A novel mutation of congenital nephrotic syndrome in a Slovenian child eventually receiving a renal transplant

Congenital nephrotic syndrome (CNS) is a rare disease defined as heavy proteinuria, hypoalbuminemia, hyperlipidemia, and edema presenting in the first three months of life. It is most commonly caused by mutations in the NPHS1 gene associated with nephrotic syndrome type 1, also known as Finnish-type CNS, which is inherited in an autosomal recessive manner. Symptomatic treatment with intravenous albumins, vitamins, minerals, nutritional, and hormonal supplementation and treatment of complications are mandatory. Children refractory to the symptomatic treatment are recommended to undergo nephrectomy and renal replacement therapy, preferably renal transplantation. We report on a child with Finnish type CNS with a NPHS1 mutation, which is the first case confirmed by genetic study in Slovenia. We showed for the first time that homozygous mutation c.2928-3del in the NPHS1 gene caused exon 22 skipping, leading to a truncated protein and Fin-minor phenotype.

Congenital nephrotic syndrome (CNS) is a rare disease defined as heavy proteinuria, hypoalbuminemia, hyperlipidemia, and edema presenting in the first three months of life. It is most commonly caused by mutations in the NPHS1 gene associated with nephrotic syndrome type 1, also known as Finnish-type CNS, which is inherited in an autosomal recessive manner. Symptomatic treatment with intravenous albumins, vitamins, minerals, nutritional, and hormonal supplementation and treatment of complications are mandatory. Children refractory to the symptomatic treatment are recommended to undergo nephrectomy and renal replacement therapy, preferably renal transplantation. We report on a child with Finnish type CNS with a NPHS1 mutation, which is the first case confirmed by genetic study in Slovenia. We showed for the first time that homozygous mutation c.2928-3del in the NPHS1 gene caused exon 22 skipping, leading to a truncated protein and Fin-minor phenotype.
Congenital nephrotic syndrome (CNS) is a rare form of nephrotic syndrome, presenting in the first three months of life. In most cases, it is caused by monogenic mutations of structural proteins that form the glomerular filtration barrier in the kidneys, such as gene NPHS1 (1).
Pathogenic variants in the NPHS1 gene are associated with nephrotic syndrome type 1, also known as Finnish-type CNS, which is inherited in an autosomal recessive manner, with the mutations being homozygous or compound heterozygous. The syndrome is characterized by a severe steroid-resistant nephrotic syndrome apparent at birth, with rapid progression to end-stage renal failure (1,2). We report on a child with Finnish-type CNS with a NPHS1 mutation, which is the first case confirmed by a genetic study in Slovenia. The reported mutation was confirmed for the first time to be pathogenic.

CASE REPORT
The patient was a girl born after 35 weeks gestation, with normal body measurements. On the first day of life, edema of the lower extremities was observed. Her mother has hereditary leyomiomatosis and renal cell cancer (HLRCC), with a mutation in gene FH (c.1189G>A;p.Gly397Arg), and essential thrombocythemia, with a mutation in gene JAK2. The mother's brother also had HLRCC and died from kidney carcinoma.
At the age of three days, blood tests revealed hypoalbuminemia, hypoproteinemia (30 g/L), and hyperlipidemia. Renal function was normal. Proteins in urine were 4+, with erythrocytes 3+. Perinatal infections were excluded. Abdominal ultrasound showed enlarged hyperechogenic kidneys.
Blood was taken for genetic analysis with next generation sequencing technologies, which revealed a previously unreported homozygous variant of uncertain significancec.2928-3del, in the NPHS1 gene. The diagnosis of the CNS of Finnish type was very likely. Both parents are heterozygous for this variant. The girl was also heterozygous for HL-RCC, with a mutation in gene FH, previously found in the mother.
Because of massive loss of proteins (up to 11 656 g proteins per mol creatinine) and hypoalbuminemia (11-20 g/L), the patient received albumin parenterally once a day and 4 g/kg of proteins per day in her diet. She was administered indomethacin, later changed to ibuprofen due to vomiting, and captopril. Excessive proteinuria persisted despite treatment.
To maintain adequate diuresis, she received furosemide. Immunoglobulin supplements were given parenterally due to hypogammaglobulinemia.
At the age of 1.5 months, anemia and severe neutropenia (378 cells/mm 3 ) were observed. Low serum iron, transferrin, thyroid hormone, and erythropoietin levels were detected, so she received hormones, together with iron, vitamin, and mineral supplements. Additionally, the total copper serum level and ceruloplasmin were decreased, so oral supplementation with copper was started, up to 15 mg per day. During the copper therapy, the neutrophil count normalized (Figure 1).
A low antithrombin III level was observed. Because of the increased risk of thrombosis, she received acetylsalicylic acid and had no thromboembolic events.
She had no appetite and received a hypercaloric diet through a nasogastric tube. Despite the disease severity, normal growth, weight gain, and development were observed.
Because the child was refractory to the applied therapy, and massive proteinuria persisted with many complications, a unilateral nephrectomy was performed at the age of 7.5 months, and the remaining kidney was removed at FiGuRE 1. improvement in neutrophil count after the initiation of copper therapy in a child with Finnish type congenital nephrotic syndrome with a NPHS1 mutation the age of 9 months, and she started automated peritoneal dialysis.
Histological examination of the resected kidneys revealed immature glomeruli showing diffuse mesangial sclerosis and mesangial hypercellularity. Some glomeruli were globally and segmentally sclerosed. Focal tubular microcystic dilatation and moderate interstitial fibrosis were observed. Immunohistochemical stain for nephrin was negative (Figure 2A,B). Electron microscopy study showed diffuse podocyte foot process effacement and an expanded mesangial matrix, with proliferation of mesangial cells ( Figure 2C).
RNA was obtained from the kidney sample after nephrectomy from the patient, as well as from another unaffected patient as a control, and reverse transcribed into cDNA.
In both patients, all except one cDNA fragment were appropriately long. In the affected patient, a homozygous shorter amplicon of NPHS1 cDNA was observed, corre-sponding to exons 20-24. Both fragments were used for Sanger sequencing, which confirmed exon 22 skipping and revealed shorter translated cDNA (985 amino-acid long protein, instead of 1241) ( Figure 2D). This is the first confirmation that this mutation affects cDNA and, consequently, protein translation. We additionally used the Human Gene Mutational Database to search for this mutation. It was found that substitution of C for G at c.2928-3 (which takes place in the case of deletion of C) is a disease-causing mutation that is predicted to induce a large splicing change.
At the age of 22 months, the patient received a deceaseddonor kidney transplant (Table 1). One month after transplantation, donor-specific antibodies were identified in serum, which spontaneously decreased without specific treatment. One year and a half after transplantation, she is doing well, with normal renal function and no proteinuria detected.

DiSCuSSiON
We reported on a patient with a typical clinical course of CNS, genetically confirmed as Finnish type with a novel homozygotic mutation in gene NPHS1. We observed not only massive proteinuria, severe hypoproteinemia, loss of immunoglobulins, and leakage of hormones but also severe neutropenia, which was attributed to low plasma copper and ceruloplasmin levels because the neutrophil level normalized after oral supplementation of copper, as already described (3).
In contrast to some reports on developmental delay and failure to thrive in patients with CNS (4), our patient experienced normal development, growth, and weight gain, which was very likely the result of early aggressive management.
Proteinuria did not significantly decrease after captopril and NSAID treatment, although some studies reported encouraging results in this regard (5,6).
Nephrotic syndrome did not recur in our patient. A recurrence of nephrotic syndrome soon after transplantation has been reported in 25% to 34% of patients with Finnish-type CNS, and anti-nephrin antibodies have been detected in the majority of patients with Fin-major homozygotes (7)(8)(9). In Fin-major homozygotes, the mutation stops gene reading very early, leading to a truncated protein of 90 amino acids as opposed to a truncated protein of 1109 amino acids in Fin-minor homozygotes and 1241 amino acids in a long normal nephrin molecule. Fin-major homozygotes have no immunological tolerance to nephrin, which leads to an increased probability of the development of anti-nephrin antibodies and a NS recurrence after transplantation. We showed for the first time that the mutation resulted in the lack of exon 22, as shown on a cDNA level, leading to a preliminary stop codon and truncated 985 amino-acid-long protein. Such a protein lacks a part of the intracellular component, similar to Fin-minor homozygotes. We do not therefore expect NS to recur in our patient (8-10).