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The Journal of Clinical Endocrinology & Metabolism Vol. 84, No. 9 3351-3354
Copyright © 1999 by The Endocrine Society

Original Studies

Molecular Analysis in Familial Neurohypophyseal Diabetes Insipidus: Early Diagnosis of an Asymptomatic Carrier

B. Calvo, J. R. Bilbao, A. Rodríguez, M. D. Rodríguez-Arnao and L. Castaño

Endocrinology and Diabetes Research Group, Department of Endocrinology, Hospital de Cruces, Barakaldo, Basque Country 48903; and the Department of Pediatric Endocrinology, Hospital Gregorio Marañón (A.R., M.D.R.-A.), Madrid 28007, Spain

Address all correspondence and requests for reprints to: Dr. Luis Castaño, Endocrinology and Diabetes Research Unit, Hospital de Cruces, 48903 Barakaldo, Bizkaia, Spain. E-mail: lcastano{at}hcru.osakidetza.net


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Familial neurohypophyseal diabetes insipidus (FNDI) is an inherited deficiency of the hormone arginine vasopressin (AVP) and is transmitted as an autosomal dominant trait. In the present study we have analyzed the AVP-neurophysin II (AVP-NPII) gene in a Spanish kindred. Studies were performed on seven members (four clinically affected) of the family. Patients were diagnosed at the Hospital Universitario Gregorio Marañón (Madrid, Spain). The entire coding region of the AVP-NPII gene of all family members was amplified by PCR and sequenced. All affected individuals presented a missense mutation (G1757->A) that replaces glycine at position 23 with arginine within the NPII domain. The substitution was confirmed by restriction endonuclease analysis and was present in heterozygosis. Additionally, one of the asymptomatic relatives (a girl 8 months old at the time of study) was identified as carrier of the same mutation and developed the disease 3 months later. The alteration found in the second exon of the gene in this family seems to be responsible for the disease, as all individuals harboring the mutation had been previously diagnosed or have eventually developed FNDI. Identification of the molecular defect underlying FNDI in affected families is a powerful tool for early asymptomatic diagnosis in infants.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
FAMILIAL (or autosomal dominant) neurohypophyseal diabetes insipidus (FNDI) is characterized by progressive postnatal deficiency of the peptide hormone arginine vasopressin (AVP). The clinical phenotype of polyuria and polydipsia typically develops at 1–6 yr of age (1, 2, 3, 4).

AVP is encoded by the 2.5-kb AVP-neurophysin II (AVP-NPII) gene in chromosome 20p13 (5, 6) and consists of three exons (7, 8). The gene product is synthesized in the magnocellular neuron cell body as a precursor polypeptide (prepro-AVP-NPII) that undergoes posttranslational processing during axonal transport from the supraoptic and paraventricular nuclei to the posterior pituitary, where it yields its three functional peptides: AVP, NPII, and the C-terminal glycopeptide (copeptin) (9).

After the establishment of the genetic linkage between the AVP-NPII gene and the disease (6) and identification of the first mutation associated in 1991 (10), at least 30 different mutations have been described in families with FNDI. Most of them fall within the coding sequence for NPII, a few of them within the signal peptide in exon 1, and 1 mutation has been found in the nonapeptide AVP itself (11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22).

In the present study we analyzed the AVP-NPII gene in a Spanish pedigree using automated DNA sequencing and identified a single base substitution in exon 2 resulting in a glycine to arginine missense mutation. Molecular analysis performed in all members of the family revealed an asymptomatic carrier of the same mutation, stressing the importance of these studies in families with a background of FNDI for early diagnosis, especially in infants.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Patient 1

An 11-yr-old girl was diagnosed with diabetes insipidus at the Hospital Gregorio Marañón (Madrid, Spain) at 9 months of age after a 3-month history of polyuria and polydipsia. Basal urinary osmolality was 65 mosmol/kg (basal plasma osmolality, 283 mosmol/kg), rising to 108 mosmol/kg after a 3-h water deprivation test with a weight loss of 3%. At this point, plasma osmolality was 309 mosmol/kg.

After intranasal administration of DDAVP (1-desamino-8-D-arginine vasopressin; 10 µg), urinary osmolality rose to 499 mosmol/kg (plasma osmolality, 299 mosmol/kg). The patient’s father and aunt had also been diagnosed with early central diabetes insipidus (data not available).

Patient 2

A 5-yr-old female cousin of patient 1 was diagnosed with the same disorder at age 11 months after a 1-month history of polyuria and polydipsia. After a 6-h water deprivation test with a weight loss of 3.5%, urinary osmolality rose from 192 to only 198 mosmol/kg (plasma osmolality, 295 and 308 mosmol/kg before and after the test, respectively). After intranasal administration of DDAVP (10 µg), urinary osmolality rose to 602 mosmol/kg (plasma osmolality, 296 mosmol/kg). Her mother and uncle (father of patient 1) also suffer from FNDI.

Magnetic resonance imaging showed no T1 hyperintensity in the neurohypophyseal area, indicative of the absence of neurosecretion or hormonal accumulation in the posterior pituitary.

Patient 3

A sister of patient 2 was 8 months old at the time of the molecular analysis and was clinically asymptomatic, with normal diuresis, no polyuria or polydipsia, and normal biochemical parameters. At age 11 months, her parents reported signs of polyuria/polydipsia during the previous week, and biochemical determinations (plasma and urinary osmolality after a 3-h water deprivation test were 278 and 112 mosmol/kg, respectively) confirmed the diagnosis of diabetes insipidus. She is currently receiving treatment with DDAVP.

Peripheral whole blood was extracted from all the members of the family after informed consent.

Amplification of the AVP-NPII gene

Genomic DNA was purified from peripheral whole blood following standard methods (23). Two independent 100-µL PCR reactions and four primers were used to amplify the entire AVP-NPII-coding region as previously described (10). 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 biotinylated 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 microplate format using T7 DNA polymerase and internal Cy5-labeled primers (21). Electrophoresis of the sequencing reactions was carried out in an ALFexpress (Pharmacia Biotech, Uppsala, Sweden) automated fluorescent DNA sequencing apparatus.

Restriction endonuclease analysis

Purified exon 2 amplicons were digested with ApaI according to the manufacturer’s instructions. Restriction fragments were separated by agarose gel electrophoresis and visualized by ethidium bromide staining.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
The coding region of the AVP-NPII gene in all affected individuals as well as in the asymptomatic relatives (as controls) was PCR amplified. Direct sequencing of the PCR products revealed a heterozygous G to A transition in nucleotide 1757 (exon 2), in accordance with an autosomal dominant mode of inheritance. The mutant nucleotide sequence encodes arginine instead of glycine in codon 23 of the NPII carrier protein, and no other alteration was observed in the remaining coding region (Fig. 1Go). The mutation abolishes a restriction site for ApaI, resulting in a different restriction fragment length polymorphism pattern of the mutant allele (Fig. 2Go).



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  		Figure 1. Partial sequence data of exon 2 of the AVP-NPII gene in a normal healthy individual (A) compared to mutated sequence from a patient with FNDI harboring a heterozygous missense mutation (G to A) in codon 23 of the NPII protein, at position 1757 of the AVP-NPII gene, resulting in a Gly->Arg substitution (B). R: G/A heterozygote.

 


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  		Figure 2. Agarose gel electrophoresis of ApaI-digested (d) and undigested (u) amplified exon 2 of AVP-NPII gene, showing the presence of the mutation among FNDI-affected members in the pedigree. Agarose gel electrophoresis of the ApaI-digested 337-bp amplification of exon 2 shows 223- and 114-bp fragments in normal alleles. The G1757A substitution eliminates one of the ApaI restriction sites. All affected individuals (filled symbols) show the two fragments of the normal allele and a 337-bp fragment corresponding to the undigested mutant allele. One of the two sisters (arrow), asymptomatic at time of study, was identified as a carrier of the same mutation and developed the disease 3 months later.

 
The mutation was present in all affected members of the family; additionally, patient 3, identified as carrier of the same mutation, developed the disease 3 months later. These results confirm that the mutation segregates completely with the FNDI phenotype in this family.


   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
AVP is synthesized as a large precursor molecule in magnocellular neurons of the supraoptic and paraventricular nuclei of the hypothalamus. It is subsequently transported through axons to the posterior pituitary and secreted into the circulation (9, 24). The role of AVP is to regulate body water homeostasis, and partial or complete absence of this circulating hormone results in the syndrome of diabetes insipidus (1, 3). The hereditary form of the disease is termed FNDI and is transmitted as an autosomal dominant trait (1, 2, 3, 25, 26).

Affected individuals are normal at birth, but usually develop symptoms at 1–6 yr of age (12), although cases of earlier clinical manifestation have been described (1, 3, 20, 27). It is also known that the disease shows clinical variability both within and between families with regard to the onset and severity of symptoms (11, 27). A recent report (20) described heterogeneity in the age of onset and the severity of the disorder among affected members of two families with the same mutation, suggesting the presence of additional factors that might modulate the rate and extent of progression of the neurodegenerative process.

Over the past years, at least 30 different mutations in the gene encoding the hypothalamic prepro-AVP-NPII precursor protein have been described as a cause of FNDI, and with the exception of a mutation in the AVP nonapeptide (22), they tend to cluster within the coding sequence for the signal peptide of the precursor or in the NPII domain (28). The former seems to affect the correct cleavage by signal peptidases (12, 21, 28, 29, 30, 31, 32), and it has been shown that an alteration at position -1 of the signal peptide in the prepro-AVP-NPII results in an abnormally processed precursor protein that remains associated with the endoplasmic reticulum and is believed to be cytotoxic (31). In a recent report (33), it has been suggested that mutant AVP precursors induce neuronal cell death as a result of their accumulation within the endoplasmic reticulum.

On the other hand, it has been speculated that NPII, the carrier protein for AVP, could be responsible for preventing proteolytic degradation of AVP during transport and storage of the hormone in neurosecretory granules (34). NPII forms dimer or tetramer complexes that can associate to form higher oligomers (16, 35, 36), and in this way, mutations occurring within the NPII-coding sequence will produce conformational changes that may decrease or completely abolish its ability to bind AVP.

In this sense, the G1757A transition described here affects residue 23 of NPII, which lies within the AVP-binding pocket (19, 20, 36), and it has also been suggested that a Gly23 substitution may be related to the early age of onset of the disorder in this family. It is believed that improper binding of AVP to NPII prevents the establishment of Cys-Cys disulfide bridges, which are essential for maintaining adequate protein conformation (19). Consequently, the misfolded protein is inefficiently processed and is retained in the endoplasmic reticulum, where it may bind to chaperone proteins (37, 38) and undergo proteolytic degradation or accumulate as large aggregates, which eventually destroy the cell (18).

Our study confirms the existence of an alteration involved in the misprocessing or misfolding of the precursor and emphasizes the importance of molecular studies in families with a background of diabetes insipidus to identify the molecular defect underlying FNDI for early asymptomatic diagnosis in infants, as some mutations can cause the disease even before 1 yr of age.

Received January 14, 1999.

Revised June 1, 1999.

Accepted June 6, 1999.


   References
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 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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