present study two kindreds with familial central diabetes insipidus were examined. The entire coding region of the arginine vasopressin-neurophysin II gene of one affected subject of each family was amplified by PCR and subcloned into a pUC 18 plasmid, and six positive clones were sequenced. After identification of the mutation, direct sequencing was performed on the respective sequence of family members and 28 healthy control subjects. In family A, a missense mutation (CT) at nucleotide position 280 was detected, predicting the substitution of alanine by valine at position -1 of the signal peptide. All affected subjects were heterozygote for the mutation, whereas none of the unaffected family members or control subjects displayed the mutant sequence. In family B, a missense mutation within the neurophysin II-coding sequence was identified (nucleotide 1757, GC), predicting the substitution of glycine by arginine at position 23. Again, affected family members were found to be heterozygote for the mutation, which was not observed in unaffected family members or in control subjects. Although the mutation of family A was recently described in 3 other kindreds as well, the mutation within the neurophysin II-coding region represents a novel mutation of the AVP-NP II gene.

	Introduction

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FAMILIAL central diabetes insipidus (FDI) is transmitted as an autosomal dominant trait (1). The disease is characterized by a deficiency of arginine vasopressin (AVP) (1, 2, 3). The gene encoding the precursor hormone, prepro-AVP-neurophysin II (NP II) is located on chromosome 20p13 (4). It consists of three exons encoding a signal peptide, AVP, NP II, and a glycopeptide of unknown function (5). FDI is associated with various mutations within the coding region of signal peptide, AVP, or NP II (see Ref. 6 for a recent summary of reported mutations and Refs. 7–12 for subsequent reports). All affected subjects reported to date exhibited heterozygote missense-, nonsense-, or deletion-type mutations. We studied two German families with FDI and identified two mutations of the AVP-NP II gene, one representing a novel mutation.

	Subjects and Methods

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Subjects

Two families (A and B) with FDI were studied. The pedigrees of the families are shown in Fig. 1. Both pedigrees show an autosomal dominant inheritance pattern of clinical overt diabetes insipidus, with complete relief of symptoms by treatment with dDAVP. In family A, the parents of the affected subjects II.2 and II.3 reported that symptoms developed within the first year of life. Both subjects II.2 and II.3 of family B are treated with a nasal formulation of 10 µg dDAVP twice daily, under which fluid intake ranges between 1.5–2.5 L. Without dDAVP, fluid intake ranges between 5–7 L. The father, subject I.1, who is now 85 yr old, reported that until 3–4 yr ago he required 10 µg dDAVP twice daily to avoid polydipsia. Three to 4 yr ago he was able to decrease the treatment dose to the single administration of 10 µg in the evening without experiencing increasing polydipsia or polyuria.

View larger version (10K): [in this window] [in a new window]   Figure 1. Pedigrees of the families studied. Affected subjects are shown as solid symbols. Arrows indicate the family members studied. Asterisks refer to subjects for whom the entire coding region of the AVP NP-II gene was amplified, subcloned, and sequenced.

Mutation analysis was performed on 7 members from three generations (family A) and on 7 members from 2 generations (family B). Two healthy spouses, 1 in each family, were studied as well as 28 healthy control subjects. Informed consent was obtained from every subject who participated in the study, and the study was approved according to the guidelines of the University Hospital of Cologne.

Methods

Genomic DNA was extracted from whole blood using the Qiagen kit (Hilden, Germany). Oligonucleotides A, B, C, and D were synthesized according to the method of Ito et al. (13). Oligonucleotides E (5'-AGCAGTGCTGCATACGGGGTCCACCTGTGT-3') and F (5'-ACTCCCGGCTCCCCTCCTCCCGCTCACCCC-3') correspond to nucleotide positions 187–216 and 1674–1703, respectively. Nucleotide and amino acid numbering are according to Sausville et al. (5). To construct oligonucleotides, a DNA synthesizer (Gene Assembler Plus, Pharmacia, Freiburg, Germany) was used. Primers A and B served to amplify exon 1; primers C and D were used for exons 2/3. Synthesized primers were purified by electrophoresis over a denaturing polyacrylamide gel followed by electroelution (Biotrap BT 1000, Schleicher and Schuell, Dassel, Germany).

PCR. Exons 1 and 2/3 were amplified by PCR in a 100-µL reaction volume. PCR was performed according to the method of Saiki et al. (14) with minor modifications. In detail, 50 pmol of each primer, 2.5 mmol/L of each deoxynucleotide triphosphate, 0.7 mmol/L MgCl₂, 1 µg purified DNA, and 1 µL of an enzyme mixture containing Taq DNA polymerase and Pwo polymerase (Expand PCR System, Boehringer, Mannheim, Germany) were used. As the AVP-NP II gene exhibits a high GC content, PCR was performed with dimethylsulfoxide at a final concentration of 10%.

Samples were subjected to 32 cycles, consisting of 94 C for 1 min, 60 C for 2 min, and 72 C for 3 min followed by 7 min at 72 C in a MiniCycler (MJ Research, Watertown, MA).

Subcloning of PCR products and sequencing procedure. PCR products of exons 1 and 2/3 from one subject of each family (Fig. 1) were extracted from 1.5% agarose gel and subcloned into a pUC 18 plasmid (Pharmacia). Six positive clones from each subject were sequenced using T7 DNA polymerase (Pharmacia) according to the dideoxy chain termination method described by Sanger et al. (15).

Direct sequencing and restriction analysis. For direct sequencing, 300 ng of previously chloroform-extracted PCR products were used. Three picomoles of the nested primers E or F were 5'-labeled with [-³²P]ATP. Direct sequencing was performed as recommended by the manufacturers (fmol DNA Sequencing System, Promega, Madison, WI), adding 10% dimethylsulfoxide. Amplification was carried out with 30 s at 95 C and 1 min at 70 C for 30 cycles. Restriction digest was performed in a 25-µL volume containing 500 ng PCR-amplified DNA of the appropriate exon and 20 U BstUI (family A) or Bsp120I (family B). Enzymes were purchased from New England Biolabs (Schwalbach, Germany).

	Results

Top Abstract Introduction Subjects and Methods Results Discussion References

 
The coding region of the AVP-NP II gene of one affected subject from each family (Fig. 1) was amplified and subcloned, and six positive clones were sequenced. PCR amplification of exons 1 and 2/3 resulted in single bands of the expected sizes on 1.5% agarose gel (data not shown). Subcloned fragments of exons 1 and 2/3 were sequenced, including 10 bp of the adjacent intronic sequence.

Family A

A missense mutation was detected in exon 1 at nucleotide position 280 (CT) in two of six sequenced fragments (data not shown). The mutant nucleotide sequence encodes a valine instead of an alanine at amino acid position -1 of the signal peptide. No further mutations were detected in exon 1, 2, or 3. Direct sequencing of affected and healthy family members and of the control subjects was performed using oligonucleotide E, located 94 bp upstream of the detected missense mutation. At position 280, all of the family members with FDI displayed a signal in both C and T lanes that was not found in unaffected family members or in the control subjects (Fig. 2a). The mutant sequence abolishes a restriction site for BstUI. Accordingly, the restriction pattern of the PCR-amplified mutant DNA shows a different restriction pattern, with an additional 266-bp fragment derived from the mutant allele (Fig. 2b).

View larger version (33K): [in this window] [in a new window]   Figure 2. a, Direct sequencing showing a heterozygous missense mutation (CT) at nucleotide position 280. b, The mutation destroys a BstUI site. Restriction analysis shows an additional band of 266 bp derived from the mutant allele compared to the restriction pattern in a healthy control subject. Lanes 1 show the unrestricted PCR-amplified fragment; lanes 2 show the BstUI-restricted fragment of a healthy control subject (normal) and the affected family member II.3 (mutant).

A single base exchange at position 1757 (GC) was detected in three of six sequenced clones. The mutation predicts an exchange of glycine to arginine at amino acid position 23 of the NP II peptide. No further mutations within the coding region or adjacent splice junctions were identified.

Direct sequencing with primer F located at a distance of 84 bp from the mutation confirmed heterozygosity for the mutation. At this location, a signal was found in both G and C lanes (Fig. 3a). PCR-amplified fragments of all remaining family members and control subjects were subjected to direct sequencing. All affected subjects were heterozygote for the mutation, whereas none of the healthy family members or control subjects displayed the mutant nucleotide sequence. Independent confirmation of the sequence change was obtained by restriction analysis. The mutation in family B destroys a Bsp120I site (Fig. 3b).

View larger version (38K): [in this window] [in a new window]   Figure 3. a, Direct sequencing analysis of the heterozygous missense mutation (GC) at nucleotide position 1757 identified in family B. b, The mutation destroys a Bsp120I site; thereby, a new restriction fragment is generated (362 bp). Although the new fragment cannot be readily distinguished from the 350-bp band, the higher intensity of the 350/362-bp band as well as the fainter 247-bp band in the mutant DNA derived from subject III.4 confirm the heterozygous mutation. Lanes 1, Unrestricted PCR-amplified fragment; lanes 2, Bsp120I restriction.

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

In the other kindred, the heterozygous missense mutation was located within the NP II-coding region, predicting the substitution of nonpolar glycine by basic arginine at amino acid position 23 of the NP II moiety. This mutation is a novel mutation of the AVP-NP II gene. The substituted amino acid lies within the AVP-binding pocket (18).

AVP is synthesized as the precursor hormone prepro-AVP-NP II in the magnocellular neurons of the paraventricular and supraoptic nuclei of the hypothalamus. Mutations within the signal peptide-coding region of the AVP-NP II gene affect processing of the prepro-hormone through inefficient or abolished cleavage by a signal peptidase (16).

The NP II monomers self associate and form dimers or oligomers (18). Mutations located within the NP II-coding region may impair correct folding of the NP II protein and its ability to self associate (6, 18). It has been suggested that expression of the disease is due to a cytotoxic effect of the mutant protein (6, 16, 19), possibly due to inefficient folding of the precursor protein (6). Affected subjects retain one intact allele of the gene, and expression of the phenotype is thus due to a detrimental effect of the mutant protein on the cells rather than to diminished AVP synthesis alone. In vitro, missense- and nonsense-type mutations of different domains of the AVP-NP II gene lead to the accumulation of the mutant precursor protein in the endoplasmatic reticulum accompanied by decreased cell viability (19). In humans, a comparable mechanism is likely to occur, considering the dominant expression of the phenotype, the later age of onset occurring months to years after birth (2, 5, 8, 12, 17, 22, 23, 24), and the finding that patients with FDI show degenerative changes in hypothalamic magnocellular neurons (20, 21).

The two families studied reported a uniform clinical picture with regard to the age of onset of symptoms. Symptoms of AVP deficiency occurred within the first year of life in family A and between the sixth and ninth months of life in family B. Although in the majority of kindreds with FDI, offsprings do not present symptoms before the age of 1 yr (5, 8, 12, 17, 22, 23), cases of early clinical manifestation have been described previously (2, 22, 24). However, the age of onset of symptoms and the severity of the disease appear to depend not solely on the genotype. Repaske et al. (12) recently reported considerable individual variation in the age at which symptoms were first noted within one kindred harboring the same missense mutation that we identified in family A. Subject I.1 of family A reported a decrease in symptoms at the age of approximately 81 yr and was able to reduce the dDAVP dose from 20 to 10 µg/day. A decrease in symptoms occurring at an advanced age is a well known phenomenon in patients with FDI (2, 9, 17) and is possibly due to an age-related decrease in the glomerular filtration rate (25).

In summary, we identified two missense mutations in two kindreds with FDI, one of them representing a novel mutation of the AVP-NP II gene.

	Footnotes

 
¹ Present address: National Institute of Diabetes, Metabolism, and Digestive and Kidney Diseases, National Institutes of Health, Building 10, Room 9C101, Bethesda, Maryland 20892.

Received December 2, 1996.

Revised June 27, 1997.

Revised October 17, 1997.

Accepted October 22, 1997.

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Top Abstract Introduction Subjects and Methods Results Discussion References

 

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This Article Abstract Full Text (PDF) Submit a related Letter to the Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Heppner, C. Articles by Müller-Wieland, D. Search for Related Content PubMed PubMed Citation Articles by Heppner, C. Articles by Müller-Wieland, D. Pubmed/NCBI databases Gene GEO Profiles HomoloGene OMIM UniGene Compound via MeSH Substance via MeSH Medline Plus Health Information Diabetes Insipidus Diabetes Medicines