Gene mutation analysis in 12 Chinese children with congenital nephrotic syndrome

Congenital nephrotic syndrome (CNS) is a rare kidney disorder characterised by a high degree of proteinuria, hypoproteinemia, and edema occurring within 3 months after birth [1]. Based on its aetiology, CNS can be divided into primary and secondary disease [2]. The secondary disease is mainly caused by various pathogen infections, such as toxoplasma, syphilis, rubella virus, cytomegalovirus, and herpes simplex virus [1, 2]. The majority of patients with CNS show mutations in genes encoding key podocyte proteins that constitute the slit diaphragm (NPHS1 and NPHS2); others are expressed in the podocyte membrane (PLCE1), mitochondria, (COQ6, COQ2) or glomerular basement membrane (LAMB2), and other genes encode transcription factors that are necessary for normal development (WT1, LMX1B) [2‐11]. NPHS1 and NPHS2 mutations are the main causes of CNS, but LAMB2 and WT1 mutations have been detected in a few CNS patients in Europe and America [3, 12]. The NPHS1 mutation is also a major cause of CNS in Japan, but NPHS2 mutations have been found in few CNS patients, and no WT1 mutations have been identified in patients with CNS [13]. A study from South Korea demonstrated that NPHS1 and WT1 mutations are the main causes of CNS and that NPHS2 and LAMB2 mutations are only found in a few patients [14]. In China, mutations of NPHS1, WT1, and LAMB2 were identified in sporadic patients with CNS [15‐18], and no other gene mutations were reported in patients with CNS. Case studies involving mutational analyses of NPHS1 and LAMB2 are limited, and all of these investigations are case reports [15‐17]; in addition, an analysis of a single WT1 mutation was performed in patients with steroid-resistant nephrotic syndrome (SRNS) [18], so it is not clear which gene is the major cause of CNS. In this study, we performed a systematic investigation of genes in 12 children with CNS from 12 unrelated families and from 10 provinces and autonomous regions in China.

The frequency of the NPHS1 gene mutation varies among different ethnic groups. In 98% of children with CNS in Finland, the disease is caused by NPHS1 mutations [21]. Lenkkeri et al. reported that 80% (28/35) of CNS patients from North America, Europe, and North Africa have been identified to harbour mutations in the NPHS1 gene [3]. A study from Europe showed that 84.8% (39/46) of paediatric CNS cases were explained by mutations in four genes. The distribution among these four genes was as follows: NPHS1, 39.8%; NPHS2, 39.8%; WT1, 2.2%; and LAMB2, 4.4% [12]. Another study from Europe revealed that 100% (15/15) of paediatric CNS cases were caused by mutations in the NPHS1, NPHS2, and WT1 genes, with frequencies of 80, 7, and 13%, respectively [22]. Furthermore, no mutations of PLCE1 and LAMB2 have been detected [22]. Therefore, NPHS1 is the main gene that causes CNS in Europe and North America. In 2005, a Japanese study showed that 4 out of 15 patients had NPHS1 gene mutations (2 homozygous, 2 heterozygous), 2 patients had NPHS2 gene mutations (1 homozygous, 1 heterozygous), and no patients had WT1 gene mutations [13]. In 2009, another Japanese study reported that 5 out of 5 patients had NPHS1 gene mutations, and the authors considered NPHS1 to be the main causative gene of CNS in Japan [23]. A study from North Korea demonstrated that NPHS1 and WT1 gene mutations were the major causes of CNS and that NPHS2 and LAMB2 gene mutations were found in only a few CNS patients [14]. An NPHS1 mutation analysis was performed as a case study in China [15, 16], with no NPHS2 mutations reported in the Chinese CNS patients. The results of a single-gene analysis in Chinese SRNS patients showed that 1 out of 5 patients with CNS had a WT1 mutation [18]. The number of studies reporting gene analyses of CNS patients is limited in China. Therefore, it is not clear which gene is the main cause of CNS in Chinese patients.

CNS is very rare in China. Mutations of NPHS1, WT1 and LAMB2 were identified in sporadic patients with CNS, and all of these investigations are case reports [15‐17]. In this study, we performed analyses of NPHS1, NPHS2, PLCE1, WT1, LAMB2, LMXIB, and COQ2 genes by direct sequencing in six Chinese children with CNS and whole exon sequencing (WES) in the other six children. The 12 children were from 12 unrelated families and 10 provinces and autonomous regions in China. Our results showed that eight patients had two heterozygous mutations in the NPHS1 gene, one patient had a homozygous mutation in the COQ6 gene, and one patient had a heterozygous mutation in the WT1 gene. No mutations in any genes associated with nephrotic syndrome and proteinuria syndrome were detected in another two children by WES. The mutations identified by WES were all confirmed by Sanger sequencing. Mutations in the NPHS1 and COQ6 genes in patients were determined to arise from their parents by family analyses. Therefore, eight patients had compound heterozygous mutations in the NPHS1 gene. This finding suggests that the NPHS1 mutation is a major cause of CNS in Chinese patients. Our study only had 12 patients, but this group represented the largest cohort to date in China. An international cohort study in 2015, the largest cohort study performed thus far, revealed that a single-gene caused 69.4% of cases of SRNS that manifested in children in the first 3 months of life, and it revealed that the distribution of the causative genes was as follows: 40% for NPHS1, 10.6% for NPHS2, 8.5% for WT1, 5.5% for LAMB2, and 4.7% for all of the other genes combined [24]. Although there is a difference between our study and the international cohort study on the distribution of causative genes, NPHS1 is a major causative gene of CNS in children in both studies.

CNS is a recessively inherited disorder that is characterised by a high degree of proteinuria at birth, a large placenta, and a marked oedema within the first 3 months of life [1, 25]. NPHS1 was identified by positional cloning more than two decades ago. The Fin-major (c.121delCT; p.L41 fs) and Fin-minor (c.3325C > T; p.R1109X) mutations account for 78 and 16% of the mutated alleles, respectively, in Finnish cases; however, these mutations are rarely found in other ethnic groups [3, 4, 26]. In 2002, Koziell et al. reported the incidence of a Turkish CNS patient with a Fin-minor mutation [26]. To date, the Fin-minor mutation has only been reported in the populations of Finland and Turkey. Herein, we also documented the first occurrence of Fin-minor mutations outside of Finland and Turkey. Patient 5 had heterozygous IVS26DS-2A > T and Fin-minor mutations in intron and exon 26 of NPHS1, which arose from the patient’s father and mother, respectively. The IVS26DS-2A > T mutation was in the splicing junctions and was not found in the 100 control subjects in our study; furthermore, the mutation was in the highly conserved splice signals (‘AG’), and the prediction software programmes showed that it had a great effect on the splicing junctions (http://​www.​cbs.​dtu.​dk/​services/​NetGene2). Therefore, we deduced that the mutation affects the splicing process at the cDNA level. Patient 5 also had the typical symptoms of CNS, such as prematurity, a large placenta, a high degree of proteinuria, and rapid progression to ESRD (within 3 years). Two heterozygous nonsense mutations, Fin-minor and p.A1160X, were identified in patient 3. The p.R1160X mutation, a specific exon 27 nonsense mutation, is predicted to form a truncated protein that lacks the C-terminal 82 amino acids that are implicated in the interaction with podocin. However, patients with CNS caused by the p.R1160X mutation have an unexpectedly milder phenotype, and most of these individuals are females, suggesting a gender effect [27]. Surprisingly, all affected patients are, reportedly, homozygous for this mutation and, among those in whom a renal biopsy is performed, the histologic findings are consistent with CNS [27]. Nevertheless, these patients either have a mild degree of proteinuria or go into remission between the ages of 5- and 19-years-old [27]. Patient 3 had serious features of nephrotic syndrome, and no improving trend of the disease during the follow-up period (3 months). He might have had a poor prognosis because of his heterozygous p.R1160X and Fin-minor mutations, which is different from patients with homozygous p.R1160X mutations. Our study showed that a Fin-minor mutation was detected in two of the eight CNS patients (25%) with a NPHS1 mutation. Although our patients were different from those in the Finnish study on race[], a Fin-minor mutation is also a common mutation in our patients.

Of these 12 children, one child (case 12) with extrarenal symptoms had a homozygous c.1078C > T (p.R360W) mutation in the COQ6 gene. The homozygous p.R360W mutation has been not reported before and is damaging for the COQ6 protein, as predicted by the PolyPhen and SIFT software. The COQ6 protein was expressed almost exclusively in the glomeruli, rather than in the tubules [28]. The COQ6 gene mutations that cause a CoQ10 deficiency led to the nephrotic syndrome [28]. The child had been treated with CoQ10 since his genetic diagnosis when he was one year old. His proteinuria was observed to be in complete remission at three months after treatment with 30 mg/ (kg.d) CoQ10. It was first instance of a reported mutation in the COQ6 gene in Chinese children with CNS. A heterozygous c.1334C > T mutation in the WT1gene was detected in case 11 by WES, which was confirmed by Sanger sequencing. Sanger sequencing in the family of case 12 showed that the c.1334C > T (p.R445Q) mutation was de novo, which had been not reported beforehand. Prediction by the PolyPhen and SIFT software displayed the c.1334C > T mutation as being damaging for the WT1 protein. Case 11 progressed to end-stage renal disease shortly after the diagnosis of CNS, and no incidences of sex differentiation disorder, Wilms tumour, and gonadoblastoma was found in this patient to date. Patients with the WT1 mutation not only more frequently presented with chronic kidney disease and hypertension at the time of diagnosis and exhibited a more rapid disease progression but also more frequently had incidences of sex reversal and/or urogenital abnormalities, Wilms tumour, and gonadoblastoma [29]. Therefore, more attention should be paid to this patient, in order to find an occurrence of Wilms tumour and gonadoblastoma in a timely fashion.

Based on our data, which showed that the NPHS1 mutation is the major cause of CNS in Chinese patients, we recommend a NPHS1 analysis in Chinese patients with CNS. Because NPHS1 mutations are distributed among different exons and splicing mutations, a mutational analysis should be performed in all NPHS1 exons, as well as at the boundaries of the introns and exons. However, mutations in several genes can cause CNS. With a decline in sequencing costs, next generation sequencing is an optimal choice for gene screening in patients with CNS, with next generation sequencing including disease targeted sequencing and WES.

Our study demonstrates that the majority of CNS cases are caused by genetic defects, and the NPHS1 mutation is the most common cause of CNS in Chinese patients. A mutational analysis of NPHS1 should be recommended in Chinese patients with CNS in all exons of NPHS1 and in the intron-exon boundaries.