NPHS2 R229Q Polymorphism in Steroid Resistant Nephrotic Syndrome: Is it Responsive to Immunosuppressive Therapy?

Abstract

Steroid-resistant nephrotic syndrome (SRNS) patients with NPHS2 gene mutations have been reported as non-responsive to immunosuppressive therapy. Inter-ethnic differences can have influence over the frequency of mutations. The present study was undertaken to find out the incidence and treatment response. Mutational analysis of NPHS2 gene was performed in 20 sporadic idiopathic SRNS, 90 steroid-sensitive nephrotic syndrome (SSNS) and 50 normal controls. NPHS2 gene analysis showed R229Q polymorphism in six SRNS (30%), four SSNS (4.4%) and 13 controls (26%). The polymorphism (G→A) showed Hardy–Weinberg distribution and risk allele (G) had strong association with the disease (odds ratio 3.14, 95% CI 1.33–7.43) than controls. Five cases of SRNS having polymorphism showed partial remission to cyclosporine and prednisolone. Overall, partial remission was achieved in 14(70%), complete remission in four (20%), one(5%) patient had no response and one(5%) died. Thus, NPHS2 gene showed R229Q polymorphism and patients achieved partial remission to therapy.

mutations, nephrotic syndrome, NPHS2 gene, treatment response

Introduction

About 10–15% of nephrotic syndrome in children does not respond to steroid and is termed as steroid-resistant nephrotic syndrome (SRNS) [1]. Renal histology shows focal and segmental glomerulosclerosis (FSGS), minimal change disease (MCD) or other glomerulopathies. The incidence of FSGS has shown an increasing trend in these patients [2, 3]. The therapeutic response to immunosuppressive treatment also depends upon their genetic heterogeneity; as patients having genetic mutations do not achieve remission with immunosuppressive drugs [4, 5].

The podocin is a protein localized at the slit diaphragm and required for structural organization of glomerular filtration barrier. Patients having NPHS2 gene, encoding for podocin, mutations have structurally defective podocytes or basement membrane lacking permselectivity and cause proteinuria [6]. There is a wide variability in the incidence of NPHS2 gene mutations among different populations of the world ranging between 6.4% and 28% of sporadic SRNS from Europe, Middle East and North America [4, 7–9]. Conversely in Asia, the incidence was found to be either low as 4.3% in Chinese [10] and 4% in Indian [11] or not observed in Korean [12] and Japanese [13] populations.

In view of marked heterogeneity in the incidence of NPHS2 gene mutations in SRNS, the present study was undertaken with objectives to detect the NPHS2 mutations in our patients of sporadic SRNS, and also to find out the response to immunosuppressive therapy.

Materials and Methods

Selection of subjects

The study subjects were selected from the patients attending to Out Patients Department and/or Pediatric Nephrology Clinic of Pediatrics during the period of October 2011 to August 2013. A total of 20 patients of sporadic idiopathic SRNS, aged 1–14 years, were enrolled. Another 90 cases of steroid-sensitive nephrotic syndrome (SSNS) and 50 normal healthy children in the same age group were also included to serve as controls. The protocol of the study was approved by the Institute Ethical Committee and it was in adherence with the Declaration of Helsinki. Informed consent was taken from parent of each study subject.

Treatment protocol

The drug prednisolone was given at the dose of 60 mg/m²/day or 2 mg/kg/day and the patients who achieved remission (proteinuria 0/trace by heat precipitation method or urine protein/creatinine ratio of <0.2 for consecutive 3 days) within 4 weeks of start of treatment were defined as SSNS. The patients who did not achieve remission by 4 weeks of prednisolone were classified as SRNS. The SRNS patients were subjected to renal biopsy and the tissues were examined under the light and immunofluorescence microscopy by the same renal pathologist.

The treatment protocol of SRNS was as per Indian Society of Paediatric Nephrology Guidelines [14]. Cyclosporine 4–6 mg/kg/day in two divided doses or cyclophosphamide 500–750 mg/m² as intravenous infusion once every month for 6 months were given along with prednisolone (40 mg/m² or 1.5 mg/kg on alternate days for 4 weeks, and then in gradual tapering doses with minimum 0.5–0.75 mg/kg for 12–18 months). Mycophenolate mofetil (25 mg/kg/day) and oral cyclophosphamide (2 mg/kg/day) were also used. Other measures included salt restriction, furosemide (1–2 mg/kg/day) and ramipril (0.2 mg /kg/day). Patients were observed for response as complete (urine protein/creatinine ratio <0.2 mg/mg or urine protein absent/traces by heat precipitation test) or partial remission (urine protein/creatinine ratio between 0.2 and 2 or urine protein ++ or more by heat precipitation test for consecutive 3 days) after start of therapy.

Mutation analysis

The NPHS2 gene mutations were tested in all the exons in patients of SRNS, SSNS and controls. Five millilitres of blood samples from patients, controls and their parents were collected in heparinised sterile vials through venepuncture and stored at −20°C. Thereafter, following procedures were undertaken for mutational analysis.

Isolation of DNA

Isolation of genomic DNA was accomplished by salting-out procedure from peripheral blood lymphocytes.

Genomic DNA quantification

After isolation of DNA, the genomic DNA was quantified spectrophotometrically using ‘Nanodrop’, which quantitates and determines the purity of DNA (260/280 ≥1.8) and 1 μl of the genomic DNA was used.

Gel electrophoresis

To check the quality of the DNA, the gel electrophoresis was done to confirm the integrity and properly dissolved in TE (Tris–EDTA buffer).

Primer designing

Done with the help of software on link: Primer 3 Input (version 0.4.0)

PCR amplification of DNA

The good qualities of DNA were used to amplify by using gene specific pair of primer.

PCR master mix composition

Each PCR reaction was performed in a final 15 µl reaction volume, where 100 ng of DNA template, 1 µl of each primer, 2× PCR master mix, magnesium chloride, MQ water and DNA template were added.

Restriction digestion of NPHS2 gene

After agarose gel electrophoresis, the remaining PCR amplified products were used for restriction digestion.

ClaI digestion of exon 5 PCR products (545 bp) normally produces two fragments of 364 and 181 bp (Fig. 1a). A R229Q mutation (G→A transition at nucleotide 755) was detected by a loss of the ClaI digestion site. Restriction digestion product of exon 5 PCR (545 bp) showed partial digestion, digestion of one allele into 364 and 181 bp, while the other allele was undigested (545 bp) because of a loss of the ClaI digestion site (heterozygous mutation) [(Fig. 1b), and detected only in exon 5].

Fig. 1.

(a) Restriction digestion- ClaI digestion of Exon 5 PCR product (545 bp) produced two fragments of 364 and 181 bp. (b) Restriction digestion product of Exon 5 PCR (545 bp) showed partial digestion, digestion of one allele into 364 bp and 181 bp, while other allele was undigested (545 bp) showing heterozygous mutation.

Statistical analysis

The data were analysed using SPSS 16.0 version software. Student’s t-test was applied for comparison of variables showing normal distribution and Mann–Whitney U test for non-Gaussian distribution. Chi-square test was applied for comparison of proportions. Odds ratio was calculated to find out the risk allele for the disease. A P-value <0.05 was considered as significant.

Results

The clinical and biochemical parameters of SSNS and SRNS are presented in Table 1. The SRNS patients were significantly older in age and had higher mean weight, height, systolic and diastolic blood pressure and median urine protein/creatinine ratio in comparison to SSNS. There were significantly more number of males in SSNS than SRNS group. The serum sodium, potassium, urea, creatinine, total protein, albumin, cholesterol and glomerular filtration rate (GFR) were comparable between the two groups.

Table 1

Clinical and biochemical parameters of study subjects

Parameters	SSNS n = 90	SRNS n = 20	P-value
Age(years)	4 (3,7)	10 (5,12)	<0.001a
Male/Female	68/22	10/10	0.023b
Systolic BP (mmHg)	105.5 ± 10.5	119.8 ± 14.6	<0.001c
Diastolic BP (mmHg)	70.5 ± 8.5	80.5 ± 10.2	<0.001c
Weight (kg)	18.0 ± 6.4	22.9 ± 8.3	0.004c
Height (cm)	102.2 ± 16.2	115.5 ± 24.5	0.003c
Sodium (meq/l)	135.5 ± 6.15	134.9 ± 5.5	0.66c
Potassium (meq/l)	4.0 ± 0.7	4.28 ± 1.1	0.172c
Urea (mg/dl)	26.5(22.2,36.8)	27(22.5,37.2)	0.807a
Creatinine (mg/dl)	0.8(0.5,1)	0.8(0.5,1)	0.139c
Serum total protein (g/dl)	4.7 ± 1.1	4.3 ± 0.74	0.095c
Serum albumin (g/dl)	2.0 ± 0.7	1.7 ± 0.4	0.173c
Serum cholesterol (mg/dl)	416.6 ± 177.1	514.4 ± 225.5	0.085c
Protein-creatnine ratio (mg/mg)	21.2 (12.9,25.2)	32.0 (8.0,58.3)	0.03a
GFR ml/min/1.73m2	109.3 ± 39.2	99.5 ± 42.0	0.31c

n, number of cases.

^aMann –Whitney U test.

^bChi-Square test.

^cStudent’s t-test.

Detailed demographic, renal function and mutation status of SRNS patients are presented in Table 2. The mean age, systolic and diastolic blood pressure, serum urea, creatinine and GFR were comparable in SRNS with and without R229Q variant. The histopathology demonstrated MCD in three, FSGS in two and membranoproliferative glomerulonephritis in one patient having R229Q polymorphism.

Table 2

Demographic, renal function and mutation status in SRNS patients

Case no.	Age at presentation (years)	Gender	Blood pressure (mm Hg)	Hypertension	Hematuria	Serum urea (mg/dl)	Serum creatinine (mg/dl)	GFR (ml/min/1.73 m2)	Histopathology	Response	NPHS2 mutation
1.	4	Male	120/70	Yes	No	24	0.4	123.7	MCD	Partial	–
2.	13	Female	110/72	No	No	20	0.79	92.1	FSGS	Partial	–
3.	2	Male	114/84	Yes	No	40.5	0.3	141.1	MCD	Complete	–
4.	10	Female	120/70	No	No	68	1.02	75.4	MPGN	Partial	–
5	11	Female	110/70	No	No	24.5	0.69	98.5	MCD	No	–
6	10	Male	140/96	Yes	No	29.4	0.4	169.1	FSGS	Partial	–
7	1	Male	92/58	No	Yes	20.8	0.4	93.5	FSGS	Complete	–
8	12	Female	120/80	No	No	31	0.97	86.3	FSGS	Partial	–
9	12	Female	136/90	Yes	No	21	0.79	104.4	MPGN	Complete	–
10	9	Female	130/92	Yes	No	31	0.5	149.6	DMP	Partial	–
11	4	Male	94/70	No	No	40	1.0	51.7	FSGS	Complete	–
12	11	Female	150/92	Yes	No	33	1.08	66.7	FSGS	Partial	–
13	9	Female	102/74	No	No	23.8	0.8	86.9	MCD	Complete	–
14	7	Female	96/68	No	No	36	1.0	92.3	MCD	Complete	–
Mean ± SD	8.2 3.9		116.7/77.5 17.7/11.4			31.6 12.5	0.72 0.27	102.2 32.8
15	10	Male	100/70	No	Yes	26	0.9	92.1	MPGN	Partial	R229Q
16	12	Male	106/76	No	No	42	0.7	88.2	MCD	Partial	R229Q
17	4	Male	94/68	No	No	32	1.0	90.4	MCD	Partial	R229Q
18	5	Female	98/70	No	No	19.6	0.7	93.7	MCD	Partial	R229Q
19	8	Male	106/72	No	Yes	28	0.82	85.4	FSGS	Partial	R229Q
20	3	Female	132/80	Yes	No	75	1.1	95.5	FSGS	Death	R229Q
Mean ± SD	7.0 3.5		106/72.6 13.5/4.5			37.1 19.9	0.87 0.16	90.8 3.7
Pa	0.737		0.912/0.907			0.288	0.069	0.891

^aStudent’s t- test.

MPGN, Membranoproliferative glomerulonephritis.

DMP, Diffuse mesangial proliferation.

The mutational analysis of NPHS2 demonstrated heterozygous mutation of R229Q in exon 5 in six SRNS (30%), four SSNS (4.4%) and 13 controls (26%). Heterozygous R229Q mutation (G→A transition at nucleotide 755) was detected by a loss of ClaI digestion site. Genotype and allele frequency of both cases and controls are presented in Table 3. It showed association only under dominant model (P = 0.0047). The polymorphism (G→A) had Hardy–Weinberg distribution and the risk allele (G) showed strong association with the disease (odds ratio of 3.14, 95% CI 1.33–7.43) than controls.

Table 3

Genotype and allele frequency

Genotype/Allele	Case frequency n = 110a (%)	Control frequency (%) n = 50
GG	100 (90.91)	37 (74)
GA	10 (9.09)	13 (26)
AA	–	–
G	210 (95.45)	87 (87)
A	10 (4.55)	13 (13)

n, number of cases.

^a90 steroid sensitive +20 steroid-resistant nephrotic syndrome.

Treatment response (Table 4)

Six cases of SRNS showed R229Q polymorphism; of these five had partial remission and one died during follow up; treated with cyclosporine and prednisolone in FSGS and MCD and prednisolone along with ramipril in membranoproliferative glomerulonephritis. Serum creatinine levels remained stable during the therapy period in all the patients. Overall, partial remission was achieved in 14(70%), complete remission in four (20%), one (5%) case had no response and one (5%) died.

Table 4

Histopathology and treatment response in steroid resistant nephritic syndrome

Histopathology	Treatment	Follow up (months)	Outcome
			Remission
			No	Partial	Complete	Death
Focal and segmental glomerulosclerosis (FSGS, n = 8)	Prednisolone + Cyclosporine + Ramipril (n = 7)	21.4 ± 2.4 (18–24)	–	4a	2	1
	Prednosilone + MMF + Ramipril (n = 1)			1
Minimal change disease (MCD, n = 8)	Prednisolone + Cyclosporine + Ramipril (n = 7)	13.1 ± 5.3 (7–22)	1	5a	1	–
	Prednisolone+ Cyclosporine + Ramipril for 9 months (n = 1) ↓ Prednisolone + Mycophenolate mofetil for 18 months ↓ Prednisolone + Cyclophosphamide For 12 weeks	30		– – 1	– – –	– – –
Membrano-proliferative glomerulonephritis (n = 3)	Prednisolone+Ramipril	9 ± 1 (8–10)	–	2a	1	–
Diffuse mesangial proliferation (n = 1)	Intravenous Cyclophosphamide + Prednisolone + Ramipril	9	–	1		–
Total (n = 20)	–	–	1(5)	14(70)	4(20)	1(5)

n, number of cases, data mean ± SD (range), MMF, mycophenolate mofetil.

^aTwo of FSGS, three of MCD and one of membranoproliferative glomerulonephritis had R229Q polymorphism.

Discussion

The SRNS patients were significantly older in age and had higher mean blood pressure and median protein/creatinine ratio than SSNS. Other authors [3, 7] have reported that SRNS patients had delayed onset of the disease. However, Ruf et al. [5] found lower median age for SRNS in comparison to SSNS. The significantly higher blood pressure was due to presence of significant histopathological lesions in which hypertension is more commonly associated. Higher level of proteinuria in SRNS has been observed in our previous observation also [15]. Bazzi et al.[16] reported higher level of daily proteinuria in FSGS (7 g) in comparison to MCD (4.9 g). This could explain higher level of proteinuria in our SRNS, as 40% of the cases had FSGS histology.

The studies from Asia showed either no [12] or very low incidence rate (3.4–4.3%) of NPHS2 mutations [10, 11, 17]. We found simple heterozygous missense nucleotide substitution G→A transition. Heterozygous mutations of R229Q were found in only exon 5. The polymorphism (G→A) shows Hardy–Weinberg distribution with risk allele (G) having strong association with the disease. Weber et al. [9] reported homozygous R229Q mutation with higher frequency in nephrotic children than general populations (5.13% vs. 3.75%).

Tsukaguchi et al. [18) reported that its allele frequency in normal healthy population was 3.6%. Authors also found that affected individuals were compound heterozygous for a non-conservative R229Q amino acid substitution and concluded that this could be associated with development of FSGS. Abid et al. [17] found homozygous R229Q mutation in the NPHS2 gene in two children with childhood-onset nephrotic syndrome. Pereira et al. [19] found its allele frequency of 2.76% and authors further emphasized that there was a strong association between R229Q and microalbuminuria. We found highest percentage of this polymorphism (30%) reported till date. However, this may be because of small sample size studied.

It appears that when this R229Q substitution is present in homozygous or compound heterozygous state, it could be pathogenic in nephrotic syndrome leading to altered podocin protein. In vitro, it has been demonstrated that this podocin shows decreased binding to its interacting protein partner nephrin hence affecting the glomerular filtration barrier [20]. However, Tsukaguchi et al. [18] hypothesized that R229Q mutation alone is insufficient to cause FSGS, but appears to enhance susceptibility to renal injury in the compound heterozygote state in association with a second mutant NPHS2 allele and there is a possibility that other mutant genes may modify the phenotypic effect R229Q [21]. Therefore, further screening for another gene should be done, and nephrotic syndrome unresponsive to immunosuppressive drugs should not be considered to be related with this mutation [22, 23]. It appears that when R229Q variant is present, it does not affect the therapeutic response and renal function in these patients.

In conclusion, patients with R229Q polymorphism showed partial response to therapy. Therefore it is judicious to give immunosuppressive treatment to patients having this variant to reduce heavy proteinuria and its further complications. However, a study on larger sample size is required to know its incidence and clinical significance in our population.

Acknowledgements

O.P.M., N.K., A.K.S., A.A., R.P.- were involved in the study design, conduction, data analysis and drafted the manuscript, GN- performed the mutational analysis and VVB- carried out the histopathological examination of renal tissues.

Funding

The present study was supported by the Department of Science and Technology (DST) Purse Grant, Government of India through Institute of Medical Sciences, Banaras Hindu University, Varanasi, India.