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Original Studies |
Department of Pediatrics, Gunma University School of Medicine (Y.N., K.O., T.N., S.Y., K.N., A.M.), Maebashi, Gunma 371; and the Department of Metabolism and Endocrinology, Saitama Children’s Medical Center (H.M.), Iwatsuki, Saitama 339, Japan
Address all correspondence and requests for reprints to: Yoko Nomura, M.D., Department of Pediatrics, Gunma University School of Medicine, 3–39-22 Showa-machi, Maebashi, Gunma 371, Japan.
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Abstract |
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We present a family with congenital NDI. Three male members were diagnosed with NDI, and examination of their V2R gene revealed a G inserted at nucleotide 804 of the open reading frame. Three female individuals display different degrees of symptoms of NDI, and all of them possess both the normal and abnormal genes.
The X-inactivation patterns of the female members were investigated via the detection of methylated trinucleotide repeat in the human androgen receptor gene. The grandmother showed extremely skewed methylation of one X chromosome, and the mother revealed moderately skewed methylation. The daughter of the grandmother’s sister, who has no symptoms of NDI, showed random methylation.
The highly skewed X-inactivation pattern of the grandmother suggests that her NDI phenotype is caused by dominant methylation of the normal allele of V2R gene.
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Introduction |
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Most cases of congenital NDI are transmitted in an X-linked recessive manner. A mutation in the vasopressin type 2 receptor (V2R) gene on the long arm of the X chromosome (Xq28) has been detected in an affected individual (2) and is thought to be the cause of the disease. Other modes of inheritance have also been described (3, 4, 5, 6). Mutations in the aquaporin 2 (AQP2) gene have been found in families with autosomal recessive inheritance (7, 8). In cases of mutations in the V2R gene, female carriers are usually asymptomatic. However, in some cases, female carriers show a clinical phenotype similar to that of male NDI patients (9, 10).
Gene dosage compensation in humans is achieved through random inactivation of one of the two X chromosomes in the cells of normal females (11). When the X chromosome with a normal allele is inactivated preferentially, female carriers may be affected (12, 13, 14, 15). Methylation-sensitive restriction enzymes may be used to verify the skewed X-inactivation. The HpaII and HhaI cleavage sites in the first exon of the human androgen receptor (AR) gene, which is one of the methylation sites in the inactive X chromosome, are highly polymorphic, and the paternal and maternal X chromosome activation status is detectable (16, 17).
Three females in a NDI family were examined in the present study. These women display different degrees of symptoms of NDI. Genetic analysis of the V2R gene showed that all three are heterozygous for a V2R mutation. In addition, the X-inactivation patterns of these three individuals were examined using methylation analysis of the polymorphic CAG repeat of the AR gene.
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Subjects and Methods |
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)The male Japanese patient (III-4) was born at term after an uncomplicated pregnancy. His parents (II-4 and II-5) were nonconsanguineous. On the 55th day of life, he was admitted to hospital with vomiting and high fever and was cured by infusion with electrolyte solution. His grandmother (I-9) suffers from polyuria and polydipsia, but had never sought medical attention. I-1 died of unknown causes. I-5 died of a brain tumor. II-1, II-2, II-3, II-4, II-5, III-1, and III-3 present no NDI symptoms, and III-2 and III-5 suffer from polyuria, polydipsia, and hypernatremia.
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) showed that the male patients
(III-2, III-4, and III-5) and the grandmother (I-9) did not concentrate
their urine osmolality even after the infusion of vasopressin. The
mother (II-5) revealed an incomplete antidiuretic response to
vasopressin loading. The activity of factor VIII coagulant was
evaluated in the grandmother and the mother to reveal the extrarenal
response of V2R. Factor VIII activity did not increase in the
grandmother (108%) after vasopressin infusion, but increased
moderately in the mother (280%).
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V2R and AQP2 were examined in patients I-9, II-1, II-2, II-5, III-2, III-4, and III-5. Genomic DNA was extracted from a peripheral blood sample from each patient using the phenol-chloroform method.
The samples were amplified using the PCR. Six sets of primers designed for the V2R gene were used and have been described previously (18). The PCR conditions were 30 s at 94 C, 30 s at 60 C, and 2 min at 72 C for 30 cycles. Each exon of the AQP2 gene was amplified through 30 cycles of 95 C for 1 min, 60 C for 1.5 min, and 72 C for 1.5 min, using previously described primers (19). The existence of the mutation was screened by PCR-single strand conformation polymorphism (SSCP) using 5% Hydrolink-MDE gel (AT BioProducts, Malvern, PA) with 5% glycerol. The electrophoresis was performed at 200 V at 4 C for 6 h. Cycle sequencing reactions were performed using the TaqDyeDeoxy termination Cycle Sequencing Kit (Applied Biosystems, Foster City, CA).
Semiquantitation of X-inactivation
DNA samples from the carrier females (I-9, II-2, and II-5) were digested using methylation-sensitive restriction enzymes (10 U HpaII and 12 U HhaI) in a reaction volume of 25 µL at 37 C for 2 h. The PCR primers used for the AR locus, that included the HpaII and HhaI cleavage sites and the CAG repeat element, were previously described (16). The 5'-end of the forward primer was modified with fluorescein (6'-FAM). Five microliters of digested and undigested samples were amplified for 30 cycles of 95 C for 45 s, 60 C for 30 s, and 72 C for 30 s.
When the DNA was initially digested, amplification only occurred if the restriction sites were methylated. The PCR products were electrophoresed with ROX mol wt markers for loading onto a ABI 310 autosequencer (Applied Biosystems, Foster City, CA). Peak heights were analyzed using GeneScan software (Applied Biosystems). The major peak heights of two alleles were seen in the undigested PCR products, and to compensate for unequal amplification of alleles, a correction factor was defined by calculating the ratio between the two alleles. The peak heights of corresponding alleles in the digested products were measured, and the values were corrected using this factor. The sum of the two alleles was defined as 100% X-inactivation, and the degree of X-inactivation for each allele was expressed as a percentage of inactive X chromosomes.
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Results |
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PCR-SSCP of samples from three male (III-2, III-4, and III-5) and
three female (I-9, II-2, and II-5) patients suggested the existence of
a mutation in V2R gene (data not shown). The DNA sequence of these
samples showed that a G had been inserted at nucleotide position 804,
causing a frame shift that resulted in a premature stop codon at amino
acid 258. The females had both normal and abnormal alleles (Fig. 1
).
The sequences of other exons of the V2R gene revealed no mutation.
No abnormalities of the AQP2 gene were observed in any individual using PCR-SSCP and direct sequencing.
Assay for X-inactivation
Peak height ratios for the two alleles in undigested samples were
(CAG)20/(CAG)22 = 1.59 in I-9,
(CAG)22/(CAG)24 = 1.03 in II-2, and
(CAG)21/(CAG)23 = 0.86 in II-5 (Fig. 2). Correcting the results for digested
samples yielded values of relative X-inactivation for one allele of
94.3% in I-9, 74.9% in II-2, and 65.8% in II-5.
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Discussion |
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The 804insG mutation, which causes a stop codon at amino acid 258 (3), results in no protein synthesis (20) and is thought to be responsible for the dysfunction of V2R.
Lieburg et al. described the clinical phenotype of NDI in female carriers with the V2R mutation (10). In three families, four females showed clinical features resembling the phenotype in male NDI patients. All affected females and two asymptomatic female members were shown to be heterozygous for the V2R mutation. Another female NDI case with marked vasopressin resistance was reported by Moses et al. (9). In that study, mother and daughter had both normal and abnormal alleles. The mother revealed a total lack of antidiuretic response and half of the normal factor VIII response. The daughter had responses similar to asymptomatic female carriers of NDI. They speculated that the difference between the phenotypes of the mother and daughter may be due to extreme lyonization of X chromosome inactivation in renal tubules.
The grandmother in the family in the present study displays severe NDI symptoms and the absence of coagulant factor VIII response to vasopressin, whereas the mother has only a mild disturbance in the ability to concentrate urine, and coagulant factor VIII rises normally in the vasopressin loading test. Several mechanisms by which female patients express complete NDI symptoms are possible and include 1) homozygous or compound heterozygous mutation of the V2R gene, 2) mutation of the AQP2 water channel gene, 3) abnormality in signal transduction cascade between receptor and water channel, 4) abnormal receptor protein that has a dominant negative effect, and 5) X-inactivation occurring preferentially to normal V2R allele. In the present study, because the exons of the V2R gene, other than the one containing the 804insG mutation, and the exons of the AQP2 gene had no mutations, mechanisms 1 and 2 above were excluded. Although mechanisms 3 and 4 cannot be excluded, no reports of these mechanisms at work have been published to date. The results of our cases and a previously reported case (3) suggest that females with the same heterozygous mutation of the V2R gene (804insG) may present variable phenotypes. The skewed inactivation of the X chromosome may be an appropriate explanation for this variable expression.
In the present study, the X-inactivation status of DNA isolated from the peripheral blood of female carriers was examined using the polymorphic CAG repeat of AR locus. In the grandmother (I-9), who has the complete congenital NDI phenotype with extrarenal involvement, methylation of the X chromosome was extremely skewed. The mother, who had mild disturbance in the ability to concentrate urine in the water deprivation test, but no subjective symptoms, showed moderately skewed X-inactivation. The daughter of the grandmother’s sister displayed a normal increase in urine osmolality and random methylation of X chromosomes. These findings suggest that the degree of nonrandom X-inactivation correlated with the degree of expression of the NDI phenotype. As the V2R locus is on Xq28, and the AR locus is on Xcen-ql3, crossing over most likely occurs between these loci (21). When translocation occurs, the V2R locus and the AR locus in the mother exist on different chromosomes. In fact, the AR trinucleotide repeat assay indicated that the proband has the (CAG)24 allele (data not shown), and that the grandmother has the (CAG)20 and (CAG)22 alleles. The results of GeneScan analysis suggested that the grandmother has the mutation allele of V2R, which is preferentially active, on the same chromosome as the (CAG)20 allele. These findings suggest that the mutation allele of V2R moved to a different allele via translocation. To obtain definitive proof that methylation in the normal allele of female NDI patient is dominant, the methylation of the locus adjacent to the V2R locus must be examined. However, other such suitable loci cannot be defined at present. Furthermore, extremely skewed X-inactivation in the X chromosomes of the grandmother indicates that NDI symptoms would be expressed in a female carrier when the normal allele is inactivated preferentially. Although the mechanism by which preferential methylation of the normal allele occurs is not clear, the possibility that the mutation affects the process of X-inactivation cannot be excluded. However, given that excessive lyonization (>90%) has been detected in 9% of healthy females (22), the skewed X-inactivation observed in this family may have occurred by chance.
In conclusion, the X-inactivation pattern of female carriers suggests that the variable NDI phenotype expressed by these carriers is caused by the degree of methylation of the normal V2R gene.
Received April 28, 1997.
Revised June 30, 1997.
Accepted July 2, 1997.
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