Following ethics approval and informed parental consent, clinical details and blood samples were collected from patients with congenital NS, diagnosed at seven tertiary care centres in the country. Diagnosis required the confirmation of nephrotic range proteinuria (spot urine protein to creatinine ratio >2.0 mg/mg or dipstick 3+/4+ on three occasions), hypoalbuminemia (serum albumin <3.0 g/dL) and edema beginning below 3-months of age. Intrauterine infections and structural renal anomalies were excluded by appropriate serology and ultrasonography, respectively. In consonance with current practice worldwide, kidney biopsy was not performed and echocardiography was performed if cardiac examination was abnormal. Management involved the use of furosemide (1-2 mg/kg daily, as indicated), enalapril (0.3-0.4 mg/kg/day orally), intravenous infusions of albumin (1-2 g/kg once every 7-14 days), and supplements of thyroxine (5-10 µg/kg/day) and vitamins, while ensuring adequate nutrition. Parents were counselled regarding outcomes including risk of progression to end stage kidney disease, and families opted for a palliative care plan due to costs of kidney replacement therapy.

The methodology of NGS, performed at Institute of Genomic and Integrative Biology, Delhi, is detailed in Supp. Methods. Whole exome sequencing (WES) was performed using the Illumina HiSeq2000 or NovaSeq platforms, sequenced reads were mapped and aligned to the reference genome (GRCh37; hg19), and called and annotated variants in 89 genes associated with nephrotic syndrome (Supp. Table SI) [3,11-14] were prioritized based on rarity (minor allele frequency, MAF <0.1%), novelty in population databases [15-17], prediction of deleteriousness by in silico tools, and if previously reported with disease [18]. Only pathogenic and likely pathogenic variants, according to criteria of the American College of Medical Genetics and Genomics (ACMG) 2015 guidelines [18,19] were considered causative, and were validated by Sanger sequencing. Sanger sequencing on parents’ samples was used to confirm allele segregation for compound heterozygous variants. Haplotype studies were performed to determine if the　NPHS1　variant c.2600G>A (p.Gly867Asp) that segregated in 10 of 34 patients　occurred on a common genetic background, suggesting inheritance from a common ancestor (founder mutation) (Supp. Methods) [20].

Statistical analyses: Data was summarized as median (interquartile range, IQR) for continuous variables and percentage with 95% confidence interval (CI) for dichotomous variables. Assuming 70% prevalence of pathogenic or likely pathogenic variations in genes encoding key podocyte proteins in patients with congenital nephrotic syndrome [1,3,12,13], 21 patients were required to be enrolled for a precision of 20%, at power of 80% and alpha error of 5%.

Samples were collected from 34 unrelated patients (53% boys) with congenital NS diagnosed at 7 centers across India. Onset of edema was at median age of 20 (IQR 15-45) days of life, and was associated with anasarca (91.2%), oliguria (41.2%), poor feeding (35.3%), seizures (32.3%), hypovolemia (23.5%), severe infections (20.6%) and/or lethargy (8.8%). Ten (29.4%) patients were born premature and 13 (38.2%) had low birth weight (Supp. Table SII). Consanguinity and similar illness in siblings were reported in 11 (32.4%) cases, each.

Median weight for age standard deviation score (SDS) was -3.1 (IQR -4.1, -1.9), length for age SDS was -3.9 (IQR -4.6, -2.2) and head circumference SDS was -3.2 (IQR -4.5, -2.2). Seven (20.6%) patients had hypertension. Isolated extrarenal features were observed in 9 patients (Supp. Table SII), while one patient each had features of Galloway-Mowat and Pierson syndrome. One patient had albinism and microcephaly and history of sibling death with similar symptoms.

WES with mean coverage of ³30x (Web Table SIII) returned 16804 variants, of which 1370 variants were present in one or more of the targeted genes (Fig. 1). After filtering, 91 variants were shortlisted (Supp. Table SIV), of which 22 variants were prioritized in 28 patients (Table I; Supp. Fig. S1). Pathogenic and likely pathogenic variants were inherited as homozygous and compound heterozygous variations in 20 and 8 patients, respectively. A monogenic cause was thus established in 82.4% (95% CI 66.9% to 92.5%) of 34 patients with congenital NS. Most variants were conserved across species (Web Fig. S2).

Variants in NPHS1 were most common, including 16 reported [11,12,14,15,22-35] and two novel variants, segregated in 24 patients as homozygous (n=16) and compound heterozygous (n=8) variants (Table I). Reported variations included 7 pathogenic and 9 likely pathogenic variants. One novel homozygous variant in ID#181 was classified as likely pathogenic, while another novel NPHS1 variant that segregated as compound heterozygous in ID#8, was assigned as pathogenic. Fig. 2 indicates the distribution of defects in NPHS1 across the structure of nephrin.

Fig. 2 Localization of novel variations and known mutations in the translated nephrin protein, comprised of eight extracellular immunoglobulin (Ig) -like domains (semi-circles), a fibronectin type III-like module (hexagon), a transmembrane domain (black rectangle) and a C-terminal (C) cytoplasmic domain (curled line). The bottom panel indicates the exons coding for the corresponding protein domains. Note that the 18 variations observed were spread throughout the protein. The variations with dotted lines are known or speculated to be founder mutations.

One previously reported [11,14] likely pathogenic NPHS1 variant in exon 19 (c.G2600A; p.Gly867Asp) was inherited as homozygous in 7 and heterozygous in 3 patients from different ethnic and regional backgrounds, without any specific phenotype (Tables I and SII). Two other reported variations, p.Arg1160Ter [11,14,27] and p.Arg367Cys [14,25,27], were common to three patients each (Table I). In patients with NPHS1 variants, atrial septal defect was seen in two patients, and developmental delay, facial dysmorphism, clubbing, café au lait spots, hirsutism and aqueductal stenosis in one patient each (Supp. Table SII).

One patient each had homozygous likely pathogenic variants in NPHS2 [34] and OSGEP [36], associated with an atrial septal defect and Galloway-Mowat syndrome, respectively. One patient each had novel pathogenic homozygous variations in PLCE1 and LAMB2 genes; the latter was associated with phenotype consistent with Pierson syndrome.

No variants were prioritized in two patients; four patients had heterozygous variations that were of unknown significance (Supp. Table SIV). Patients with causative variations also had additional heterozygous variations (Supp. Table SIV).

There were no differences in sex ratio, age at onset of symptoms, levels of serum albumin or estimated GFR between patients with NPHS1 variations and those with other or no significant variations (P>0.05 each).

Forty-four of 900 single nucleotide polymorphisms (SNPs) (Supp. Table SV) in the region (±500 kbp) flanking the c.2600G>A (Gly867Asp) were selected for haplotype analysis in 33 patients. All 17 alleles carrying the c.2600G>A variant (homozygous in 7 and heterozygous in 3 patients) shared a core haplotype in the 500 kbp region between rs2230181 to rs466452 (Supp. Table SVI). Thirteen of 17 alleles also shared a core haplotype extending to 800 kbp length. The 500 kbp core haplotype was observed in only one of 49 non-mutant chromosomes, suggesting a founder effect.

There is significant heterogeneity in prevalence of inherited defects across studies (Supp. Table SVII) [6-10,12,22-26,34]. Variants in NPHS1 predominate even in non-Finnish cohorts, and contributions by NPHS2, WT1 and LAMB2 defects differ widely across populations. In the present study, the use of NGS enabled a diagnosis in 82% of 34 patients. These findings are unlike previous studies from non-Caucasian populations that report lower rates of inherited defects, perhaps due to focused testing including a few genes (Supp. Table SVII).

Two founder deletion mutations in NPHS1, accounting for the majority of cases of Finnish type of congenital nephrotic syndrome, were not observed in our patients, similar to reports from non-Finnish populations [2,3,6-10]. Over 200 NPHS1 mutations are described worldwide in non-Finnish populations [29,32]. In our report, homozygous and compound heterozygous mutations in NPHS1 accounted for 70.6% of cases of congenital NS, and 85.7% of cases with an identified genetic etiology. This proportion is higher than previous reports from Asia, in which NPHS1 mutations accounted for 22-67% of cases, but similar to proportions reported in series including non-Finnish populations (Supp. Table SVII).

As shown in Fig. 2, variants in NPHS1 were distributed all over the protein. Three patients shared the variant p.Arg1160Ter, responsible for premature truncation of protein in the intracellular domain that interacts with podocin. This variant, a founder mutation in Maltese patients, is associated with a different allele in Asian patients [25]. While Koziell, et al. reported a mild phenotype in affected girl infants [25], we and other authors [24,35] found a severe phenotype, irrespective of gender, indistinguishable from other NPHS1 mutations. Three patients carried a variant (c.1099C>T; p.Arg367Cys), reported previously as a founder mutation from India [12]. One NPHS1 variant, c.2600G>A (p.Gly867Asp), that translates into a change in the immunoglobulin-like domain 8, found in 10 unrelated patients from five states in north India (Supp. Table SII, Table I and Fig. 1), has been reported from India, Pakistan and Saudi Arabia [8,11,14,37], but not from east Asia [6,7,9] or Europe. Using statistical tools considered more efficient that conventional haplotyping [20], we show that c.2600G>A is possibly a founder mutation, as suggested by the lack of genetic variation in the 500-800 kbp length flanking regions [38]. The differences in frequency of the shared haplotype in various ethnicities in the 1000 genome database suggests a European origin for the mutation (Supp. Table SVIII) [15]. Our hypothesis requires confirmation by examining for the same shared haplotype in previously reported patients with the　p.Gly867Asp mutation.

Mutations in NPHS2 and WT1 account for 0-51% and 0-40% cases, respectively, across populations, though NPHS2 variants are uncommon in Asia (Supp. Table SVII). In this cross-sectional study, only one patient had homozygous mutations in NPHS2, and none had variants in WT1. Given the small study size, these findings have limited generalisability.

Confirming previous findings, we failed to find specific phenotypic associations in patients with NPHS1, NPHS2 and PLCE1 mutations [4,26,39]. The lone patient with homozygous LAMB2 variant had findings of Pierson syndrome while another had Galloway-Mowat syndrome secondary to OSGEP mutation [36]. The latter patient had the same mutation and phenotype as an infant of Pakistani ethnicity described previously [36].

The present series underscores the utility of providing a genetic etiology in patients with congenital NS, thereby facilitating prenatal counseling and testing in subsequent pregnancies. One NPHS1 mutation is hypothesized to have a founder effect in Indian population. Information on long term outcomes, including post-transplantation, is lacking since most children were lost to follow up after families chose a palliative care plan. Despite being a multicenter study, the findings of the relatively small sample size might not be generalizable.

Note: Supplementary material related to this study is available with the online version at www.indianpediatrics.net

Acknowledgments: We thank the following colleagues participating in the NephQuest network who contributed samples and details of their patients: SP Veeturi, Rainbow Children Hospital, Hyderabad; KL Tiewsoh, Postgraduate Institute of Medical Education and Research, Chandigarh; A Mittal, All India Institute of Medical Sciences, Jodhpur; S Krishnamurthy, Jawaharlal Institute of Postgraduate Medical Education and Research, Puducherry; M Mantan, Maulana Azad Medical College, New Delhi; M Kumar and K Mishra, Chacha Nehru Bal Chikitsalaya, Delhi.

Ethics approval: Ethics committees at CSIR Institute of Genomics and Integrative Biology, Delhi and All India Institute of Medical Sciences, New Delhi; Sanction no. IECPG-616/21.12.2016. RT-33/22.03.2017 and 6/GAP127/CSIR-IGIB/2017

Contributors: All authors contributed to the study conception and design. AJ, AS, AS, MF, AB: material preparation, data collection and analysis were performed; AJ, AS: The first draft of the manuscript was written jointly. All authors commented on the manuscript, and approved the final manuscript.

Funding: Department of Biotechnology, Government of India (BT/11030/MED/30/1644/2016).

Competing interest: None stated.

1. Jalanko H. Congenital nephrotic syndrome. Pediatr Nephrol 2009; 24: 2121-28

2. Norio R. Heredity in the congenital nephrotic syndrome. A genetic study of 57 Finnish families with a review of reported cases. Ann Paediatr Fenn 1996;12:S27:1-94

3. Ha TS. Genetics of hereditary nephrotic syndrome: a clinical review. Korean J Pediatr 2017;60:55-63

4. Hinkes BG, Mucha B, Vlangos CN, et al; Arbeitsgemeinschaftfür Paediatrische Nephrologie Study Group. Nephrotic syndrome in the first year of life: Two thirds of cases are caused by mutations in 4 genes (NPHS1, NPHS2, WT1, and LAMB2). Pediatrics 2007;119: e907-19

5. Wang JJ, Mao JH. The etiology of congenital nephrotic syndrome: Current status and challenges. World J Pediatr 2016;12:149-58

6. Li GM, Cao Q, Shen Q, et al. Gene mutation analysis in 12 Chinese children with congenital nephrotic syndrome. BMC Nephrol 2018;19:382.

7. Sako M, Nakanishi K, Obana M, et al. Analysis of NPHS1, NPHS2, ACTN4, and WT1 in Japanese patients with congenital nephrotic syndrome. Kidney Int 2005;67: 1248-55

8. Sharief SN, Hefni NA, Alzahrani WA, et al. Genetics of congenital and infantile nephrotic syndrome. World J Pediatr 2019;15:198-203

9. Nishi K, Inoguchi T, Kamei K, et al. Detailed clinical manifestations at onset and prognosis of neonatal-onset Denys-Drash syndrome and congenital nephrotic syndrome of the Finnish type. Clin Exp Nephrol 2019;23:1058-65

10. Sinha R, Vasudevan A, Agarwal I, et al. Congenital nephrotic syndrome in India in the current era: A multicenter case series. Nephron 2019;144:1-9

11. Bierzynska A, McCarthy HJ, Soderquest K, et al. Genomic and clinical profiling of a national nephrotic syndrome cohort advocates a precision medicine approach to disease management. Kidney Int 2017;91:937-47

12. Sadowski CE, Lovric S, Ashraf S, et al; SRNS Study Group, Hildebrandt F. A single-gene cause in 29.5% of cases of steroid-resistant nephrotic syndrome. J Am Soc Nephrol 2015;26:1279-89

13. Trautmann A, Lipska-Ziêtkiewicz BS, Schaefer F. Exploring the clinical and genetic spectrum of steroid resistant nephrotic syndrome: The PodoNet registry. Front Pediatr 2018;6:200

14. Lovric S, Fang H, Vega-Warner V, et al; Nephrotic Syndrome Study Group. Rapid detection of monogenic causes of childhood-onset steroid-resistant nephrotic syndrome. Clin J Am Soc Nephrol 2014;9:1109-16

15. 1000 Genomes Project Consortium; Auton A, Brooks LD, Durbin RM, et al. A global reference for human genetic variation. Nature 2015; 526: 68-74

16. Karczewski KJ, Weisburd B, Thomas B, et al; The Exome Aggregation Consortium, Daly MJ, MacArthur DG. The ExAC browser: Displaying reference data information from over 60000 exomes. Nucleic Acids Res 2017;45 (D1): D840-5

17. Karczewski KJ, Francioli L, Tiao G, et al. The mutational constraint spectrum quantified from variation in 141,456 humans. Nature 2022;581:434-43.

18. Hamosh A, Scott AF, Amberger JS, Bocchini CA, McKusick VA. Online Mendelian Inheritance in Man (OMIM), a knowledgebase of human genes and genetic disorders. Nucleic Acids Res 2005;33:D514-7

19. Richards S, Aziz N, Bale S, et al; ACMG Laboratory Quality Assurance Committee. Standards and Guidelines for the Interpretation of Sequence Variants: A Joint Consensus Recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Med 2015;17:405-24

20. Stephens M, Smith NJ, Donnelly P. A new statistical method for haplotype reconstruction from population data. Am J Hum Genet 2001;68:978-89

21. Schwartz GJ, Muñoz A, Schneider MF, et al. New equations to estimate GFR in children with CKD. J Am Soc Nephrol 2009;20:629-37

22. Schoeb DS, Chernin G, Heeringa SF, et al; Gesselschaftfür Paediatrische Nephrologie Study Group. Nineteen novel NPHS1 mutations in a worldwide cohort of patients with congenital nephrotic syndrome (CNS). Nephrol Dial Transplant 2010;25:2970-6

23. Lee JH, Han KH, Lee H, et al. Genetic basis of congenital and infantile nephrotic syndromes. Am J Kidney Dis 2011;58:1042-3

24. Warejko JK, Tan W, Daga A, et al. Whole exome sequencing of patients with steroid-resistant nephrotic syndrome. Clin J Am Soc Nephrol 2018;13:53-62

25. Koziell A, Grech V, Hussain S, et al. Genotype/phenotype correlations of NPHS1 and NPHS2 mutations in nephrotic syndrome advocate a functional inter-relationship in glomerular filtration. Hum Mol Genet 2002;11:379-88

26. Machuca E, Benoit G, Nevo F, et al. Genotype-phenotype correlations in non-Finnish congenital nephrotic syndrome. J Am Soc Nephrol 2010;21:1209-17

27. Lenkkeri U, Männikkö M, McCready P, et al. Structure of the gene for congenital nephrotic syndrome of the Finnish type (NPHS1) and characterization of mutations. Am J Hum Genet 1999;64:51–61

28. Kestilä M, Lenkkeri U, Männikkö M, et al. Positionally cloned gene for a novel glomerular protein-nephrin-is mutated in congenital nephrotic syndrome. Mol Cell 1998;1:575-82

29. Beltcheva O, Martin P, Lenkkeri U, Tryggvason K. Mutation spectrum in the nephrin gene (NPHS1) in congenital nephrotic syndrome. Hum Mutat 2001;17: 368-73

30. Ulinski T, Aoun B, Toubiana J, Vitkevic R, Bensman A, Donadieu J. Neutropenia in congenital nephrotic syndrome of the Finnish type: Role of urinary ceruloplasmin loss. Blood 2009;113:4820-1

31. Bolk S, Puffenberger EG, Hudson J, Morton DH, Chakravarti A. Elevated frequency and allelic heterogeneity of congenital nephrotic syndrome, Finnish type, in the old order Mennonites. Am J Hum Genet 1999; 65: 1785-90

32. Heeringa SF, Vlangos CN, Chernin G, et al; Members of the APN Study Group. Thirteen novel NPHS1 mutations in a large cohort of children with congenital nephrotic syndrome. Nephrol Dial Transplant 2008;23:3527-33

33. Ashton E, Bockenhauer D, Boustred C, Jenkins L, Lench N. ASHG 2013 Poster: Development of a rapid genetic testing service for nephrotic syndrome using next generation sequencing. Conference: American Society of Human Genetics, At Boston, USA 2013; available at https://www.researchgate.net/publication/258860074_ASHG_ 2013_Poster_Development_of_a_rapid_genetic_ testing_ service_for_nephrotic_syndrom e_using_next_generation_ sequencing; last accessed 21 February 2020

34. Sen ES, Dean P, Yarram-Smith L, et al. Clinical genetic testing using a custom-designed steroid-resistant nephrotic syndrome gene panel: Analysis and recommendations. J Med Genet 2017;54:795-804.

35. Ovunc B, Ashraf S, Vega-Warner V, et al; Gesellschaft für Pädiatrische Nephrologie (GPN) Study Group. Mutation analysis of NPHS1 in a worldwide cohort of congenital nephrotic syndrome patients. Nephron Clin Pract 2012;120:c139-46

36. Domingo-Gallego A, Furlano M, Pybus M, et al. Novel homozygous OSGEP gene pathogenic variants in two unrelated patients with Galloway-Mowat syndrome: Case report and review of the literature. BMC Nephrol 2019;20:126

37. Abid A, Khaliq S, Shahid S, et al. A spectrum of novel NPHS1 and NPHS2 gene mutations in pediatric nephrotic syndrome patients from Pakistan. Gene 2012;502:133-7

38. Zlotogora J. High frequencies of human genetic diseases: founder effect with genetic drift or selection? Am J Med Genet 1994;49:10-3

39. Schultheiss M, Ruf RG, Mucha BE, et al. No evidence for genotype/phenotype correlation in NPHS1 and NPHS2 mutations. Pediatr Nephrol 2004;19:1340-8.