Pediatric Nephrology

, Volume 24, Issue 2, pp 281–285

Exclusion of homozygous PLCE1 (NPHS3) mutations in 69 families with idiopathic and hereditary FSGS


    • Department of PediatricsDuke University Medical Center
    • Center for Human GeneticsDuke University Medical Center
  • Bartlomiej Bartkowiak
    • Department of PediatricsDuke University Medical Center
    • Center for Human GeneticsDuke University Medical Center
  • Peter J. Lavin
    • Department of MedicineDuke University Medical Center
    • Center for Human GeneticsDuke University Medical Center
  • Nirvan Mukerji
    • Department of MedicineDuke University Medical Center
    • Center for Human GeneticsDuke University Medical Center
  • Guanghong Wu
    • Department of MedicineDuke University Medical Center
    • Center for Human GeneticsDuke University Medical Center
  • Brandy Bowling
    • Department of MedicineDuke University Medical Center
    • Center for Human GeneticsDuke University Medical Center
  • Jason Eckel
    • Department of MedicineDuke University Medical Center
    • Center for Human GeneticsDuke University Medical Center
  • Tirupapuliyur Damodaran
    • Department of MedicineDuke University Medical Center
    • Center for Human GeneticsDuke University Medical Center
    • Department of MedicineDuke University Medical Center
    • Center for Human GeneticsDuke University Medical Center
Original Article

DOI: 10.1007/s00467-008-1025-5

Cite this article as:
Gbadegesin, R., Bartkowiak, B., Lavin, P.J. et al. Pediatr Nephrol (2009) 24: 281. doi:10.1007/s00467-008-1025-5


Focal and segmental glomerulosclerosis (FSGS) is the most common glomerular cause of end-stage kidney disease (ESKD). Although the etiology of FSGS has not been fully elucidated, recent results from the positional cloning of genes mutated in nephrotic syndromes are now beginning to provide insight into the pathogenesis of these diseases. Mutations in PLCE1/NPHS3 have recently been reported as a cause of nephrotic syndrome characterized by diffuse mesangial sclerosis (DMS) histology. One single family with a missense mutation had late onset of the disease that was characterized by FSGS. To further define the role of PLCE1 mutations in the etiology of FSGS, we performed mutational analysis in 69 families with FSGS. A total of 69 families with 231 affected individuals were examined. The median age of disease onset was 26 years (range 1–66 years). Onset of ESKD was at a median age of 35.5 years. Seven variants leading to non-synonymous changes were found, of which only two are new variants (exon 4 c.1682 G>A R561Q, exon 31 c.6518A>G K2173R). No known disease-causing mutations were identified in the families screened. PLCE1/NPHS3 mutations are not a cause of FSGS in this cohort. The absence of mutations in PLCE1/NPHS3 in this study indicates that there are additional genetic causes of FSGS and that hereditary FSGS is a heterogeneous disease. Kindreds appropriate for genome-wide screening are currently being subjected to analysis with the aim of identifying other genetic causes of FSGS.


Familial focal segmental glomerulosclerosisFamilial nephropathyFSGSGeneticsHereditaryPLCE1 mutation


Focal and segmental glomerulosclerosis (FSGS) is a clinicopathological entity that is characterized by the nephrotic syndrome and is often steroid resistant. Progression to end-stage kidney disease (ESKD) is frequent. Histologically, the lesion is characterized by focal glomerulosclerosis or tuft collapse, segmental hyalinosis and, occasionally, immunoglobulin (Ig)M staining on immunofluorescence and effacement of foot processes on electron microscopy [1]. It is responsible for 2–20% of all cases of ESKD in the USA and is the most common cause of glomerular disease in this patient subset [2, 3]. The incidence is higher in blacks than in whites, with the former showing a striking difference in age distribution pattern, with the highest incidence of the disease occurring in the 40- to 49-year-old age group [2, 3].

Current doctrine is that the primary defect in FSGS is in the filtration barrier of the glomerulus. This barrier is composed of fenestrated endothelium, the glomerular basement membrane, and podocytes which have a slit diaphragm between their interdigitating foot processes. Disruption of the filtration barrier results in the loss of permselectivity and albuminuria. The pathogenesis of FSGS has not been fully elucidated; however, recent data from studies of familial cases reveal mutations in genes that encode the slit diaphragm and podocyte cytoskeletal proteins, suggesting that FSGS is a primary disease of the podocyte. The first major breakthrough was the cloning of nephrin (NPHS1) as a cause of congenital nephrotic syndrome of the Finnish type [4]. Since then, four additional genes have been identified, including podocin (NPHS2), actinin-α4 (FSGS1), transient receptor potential cation channel, type 6 (TRPC6/FSGS2), and CD2-associated protein (FSGS3), as causes of FSGS and hereditary nephrotic syndromes [5-8].

Mutations in phospholipase c epsilon-1 (PLCE1/NPHS3) have recently been reported as a cause of early onset nephrotic syndrome that is characterized by histology of diffuse mesangial sclerosis (DMS) [9]. The PLCE1 gene is on chromosome 10q23. PLCε1 is a member of the phospholipase family of enzymes that catalyzes the hydrolysis of polyphosphoinositides to generate second messengers, such as inositol-1, 4, 5 trisphosphate and diacylglycerol [9]. These second messengers are involved in cell growth and differentiation [9]. PLCε1 is expressed in the podocyte, and although the mechanism by which it causes DMS has not been fully elucidated, in vitro data suggest that it may act as a scaffolding protein in the glomerular slit diaphragm [9]. All of the mutations reported in children with DMS to date have been loss-of-function mutations. Interestingly, two of the children in the original report had the histology of FSGS; in addition, they both had later onset disease and a missense mutation, which is in contrast to children with DMS who all had early onset disease and truncating/loss-of-function mutations. These findings suggest that PLCE1 mutations may have a role in the pathogenesis of FSGS either as the cause of the disease or at least as a modifier gene acting in concert with other mutations and environmental factors.

To further define the role of PLCE1 mutations in the etiology and pathogenesis of familial FSGS, we have performed mutational analysis in a world-wide cohort of 69 families with FSGS.


Clinical data

Institutional Review Board approval for this study was obtained from Duke University Medical Center (Durham, NC, USA). The Methods of subject recruitment have been previously reported [10]. Inclusion criteria in this study were (1) diagnosis of biopsy-proven FSGS in at least two family members with multiple affected members; in cases of idiopathic FSGS, a diagnosis of biopsy-proven FSGS was also required; (2) exclusion of secondary causes of FSGS, such as human immunodeficiency virus infection, hepatitis, and obesity; (3) exclusion of mutations in NPHS1, NPHS2, ACTN4, and TRPC6. Clinical data obtained included a complete family history, other associated morbidities, age at onset of disease, and age at ESKD; urinalysis and serum creatinine were measured as appropriate.

Mutational analysis

Genomic DNA was extracted from whole blood using the Puregene kit (Qiagen, Hilden, Germany). Mutational analysis was carried out by sequencing both strands of all exons of PLCE1 using exon flanking primers. All sequences were analyzed with the Sequencher software (Gene Codes Corp, Ann Arbor, MI).

Data analysis

The clinical data and frequency of mutation and sequence variants were compared between the single- and multi-generation families. All categorical data were compared by means of χ2 test, and continuous variables were assessed using the Student t test if they were normally distributed and the Kruskal–Wallis test for continuous data that were not normally distributed.


Clinical phenotype

We studied 231 affected individuals from 69 families. Of these families, ten (14.5%) had only one affected person, 19 (27.5%) had two or more affected individuals in one generation, and 40 (58%) had two or more affected individuals in at least two generations. The kindreds with only one affected member were classified as idiopathic (10/69 or 14.5%), those with two or more individuals in only one generation were classified as single generation (SG: 19/69 or 27.5%), and those with affected individuals in at least two generations were classified as multi-generation (MG: 40/69 or 58%). Kindreds with more than one affected individual (SG) in one generation were assumed to be autosomal recessive. Kindreds with affected individuals in more than one generation and male-to-male transmission (MG) were assumed to be autosomal dominant. None of the families are known to be consanguineous. The racial distribution, age at onset of disease, and age at ESKD were not different between the SG and the MG group (Table 1). At least one individual in 52.6% (10/19) of the SG families and 55% (22/40) of the MG families are known to have had a kidney transplant. Two families had one individual each with recurrence of FSGS in their renal allograft, and both were in the MG group.
Table 1

Clinical characteristics of 69 families with idiopathic and familial focal and segmental glomerulosclerosis


Idiopathic (n = 10)

SG+ (n = 19)

MG+ (n = 40)

Race (%)






  African American








Median age at onset, years (range)

36.5 (16–45)

19 (2.5–49)

26 (1–66)

Median age ESKD, years (range)

35 (17–45)

5.2 (1–10)

31.75 (6–50)

Proteinuria median, g/24 h (range)

5.8 (2.4–15)

39.5 (10–63)

2.5 (1–10)

Response to therapy

0/2 (0%)

0/4 (0%)

2/9 (22.2%)


3/10 (30.0%)

10/19 (52.6%)

22/40 (55%)

Transplant recurrence

0/3 (0%)

0/10 (0%)

2/22 (9.1%)

ESKD End-stage kidney disease; SG+ single generation MG+ multi-generation

Mutation analysis

We found no new or previously documented disease causing mutations in any of the 69 families studied. Eight non-synonymous changes were documented in the 69 families (Table 2), six of which are known single nucleotide polymorphisms. Two heterozygous missense sequence variations (exon 4 c.1682G>A R561Q and exon 31 c.6518A>G K2173R) found in two separate families are novel variants that have not been previously reported.
Table 2

Non-synonymous variants in PLCE1 in 69 families with familial focal and segmental glomerulosclerosis


Nucleotide change

Amino acid change

SNP number


















4724 G>C















SNP Single nucleotide polymorphism


The molecular etiology and pathogenesis of FSGS is still under investigation, with new information being generated from positional cloning. Mutations in PLCE1 have recently been reported as a cause of early onset nephrotic syndrome characterized mainly by DMS histology pattern, although two individuals were found to have FSGS, implying that mutations in this gene may be a cause of some cases of FSGS [9]. In our study we performed mutation analysis in PLCE1 in 69 families, 59 of which have at least two or more affected individuals and only ten have one affected individual. Our cohort therefore predominantly comprises families with FSGS inherited in a Mendelian fashion, which is likely due to a single gene defect. It is possible that the ten idiopathic cases may represent individuals with polygenic inheritance.

Two individuals from separate families had recurrence of FSGS in their renal allograft. One is from a large Central European family with 13 affected individuals spread across four generations, while the other is from a North American family with affected individuals in two generations. The source of the kidney transplant from one person is unknown, but the second individual had a living related donor (LRD) kidney transplant. The disease recurrence rate in families with familial FSGS in this cohort is low when compared to the known recurrence rate for idiopathic FSGS [11]. The low recurrence risk seen in familial cases in this series is, however, similar to that previously reported in subjects with NPHS2 mutations, where it was found that the rate of recurrence of disease in renal allografts was considerably lower than in those in subjects without NPHS2 mutations [12]. There are a number of possible explanations for disease recurrence in the two subjects in our study. The defective gene and its product may not necessarily be localized to the kidney or the podocyte, or the individual who received an LRD kidney may have obtained it from an obligate heterozygote who may have been asymptomatic at the time of the transplant. It is also possible that the two individuals are phenocopies with idiopathic FSGS and that their disease may not be due to the genetic defects in the family.

We did not find disease causing homozygous or compound heterozygous mutations in any of the families studied. This result is similar to that reported in a recent Dutch study that found no mutations in PLCE1 in 19 cases of childhood-onset FSGS [13]. In contrast, in a study of 57 families with autosomal recessive FSGS, Nevo et al. reported truncating and missense mutation in six families [14]. All of the affected families in the study had rapidly progressive disease leading to ESKD before the age of 7 years. The reason for the difference between our findings and the observations in previous studies is not clear. It is possible that most of the families in our study have autosomal dominant disease because there are more than two generations affected with male-to-male transmission. However, even in the subset with apparent autosomal recessive inheritance, we did not detect any mutation. The two individuals reported in the original description of PLCE1 mutations are from a consanguineous Turkish family [9], while the ethnic origin of the families from the Nevo et al. study [14] is unknown. In our series, none of the families are known to be consanguineous, and most of the families are from North America and Central Europe. It is therefore possible that PLCE1 mutations are rare in this population. The other difference between our series and the two previous reports is the late onset of disease in our cohort; this finding will be in keeping with the initial observation that PLCE1 may be important during glomerulogenesis by affecting capillary development and scaffolding in the slit diaphragm and will, therefore, cause early onset disease. We found two novel heterozygous variants in two different families (exon 4 c.1682G>A R561Q and exon 31 c.6518A>G K2173R). Exon 4 of the gene encodes for the Ras binding domain of PLCε1, and exon 31 encodes for the C-terminal Ras-associating (RA) domain of the protein. Both domains are important for the ability of PLCε1 to function as an initiator and recipient of activated G protein signals [15]. The two variants are conserved in evolution down to the zebrafish (R561Q change) and to drosophila (K2173R change). However, despite this impressive conservation in evolution, we did not find this change in all of the affected individuals, and we also found the same change in some unaffected members of the families, which implies that these changes are unlikely to be a cause of disease. The amino acid change in the exon 4 variant is from arginine (a basic amino acid) to glutamine (a neutral amino acid), while the variant resulting in the exon 31 change are both basic amino acids. Analysis of the amino acid substitution using the software developed by Sunyaev et al. showed that the two amino acid substitutions are unlikely to be deleterious to the functions of PLCε1 [16].

In conclusion, we did not find PLCE1 mutations in this cohort, suggesting that the mutation in this gene is a very rare cause of FSGS in this population. Routine screening for mutations in individuals with hereditary autosomal dominant FSGS is therefore not warranted, and mutational analysis in this gene should probably be limited to disease with DMS histology. The absence of mutations in PLCE1 and other known FSGS genes is an indication that there are additional genetic causes of FSGS. We are currently subjecting the families in this study to a genome-wide search to identify other causes of FSGS.


This research was funded by the National Institutes of Health R01 DK074748-01 to MPW and grants from Nephcure foundation to RG. We would like to thank the personnel of the Center for Human Genetics core facilities and especially the family members of the Duke FSGS project.

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© IPNA 2008