Integration of Genetic Testing and Pathology for the Diagnosis of Adults with FSGS : Clinical Journal of the American Society of Nephrology

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Original Articles: Genetics

Integration of Genetic Testing and Pathology for the Diagnosis of Adults with FSGS

Yao, Tony1,2; Udwan, Khalil1,2; John, Rohan3; Rana, Akanchaya1,2,4; Haghighi, Amirreza1; Xu, Lizhen5; Hack, Saidah1,2; Reich, Heather N.1,2,4,6; Hladunewich, Michelle Adrienne7; Cattran, Daniel C.1,2,4,6; Paterson, Andrew D.4,6,8,9; Pei, York1,2,4,6; Barua, Moumita1,2,4,6

Author Information
Clinical Journal of the American Society of Nephrology 14(2):p 213-223, February 2019. | DOI: 10.2215/CJN.08750718
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FSGS is a clinicopathologic entity characterized by proteinuria, with or without features of nephrotic syndrome, glomerulosclerosis and podocyte foot process effacement. It is divided into primary and secondary forms, but overlapping clinical and histopathologic features make precise etiologic diagnosis challenging. As a result, many cases of FSGS treated with immunosuppression are unresponsive, progressing to ESKD necessitating dialysis and/or transplantation (1).

The study of hereditary forms of FSGS has helped inform our understanding of its molecular pathogenesis (2–4). Most genes in which specific variants are reported to cause FSGS have protein products that are important regulators of the podocyte actin cytoskeleton or slit diaphragm components, in keeping with the current paradigm that FSGS represents a primary podocyte disorder (2–6). Much of the literature where this viewpoint arises is focused on pediatric patients and limited to sequencing gene panels composed mostly of genes associated with disease that are also expressed in the podocyte. However, such a list can quickly become incomplete in the next-generation era of rapid disease gene discovery.

Several reports have implicated pathogenic variants in COL4A3/A4/A5 in FSGS, with at least one indicating that it is the leading single gene (i.e., monogenic) cause in adults with disease (7–13).These reports consist of smaller sample sizes, cohorts comprising many related individuals, and examined only select genes. Previous work has also demonstrated that pathogenic variants in genes associated with other kidney diseases, such as congenital abnormalities of the kidney and urinary tract (CAKUT) and not expressed in the podocyte, are also found in individuals clinically diagnosed with FSGS (14,15).

We performed whole-exome sequencing in a large cohort of mostly unrelated cases comprising 193 adults from 179 families with FSGS. We examined an expanded subset of 109 genes associated with FSGS, basement membrane abnormalities, as well as causes of pediatric ESKD including CAKUT and nephronophthisis, to determine the distribution of pathogenic variants in a genetically determined, predominantly European, adult population.

Materials and Methods

Patient Ascertainment

Patients were recruited at University Health Network, Toronto, Ontario, Canada, with the majority from the Toronto GN Registry from 1976 to 2017, after receiving informed consent in accordance with the hospital Research Ethics Board. We obtained longitudinal clinical data and eventually blood, saliva, or isolated DNA. Clinical information was obtained from telephone interviews, questionnaires, and physician reports. Genomic DNA was extracted from blood or saliva samples using standard procedures.

Clinical Definitions

Study participants were probands with a pathologic diagnosis of FSGS as a result of (1) segmental and/or global glomerular sclerosis, (2) podocyte effacement, or (3) nonspecific immunofluorescence, and without any other diagnoses such as SLE nephritis, Henoch–Schonlein purpura/IgA, membranoproliferative GN/C3, membranous nephropathy, and HIV-associated nephropathy. Relatives of probands with >500 mg of protein excretion per day were considered as affected and also included. Familial cases were defined as two or more affected individuals that were not separated by more than two meiotic events. Complete remission was defined as reduction of proteinuria to <0.3 g/d, associated with stable kidney function (change in serum creatinine <25% from baseline) after 3–6 months of immunosuppressive treatment including prednisone, cyclophosphamide, cyclosporine, tacrolimus, mycophenolate mofetil, azathioprine, and rituximab, either alone or in combination. Partial remission was defined as a reduction of proteinuria to 0.3–3 g/d and stable serum creatinine. Resistance was defined as no complete or partial remission. Clinical data are reported as percentage of all individuals with available information.

Clinicopathologic parameters were compared in pairwise combinations of patients with pathogenic type 4 collagen variants, patients with other pathogenic variants, and patients with no proven genetic cause. For all percentages shown, the denominator was adjusted to reflect missing data. Statistical significance was determined by the two-sample t test (two-tailed) and two-proportion Z test (two-tailed). The Kaplan–Meier method was used to analyze censored events over time to estimate mean age at disease onset and ESKD. Kaplan–Meier survival plots were generated with IBM SPSS Statistics version 25.0 (IBM, Armonk, NY). Statistical significances were determined by the log rank (Mantel–Cox) test.

Exome Capture and Next-Generation Sequencing

Whole-exome sequencing was performed by The Centre for Applied Genomics, The Hospital for Sick Children, Toronto, Ontario, Canada. A shotgun library was made from each sample and captured using the Agilent SureSelect Human All Exon V5 (Santa Clara, CA) according to protocol. The manufacturer’s specifications state that the capture regions total approximately 180,000 exons from approximately 18,700 genes or 54 Mb. Enriched libraries were then sequenced by 150 bp paired-end read sequencing on Illumina HiSeq 2500 (Illumina Inc., San Diego, CA).

In Silico Data Processing

Reads were mapped to the hg19 reference sequence using the BWA-backtrack algorithm from BWA v0.5.9 (16). Duplicate reads were removed using MarkDuplicates from Picard v1.79. Local read realignment around insertions and deletions (indels), base quality score recalibration, and variant calling with UnifiedGenotyper, were accomplished using GATK v1.1–28 (17,18). Single nucleotide polymorphism calls were subjected to variant quality score recalibration. Indels were discarded if they overlapped repeat masked regions, and hard-filtered by variant call annotations QualByDepth (QD <10.0), ReadPosRankSumTest (ReadPosRankSum <−20.0), and Strand Bias (SB >−0.01). Base calling was performed using CASAVA v1.8.2. Copy number variants were identified using XHMM after filtering out regions with extreme GC-content and repeat-masked regions (19,20).

Genetic ancestry of the study cohort was analyzed using iAdmix software ( with population allele frequencies from HapMap Phase III data ( (21,22).For each individual, admixture proportions were calculated then summed into three continental groups: European, African, and East Asian. Individuals with estimated >90% ancestry from one continental population were classified as belonging to the respective continental group, whereas the remaining individuals were considered admixed.

Variant calls were compared against ethnically matched controls. The following minor allele frequency cut-offs, as determined in gnomAD (, were used for dominant and recessive disease genes respectively: 0.00005, and 0.005 (accessed February 22, 2018) (23). In addition, variants meeting these criteria and that were shared by affected relatives where applicable were also kept. Copy number variants were kept if absent from the Database of Genomic Variants (; accessed June 25, 2018) (24).

Assignment of Pathogenicity Categories

Recommendations by the American College of Medical Genetics (ACMG) were followed to categorize variants (25). Variants in 109 genes associated with FSGS, nephrotic syndrome, CAKUT, or nephronophthisis were examined. Zygosity of each variant in the patient had to match the corresponding gene’s known pattern of inheritance. Missense variants predicted to be deleterious by more than half of all in silico algorithms in dbNSFP v3.0 were considered “consistently predicted to be deleterious” (26,27). Human Splicing Finder 3.1 ( was used to analyze the effect of splice site variants (28).

The following types of variants were considered “definitely pathogenic”: variants previously reported to cause FSGS or related phenotypes, and de novo variants. The following types of variants were considered “likely pathogenic”: nonsense, frameshift, and canonical splice site variants in genes in which loss of function is a known pathogenic mechanism; or variants affecting the same codon as a previously reported pathogenic mutation and consistently predicted to be deleterious by in silico algorithms. The following types of variants were considered “possibly pathogenic”: variants residing in well established functional domains that contain previously reported pathogenic mutations and consistently predicted to be deleterious by in silico algorithms, or variants intrans with a previously reported pathogenic mutation and consistently predicted to be deleterious by in silico algorithms.

The remaining rare variants were considered variants of unknown significance.

Our definitions of possibly pathogenic falls within likely pathogenic variant ACMG criteria. However, we have added the category of possibly pathogenic to reflect the different levels of evidence that is being satisfied.

De Novo Variation and Confirming Biologic Identity of Parents

The PCR AmpFLSTR Identifiler kit was used to test 15 short tandem repeats. The number of repeats for each specified locus was evaluated for concordance with parental genotypes.



The cohort was composed of 193 individuals from 179 families. Forty three individuals were familial cases belonging to 29 pedigrees, with the remaining 150 cases being sporadic. Sex distribution within the cohort was 56% male (Supplemental Table 1, Table 1). In familial cases, 51% were male, whereas in sporadic cases, 57% were male (P=0.49). Population substructure revealed that 65% were European, 4% were African, 12% were East Asian, and 19% were of admixed descent (Table 1). The mean age of onset was 34±16 (SD) years, and most (78%) individuals presented with normal kidney function (Table 1). A total of 49% of patients developed ESKD at mean age of 47±17 years at the time of last follow-up (Table 1). Of these, 59% underwent kidney transplantation, with 10% showing disease recurrence (Table 1).

Table 1. - Baseline characteristics including pathologic features of the sequenced cohort
Characteristic Percentage (%) of Available Data No. of Patients
Sex 193
 Male 56 109
 Female 44 84
Ethnicity 193
 European 65 125
 African 4 7
 East Asian 12 24
 Admixed 19 37
Pathology where electron microscopy is available 126
Glomerular basement membrane abnormalities 30 37
 Podocyte effacement >50% 73 91
Patients on therapy 154
 Prednisolone alone 23 35
 Other immunosuppression alone 4 6
 Prednisolone and other immunosuppression 43 66
 ACEi+ARB alone 31 47
Remission rate on steroid or immunosuppression throughout the course of the disease 82
 Partial remission 45 37
 Complete remission 29 24
 No remission 26 21
Patients with known status for ESKD 147
 Progression to ESKD 49 72
Kidney transplant (% of transplant in patients with ESKD) 59 42
 Recurrence of disease after kidney transplant (within the first yr) 10 4
Mean age±SD (years) No. of Patients
Onset of kidney disease 34±16 161
ESKD 47±17 70
A total of 193 individuals from 179 families had exome sequencing performed but data were not available for all participants. Number of individuals in which data are available or satisfying the criteria is indicated in the last column. ACEi, angiotensin converting enzyme inhibitors; ARB, angiotensin receptor blockers.

One hundred forty-eight participants had at least one kidney biopsy, with a pathologic diagnosis that was consistent with FSGS. Nineteen individuals were affected relatives to biopsied probands, with no kidney biopsy. Of biopsied individuals, 145 reports were available for review, 126 of which had electron micrograph descriptions/interpretations. Thirty percent had some comment on glomerular basement membrane abnormalities, typically focal thinning, and 73% had >50% podocyte foot process effacement (Table 1). Where data were available, we found that 69% (107 out of 154) of patients were treated with some form of immunosuppressive medication (Table 1). Of the 107 treated patients, the remission status was known for 82 individuals. A partial and complete remission was reported in 45% (37 out of 82) and 29% (24 out of 82), respectively, and no remission was reported in 26% (21/82).

Pathogenic Variants

Massively parallel sequencing resulted in an average of 52,378,976 uniquely aligned 150 bp paired-end reads per individual, with a mean target coverage of 88× and 89% exomic coverage. An examination of 109 genes associated with FSGS, basement membrane abnormalities, CAKUT, and nephronophthisis was done with gene-specific mean depth of coverage across all samples provided in Supplemental Table 2. Most pathogenic variants were categorized as such because they were previously reported in the literature and had a higher prevalence in cases compared with controls, as per ACMG guidelines (25). In one case, we were able to identify a de novo variant in INF2, after confirming parental status with short tandem repeat profiling.

The overall genetic diagnostic rate was 11% (20 out of 179). Genetic testing yielded a diagnosis in 28% (eight out of 29) of families with kidney disease and in 8% (12 out of 150) of sporadic cases (Supplemental Figure 1). Pathogenic variants in COL4A3/A4/A5 accounted for 55% (11 out of 20) of cases attributed to a single gene cause, with most found in COL4A5 (Figure 1, Table 2) . Interestingly, we found COL4A5 pathogenic variants in a nearly equal distribution of males and females. Pathogenic variants in podocyte genes accounted for 40% (eight out of 20) of cases with a genetic cause, including in those frequently reported in pediatric patients such as NPHS1, NPHS2, and LMX1B (Figure 1, Table 3). One case was found to be due to a pathogenic variant in BMP4, which has been associated with CAKUT (Figure 1, Table 3).

Figure 1.:
Distribution of gene groups in definitely and likely pathogenic variants reveal that COL4A is leading single gene cause. The overall genetic diagnostic rate was 11% (20 out of 179) in the case series when only considering definitely pathogenic variants. There were (A) 20 definitely pathogenic, (B) 18 likely pathogenic, and (C) 15 possibly pathogenic variants identified. Of the (A) definitely pathogenic and (B) likely pathogenic variants, the highest percentage was in COL4A3/A4/A5, followed by podocyte then CAKUT genes. A minority of likely pathogenic and (C) possibly pathogenic variants were also found in the NPHP genes. Numbers in graph represent percentage.
Table 2. - Pathogenic variants in COL4A genes
Patient ID (Family ID) Sex Ethnicity Age at Disease Onset Age at ESKD Exon Number Nucleotide Change Protein Effect Allele Frequency Zygosity References
Pathogenic COL4A3 variants
 7215 (F23) F EUR Early teens n/a (34) 31 c.2452G>A Gly818Arg 1.17E-05 Het S17, S18
 2555 (s) F EUR 36 48 42 c.3655G>T Gly1219Cys 0 Het S19
 6062 (s) F EUR 30 unknown 21 c.1219G>C Gly407Arg 0 Het S20
Pathogenic COL4A4 variants
 6329 (F14) M EUR 42 n/a (48) 32 c.2906C>G Ser969stop 6.49E-05 Het S18, S21–S24
Pathogenic COL4A5 variants
 5515 (F8) M EUR 32 n/a (42) 50 c.4946T>G Leu1649Arg 0 Hemi S25
 5519 (F8) M EUR 25 n/a (44) 50 c.4946T>G Leu1649Arg 0 Hemi S25
 2594 (F16) F EUR 28 40 20 c.1276G>A Gly426Arg 0 Het S26, S27
 1590 (s) F EUR 28 n/a (56) 35 c.3017G>T Gly1006Val 0 Het S28
 2480 (s) F Admixed Unk 30 33 c.2804G>A Gly935Asp 0 Het S27
 4976 (s) M EUR 41 n/a (56) 25 c.1781G>A Gly594Asp 0 Hemi S29
 6223 (s) F EUR Unk unknown 31 c.2605G>A Gly869Arg 0 Het S26, S30–S35
 5269 (s) M EUR 57 66 39 c.3508G>A Gly1170Ser 0 Hemi S17, S36–S39
The following minor allele frequency (MAF) cut-offs as determined in gnomAD ( were used for dominant and recessive disease genes respectively: 0.00005 and 0.005 (accessed February 22, 2018). The MAF of the only COL4A4 variant exceeds the cut-off, but it is a well established founder mutation. Patients 5515 and 5519 are brothers. (F) designates family pedigree number whereas (s) indicates a sporadic case. Het indicates heterozygous and hemi indicates hemizygous. All references refer to the supplemental reference list, which can be found in the Supplemental Material, and indicate previous reports of the variant. Age at ESKD was indicated as n/a for patients without ESKD, followed by their age at time of analysis in parentheses.. F, female; EUR, European; M, male.

Table 3. - Pathogenic variants in non-COL4A genes
Patient ID (Family ID) Sex Ethnicity Age at Disease Onset Age at ESKD Gene Symbol Inheritance Exon Number Nucleotide Change Protein Effect Allele Frequency Zygosity References
Pathogenic podocyte gene variants
 2517 (F4) M Admixed unk 14 LMX1B AD 4 c.668G>A Arg223Gln 0 Het S40–S44
 2518 (F4) M Admixed unk 18 LMX1B AD 4 c.668G>A Arg223Gln 0 Het S40–S44
 5496 (F7) M EAS 16 22 COQ8B AR 15 c.1356_1362del Gln452Hisfs 0 Homo S45
 5497 (F7) F EAS 21 n/a (30) COQ8B AR 15 c.1356_1362del Gln452Hisfs 0 Homo S45
 5935 (F9) F EUR unk unk INF2 AD 2 c.312C>G Cys104Trp 0 Het S46
 5936 (F9) F EUR unk unk INF2 AD 2 c.312C>G Cys104Trp 0 Het S46
 6996 (F21) M Admixed 25 n/a (51) NPHS1 AR 22 c.2928G>T Arg976Ser 4.87E-05 Homo S47–S51
 2378 (s) M EUR 22 23 INF2 AD 2 c.317G>C Arg106Pro 0 Het S46
 2745 (s) F EAS 11 n/a (20) LMX1B AD 4 c.737G>A Arg246Gln 0 Het S52–S54
 5601 (s) F EUR 24 unk LMX1B AD 4 c.737G>A Arg246Gln 0 Het S52–S54
 6251 (s) F EUR 40 n/a (49) NPHS2 AR 7 c.868G>A Val290Met 1.20E-04 Homo S55–S58
Pathogenic CAKUT gene variants
 7942 (s) M EUR 55 n/a (59) BMP4 AD 2 c.272C>G Ser91Cys 1.90E-04 Het S59, S60
The following minor allele frequency (MAF) cut-offs as determined in gnomAD ( were used for dominant and recessive disease genes respectively: 0.00005 and 0.005 (accessed February 22, 2018). The INF2 variant in 2378 was shown to be de novo by examining parental sequence data. The MAF of the BMP4 variant exceeds the cut-off, but it has been shown to be functionally hypomorphic. Patients 2517 and 2518 are son and father. Patients 5496 and 5497 are siblings. The relationship of patients 5935 and 5936 is unknown. (F) designates family pedigree number whereas (s) indicates a sporadic case. Het indicates heterozygous and homo indicates homozygous. All references refer to the supplemental reference list, which can be found in the Supplemental Material, and indicate previous reports of the variant and indicate previous reports of the variant. Age at kidney disease was indicated as n/a for patients without kidney disease, followed by their age at time of analysis in parentheses. Unk indicates that data were unavailable. M, male; EAS, East Asian; F, female; EUR, European.

Likely Pathogenic, Possibly Pathogenic, and Digenic Variants

Eighteen likely pathogenic variants were identified in 17 patients, which is an additional 9% (17 out of 179) of the cohort. Of these, 14 were sporadic and three were familial cases (Supplemental Tables 3 and 4). Thirty nine percent (seven out of 18) of variants were in COL4A3/A4/A5 (Figure 1). Possibly pathogenic variants were found in further 6% (11 out of 179) of the cohort (Supplemental Table 5). Ten were in sporadic cases and one was a familial case.

Three cases with two definitely or likely pathogenic variants (called digenic) were discovered. Patient 2378 had a definitely pathogenic variant in INF2 (c.317G>C, p.R106P) and a likely pathogenic variant in COL4A3 (c.2172delA, p.G724fs). Patient 7939 had two likely pathogenic variants in LMX1B (c.879delG, p.L293fs) and COL4A3 (c.84delC, p.S28fs). Patient 7276 had a likely pathogenic variant in LAMA5 (c.6065–1G>T, abolished exon 46 splice acceptor) and a possibly pathogenic variant in INF2 (c.500A>C, p.H167P).

Seven cases were found to be carriers of previously reported pathogenic variants in genes associated with autosomal recessive inheritance patterns (Supplemental Table 6). Two of these cases had an additional definitely, likely, or possibly pathogenic variant (Supplemental Table 6). Patient 2517, a familial case with a segregating definitely pathogenic variant in LMX1B (c.668G>A, p.R223Q), was also found to be heterozygous for a reported definitely pathogenic variant in ZMPSTE24 (c.794A>G, p.N265S) that was not found in his affected father. Patient 7596 had possibly pathogenic variants in CUBN (c.6928_6934delTAACCTC, p.E2310Cfs; c.7968_7969delinsGTTATATAAGGTATAACA, p.L2656_P2657delinsFVIPYIT) and a heterozygous definitely pathogenic variant in NPHS1 (c.1868G>T, p.C623F).

Copy number analysis from exome data revealed a heterozygous deletion not in other cases or controls spanning intron 16 to intron 30 of COL4A4 in patient 6084, who additionally has a variant of uncertain significance in COL4A4 (c.4708G>A, p.E1570K). The breakpoints of the deletion could not be determined, limited by having only coding sequence.

Clinical and Pathologic Characteristics associated with Definitely and Likely Pathogenic Variants

Clinical features were analyzed in patients with definitely pathogenic variants in COL4A, podocyte, CAKUT, and nephronophthisis genes (Supplemental Table 7, Table 4). Patients with definitely pathogenic variants in genes expressed in podocytes or associated with developmental defects had a trend toward earlier age at disease onset (26 years; 95% confidence interval [95% CI], 17 to 37) than both those with COL4A (36 years; 95% CI, 29 to 42) and no proven genetic basis (34 years; 95% CI, 32 to 37) subgroups (P=0.15 and 0.11, respectively; Figure 2, Table 4); and similarly, an earlier estimated mean survival/age of ESKD (mean age, 43 years; 95% CI, 31 to 55) than COL4A (58 years; 95% CI, 49 to 69) and no proven genetic basis (62 years; 95% CI, 58 to 66) subgroups (P=0.26 and P=0.06, respectively; Figure 2, Table 4). An earlier age of estimated mean survival/age of ESKD was also observed for COL4A (58 years; 95% CI, 49 to 69) compared with the no proven genetic basis (62 years; 95% CI, 58 to 66) subgroup (P=0.06). None of the comparisons were significant but the lowest P values achieved were for ESKD with podocyte/developmental defects compared with no proven genetic basis subgroup (P=0.06), and COL4A compared with no proven genetic basis subgroup (P=0.06). The analysis was also performed evaluating males and females separately (Supplemental Table 8).

Table 4. - Clinical characteristics of COL4A, other genetic cause, and no proven genetic cause subgroups
Clinical characteristic COL4A (n=12) Other Genetic (n=12) No Proven Genetic (n=169)
% Male 42 67 58
Mean age of onset of kidney disease (95% CI) 36 (29 to 42) 26 (17 to 37) 34 (32 to 37)
% with hematuria 60 25 29
% of patients with family history of kidney disease 46 44 12.2
% Partial remission 29 30 26
% Complete remission 0 0 20
% No remission 14 20 15
% Unknown status of remission 57 50 39
% ESKD 46 44 50
% with only global glomerulosclerosis on light microscopy 11 50 9
% Glomerular basement membrane abnormalities on electron microscopy 56 25 27
% with >50% podocyte foot process effacement on electron microscopy 100 75 72
No. of patients where pathology report with electron microscopy description is available 9 4 113
Mean age at ESKD (95% CI) 58 (49 to 69) 43 (31 to 55) 62 (58 to 66)
% Kidney transplant in patients with ESKD 100 75 54
% of recurrence of disease after kidney transplant (within the first year) 0 0 13
Patients in the COL4A, podocyte/kidney development (called other), and no proven genetic basis subgroups had disease onset at 36 years (95% CI, 29 to 42), 26 years (95% CI, 17 to 37), and 34 years (95% CI, 32 to 37). The estimated mean survival/age at ESKD was 58 years (95% CI, 49 to 69), 43 years (95% CI, 31 to 55), and 62 years (95% CI, 58 to 66). The only statistically significant P values were in comparing COL4A with the no proven genetic basis subgroup for hematuria (P=0.02), glomerular basement membrane abnormalities (P=0.03), and >50% effacement (P=0.03). Number of patients where the pathology report was available is indicated. Percentages represent of available data. Absolute numbers are presented in Supplemental Table 9. 95% CI, 95% confidence interval.

Figure 2.:
Kaplan-Meier plots demonstrating that patients with pathogenic variants in genes expressed in the podocyte or associated with development defects had a trend toward an earlier age at disease onset and ESKD compared to other subgroups, although none of the pairwise comparisons were significant. The lowest P values achieved were for age at ESKD for podocyte/developmental defects compared with no proven genetic basis subgroup (P=0.06), and COL4A compared with no proven genetic basis subgroup (P=0.06). COL4A, COL4A subgroup; NG, no proven genetic basis subgroup; Podocyte, podocyte and kidney development defect subgroup.

For other clinicopathologic characteristics, the statistically significant comparisons were for COL4A compared with the no proven genetic basis subgroup for hematuria (P=0.02), glomerular basement membrane abnormalities (P=0.03), and >50% podocyte effacement (P=0.03) (Figure 3, Table 4). No individuals with an identified genetic cause had a complete remission when treated with immunosuppression (Table 4). Those with likely pathogenic variants in COL4A revealed GBM abnormalities in one of four cases where pathology was available, supportive of a deleterious role of at least some associated likely pathogenic COL4A variants (Figure 4).

Figure 3.:
Kidney pathology in COL4A -associated FSGS often demonstrate glomerular basement membrane abnormalities. Female patient (6062) with COL4A3-associated disease showing (A) segmental scarring (periodic acid–Schiff staining, ×20 magnification), and on (B) ultrastructural examination, diffusely thin glomerular basement membranes, and extensive podocyte foot process effacement, at approximately 80% (×10,000 magnification). Female patient (6251) with NPHS2-associated disease showing (C) enlarged glomeruli (periodic acid–Schiff staining, ×10 magnification) and one glomerulus with a segmental scar (not shown). (D) Ultrastructural examination shows normal thickness glomerular basement membranes and diffuse podocyte foot process effacement, at approximately 90% (×6000 magnification).
Figure 4.:
Genetic testing and pathology can be complementary tests to improve interpretation. Kidney pathology in a female (7939) with a likely pathogenic variant in COL4A3. (A) The glomeruli show mildly thickened capillary walls and no segmental scarring (in 53 glomeruli available for light microscopy; periodic acid-Schiff staining, x40 magnification). (B) Ultrastructural examination confirms thickened glomerular basement membranes, at approximately two-fold, and demonstrates extensive podocyte foot process effacement, at approximately 80% (×4000 magnification).


We examine a large panel of genes associated with FSGS, glomerular basement membrane disease, CAKUT, and nephronophthisis available from exome data in 193 mostly sporadic adult FSGS cases, achieving an overall genetic diagnostic rate of 11%. However, we are likely underestimating genetic contribution by the stringency of the criteria we use. Our findings demonstrate FSGS is commonly the end point of COL4A disorders as well as podocyte and kidney developmental defects.

The majority of kidney pathologies associated with definitely pathogenic COL4A3/A4/A5 variants had evidence of glomerular basement membrane abnormalities including thinning, thickening, irregularities, and lamellation, usually focal and sometimes subjectively described as minor and presumed not to be clinically significant. However, we also identified a minority of definitely pathogenic COL4A3/A4/A5 variants that do not have any obvious glomerular basement membrane abnormalities. Furthermore, we found cases with no proven genetic cause but that are associated with extensive glomerular basement membrane abnormalities. This suggests that our method for genetic testing has missed some definitely pathogenic variants and/or that morphologic glomerular basement membrane abnormalities can be nonspecific. Subgenomic capture strategy, as is used for exome sequencing, does not capture the entirety of the coding sequence; in our case, 95% (with at least 10× coverage) of COL4A. Variants and copy number changes in intronic regions will also not be detected with this methodology. Additionally, in cases with likely pathogenic variants in COL4A not considered in the diagnostic rate, we found one case with glomerular basement abnormalities, supporting a possible deleterious role for at least some of these variants.

Our genetic–pathologic correlation supports the notion that diffuse podocyte effacement can occur as a result of GBM abnormalities. In the absence of a complementary test (i.e., genetic testing) at the time of presentation, as well as other clinical characteristics that decreased suspicion for a collagen 4A defect such as a lack of family history, being female, and diffuse podocyte foot effacement on pathology, many of these individuals were tried on immunosuppressive therapies and were unresponsive. For other genetic causes such as CAKUT, biopsy features including glomerulomegaly could similarly raise suspicion for likely pathogenic variant disease causality. Thus, our experience highlights the complementary nature of pathology and genetic testing to improve interpretation of each test.

A previous report suggests that the risk of developing ESKD in heterozygous COL4A females is low before 40 years of age, but increases after 60 years of age (29). Twice as many women are affected by X-linked diseases, but are commonly undiagnosed (30). Patients with pathogenic variants in genes expressed in podocytes or associated with developmental defects had an earlier age at disease onset and ESKD than both the COL4A and no proven genetic basis subgroups. Our estimated mean survival/age for ESKD in COL4A5 was 58 years (95% CI, 46 to 70), which is later than the 24 years reported in the literature, which is likely driven by the preponderance of females with deleterious COL4A5 variants, influencing a milder clinical course than what has been previously reported for collagen 4A disorders (31).

COL4A5 disease has been reported to have X-linked recessive inheritance. We report COL4A5 variants that have been found in other cases, regardless of sex, but cannot definitively conclude causality as we did not perform X-chromosome inactivation studies. Although X-inactivation ratios of 50:50 are expected in a normal population of cells, ratios can deviate from modifier genes or variant selection advantages (32). The Genotype-Tissue Expression (GTEx project) includes high-coverage RNA-sequencing data from diverse human tissues to investigate male–female differences in expression and shows that COL4A5 is indeed subject to inactivation (33).

The cases included in this study are patients recruited from specialty clinics at a quaternary-level hospital in Toronto, Ontario, Canada, and may not be representative of a population-based study. Furthermore, we have acquired a predominantly case cohort, recruiting only affected individuals. As a result, we are unable to determine parental phase for two variants in the same individual, identify de novo variants, and define the penetrance of rare variants that are found. Additionally, for COL4A cases, we do not have formalized ocular and auditory assessments to report.

Our experience suggests that genetic–pathologic correlation improves diagnostic accuracy in FSGS. Nearly all variants we designated as definitely pathogenic are the exact same variants that have been previously reported. Inherently, designation of pathogenicity is biased toward the most studied genes. We suspect that we are still underestimating genetic contribution because of our inability to resolve the significance of many rare variants, highlighting the need for genetic, clinical, and pathologic data sharing as well as large-scale functional libraries.



T.Y., K.U., Y.P., and M.B. contributed equally to this work

Published online ahead of print. Publication date available at


The authors thank the patients and their families for their participation; Sergio Pereira, Daniel Merico, Wilson Sung, Liz Weili, and Bhooma Thiruvahindrapuram of The Centre for Applied Genomics, The Hospital for Sick Children, Toronto, Ontario, Canada for assistance with sequencing and variant calling; and Winnie Chan, Xuewen Song, and Ning He for assistance with patient recruitment and technical advice.

M.B. received a University of Toronto McLaughlin Centre Accelerator grant in Genomic Medicine in 2017, a NephCure Kidney International-Neptune Ancillary Studies grant in 2016, and a health research grant (14-04) from Physician’s Services Incorporated in 2015, which funded this work. M.B. is supported by a new investigator award from the Kidney Research Scientist Core Education and National Training Program.

Supplemental Material

This article contains the following supplemental material online at

Supplemental Table 1. Additional baseline clinical characteristics of the sequenced cohort.

Supplemental Table 2. List of genes associated with FSGS and related phenotypes.

Supplemental Table 3. Likely pathogenic variants in COL4A genes.

Supplemental Table 4. Likely pathogenic variants in non-COL4A genes.

Supplemental Table 5. Possibly pathogenic variants.

Supplemental Table 6. Previously reported variants with uncertain pathogenic significance.

Supplemental Table 7. Additional clinical characteristics for patient subgroups.

Supplemental Table 8. Tables demonstrating ages at disease onset and estimated mean survival/age of kidney failure by subgroup, with sexes analyzed separately.

Supplemental Table 9. Clinical characteristics of COL4A, other genetic cause, and no proven genetic cause subgroups.

Supplemental Figure 1. Pedigrees of families with definitely pathogenic variants.


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    FSGS; idiopathic nephrotic syndrome; human genetics; type 4A collagen; renal development; Glomerular Basement Membrane; Podocytes; nephrotic syndrome; Whole Exome Sequencing; Urogenital Abnormalities; vesico-ureteral reflux; kidney; glomerulonephritis; Kidney Failure, Chronic; Renal Insufficiency; Genetic Testing; Registries; Cohort Studies

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