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Clinical Studies |
Val in the Signal Peptide of the Arginine Vasopressin/Neurophysin II/Copeptin Precursor1
Division of Endocrinology, Children’s Hospital Medical Center (D.R.R., E.K.G), Cincinnati, Ohio 45229-3039; the Department of Endocrinology, Hotel-Dieu de France (R.M., G.H.), Beirut, Lebanon; Department of Pediatrics, Vanderbilt University School of Medicine (M.R.S.K., J.A.P), Nashville, Tennessee 37232-2578; and the Division of Endocrinology, St. Luke’s Medical Center (J.W.F.), Milwaukee, Wisconsin 53215
Address all correspondence and requests for reprints to: David Repaske, Ph.D., M.D., NWM-1 TCHRF, Children’s Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, Ohio 45229-3039. E-mail: repaskdr{at}ucunix.san.uc.edu
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Abstract |
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 Top Abstract IntroductionSubjects and MethodsResultsDiscussionReferences |
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T) in the coding sequence for
the signal peptide of the prepro-AVP-NPII precursor in both families.
This mutation encodes an AlaVal substitution at the C-terminus of
the signal peptide (-1 amino acid). This mutation predicts the
complete inability of signal peptidase to cleave the signal peptide
from the preproprecursor and supports the hypothesis that the
progressive neural degeneration that underlies ADNDI is caused by
accumulation of malprocessed precursor. However, considerable
heterogeneity in the age of onset (1–28 yr of age) and the severity of
diabetes insipidus among affected members of these two families
suggests that additional factors modulate the rate and extent of
progression of the neurodegeneration that results from this one
specific ADNDI mutation. |
Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Genetic linkage was established between ADNDI and the AVP-NPII gene (19), and subsequently, 22 different mutations in this gene have been shown to cause ADNDI (20, 21). Surprisingly, most mutations fall within the coding sequence for NPII, a few fall within the signal peptide, and none falls within the coding sequence for AVP itself. The signal peptide mutations include a single base deletion that destroys the codon for the initiation methionine and disrupts the signal peptide, presumably forcing use of the subsequent methionine as the initiation amino acid (10). Another mutation disrupts the codon for the -3 amino acid of the signal peptide, substituting Phe for Ser (20), and two different mutations have been described that disrupt the codon for the -1 amino acid substituting Thr (4, 22, 23) and Val (20) for Ala.
Although the number of AVP-NPII mutations reported to cause ADNDI has grown rapidly, most of these reports have included relatively little information on the clinical manifestations of ADNDI. We have evaluated the AVP-NPII genes in two additional, large families with ADNDI and have identified a mutation that encodes a Val for Ala substitution at the -1 position of the signal peptide in both families. This mutation predicts the complete inability of signal peptidase to cleave the signal peptide from the preproprecursor and thus predicts disruption of the initial step of precursor processing. A surprising extent of significant clinical variability is seen among individuals heterozygous for the same mutation within each family and between the two families, suggesting that additional factors influence the course of vasopressinergic neuron degeneration in ADNDI.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Two families with inherited diabetes insipidus were identified.
Nine members of 3 generations of the Ma family from the United States
and 33 members of 3 generations of the Mo family from Lebanon were
evaluated (see Fig. 1
). In both families, inheritance of
symptomatic diabetes insipidus (DI) is consistent with autosomal
dominant transmission. This protocol was approved by the institutional
review board of the Children’s Hospital Medical Center, and informed
consent was obtained from all subjects or their parents.
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Formal assessment of the diagnosis of diabetes insipidus was carried out in the hospital. A standard water deprivation test (24) was administered, with hourly measurement of plasma and urine osmolality. The test was concluded when plasma osmolality exceeded 295 mmol/kg. If the maximal urine concentration was between 250–600 mmol/kg, a hypertonic (5%) saline infusion (0.06 mL/kg·min) was administered for 120 min to raise the plasma osmolality to above 300 mmol/kg, and plasma AVP was then measured. Aqueous vasopressin (5 IU) was administered to all subjects sc at the conclusion of the test, and urinary osmolality was remeasured 2 h later.
Individuals were classified as having partial DI if their maximal urinary concentration fell in the range of 250–600 mmol/kg. All of these patients had a plasma AVP level less than 3 pg/mL after hypertonic saline infusion, confirming the diagnosis of partial DI. Individuals were classified as having complete DI if urineary osmolality did not rise above 250 mmol/kg despite an increase in plasma osmolality above 295 mmol/kg. All patients had more than a 50% increase in urinary osmolality after the administration of exogenous AVP, confirming the diagnosis of neurohypophyseal diabetes insipidus.
Nucleotide sequences of exons of AVP-NPII gene
Genomic DNA was isolated from peripheral blood leukocytes, and each of the three exons of the AVP-NPII gene (16) or the entire gene (15) was amplified by PCR in 10% dimethylsulfoxide. The nucleotide sequence of both strands of the PCR products was determined directly by thermocycle sequencing as previously described (1, 3).
Restriction endonuclease analysis to confirm mutations
The AVP-NPII genes from Ma family members were PCR amplified as described above to produce a 280-bp PCR fragment containing the entire exon I. The AVP-NPII genes from Mo family members were PCR amplified, producing 158-bp PCR fragments (nucleotides 142–300) containing a portion of exon I including the mutation site. The PCR products were agarose gel purified and digested with restriction endonuclease BstUI according to the manufacturer’s recommendations. Digestion products were visualized after electrophoresis in a 3% NuSieve (FMC Bioproducts, Rockland, ME) gel and staining with ethidium bromide as previously described (16).
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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The Ma family index case (II-1) is currently 54 yr old and had untreated polyuria and polydipsia "all her life." She had nocturia three or four times nightly and drank water frequently, including several liters nightly. Water deprivation testing indicated that she has complete DI. She currently is taking the long-acting vasopressin analog, desmopressin, and has had a dramatic reduction in her polyuria and polydipsia. By history, her father and many of his siblings have symptoms of DI. Two of her three children have symptoms of DI, both with onset at 2 yr of age. Two of the three children of her symptomatic son (III-1) also have symptoms of DI, with onset at 1 yr of age (IV-1) and 2 yr of age (IV-3).
The Mo family includes 17 individuals symptomatic with polyuria and
polydipsia from 4 generations. Table 1 summarizes their
biochemical status based on formal water deprivation testing, their
clinical status based on qualitative assessment of daily urine volume,
and the age at onset of clinical diabetes insipidus. Note the wide
range in age of onset of DI and that those that have earlier onset of
DI tend to progress to more severe DI. Two affected family members
(III-1 and III-6) have had magnetic resonance imaging scans of the
hypothalamus and pituitary, and both scans demonstrate the absence of
the T-1 weighted image posterior pituitary bright spot.
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Genomic DNA was isolated from 9 members of the Ma family (5
clinically affected and 4 clinically unaffected) and 14 members of the
Mo family (8 clinically affected and 6 unaffected; see Table 1
). Each
of the 3 exons of the AVP-NPII gene from each of the Ma family members
and the entire gene of 1 affected and 1 unaffected member of the Mo
family were PCR amplified, and the nucleotide sequences of these PCR
products were determined. For all affected individuals in the Ma family
(Fig. 2) and for the affected individual in the Mo
family (data not shown), the DNA sequence indicated heterozygosity for
the normal sequence and a CT transition mutation at nucleotide 280
[according to the numbering system of Sausville et al. (11, 25)]. This mutation occurs at a CpG dinucleotide and encodes an
AlaVal substitution at the -1 amino acid of the prepropeptide
(i.e. the C-terminus of the signal peptide). Heterozygosity
for this mutation in all affected individuals is consistent with an
autosomal dominant mode of inheritance. Samples from all clinically
unaffected individuals from the Ma family and 1 representative
unaffected individual from the Mo family demonstrated only the normal
DNA sequence for all 3 exons of the AVP-NPII gene. These results
demonstrate that the mutation segregates completely with the ADNDI
phenotype in the Ma family.
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The mutation detected by direct sequencing of the PCR-amplified
AVP-NPII gene was confirmed by restriction endonuclease analysis.
Genomic DNA from one affected (III-3) and one unaffected member of the
Ma family was first PCR amplified to produce a 280-bp DNA product
containing the entire AVP-NPII gene exon I. This 280-bp fragment
normally has two BstUI restriction sites (CGCG) that define
three restriction fragments of 9, 107, and 164 bp. The
C280T mutation destroys one BstUI restriction
site and predicts restriction fragments lengths of 107 and 164 bp. DNA
from the affected family member produced fragments of 107, 164, and 271
bp (Fig. 3A), confirming heterozygosity for this
mutation. The 9-bp fragment is not visible by this technique.
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T mutation destroys
this BstUI site and yields a full-length 158-bp product
after digestion. Molecular diagnosis was performed on the Mo family
members (Fig. 3B) by the presence of only 138-bp fragments (normal) or
the heterozygous presence of both 138- and 158-bp fragments (ADNDI).
These results are summarized in Table 1 and demonstrate that, except
for III-10 (see Discussion), the mutation segregates
completely with the ADNDI phenotype in the Mo family. Again, the
generation of two restriction fragments from DNA of affected
individuals confirms heterozygosity for the mutation, consistent with
autosomal dominant inheritance. |
Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Val substitution does not provide clinical data
about affected individuals (20). In this study, the AVP-NPII genes were analyzed from individuals in two nonrelated kindreds with ADNDI. Heterozygosity for an identical novel mutation was identified in all affected individuals and was confirmed by restriction endonuclease digestion. This mutation changes the nucleotide sequence at the site of a CpG dinucleotide (26) from CpG to TpG. The fact that the site of this mutation is a CpG mutational hot spot may explain the recurrence of this mutation in three independent families.
The Ma family demonstrates a typical clinical presentation of ADNDI, with onset of symptoms at 1–2 yr of age in all cases. Individuals from the Mo family, on the other hand, have the same mutation as that in the Ma family, but show a very different spectrum of clinical presentation in both age of onset and severity of DI. IV-2 presented typically at the age of 3 yr, but 12 of the 16 individuals with known age of onset presented atypically at greater than 6 yr of age. Several presented at greater than 20 yr of age, and 1 28-yr-old (III-10), who has a positive molecular diagnosis and partial DI on formal water deprivation testing, still does not have clinical symptoms that she identifies as consistent with DI. Interestingly, those that presented at the oldest ages also have the mildest DI. Thus, III-17, who presented at 23 yr of age; III-5 who presented at 22 yr of age; III-18, who presented at 17 yr of age; and III-10, who has yet to present at 28 yr of age, all have clinically mild DI and the 3 subjects who have been formally tested have partial DI. This is not simply due to a shorter duration of illness in those who presented later in life, as III-5 has been symptomatic for 14 yr, and III-18 has been symptomatic for 4 yr. No apparent sex difference in presentation exists, as individuals with partial DI include both males and females. However, duration of symptoms may play a role in the severity of the DI in those that presented at earlier ages. Of those that presented at 15 yr of age or less, all that have had DI for greater than 5 yr (II-1, II-4, III-1, III-6, III-8, III-11, III-15, III-16, and IV-2) have severe DI, and 3 of 4 that have had DI for 3 yr or less (IV-3, IV-4, IV-6, and IV-8) have only mild DI. With time, the rest of these individuals with early onset mild DI may progress to more severe DI. In some families there are reports of spontaneous remission of clinical DI in the third to fourth decade despite continued vasopressin deficiency (1, 4, 27, 28). Perhaps the late presentation in the Mo family is related to masking of symptoms by early onset of this remission phenomenon, but this is unlikely, as none of the individuals in these kindreds report remission or even improvement in symptoms.
The molecular pathogenesis of ADNDI remains elusive (4, 20, 22, 23, 25, 29, 30, 31, 32). The mutations that fall within NPII have been hypothesized to
alter the AVP binding pocket and may diminish the ability of NPII to
protect AVP from proteolytic degradation. A mutation that results in an
amino acid substitution at the last amino acid of the signal peptide
(Ala-1Thr) has been shown to inhibit the initial step
in processing of the preproprecursor (22).
The mutation described in this report substitutes a valine for an
alanine at the C-terminus of the signal peptide. Valine does not occur
in this position in eukaryotic signal peptides (33), and when placed in
this position in preproapolipoprotein, cleavage of the signal peptide
at the normal site was completely eliminated (34). Thus, we hypothesize
that the valine for alanine substitution associated with the
C280T mutation in these two families completely
eliminates normal processing of the prepropeptide. As affected
individuals are not symptomatic in their early life, this processing
defect per se does not block AVP production. However, the
progressive accumulation of misprocessed precursor in magnocellular
neurons may ultimately lead to degeneration of these AVP-producing
neurons, thus preventing the production of AVP from even the normal
allele. If slow accumulation of misprocessed protein precursor is
confirmed to cause the neuronal death that underlies ADNDI, then this
disease may serve as a useful model for the study of the pathogenesis
of other human neurodegenerative diseases.
What remains a mystery is the variability in age of onset and severity of disease observed within and between families. The gene defect should not allow any production of vasopressin from the defective allele, but the length of time that the normal allele is capable of producing vasopressin and the amount of vasopressin produced can be variable. If malprocessed AVP-NPII precursor accumulation causes magnocellular neuron death, perhaps the toxic effect is modulated by the amount of AVP that these cells produce. Possibly, individuals with different environmental or genetic factors, such as a more sensitive thirst mechanism, may preserve the vasopressinergic magnocellular functioning for longer due to less need to produce AVP. Alternatively, different individuals may have different activities of the chaperone proteins that help prevent irreversible denaturation of malprocessed protein precursors in the endoplasmic reticulum or different activities of the pathway that successfully clears irreversibly denatured AVP-NPII precursors from the endoplasmic reticulum. Any of these mechanisms may help preserve magnocellular neuron functioning and result in variable clinical expression of clinical DI.
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Acknowledgments |
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Footnotes |
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Received July 15, 1996.
Revised August 30, 1996.
Accepted September 9, 1996.
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References |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Leu) is associated with onset and transmission of
autosomal dominant neurohypophyseal diabetes insipidus. J Clin
Endocrinol Metab. 79:421–427.[Abstract]
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