Nephrogenic diabetes insipidus results from a novel in-frame deletion of AVPR2 gene in monozygotic-twin boys and their mother and grandmother

Introduction

X-linked nephrogenic diabetes insipidus (XL-NDI, OMIM: 304800) is a water unbalance disorder. More than 90 % are caused by mutations in the AVPR2 gene located in Xq28 [1]. The AVPR2 gene encodes arginine vasopressin receptor 2 (V2R), which is distributed in the renal collecting ducts and plays a role in maintaining the body’s water balance [2]. The main characteristics of XL-NDI patients are polydipsia, polyuria, and dysphoria. The main clinical phenotypes include macrocystis, polyuria, irritability, diabetes insipidus, intellectual deficiency, seizures, inability to grow, unexplained fever, polydipsia, hypertonic dehydration, vomiting, constipation, hypernatremia, neonatal-onset, short stature, and feeding difficulties in infancy.

NDI is a rare X-linked recessive genetic disorder. Although the exact prevalence of NDI is unknown, it is estimated that in Quebec, Canada, there are 8.8 cases per 1 million men [3]. Due to the founder effect, certain areas, such as Utah and Nova Scotia have seen an increased incidence [4]. The mutation is typically passed from the mother to her male offspring, leading to a much higher incidence in males. Female carriers exhibit clinical heterogeneity due to varying X-chromosome inactivation statuses [5].

A thorough analysis and identification of the genetic variant have significant clinical implications, especially in managing and counseling affected families. NDI patients should receive timely treatment and management. The water intake should not be restricted, and using normal saline (0.9 % NaCl) to treat dehydration can pose serious risks. Heterozygous women with this X-linked condition may be asymptomatic or may experience varying degrees of polyuria and polydipsia. If a pathogenic variant of the AVPR2 gene was identified in an affected family member, conducting molecular genetic testing on at-risk, asymptomatic female relatives can offer valuable insights into their genetic status. Genetic counseling is essential, as it involves discussions about potential risks to offspring and available reproductive options for affected patients, heterozygotes, and young adults who may carry the risk of becoming heterozygotes.

Here, we present identical twin boys with typical NDI who carried a novel in-frame hemizygous deletion. This research aims to evaluate the pathogenesis of the variant, find the cause of clinical heterogeneity of female carriers, and provide adequate genetic counseling for them.

Materials and methods

Results

Sequencing results

The proband was identified with a novel hemizygous variant of the AVPR2 gene (chrX: 153171373–153171375, NM_000054; exon3, c.413_415delACC(p.H138del). Family verification confirmed that the proband’s twin brother shared the same hemizygous variant, and the mother, grandmother, and maternal aunt were all found to be heterozygous, while the wild type in father and grandfather. For more details, please refer to the Sanger sequencing diagram (Figure 1) and family diagram (Figure 2).

Figure 1:

Sanger sequencing of AVPR2 gene. The proband and twin brother shared the same hemizygous variant, and the mother, grandmother, and maternal aunt were all found to be heterozygous, while the wild type in father and grandfather. Arrows indicated the variants.

Figure 2:

Family diagram of AVPR2 gene variant. (-): Wild type; (+): Mutant type; (%): X chromosome inactivation ratio; (→): The proband.

The results of X chromosome inactivation

The analysis of X chromosome inactivation revealed that the blood XCI of the mother, grandmother, and maternal aunt were randomly inactivated at a rate of 36.18 , 48.37, and 49.30 %, respectively, as shown in Figure 2 and Figure 3.

Figure 3:

The electrophoretogram of blood XCI in the family. The vertical and horizontal axes represent fluorescence intensity and fragment length, respectively, with the corresponding values below the fluorescence peak. Three control fragments were analyzed from left to right to evaluate enzyme digestion efficiency, PCR amplification, and the copy number of the X chromosome. Before digestion, heterozygous genetic markers, including XpA, XpB, XpC, and XqA, were used to determine the status of X chromosome inactivation (XCI). The allele with the highest fluorescence peak after digestion is labeled D1, while its corresponding undigested peak is labeled U1. The other alleles are designated as D2 and U2, respectively. To calculate the XCI rate, we use the formula: (d1/u1)/(d1/u1+d2/u2) *100 %. A bias in XCI is indicated if the XCI ratio exceeds 75 %. The results of XCI revealed that the blood XCI of the mother, grandmother, and maternal aunt were randomly inactivated at a rate of 36.18 , 48.37, and 49.30 %, respectively. DE: digestation of enzyme.

The results of X chromosome haplotype and X recombination analysis

The proband and his identical twin brother inherited an allelic variant from their mother. The X haplotype results showed that the proband’s mother and grandmother had the variant on each active X chromosome(Xa), while the maternal aunt’s on her inactivated X chromosome(Xi). The X-chromosome haplotype structure of the mother and maternal aunt revealed several recombinations between the grandmother’s homologous X chromosomes and an additional Xqters exchange from the grandmother and grandfather in the maternal aunt’s X chromosomes. For more details, please refer to Figure 4.

Figure 4:

The diagram of X chromosome haplotype and X recombination of the family. (A) The proband and his twin brother had identical short tandem repeat (STR) loci, and both inherited an allelic variant of the AVPR2 gene (marked with a red star) from their mother and the mother from her mother. The variant of the grandmother and mother was on each active X chromosome(Xa), indicated by the green box. (B) The AVPR2 variant of the maternal aunt was on the inactivated X chromosome(Xi). Her X chromosome haplotype hinted several recombinations from her mother’s X-homologous chromosomes (the blue dashed line indicated the recombination breakpoints) and additional Xqters exchange (red dashed line and red cross) from her parents.

Discussion

In this study, both the male proband and his identical twin sibling showed typical symptoms and signs of NDI, such as polydipsia, hypernatremia, hyperchloremia, hypotonia, growth delay, repeated hypothermia, and vomiting. And they shared a hemigamous mutation of AVPR2 exon3 (NM_000054) c.413_415delACC(p.H138del) (GRch37 chrX: 153171372–153171375). HGMD has reported more than 300 variants of the AVPR2 gene result in NDI. Most AVPR2 mutations of NDI are missense mutations, followed by nonsense mutations, and fewer are in-frame deletions. However, the latter two may cause more serious diseases [8]. Bicchet et al. [2] showed that the AVPR2 receptor protein has seven transmembrane domains, connecting three extracellular domains (EC-1, EC-2, EC3) and three intracellular loops (IC-1, IC-2, IC-3). In HGMD, several cases of NDI with in-frame deletion of AVPR2 have been reported, such as CD2115497: 380_382delCCT (Ser127del), CD001867: 382_384delTAC(Tyr128del), CD001870: 526_528delTTC (Phe176del), CD983639: 606_608delTCG(Arg203del). Meanwhile, previous studies reported some NDI cases of AVPR2 in-frame deletion, including single or multiple amino acids. For details, please see Table 1. The clinical phenotypes of some patients were mild, and others were severe. The severity might vary with the different domains involved in deletion. In our patient, c.413_415delACC (p.H138del) is an in-frame deletion of a single amino acid. Although there still have been no familiar cases reported, p.H138 is in an important functional domain, IC-2, which is highly conserved in different species [14]. We speculated the variant might affect the protein function. According to the population frequency, highly-specific phenotype, hereditary mode, the variant is classified as likely pathogenic.

The grandmother, mother, and maternal aunt all had the heterozygous variant, and we have clarified their clinical variability. Compared to women without the variant, the grandmother and mother experienced significant increases in both take-in water and urine volume, along with frequent nighttime urination, 3 to 4 times per night. They often felt tired and thirsty, preferred cold drinks, experienced easy fatigue, and occasionally felt sleepy. Additionally, they faced menstrual irregularities, while the aunt did not exhibit any of these symptoms. Although only about 1 % of women with the AVPR2 heterozygous variant have the same severe phenotype as men with NDI [3], a higher proportion of female carriers have significant NDI-related symptoms. In a Japanese study, 25 % of female carriers developed NDI [20], and a Spanish study showed a 50 % incidence of female carriers [21]. Like 75 % of genes on the X chromosome, the AVPR2 is a gene subject to X inactivation [22]. The clinical phenotype of female individuals carrying the same AVPR2 heterozygous variant ranges from severe to mild or even normal phenotype, is associated with variations in underlying environmental and epigenetic factors or differences in complex recombination of the X chromosomes. At first, XCI is the well-known influence factor. The different XCI statuses varied with age and tissue [5], [22], [23], [24], [25]. In our study, preferential X inactivation was not shown in the peripheral blood of the mother and grandmother with NDI clinical phenotype, consistent with the previous reports that blood lymphocytes of female NDI patients with random X inactivation [24], 26]. Although X chromosome inactivation (XCI) levels in their blood tissues appeared randomly inactivated, XCI levels may vary, particularly in renal collecting ducts cells. Given the severity of their clinical phenotypes, we hypothesize that the renal collecting ducts cells of the mother and grandmother may have skewed XCI, whereas the aunt likely has random XCI. Unfortunately, renal collecting ducts cells samples from family members are unavailable for further testing. Secondly, X chromosome recombination or exchange is another factor affecting clinical heterogeneity. The X-chromosome haplotype structure of the mother and maternal aunt revealed several recombinations between the grandmother’s homologous X chromosomes and an additional Xqters exchange from the grandmother and grandfather in the maternal aunt’s X chromosomes. The recombination rate of the X chromosome is much higher than that of autosomes [27]. Recombination factors should also be considered in X-chromosome-related diseases, the same as in the study of kinship [28]. Previous studies have estimated that the recombination rate of PAR1 is 20 times the genomic average [29], and that of PAR2 is five times [30]. Although the recombination rate of PAR2 is much lower than PAR1, it is still higher than the average recombination rate of the rest parts of the X chromosome [31]. In addition, non-homologous recombination at the pseudoautosomal boundary of X is also a common situation [29]. The AVPR2 gene was only about 1.80 Mb away from the PAR2 region, so we speculated the AVPR2 gene exchange of the maternal aunt was related to the PAR2 region. When the PAR2 boundary region was recombined or exchanged, the maternal AVPR2 variant transferred into another Xi, which inactivated the variant AVPR2 gene, presenting no NDI phenotype. On the contrary, the AVPR2 variant of the grandmother and mother was on the Xa, and their mutation effects showed up.

Our study compares initially the clinical severity of the novel in-frame deletion variants of the AVPR2 gene identified here with those reported previously. We found that the inconsistencies in clinical severity among these variants may be related to their involvement in different domains. In future studies, we will further investigate the impact of these variants on gene function and conduct additional functional assessments.

XCI is one of the pivotal clinical reference indicators for some X-linked diseases in female carriers. Several issues should be in mind in XCI testing. Firstly, we should choose the appropriate method of XCI detection. The methylation status of a polymorphic locus in the androgen receptor gene (HUMARA assay) [32], 33] is a popular method. The STR heterozygosity of the AR gene in the population is about 90 % [34], 35], and the remaining 10 % homozygous locus of female individuals cannot be detected. Therefore, the application range of this method in scientific research and clinical practice is limited. In this study, four polymorphic genetic marker loci, including the AR gene, were simultaneously subjected to methylation-sensitive restriction enzyme digestion. According to the kit instructions, the detection rate is as high as 99 %, with at least one locus as heterozygous. Secondly, the related tissue or functional cells should be detected when evaluating the XCI status. Although both peripheral blood and urinary system cells are of mesodermal origin, we cannot predict the clinical phenotype of female carriers by evaluating the XCI of peripheral blood leukocytes instead of renal collecting ducts cells, which affect urine reabsorption. Thirdly, the recombination of sex chromosomes is frequent, so haplotype analysis of X chromosomes should be performed when analyzing X-related diseases. When adopting the X-STR marker, it is crucial to consider linkage disequilibrium [28], 36], as the STR of the X chromosome shows more serious linkage disequilibrium than those on autosomes [36], [37], [38].

Primary manifestations and complications can be prevented through timely diagnosis and clinical intervention in suspected NDI patients during their early life [39]. Patients need lifelong treatment. Treatment methods mainly include general symptomatic treatment and individualized drug treatment: the former limited sodium and unlimited water intake, adequate care, and sufficient nutrition, and the latter hydrochlorothiazide and indomethacin, and so on. Meanwhile, we recommend enhanced surveillance and management for female carriers. During pregnancy, heterozygous women with AVPR2-NDI may experience a slight increase in urine production and thirst. In a small number of pregnancies involving fetuses affected by NDI, excessive amniotic fluid may occur, necessitating frequent drainage to alleviate discomfort for the mother, particularly in cases of severe hyperamnios [40]. If pathogenic variants of the AVPR2 gene are identified in affected family members, conducting molecular genetic testing on asymptomatic at-risk female relatives is highly beneficial. Maternal aunts of male proband may be at risk of being heterozygous carriers of pathogenic variants, and their offspring may also be at risk for male patients or heterozygosity.