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Identification of Genetic Causes of Focal Segmental Glomerulosclerosis Increases With Proper Patient Selection

Open AccessPublished:June 10, 2021DOI:https://doi.org/10.1016/j.mayocp.2021.01.037

      Abstract

      Objective

      To increase the likelihood of finding a causative genetic variant in patients with a focal segmental glomerulosclerosis (FSGS) lesion, clinical and histologic characteristics were analyzed.

      Patients and Methods

      Individuals 18 years and older with an FSGS lesion on kidney biopsy evaluated at Mayo Clinic from November 1, 1999, through October 31, 2019, were divided into 4 groups based on clinical and histologic characteristics: primary FSGS, secondary FSGS with known cause, secondary FSGS without known cause, and undetermined FSGS. A targeted gene panel and a customized gene panel retrieved from exome sequencing were performed.

      Results

      The overall rate of detection of a monogenic cause was 42.9% (21/49). Individuals with undetermined FSGS had the highest rate of positivity (87.5%; 7/8) followed by secondary FSGS without an identifiable cause (61.5%; 8/13) and secondary FSGS with known cause (33.3%; 5/15). Four of 5 (80%) individuals in the latter group who had positive genetic testing results also had a family history of kidney disease. Univariate analysis showed that family history of kidney disease (odds ratio [OR], 13.8; 95% CI, 3.7 to 62.4; P<.001), absence of nephrotic syndrome (OR, 8.2; 95% CI, 1.9 to 58.1; P=.004), and female sex (OR, 5.1; 95% CI, 1.5 to 19.9; P=.01) were strong predictors of finding a causative genetic variant in the entire cohort. The most common variants were in the collagen genes (52.4%; 11/21), followed by the podocyte genes (38.1%; 8/21).

      Conclusion

      In adults with FSGS lesions, proper selection of patients increases the rate of positive genetic testing significantly. The majority of individuals with undetermined FSGS in whom the clinical presentation and histologic parameters are discordant had a genetic diagnosis.

      Abbreviations and Acronyms:

      eGFR (estimated glomerular filtration rate), EM (electron microscopy), FPE (foot-process effacement), FSGS (focal segmental glomerulosclerosis), GBM (glomerular basement membrane), IFTA (interstitial fibrosis of tubular atrophy), NS (nephrotic syndrome), OR (odds ratio), PR (partial remission), VUS (variants of uncertain significance)
      Focal segmental glomerulosclerosis (FSGS) is a histologic lesion defined by the presence of a sclerotic lesion in parts (segmental) of some (focal) glomeruli on light microscopy.
      • Sethi S.
      • Glassock R.J.
      • Fervenza F.C.
      Focal segmental glomerulosclerosis: towards a better understanding for the practicing nephrologist.
      ,
      • D'Agati V.
      Pathologic classification of focal segmental glomerulosclerosis.
      The FSGS results from a variety of different pathogenic mechanisms that predominantly cause injury to podocytes, leading to the loss of selectivity of the glomerular filtration barrier and the development of proteinuria.
      • Fogo A.B.
      Causes and pathogenesis of focal segmental glomerulosclerosis.
      ,
      • Deegens J.K.
      • Dijkman H.B.
      • Borm G.F.
      • et al.
      Podocyte foot process effacement as a diagnostic tool in focal segmental glomerulosclerosis.
      On electron microscopy (EM), foot processes show variable degrees of effacement, depending on the underlying pathogenic mechanism.
      • Deegens J.K.
      • Dijkman H.B.
      • Borm G.F.
      • et al.
      Podocyte foot process effacement as a diagnostic tool in focal segmental glomerulosclerosis.
      ,
      • Campbell K.N.
      • Tumlin J.A.
      Protecting podocytes: a key target for therapy of focal segmental glomerulosclerosis.
      Currently, FSGS is divided into primary, secondary, and genetic forms.
      • Ahn W.
      • Bomback A.S.
      Approach to diagnosis and management of primary glomerular diseases due to podocytopathies in adults: core curriculum 2020.
      • De Vriese A.S.
      • Sethi S.
      • Nath K.A.
      • Glassock R.J.
      • Fervenza F.C.
      Differentiating primary, genetic, and secondary FSGS in adults: a clinicopathologic approach.
      • Lepori N.
      • Zand L.
      • Sethi S.
      • Fernandez-Juarez G.
      • Fervenza F.C.
      Clinical and pathological phenotype of genetic causes of focal segmental glomerulosclerosis in adults.
      • Shabaka A.
      • Tato Ribera A.
      • Fernández-Juárez G.
      Focal segmental glomerulosclerosis: state-of-the-art and clinical perspective.
      Such distinction is important because it has both prognostic and therapeutic implications.
      • De Vriese A.S.
      • Sethi S.
      • Nath K.A.
      • Glassock R.J.
      • Fervenza F.C.
      Differentiating primary, genetic, and secondary FSGS in adults: a clinicopathologic approach.
      ,
      • Lepori N.
      • Zand L.
      • Sethi S.
      • Fernandez-Juarez G.
      • Fervenza F.C.
      Clinical and pathological phenotype of genetic causes of focal segmental glomerulosclerosis in adults.
      Genetic forms of FSGS may happen sporadically or be familial and are caused by variants in genes that encode proteins that are expressed in either podocytes or the glomerular basement membrane (GBM).
      • Lepori N.
      • Zand L.
      • Sethi S.
      • Fernandez-Juarez G.
      • Fervenza F.C.
      Clinical and pathological phenotype of genetic causes of focal segmental glomerulosclerosis in adults.
      Currently, more than 50 genes are known to be involved in FSGS, such as the podocyte-related genes NPHS1, NPHS2, TRPC6, and INF2 and the GBM-related genes such as COL4A3/A4/A5 (for expansion of gene symbols, use search tool at www.genenames.org).
      • Lepori N.
      • Zand L.
      • Sethi S.
      • Fernandez-Juarez G.
      • Fervenza F.C.
      Clinical and pathological phenotype of genetic causes of focal segmental glomerulosclerosis in adults.
      ,
      • Snoek R.
      • Nguyen T.Q.
      • van der Zwaag B.
      • et al.
      Importance of genetic diagnostics in adult-onset focal segmental glomerulosclerosis.
      Typically, the genetic forms have an early onset of symptoms. However, with widespread availability of genetic testing, there is increased recognition of adult-onset genetic FSGS. In individuals who present with steroid-resistant nephrotic syndrome (NS), a causative genetic variant has been identified in 10% of adult-onset FSGS, and this rate is as high as 43% in familial cases.
      • Santin S.
      • Bullich G.
      • Tazon-Vega B.
      • et al.
      Clinical utility of genetic testing in children and adults with steroid-resistant nephrotic syndrome.
      Identifying patients who have a genetic cause of FSGS is important because it allows for selecting the appropriate treatment strategy and proper family counseling and provides information regarding kidney transplantation and donor selection of family members. However, the challenge for nephrologists is to select which individuals are the best candidates for genetic testing. Despite the widespread availability of next-generation sequencing and the reduction in cost, there are still individuals who are not selected for genetic testing and are mislabeled as having primary or secondary forms of FSGS.
      • Lepori N.
      • Zand L.
      • Sethi S.
      • Fernandez-Juarez G.
      • Fervenza F.C.
      Clinical and pathological phenotype of genetic causes of focal segmental glomerulosclerosis in adults.
      This is partly because in patients with adult-onset FSGS, clinical and histologic features are heterogeneous and are rarely specific.
      • De Vriese A.S.
      • Sethi S.
      • Nath K.A.
      • Glassock R.J.
      • Fervenza F.C.
      Differentiating primary, genetic, and secondary FSGS in adults: a clinicopathologic approach.
      ,
      • Lepori N.
      • Zand L.
      • Sethi S.
      • Fernandez-Juarez G.
      • Fervenza F.C.
      Clinical and pathological phenotype of genetic causes of focal segmental glomerulosclerosis in adults.
      ,
      • Bierzynska A.
      • McCarthy H.J.
      • Soderquest K.
      • et al.
      Genomic and clinical profiling of a national nephrotic syndrome cohort advocates a precision medicine approach to disease management.
      To overcome this limitation, there is a need for a comprehensive evaluation of demographic, clinical, and histologic features of patients with genetic FSGS to distinguish it from other forms. In this study, we aimed to identify clinical and histologic factors that would be predictive of identifying a genetic diagnosis in individuals who were clinically categorized as having an FSGS lesion on kidney pathology.

      Patients and Methods

      The study was approved by Mayo Clinic Institutional Review Board. All participants who underwent research testing provided written informed consent.
      Patients were evaluated in the Mayo Clinic Nephrology and Hypertension Division. All patients were 18 years or older at the time of their first kidney biopsy from November 1, 1999, through October 31, 2019. Pathology had to show an FSGS lesion on light microscopy in the absence of any other glomerular finding (eg, diabetes related) with a negative immunofluorescence study. All biopsies were reviewed and diagnosis was confirmed by a renal pathologist at Mayo Clinic in Rochester, Minnesota. Data regarding demographic characteristics, laboratory results, histology, immunosuppressive therapy, and kidney outcomes were obtained from review of electronic medical records.
      Based on clinical presentation and kidney biopsy findings, patients were divided into 4 groups. Group 1 included individuals with primary FSGS; group 2, individuals with secondary FSGS with an identifiable cause; group 3, individuals with secondary FSGS without a clear cause; and group 4, individuals with undetermined FSGS.

      Clinical Definitions

      Primary FSGS was defined as the presence of NS, defined as urinary protein excretion greater than 3.5 g per 24 hours and serum albumin level less than 3.5 g/dL (to convert to g/L, multiply by 10), in addition to 80% or greater foot-process effacement (FPE) on EM. Complete remission was defined as reduction of proteinuria to protein excretion less than 0.3 g per 24 hours after immunosuppressive therapy with prednisone only. Partial remission (PR) was defined as reduction of proteinuria to protein excretion of 0.3 to 3 g per 24 hours and 50% reduction in proteinuria from baseline. Resistance was defined as not achieving complete remission or PR after at least 16 weeks of immunosuppression therapy with prednisone only. Of note, 76.9% (10/13) of patients who were resistant to prednisone were also resistant to other forms of immunosuppressive therapy.
      Secondary FSGS was defined as absence of NS (serum albumin ≥3.5 g/dL with any degree of proteinuria) and less than 80% FPE on EM. Identifiable cause of secondary FSGS included obesity (body mass index [calculated as the weight in kilograms divided by the height in meters squared] >30 kg/m2), presence of sleep apnea,
      • Rosenberg A.Z.
      • Kopp J.B.
      Focal segmental glomerulosclerosis.
      partial or complete nephrectomy, or reflux nephropathy.
      Individuals with undetermined FSGS were defined as those in whom the clinical presentation and histologic manifestation did not match; for example, individuals with NS but with less than 80% FPE on EM or absence of NS but with FPE of 80% or greater on EM. Abnormal GBM was defined as the presence of any alteration, such as irregularity, thickening, thinning, lamellations, scalloping, microparticles, or wrinkling.
      Positive family history was defined as the presence of first-degree relatives with kidney disease associated with proteinuria or other known genetic causes such as Alport syndrome or FSGS or unknown cause that resulted in end-stage kidney disease. End-stage kidney disease was defined as estimated glomerular filtration rate less than 15 mL/min/1.73 m2 or need for dialysis or kidney transplantation. Age of onset was the age at which the first symptom of kidney disease was noted. This included the first identification of protein or blood on the urine studies or detection of impaired estimated glomerular filtration rate on blood testing.

      Genetic Testing and Variant Interpretation

      Genomic DNA was isolated from whole blood or buccal swab samples. A targeted gene panel including 55 genes associated with FSGS and a customized gene panel retrieved from exome sequencing data including 346 genes associated with kidney-related genes (Supplemental Table 1, available online at http://www.mayoclinicproceedings.org) were performed in a Clinical Laboratory Improvement Amendments–certified laboratory. Research exome sequencing was performed either at the Mayo Clinic Medical Genome Facility or the same laboratory that did the clinical testing when the initial panel was negative or if it could not be done on a clinical basis. Information on sequencing methodology can be found in Supplemental Appendix 1 (available online at http://www.mayoclinproceedings.org).
      Variants were classified according to the American College of Medical Genetics recommendations.
      • Richards S.
      • Aziz N.
      • Bale S.
      • et al.
      ACMG Laboratory Quality Assurance Committee
      Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology.
      Individuals with pathogenic and/or likely pathogenic variants in genes that were associated with FSGS were considered as having a genetic diagnosis. In addition, after careful review by geneticists and nephrologists, some variants of uncertain significance (VUS) were deemed of higher likelihood of being causative for individuals’ phenotypes, though without enough evidence to change the classification based on the American College of Medical Genetics 2015 guidelines. These individuals were labeled as having “relevant” VUS. Variants found in research testing were confirmed in a Clinical Laboratory Improvement Amendments–certified laboratory. Family segregation was pursued when possible and aided in reclassifying VUS.

      Statistical Analyses

      Results are expressed as mean ± SD for quantitative data or absolute numbers with percentage for qualitative data. Statistical significance was determined using unpaired t test (2 tailed) and Fisher or χ2 (2-sided) test, respectively. A logistic regression model was used when evaluating the probability of finding a causative genetic variant. Odds ratios (ORs) and 95% CIs were presented. All analyses were performed using JMP software, version 14.0 (SAS Institute Inc). P<.05 was considered to be statistically significant.

      Results

      Baseline Clinical Characteristics

      There were 49 patients recruited for this study. The average age of the cohort at the time of kidney biopsy was 43.9 ± 15.4 years, with 33 men (67.3%). Patients were divided into 4 groups of primary FSGS (n=13), secondary FSGS with known cause (n=15), secondary FSGS without a known cause (n=13), and undetermined FSGS (n=8; Figure 1). The detailed baseline characteristics and histologic findings of each group are shown in Table 1. Overall age of onset was 37.7 ± 15.4 years, and on average, symptoms presented 6.2 years before the patient underwent a kidney biopsy. The gap was longest in patients with secondary FSGS without an identifiable cause (10.6 years). Patients with secondary FSGS due to a known cause were older (age at biopsy, 50.5 ± 11.6 years) and had higher baseline creatinine levels (2.2 ± 1.8 mg/dL [to convert to μmol/L, multiply by 88.4), higher body mass index (33.2 ± 3.8 kg/m2), and higher rate of sleep apnea (53.3% [8/15]). In the overall cohort, 20 patients (41.7% [20/48]) had a positive family history. The highest rate of positive family history was in patients with secondary FSGS without an identifiable cause (84.6% [11/13]), followed by the undetermined FSGS group (37.5% [3/8]). Of the 49 patients, 17 (34.7% [17/49]) developed end-stage kidney disease, with the highest rate in the undetermined FSGS group (62.5% [5/8]).
      Figure thumbnail gr1
      Figure 1Focal segmental glomerulosclerosis (FSGS) subgroups by clinic evaluation and genetic testing results.
      Table 1Individuals’ Baseline Clinical and Histologic Characteristics
      eGFR, estimated glomerular filtration rate; FSGS, focal segmental glomerulosclerosis; IFTA, interstitial fibrosis of tubular atrophy.
      ,
      SI conversion factors: To convert serum creatinine values to μmol/L, multiply by 88.4; to convert albumin values to g/L, multiply by 10; to convert cholesterol values to mmol/L, multiply by 0.0259.
      Baseline CharacteristicTotal Cohort (n=49)Primary (n=13)Known-Cause Secondary (n=15)Unknown-Cause Secondary (n=13)Undetermined (n=8)
      Male sex, no. (%)33 (67.3)8 (61.5)14 (93.3)8 (61.5)3 (37.5)
      Age at 1st biopsy (y), mean ± SD43.9 ± 15.442.6 ± 13.750.5 ± 11.638.9 ± 16.542 ± 20.2
      Age at disease onset (y), mean ± SD37.7 ± 15.441.8 ± 14.740.3 ± 13.128.3 ± 13.241.3 ± 19.7
      Body mass index (kg/m2), mean ± SD29.6 ± 5.329.9 ± 4.033.2 ± 3.826.2 ± 2.527.4 ± 8.7
      Serum creatinine (mg/dL), mean ± SD1.7 ± 1.11.4 ± 0.52.2 ± 1.81.6 ± 0.41.4 ± 0.6
      eGFR (mL/min/1.73 m2), median (25%, 75% percentile)49.7 (37.3, 63.3)58 (38.0, 77.5)48.9 (26.7, 63.8)48 (38.5, 69.0)50.5 (34.0, 60.0)
      Albumin (g/dL), mean ± SD3.4 ± 0.92.3 ± 0.54.0 ± 0.44.1 ± 0.33.2 ± 0.5
      Proteinuria (g/24 h), mean ± SD6.2 ± 6.113.8 ± 7.63.7 ± 2.12.8 ± 1.84.4 ± 1.5
      Total cholesterol (mg/dL), mean ± SD229.5 ± 90.0308.8 ± 125.3187.9 ± 51.9212.2 ± 60.2216.8 ± 53.9
      Low-density lipoprotein cholesterol (mg/dL), mean ± SD124.0 ± 55.9160.2 ± 78.2100.9 ± 44.3120.3 ± 44.1120.5 ± 34.5
      Nephrotic syndrome, no. (%)15 (30.6)13 (100)0 (0.0)0 (0.0)2 (25.0)
      Hypertension, no. (%)41 (83.7)9 (69.2)14 (93.3)12 (92.3)6 (75.0)
      Sleep apnea, no. (%)14 (28.6)2 (15.4)8 (53.3)0 (0.0)4 (50.0)
      Family history (positive), no. (%)
      Unknown family history in 1 individual in the group of known-cause secondary FSGS.
      20 (41.7)1 (7.7)5 (35.7)11 (84.6)3 (37.5)
      Kidney histology
       Global sclerosis (%), mean ± SD27.9 ± 19.316.1 ± 18.632.9 ± 21.432.1 ± 15.331.1 ± 16.4
       Segmental sclerosis (%), mean ± SD17.0 ± 8.516.2 ± 10.616.6 ± 4.317.1 ± 10.719.3 ± 8.6
       Glomerulomegaly (yes), no. (%)19 (38.8)3 (23.1)9 (60.0)6 (46.2)1 (12.5)
       Foot-process effacement, no. (%)
      Data are unavailable for 3 individuals who have known-cause secondary, unknown-cause secondary, and undetermined FSGS group, respectively.
      <40%13 (28.3)0 (0.0)6 (42.9)7 (58.3)0 (0.0)
      40%-79%16 (34.8)0 (0.0)8 (57.1)5 (41.7)3 (42.9)
      ≥80%17 (37.0)13 (100)0 (0.0)0 (0.0)4 (57.1)
       Glomerular basement membrane (abnormal), no. (%)
      Data are unavailable for 3 individuals who have known-cause secondary, unknown-cause secondary, and undetermined FSGS group, respectively.
      23 (50.0)5 (38.5)7 (50.0)5 (41.7)6 (85.7)
       IFTA, no. (%)
      Data are unavailable in 1 individual in the group of undetermined FSGS.
      None/mild45 (93.8)13 (100)13 (86.7)13 (100)6 (85.7)
      Moderate/severe3 (6.3)0 (0.0)2 (13.3)0 (0.0)1 (14.3)
       Arteriolar hyalinosis, no. (%)
      None27 (55.1)7 (53.8)7 (46.7)7 (53.8)6 (75.0)
      Mild12 (24.5)6 (46.2)2 (13.3)4 (30.8)0 (0.0)
      Moderate/severe10 (20.4)0 (0.0)6 (40.0)2 (15.4)2 (25.0)
       Arteriosclerosis, no. (%)
      None18 (36.7)7 (53.8)3 (20.0)5 (38.5)3 (37.5)
      Mild17 (34.7)3 (23.1)5 (33.3)6 (46.2)3 (37.5)
      Moderate/severe14 (28.6)3 (23.1)7 (46.7)2 (15.4)2 (25.0)
      Kidney and genetic results
       End-stage kidney disease, no. (%)17 (34.7)3 (23.1)5 (33.3)4 (30.8)5 (62.5)
       Treatment with immunosuppressants, no. (%)22 (44.9)13 (100)3 (20.0)2 (15.4)4 (50.0)
       Response to immunosuppression, no. (%)
      Complete/partial9 (40.9)8 (61.5)0 (0.0)0 (0.0)1 (25.0)
      Resistant13 (59.1)5 (38.5)3 (100)2 (100)3 (75.0)
       Genetic testing (positive), no. (%)21 (42.9)1 (7.7)5 (33.3)8 (61.5)7 (87.5)
       Causative genesCOL4A3 (n=7)NPHS2 (n=1)COL4A3 (n=2)COL4A3 (n=2)COL4A3 (n=3)
      COL4A5 (n=2)COL4A4 (n=1)COL4A5 (n=2)NPHS2 (n=2)
      COL4A4 (n=2)NPHS1 (n=1)COL4A4 (n=1)INF2 (n=1)
      NPHS2 (n=3)TRPC6 (n=1)INF2 (n=2)SMARCAL1 (n=1)
      INF2 (n=3)UMOD (n=1)
      NPHS1 (n=1)
      TRPC6 (n=1)
      UMOD (n=1)
      SMARCAL1 (n=1)
      a eGFR, estimated glomerular filtration rate; FSGS, focal segmental glomerulosclerosis; IFTA, interstitial fibrosis of tubular atrophy.
      b SI conversion factors: To convert serum creatinine values to μmol/L, multiply by 88.4; to convert albumin values to g/L, multiply by 10; to convert cholesterol values to mmol/L, multiply by 0.0259.
      c Unknown family history in 1 individual in the group of known-cause secondary FSGS.
      d Data are unavailable for 3 individuals who have known-cause secondary, unknown-cause secondary, and undetermined FSGS group, respectively.
      e Data are unavailable in 1 individual in the group of undetermined FSGS.

      Genetic Testing Results

      Twenty-one individuals had a monogenic cause for their FSGS (42.9% [21/49]). Nineteen patients had pathogenic and/or likely pathogenic variants and 2 patients had relevant VUS that were deemed causative. Pathogenic variants occurred in collagen genes (COL4A3/A4/A5) in 52.4% of the cases (11/21); podocyte genes (NPHS1, NPHS2, INF2, and TRPC6), in 38.1% of cases (8/21); and other genes (SMARCAL1 and UMOD), in 9.5% of cases (2/21). Relevant VUS deemed causative were in the podocyte genes INF2 and NPHS1 (Table 2).
      Table 2Genes With Causative Genetic Variants Detected
      Genes
      Collagen, no. (%)11 (52.4)
      COL4A37
      COL4A42
      COL4A52
      Podocyte, no. (%)8 (38.1)
      NPHS23
      INF2
      INF2 variant: rare, predicted deleterious, in a region where other pathogenic variants have been reported, and segregates with the disease in the family.
      3
      TRPC61
      NPHS1
      NPHS1 variants: rare, predicted deleterious, lacking phase.
      1
      Other, no. (%)2 (9.5)
      SMARCAL11
      UMOD1
      Total, no.
      In the total cohort, genetic diagnostic rate is 42.9% (21/49).
      21
      a INF2 variant: rare, predicted deleterious, in a region where other pathogenic variants have been reported, and segregates with the disease in the family.
      b NPHS1 variants: rare, predicted deleterious, lacking phase.
      c In the total cohort, genetic diagnostic rate is 42.9% (21/49).
      The detailed clinical characteristics and histologic findings of individuals with COL4A genetic variants vs non-COL4A genetic variants are shown in Supplemental Table 2 (available online at http://www.mayoclinicproceedings.org), and the EM findings in individuals with COL4A genetic variants are shown in Supplemental Table 3 (available online at http://www.mayoclinicproceedings.org).
      Detailed information on the causative genetic variants is shown in Supplemental Table 4 (available online at http://www.mayoclinicproceedings.org). Additional variants were found in 7 individuals with a genetic diagnosis and are reported in Supplemental Table 5 (available online at http://www.mayoclinicproceedings.org). Of the 28 patients who were not considered to have a genetic diagnosis, 14 had VUS (Supplemental Table 6, available online at http://www.mayoclinicproceedings.org). One individual had two VUS in DLC1 that were not considered causative because there is no definitive gene-disease association currently. Fourteen individuals did not have a genetic variant reported by the clinical laboratory.
      Individuals with undetermined FSGS had the highest rate of genetic diagnosis, with 7 of 8 (87.5%) having pathogenic/likely pathogenic variants. The next group with the highest rate of positive results was individuals with secondary FSGS without an identifiable cause, for whom 8 of 13 (61.5%) had a genetic diagnosis. Five of 15 individuals (33.3%) with FSGS with an underlying cause had positive genetic testing results. Four of them had a positive family history of kidney disease.

      Clinical Predictors for Positive Genetic Results

      A comparison of patients' baseline characteristics in those with negative genetic testing results (n=28) and those with positive genetic testing results (n=21) is shown in Table 3. In univariate analysis, the strongest predictor of finding a causative genetic variant was the presence of a family history (OR, 13.8; 95% CI, 3.7 to 62.4; P<.001), followed by the absence of NS (OR, 8.2; 95% CI, 1.9 to 58.1; P=.004), female sex (OR, 5.1; 95% CI, 1.5 to 19.9; P=.01), and higher albumin level (OR, 2.1; 95% CI, 1.1-4.9; P=.04). The higher proteinuria level predicted the lower likelihood of finding a genetic diagnosis (OR, 0.8; 95% CI, 0.6 to 0.9; P=.02). Clinical parameters including hypertension and sleep apnea and histologic findings such as degree of FPE, presence of GBM abnormalities, and presence of glomerulomegaly were not predictors of a genetic diagnosis (P=.21, P=.76, and P=.93, respectively; Table 4).
      Table 3Comparison of Patients’ Characteristics in Patients With Positive vs Negative Genetic Testing Results
      eGFR, estimated glomerular filtration rate; IFTA, interstitial fibrosis of tubular atrophy.
      ,
      SI conversion factors: To convert serum creatinine values to μmol/L, multiply by 88.4; to convert albumin values to g/L, multiply by 10; to convert cholesterol values to mmol/L, multiply by 0.0259.
      Baseline CharacteristicPositive (n=21)Negative (n=28)P
      P values derived from a 2-sample test, either t test or χ2/Fisher exact test for comparison of characteristics in patients with positive vs negative genetic test results.
      Male sex, no. (%)10 (47.6)23 (82.1).02
      Age at 1st biopsy (y), mean ± SD42.7 ± 16.444.9 ± 14.7.60
      Age at disease onset (y), mean ± SD33.8 ± 13.240.8 ± 16.3.09
      Body mass index (kg/m2), mean ± SD28.6 ± 6.130.3 ± 4.6.31
      Serum creatinine (mg/dL), mean ± SD1.4 ± 0.41.9 ± 1.4.09
      eGFR (mL/min/1.73m2), mean ± SD57.9 ± 30.852.5 ± 25.6.52
      Albumin (g/dL), mean ± SD3.7 ± 0.63.2 ± 1.0.03
      Proteinuria (g/24 h), mean ± SD3.6 ± 1.98.2 ± 7.4.005
      Total cholesterol (mg/dL), mean ± SD221.8 ± 55.2235.5 ± 110.6.60
      Low-density lipoprotein cholesterol (mg/dL), mean ± SD122.3 ± 42.5125.4 ± 65.9.80
      Nephrotic syndrome, no. (%)2 (9.5)13 (46.4).01
      Family history (positive), no. (%)
      Unknown family history in 1 individual in positive group.
      15 (75.0)5 (17.9)<0.001
      Hypertension, no. (%)18 (85.7)23 (82.1)>.99
      Sleep apnea, no. (%)8 (38.1)6 (21.4).22
      Kidney histology
       Global sclerosis (%), mean ± SD30.5 ± 16.125.9 ± 21.3.41
       Segmental sclerosis (%), mean ± SD18.5 ± 9.316.0 ± 7.9.40
       Glomerulomegaly (yes), no. (%)8 (38.1)11 (39.3)>.99
       Foot-process effacement, no. (%)
      Data are unavailable in 2 individuals in positive group and 1 individual in negative group, respectively.
      <40%6 (31.6)7 (25.9).45
      40%-79%8 (42.1)8 (29.6)
      ≥80%5 (26.3)12 (44.4)
       Glomerular basement membrane (abnormal), no. (%)
      Data are unavailable in 2 individuals in positive group and 1 individual in negative group, respectively.
      10 (52.6)13 (48.1)>.99
       IFTA, no. (%)
      Data are unavailable in 1 individual in positive group.
      None/mild19 (95.0)26 (92.9).71
      Moderate/severe1 (5.0)2 (7.1)
       Arteriolar hyalinosis, no. (%)
      None13 (61.9)14 (50.0).67
      Mild4 (19.0)8 (28.6)
      Moderate/severe4 (19.0)6 (21.4)
       Arteriosclerosis, no. (%)
      None9 (42.9)9 (32.1).71
      Mild7 (33.3)10 (35.7)
      Moderate/severe5 (23.8)9 (32.1)
      Kidney outcomes
       End-stage kidney disease, no. (%)8 (38.1)9 (32.1).77
       Treatment with immunosupressant, no. (%)7 (33.3)15 (53.6).25
      Response to immunosuppression, no. (%)
       Complete/partial1 (14.3)8 (53.3).17
       Resistant6 (85.7)7 (46.7)
      a eGFR, estimated glomerular filtration rate; IFTA, interstitial fibrosis of tubular atrophy.
      b SI conversion factors: To convert serum creatinine values to μmol/L, multiply by 88.4; to convert albumin values to g/L, multiply by 10; to convert cholesterol values to mmol/L, multiply by 0.0259.
      c P values derived from a 2-sample test, either t test or χ2/Fisher exact test for comparison of characteristics in patients with positive vs negative genetic test results.
      d Unknown family history in 1 individual in positive group.
      e Data are unavailable in 2 individuals in positive group and 1 individual in negative group, respectively.
      f Data are unavailable in 1 individual in positive group.
      Table 4Univariate Association Between Clinical and Histologic Characteristics and Positive Genetic Testing Results
      SI conversion factor: To convert albumin values to g/L, multiply by 10.
      Clinical PredictorOdds Ratio (95% CI)
      Odds ratios with 95% CIs and P values calculated using logistic regression model for evaluating association between the variable and the probability of finding a causative genetic variant.
      P
      Positive family history13.8 (3.7-62.4)<.001
      Female sex (vs male)5.1 (1.5-19.9).01
      Proteinuria (g/24 h)
      Odds ratios for continuous variables are per unit change in regression.
      0.8 (0.6-0.9).02
      Albumin (g/dL)
      Odds ratios for continuous variables are per unit change in regression.
      2.1 (1.1-4.9).04
      Absence of nephrotic syndrome8.2 (1.9-58.1).004
      Hypertension1.3 (0.3-7.0).74
      Sleep apnea2.3 (0.6-8.3).20
      Foot-process effacement
       <80%Reference.21
       ≥80%0.5 (0.1-1.6)
      Glomerular basement membrane
       NormalReference.76
       Abnormal1.2 (0.4-3.9)
      Glomerulomegaly
       NoReference
       Yes1.0 (0.3-3.0).93
      a SI conversion factor: To convert albumin values to g/L, multiply by 10.
      b Odds ratios with 95% CIs and P values calculated using logistic regression model for evaluating association between the variable and the probability of finding a causative genetic variant.
      c Odds ratios for continuous variables are per unit change in regression.

      Discussion

      This is the first study to comprehensively evaluate the clinical and histologic parameters that increase the likelihood of identifying a monogenic disease in adult patients with FSGS lesion on the kidney biopsy. The prevalence of genetic FSGS in the adult population has ranged between 10% and 43%, depending on which individuals have been chosen for testing and which genes have been tested.
      • Santin S.
      • Bullich G.
      • Tazon-Vega B.
      • et al.
      Clinical utility of genetic testing in children and adults with steroid-resistant nephrotic syndrome.
      ,
      • Sen E.S.
      • Dean P.
      • Yarram-Smith L.
      • et al.
      Clinical genetic testing using a custom-designed steroid-resistant nephrotic syndrome gene panel: analysis and recommendations.
      ,
      • Sadowski C.E.
      • Lovric S.
      • Ashraf S.
      • et al.
      A single-gene cause in 29.5% of cases of steroid-resistant nephrotic syndrome.
      In the current study, we were able to find a genetic diagnosis for almost half of the overall cohort. Although a very comprehensive panel including 346 genes associated with kidney diseases was used, only 9 genes were found to have definitive causative variants for individuals’ phenotypes.
      Most studies that have reported the diagnostic yield of genetic testing in FSGS have focused on the evaluation of individuals with primary FSGS who were resistant to therapy.
      • Santin S.
      • Bullich G.
      • Tazon-Vega B.
      • et al.
      Clinical utility of genetic testing in children and adults with steroid-resistant nephrotic syndrome.
      ,
      • Sen E.S.
      • Dean P.
      • Yarram-Smith L.
      • et al.
      Clinical genetic testing using a custom-designed steroid-resistant nephrotic syndrome gene panel: analysis and recommendations.
      ,
      • Sadowski C.E.
      • Lovric S.
      • Ashraf S.
      • et al.
      A single-gene cause in 29.5% of cases of steroid-resistant nephrotic syndrome.
      In this study, we categorized affected individuals into 4 groups based on clinical presentation, complementary examinations, and histologic characteristics of the kidney biopsy specimens. In our cohort, only 1 of 13 individuals with primary FSGS had a genetic diagnosis (7.7%). When evaluating the entire cohort of 49 patients, 22 individuals received immunosuppressant treatment and 13 were treatment resistant. Of those who were resistant, 6 (46.2% [6/13]) had a genetic diagnosis. Among the patients who responded to immunosuppressive therapy, only 1 (from the undetermined FSGS group) with temporary PR following combined therapy with prednisolone and tacrolimus had a genetic diagnosis.
      Noteworthy, individuals in the undetermined group had the highest rate of genetic diagnosis (87.5% [7/8]). All 4 individuals who did not have NS but had severe FPE were found having genetic FSGS. In addition, only 1 individual in the undetermined group with NS but without severe FPE was found having genetic FSGS. Of note, for this individual, repeat serum albumin levels oscillated from values just below the lower limit of normal to values in the normal range in most readings.
      It is likely that previous studies have wrongly categorized patients as having “primary” FSGS because thorough evaluation with EM was not performed and serum albumin levels were not routinely recorded.
      • Santin S.
      • Bullich G.
      • Tazon-Vega B.
      • et al.
      Clinical utility of genetic testing in children and adults with steroid-resistant nephrotic syndrome.
      ,
      • Sen E.S.
      • Dean P.
      • Yarram-Smith L.
      • et al.
      Clinical genetic testing using a custom-designed steroid-resistant nephrotic syndrome gene panel: analysis and recommendations.
      This difference in properly categorizing the patients likely accounts for the lower rate of positive genetic results in patients who had steroid-resistant primary FSGS in our cohort. The second group that had the highest yield of positive genetic testing results were individuals with secondary FSGS for whom an underlying cause was not readily identifiable. These individuals had no evidence of reduced nephron mass based on clinical history or imagining and had no evidence of increased demand on the kidneys (such as obesity or sleep apnea). Despite the absence of an obvious cause, they had similar clinical and histologic biomarkers as others with a known cause of secondary FSGS. In our cohort, this group had the highest rate of positive family history (84.6%; 11/13), making this strong evidence for a genetic diagnosis. In individuals with an underlying cause for secondary FSGS, one-third had a genetic FSGS and most had a positive family history. These individuals with a known cause for secondary FSGS are often excluded in studies that have evaluated the diagnostic rate of genetic testing in FSGS.
      • Buscher A.K.
      • Konrad M.
      • Nagel M.
      • et al.
      Mutations in podocyte genes are a rare cause of primary FSGS associated with ESRD in adult patients.
      ,
      • Zhang Q.
      • Ma J.
      • Xie J.
      • et al.
      Screening of ACTN4 and TRPC6 mutations in a Chinese cohort of patients with adult-onset familial focal segmental glomerulosclerosis.
      Our results suggest that in patients with known-cause FSGS in whom there is a positive family history, genetic testing should be considered.
      As expected, a positive family history was the strongest predictor of a genetic diagnosis, with 75% (15/20) of individuals with positive genetic testing results having a first-degree relative with kidney disease. The importance of a positive family history has been highlighted in prior studies.
      • Snoek R.
      • Nguyen T.Q.
      • van der Zwaag B.
      • et al.
      Importance of genetic diagnostics in adult-onset focal segmental glomerulosclerosis.
      However, almost one-third of individuals with a genetic form of FSGS did not have a family history of kidney disease. The disease might be caused by germline mosaicism, recessive inheritance, de novo variants, or incomplete penetrance.
      • Santin S.
      • Bullich G.
      • Tazon-Vega B.
      • et al.
      Clinical utility of genetic testing in children and adults with steroid-resistant nephrotic syndrome.
      ,
      • Gbadegesin R.A.
      • Lavin P.J.
      • Hall G.
      • et al.
      Inverted formin 2 mutations with variable expression in patients with sporadic and hereditary focal and segmental glomerulosclerosis.
      ,
      • Hildebrandt F.
      Genetic kidney diseases.
      This highlights that absence of a family history of kidney disease should not dissuade the clinician to pursue genetic testing if there are other clinical and histologic features that suggest a potential genetic cause.
      In our study, female sex was a strong predictor of finding a genetic variant in patients with an FSGS lesion. The exact reason for this is unclear. It is possible that this may be the result of the small sample size of our study, patient selection bias, increased risk for developing an FSGS lesion in men with obesity and sleep apnea, or other unknown reasons that require further investigation in a large cohort study. To our knowledge, there is no report to date about the sex distribution in genetic FSGS vs nongenetic FSGS.
      Overall, individuals with a genetic diagnosis in our cohort were younger than those who had negative genetic testing results.
      • Yao T.
      • Udwan K.
      • John R.
      • et al.
      Integration of genetic testing and pathology for the diagnosis of adults with FSGS.
      However, several individuals with genetic FSGS presented with proteinuria after 40 years of age, especially individuals with COL4A variants, similar to prior reports.
      • Lepori N.
      • Zand L.
      • Sethi S.
      • Fernandez-Juarez G.
      • Fervenza F.C.
      Clinical and pathological phenotype of genetic causes of focal segmental glomerulosclerosis in adults.
      ,
      • Vivante A.
      • Hildebrandt F.
      Exploring the genetic basis of early-onset chronic kidney disease.
      In our cohort of adult probands, lower degree of proteinuria and absence of NS were predictors of a positive genetic testing result. Usually children with genetic forms of FSGS present with steroid-resistant NS and variants in NPHS1 and NPHS2, which are autosomal recessive genes with full penetrance, whereas COL4A5 is the most common genetic cause in adults.
      • Trautmann A.
      • Bodria M.
      • Ozaltin F.
      • Consortium PodoNet
      • et al.
      Spectrum of steroid-resistant and congenital nephrotic syndrome in children: the PodoNet registry cohort.
      • Rood I.M.
      • Deegens J.K.
      • Wetzels J.F.
      Genetic causes of focal segmental glomerulosclerosis: implications for clinical practice.
      • Cil O.
      • Perwad F.
      Monogenic causes of proteinuria in children.
      Similar to previous reports,
      • Yao T.
      • Udwan K.
      • John R.
      • et al.
      Integration of genetic testing and pathology for the diagnosis of adults with FSGS.
      52.4% (11/21) of the affected individuals had a variant in a COL4A gene, with COL4A3 being the most common gene (63.6%; 7/11) and podocyte genes (INF2, NPHS2, TRPC6, and NPHS1) accounted for 38.1% (8/21) of the cases.
      We compared clinical and histologic features between the individuals with COL4A genetic variants and those with non-COL4A genetic variants. Apart from more glomerulomegaly (63.6% [7/11] vs 10% [1/10]; P=.02) in the group of COL4A genetic variants than that in the group of non-COL4A genetic variants, other clinical and histologic characteristics such as GBM abnormalities, podocyte FPE degree, and interstitial fibrosis of tubular did not differ between the 2 groups (Supplemental Table 2). Similar results have been reported by Yao et al.
      • Yao T.
      • Udwan K.
      • John R.
      • et al.
      Integration of genetic testing and pathology for the diagnosis of adults with FSGS.
      Of note, GBM abnormality was not predictive of positive genetic testing results. In addition, patients in the current study did not have Alport syndrome clinically or histologically diagnosed before genetic testing. Furthermore, 40% (4/10) of the individuals who had a COL4A variant had a normal GBM (Supplemental Table 3), a finding previously reported by Gast et al.
      • Gast C.
      • Pengelly R.J.
      • Lyon M.
      • et al.
      Collagen (COL4A) mutations are the most frequent mutations underlying adult focal segmental glomerulosclerosis.
      Therefore, absence of GBM changes should not dissuade the clinician from ordering genetic testing for an adult individual with FSGS.
      Podocyte injury and FPE play a critical role in the pathogenesis of proteinuria in individuals with FSGS,
      • Fogo A.B.
      Causes and pathogenesis of focal segmental glomerulosclerosis.
      ,
      • Deegens J.K.
      • Dijkman H.B.
      • Borm G.F.
      • et al.
      Podocyte foot process effacement as a diagnostic tool in focal segmental glomerulosclerosis.
      and severe FPE (≥80%) is a universal finding in patients with primary FSGS.
      • Hommos M.S.
      • De Vriese A.S.
      • Alexander M.P.
      • et al.
      The incidence of primary vs secondary focal segmental glomerulosclerosis: a clinicopathologic study.
      Our study is the first to evaluate the degree of FPE in individuals with genetic FSGS in adults. Most of the patients with genetic FSGS (42.1% [8/19]) had FPE ranging between 40% and 79%; however, 26.3% [5/19] had FPE of 80% or greater, similar to patients with primary FSGS (Table 3; Figure 2). Moreover, only 1 of the 5 individuals with diffuse FPE had NS that was resistant to treatment, and the other 4 patients did not have NS. This highlights the importance of correlating histologic findings with clinical information.
      Figure thumbnail gr2
      Figure 2Association between proteinuria and foot-process effacement in individuals with positive (shown in blue) and negative (shown in green) genetic results.
      Of the 5 individuals with FPE of 80% or greater, 3 had causative variants in NPHS2 and 2 had a COL4A3-related disease. The degree of FPE in individuals with genetic FSGS is likely related to the causative gene whether it affects the podocyte or the collagen in the GBM. Podocin is a hairpin-like protein at the slit diaphragm, and NPHS2 disruption often causes widespread FPE. Individuals with pathogenic variants in INF2, which encodes a member of the formin family of actin-regulating proteins, usually present with segmental FPE and preserved foot processes that focally appear irregular and jagged, often with unusually prominent longitudinal actin bundles.
      • Brown E.J.
      • Schlondorff J.S.
      • Becker D.J.
      • et al.
      Mutations in the formin gene INF2 cause focal segmental glomerulosclerosis.
      In our study, none of the affected individuals with variants in INF2 had FPE of 80% or greater. Furthermore, none of the individuals with a genetic diagnosis had proteinuria with protein excretion greater than 7 g per 24 hours. A summary of recommendations for which individuals with FSGS lesion should be considered for genetic testing is outlined in Box 1.
      BoxRecommendations for Genetic Testing in FSGS
      • Individuals with:
      • Primary FSGS resistant to immunosuppressant
      • Secondary FSGS due to a “known cause” with positive family history of kidney disease
      • Secondary FSGS without an identifiable underlying cause
      • Discordant clinical and histologic findings for whom the type of FSGS cannot be determined
      FSGS, focal segmental glomerulosclerosis.
      It is important to note that due to technical limitations and evolving knowledge about genes and variants, individuals with negative genetic testing results still may have an unrevealed genetic form of FSGS. For example, 1 individual in our cohort had a variant that is predicted to be deleterious in COL4A1 because it affects a glycine residue in a collagen triple helix. This gene has been associated with a syndrome that causes angiopathy, nephropathy, aneurysms, and muscle cramps, which this individual does not present. However, there is a report of an individual with glomerulopathy with the same type of COL4A1 variant.
      • Bullich G.
      • Domingo-Gallego A.
      • Vargas I.
      • et al.
      A kidney-disease gene panel allows a comprehensive genetic diagnosis of cystic and glomerular inherited kidney diseases.
      At this time, there is not enough evidence to determine this gene as causal for FSGS only, but as other reports become available, we might be able to reclassify this individual’s variant. Furthermore, the only individual without a definitive genetic cause in the undetermined group was a compound heterozygote for relevant VUS in DLC1. This gene has been associated with NS in 4 families in only a single publication. Therefore, more evidence is needed to determine that DLC1 is definitively an NS-related gene.
      • Ashraf S.
      • Kudo H.
      • Rao J.
      • et al.
      Mutations in six nephrosis genes delineate a pathogenic pathway amenable to treatment.
      Variants in noncoding regions are usually not covered by multigene panels and exome sequencing, and small exonic deletions and duplications are not reliable covered by the technology used in this study. Furthermore, variants in multiple genes might contribute to render podocytes susceptible to injury, adding another layer of complexity to the interpretation of genetic results.
      • Weber S.
      • Buscher A.K.
      • Hagmann H.
      • et al.
      Dealing with the incidental finding of secondary variants by the example of SRNS patients undergoing targeted next-generation sequencing.
      Thus, when a multigene panel is negative and a genetic form of FSGS is suspected based on clinical and histologic parameters, the clinician should pursue the genetic investigation including a more comprehensive test, such as a larger panel, exome or genome sequencing, and a reliable analysis for copy number variants.

      Conclusion

      This is the first study to comprehensively evaluate clinical presentation and identify the predictors of finding a genetic diagnosis in individuals with an FSGS lesion on the kidney biopsy specimen. Overall, individuals with a family history of kidney disease, unknown cause for FSGS, and discordant clinical and histologic findings are more likely to have a genetic form of FSGS. Moreover, careful evaluation of the kidney biopsy using EM is essential when caring for individuals with an FSGS lesion.

      Acknowledgments

      We would like to thank the patients and their families for participating in this study. Also, we would like to thank the Mayo Clinic Center for Individualized Medicine for their support. Drs Maio and Pinto e Vairo contributed equally to this work.

      Supplemental Online Material

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