The Role of p.Ser1105Ser (in NPHS1 Gene) and p.Arg548Leu (in PLCE1 Gene) with Disease Status of Vietnamese Patients with Congenital Nephrotic Syndrome: Benign or Pathogenic?

Background and Objectives: Congenital nephrotic syndrome (CNS), a genetic disease caused by mutations in genes on autosomes, usually occurs in the first three months after birth. A number of genetic mutations in genes, which encode for the components of the glomerular filtration barrier have been identified. We investigated mutations in NPHS1, NPHS2, PLCE1 (NPHS3), and WT1 genes that relate to the disease in Vietnamese patients. Materials and Methods: We performed genetic analysis of two unrelated patients, who were diagnosed with CNS in the Vietnam National Children’s Hospital with different disease status. The entire coding region and adjacent splice sites of these genes were amplified and sequenced using the Sanger method. The sequencing data were analyzed and compared with the NPHS1, NPHS2, PLCE1, and WT1 gene sequences published in Ensembl (ENSG00000161270, ENSG00000116218, ENSG00000138193, and ENSG00000184937, respectively) using BioEdit software to detect mutations. Results: We detected a new variant p.Ser607Arg and two other (p.Glu117Lys and p.Ser1105Ser) in the NPHS1 gene, as well as two variants (p.Arg548Leu, p.Pro1575Arg) in the PLCE1 gene. No mutations were detected in the NPHS2 and WT1 genes. Patient 1, who presented a heterozygous genotype of p.Ser1105Ser and p.Arg548Leu had a mild disease status but patient 2, who presented a homozygous genotype of these alleles, had a severe phenotype. Conclusions: These results suggest that variants p.Ser1105Ser (in NPHS1 gene) and p.Arg548Leu (in PLCE1 gene) in the homozygous form might play a role in the development of the disease in patients.


Study Subjects
Patient 1 is a 2.5-month-old girl, who was hospitalized with clinical features such as moderate edema, pneumonia, jaundice, infected with hepatitis B virus, and elevated hepatic enzyme. She is the first child in her family. She was a full-term normal delivery and the weight of the placenta was unknown. Her mother was infected with hepatitis B virus. The biochemical indices of the blood serum revealed 49 mg/dL protein (normal is 6-24 mg/dL), 4.5 mg/dL creatinine (normal is 0.5-1.2 mg/dL), and 12 g/L albumin (normal is 35-50 g/L). The results of electrolysis showed that blood sodium, potassium, and calcium were 129 mmol/L (normal is 135-145 mmol/L), 3.9 mmol/L (normal is 3.5-4.5 mmol/L), and 1.9 mmol/L (normal is 2.2-2.6 mmol/L), respectively. In addition, biochemical indices of the urine revealed 11.2 g/24 h protein (normal is 0-0.2 g/24 h). She was diagnosed with CNS in the Vietnam National Children's Hospital. Her parents had a normal phenotype.
Patient 2 is a 7-day-old boy, who was hospitalized with clinical features such as moderate edema, severe pneumonia. He is the second child in the family with an older brother who died of CNS at one-month-old. However, we did not collect a sample from his brother for genetic analysis. He was a full-term normal delivery and the weight of the placenta was unknown. The biochemical indices of the blood serum revealed 29 mg/dL protein (normal is 6-24 mg/dL), 3.5 mg/dL creatinine (normal is 0.5-1.2 mg/dL), and 6.8 g/L albumin (normal is 35-50 g/L). The results of electrolytes showed that blood sodium, potassium, and calcium were 132 mmol/L (normal is 135-145 mmol/L), 2.5 mmol/L (normal is 3.5-4.5 mmol/L), and 1.9 mmol/L (normal is 2.2-2.6 mmol/L), respectively. In addition, biochemical indices of the urine revealed 6.8 g/24 h protein (normal is 0-0.2 g/24 h). He was diagnosed with CNS in the Vietnam National Children's Hospital. His parents had a normal phenotype.
The written consent form were obtained from the patients.

Genetic Analysis
The genomic DNA was isolated from peripheral blood samples (including a sample from patients and their families) using a Qiagen DNA blood mini kit (QIAamp DNA Blood Mini preparation kits, Qiagen, Hilden, Germany) following the manufacturer's guidelines. The DNA concentration was determined using a Thermo Scientific NanoDrop spectrophotometer (Waltham, MA, USA). All of the exons and exon-intron boundaries of the NPHS1, NPHS2, PLCE1, and WT1 genes were amplified and analyzed by direct sequencing. The oligonucleotide primers were synthesized and purchased from IDT (Integrated DNA Technologies, Inc., Coralville, IA, USA) [11,28,32,35] for amplifying the NPHS1, NPHS2, PLCE1 and WT1 genes, respectively. Fifty nanograms of genomic DNA was subjected to 35 cycles of PCR amplification in a 25 µL volume consisting of 10X PCR buffer (Thermo), 10 µM concentration of each primer, 20 mM MgCl 2 , 10 µM dNTPs, and 5 U Dream Taq DNA polymerase (Thermo). DNA was denatured at 95 • C for 12 min followed by 35 cycles of denaturation for 1 min at 95 • C, annealing for 1 min at 53-65 • C, and extension for 1 min at 72 • C, with a final extension for 7 min at 72 • C. The PCR amplification was carried out on an Eppendorf Mastercycler EP gradient (USA Scientific, Inc., Ocala, FL, USA).
The DNA sequencing was performed in both directions, initiated from forward and reverse primers, which had been used in an initial PCR reaction. The PCR products were purified with the Qiagen Purification kit (QIAquick PCR Purification Kit, Qiagen, Hilden, Germany) and sequenced on an ABI PRISM 3500 Genetic Analyser machine (Applied Biosystems Thermo Scientific, Foster, CA, USA). The Sequencing data were analyzed and compared with the NPHS1, NPHS2, PLCE1 and WT1 gene sequence published on Ensembl (ENSG00000161270, ENSG00000116218, ENSG00000138193, and ENSG00000184937, respectively) by using the BioEdit software version 7.0.9.0 to detect mutations.

Ethical Approval
All experiments performed in accordance with relevant guidelines and regulations based on the experimental protocol on human subjects which was approved by the Scientific Committee of Institute of Genome Research, Vietnam Academy of Science and Technology under reference number 15/QD-NCHG.

NPHS1 Analysis
We detected a new variant p.Ser607Arg, and two other (p.Glu117Lys and p.Ser1105Ser) in the NPHS1 gene from two unrelated patients with CNS. The nucleotide change c.3315G>A (p.Ser1105Ser), at the fourth nucleotide on the 5'-end of exon 26, was found in both of the patients in our study. The variants p.Glu117Lys (c.349G>A, in exon 3) and p.Ser607Arg (c.1821C>A, in exon 14) in the NPHS1 gene were found in patient 2. All of these variants were heterozygous, except p.Ser1105Ser in patient 2. These variants were inherited from their parents. Among them, variant p.Ser607Arg was identified as a novel variant and has not yet been published in [36,37].

PLCE1 Analysis
For the PLCE1 gene, two variants (p.Arg548Leu and p.Pro1575Arg) were detected in the patients. The variant p.Arg548Leu (c.1643G>T) in exon 5 was identified in patient 1 (in heterozygous form) and patient 2 (in homozygous form). Patient 2 also carried another variant p.Pro1575Arg (c.4724C>G) in exon 19.

NPHS2 and WT1 Analysis
No NPHS2 and WT1 mutations were detected in any of the four CNS patients in this study.

Patient 1
Patient 1 was a mild case and the patient's condition had been stable after the treatment time. Genetic analysis showed that she carried simultaneously, a heterozygous variant p.Ser1105Ser (c.3315G>A) in the cytosolic domain of NPHS1, a heterozygous variant p.Ser607Arg in NPHS1 ( Figure 1) and another variant, p.Arg548Leu, in PLCE1 ( Figure 2). A prediction of the pathogenicity of the variants was done by using PolyPhen 2 and Mutation Taster tools [38,39]. The results showed that this variant was benign, with a score of 0.26 (by PolyPhen 2 tool), and potentially pathogenic, with a score of 110 (by Mutation Taster tool). Research by Liu et al. [40] showed that mutations leading to amino acid changes may be the cause of folding errors of the protein molecule, which affected the intracellular transport function of the NPHS1 protein. In the study of Liu et al. [40], most of the CNS patients carried heterozygous mutations in the Ig extracellular region of the protein molecule. The results of this study indicated that mutations in the Ig functional regions have important effects on the function of the NPHS1 protein. However, there is no spatial structure of the nephrin protein, so it is not possible to predict the effect of this mutation on protein folding.  The common variant, p.Ser1105Ser (c.3315G>A), which does not result in an amino acid substitution, has previously been found in Finnish, Chinese and Japanese subjects [5,[41][42][43]. Research by Machuca et al. [44] showed that milder cases, resulting from mutant NPHS1, either had two mutations in the cytoplasmic tail or two missense mutations in the extracellular domain, including at least one that preserved structure and function. These observations also suggested that mutations in the cytosolic region may have less effect on protein function. This result could explain the mild phenotype of the patient and the normal phenotype of her father (Table 1).  The common variant, p.Ser1105Ser (c.3315G>A), which does not result in an amino acid substitution, has previously been found in Finnish, Chinese and Japanese subjects [5,[41][42][43]. Research by Machuca et al. [44] showed that milder cases, resulting from mutant NPHS1, either had two mutations in the cytoplasmic tail or two missense mutations in the extracellular domain, including at least one that preserved structure and function. These observations also suggested that mutations in the cytosolic region may have less effect on protein function. This result could explain the mild phenotype of the patient and the normal phenotype of her father (Table 1). The common variant, p.Ser1105Ser (c.3315G>A), which does not result in an amino acid substitution, has previously been found in Finnish, Chinese and Japanese subjects [5,[41][42][43]. Research by Machuca et al. [44] showed that milder cases, resulting from mutant NPHS1, either had two mutations in the cytoplasmic tail or two missense mutations in the extracellular domain, including at least one that preserved structure and function. These observations also suggested that mutations in the cytosolic region may have less effect on protein function. This result could explain the mild phenotype of the patient and the normal phenotype of her father (Table 1).

Patient 2
A heterozygous polymorphism p.Glu117Lys in exon 3, a heterozygous polymorphism p.Ser607Arg in exon 14, and a homozygous polymorphism p.Ser1105Ser (c.3315G>A) in exon 26 of the NPHS1 gene were identified in patient 2 ( Figure 3). p.Glu117Lys was reported by Lenkkeri et al. [11] as a single nucleotide polymorphism in a CNS cohort and has now been accepted as a well-known polymorphism. The patient also carried a homozygous variant c.3315G>A, located in the 4th position in exon 26 of the NPHS1 gene, which can affect the pre-mRNA splicing process. This theory has been reinforced by the investigations of Wu and Hurst [45] who revealed that 5'-pathogenic single nucleotide polymorphisms (SNPs) might be more common and they may have a more drastic effect on the subsequent protein.

Patient 2
A heterozygous polymorphism p.Glu117Lys in exon 3, a heterozygous polymorphism p.Ser607Arg in exon 14, and a homozygous polymorphism p.Ser1105Ser (c.3315G>A) in exon 26 of the NPHS1 gene were identified in patient 2 ( Figure 3). p.Glu117Lys was reported by Lenkkeri et al. [11] as a single nucleotide polymorphism in a CNS cohort and has now been accepted as a wellknown polymorphism. The patient also carried a homozygous variant c.3315G>A, located in the 4th position in exon 26 of the NPHS1 gene, which can affect the pre-mRNA splicing process. This theory has been reinforced by the investigations of Wu and Hurst [45] who revealed that 5'pathogenic single nucleotide polymorphisms (SNPs) might be more common and they may have a more drastic effect on the subsequent protein. Patient 2 also carried two of genetic changes in the PLCE1 gene, including a homozygous variant p.Arg548Leu and a heterozygous variant p.Pro1575Arg (Figure 4). PLCE1 belongs to the Ras family genes, which encode membrane-associated molecular switches that bind GTP and GDP and slowly hydrolyze GTP to GDP [46]. A homozygous polymorphism p.Arg548Leu was located in a Ras-GEF domain, which may thus affect its capacity to activate the Ras protein. p.Arg548Leu and p.Arg1575Pro were also detected by Machuca et al. [44] in CNS patients and they were identified to play a role in the pathogenesis in these patients. In addition, Yilmaz et al. [47] reported a case with CNS, which had a homozygous variant p.Glu117Lys in NPHS1 as the only cause of the disease. Patient 2 also carried two of genetic changes in the PLCE1 gene, including a homozygous variant p.Arg548Leu and a heterozygous variant p.Pro1575Arg (Figure 4). PLCE1 belongs to the Ras family genes, which encode membrane-associated molecular switches that bind GTP and GDP and slowly hydrolyze GTP to GDP [46]. A homozygous polymorphism p.Arg548Leu was located in a Ras-GEF domain, which may thus affect its capacity to activate the Ras protein. p.Arg548Leu and p.Arg1575Pro were also detected by Machuca et al. [44] in CNS patients and they were identified to play a role in the pathogenesis in these patients. In addition, Yilmaz et al. [47] reported a case with CNS, which had a homozygous variant p.Glu117Lys in NPHS1 as the only cause of the disease.
In our study, the combination of the two variants p.Ser1105Ser (in NPHS1 gene) and p.Arg548Leu (in PLCE1 gene), in the homozygous form, could be the cause of his severe symptoms. Thus, these variants in the heterozygous forms created only a mild phenotype in patient 1, and did not indicate the phenotype in their parents, but in the homozygous form had created a very severe phenotype in patient 2. These results suggested that a variant can become pathogenic when in a homozygous form. In our study, the combination of the two variants p.Ser1105Ser (in NPHS1 gene) and p.Arg548Leu (in PLCE1 gene), in the homozygous form, could be the cause of his severe symptoms. Thus, these variants in the heterozygous forms created only a mild phenotype in patient 1, and did not indicate the phenotype in their parents, but in the homozygous form had created a very severe phenotype in patient 2. These results suggested that a variant can become pathogenic when in a homozygous form.

Conclusions
In our study, we detected three variants (p.Glu117Leu, p.Ser607Arg, and p.Ser1105Ser) in the NPHS1 gene and two variants (p.Arg548Leu and p.Pro1575Arg) in the PLCE1 gene. Among these, a variant (p.Ser607Arg in exon 14) was novel in the NPHS1 gene. No mutations were detected in the NPHS2 and WT1 genes. Patient 1, with a heterozygous genotype of p.Ser1105Ser and p.Arg548Leu had a mild disease status but patient 2, with a homozygous genotype of these alleles, had a severe phenotype. The results in our study suggested that variants p.Ser1105Ser (in NPHS1 gene) and p.Arg548Leu (in PLCE1 gene) in the homozygous form might play a role in the development of the disease in the patients with CNS.

Conclusions
In our study, we detected three variants (p.Glu117Leu, p.Ser607Arg, and p.Ser1105Ser) in the NPHS1 gene and two variants (p.Arg548Leu and p.Pro1575Arg) in the PLCE1 gene. Among these, a variant (p.Ser607Arg in exon 14) was novel in the NPHS1 gene. No mutations were detected in the NPHS2 and WT1 genes. Patient 1, with a heterozygous genotype of p.Ser1105Ser and p.Arg548Leu had a mild disease status but patient 2, with a homozygous genotype of these alleles, had a severe phenotype. The results in our study suggested that variants p.Ser1105Ser (in NPHS1 gene) and p.Arg548Leu (in PLCE1 gene) in the homozygous form might play a role in the development of the disease in the patients with CNS.