ResultsNPHS2 : Non-synonymous Variants
We directly sequenced the entire coding region of the NPHS2 gene in PCR amplified DNA from 371 individuals, 122 of whom also had at least 1 relative with FSGS. We identified fifteen alleles that changed the predicted amino acid sequence in 63 patients (or 17% of the samples screened). These were either missense (p.R10T (c.29G>C), p.V127W (c.379G>T), p.R138Q (c.413G>A), p.V180M (c.538G>A), p.R229Q (c.686G>A), p.T232I (c.694C>T), p.E237Q (c.709G>C), p.A242V (c.725 C>T), p.L270F (c.810G>T), p.A284V (c.851C>T), p.L312V (c.934C>G) and p.L327F (c.1048C>T), truncation (p.Q215X (c.643C>T) or frameshift variants (397delA and 855-856delAA) ( Table 1 ). Twelve of these 63 patients had non-synonymous variants in two alleles; 3 were homozygous for p.V127W, p.R138Q or p.V180M, and the remaining 9 were compound heterozygous for p.R138Q and/or p.R229Q and a variety of other alleles ( Table 2 ).
Homozygous Non-synonymous and Compound Heterozygous Variants
In 3 of the 122 families studied (2.5%), the p.R138Q, p.V127W and p.V180M alleles were demonstrated in a homozygous state ( Table 2 ). In families with multiple affected individuals available for genetic analysis, these alleles segregated in a pattern consistent with autosomal recessive transmission. We identified 9 compound heterozygous events. A majority (7/9 or 78%) of these consisted of p.R229Q, inherited together either with a rarer allele (n = 4), p.R138Q (n = 2) or p.A284V (n = 3). These mutations were identified in 2 index cases with a family history of disease and 7 sporadic cases ( Table 2 ). In the proband's family, the alleles segregated with disease in an autosomal recessive manner. In addition, we identified p.R229Q and p.A242V in homozygous and compound heterozygous states. The p.R229Q and p.A242V homozygous events were identified in two unrelated sporadic patients (CPMC-96 and FG-GC-1112 respectively), and the p.R229Q/p.A242V compound heterozygous state was found in another unrelated sporadic patient (CH-1).
The p.R10T, p.V127W, p.Q215X and p.L270F alleles (present in a compound heterozygous state with either p.R138Q or p.R229Q ( Table 2 ) were not observed in 362 non-diseased control alleles. Only 1 proband with 2 deleterious NPHS2 alleles other than p.R229Q developed disease in adulthood ( Table 2 ). The 4 other probands with adult onset disease and significant NPHS2 variants in both alleles had one p.R229Q variant. This supports the notion that in the absence of a p.R229Q NPHS2 variant, NPHS2 defects are unlikely to be the cause of disease in an adult with FSGS undergoing genetic analysis.
Fifty-one patients had a single heterozygous NPHS2 allele predicted to alter the encoded protein. These variants (and the respective number of patients they were demonstrated in) were: p.R138Q (n = 3), p.R229Q (n = 32), p. T232I (n = 1), p.E237Q (n = 1), p.A242V (n = 13) and p.L312V (n = 1). Both the p.T232I (c.694C>T) and p.L312V (c.934C>G) alleles were absent in 362 non-diseased control alleles sequenced, while all the others have been demonstrated by previous studies.[6,7,8,12,13,14,15,16,17,18,19,20,21,22,23,24] The p.T232I variant was identified in an 8 year-old African-American boy (UAB-023) with no family history who developed FSGS at the age of 2.5 years. The p.L312V mutation was identified in 2 of 4 siblings in a heterozygous state, both of whom had biopsy-confirmed FSGS (Family FG-DK). No second mutant allele was found. Both parents were deceased, the father from kidney failure secondary to prostrate cancer and the mother for unknown reasons.
The p.R229Q and p.A242V variants were the most common non-synonymous variants demonstrated in our FSGS patient cohort (with allele frequencies of 0.02 and 0.06 respectively). The frequency of p.R229Q did not differ between the FSGS races studied, whereas the p.A242V variant was present in a higher proportion of African Americans with FSGS compared to other races (chi-square with Yates correction 9.79, p = 0.00057). We were interested in whether both the p.R229Q and p.A242V variants might contribute to the risk of renal impairment in the general population by investigating whether these variants associate with increased urinary albumin/creatinine ratio, an early marker of renal disease.
We genotyped the p.R229Q variant in a cohort of 2,596 women aged over 50 years who were enrolled in 1976 to participate in the Nurses' Health Study I (of which 97% were of Western european descent). We genotyped the p.A242V variant in 1559 participants in the Nurses' Health Study II (age 44 or greater; 94% of Western european descent). The allele frequencies of the p.R229Q and p.A242V variants in these cohorts were 0.0352 and 0.034, respectively. The genotype frequencies of p.R229Q and p.A242V heterozygotes were 6.5% and 7.0% respectively. Homozygous events were rare for both variants, with genotype frequencies of 0.1% and 0.26% (for p.R229Q and p.A242V respectively). We observed no association between the presence of p.R229Q and p.A242V in either of the homozygous or heterozygous states with increased urinary albumin/creatinine ratio.
We were also interested in investigating whether p.R229Q might represent a modifying allele for renal disease in a more common cause of renal impairment, diabetic nephropathy. The p.R229Q variant has been associated with microalbuminuric events (designated by semi quantitative protocol) in an urban population isolated from Brazil and has shown to cause FSGS in the compound heterozygous state along with rarer non-synonymous variants. We genotyped p.R229Q in 1988 diabetic patients from 3 different cohorts and investigated for associations between p.R229Q with end-stage renal disease and proteinuria ( Table 3 ).
Two hundred and eighty patients with either type 1 diabetes (n = 41, 15%) or type 2 diabetes (n = 239, 85%) were studied. Twenty patients (49%) with type 1 diabetes and 89 patients (37%) with type 2 diabetes had end-stage renal disease. Diabetic patients without end-stage renal disease were used as controls (21/41, 51% of type 1 diabetics; 150/239, 63% of type 2 diabetics). The allele frequency of R229Q in the diabetic cases with end-stage renal disease was 0.036, and 0.030 in the controls without ESRD ( Table 3 ). No associations between p.R229Q and end-stage renal disease in either type 1 or type 2 diabetes were found (chi-square 3.39 and 0.121 with p values of 0.07 and 0.73 respectively).
One thousand two hundred and seventy-nine patients with 10 or more years of type 1 diabetes from the Genetics of Kidney Disease in Diabetes (GoKinD) were genotyped for p.R229Q. Four hundred and fifty-five of these subjects were "case" samples with end-stage renal disease, while the remaining 824 without end-stage renal disease served as "control" subjects. p.R229Q was found to have an overall allele frequency of 0.04 in the entire set of GoKinD samples. The allele frequency of the variant in "cases" with end-stage renal disease was 0.0297 and 0.039 in the control type 1 diabetics without nephropathy. There was no association between p.R229Q in the heterozygous state and proteinuria or end-stage renal disease in the case diabetics compared to normoalbuminuric control diabetics (chi-square 1.68, p = 0.20). In addition, the presence of a p.R229Q allele was not seen at greater frequency in subjects with elevated creatinine. p.R229Q Heterozygous individuals also did not have a higher incidence of abnormal creatinine (chi-square 1.314, p = 0.252) than individuals lacking this variant.
Renal disease is an established risk factor for cardiovascular disease. p.R229Q was not seen at greater frequency in individuals with cardiovascular complications (chi-square 0.21 p = 0.65), hypertension (chi-square 1.31, p = 0.25), nor with subjects taking an angiotensin converting enzyme inhibitor (ACEi), or other antihypertensive medications (chi-square 0.002 and 0.99 with corresponding p values of 0.97 and 0.32).
We also found 3 samples homozygous for p.R229Q, but these were excluded from all our association studies. The homozygotes with p.R229Q had a 2.15 fold increase of developing proteinuria or ESRD but this did not reach statistical significance. This may be due to the very small number of subjects having this homozygous genotype (95% confidence interval 0.19–24.1), or it may be due to chance.
Four hundred and twenty-four patients with median diabetes duration of 20 years (range 3-to-58 years) enrolled through the Australian endocrine diabetes study were also studied. Sixty-seven had type 1 diabetes (51 with an urinary albumin excretion rate (AER) <20 μg/min, 10 with an AER between 20 μg/min and 200 μg/min, and the remaining 6 with an AER >20 μg/min) and the remaining 357 had type 2 diabetes (228 with an AER<20 μg/min, 83 with an AER between 20 μg/min and 200 μg/min, and the remaining 46 with an AER >200 μg/min). The p.R229Q variant had an allele frequency of 0.044 in the Australian endocrine diabetics. The allele frequency in cases with "nephropathy" (defined by an AER >20 μg/min) was 0.041 and 0.047 in the controls (subjects with an AER <20 μg/min) ( Table 3 ). p R229Q did not associate with "nephropathy" (defined by an AER >20 μg/min) in the Australian endocrine diabetics (chi-square= 0.13, p = 0.72), micro or macroalbuminuria in type 1 diabetes (chi-square 1.39 and 0.5 with p values of 0.24 and 0.48 respectively), nor did it associate with micro or macroalbumiuria in type 2 diabetes (chi-square 0.44 and 0.04 with p values of 0.51 and 0.84 respectively).
When we pool the results from all 3 cohorts together and define a "case" diabetic as one with end-stage renal disease or abnormal proteinuria and a "control" as having no end-stage renal disease and normal urinary protein, no associations are present between p.R229Q and "diabetic nephropathy" (chi-square 0.67 and p value of 0.41).
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Cite this: NPHS2 Variation in Focal and Segmental Glomerulosclerosis - Medscape - Sep 29, 2008.