NPHS2 Variation in Focal and Segmental Glomerulosclerosis

Stephen J Tonna; Alexander Needham; Krishna Polu; Andrea Uscinski; Gerald B Appel; Ronald J Falk; Avi Katz; Salah Al-Waheeb; Bernard S Kaplan; George Jerums; Judy Savige; Jennifer Harmon; Kang Zhang; Gary C Curhan; Martin R Pollak

Disclosures

BMC Nephrology 

In This Article

Discussion

We resequenced the coding sequence of NPHS2 in 371 individuals with FSGS and a median age of onset of disease of 25 years and found that 63 (or 17%) of the patients have at least one allele that alters the NPHS2 coding sequence, and of these, 12 (or 3.2% of the 371 screened) had these events in both NPHS2 alleles. Likely disease-causing mutations (homozygous or compound heterozygous) were identified in 4% (5/122) of the families and 2.8% (7/249) of the sporadics (excluding the homozygous and compound heterozygous events of p.R229Q and/or p.A242V).

We previously screened 30 multiplex families for NPHS2 mutations with adolescent or adult onset FSGS and identified 7 (23%) homozygous (n = 1) or compound heterozygous (n = 6) patients.[6] When these families are added to those screened here, we have identified likely disease-causing mutations in 8% of families (12 of 154 families). Our data show a non-trivial frequency of homozygous or compound heterozygous alleles in NPHS2 in late onset FSGS-affected individuals. Such genotypes are more frequent in patients with at least 1 other affected family member compared to sporadic FSGS patients (8% vs 2.8% respectively). These percentages are significantly smaller than most of the previous large studies which have focused on pediatric disease.[15,17] Several of the novel mutations identified in this study that both lead to amino acid substitutions and are present in the compound heterozygous state, with other previously known variants, are predicted to affect protein function of podocin using two SNP prediction algorithms; Sorting Intolerant From Tolerant (SIFT) and Polymorphism Phenotyping (PolyPhen). The p.R10T variant is predicted to be tolerated by SIFT and benign using Polyphen, p.V127W is predicted to be not tolerated by SIFT and possibly damaging by PolyPhen and p.L270F is predicted to be not tolerated by SIFT and possibly damaging using PolyPhen.

The finding of mutant NPHS2 alleles in sporadic cases confirms that disease that appears to be sporadic may in fact be inherited as a result of inheritance of mutant alleles from both parents, even in the absence of a positive family history of disease.[27] This may mean that homozygous or compound heterozygous events in other not-yet-identified genes that cause FSGS by recessive inheritance may underlie disease in these patients.

We identified 51 patients (51/371, 14%) that had a heterozygous allele that altered the amino acid sequence without any other identified NPHS2 allele. The majority of these were the p.R229Q and p.A242V alleles ( Table 1 ). Two of these alleles (p.T232I (c.694C>T) and p.L312V (c.934C>G)) are private non-conservative alleles that have not been identified in previous NPHS2 resequencing studies. The contribution of these rare alleles to disease is unclear. Neither was observed in any of the control alleles genotyped. The p.T232I allele was demonstrated in a subject with sporadic disease. The p.L312V allele was present in all affected siblings in a family with biopsy-confirmed FSGS. We cannot out rule the possibility of autosomal dominant disease in this family, as the father reportedly died from kidney impairment secondary to prostate cancer and the mother died of unknown causes. Interestingly, the p.T232I variant is predicted to be not tolerated by SIFT and possibly damaging by PolyPhen, and p.L327V is tolerated by SIFT and benign using PolyPhen. Rare heterozygous alleles in NPHS2 may possibly affect protein function, but these will need to be studied in cell culture to verify this.

Rare NPHS2 alleles have also been demonstrated in subjects with thin basement membrane nephropathy (TBMN). Tonna et al. 2003 identified a rare heterozygous variant (p.R224H (c.672G>A)) in only one patient with both TBMN and proteinuria, and like p.T232I the allele was not demonstrated in any control sample.[28] Without knowing the frequency with which non-proteinuric control groups carry single, rare, non-synonymous variants, it is difficult to know the clinical significance of such variants.

A small number of studies have attempted to show that heterozygous mutations in both the NPHS2 and NPHS1 genes in a single patient can cause FSGS.[7,8,16] It is unclear, however, what the frequency of such digenic events is in non-proteinuric (control) individuals. It is a straightforward hypothesis that digenic or perhaps even trigenic combinations of non-synonymous variants may occur in either of the NPHS2, ACTN4, TRPC6, CD2AP, or even PLCE1 genes in FSGS patients (especially those with sporadic disease). This may be in fact be the case in some of these patients with a single non-synonymous NPHS2 allele (especially if the allele is rare or is known to cause disease in the homozygous or compound heterozygous state), but confirming such a hypothesis will require extensive resequencing of many genes in FSGS cases as well as controls. It is however clear that 2.8% of patients with late onset, non-familial, FSGS have disease attributable to two mutant NPHS2 alleles.

The most common non-synonymous changes we identified were the p.R229Q and p.A242V variants with population frequencies of 0.02 and 0.06, respectively, in the FSGS probands. We found one FSGS patient homozygous for p.R229Q, one homozygous for p.A242V, and one with compound heterozygosity for each of these alleles. Weber et al identified 3 families (2 of which are consanguineous) and 2 sporadic cases of FSGS with homozygosity for p.R229Q,.[17] This substitution is one of the most commonly reported NPHS2 alleles[6,8,14,15,20,21,25] with a greater frequency in Europeans (0.036) compared to the frequency in African Americans and Brazilians.[6,17,25] In another study, p.R229Q heterozygotes were found to have a 2.77 fold increased risk of developing microalbuminuria compared with controls,[25] but it has been unclear whether this allele represents a genetic modifier for renal impairment in other diseases such as diabetic nephropathy.

To clarify whether these common non-synonymous variants cause or contribute to the development of albuminuria, we genotyped both of these alleles in cohorts of women from the Nurses' Health Study I and II. We observed that neither the p.R229Q nor the p.A242V allele were associated with increases in urine albumin/creatinine ratio in either the homozygous or heterozygous states. We found that p.A242V was present in an allele frequency of 0.034 in the Nurses' Health Study II, but in only 0.02 in the FSGS cohort. We note that this frequency in the control group (NHSII) is higher than that previously reported in control groups of similar ethnicity.[29] In the FSGS sample set, this variant was present in a higher frequency in persons of African descent compared to other ethnicities (chi-square with Yates correction 9.79, p = 0.00057), consistent with other studies[17]). It is not clear how the p.R229Q allele causes FSGS in the compound heterozygote state when inherited together with a second mutant allele,[6,8,14,15,16,17,19,22] but not in the homozygous state, but this study confirms earlier suspicion that p.R229Q causes disease only in conjunction with a second more detrimental allele.[6] An earlier study reported that p.R229Q associates with microalbuminuric events in the population.[25] However, in the present study, we saw no association of the p.R229Q allele with albuminuria in either the Nurses' Health Study I nor in the diabetic populations analyzed. Further, the p.R229Q and p.A242V alleles in the homozygous state do not cause FSGS. The results of the genotyping of p.R229Q and p.A242V in the Nurses' Health Study reinforces the importance of evaluating potentially pathogenic variants in large populations.

Pathogenicity of NPHS2 Mutations

 

The biological effect of twelve mutant NPHS2 alleles (p.P118L, p.R138Q, p.R138X, p.D160G, p.R168H, p.R168C, p.R168S, p.V180M, p.R238S, DelLER (aa 237–239), p.V260E, and p.R291W) and two relatively common variants (p.P20L and p.G92C) have been studied in cell culture.[30,31,32] All but two of the mutants (p.V180M and p.R238S) fail to reach the plasma membrane in contrast to wild type podocin, whereas both the p.P20L and p.G92C variants do reach the plasma membrane. The lack of proper targeting of mutant NPHS2 to the plasma membrane has been shown to affect nephrin trafficking.[30,31,32] Other variants may have less avid binding to nephrin, as has been demonstrated in the case of p.R229Q.[6] However, lacking a systematic study of the frequency and biological effects of non-synonymous variants, it remains unclear which altered functions are meaningful markers of clinical pathogenicity.

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