Fibrinogen A Alpha-Chain Amyloidosis Associated With a Novel Variant in a Chinese Family

Amyloidosis is a group of diseases characterized by extracellular deposition of insoluble fibrils resulting from abnormal folding of proteins, leading to progressive tissue disruption and organ failure. Amyloid deposits in the kidney may arise from immunoglobulin light chains (AL amyloidosis), amyloid A, fibrinogen Aα-chain, lysozyme, gelsolin, apolipoprotein A-I, apolipoprotein A-II, apolipoprotein A-IV, apolipoprotein C2, apolipoprotein C3, transthyretin and leukocyte chemotactic factor 2. Fibrinogen Aα-chain (AFib) amyloidosis is an autosomal-dominant hereditary systemic amyloidosis caused by the deposition of amyloid fibrils comprising fibrinogen Aα-chain variants induced by mutations in fibrinogen Aα-chain gene (FGA). Patients with AFib amyloidosis present with renal disease and typically progress to end-stage renal disease. Here we report a fibrinogen Aα-chain amyloidosis family with a novel FGA mutation, which was identified by DNA sequencing and mass spectrometry (MS). We also review the literature and discuss the diagnosis and treatments of AFib amyloidosis. To our knowledge, this is the first report of fibrinogen Aα-chain amyloidosis with the p.Lys558Argfs∗10 variant in a Chinese family. A 33-year-old Chinese women presenting with proteinuria and bilateral lower limb edema for 1-month duration was admitted into hospital in November 2018. She had no complaints of fever, dyspnea, skin rashes, arthralgia, or gastrointestinal symptoms. Past medical history was negative. She denied tobacco use or alcoholism. Physical examination revealed blood pressure 110/70 mm Hg, temperature 37°C, and pulse rate 68 bpm, pitting edema of bilateral lower limb, but no rash, lymphadenopathy, organomegaly, or peripheral neuropathy. Laboratory evaluation showed nephritic range of proteinuria (2230–2870 mg/24 hours) and microscopic hematuria (+). Serum creatinine (0.75 mg/dl) and estimated glomerular filtration rate of (105 ml/min/1.73) m2 were within normal range. Serum albumin was 29g/l. Lipid panel screen showed hypercholesterolemia (total cholesterol 7.95 mmol/l, LDL-C 5.76 mmol/l) with plasma levels of total triglyceride, high-density lipoprotein cholesterol and Apolipoprotein A-I within normal range. Liver function, myocardial enzyme, serum complements were within normal range. Hepatitis, HIV, and syphilis tests were negative. Immunology tests (double-stranded DNA, antinuclear antibody, antineutrophil cytoplasmic antibody, antistreptolysin O, and rheumatoid factor) were normal. Computed tomography scan of the lung, ultrasonic cardiogram, and ultrasound of abdominal organs were normal. There was no evidence of a plasma cell disorder according to sensitive serum free light chain assay, serum protein electrophoresis, and immune fixation electrophoresis. Renal biopsy was performed and 3 strips of renal cortex containing 39 glomeruli were seen under light microscope. Extensive homogeneous and periodic acid-Schiff–positive stained material was present in glomerular mesangium and subendothelium. These deposits produced apple green birefringence when stained with Congo red and viewed under polarized light. Focal tubular atrophy and mild infiltration of lymphocytes and monocytes without interstitial fibrosis were seen. Arteriolar walls were unaffected. There was no amyloid within the tubules, interstitium, or vessels. Electron microscopy showed massive amorphous deposits with medium to low electron densities in the mesangium, and with a higher power, unbranched fibrils with a diameter of 8 to 12 nm were viewed (Figure 1). Routine immunofluorescence showed nonspecific adhesion of immunoglobulins, complements, and light chains (Supplementary Figure S1). Since routine kidney biopsy tests showed non–AL amyloidosis, we asked the patient’s family history in detail and found a complicated family history of kidney disease (Figure 2). Her mother (Figure 2 III-2) and cousin(Figure 2 IV-2) had a history of renal amyloidosis and was now receiving maintenance dialysis for uremia. Her grandmother (Figure 2 II-1) and great-grandfather (Figure 2 I-1) died of uremia years earlier. To identify amyloid typing, immunohistochemical analysis of the specimen was carried out and showed strong positive staining of fibrinogen in glomeruli (Figure 1f). Immunohistochemical analysis with antibodies against λ-light chain, κ-light chain, AA amyloid, lysozyme, transthyretin and gelsolin, apolipoprotein A-I, and LECT2 were negative. Genetic analysis of the patient and her parents furthermore revealed a novel frameshift mutation p.Lys558Argfs∗10 of FGA gene in our patient and her mother (Figure 3a), resulting from a deletion of an adenine nucleotide (c.1673delA). The new reading frame created by the deletion predicted the premature termination of the protein 10 amino acids downstream from the site of mutation. There was no mutation in other genes known to be associated with renal amyloidosis including APOA1, APOA2, and LYZ. MS-based proteomic analysis was run and confirmed the deposits in glomeruli were mutant fibrinogen alpha chain (Figure 3b,c).Figure 3Amyloid typing. (a) Genetic analysis of the proband and her parents showed a deletion of adenine nucleotide resulting a novel FGA mutation in our patient inherited from her mother. (b,c) Mass spectrometry–based proteomic analysis confirmed mutant fibrinogen alpha chain deposits.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Our patient was diagnosed hereditary fibrinogen Aα-chain amyloidosis. She received valsartan 80 mg/day and tripterygium glycosides 10 mg twice a day for treatment. Although the patient’s proteinuria decreased to minimum of 1.11 g/24 hours after 8 months, she developed nephrotic range of proteinuria after we stopped using tripterygium glycosides. Her serum creatinine gradually increased to 3.76 mg/dl at 28-month follow-up (Supplementary Figure S2). She remains on the active waiting list for combined liver and kidney transplantation. Fibrinogen is a 340-kDa soluble glycoprotein produced by hepatocyte which plays a crucial role in blood coagulation cascade.1Straub PW A study of fibrinogen production by human liver slices in vitro by an immunoprecipitin method.J Clin Invest. 1963; 42: 130-136Crossref PubMed Scopus (17) Google Scholar Hereditary fibrinogen Aα-chain amyloidosis, first described in 1993 by Benson,2Benson MD Liepnieks J Uemichi T et al.Hereditary renal amyloidosis associated with a mutant fibrinogen alpha-chain.Nat Genet. 1993; 3: 252-255Crossref PubMed Scopus (187) Google Scholar is a rare autosomal-dominant inherited disorder resulting from a mutation of FGA gene. The sequences and main clinical manifestations of 16 FGA mutations identified to date are summarized in Table 1. Our case is the first report of p.Lys558Argfs∗10 variant in a Chinese family.Table 1Gene mutations and clinical features of fibrinogen Aα-chain amyloidosisNumberProtein variantSequence variant (mRNA)Codon changeClinical featuresEthnic groupDiscovery timeReference1p.Arg573Leuc.1718G>TCGT>CTTRenal failurePeruvian, African American19932Benson MD Liepnieks J Uemichi T et al.Hereditary renal amyloidosis associated with a mutant fibrinogen alpha-chain.Nat Genet. 1993; 3: 252-255Crossref PubMed Scopus (187) Google Scholar2p.Glu545Valc.1634A>TGAG>GTGRenal failureNorthern European19943Uemichi T Liepnieks JJ Benson MD Hereditary renal amyloidosis with a novel variant fibrinogen.J Clin Invest. 1994; 93: 731-736Crossref PubMed Scopus (85) Google Scholar3p.Glu543Glufs∗25c.1629delGFrame shifting mutationRenal failureAmerican19964Uemichi T Liepnieks JJ Yamada T et al.A frame shift mutation in the fibrinogen A alpha chain gene in a kindred with renal amyloidosis.Blood. 1996; 87: 4197-4203Crossref PubMed Google Scholar4p.Val541Alafs∗27c.1622delTFrame shifting mutationRenal failureFrench19975Hamidi L Liepnieks JJ Uemichi T et al.Renal amyloidosis with a frame shift mutation in fibrinogen A alpha-chain gene producing a novel amyloid protein.Blood. 1997; 90: 4799-4805PubMed Google Scholar5p.Met536_Phe540delinsGlnSerfs∗28c.1606_1620 delATGTTAGGAGAGTTTinsCAFrame shifting mutationRenal failureKorean20056Kang HG Bybee A Ha IS et al.Hereditary amyloidosis in early childhood associated with a novel insertion-deletion (indel) in the fibrinogen Aalpha chain gene.Kidney In. 2004; 68: 1994-1998Google Scholar6p.Thr544Thrfs∗24c.1632delTFrame shifting mutationRenal failureChinese20097Gillmore JD Lachmann HJ Rowczenio D et al.Diagnosis, pathogenesis, treatment, and prognosis of hereditary fibrinogen A alpha-chain amyloidosis.J Am Soc Nephrol. 2009; 20: 444-451Crossref PubMed Scopus (107) Google Scholar7p.Thr557Lysc.1670C>AACA>AAARenal failureChinese20097Gillmore JD Lachmann HJ Rowczenio D et al.Diagnosis, pathogenesis, treatment, and prognosis of hereditary fibrinogen A alpha-chain amyloidosis.J Am Soc Nephrol. 2009; 20: 444-451Crossref PubMed Scopus (107) Google Scholar8p.Glu559Valc.1676A>TGAA>GTARenal failureGerman20097Gillmore JD Lachmann HJ Rowczenio D et al.Diagnosis, pathogenesis, treatment, and prognosis of hereditary fibrinogen A alpha-chain amyloidosis.J Am Soc Nephrol. 2009; 20: 444-451Crossref PubMed Scopus (107) Google Scholar9p.Pro571Hisc.1712C>ACCT>CATRenal failureAfro-Caribbean20097Gillmore JD Lachmann HJ Rowczenio D et al.Diagnosis, pathogenesis, treatment, and prognosis of hereditary fibrinogen A alpha-chain amyloidosis.J Am Soc Nephrol. 2009; 20: 444-451Crossref PubMed Scopus (107) Google Scholar10p.Ser523Argfs∗25c.1624_1627delAGTGFrame shifting mutationNephropathyJapanese20158Yazaki M Yoshinaga T Sekijima Y et al.The first pure form of Ostertag-type amyloidosis in Japan: a sporadic case of hereditary fibrinogen Aα-chain amyloidosis associated with a novel frameshift variant.Amyloid. 2015; 22: 142-144Crossref PubMed Scopus (10) Google Scholar11p.Phe540Leufs∗28c.1620delTFrame shifting mutationNephropathyFrench20179Garnier C Briki F Nedelec B et al.VLITL is a major cross-β-sheet signal for fibrinogen Aα-chain frameshift variants.Blood. 2017; 130: 2799-2807Crossref PubMed Scopus (9) Google Scholar12p.Gly538Glufs∗30c.1611delAFrame shifting mutationRenal failureFrench2017S113p.Phe540Serfs∗27c.1619_1622delTTGTFrame shifting mutationRenal failureArab2017S114p.Glu543Lysc.1627G>AGAG>AAGRenal failureCaucasian2017S115p.Glu545Lysc.1633G>AGAG>AAGRenal failureRussian2017S116p.Arg573Hisc.1718G>ACGT>CATnon-amyloidogenicBritish2017S117p.Gly574Phec.1720_1721delGGinsTTGGT>TTTRenal failureNorwegian2017S1 Open table in a new tab Table 2Teaching pointsFibrinogen A alpha-chain amyloidosis is rare in Asian families. The main pathologic findings include massive amounts of amyloid deposition in the glomeruli without interstitium, vessels or medulla involved, negative routine immunofluorescence and 8- to12-nm unbranched fibrils under electron microscopy.Clinical awareness and suspicion of hereditary amyloidosis corroborated by laser capture microdissection–mass spectrometry and genetic analysis is valuable to avoid misdiagnosis and imperative for correct management and prognosis. Open table in a new tab Diagnosis of AFib amyloidosis is based on the occurrence of proteinuria, positive family history, and identification of amyloid deposits in affected tissues. Unlike AL amyloidosis and lysozyme amyloidosis, interstitium and vessels are usually free of amyloid accumulation in AFib amyloidosis. Immunofluorescence studies are negative. However, nonspecific smudgy glomerular staining of immunoglobulins can be present because amyloid tends to be made up of sticky proteins, just like our case. In such cases, immunohistochemical staining, MS, and genetic sequencing should be used to confirm amyloid typing. Laser capture microdissection of affected areas followed by tandem MS has become a useful technique for typing of amyloidosis in recent years. However, a major limitation of MS-based proteomics is its dependence on a well-curated database. Previously unreported variants of fibril proteins will not be identified. Some reports described that identification of the mutated fibrinogen alpha chain by MS is more difficult in the case of frameshift mutations,S2,S3 and a score-based algorithm has been designed for diagnosis of AFib from MS data that utilizes knowledge based on large renal amyloidosis data sets. In our case, laser capture microdissection-MS showed predominantly fibrinogen Aα chain in glomeruli, composed of 28 unique peptides including 3 mutant-specific peptides, consistent with the p.Lys558Argfs∗10 mutation observed in gene analysis. Therefore, sequencing of the FGA gene following the determination of AFib amyloidosis and then confirmation of deposition of the sequence by MS is the best way to determine the theoretical sequence and then confirm expression (Table 2). Patients with AFib amyloidosis often experience rapid deterioration to end-stage renal disease and begin maintenance dialysis within 5 years.7Gillmore JD Lachmann HJ Rowczenio D et al.Diagnosis, pathogenesis, treatment, and prognosis of hereditary fibrinogen A alpha-chain amyloidosis.J Am Soc Nephrol. 2009; 20: 444-451Crossref PubMed Scopus (107) Google Scholar There are currently no effective treatments that can lead to resolution of amyloid deposits. For patients without cardiovascular involvement, combined kidney and liver transplant has been performed and demonstrated to retard disease progression.S4 In our case, during the waiting time for combined liver and kidney transplantation, supportive therapy including angiotensin receptor blocker and traditional Chinese medicine showed temporary reduction of proteinuria but uncontrolled kidney function loss. In summary, we report a fibrinogen Aα-chain amyloidosis Chinese family presenting with kidney failure, associated with a novel FGA mutation variant, which was identified by MS combined with DNA sequencing. All the authors declared no competing interests. The authors declare that they have obtained consent from the patients discussed in the report. This work was supported by the National Natural Science Foundation of China (No. 81803880); Major Projects of Scientific Research and Innovation Plan of Shanghai Education Commission (No. 2017-01-07-00-07-E00009); Shanghai Medical Center of Kidney (No. 2017ZZ01015); and Shanghai Key Laboratory of Kidney and Blood Purification (No. 14DZ2260200). Download .pdf (.91 MB) Help with pdf files Supplementary File (PDF) Supplementary material on immunostaining Supplementary material on follow-up Supplementary References