Pediatric Nephrology

, Volume 22, Issue 7, pp 975–980

OCRL1 mutations in patients with Dent disease phenotype in Japan

Authors

    • Department of PediatricsGraduate School of Medicine,The University of Tokyo
    • Department of PediatricsFaculty of Medicine, The University of Tokyo
  • Kandai Nozu
    • Department of PediatricsKobe University Graduate School of Medicine
  • Rashmi Iyengar
    • Department of PediatricsGraduate School of Medicine,The University of Tokyo
  • Xue Jun Fu
    • Department of PediatricsKobe University Graduate School of Medicine
  • Masafumi Matsuo
    • Department of PediatricsKobe University Graduate School of Medicine
  • Ryojiro Tanaka
    • Division of NephrologyHyogo Prefectural Children’s Hospital
  • Kazumoto Iijima
    • Department of NephrologyNational Center for Child Health and Development
  • Emiko Matsui
    • Department of PediatricsGraduate School of Medicine,The University of Tokyo
  • Yutaka Harita
    • Department of PediatricsGraduate School of Medicine,The University of Tokyo
  • Jun Inatomi
    • Department of PediatricsGraduate School of Medicine,The University of Tokyo
  • Takashi Igarashi
    • Department of PediatricsGraduate School of Medicine,The University of Tokyo
Original Article

DOI: 10.1007/s00467-007-0454-x

Cite this article as:
Sekine, T., Nozu, K., Iyengar, R. et al. Pediatr Nephrol (2007) 22: 975. doi:10.1007/s00467-007-0454-x

Abstract

Three distinct OCRL1 mutations in three patients with the Dent disease phenotype are described. All the patients manifested an extremely high degree of low-molecular-weight proteinuria and showed no ocular abnormalities or apparent mental retardation. Urinalysis and blood chemistry showed no findings suggestive of Fanconi syndrome with renal tubular acidosis. Mutations in CLCN5 were ruled out. The mutations identified in OCRL1 are one frame-shift mutation (I127stop) and two missense mutations (R301C and R476W). R301C and R476W mutations might be hot spots in OCRL1, which develop very similar phenotypes as Dent-2.

Keywords

Dent diseaseLowe syndromeOCRL1CLCN5

Abbreviations

β2MG

β2 microglobulin

TPR

tubular reabsorption of inorganic phosphate

FEK

fractional excretion of potassium

IP

inorganic phosphate

Introduction

Dent disease is an X-linked disorder characterized by low-molecular-weight proteinuria, hypercalciuria, nephrocalcinosis, calcium nephrolithiasis, and the development of renal insufficiency after the age of 30∼40 years [1, 2]. Some patients develop rickets; however, other systemic clinical manifestations of this disease are not known. In 1996, a gene responsible for Dent disease was identified, i.e., CLCN5, which encodes voltage-dependent chloride channel 5 (CLC-5) [3]. CLC-5 is located in the endosomes of proximal tubular cells and is also expressed in the thick ascending limb of Henle and collecting duct cells [4]. CLC-5 is supposed to play important roles in the acidification of intraendosomal compartments and endosomal recycling. CLC-5 was recently revealed to function as a chloride/proton antiporter [5]. CLCN5 knockout mice manifest phenotypes nearly identical to the clinical features of Dent disease [6, 7]. However, previous genetic analyses by Hoopes et al. showed that only 60% of patients clinically diagnosed as having Dent disease possess CLCN5 mutations [8]. Thus, a genetic heterogeneity has been suggested in Dent disease [8].

Lowe syndrome is characterized by (1) ocular abnormalities, particularly congenital cataract, (2) mild to severe mental retardation, and (3) generalized solute transport dysfunction of proximal tubular cells, i.e., Fanconi syndrome including renal tubular acidosis (RTA). The gene responsible for Lowe syndrome is OCRL1, which is also localized in the X-chromosome [9]. OCRL1 encodes phosphatidylinositol 4,5-bis-phosphate (PtInsP2) 5-phosphatase, and this protein is predominantly localized in the Golgi complex, lysosomes, and endosomes [10, 11]. OCRL1 is localized ubiquitously in human tissues, including those of the eyes, kidneys, and brain [10, 11]. The pathophysiological mechanisms by which mutations in OCRL1 cause Lowe syndrome are not fully understood; the perturbation of trans Golgi network trafficking and dysregulation of actin dynamics are the mechanisms proposed at present [10, 11].

In 2005, Hoopes et al. identified OCRL1 as a second causative gene responsible for Dent disease, using a genetic linkage analysis and direct sequencing [12]. The clinical manifestations of Dent disease are distinct from those of Lowe syndrome despite the common renal proximal tubular dysfunction. Regarding tubular dysfunction, RTA is commonly observed in patients with Lowe syndrome but not in those with Dent disease, whereas hypercalciuria and nephrocalcinosis are common features in Dent disease but not in Lowe syndrome. Thus, the clinical manifestations of each disease are quite different, and the result found by Hoopes et al. was unexpected. At the moment, Dent disease caused by a CLCN5 mutation is assigned OMIN No. 300009 (Dent disease-1), whereas that caused by an OCRL1 mutation is assigned OMIN No. 300555 (Dent disease-2).

In this paper, we report three patients with mutations in OCRL1 but not in CLCN5 showing an apparent Dent disease phenotype.

Methods

Informed consent for participation in this study was obtained from patients and their family members after the purpose, methods, and potential risks were thoroughly explained. This study was approved by the Ethics Committee for Analysis of the Human Genome of Tokyo University Hospital. The inclusion of patients in this study required the following criteria to be met:
  1. 1.

    Existence of extremely high urinary β2 microglobulin (β2MG) level

     
  2. 2.

    Absence of histories or clinical data indicative of underling disease, such as mitochondrial disorders, or ingestion of nephrotoxic substances

     
  3. 3.

    Absence of cataract, which was confirmed by slit-lamp examinations by ophthalmologists

     
  4. 4.

    No apparent neurological or mental abnormalities suggestive of Lowe syndrome, such as apparent mental retardation, muscular hypotonus, or apparent behavioral abnormalities.

     

All patients in this study were clinically diagnosed as having Japanese Dent disease by pediatric nephrologists and were referred to our institute for genetic examination. Within the last 2 years, we have performed direct sequencing of all exons and exon–intron boundaries of genomic DNA of 18 patients with Dent disease, of whom eight patients showed no mutations in CLCN5. In this study, we performed direct sequencing of all exon and exon–intron boundaries of OCRL1 using genomic DNA extracted from peripheral blood leukocytes of these eight patients. The primers used for the genomic sequencing of OCRL1 are the same as those described in a previous report [9].

Case reports

Among the eight patients analyzed in this study, genomic DNA sequencing revealed OCRL1 mutations in three patients. The essential clinical findings and laboratory data of these three patients are shown in Table 1, along with the data of the mutations in OCRL1. Other clinical histories and presentations of each case are described below.
Table 1

Clinical findings and laboratory data of three patients, along with data of OCRL1 mutations

 

Patient 1 (11 years)

Patient 2 (15 years)

Patient 3 (5 years)

aBrother of Patient 3 (3 years)

Mutations in OCRL1

I127X

R301C (de novo)

R476W

R476W

Family history

(−)

(−)

(+) (Fig. 1)

(+) (Fig. 1)

Clinical manifestations

 Ocular findings

  Cataract/glaucoma

(−)/(−)

(−)/(−)

(−)/(−)

(−)/(−)

 Neurological manifestations

  Intelligence level

Normal

Normal except communication skills

Normal

Normal

  Behavioral abnormalities

(−)

(−)

(−)/(+)

(−)

 Nephrocalcinosis or urolithiasis

(+)

(−)

(−)

(−)

 Bone disease

(−)

(−)

(−)

(−)

Urinalysis and tubular function Reference value

  Proteinuria (−) or <15 mg/dl

46 mg/dl

3+ (508 mg/dl)

3+ (254 mg/dl)

3+ (174 mg/dl)

  Hematuria (−) or <5/HPF

(−)

(+) RBC 5-9/HPF

(−)

(−)

  Urinary β2MG (μg/l) <230 mg/l

103,183

15,444

22,316

161,187

  Aminoaciduria (−)

N.D.

(−)

(−)

N.D.

  Glucosuria (−)

(−)

(−)

(−)

(−)

  Urinary Ca/Cr (mg/mg) <0.21

0.75

0.12

0.43

0.31

  TPR (%) 85∼98

N.D.

75

86

82

  FEK (%) 5∼16

14.2

8.3

5.2

8.6

  Plasma HCO3 (mEq/l) 22∼26

26.1

26

22.5

20.2

  Required alkali therapy (−)

(−)

(−)

(−)

(−)

Blood biochemistry Reference value

    

  Na (mEq/l) 136∼147

141

139

140

142

  K (mEq/l) 3.6∼5.0

3.5

4.2

4.2

4.2

  C1 (mEq/l) 98∼109

106

105

107

109

  Cr (mg/dl)

0.46

1.11

0.4

0.34

  BUN (mg/dl) 6∼20

12.9

22

12

13

  UA (mg/dl) 3.7∼7.0 (male)

5.8

7.3

2.5

2.1

  IP (mg/dl) 3.6∼5.8

3.2

3.2

5.4

4.2

  CK (IU/l) 52∼305 (male)

407

446

223

303

  LDH (IU/l) 280∼550 (male)

340

267

390

295

Normal level of serum Cr is markedly age-dependent and is not included in this table.

N.D. Not determined

aFor comparison of phenotype of two brothers possessing the same OCRL1 mutation (R476W).CK Creatinine kinase, BUN Blood urea nitrogen, LDH Lactate dehydrogenase, HPF High power field, UA Uric acid

Clinical and laboratory data of the remaining five patients, in whom no mutation was identified in either CLCN5 and OCRL1, are as follows: All five patients manifested remarkably high levels of urinary β2MG. Hypercalciuria was identified in three patients. Nephrocalcinosis and/or urolithiasis were identified in four patients. Microscopic hematuria or renal insufficiency was not present in any of the five patients. Hypophosphatemia was noted in one patient. As a result, three patients out of five met the criteria of Dent disease defined by Hoopes et al. [8].

Patient 1

Patient 1, an 11-year-old boy, was incidentally diagnosed as having low-molecular-weight proteinuria when he was a neonate. Thereafter, he was routinely examined because of continuing proteinuria. He is intelligent and achieved good results in school examinations. He had neither cataract nor glaucoma. He has no history of bone fracture or evidence of rickets. At 11 years of age, his height was 125.3 cm (−2.6 SD). An ultrasound sonography of the kidney revealed nephrocalcinosis. Hypercalciuria [urine Ca (mg)/Cr (mg): 0.75] was noted. Urinalysis and serum blood chemistry showed no signs of Fanconi syndrome except the existence of an extraordinarily high β2MG level in the urine (103,183 μg/l). His serum bicarbonate level was 26 mEq/l with no alkali therapy. The serum CK level was slightly elevated (407 IU/l), although there was no history suggestive of any muscular diseases.

Patient 2

In patient 2, a 15-year-old boy, proteinuria was detected at 7 years of age in an annual urine-screening program. He was followed up as a chronic nephritis patient because of continuing hematuria and proteinuria. Renal biopsy showed minor glomerular abnormalities. He showed neither cataract nor glaucoma. His school achievement in mathematics was higher than average but was poor in Japanese literature. His family was reluctant to allow him to undergo objective intellectual tests. The evaluation of a pediatric neurologist on this patient through interview is as follows: The overall intelligence level of this patient seems to be normal. However, the communication skills appear to be lower than the normal range. Apparent behavioral abnormalities are not observed. Ultrasound sonography did not detect nephrocalcinosis or renal stones, and no hypercalciuria was present. His urinary β2MG was 15,444 μg/l. His blood gas analysis did not show metabolic acidosis (plasma bicarbonate level: 26 mEq/l). Blood analysis revealed mild impairment of renal function (Cr level: 1.11 mg/dl) and a slightly elevated CK level (446 IU/l). Height and weight were 162 cm (−1.27 SD) and 80 kg (+1.86 SD), respectively, when he is 16 years old. The estimated glomerular filtration rate (GFR) of patient 2 calculated using Schwartz equation is 90 ml/min/1.73 m2. A paternal uncle developed end-stage renal failure without any apparent features of systemic diseases. Importantly, the patient’s father does not show any renal symptoms, including low-molecular proteinuria: his renal function is normal. We consider that this family history is not related to the development of the Dent phenotype in patient 2.

Patient 3

In patient 3, a 5-year-old boy, proteinuria was detected at 3 years of age in a kindergarten urine-screening program, and the presence of an extremely high degree of low-molecular-weight proteinuria (urine β2MG: 22,316 μg/l) was noted. He showed no ocular abnormalities. Height and weight were 110 cm (±0 SD) and 18 kg (−0.3 SD), respectively, when he was 6 years old. His blood biochemistry and blood gas analysis disclosed normal electrolyte levels, renal function, and CK level, and the absence of RTA (Table 1). Although there were no signs of nephrocalcinosis by ultrasound sonography, he had hypercalciuria [urine Ca (mg)/Cr (mg): 0.43]. He did not show apparent mental retardation. From these clinical and laboratory data, he was diagnosed as having Dent disease. After we determined the presence of an OCRL1 mutation in this patient, we asked his mother questions in detail regarding behavioral abnormalities. His mother noticed that this patient sometimes adheres to certain types of play in daily life. There is an apparent family history in this patient (Fig. 1). His younger brother shows a similar clinical phenotype and was revealed to have the same mutation in OCRL1. All three male siblings of his mother show a similar phenotype (very high level of low-molecular-weight proteinuria). All uncles of patient 3 are working in companies without any difficulties in their ability to work. Their renal functions are normal. There are no extrarenal manifestations characteristic in Lowe syndrome, such as debilitating arthropathy, in any of the three uncles.
https://static-content.springer.com/image/art%3A10.1007%2Fs00467-007-0454-x/MediaObjects/467_2007_454_Fig1_HTML.gif
Fig. 1

Pedigree of patient 3. Affected individuals, in whom low-molecular-weight proteinuria was detected, are shown as closed boxes. The younger brother shows similar clinical symptoms to patient 3, and possesses the same OCRL1 mutation (R476W). A genetic analysis of three uncles was not performed. None of the affected individuals manifest apparent mental retardation or cataract

Results of DNA sequencing

Results of the DNA sequencing of the three patients are shown in Fig. 2. The mutations identified in OCRL1 are one frame-shift mutation (patient 1: I127stop) and two missense mutations (R301C in patient 2 and R476W in patient 3).
https://static-content.springer.com/image/art%3A10.1007%2Fs00467-007-0454-x/MediaObjects/467_2007_454_Fig2_HTML.gif
Fig. 2

Results of polymerase chain reaction (PCR) direct sequencing of three patients

A frame-shift mutation identified in patient 1 (8-base deletion in exon 5 resulting in I127stop) disrupted most of the latter portion (nearly 85 %) of OCRL1. The R301C mutation identified in patient 2 was identical to that described by Hoopes et al. [12]. Sequencing of the mother of patient 2 did not identify this mutation, which was revealed to be de novo. The R476W mutation in patient 3 was novel. We tested 76 normal control samples of genomic DNA, and none showed these nucleotide and amino acid changes, indicating that the R476W mutation is not a single-nucleotide polymorphism (SNP). The R476W mutation was also identified in the younger brother of patient 3, who had a similar clinical phenotype.

In all patients, an objective IQ test was not performed, because consent was not obtained from their parents. The essential clinical findings and laboratory data are described in Table 1, along with data of the mutations in OCRL1.

Discussion

One report indicates that mutations in CLCN5 are responsible for 60% of the clinically distinct Dent disease [8]. In Japan, we have performed a genetic analysis of CLCN5 on 94 unrelated patients with Dent disease and identified CLCN5 mutations in 56 patients (59.6%) (unpublished data, and references [13, 14]), which is completely the same result of the previous one. Thus, Dent disease is considered to be a genetically heterogeneous disorder. In 2005, Hoopes et al. identified OCRL1 as a second causative gene for Dent disease [12]. They identified five distinct mutations in OCRL1 in 13 families with Dent disease in which no mutations were detected in CLCN5. Their results indicate that a relatively high incidence of OCRL1 mutations exists in Dent disease. In this study, three mutations were identified in the eight patients examined (38% in those patients without a CLCN5 mutation).

The mutation identified in patient 1, i.e., I127stop, disrupts the formation of a large portion of OCRL1, and it is most probable that the protein produced in patient 1 lost its PtInsP2 5-phosphatase activity. The mutation in patient 2 is identical to a previously reported one. The patient described by Hoopes et al. [12] possessing the same mutation as patient 2 (R301C) shows mild mental retardation, and patient 2 also has poor school achievement in Japanese literature; this might be related to the same mutation in OCRL1. The amino acid residue in which the mutation was identified in patient 3, i.e., 476 arginine (R), is widely preserved among various species, including humans, mice, rats, dogs, and monkeys. In addition, tryptophan (W) is a neutral nonpolar amino acid, whereas arginine is a basic polar amino acid. We confirmed that the R476W mutation is not an SNP by analyzing 76 control genomic DNA sequences. In addition, the brother of patient 3 showed very similar phenotypes to those of patient 3 (Table 1). These data suggest that the R476W mutation alters the functional characteristics of OCRL1.1

The reason OCRL1 mutations cause two clinically distinct phenotypes remains to be elucidated. The most probable explanation of why OCRL1 mutations cause only renal manifestations is the compensation of PtInsP2 5-phosphatase activity by other proteins. OCRL1 is ubiquitously distributed among nearly all human tissues [10], whereas the clinical manifestations of Lowe syndrome are restricted to only those of the kidneys, eyes, and brain. Humans have a highly homologous gene to OCRL1, namely, the inositol polyphosphate 5-phosphatase gene (Inpp5b), which encodes phosphatidylinositol bisphosphate 5-phosphatase [1517]. This homologous phosphatase may compensate for the abrogation of OCRL1 function in several tissues. If so, there is a possibility that Inpp5b is upregulated and compensates for the abrogation of OCRL1 function in the eyes, brain, and kidneys in the patients described in this paper.

At least in the kidneys, Dent disease and Lowe syndrome show similar proximal tubular defects, i.e., impaired reabsorption of several solutes. The pathophysiological mechanisms of both disorders in proximal tubular cells are not fully understood, but both CLC-5 and OCRL1 are expressed in endosomes and thought to be related to the recycling of multiligand receptors, i.e., megalin and cubilin [18]. These functions of both proteins could cause similar defects, at least in the kidneys.

The three patients described in this study lack the cardinal manifestations of Lowe syndrome, i.e., congenital cataract and apparent mental retardation. In addition, the types of tubular dysfunction in these patients are distinct from those observed in Lowe syndrome patients. Although most Lowe syndrome patients manifest proximal renal tubular acidosis (pRTA) requiring alkali therapy, our three patients showed no pRTA. In contrast, patients 1 and 3 showed hypercalciuria, and patient 1 showed nephrocalcinosis, both of which are common in Dent disease. Thus, the phenotypes of these three patients are quite similar to those of patients with Dent disease, and we diagnosed all three patients as having Dent disease. However, when we studied in detail the clinical manifestations and data of the three patients described in this study, some points that are not common in Dent disease were recognized. Patients 1 and 2 showed a mildly elevated serum CK level; patient 3 manifested some behavioral abnormalities. These symptoms are rather compatible with Lowe syndrome. The present study might expand the clinical spectrum of Lowe syndrome; in other words, the existence of a “very mild Lowe syndrome with an OCRL1 mutation” without cataract, a cardinal sign of classical Lowe syndrome.

In conclusion, we described three distinct mutations in OCRL1 in patients clinically diagnosed as having Dent disease. OCRL1 is the second gene found to be responsible for the development of Dent disease.

Footnotes
1

During revision of this manuscript, Utsch et al. published novel OCRL1 mutations in patients showing Dent phenotype, in which R476W mutation was present [19]. R301C and R476W mutations might be hot spots in OCRL1, which develop very similar phenotypes as Dent-2.

 

Acknowledgments

This work was supported by a grant from the Japanese Ministry of Education, Culture, Sports, Science and Technology (grant 15591089). The authors are grateful for the helpful comments of Prof. Kiyoshi Hayasaya of Yamagata University and Prof. Scheinman of Upstate Medical University.

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© IPNA 2007