Scholarly article on topic 'The importance of genetic testing in adolescent-onset steroid-resistant nephrotic syndrome - Case report/ Importanţa testării genetice la adolescenţii cu sindrom nefrotic corticorezistent – Prezentări de caz'

The importance of genetic testing in adolescent-onset steroid-resistant nephrotic syndrome - Case report/ Importanţa testării genetice la adolescenţii cu sindrom nefrotic corticorezistent – Prezentări de caz Academic research paper on "Clinical medicine"

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Academic research paper on topic "The importance of genetic testing in adolescent-onset steroid-resistant nephrotic syndrome - Case report/ Importanţa testării genetice la adolescenţii cu sindrom nefrotic corticorezistent – Prezentări de caz"


Case report

DOI: 10.2478/rrlm-2014-0031

The importance of genetic testing in adolescent-onset steroid-resistant nephrotic syndrome - Case report

Importanta testärii genetice la adolescent» cu sindrom nefrotic cortico-

rezistent - Prezentäri de caz

Zsuzsanna Gall1*, Eva Kiss2, Kalman Tory3, Attila Fintha4, Carmen Duicu2

1. University of Medicine and Pharmacy Tîrgu Mures, România; 2. Department of II Pediatrics, University of Medicine and Pharmacy Tîrgu Mureç, România; 3. Department of I Pediatrics, Semmelweis University, Budapest, Hungary; 4. Department of IIPathology, Semmelweis University,

Budapest, Hungary

Approximately 10-20% of children and 40% of adults with idiopathic nephrotic syndrome are steroid resistant and progress to end-stage renal disease requiring dialysis or renal transplantation. In these cases, renal histology typically shows focal segmental glomerulosclerosis. Mutations in NPHS1, NPHS2, WT1, CD2AP and ACTN4 genes located on different chromosomes, expressed by glomerular podocytes, have been identified in patients with steroid-resistant nephrotic syndrome.

The authors report two cases of adolescent-onset steroid-resistant nephrotic syndrome. Both cases had similar clinical and histopathological manifestations, with different prognosis and evolution due to different mechanisms leading to proteinuria: an acquired and a genetic form. The first case, a 16 year old girl presented the onset of the disease with massive, generalized edema, secondary hypothyroidism and high blood pressure. Evolution was favorable under cyclosporine therapy. The second case, a 13-years-old adolescent girl, presented an insidious onset of the disease with mild edema. Genetic testing revealed a mutation in the WT1 gene. The patient developed end-stage kidney failure eight months after the onset of the disease andfollowing kidney transplant had a favorable evolution. Histological examination of the renal biopsy specimen showed focal segmental glomerulosclerosis in both cases.

Conclusions: Genetic forms of nephrotic syndrome do not respond to immunosuppressive therapy and may progress to end-stage renal disease, but after kidney transplantation relapse is not expected, in contrast to the immune form. The early genetic diagnosis in steroid-resistant nephrotic syndrome is time-consuming, but is important for proper clinical management of the patients, prognosis and genetic counseling of the families.

Keywords: steroid-resistant nephrotic syndrome; adolescent; WT1; focal segmental glomerulosclerosis

Autorii prezintä douä cazuri de sindrom nefrotic cortico-rezistent cu debut în adolescenß, cu manifestäri clinice §i histopatologice similare (sclerozä glomerularä focal-segmentalä), dar cu mecanisme diferite de



Corresponding author: Zsuzsanna Gall, University of Medicine and Pharmacy Tirgu Mures Tirgu Mures, Romania, e-mail:

producere a proteinuriei: o forma dobándita §i una genetica, avánd astfel prognostic §i evolufie diferita. Primul caz, o adolescenta de 16 ani prezinta debutul bolii brusc cu edeme masive, generalízate, cu raspuns favorabil la tratamentul cu ciclosporina. A doua pacienta, o adolescenta de 13 ani, a prezentat sindrom nefrotic cortico-rezistent cu debut insidios cu edeme u§oare. In acest caz testarea genetica a evidenfiat o mutafie in gena WT1, iar evolufia a fost rapida (in 8 luni) catre insuficienfa renala terminala, dar cu evolufie §i prognostic favorabil dupa transplant renal. Concluzii: Formele genetice ale sindromului nefrotic nu raspund la tratamentul imunosupresiv §i pot evolua catre boala renala terminala, dar dupa transplant recaderea este pufin probabila, in contrast cu formele imune. Diagnosticul genetic precoce in sindromul nefrotic cortico-rezistent este important pentru managementul optim al pacienfilor, precizarea prognosticului, respectiv pentru consilierea genetica a familiei.

Cuvinte cheie: sindrom nefrotic cortico-rezistent; adolescent; WT1; FSGS

Received: 8th May 2014; Accepted: 9th August 2014; Published: 6th September 2014.


Idiopathic nephrotic syndrome (NS) describes the collection of clinical and laboratory findings secondary to glomerular dysfunction, resulting in proteinuria [1, 2]. The annual prevalence is 2-16/100,000 [1, 3]. The diagnostic criteria are massive proteinuria (>50mg/kg/day), generalized edema, hypoalbuminemia and hy-perlipidemia [3, 4].

The majority of patients with NS respond to steroid therapy and have a good clinical prognosis. Unfortunately, approximately 10-20% of children and 40% of adults with idiopathic NS are resistant to steroid treatment (SRNS) and progress to end-stage renal disease (ESRD) requiring dialysis or renal transplantation [2-7]. In these cases, renal histology usually shows focal segmental glomerulosclerosis (FSGS) [3, 6, 7].

Based on etiology, two forms of SRNS are distinguished, an immune and a genetic form, with different treatments, thus a correct clinical diagnosis becomes essential [4, 6, 8]. In genetic forms of SRNS were identified so far mutations in more than 20 genes, most of which encode podocyte proteins. The most frequently mutated genes are NPHS2, WT1, and NPHS1 [5, 6, 8-10].

We report two cases of steroid-resistant ne-phrotic syndrome with FSGS in female adolescents. Both cases had similar clinical and his-

topathologic manifestations, but two different mechanisms leading to proteinuria: an acquired and a genetic form, and therefore a very different prognosis and evolution.

Patients and observation

The first patient, a 16 year old WT1 negative girl, with no significant medical history, was admitted to hospital with a seven week history of edema. Physical examination revealed moderate periorbital and significant lower leg edema. The 24 hour ambulatory blood pressure monitoring showed high blood pressure (above the 95th percentile).

Laboratory data (presented in Table I) revealed nephrotic-range proteinuria without hae-maturia, hypoproteinemia and hyperlipidemia. Blood urea nitrogen, creatinine, electrolytes, im-munogram and complementemia were normal, but secondary hypothyroidism was observed: FT3 1.04 pg/ml (reference range: 1.4-4.7 pg/ml), FT4 0.45 ng/dl (reference range: 0.8-2.2 ng/dl), and TSH 45 IU/ml (reference range: 0.4-4 IU/ ml).

Based on clinical and laboratory data, the diagnosis of nephrotic syndrome was established and prednisone therapy was initiated (60 mg/ m2). The evolution under steroid treatment was unfavorable with persistent massive proteinuria (2.9-5.8 g/24 hours) (Figure 1).

Following corticotherapy our patient presented side effects, showing Cushingoid appearance, hirsutism, striae on the abdomen and thighs, high BP (152/94 mmHg) and obesity (weight 86 kg, BMI: 32.6 kg/m2). The diagnosis of SRNS emerged as no remission was observed after 4 weeks of prednisone therapy and three intravenous pulses of methylprednisolone, so corticotherapy was suspended. Cyclophospha-mide treatment (3mg/kg, for 8 weeks) was introduced, but no remission was observed, than, subsequently cyclosporine therapy was initiated. In the meantime renal biopsy and genetic testing were performed.

Renal biopsy, performed at 10 months after the diagnosis of the disease, showed focal segmental glomerulosclerosis (FSGS). It reported both focal (6 glomeruli affected) and segmental (only the perihilar portion of the affected glomerulus was sclerotic in 1 juxtamedullary located glomerulus) lesions.

The coding exons and splicing junctions of NPHS2 and WT1 were directly (Sanger) sequenced for both patients. Genomic DNA was isolated from peripheral blood by standard methods, after obtaining informed consent from

the patient's parents. Exons 1-8 of NPHS2 and exons 8 and 9 of WT1 with their adjacent intronic junctions were analyzed by direct sequencing using the ABI3500 Genetic Analyzer (Applied Biosystems, Foster City, CA) in the genetic laboratory of Ist Department of Pediatrics, Semmelweis University, Budapest, Hungary. Primers are available upon request. For the sequence analysis, the Sequencher software (Gene Codes, Ann Arbor, MI) was used.

In this case no mutation was found, neither in NPHS2 and WT1, nor in the subsequently sequenced PAX2 gene.

The patient's evolution under immunosup-pressive treatment was favorable, resulting in complete remission (proteinuria <150 mg/day) after 9 weeks of therapy. No relapse has occurred since, the patient being without treatment for the past 22 months.

Our second case, a 13 year old WTlpositive girl, was admitted to hospital with a four week history of mild eye swelling and facial puffi-ness. In her medical history we noted an unin-vestigated episode of edema syndrome at the age of seven, with spontaneous resolution. Physical examination revealed mild periorbital edema

Table I. Clinical features and laboratory data at the onset of the disease

WT1 negative (first WT1 positive (second patient) patient)

Clinical features_

_Age (years)_16_13_

Weight at presentation (kg)_79_36_

Weight at discharge (kg)_69_35_

_Height (cm)_163_158_

periorbital edema moderate mild

_lower leg edema_significant_without_

Laboratory parameters_

Proteinuria (g/day)_5.8-8.7_6.35-7.2

_Serum protein (g/l)_46_50_

Serum cholesterol (mg/dl)_227_345_

Serum triglyceride (mg/dl)_181_205_

1 CyA A CyA

corticotherapy II


Time [months]

Figure 1. Laboratory test results over time. a. Evolution of the urinary protein excretion - stationary in both cases under corticosteroid treatment. Disappearance of proteinuria in WT1 negative case under immunosuppressive therapy and in the WT1 positive case after transplantation. b. Laboratory investigations in dynamics in the second WT1 positive case. (CPA cyclophosphamid, CyA - cyclosporine A)

without any extension to the legs, otherwise normal status with normal heart rate and blood pressure and a normal female phenotype. The main laboratory results are presented in table I. Based on these data the diagnosis of NS was made and corticotherapy was started, but no response was observed after 4 weeks. The side effects of corticotherapy were also noticed in this girl: moon face, hirsutism, striae, arterial hypertension 140/90 mmHg with epistaxis and transient hyperglycemia (179 mg%). Therefore, steroid treatment was discontinued and cyclosporine treatment was started. Complementary investigations (kidney biopsy, genetic test) were performed in this case too.

The renal biopsy, performed at 6 months after the diagnosis of the disease, also reported both focal (9 glomeruli affected) and segmental (perihilar portion of affected glomerulus sclerotic in 3 juxtamedullary located glomeruli) lesions.

In both cases the interstitium was widened and tubular atrophy was observed only in the vicinity of the affected glomeruli. Small and medium-sized arteries were showing specific changes for hypertension. Direct immunofluores-cent staining revealed no immune deposits: negative for IgA, IgG, IgM immunoglobulins, kappa and lambda light chains, and Complement C4 and C1q proteins. The anti-C3 antibody test was weakly positive in arteries and glomeru-

lar arterioles. Electron microscopy examination showed diffuse effacement of the epithelial cell foot (podocyte) processes (Figure 2).

In case of this patient genetic testing revealed a heterozygous mutation in the WT1 gene (c.1228+5 G>A) (Figure 3). As frequently observed, the mutation is likely to be de novo, as the father does not carry the mutation, though no

Figure 2. Light microscopy image of renal biopsy

specimen from the WT1 positive patient (with similar modifications in the first patient): a. one glomerulus with global sclerosis (black arrow) and two glomeruli with perihilar segmental sclerosis (white arrow) (PAS stain, x200). b. A glomerulus with perihilar segmental sclerosis (Jones-stain, x400)

DNA sample was available from the asymptomatic mother. To rule out the diagnosis of Frasier syndrome, generated by the mutation in the WT1 gene, a karyotype was performed, that indicated a female 46,XX chromosomal formula.

In this WT1 positive patient, no changes in the degree of proteinuria were seen after 3 months of cyclosporine therapy, therefore treatment was discontinued.

The evolution under symptomatic therapy was unfavorable with high BP (180/115 mmHg) unresponsive to antihypertensive treatment and reduction of glomerular filtration rate (Figure 1), progressing to ESRD at 8 months after the onset of the disease. She underwent transplantation two months later. There was no recurrence during the follow-up period of 14 months.

Based on clinical and laboratory data, the diagnosis of steroid-resitant nephrotic syndrome was established in both cases; in the first case, an immune form, with favorable evolution under immunosuppressive therapy, while in the second case, a genetic form, with a pathogenic mutation in the WT1 gene, with favorable evolution after renal transplantation.


In all children with SRNS, treatment is problematic and is directed against immunological abnormalities. In all cases it is recommended to perform a renal biopsy before immunosuppres-sive therapy. As a second-line drug, two options are considered: alkylating agents and calcineurin inhibitors. [1] The randomized controlled trials in children with SRNS have demonstrated that calcineurin inhibitors are more effective than placebo or no specific therapy and also alkylating agents (30-33% of patients treated with cyclosporin obtained complete remission and 70% partial remission, while with cyclophosphamide favorable response was observed in 17-28%). [1, 11]

• 443_9F_E11_05_BDxF»tSeq50_POP7_1_inj9»«:1600v Frag me

\ [\Î\f\ ^^ ^^

Figure 3. Sequencing chromatogram of the WT1 gene in the donor splice-site of intron 9: c.1228+5 G>A mutation in heterozygous state in the second patient

In this report two cases of adolescent onset SRNS are presented, with two different mechanisms leading to proteinuria and therefore with very different evolutions. Both cases presented primary resistance to steroid therapy. The first adolescent presented an acquired form of SRNS and FSGS, with favorable response to immuno-suppressive therapy with a calcineurin inhibitor. In contrast, the second case presented an adolescent onset congenital form of SRNS and FSGS, with no response to the corticosteroid and immu-nosuppressive treatment and rapid progression towards ESRD.

Identification of a pathogenic mutation in a patient with SRNS has direct clinical consequences, as it makes further immunosuppressive

treatment unnecessary and the recurrence of NS in a kidney graft unlikely [2, 4, 9, 12-15].

In patients with SRNS, WT1 mutations can lead to three distinct clinical entities [2, 9, 1315]. 1. Denys-Drash syndrome, typically characterized by the triad of infantile SRNS with male pseudo-hermaphroditism, diffuse mesangial sclerosis and Wilms tumor; this was ruled out by the clinical manifestations (adolescent onset) and the histologically evidenced FSGS. 2. Frasier syndrome, characterized by association of SRNS with male pseudohermaphroditism, excluded by normal female karyotype (46, XX). 3. isolated NS - as we found in our case.

The identified WT1 mutation is a frequent mutation in Frasier syndrome, which affects the

cleavage of exon 9 [13,14]. This form of NS does not respond to immunosuppressive therapy and patients should receive symptomatic treatment only. This mutation leads to several pathological conditions, including renal failure in the second or third decade of life. In case of our second patient the disease evolved rapidly to ESRD, within 8 months and as expected, no recurrence was seen after kidney transplantation.

The autosomal dominant transmission of this mutation means that the patient's daughters will have a 50% risk of inheriting the mutation. While male descendants will have the same risk of inheritance, they will also show associated genital disorders (intersex genitalia). If these children then develop FSGS, their disease will be more severe than that of their affected parent. The newborn child of a woman showing a mutated WT1 gene associated with isolated FSGS (like our patient) can develop a more severe phe-notype, like Denysh-Drash Syndrome or Fra-sier Syndrome [17]. If the mutation is known, targeted molecular prenatal diagnosis is possible and genetic counseling is highly recommended. Although the WT1 gene mutations predispose to Wilms tumor and gonadoblastoma, they rarely develop when the mutation affects the splice site regions [9, 13, 16, 18]. According to literature, the risk of nephroblastoma in our patient is 5% and germ cell tumors occur only in patients with sex reversal [18].

Because detecting a mutation in patients with NS decisively influences the therapeutic options and reduces the possibility of post transplant relapses, screening for mutations in these genes is justified in clinical practice. If mutations in the WT1 gene are found in a patient with a female phenotype, the gender genotype must be investigated (thus excluding the XY genotype with pseudohermaphroditism) [17].

There is an urgent need to distinguish between patients who may benefit from prolonged immunosuppressive treatment and those who

do not, nevertheless suffering from its side effects. In clinical practice, steroid therapy is initiated in all patients and genetic investigations are required only if steroid resistance is being observed. This initial steroid treatment can be avoided in patients with a family history of SRNS or in those who have a known causative mutation. In many cases, there is a tremendous delay between diagnosis and the request for genetic testing, although the genetic forms of SRNS should receive only symptomatic treatment [11].


Steroid-resistant nephrotic syndrome is not a single entity, therefore it is important to determine and understand the underlying mechanisms and diseases. The genetic form of NS does not respond to immunosuppressive therapy and may progress to ESRD, but after kidney transplantation relapse is not expected, in contrast to the immune form. As WT1 mutation is frequently found in young patients with NS, genetic screening is important for tumor prophylaxis, these patients having an increased risk to develop Wilms tumor or gonadoblastoma. These cases highlight the importance of early genetic diagnosis in SRNS. Although genetic testing is time-consuming, it is important for proper clinical management of the patients, prognosis and genetic counseling of the families.

Conflicts of interest

The authors declare that they have no conflict of interest.


BMI -body mass index

BP - blood pressure

C - complement

ESRD - end-stage renal disease

FSGS - focal segmental glomerulosclerosis FT3 - free triiodothyronine FT4 - free thyroxine NS - nephrotic syndrome NPHS2 - nephrosis 2, idiopathic, steroid-resistant (podocin) gene

NPHS1 - nephrosis 1, congenital, Finnish type (nephrin) gene

SRNS - steroid resistant nephrotic syndrome TSH - Thyroid-stimulating hormone WT1 - Wilms tumor 1 gene


We thank Dr. Attila Szabô, Dr. George Reusz, Dr. Magdolna Kardos and Dr. Eszter Jâvorszky for their help.

This research was supported in part by the Internal Research Grants of the University of Medicine and Pharmacy Tîrgu Mureç, Romania, contract no 29/11.12.2013


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