Introduction

Autosomal dominant familial neurohypophyseal diabetes insipidus (adFNDI) is a rare inherited disease characterized by polydipsia, polyuria, and dehydration caused by deficient secretion of the peptide hormone, arginine vasopressin (AVP) [1]. AVP and its corresponding carrier neurophysin II (NPII), are synthesized as a composite precursor by the magnocellular neurons of the supraoptic and paraventricular nuclei of the hypothalamus. The precursor is packaged into neurosecretory granules and transported axonally in the stalk of the posterior pituitary, En route to the neurohypophysis, the precursor is processed into the active hormone. Prepro-vasopressin has 164 amino acids and is encoded by the 2.5 kb prepro-AVP–NPII gene located in chromosome region 20p13. The AVP–NPII gene consists of three exons and two introns. Exon 1 of the AVP gene encodes the signal peptide AVP and the NH2-terminal region of NPII. Exon 2 encodes the central region of NPII, and exon 3 encodes the COOH–terminal region of NPII and the glycopeptide [2, 3].

Disease onset is typically in infancy or adolescence in the affected individuals, with symptoms worsening throughout adulthood. To date, there have been more than 50 different mutations in the AVP–NPII gene encoding the AVP preprohormone [4]. The mutations occur in different types and are located at different sites in the coding region, and affect amino acid residues that are essential for proper folding and dimerization of the neurophysin II moiety of the AVP preprohormone [5, 6]. This study analyzes a Chinese family with adFNDI associated with mutations in the AVP–NPII gene. We also report a novel mutation (c.193T>A) and a synonymous mutation (c.192C>A) identified in the AVP–NPII gene. The novel missense mutation resulted in the substitution of cysteine by serine at a highly conserved codon 65 of exon 2 of the AVP–NPII gene in all the affected individuals.

Subjects and methods

Subjects

After obtaining written informed consent, we studied only six living members of the Chinese pedigree of Han family presenting with adFNDI, as other members of the pedigree refused to participate. II.3 and II.4 had a chronic history of polyuria and polydipsia with onset of symptoms including edema of lower extremity, at about the ages 38 and 40 years, respectively. Subsequently, they were diagnosed with backache at a local hospital. The findings from color Doppler sonography revealed hydronephrosis and enlarged urinary tract, for which they consulted with an urologist at our hospital initially. The family member II.9 showed milder signs, while the other living members including III.8, III.12, IV.18 were unaffected (Fig. 1).

Fig. 1
figure 1

Pedigree of the kindred. The individuals numbered are those who were available for mutation screening of the AVP–NPII gene. Black and white symbols represent clinically affected and unaffected individuals, respectively. Genetically tested individuals are indicated by the “+” symbol. The arrow to II.3 is propositus and indicates the carrier of the 1619T>A mutation

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Methods

Clinical data and sample collection

All participants underwent full physical examination and fluid deprivation test plus desmopressin (using DDAVP to terminate the fluid restriction period used for differentiating central from nephrogenic diabetes insipidus). Urine was measured for specific gravity. All the samples of urine and serum were analyzed at the second affiliated hospital of Harbin Medical University (Harbin, China). Samples of blood were shipped to Department of Genetics, National Research Institute for Family Planning for DNA analysis (Beijing, China).

Molecular genetic procedures

Venous blood samples were obtained from six members of the pedigree and 100 normal controls. Genomic DNA was extracted from whole blood using a QIAamp DNA Blood Mini Kits (QIAGEN Science, Germantown, MD). All coding exons of AVP–NPII genes were amplified by polymerase chain reaction (PCR) with primers listed in Table 1. The PCR products were then sequenced from both directions with the ABI3730 Automated Sequence (PE Biosystems, Foster City, CA).The sequencing results were analyzed using Chromas (version 2.3) and compared with the reference sequences in the NCBI database.

Table 1 Primers used for DNA amplification by PCR
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Results

Clinical data

Analysis of the water deprivation test results was performed in the index patient, according to the standard procedures, along with the DDAVP test (Table 2). Patients voided the bladder before the administration and urinary flow was determined hourly. The water deprivation test demonstrated the urine concentration deficiencies in the affected individuals of the Chinese family: the urine specific gravity sharply increased in the affected individuals after DDAVP administration (AVP, 5 IU i.m.). The data supported the diagnosis of diabetes insipidus (DI), differentiating central from nephrogenic types [7]. However, in all the investigations, no abnormalities were seen in the three unaffected individuals of the family. Overall, the clinical findings were consistent with the diagnosis of central AVP-deficient DI. The patients were treated by DDAVP (II.3 and II.4: Minrin 0.1 mg × 4 t.i.d, p.o.; III.9: 0.1 mg × 2 t.i.d, p.o.). Although some family members refused to undergo the various tests details of family history obtained from the participants indicated seven living family members were suspected to have symptomatic vasopressin-deficient DI to varying degree. Although not tested, I.1 might be the first affected family member, and thus expected to have milder signs.

Table 2 Water deprivation and vasopressin tests in the index case
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Molecular genetic analysis

Direct sequencing of AVP–NPII gene revealed a heterozygous T>A transition at position c.193, which resulted in the substitution of cysteine by serine at the highly conserved codon 65 of exon 2, and a synonymous mutation c.192C>A at codon 64 of exon 2, changing GGC to GGA corresponding to Gly codon (Fig. 2). Meanwhile, the mutation was confirmed by restriction endonuclease analysis of PCR amplification products that contain the corresponding segment of the AVP–NPII gene. The mutation was a thymidine base substituted by adenine at nucleotide c.193(T>A) in exon 2, which altered codon 65 of the prepro-AVP–NPII sequence where the NPII moiety was derived from TGC to an AGC mutation. No other mutations were detected in exons 1, 2 or 3. The missense mutation and the synonymous mutation were found to be similar in all the affected individuals and co-segregated with the affected individuals in the family but absent in the unaffected family members as confirmed by the sequencing of 100 control chromosomes.

Fig. 2
figure 2

Automated sequencing of the AVP–NPII gene. A portion of exon 2: the two superimposed chromatographic peaks demonstrate the heterozygous mutation c.193(T>A) and nonsense mutation c.192(C>A) found in the Chinese family. The arrow represents the novel mutation identified

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Discussion

The mutations included intragenic deletions, missense and nonsense mutations affecting the signal peptide, the AVP moiety, or the AVP carrier protein (NPII). Over 50 distinct AVP–NPII gene mutations have been reported in adFNDI. However, only five nonsense mutations were reported, with almost a quarter affecting one of the seven disulfide bonds present in NPII that are important in determining its tertiary structure [5, 8, 9]. If only the mutations affecting the NPII moiety were considered, the proportion of the modified cysteine residues, forming disulfide bonds, rises to nearly 50%. Therefore, the mutations affecting the NPII play an important role in the adFNDI pathophysiology. The study demonstrated the clinical symptoms in this kindred, with a co-segregation of the novel mutation (c.193T>A) in the AVP–NPII gene. The heterozygous c.193T>A mutation identified in the neurophysin moiety corresponds with the different patterns of the other known mutations associated with initial mild symptoms worsening in severity with AVP deficiency. The novel mutation identified in the Chinese kindred resulting from the substitution of cysteine by serine at the highly conserved codon 65 of exon 2 of the AVP–NPII gene was seen in all the affected individuals. Only the affected individuals carrying a mutation showed the symptoms of the adFNDI. Therefore, c.193(T>A) is most likely a disease-causing mutation.

The pathogenesis of adFNDI is generally linked with deficient or insufficient function of mutant NPII molecules. NPII is responsible for safe axonal transport and prevention of intracellular AVP proteolysis. Mutant AVP prohormone is degraded by the cytosolic proteolytic system. The mutant precursor hormones are retained by the endoplasmic reticulum quality control mechanisms resulting in cytotoxic accumulation and protein aggregation in the neurons, or degeneration of magnocellular neurons of the supraoptic and paraventricular nuclei of the hypothalamus [2, 10–17]. The AVP peptide binds in a “pocket” formed by NPII, and thereby shielded from proteolytic degradation during the axonal transport of the secretory granule to the neurohypophysis where AVP is released, as needed [2, 18]. Therefore, heterozygous T>A transition at highly conserved codon 65 of exon 2, resulting in the substitution of cysteine by serine could disorder the NPII protein. The mutant NPII with a deficient or insufficient function interferes with the safe axonal transport and prevention of intracellular AVP proteolysis.

NPII is a cysteine-rich protein that contains seven disulfide bonds between its 14 cysteine residues. The disulfide bonds occur between codons 41–85, 44–58, 52–75, 59–65, 92–104, 98–116, and 105–110 of the prepro-AVP–NPII sequence. Mutations involving cysteine residues, which form disulfide bonds in the NPII protein, have been described: one in the fourth, three in the fifth, and four in the sixth, and three in the seventh [17, 19]. In the three-dimensional structure of neurophysin, the mutation (c.193T>A) disrupted the normal disulfide bond linking C59 with C65. Until now, Cys65Phe and other mutations involving cysteine residues in four of the seven disulfide bridges have reportedly disrupted the disulfide bonds during protein folding in the endoplasmic reticulum, leading to destabilization of protein structure and function in NPII [19, 20]. The novel mutation involving the disruption of the fourth disulfide bond in the AVP–NPII precursor protein will enable us to further investigate the pathogenesis of adFNDI, at the molecular level.

Mutations in the NPII-coding region are apparently linked with an early disease onset. Interestingly, fewer cases live to a normal age as reported here, beyond the first few decades, possibly due to adequate AVP to maintain the urine volume. A few researchers report that the pathogenesis may be associated with abnormal neuronal secretion due to improper AVP protein folding or synthesis compared with individuals possessing the normal alleles. Furthermore, the cadaver studies of ADNDI patients show that the neurocytes in the patients’ hypothalamus for AVP have selective deletion. Other autopsy studies demonstrated that although the structure of hypothalamus and the staining of supraoptic nucleus are normal, the staining of the paraventricular nuclei is abnormal [6, 10, 21–24].

In conclusion, a novel missense mutation identified in the AVP–NPII gene resulted in neurohypophyseal DI due to disruption of the fourth disulfide bond in the AVP–NPII precursor protein required for correct folding of the neurophysin moiety; a synonymous mutation (c.192C>A) was identified in the AVP–NPII gene in this Chinese family. Although the findings do not directly explain the underlying pathogenesis of the adFNDI, this mutation increases the number of genetic abnormalities in the AVP–NPII gene and provides a molecular basis for understanding the characteristics of NPII that are associated with abnormal protein translation.