Introduction

Dent disease [1] is an X-linked disorder characterized by low molecular weight proteinuria, hypercalciuria, nephrolithiasis, and nephrocalcinosis [2, 3]. It can be divided into two types: Dent disease 1 (OMIM #300009), which is caused by pathogenic variants of the CLCN5 gene, and Dent disease 2 (OMIM #300555), caused by pathogenic variants of the OCRL gene. Approximately 60% of patients with clinically diagnosed Dent disease have Dent disease 1, while 15% have Dent disease 2. In the remaining 25%, the causative gene has not been identified [4, 5].

Dent disease 1 is caused by dysfunction of the ClC-5 chloride channel due to an abnormality in the CLCN5 gene located on the X chromosome at Xp11.22; it is mainly expressed in early endosomes of the proximal tubule [6, 7]. In contrast, Dent disease 2 is caused by an abnormality of OCRL encoding phosphatidylinositol 4,5-bisphosphate 5-phosphatase, also located on the X chromosome at Xq25 [5, 8]. This protein is also expressed in endosomes of the proximal tubule like CLCN5, but is also widely expressed elsewhere, including in the brain [9].

OCRL mutations were originally described in patients with Lowe syndrome [10], which almost always comprises Fanconi syndrome, congenital cataracts, hypotonia, and severe developmental delay [11]. In addition, this condition often leads to chronic kidney disease (CKD) stage 5 [12, 13]. Despite having the same causative gene as Lowe syndrome, symptoms in Dent disease 2 are usually restricted to the kidney and are less severe than in Lowe syndrome. The reason for this difference remains unclear, but previous reports have shown that patients with any type of mutations in exons 1–7 were diagnosed with Dent disease 2 and those with truncating mutations in exons 8–24 were diagnosed with Lowe syndrome [14]. It has also been reported that Dent disease 2 is a mild form of Lowe syndrome because some patients diagnosed with Dent disease 2 have mild cataracts and/or developmental delay [15].

Although the renal manifestations of Dent disease 1 and Dent disease 2 are quite similar, some cases of Dent disease 2 with mild symptoms of Lowe syndrome suggest that there may be differences in the renal and extrarenal manifestations between Dent disease 1 and Dent disease 2, as reported previously [15, 16].

Against this background, the purpose of this study is to clarify the differences in clinical symptoms and laboratory data between pediatric patients with Dent disease 1 and Dent disease 2 who underwent gene testing at a single institution.

Methods

Subjects

We have been performing gene testing for patients clinically suspected of having Dent disease since September 2014. Among them, all male cases clinically and genetically diagnosed with Dent disease were studied. Patients with cataracts were excluded as having Lowe syndrome, even when the diagnosis by their primary doctor was Dent disease. As a result, a total of 72 cases in 64 families with Dent disease 1 and 13 cases in 10 families with Dent disease 2 were recruited. We conducted a retrospective review of these patients regarding the clinical findings and laboratory data collected from questionnaires submitted at the time of application for a gene testing. Details regarding the clinical features were obtained from the referring clinician or the patient’s hospital records by the local doctors. This report does not include cases from a previous cohort of Dent disease in Japan [17].

Assessment of clinical findings

The presence or absence of intellectual disability, autism spectrum disorder, nephrocalcinosis, metabolic acidosis, or glycosuria was determined according to the questionnaire completed by each clinician. The presence or absence of intellectual disability in this study was based on developmental tests, but for patients without developmental tests, the decision was made by their clinician. The height SDS was calculated from the standard data for the Japanese. Cr-eGFR was calculated using the formula for Japanese children and adolescents under 18 years old [18, 19]. For patients over 18 years old, the Cr-eGFR calculation formula adjusted for the Japanese was also used [20]. CKD was defined as Cr-eGFR of < 90 ml/min/1.73 m2. The presence or absence of hypercalciuria was determined using previously reported age-specific reference values [21].

Gene testing

Gene testing was performed after informed consent was received. Genomic DNA was isolated from the peripheral blood leukocytes of patients and their family members using the QuickGene-Mini80 system (Wako Pure Chemical Industries, Ltd., Tokyo, Japan), in accordance with the manufacturer’s instructions. Direct sequencing or targeted sequencing using next-generation sequencing (NGS) was conducted on genes responsible for inherited kidney diseases. NGS samples were prepared using a HaloPlex target enrichment system kit (Agilent Technologies, Santa Clara, CA, USA), in accordance with the manufacturer’s instructions. Briefly, 225 ng of genomic DNA was used for a restriction reaction and hybridized at 54 °C for 16 h with NGS probes. All indexed DNA samples were amplified by polymerase chain reaction (PCR) and sequenced using the MiSeq platform (Illumina, San Diego, CA, USA). We analyzed the data using SureCall 4.0, which is a desktop application combining algorithms for end-to-end NGS data analysis, from alignment to the categorization of mutations (Agilent Technologies). The cDNA reference number of CLCN5 in this study is NM_000084.4 and that of OCRL is NM_000276.3. Pathogenicity predictions were performed in accordance with the American College of Medical Genetics guidelines [22]. Several online predictive tools, including SIFT (https://sift.bii.a-star.edu.sg/), PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2/), Mutation Taster (http://www.pathogenicvarianttaster.org/), Human Splicing Finder (http://www.umd.be/HSF/), MaxEntScan (http://genes.mit.edu/burgelab/maxent/Xmaxentscan_ scoreseq.html), and NNSplice (http://www.fruitfly.org/seq_tools/splice.html), were used to predict the pathogenicity of the variants.

Statistical analysis

Data are shown as the median and interquartile range (IQR). Clinical findings and laboratory test results of the patients were compared using the Mann-Whitney U test and Fisher’s exact test, as appropriate. Factors considered to be related to age were adjusted for age using analysis of covariance. Statistical analysis was performed using standard statistical software (JMP version 10 for Windows; SAS Institute, Cary, NC, USA). p < 0.05 was considered statistically significant.

Results

We examined differences in clinical and laboratory data between Dent disease 1 and Dent disease 2, the results of which are shown in Table 1. The median age at which gene testing was performed was significantly lower in Dent disease 2 than in Dent disease 1. The height SDS was significantly lower in Dent disease 2 than in Dent disease 1 (−2.2 SD vs. −0.2 SD; Fig. 1a). There was no significant difference in the prevalence of intellectual disability or autism spectrum disorder.

Table 1 Comparison of clinical findings between patients with Dent disease 1 and Dent disease 2
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Fig. 1
figure 1

Comparison of height SDSand Cr-eGFR between patients with Dent disease 1 and Dent disease 2. (a) Patients with Dent disease 2 were shorter than those with Dent disease 1 (median: −2.2 SD vs. −0.2 SD, p < 0.01). (b) Cr-eGFR levels were significantly lower in patients with Dent disease 2 than in those with Dent disease 1 (median: 84 vs. 127 ml/min/1.73 m2, p < 0.01)

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Patients with Dent disease 2 had significantly lower Cr-eGFR than patients with Dent disease 1 (median 84 vs. 127 ml/min/1.73 m2) (Fig. 1b). When Cr-eGFR under 90 ml/min/1.73 m2 was defined as CKD, the prevalence of CKD was only 8% (6 out of 71) in Dent disease 1, but 58% (7 out of 12) in Dent disease 2. The six patients with CKD in Dent disease 1 consisted of five with CKD stage 2 (60–89 ml/min/1.73 m2) and one with CKD stage 3 (30–59 ml/min/1.73 m2). All seven patients with CKD in Dent disease 2 were at CKD stage 2. There were no patients with CKD stage 5 in either group.

Serum levels of creatine kinase (CK), lactate dehyrogenase (LDH), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) were significantly higher in Dent disease 2 (Fig. 2). None of the patients had metabolic acidosis. Serum potassium and phosphate levels were significantly lower in Dent disease 1, but there were no significant differences for serum sodium, calcium, uric acid (UA), and alkaline phosphatase (ALP). After adjustment for age, serum level of uric acid was significantly lower in Dent disease 1.

Fig. 2
figure 2

Comparison of (a) CK, (b) LDH, (c) AST, and (d) ALT between patients with Dent disease 1 and Dent disease 2. All parameters were significantly higher in patients with Dent disease 2 than in those with Dent disease 1

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The findings from comparing the urinalysis results are shown in Fig. 3. Urine protein/creatinine ratio and urine calcium/creatinine ratio were significantly higher in Dent disease 2. However, there was no significant difference in urine β2-microglobulin and the prevalence of hypercalciuria in relation to the age-specific baseline values for children. The number of patients with glycosuria was low, and there was no significant difference in this regard between the two groups. Nephrocalcinosis was observed only in Dent disease 1, but the difference did not reach significance due to the small number of Dent disease 2 cases.

Fig. 3
figure 3

Comparison of urine protein/creatinine ratio (urine Pro/Cre), urine β2-microglobulin (urine β2MG), and urine calcium/creatinine ratio (urine Ca/Cre) between patients with Dent disease 1 and Dent disease 2. (a) Urine protein/creatinine ratio was significantly higher in patients with Dent disease 2 (median: 3.5 vs. 1.6 mg/mg, p = 0.01). (b) However, there was no significant difference in urine β2-microglobulin. (c) Urine calcium/creatinine ratio was significantly higher in patients with Dent disease 2 (median: 0.69 vs. 0.25 mg/mg, p < 0.01)

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The frequency of major renal manifestations of Dent disease is summarized in Table 2, along with the results of previous studies comparing Dent disease 1 and Dent disease 2.

Table 2 Comparison of major renal manifestations of Dent disease 1 and Dent disease 2 in previous and our reports
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All genetic variants detected in this study are shown in Supplementary Table S1. Evaluation of novel missense mutations using in silico analysis and population database are shown in Supplementary Table S2. We detected 64 families with CLCN5 mutation, comprising 32 missense, 17 nonsense, and 10 splice-site mutations, 4 small deletions, as well as 1 small insertion. Of these, 28 were novel mutations. We also identified OCRL mutations in 10 families, 5 of which were novel. The mutations included 5 missense, 1 nonsense, and 2 splice-site mutations, as well as 2 small deletions.

Discussion

Dent disease does not usually cause extrarenal symptoms, and it is difficult to distinguish between Dent disease 1 and Dent disease 2 based on clinical symptoms alone. A gene testing is required to distinguish them. However, several reports have been published showing differences in the clinical features of Dent disease 1 and Dent disease 2 [15, 16].

In Japan, Dent disease is usually detected as asymptomatic proteinuria at the time of urine screening, such as urinalysis routinely performed at the age of 3 or at school. In other words, the age group differs from that in cohort studies in other countries because it is often detected much earlier in Japan than in Europe and the US. Therefore, it is common for there to be no extrarenal symptoms at the time of diagnosis, and there are no patients with CKD stage 5 among the subjects. However, it is unclear how many patients will eventually develop CKD stage 5. As pointed out in a report on a large study in Japan [17], Japanese cases may have milder symptoms of Dent disease.

Table 2 compares the frequency of major renal manifestations of Dent disease in this study with those in previous large cohorts; some symptoms in this study are similar to those in previous reports, while others are not. Although subtle differences in the definitions of hypercalciuria and kidney dysfunction in each study may have contributed to this, our reported findings are relatively similar to those in a previous Japanese cohort [17], so the renal manifestations of Dent disease may vary from country to country.

A previous large French cohort showed no difference in GFR decline between Dent disease 1 and Dent disease 2 [12], and another report showed a trend toward lower eGFR in Dent disease 2, but the difference was not significant [15]. Another Chinese cohort, without statistical analysis, noted a mean eGFR of 82.6 mL/min/1.73 m2 in Dent disease 2 versus 110.7 mL/min/1.73 m2 in Dent disease 1 [23].

In our study, Dent disease 2 had significantly lower eGFR than Dent disease 1, and it was proven that kidney dysfunction was shown from an earlier age in Dent disease 2.

There may also be differences in other renal manifestations characteristic of Dent disease. In this study, although there was no significant difference in urine β2-microglobulin, there was a significantly higher urine protein/creatinine ratio reflecting the amount of low molecular weight proteinuria in Dent disease 2.

Hypercalciuria is a common symptom in both disorders. In a previous Japanese report, the incidence of hypercalciuria was significantly higher in Dent disease 2 than in Dent disease 1 [17]; however, this is not always the case in other reports. Hypercalciuria in Dent disease is thought to be caused by increased absorption of calcium from the gastrointestinal tract [24]. The activity of TRPV6, an intestinal calcium channel, is suppressed by OCRL protein, and mutations in the OCRL gene alleviate this suppression [25]. Thus, intestinal calcium absorption may be enhanced in cases of Dent disease 2. In addition, in our study, urine calcium/creatinine ratio was significantly higher in patients with Dent disease 2, even after adjusting for age. However, there was no significant difference in the prevalence of hypercalciuria. Urine calcium/creatinine ratio varies widely in younger children, so the result for this variable would have been influenced by age differences between the two groups.

The incidence of nephrocalcinosis was 0% in Dent disease 2 and 22% in Dent disease 1, although there was no significant difference in this study. In five previous reports [12, 15,16,17, 26], the incidence of nephrocalcinosis appears to have been lower in patients with Dent disease 2. This was contrary to the expected result from our study that the calcium creatinine ratio was significantly higher for Dent disease 2 than for Dent disease 1. In other words, hypercalciuria may not necessarily be a direct risk for nephrocalcinosis.

Some of the symptoms of Fanconi syndrome are observed in Dent disease [12]. Abnormalities observed in Fanconi syndrome such as hypokalemia, hypouricemia, and hypophosphatemia were also examined in this study. None of the patients in our study had hypophosphatemia or hypokalemia that required medication, but serum inorganic phosphorus and potassium levels were significantly lower in patients with Dent disease 1 than in those with Dent disease 2. After adjustment for age, in addition to these two parameters, serum uric acid levels were lower in patients with Dent disease 1. Although Fanconi syndrome is rare in patients with Dent disease and often only some of its symptoms are exhibited, it is possible that electrolyte abnormalities associated with Fanconi syndrome are observed more in patients with Dent disease 1.

In a previous Korean cohort, it has also been reported that CK, LDH, and AST are higher in Dent disease 2 than in Dent disease 1 [16]. This was again confirmed in our investigation. In Lowe syndrome, which is caused by the same OCRL gene abnormality as Dent disease 2, muscle weakness is one of the major symptoms and the levels of muscle enzymes are elevated. If Dent disease 2 can be categorized as a mild form of Lowe syndrome, it is reasonable to assume that muscle enzyme elevation is also observed in Dent disease 2. However, the precise reason for the muscle enzyme elevation in Lowe syndrome is unknown.

As in our study, patients with Dent disease 2 were previously reported to be significantly shorter than those with Dent disease 1 [15]. In addition, the lack of growth hormone in patients with Dent disease 2 has been demonstrated elsewhere [27]. It remains unclear whether growth hormone should be given to patients with Dent disease, but we will need to evaluate laboratory data such as growth hormone and insulin-like growth factor-1 levels to determine this.

There were no significant differences in intellectual disability or autism spectrum disorder between the two groups in this study, but those with Dent disease 2 tended to show a higher prevalence of these abnormalities. In this study, the rate of intellectual disability or autism spectrum disorder was also high in Dent disease 1: 5 out of 69 patients (7%). Two of them were twins, and another two had mild delays with developmental quotients (DQ) of 78 and 82. However, this high rate of intellectual disability may have been coincidental. To obtain more definitive findings, it is desirable to increase the number of cases and to collect detailed scoring data such as on DQ or intelligence quotient (IQ).

In this study, we were able to identify a number of novel mutations in addition to those previously reported. Mutations in CLCN5 were detected at various points throughout the whole gene. Missense mutations accounted for 50%, nonsense mutations 26%, and splice-site mutations 15%. In our study, the rate of missense mutations was higher, and the rate of frameshift mutations was lower than in two previous reports [26, 28], but they were similar to those in a previous cohort from the Japanese population [17]. Among OCRL mutations, missense mutations were also the most common at 50%, which is similar to the rates in some previous reports [17, 26, 29]. Regarding the other types of mutation in OCRL, comparison was difficult since the number of cases was small. However, as indicated previously [14, 30], frameshift and nonsense mutations in Dent disease 2 were also specifically identified in exons 1–7.

As a limitation of this study, data may initially have been collected from questionnaires by the patient’s local doctor. In other words, there may have been a lack of objectivity regarding the classification of nephrocalcinosis and intellectual disability. Another problem is that the distinction between Dent disease and Lowe syndrome is unclear. In this study, patients with cataracts were ruled out from this study of Dent disease as having Lowe syndrome because, in our experience, most patients without cataracts do not have representative symptoms of Lowe syndrome, such as Fanconi syndrome and severe developmental delay. However, this definition includes patients without Fanconi syndrome or cataracts but with mild mental or developmental disability, such as children who are unable to keep up with their academic studies at school. As mentioned earlier, it is necessary to clearly differentiate the presence or absence of intellectual disability based on objective indicators such as IQ.

Despite these limitations, this work provides important findings regarding the differences in clinical manifestations, especially kidney function, between patients with Dent disease 1 and Dent disease 2 at a single institution.