Pathologic-Genomic Correlation Identified a Novel Variant in FN1 and Established the Diagnosis of Recurrent Fibronectin Glomerulopathy in the Kidney Allograft

Abstract

Fibronectin glomerulopathy is a rare inherited kidney disease, characterized by abnormal accumulation of fibronectin in the glomeruli. We report an exceptional case of recurrent fibronectin glomerulopathy first diagnosed in the kidney allograft. The presence of IgA staining in the native kidney biopsy and the reported family history of IgA nephropathy had led to initial pre-transplant diagnosis of IgA nephropathy.

Four and a half years post-transplantation, the patient presented with kidney insufficiency and minimal proteinuria. The allograft biopsy revealed glomerular deposits with very weak staining for immunoglobulins and vague filamentous material. Immunostaining for fibronectin was positive and genetic studies showed a variant of unknown significance in the fibronectin-1 gene. Proteomic analyses of the glomeruli in the native kidney biopsy demonstrated large amount of fibronectin with abundant accumulation of the peptide synthesized by the detected variant. These findings established the diagnosis of recurrent fibronectin glomerulopathy secondary to a novel variant in the fibronectin-1 gene. This report sheds light on recurrent fibronectin glomerulopathy post-transplantation, highlights the diagnostic pitfalls of the disease, and underscores the importance of pathologic-genomic correlation to establish the correct diagnosis.

Introduction

Fibronectin glomerulopathy (FNG) is a rare, autosomal-dominant, and slowly progressive kidney disease, caused by glomerular deposition of plasma-derived fibronectin (FN)¹. We present a case of FNG that was first diagnosed post-transplantation from a patient with apparent family history of IgA nephropathy (IgAN), which complicated the pathologic diagnosis in the native kidney.

Case report

A twenty-three-year-old Asian-American female presented with nephrotic range proteinuria and negative serologic work-up. The patient had a history of hypertension and proteinuria for several years and a family history of proteinuria in the mother and sister, with the sister’s biopsy reportedly showed IgAN. Laboratory work-up revealed serum creatinine (sCr): 1.1 mg/dL and urine protein-to-creatinine: 5.3 g/g. The native kidney biopsy demonstrated membranoproliferative pattern of injury, immunofluorescence staining including IgA, and massive mesangial and subendothelial electron dense deposits (Figure 1). Given the family history of IgAN, these biopsy findings were interpreted as IgAN, which was treated with Losartan-Hydrochlorothiazide and alternate-day corticosteroids. Due to subsequent increase of sCr to 2.5 mg/dL, a second biopsy was performed 4 years later and also revealed membranoproliferative features with IgA staining. The patient was enrolled in the CKD genetic study at Columbia University and whole exome sequencing was interpreted as non-diagnostic. Kidney function continued to deteriorate and the patient commenced dialysis after 1.5 years.

Figure 1:

Histologic findings in the first native kidney biopsy (A) Large glomerulus with lobulated appearance and prominent membranoproliferative features (Periodic Acid-Schiff, original magnification x200). (B) A glomerulus showing mesangial and subendothelial expansion by silver-negative material (Jones methenamine silver, original magnification, ×400). (C) A glomerulus revealing smudgy IgA staining (1–2+) with somewhat segmental distribution (immunofluorescence, original magnification x200). Other immunofluorescence stains, which are similar in nature, included IgG (1–2+), IgM (2+), kappa (2+), and lambda (1+). Of note, the second native kidney biopsy also showed similar pattern of immunofluorescence staining for IgA (2+), IgG (2+), IgM (1+), kappa (2+), and lambda (1+). (D) Ultrastructural examination demonstrating mesangial and subendothelial expansion by electron dense deposits (Electron microscopy, original magnification x5000). Given the high density of the deposits, it was more challenging to find foci showing the vague filamentous appearance of the deposits (inset, Electron microscopy, original magnification x40,000).

At age 31, the patient received a four-antigen mismatched kidney allograft from a living-unrelated donor. She was induced by thymoglobulin, and maintained on tacrolimus and mycophenolate mofetil. Four years post-transplantation, the patient presented with increase of sCr from 0.9 mg/dL to 1.6 mg/dL over a six-month period. Additional laboratory work-up revealed urine protein-to-creatinine: 0.2 g/g, serum albumin: 4.8 g/dL, hemoglobin A1C: 6.1%, and bland urine sediment.

Kidney allograft biopsy

Sampling for light microscopy contained 19 glomeruli, of which 4 were globally sclerotic and 2 were segmentally sclerotic. The remaining glomeruli showed mild to moderate mesangial hypercellularity and expansion by Periodic acid-Schiff (PAS)-positive and silver-pale material, and rare double contours (Figure 2A-B). The biopsy also demonstrated moderate tubulointerstitial scarring without glomerulitis or peritubular capillaritis. Immunofluorescence showed weak (trace to 1+) smudgy staining for IgG, kappa, and lambda, negative IgA staining, and negative C4d staining in peritubular capillaries. Congo red staining and immunofluorescence staining of pronase-digested paraffin-embedded tissue were all negative. Ultrastructural evaluation revealed granular to vague filamentous material in the mesangium (Figure 2C). These findings prompted us to stain for plasma-derived FN, which showed intense glomerular staining (Figure 2D), supporting a diagnosis of FNG.

Figure 2:

Histologic findings in the allograft biopsy. (A) A large glomerulus with lobulated expanded mesangium by Periodic Acid-Schiff positive material that is associated with mild mesangial hypercellularity and rare glomerular capillary double contours (arrowhead) (Periodic Acid-Schiff, original magnification x400). (B) Same glomerulus showing mesangial expansion by silver-negative material (Jones methenamine silver, original magnification ×400). (C) Ultrastructural examination demonstrating mesangial extension by extracellular material (Electron microscopy, original magnification x5000) that have vague filamentous structures on high power view (inset, Electron microscopy, original magnification x60,000). (D) A glomerulus showing global staining for plasma-derived FN (polyclonal rabbit, Agilent Technologies, Santa Clara, CA) in the mesangium as well as in the glomerular capillaries (FN immunoperoxidase stain, original magnification x400).

To explore the possibility of recurrent disease, the first native biopsy was stained for FN, which revealed positive glomerular staining (Figure 3A). Additionally, the tissue block was sent for mass spectrometry-based analysis after glomerular dissection², which demonstrated abundant spectra corresponding to FN and fibulin-1, consistent with FNG³ (Figure 3B).

Figure 3:

Pathologic confirmation of FNG in the first native kidney biopsy (A) A glomerulus showing global staining for plasma-derived FN (polyclonal rabbit, Agilent Technologies, Santa Clara, CA) in the mesangium as well as in the glomerular capillaries (FN immunoperoxidase stain, original magnification x400). Of note, the FN staining had lower intensity than the allograft biopsy, probably reflecting loss of antigenicity given that the native biopsy was performed 12 years earlier. (B) Liquid chromatography tandem mass spectrometry (LC MS/MS) was performed on peptides extracted from micro-dissected glomeruli of the paraffin-embedded kidney biopsy tissue. Proteins highlighted with blue stars are validated biomarkers of glomerular tissue. Numbers in green boxes represent the total number of unique spectra matching to the protein in corresponding sample. The analysis showed abundant spectra corresponding to FN and fibulin-1 consistent with FNG. (C) A representative MS/MS spectrum matching to the peptide containing the mutant amino acid p.Trp1017Arg is shown. Red asterisk represents the mutated amino acid location in the sequence. (D) The spectra counts of mutant and wild type peptide.

Consequently, the initial research exome data were re-analyzed, with a focus on the FN1 gene and identified a missense variant (c.3049T>C; p.Trp1017Arg), which was absent from variant databases and the gnomAD dataset. In-silico prediction tools provided strong support for pathogenicity (REVEL=0.924). The variant was subsequently confirmed by genetic testing in a clinical laboratory using the Natera Renasight Kidney gene panel. Following the ACMG interpretation guidelines, the clinical laboratory classified this finding as a variant of uncertain significance (VUS). The patient’s family was not available for cascade genetic testing.

To provide evidence for the potential pathogenic role of this variant, we performed proteomic assessment of the dissected glomeruli, searching for the mutated form of FN⁴. We detected p.Trp1017Arg peptide, which was more abundant than its wild type counterpart (Figure 3C-D), supporting likely pathogenic role of this peptide.

Because of the classic slow progression of FNG, we have continued her tacrolimus and mycophenolate mofetil and the patient is considering therapy with RAAS blockade or SGLT2 inhibition. Nine months after biopsy, she had sCr: 1.4 mg/dl and urine protein-to-creatinine: 0.2 g/g.

Discussion:

FNG is a rare slowly progressive kidney disease⁵. Patients usually present between 2^nd and 4^th decade of life, with nephrotic range proteinuria, negative serologic work-up, and, less often, microhematuria and hypertension^6–8. Unfortunately, FNG lacks specific therapy and tends to progress to kidney failure several years after diagnosis⁸. Our patient had prolonged history of proteinuria that progressed to nephrotic range at age 23 and native kidney failure 5.5 years later.

FNG is an autosomal-dominant disorder¹, which is associated with variants in the FN1 gene in up to half of affected patients⁹. Given the rarity of the disease, only a few pathogenic variants have been identified so far^9,10. These variants can cause structural defects in plasma-derived FN, which reduce its solubility⁹ and facilitate its accumulation in the glomeruli^6,11. Although the missense variant detected in our patient (p.Trp1017Arg) has not been reported in the literature, the additional demonstration of abundant amount of its synthesized peptide within the glomerular deposits was consistent with a likely pathogenic role of this variant.

Regrettably in our case, we could not perform cascade genetic testing and segregation analyses on the patient’s family members. However, it is worth noting that a different previously reported amino acid change at the same site (p.Trp1017Cys) segregated with FNG in one family, and was considered pathogenic for the disease¹².

The glomeruli in FNG typically show glomerular expansion by PAS-positive material that have granular and filamentous appearance by electron microscopy⁶. FNG typically lacks immunoglobulins staining by immunofluorescence, although some trapping, which frequently tends to be smudgy and segmental in nature, may be observed⁸. All these findings were observed in our patient. However, the substantial immunoglobulin trapping, the high density of the deposits on ultrastructural examination, and the reported family history of IgAN, led to misinterpretation of the native biopsy findings as IgAN.

Only a few cases of post-transplant FNG has been reported in the English medical literature^1,13, rarely with detailed clinicopathologic description^9,13–15 (Table 1). These limited data suggest that recurrent FNG often occurs after the first year of transplantation, presents with mild proteinuria, and is slowly progressive.

Table 1:

Detailed published cases of post-transplant fibronectin glomerulopathy

Cases	Case 1	Case 2	Case 3	Case 4	Case 5
Authors	Gemperle et al. (1996)	Castelletti et al. (2008)	Otsuka et al. (2012)	Wei et al. (2022)	Batal et al. (current report)
Age	47	41	52	66	35
Gender	Male	NA	Female	Female	Female
Race	NA	NA	Asian	NA	Asian
Allograft source	Deceased	NA	Living-unrelated	Deceased	Living-unrelated
Time post-transplant	23 months	3 years	1st year	27 months	4 years
sCr at biopsy (mg/dl)	1.6	1.2	1.5	1.4	1.6
Proteinuria	2.6 g/day	0.2 g/day	0.4 g/day	1+	0.2 g/g
Treatment	NA	NA	NA	Losartan potassium, β receptor blocker	No change
Outcome	Died with functioning graft (post-transplant time NA)	NA	NA	Functioning (~3 years after transplant)	Functioning (5.25 years after transplant)

Abbreviations: NA, not available; sCr, serum creatinine

In conclusion, we reported an exceptional case of recurrent FNG in the kidney allograft. Greater familiarity with the defining histologic features and pathologic pitfalls and close correlation of pathology and genomic findings are needed to ensure correct classification of this rare disease.

Abbreviations:

FN

Fibronectin

FNG

Fibronectin Glomerulopathy

IgAN

IgA nephropathy

sCr

serum creatinine

VUS

Variant of uncertain significance