Diverse Phenotypic Expression of NPHP4 Mutations in Four Siblings

SUMMARY

Nephronophthisis (NPHP) is an autosomal recessive disease characterized by renal tubular basement membrane disruption, interstitial fibrosis, and tubular cysts that progresses to end-stage kidney disease (ESKD). There are characteristic extrarenal manifestations. Mutations of more than thirteen genes have been identified that can cause NPHP. We herein report four siblings from a consanguineous family, who carried the same NPHP4 mutations but presented with different disease phenotypes ranging from enuresis nocturna to ESKD. Diluted urine and echogenic kidneys in ultrasound examination were consistent, which is typical for 100 % of the NPHP cases that have been described. Chronic kidney disease developed in the older two brothers. The observed phenotypic differences are likely to be related to environmental and epigenetic factors, oligogenic inheritance and modifier genes affecting their ages at presentation. NPHP should be considered as an important cause of ESKD in children, which insidiously progresses to ESKD and no specific therapy is available.

Introduction

Nephronophthisis (NPHP) is an autosomal recessive chronic tubulointerstitial nephritis that constitutes the most frequent genetic cause of the ESKD in children [1]. Thirteen causative genes have been identified. They encode proteins expressed in the primary cilia or centrosomes of renal epitelial cells. The NPHP1 encodes nephrocystin-1; NPHP2, inversin; NPHP3, nephrocystin-3; and NPHP4, nephrocystin-4 (nephroretinin) [2]. Three clinical variants have been described: infantile, juvenile and adolescent forms [3]. Juvenile NPHP is the most common form, which accounts for nearly 5–10% of sporadic cases of ESKD in children [4]. The first symptoms, polyuria and polydipsia, generally develop around 4–6 years of age [5]. Late symptoms are related to the progressive renal insufficiency including anemia, metabolic acidosis, nausea, anorexia and growth retardation. ESKD develops around 13 years but can also occur much later [6,7].

The gene product of NPHP1, nephrocystin, encodes a docking protein that interacts with components of cell-cell and cell-matrix signaling [8]. It also interacts with the gene product of NPHP4, nephrocystin-4. The mutations of NPHP1 or NPHP4 are associated with the juvenile type of NPHP [9]. The NPHP4 located on chromosome 1p36 encodes a 1,426 amino acid protein called nephrocystin-4/nephroretinin [10].

We herein report a family (F1270) with four children that carry the same homozygous NPHP4 mutation but exhibit heterogenous phenotypes. The first case was referred to our clinic with mild renal insufficiency at the age of 13. His brother had chronic kidney disease (CKD) stage IV when he presented. Two siblings have only enuresis at the ages of 13 and 10, respectively.

Case 1

A 13-year-old boy who referred to our clinic with the complaints of polyuria, polydipsia, enuresis and growth retardation. Past medical history was unremarkable except for one-year growth hormone (GH) therapy two years before. There was a parental consanguinity.

On admission, physical and eye examination were normal. Blood pressure (BP) was: 100/60 mmHg. Body weight (BW) was 34,5 kg (5–10 percentile), Height: 151 cm (25–50 percentile). Complete blood count (CBC) and peripheral smear were normal. He had mild metabolic acidosis (pH: 7.25, HCO₃: 19.6, BE: −4.0). Urinalysis showed a specific gravity (SG) of 1005, pH:7, 3–5 erythrocytes and 1–2 leukocytes/HPF. He had no proteinuria. Serum creatinine (Cr) was 1.1 mg/dl and creatinine clearance (CCr): 75 ml/min/1.73 m². Serum complement component (C) 3 and C4 levels were normal and anti-double stranded DNA (anti-dsDNA) and anti-nuclear antibody (ANA) were negative. His parathormone level was 316 pg/ml (N: 12–75) ferritin: 48,52 ng/ml (12–200). Abdominal ultrasonography (US) revealed normal sized, moderately echogenic kidneys (grade 2–3). There was no vesico-ureteral reflux.

Since he had stage II CKD, renal biopsy was performed for investigating underlying renal disease. Biopsy revealed chronic tubulointerstitial damage and periglomerular fibrosis which can be suggestive of NPHP (Fig. 1).

Figure 1.

Light microscopy: 12 of 27 glomeruli were sclerotic. Some of the nonsclerotic glomeruli showed periglomerular fibrosis, mild mesangial matrix increase and minimal mesangial hypercellularity. Interstitium was characterized by prominent fibrosis and accompanying mononuclear inflammatory cell infiltrate. Tubuli showed focal atrophy with prominent basement membrane thickening. Some tubuli were dilated.

His genetic examination gave negative result for NPHP1 mutation, but a homozygous mutation on NPHP4 (c. 2368 G>T (p.E790X)) was detected [9]. He has been on symptomatic treatment for CKD (calcitriol, calcium acetate and bicarbonate). In his last examination serum Cr level was 3.4 mg/dl and CCr was 27.5 ml/min/m² at the age of 20. The pedigree is shown in Fig. 2. The patient has 4 siblings: 2 affected younger sisters (Cases 2 and 3) and 1 affected older brother (Case 4).

Figure 2.

Pedigree. The parents are consanguineous in the 2nd generation. Solid symbols denote affected children; circles denote females; squares denote males.

Case 2

Patient 2 is the younger sister of the patient (13 years-old). She was evaluated for the complaints of polyuria, polidypsia, enuresis and growth retardation. Past medical history was unremarkable except for one year GH therapy, two years ago. On admission, her BP was 110/70 mmHg. BW was 35 kg (5–10 percentile) and height 157 cm (50–75 percentile). Laboratory tests including CBC, renal function tests, CCr were normal. Urine analysis was normal except a decreased SG (1006). The abdominal ultrasonography showed increased renal echogenicity (grade 1). The same mutation of the NPHP4 was detected.

Case 3

Patient 3 the youngest sibling, a 10-year-old girl, had the same complaints as case 2 except growth retardation. Past medical history was unremarkable. On admission, her physical and eye examination and BP were normal. BW was 40 kg (75–90 percentile) and height 145 cm (75–90 percentile). There was no anemia or uremia. 24-h urine protein excretion and CCr were normal. The abdominal ultrasonography showed increased echogenicity up to grade 1. Mutation analysis for NPHP4 revealed the same homozygous mutation.

Case 4

Patient 4 is the oldest sibling of this family (24 years old). He was diagnosed with CKD stage IV with unknown etiology in another center when he was 15. He had been enuretic. He had no growth retardation but bilateral small and echogenic kidneys. On admission, he had hypertension, diluted urine, mild proteinuria and anemia. Serum complement C3, C4 levels were normal and ANA, anti-DNA were negative. He had been biopsied when his creatinine level was 3.3 mg/dl. Biopsy revealed 30 glomeruli, 75% of which showed global sclerosis. Mesangial proliferation was present in 8 nonsclerotic glomeruli. Interstitial fibrosis and tubular atrophy were present in large areas. These findings were interpreted as chronic renal damage possibly secondary to chronic tubulointerstitial nephritis. Due to the progression to ESKD in 18 months, a preemptive kidney transplantation from his father was performed in another center. His mutation analysis for NPHP4 revealed the same homozygous mutation as in the other siblings.

Discussion

Nephronophthisis is a recessive cystic kidney disease leading to ESKD in the first three decades of life. The corresponding proteins encoded by the NPHP genes, the nephrocystins, are expressed in the kidney, brain and eye, and suggested to participate in the formation of complex protein networks [11]. The nephrocystins are being found at cell–cell junctions, centrosomes and primary cilia of renal epithelial cells and may have different functions at various sites due to the variety of extrarenal symptoms, such as retinal dystrophy, cerebellar hypoplasia, mental retardation, situs inversus, polydactyly and hepatic cysts [12].

Positional cloning and candidate gene approaches led to the identification of the causative 13 genes, NPHP 1,2,3,4,5,6,7,8,9,10,11,12,13 [2]. Clinical and histological features of patients with NPHP4 mutation were first reported by Hoefele et al [13]. After a 20-year follow-up of an index family with three affected siblings, mapping of the NPHP4 had been possible [14]. Our index patient had CKD stage II when he was 13 years-old and now, has stage IV CKD at the age of 21 years, wich is a typical clinical course of juvenile NPHP. However, his older brother developed ESKD when he was younger than him, 16.5 years-old. This may be partly explained by the success in medical management strategies for CKD (management of anemia, hyperparathyroidism and metabolic acidosis, providing patient adherence to medications and diet, maintaining a good volume control, etc.), and close follow up of our index patient, which may delay the progression of CKD to ESKD. The other two siblings, had normal renal function at the ages of 10 and 13. Diverse phenotypic expression of the disease among siblings who carry the same mutation in the NPHP4 (Fig. 2) can partly be explained by oligogenic inheritance, which shown in polycyctic kidney disease and NPHP [15,16]. Oligogenic inheritance in NPHP was confirmed by Hoefele et al. [15] in 2007 by detecting two mutations in one of the NPHP genes in combination with a third mutation in another NPHP gene in six different families with NPHP. In our study, NPHP1 mutation analysis was performed only in Case 1. After obtaining a negative result for NPHP1, NPHP4 was studied and mutation was detected in the index case first and then in three siblings. Since mutational analysis was not performed in all known NPHP genes, the possibility of oligogenicity can not be ruled out. Therefore a new mutational screening for all identified NPHP genes might be helpful for the explanation of intrafamilial variability in our cases [2]. Environmental factors might be another cause for the intrafamilial variability, since Case 1 and Case 4 grown up in a different city far away from their sisters. Alternatively, modifier genes segregating independently among the siblings may be postulated to explain the variable age of onset, as previously proposed by Hoefele [13].

The common initial complaints of our four siblings were urination symptoms like polyuria, polydipsia and enuresis, while anemia was not a prominent finding. Similarly, growth retardation was not detected in any of the siblings. This may partly be explained by GH therapy in the medical history of the two siblings. Echogenic kidneys in different severity, even if in the absence of the cysts in US examination, and diluted urine were the only consistent findings in all of our four siblings. If a child presents with polyuria, enuresis, growth failure, renal insufficiency without hematuria or proteinuria and normal BP, NPHP could be a possible diagnosis and molecular analysis should be proceeded. Whenever the NPHP1 mutation analysis is negative, NPHP4 and if possible all known genes should be screened for disease causing mutations, even if full blown clinical picture was absent. On the other hand, it should be kept in mind that, the frequency of homozygous or compound heterozygous NPHP4 mutations was quite low, 2.4% among 250 patients with NPHP [17]. However, genetic study is the only way to make a definitive diagnosis in this rare disease group.

In children with recessive mutations in the NPHP 1,2,3 and 4, retinitis pigmentosa occurs approximately 10% of all affected families. Since there is no genotype/phenotype correlation for extrarenal manifestations [17] in cases of NPHP4 mutation; such manifestation could be absent, as in our patients. There was no retinal finding in ophtalmologic examination in any of the siblings.

In conclusion, NPHP is an important cause of CKD in children, which insidiously progresses to ESKD. Since there is no specific therapy of the disease, early diagnosis, close follow-up, prevention and treatment of the complications of progressive renal insufficiency are the mainstays of the management.