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HuGENet Review
“The findings and conclusions in this review are those of the author(s) and do not
necessarily represent the views of the funding agency.”
This HuGE Review was published in Genetics in Medicine 2006; 8(2):63-75
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NPHS2 gene, nephrotic syndrome and focal segmental glomerulosclerosis
Franceschini, Nora MD, MPH1; North, Kari E. PhD1; Kopp, Jeffrey B. MD2; Mckenzie, Louise PhD3; Winkler, Cheryl PhD3

1 Department of Epidemiology, School of Public Health, University of North Carolina, Chapel Hill, NC
2 Kidney Disease Section, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health,       Bethesda, MD;
3 Laboratory of Genomic Diversity, SAIC-Frederick, National Cancer Institute, Frederick, MD.

Nora Franceschini, MD, MPH, University of North Carolina, Department of Epidemiology, Bank of America Center, 137 E. Franklin Street, suite 306 CB#8050, Chapel Hill, NC 27514-3628.

Received September 4, 2005. Accepted November 15, 2005.


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 ABSTRACT

Nephrotic syndrome, characterized by edema, proteinuria, hyperlipidemia and low serum albumin, is a manifestation of kidney disease involving the glomeruli. Nephrotic syndrome may be caused by primary kidney disease such as focal segmental glomerulosclerosis. Mutations in the podocin gene, NPHS2, have been shown in familial and sporadic forms of steroid-resistant nephrotic syndrome, including focal segmental glomerulosclerosis. Podocin is an integral membrane protein located at the slit diaphragm of the glomerular permeability barrier. Complete information is lacking for the population frequency of some NPHS2 variants for all racial and ethnic groups. The most frequently reported variant, R229Q, is more common among European-derived populations than African-derived populations. We calculated crude odds ratios and 95% confidence intervals of childhood nephrotic syndrome and focal segmental glomerulosclerosis associated with R229Q heterozygosity using data from five studies. The R229Q variant is not associated with focal segmental glomerulosclerosis in the US population of African descent. In contrast, the R229Q variant is associated with a trend toward increased focal segmental glomerulosclerosis risk in European-derived populations, with an estimated increased risk of 20-40%. Our insight into the association between NPHS2 variants and nephrotic disease is hampered by the limitations of the existing studies, including small numbers of affected individuals and suboptimal control groups. Nevertheless, the available data suggest that large epidemiological case-control studies to examine the association between NPHS2 variants and nephrotic syndrome are warranted.

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 GENE

The NPHS2 gene (OMIM number 604766) is located at chromosome 1q25-q31 and was first mapped by linkage analysis in families with autosomal recessive steroid-resistant nephrotic syndrome.(1) The NPHS2 gene was subsequently identified by positional cloning.(2) The NPHS2 coding region is 1,149 bp in length and is followed by a 635-bp 3′ UTR containing atypical polyadenylation signals (AATTAAA) situated 13 nt upstream of the poly (A) tail. The gene has 8 exons and encodes the 42 kD integral membrane protein podocin which is expressed in both fetal and mature kidney. Podocin is homologous to stomatins (band-7 proteins), sharing 46% amino acid identity to human stomatin and 40% identity to MEC-2 and Unc1.(3) Stomatin is an integral membrane protein of unknown function. Defects in stomatin trafficking leading to its absence in red blood cell membranes cause hereditary stomatocytosis, a rare form of hemolytic anemia associated with transmembrane leak of sodium and potassium.(4) MEC-2 mutations cause peripheral touch sensation dysfunction by interfering with a neuronal transduction mechanism associated with an ion channel.(4) Sequence analysis of podocin indicates that it consists of 383 amino acids, with cytosolic C- and N-terminal domains and one short transmembrane domain, forming a hairpin-like structure.(5) Similar to stomatins, podocin forms homo-oligomeric complexes that directly interact with cholesterol in specialized microdomains of the plasma membrane called lipid rafts.(6)

By in situ hybridization, podocin has been localized exclusively in glomeruli, with transcript expression restricted to the podocytes.(2) Podocin is located at the foot process of the podocytes in the slit diaphragm, the site responsible for size and charge selectivity of filtration.(7,8) It localizes to the lipid rafts which have high concentration of signal transduction molecules.(8,9) There, podocin interacts with nephrin and CD2AP, among other proteins in the slit diaphragm complex. Mutations of the nephrin gene, NPHS1, cause congenital nephrotic syndrome of the Finnish type, a form of severe nephrotic syndrome first manifested in utero and characterized by immature glomeruli and rapid progression to kidney failure.(10) Podocin is necessary for recruitment of nephrin into the lipid rafts.(9) In fact, some podocin mutations result in changes in the distribution of nephrin and other proteins in podocytes.(11) Homozygous mutations of podocin have been implicated in few cases of congenital nephrotic syndrome.(9,12,13) In addition, podocin-deficient NPHS2 knock-out mice present with massive proteinuria at birth.(14) Thus, it is clear that podocin plays an essential role in the maintenance of the glomerular permeability barrier.

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 GENE VARIANTS

We searched Medline and PubMed using the combination of the keywords NPHS2, podocin, genetics and epidemiology to identify studies for the estimation of the prevalence of NPHS2 polymorphisms in different populations. In addition, we searched the combination of terms podocin, NPHS2 and nephrotic syndrome, focal segmental glomerulosclerosis or steroid-resistant nephrotic syndrome to identify population-based studies describing the association of the NPHS2 gene and kidney disease. Papers published in English between 2000 and June, 2005 were reviewed since the gene was first identified in 2000. Reference lists from published articles were also reviewed. We also searched for relevant abstracts published by the American Society of Nephrology, International Society of Nephrology, European Renal Association and European Dialysis and Transplant Association, and the International Genetic Epidemiology Society from 2002 to November, 2004. Additional information regarding podocin variant frequencies in control subjects was obtained by direct contact with study authors. Unpublished data of the National Institutes of Health (NIH) FSGS Genetic Study were provided by three of the coauthors (JK, LM, and CW). We selected for this review only studies that described the allele or genotype frequency of the NPHS2 polymorphisms in a minimum of 40 control participants. Allele frequencies were obtained from published studies and the 95% confidence intervals (CI) were calculated using standard methods and using Excel software (Microsoft, Bellevue, WA).(15) Pooled allele frequencies and their 95% confidence intervals (CI) were calculated on the control sample stratified by ethnicity using standard methods(15) and SAS statistical sofware (SAS Institute, NC). When available, allele frequencies were used to calculate expected genotypic proportions according to Hardy-Weinberg equilibrium (HWE). Chi-square statistics were used to determine if the expected number of persons with a given genotype deviated from expectation. For the association analysis, all studies displaying deviations from HWE controls were excluded.

Most published studies included cases of individuals with steroid-resistant nephrotic syndrome (lacking a kidney biopsy) and biopsy-proven focal segmental glomerulosclerosis (FSGS). They described new mutations of the NPHS2 gene in families or in individual cases (sporadic). Studies usually included ethnic-matched controls consisting of blood donors, spouses of affected individuals or other unrelated individuals defined as healthy controls or blood donors (Table 1). Since FSGS is uncommon and often symptomatic, it is likely that these control groups did not have FSGS. In general, studies described the allele frequency of polymorphisms and less often genotype frequencies.

Table 1: Allele frequency of non-synonymous and synonymous NPHSvariants and intronic variants in normal controls by geographic area and by study population.

Table 1 displays the allele frequencies of non-synonymous and synonymous variants in exons and introns observed in control subjects. Limited data are available on race and geographic distribution for many of the podocin alleles (Table 1). The majority of the non-synonymous minor alleles have allele frequencies < 1%.
Figure 1 shows the gene location of the polymorphisms. Four non-synonymous variants are present more frequently: R229Q (range: 0.5% to 7%), G34E (2% in Japanese), A61V (1.6% in African Americans (AA)) and A242V (4% African descent in Europe and 6 to 8.7% in AA). For those studies reporting HWE expectations, only one non-synonymous polymorphism was out of HWE. This study observed one 34E/E in 44 normal donors, but no heterozygotes were observed.(16) Among the six synonymous polymorphisms, one each is observed in exons 1, 2, and 7 and three occur in exon 8. The allele frequencies vary among racial/ethnic groups (range 0.03 to 50%) but tend to be more frequent than the non-synonymous polymorphisms. Polymorphisms in non-coding regions of the NPHS2 gene have been reported in selected populations (Table 1).

Figure 1: Podocin polymorphisms (1% or more minor allele frequency in control populations) by gene exon. Exons are represented by boxes with corresponding numbers above and introns by lines between the exons. Non-synonymous and synonymous changes in podocin protein are shown below the exons. Exons are proportional to their lenght but introns and UTRs are not. For polymorphisms involving introns and untranslated regions refer to Table 1. Adapted from Dusel et al.(18)

R229Q in one of the most commonly reported NPHS2 polymorphism (Table 1 and Table 2). A G to A nucleotide exchange at position 686 in exon 5 results in an arginine to glutamine substitution at codon 229. The allele frequency was reported in nine published studies and one large unpublished ongoing study at the National Institutes of Health (NIH). One study had controls recruited from a population-based source.(17) In general, the polymorphism is more common in people of European descent (allele frequency varies from 2 to 7%) and is less common in people of African descent from the US and Brazil (0.5 to 2.5%) (Table 1). The allele frequency in Africa and Asia is unknown. Based on available data, we estimated the R229Q minor allele frequency and 95% CI in controls by combining the number of alleles of individuals with similar ethnic background from different studies. Figure 2 shows the R229Q minor allele frequency in individual studies and the results of the pooled analysis of the allele frequency by ethnicity using the whole sample. These results confirm that the 'A' allele frequency is more common in populations European descent (0.03, 95% CI, 0.02-0.04) compared to African descent (0.016, 95% CI, 0.00-0.02).

Table 2: Allele frequency of non-synonymous and synonymous NPHSvariants and intronic variants in normal controls by geographic area and by study population.

Figure 2: Allele frequency of R229Q polymorphism in controls, by ethnic group. Results are the estimates and 95% confidence interval from individual studies and the pooled estimates and 95% confidence interval by ethnicity using combined data from the studies. Numbers under the x-axis are the studies' references. Studies included in the pooled analysis include patients of African descent (Tsukaguchi et al.(3)(N = 32), National Institutes of Health cohort study (unpublished, N = 634), Pereira et al.(17) (N = not reported), Dusel et al.(18) (N = 96)), and European descent (Tsukaguchi et al.(3) (N = 124), National Institutes of Health cohort study (N = 272), Pereira et al.(17) (N = not reported), Karle et al.(21) (N = 100), Aucella et al.(34) (N = 124), Ruf et al.(35) (N = 80), Caridi et al.(38) (N = 260)). We were unable to calculate allele frequency confidence interval for the study by Pereira et al. because the number of screened individuals by ethnicity was not reported.

Eight studies described the genotype frequency distribution of R229Q polymorphism (Table 2). All but one of the studies reporting genotype frequency for this polymorphism had more than 100 individuals tested. The genotype distribution of R229Q conformed to HWE in control populations, although three studies had a mixed ethnic population (Table 2). The estimated frequency of heterozygotes varied from 0.03 to 0.13 in Caucasians and it was 0.025 in a single study of African Americans (Table 2).

Another non-synonymous polymorphism in exon 5 is A242V which results from a change of C to T at the nucleotide 725. A242V is more common in subjects of African descent in the US and Europe, with an allele frequency varying from 4 to 8.7%, and is uncommon in populations of European descent (Table 1). The genotype frequency of this polymorphism was described in only one European study.(13) Among 160 control subjects of French descent and African descent, 1% carried A242V C/T heterogosity, and 0% had the T/T genotype. In the 75 subjects of African descent, 8% had the C/T genotype and 0% had the T/T genotype.(13)

The G34E polymorphism results from a G to A nucleotide substitution at position 101 of exon 1 and has only been reported in one study from Japan.(16) The A61V polymorphism in exon 1 was described in a US study of African descent controls but was not observed in controls of African descent in France.(18,13)

The synonymous polymorphisms A318A, S96S and L346L were initially described in China(19,20) and later by others.(16,18,21) A138A results from a T to C nucleotide exchange at position 954 in exon 8. The minor allele frequency is similar among African-American and Asian populations. The S96S polymorphism is a result of a nucleotide change from C to T at position 288 on exon 2. This polymorphism has similar frequency among Europeans and Asians and higher allele frequency among subjects of African descent in the US. The L346L polymorphism is a product of an A to G nucleotide substitution at position 1038 in exon 8. The allele frequency is higher in those of African descent compared to Asians or Europeans.

The A44E, G34G and A297A polymorphisms have been described in a study of non-diabetic African-American patients with end-stage renal disease (ESRD).(18) A317A has only been described in an Italian study.(22)

A small number of studies have described polymorphism in untranslated regions including intronic positions (Table 1). Further studies are necessary to determine the allele and genotype frequencies across different population.

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 DISEASE

Idiopathic nephrotic syndrome is characterized by edema, increased urinary protein excretion, hyperlipidemia and low serum albumin. Nephrotic syndrome results from a group of heterogeneous diseases affecting the glomeruli (the filtration units of the kidney). Nephrotic syndrome can be due to systemic diseases, such as diabetic mellitus and lupus, and to primary renal diseases, including minimal change nephropathy and focal segmental glomerulosclerosis (sporadic FSGS). In adults, sporadic FSGS is the most common forms of primary nephrotic syndrome in the US.(23) In children, who frequently do not undergo renal biopsy, nephrotic syndrome is classified based on response to therapy in steroid-sensitive (SSNS) and steroid-resistant nephrotic syndrome (SRNS). Most SSNS is due to minimal change nephropathy and most SRNS is due to FSGS.

Acquired forms of FSGS include post-adaptive FSGS (following renal adaptation consisting of glomerulomegaly and hyperfiltration), medication-associated FSGS (pamidronate, interferon-alpha, cyclosporine, tacrolimus, lithium) and idiopathic FSGS (unknown cause). Patients with FSGS may present with asymptomatic proteinuria or with edema, and may manifest nephrotic proteinuria (>3.5 g/day) or subnephrotic proteinuria.(24-26) Collapsing glomerulopathy is often considered to be a subset of FSGS, although its distinctive histology features argue for a separate classification. Compared to patients with FSGS, patients with collapsing glomerulopathy tend to have heavier proteinuria, more steroid-resistance, and worse prognosis. Acquired forms of collapsing glomerulopathy include HIV-associated collapsing glomerulopathy (also termed HIV-associated nephropathy), idiopathic collapsing glomerulopathy, and medication-associated collapsing glomerulopathy. Most papers have not distinguished between FSGS and collapsing glomerulopathy, and so in the present manuscript FSGS will often stand for both syndromes.

Individuals of African descent are at increased risk for FSGS (4-fold), HIV-associated collapsing glomerulopathy (14-fold), and idiopathic collapsing glomerulopathy (risk not determined) compared to European-Americans.(27) African-Americans with HIV-associated collapsing glomerulopathy are 5.4 times more likely to have a family history of ESRD compared to HIV controls without renal dysfunction (95% CI, 1.6, 18.4), suggesting a genetic risk for progressive renal injury in response to a range of renal insults.(28)

The diagnosis of FSGS is made by a renal biopsy. Because renal biopsy is rarely performed among patients presenting with low levels of proteinuria and in those with advanced kidney failure, the incidence and prevalence of FSGS are likely underestimated by studying renal biopsy data.(29) The annual incidence of FSGS based on biopsy records is estimated to be 1.4 per million in Asia (Singapore), 2.3 to 12 per million in Europe and 21 per million in Oceania (Australia).(29) The incidence of the disease in the US is unknown.

FSGS has poor renal outcome, with approximately 50% kidney survival over 10 years.(30,31) The disease is currently the most common cause of incident ESRD due to primary glomerulonephritis. Glomerulonephritis are the third leading cause of ESRD in the US after diabetes and hypertension. FSGS compromises 2-3% of overall cases of incident ESRD in the US.(29) African-Americans may be at increased risk of progression to renal failure and are over-represented in ESRD groups. During the period 1995 to 1999, the annual incidence rate of ESRD due to FSGS was 7 cases/million for the general US population, with five cases per million in whites and 20 cases/million in African-Americans.(29) In addition, the highest annual incident rates of ESRD in African-Americans occur at younger ages compared to other races (ages 40-49 years for African-Americans and ages 70-79 years for other races), contributing for the increased burden of the disease in this ethnic group.29 Risk factors associated with progression to ESRD are nephrotic syndrome and elevated serum creatinine at biopsy, lack of remission of the nephrotic syndrome (or resistance to therapy), and possible African descent.(32) Genetic factors associated with progression of FSGS have not been investigated.

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 GENOTYPE-PHENOTYPE CORRELATIONS

Association studies

NPHS2 polymorphisms associated with nephrotic syndrome and FSGS

Studies that report genotype frequency among affected individuals and controls were systematically identified (see section Gene Variants). The studies were designed to identify mutations of the NPHS2 gene in affected families or individuals presenting with steroid-resistant nephrotic syndrome and/or focal segmental glomerulosclerosis (FSGS). Controls were selected from the same ethnic population of affected individuals in order to determine allele frequency of polymorphisms in the population. For the association analysis, we excluded studies that described multiple affected individuals in the same family (familial disease). Some studies have pooled data from individuals with familial and sporadic nephrotic syndrome. We included these studies in the analysis if the available data differentiated familial from sporadic cases. We calculated the crude odds ratio of disease and 95% CI by ethnicity according to Mantel-Haenszel method using SAS software.

Most studies have only described the R229Q polymorphism; thus insufficient data were available for consideration of other NPHS2 polymorphisms in subsequent analyses. This polymorphism has been described in familial and sporadic SRNS and in FSGS (Table 3). Affected individuals are usually homozygous, compound heterozygous or heterozygous for the R229Q polymorphism. (3,13,21,33-35) Compound heterozygous individuals have a second mutation in the NPHS2 gene. However, heterozygous and homozygous R229Q genotypes have also been described in unaffected individuals.(13,17) The arginine residue at position 229 is highly conserved in sequence analyses from NPHS2 orthologs. In addition, in vitro studies have shown that the R229Q polymorphism may cause reduced binding to nephrin.(3) Therefore, this polymorphism is a likely candidate for genetic susceptibility to diseases presenting with proteinuria.

Table 3: Findings from studies of the relation between NPHS2 polymorphism R229Q (686G/A) and nephrotic syndrome or focal segmental glomerulosclerosis.

Seven studies met the selection criteria (Table 3). Most of these studies included primarily Caucasian individuals, with exception of the unpublished NIH study that included individuals of both African and European descent. Four European studies included children with SRNS, of which most had FSGS at renal biopsy. Three studies included adults with FSGS. Because few individuals had the A/A genotype among cases and controls, we estimated the risk of disease for individuals heterozygous (G/A) or compound heterozygous for the R229Q variant and some other NPHS2 variant.

Table 3 shows the study characteristics and the risk estimates of disease for R229Q heterozygosity. The study by Karle et al. did not find any R229Q heterozygous among 25 children with nonfamilial SRNS from Europe.(21) The authors expanded the analysis to 165 families with SRNS and 120 families with SSNS, mostly of European descent.(35) Controls were healthy individuals. Combined results of homozygous, heterozygous and compound heterozygous were reported and, therefore, we were unable to calculate an estimate of the disease risk from this study.

Five of the above studies had complete data or at least one individual with the G/A genotype in cases and controls for association analysis. The number of individuals with the G/A genotype was small among the cases and controls and estimates were imprecise as reflected by wide confidence intervals. Caridi et al. studied 120 Italian children with sporadic SRNS and 59 with sporadic SSNS, most of them with FSGS.(33) Controls were blood donors. Weber et al. studied 172 children with sporadic SRNS mainly from France but also included a few children from North Africa.(13) Controls were individuals of French and African descent. The odds of sporadic SRNS were 0.9 (95% CI 0.4-0.9) and 1.2 (95% CI 0.4-3.8), respectively, compared to controls in the two above described European studies but 1.8 (95% CI 0.5-6.4) for children with SSNS.(33,13)

One European study and two US studies described podocin variants among adult patients with sporadic FSGS. In the Italian study, blood donor controls were screened for the R229Q polymorphism.(34) The study by Tsukaguchi et al. had ethnic matched controls screened for podocin variants. The NIH cohort study recruited patients with biopsy-proven FSGS from 22 medical centers throughout the US, in two subcohorts of European and African descent (unpublished). Cases of HIV-1 associated collapsing FSGS were included in the African American cohort. Control subjects included African American HIV positive subjects without renal disease after at least eight years of exposure recruited from the AIDS Link to Intravenous Experience cohort (ALIVE) and randomly recruited blood donors from the NIH. The European descent controls were blood donors from the NIH and healthy volunteers.

FSGS disease risk estimates for R229Q heterozygosity are shown in Table 3. In adults with sporadic FSGS, the risk of disease was increased by 20 to 70% in studies that included mainly Caucasian patients, although the results were not statistically significant (Table 3). The highest risk was found in the Italian study that had a small number of cases (OR 1.7). Larger studies of Caucasians from the US showed a smaller risk although the estimated risk of one of the studies could be confounded by population admixture.(3) Estimates including compound heterozygous individuals were higher compared to those including individuals without a second NPHS2 mutation (Table 3). R229Q heterozygosity was not associated with increased risk of FSGS among those of African descent in the NIH study, the only one to include this population.

The results reported here were from unadjusted analysis and should be interpreted with caution. Several important potential biases should be considered including population admixture, selection bias in control participants and lack of important data to evaluate confounding. For example, some studies included persons of different ethnicity.(3,13,35) Moreover, although populations of African descent are at higher risk of developing FSGS, the majority of the studies included mainly Caucasian populations.(3,34) In addition, studies were not designed to address the effect of the polymorphism on disease and therefore, controls were not always selected from population-based sources. (3,13,21,33,34) Data on potential environmental confounders were also not available.

Overall, the available studies were not designed to answer the question of the role of NPHS2 gene polymorphisms on genetic susceptibility to disease. Based on the available data, it is likely that R229Q polymorphism in isolation is not associated with FSGS in US populations of African descent. In contrast, European descent populations appear to have a non-significant increased risk of FSGS by 20 to 70%. Further studies with larger number of patients are required to answer this question. Small number of affected individuals and inadequate controls are some of the problems that need to be addressed in future case-control studies of this variant and the disease. Clearly, more large case-control studies are warranted and will be required to properly evaluate the association between NPHS2 gene variants and FSGS and other podocyte diseases.

Other related traits

One study has investigated the association of the polymorphism R229Q with microalbuminuria.(17) Microalbuminuria, defined as the daily urine albumin excretion of 30-300 mg, affects 5% of the general population and is a marker of increased glomerular capillary permeability. In diabetic subjects microalbuminuria is associated with the subsequent development of overt renal disease, but microalbuminuria does not precede FSGS. Microalbuminuria also correlates with extra-renal endothelial dysfunction and is a risk factor for cardiovascular disease. Subjects, aged 25 to 64, were recruited from a cross-sectional study of risk factors for cardiovascular disease in Vitoria, Brazil.(17) Eighty-five of 1577 individuals were heterozygous for R229Q and one was homozygous for R229Q. Eighty-five heterozygous individuals were compared to 1491 subjects without the polymorphism. Individuals with R229Q had similar age and gender distribution to those without it. Only 1027 individuals had information regarding microalbuminuria, defined as a urine albumin higher than 2 mg/dL. The odds ratio of microalbuminuria was 2.8 (95% CI 1.2, 6.3) for those with the allele R229Q compared to those without it, adjusting for age, ethnicity, hypertension, obesity, and diabetes.(17) The increased risk of microalbuminuria was present in obese subjects but not in non-obese patients. This study failed to consider other NPHS2 polymorphisms in the analysis. In addition, although they have reported differences in the allele frequency among different ethnic groups, the study used pooled data from subjects of different racial backgrounds in their analysis.

A recent published case-control study compared NPHS2 variants among African-American non-diabetic ESRD patients to population-based controls.(18) The R229Q polymorphism was not identified among 96 cases. The majority of the variants were not associated with ESRD but an intronic variant, IVS3 + 9insA, present in 10 of 288 cases and 1 of 278 controls (allele frequency in controls of 0.002), was significant associated with ESRD (P = 0.02).(18) Haplotype analysis was conducted. The haplotype of 5 SNPs tagging this intronic variant was associated with non-diabetic ESRD (P = 0.012). The most common polymorphism was the A242V variant (allele frequency of 0.087 in controls) and it was not associated with ESRD. Given the low allele frequency of IVS3 + 9insA in African-American controls, it is unlikely that this variant is a major cause of ESRD. Nonetheless, these data represent interesting preliminary data and demonstrate the importance of conducting much larger case-control studies so that rare variants may be considered with adequate power to detect genetic effects.

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 GENE-GENE INTERACTIONS

There are no population-based studies of gene-gene interaction including NPHS2 polymorphisms and nephrotic syndrome. However, four studies described combined mutations or polymorphisms of the NPHS2 and NPHS1 genes. The NPHS1 gene, mapped at chromosome 19q13.1, encodes nephrin, a podocyte protein. Nephrin and podocin may interact directly or indirectly and are important for maintenance of the glomerular capillary permeability barrier. Koziel et al. examined the genotype/phenotype correlation of NPHS1 and NPHS2 gene mutations in patients with congenital nephrotic syndrome.(12) Four patients were found to have NPHS1 and NPHS2 mutations. Patients had either homozygous mutations in NPHS1 and heterozygous mutation in NPHS2 or homozygous mutations in NPHS2 and heterozygous mutation in NPHS1. The two NPHS2 homozygous mutations were 436delA frameshift and R138Q. One patient had R229Q heterozygous NPHS2 polymorphism in association with a homozygous aberrant splicing mutation of nephrin. Parents that were compound heterozygous carriers of the NPHS1 and NPHS2 mutations were phenotypically normal.(12) Others have also described SRNS associated with homozygous NPHS2 R138Q mutations and a heterozygous splice mutation of NPHS1 (13) and compound heterozygous A297V/R229Q of NPHS2 and heterozygous mutations of NPHS1 (33) However, one study has described a lack of genotype/phenotype correlation among five patients with NPHS1 and NPHS2 mutations; although most of the patients had the R229Q variant.(36) Therefore, triallelic mutations of podocin and nephrin may exhibit a phenotype that resembles the phenotype of nephrin homozygotes, although this remains controversial.

There are no population studies of gene-environment interaction including the NPHS2 gene. A study of genetic polymorphism in subjects of African descent with FSGS and collapsing glomerulopathy is currently ongoing in the US (Kopp et al., unpublished).

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 LABORATORY TESTS

The extraction of genomic DNA from peripheral blood cells was completed using standard techniques.(2) Mutational analysis was usually performed by direct resequencing of the two strands of all eight exons of the NPHS2 gene. A few studies have also performed sequence analysis of the intronic and promoter regions of the gene.(13,18,22) For analysis of the polymorphisms, DNA from healthy controls, blood donors and volunteers were also resequenced.

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 POPULATION TESTING

Currently, genotype analysis of NPHS1 and NPHS2 is commercially available (Athena Diagnostics, Worcester, MA). Bi-directional resequencing of coding region of NPHS1 and NPHS2 can be ordered individually or in combination. In addition, these tests are also available in research facilities at Harvard University, University of Michigan, and the National Institutes of Health at no cost. Because some NPHS2 mutations are associated with steroid-resistance, some have advocated the screening of patients with familial nephrotic syndrome and childhood sporadic FSGS for podocin mutations in order to spare drug toxicity or prevent delay of more effective therapy. (37) Current available data are insufficient to provide recommendations. In addition, there are insufficient data to determine whether NPHS2 polymorphisms such as R229Q cause FSGS. Given the complexity of FSGS, it is unlikely that a single nucleotide polymorphism (SNP) in a gene will be sufficiently implicated to warrant genetic testing.

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 COMMENTARY

Studies to date have focused primarily on identifying gene loci for familial and idiopathic nephrotic syndrome and in characterizing mutations of known causal genes such as NPHS1 and NPHS2. Although several podocin polymorphisms have been described, the allele and genotype frequencies are often not reported, which limits their use in genetic association studies. In some cases, published studies have relied on descriptive analysis of allele frequency among affected and controls without considering gene-gene interaction and environmental risk factors.

Several questions should be considered in future work. For example, do NPHS2 variants 1) increase the risk for sporadic FSGS or collapsing glomerulopathy, including idiopathic, postadaptive, virus-associated, and medication-associated forms, 2) increase the risk to develop other renal diseases, including diabetic nephropathy, 3) increase the risk for progression to ESRD with any renal disease and 4) interact with other podocyte gene variants in any of these foregoing renal conditions. For evaluation of these hypotheses, large well-characterized patient populations, with uniform assessment of renal histology and clinical findings, are required to establish standardize phenotypic criteria. These patient populations must be ethnically-matched to control subjects. Sophisticated analytic techniques will be required to test multiple disease subgroups while maintaining statistical power. Increasingly, it is apparent that such studies will require thousands of affected subjects and control subjects, and this will require of collaboration among investigators.

In summary, we have reviewed the NPHS2 gene variants and its association with nephrotic syndrome and FSGS across different ethnic populations. We have found that R229Q is one of the best characterized polymorphism, although with low frequencies across multiple ethnic groups. There is insufficient evidence implicating R229Q in the etiology of FSGS among Caucasians and our unadjusted analysis suggests small increased risk among this population. Clearly, large case-control studies will be required to evaluate the role of this polymorphism in disease. The R229Q variant is noticeably rare in populations of African descent, and was not associated with FSGS in African American subjects. Nonetheless, given the disparity of prevalence of FSGS among African American patients and the lack of studies evaluating NPHS2 variants with FSGS, further evaluation of this gene in the etiology of FSGS is warranted.

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 ACKNOWLEDGEMENTS

We would like to thank the following researchers for sharing unpublished data from studies: Dr Gianluca Caridi, Laboratory and Unit of Nephrology, G. Gaslini Institute, Genoa, Italy and Dr Martin R Pollak, Renal Division, Brigham and Women's Hospital, Boston, Massachusetts. This work was supported by the NCI and NIDDK Intramural Research Programs. Dr Franceschini is supported by the Department of Epidemiology, University of North Carolina School of Public Health.

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 REFERENCES

List of References

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Page last reviewed: February 24, 2006 (archived document)
Page last updated: November 2, 2007
Content Source: National Office of Public Health Genomics