BRIEF COMMUNICATION www.jasn.org Phospholipase A2 Receptor–Related Membranous Nephropathy and Mannan-Binding Lectin Deficiency †‡ | Stéphane Bally,* Hanna Debiec, Denise Ponard,§ Frédérique Dijoud, John Rendu,¶ †‡ Julien Fauré,¶ Pierre Ronco, ** and Chantal Dumestre-Perard§ *Service de Néphrologie Dialyse, Centre Hospitalier Métropole Savoie, Chambery, France; †Sorbonne Universités, Universitè Pierre and Marie Curie University, Paris 06, Paris, France; ‡Institut National de la Santé et de la Recherche Médicale, Unité Mixte de Recherche UMR_S1155, Paris, France; §Laboratoire d’Immunologie, Pôle de Biologie, Centre | Hospitalier Universitalier de Grenoble, Grenoble, France; Centre de Pathologie Est, Hôpitaux de Lyon, Bron, France; BRIEF COMMUNICATION ¶Département de Biochimie Pharmacologie, Biochimie et Génétique Moléculaire, Université de Grenoble Alpes Grenoble, France; and **Assistance Publique-Hôpitaux de Paris, Néphrologie et Dialyses, Hôpital Tenon, Paris, France ABSTRACT Most patients with idiopathic membranous nephropathy (IMN) have IgG4 autoan- pathway. The concomitant presence of tibodies against phospholipase A2 receptor (PLA2R). C3 and C5b-9 are found in IgG1, especially in early immune deposits, immune deposits of IMN kidney biopsy specimens, but the pathway of complement could possibly be responsible for classic activation in IMN remains elusive. We report the case of a patient who developed pathway activation.9 However, most pa- IMN with intense staining for PLA2R, IgG4, C3, C5b-9, factor B, and properdin and tients with IMN have very weak or unde- very weak staining for C1q, C4d, and IgG1. Measurement of mannan binding lectin tectable C1q deposits. These observations (MBL) antigenic level and activity revealed MBL deficiency. Genotyping revealed a suggest that the alternative and/or lectin heterozygous (A/C) polymorphism in codon 57 of MBL2 exon 1 associated with pathways might be predominantly in- homozygous and heterozygous variations in the promoter region at 2550 (L/L) volved in complement activation and for- and 2221 (X/Y), respectively, suggesting that the patient harbored the LXA/LYC mation of the C5b-9 complex. The first haplotypes linked to MBL deficiency. Genetic sequencing in 77 consecutive patients case series of MBL deficiency reported be- with IMN identified four patients with MBL2 promoter and coding region variations low emphasizes the role of alternative associated with MBL deficiency and the same complement pattern in immune de- pathway complement activation. posits as the index patient. In contrast, patients with wild-type MBL2 had immune A 25-year-old man was referred be- deposits with intense Cd4 staining. Thus, IMN can develop in patients with complete cause of the fortuitous discovery of pro- MBL deficiency, with complement activated mainly by the alternative pathway, teinuria (2.5 g/d) with normal urinary whereas the lectin pathway is also activated in those with wild-type MBL2. sediment. Serum albumin was 3.8 g/dl; serum cholesterol was 5.78 mmol/L J Am Soc Nephrol 27: 3539–3544, 2016. doi: 10.1681/ASN.2015101155 Received October 21, 2015. Accepted March 16, Membranous nephropathy (MN) is one of podocyte antigens, such as the M-type re- 2016. the most common causes of the nephrotic ceptor for secretory phospholipase A2 2 S.B., H.D., P.R., and C.D.-P. contributed equally to syndrome in adults who are not diabetic, (PLA2R) and in rare cases, the thrombo- this work. accounting for up to one third of biopsy spondin type 1 domain 7A.3 Complement Published online ahead of print. Publication date diagnoses. It can be secondary to various plays an important role, even if mecha- available at www.jasn.org. conditions; however, in 70%–80% of pa- nisms of activation have not been clarified tients, MN is a primary (idiopathic) dis- yet. Experimental models have shown that Correspondence: Dr. Stéphane Bally, Service de Néphrologie Dialyse, Centre Hospitalier Métropole ease, with a peak incidence during the C5b-9, the membrane attack complex of Savoie, Place Lucien Biset, 73000 Chambery, France, fourth and fifth decades of life and a complement, is the major mediator of or Dr. Hanna Debiec, Unité Institut National de la men-to-women ratio of 2–3:1.1 proteinuria.4,5 IMN is characterized by Santé et de la Recherche Médicale, Unité Mixte de Recherche UMR_S1155, Hôpital Tenon, 4 rue de la Recent advances have shown that prevailing IgG4 deposits with usually low Chine, 75020 Paris, France. Email: stephane.bally@ idiopathic membranous nephropathy amounts of IgG1.6–8 IgG4 is unique ch-metropole-savoie.fr or [email protected] – fi (IMN) is a kidney speci c autoimmune among IgG subclasses, because it does Copyright © 2016 by the American Society of disease induced by antibodies specificfor not activate the classic complement Nephrology J Am Soc Nephrol 27: 3539–3544, 2016 ISSN : 1046-6673/2712-3539 3539 BRIEF COMMUNICATION www.jasn.org (0.223 g/dl). Renal function was normal, with serum creatinine of 75 mmol/L (0.85 mg/dl; Chronic Kidney Disease Epidemi- ology Collaboration .120 ml/min). Renal ultrasound imaging was unremarkable. Clinical examination was normal, and the BP was 120/60 mmHg. The patient did not report any personal or family his- tory of renal or autoimmune disease, toxic exposure, medication intake, and consan- guinity. However, he mentioned very frequent and severe respiratory tract infec- tions during childhood, which were also reported by his mother. A kidney biopsy was performed. Light microscopy showed 24 permeable glomer- uli, with thickening of the glomerular basement membrane and spikes delimit- ing subepithelial deposits. There was no mesangial hypercellularity, extracapillary proliferation, or tubulointerstitial abnor- mality. Glomeruli showed finely granular deposits of IgG (Supplemental Figure 1). Immunofluorescence examination re- vealed intense staining for PLA2R and IgG4, whereas IgG1 was weak (Figure 1). There was no staining for IgG2 or IgG3. Complement components C3 and C5b-9 were present within the subepithelial de- posit, whereas staining for C1q and C4d was graded as very weak (Figure 1). In addition, factor B and properdin were identified within the subepithelial de- posits. Anti-PLA2R antibodies were not detected by ELISA (Euroimmun AG). Search for a secondary cause of MN remained negative throughout evolu- tion. Because of the past history of severe respiratory tract infections, antigenicity and activity of complement components were investigated. Complement hemo- lytic activity of 50% was normal (83%; normal range =82%–126%). C3 compo- nent was mildly decreased at 715 mg/L Figure 1. Detection of PLA2R antigen and characterization of immune deposits in kidney biopsy – (normal range =880 1650 mg/L). Factor specimen of the index patient. (A) Immunofluorescence shows the presence of PLA2R in sub- B and C4 component were normal at 246 epithelial deposits along glomerular capillary loops. Immunostaining for IgG subclasses shows mg/L (normal range =216–504 mg/L) (B) a weak fluorescence for IgG1 and (C) a bright fluorescence for IgG4. Complement compo- and 160 mg/L (normal range =100–400 nents, including (D) C3 and (E) C5b-9, are present along glomerular capillary loops. Note the very mg/L), respectively. Alternative path- weak and segmental staining for (F) C1q and (G) C4d and pseudolinear deposition of (H) factor B way activity was high at 225% (normal and (I) properdin along the capillary wall. The images shown in A and G–Iarefromparaffin range =84%–150%). sections, and those shown in B–F are from cryostat sections. Original magnification, 3400. J Exploration of the lectin pathway illustrates the three different pathways of complement activation. The very weak staining for fi revealed a severe quantitative and func- IgG1, C1q, and C4d and the absence of IgG2 and IgG3 deposits argue against signi cant ac- tivation of the classic and MBL pathways. The staining for factor B and properdin favors activation tional mannan binding lectin (MBL) of the alternative pathway. AP, alternative pathway; MASP1, MBL–associated serine protease 1. deficiency. MBL antigen level assayed 3540 Journal of the American Society of Nephrology J Am Soc Nephrol 27: 3539–3544, 2016 www.jasn.org BRIEF COMMUNICATION by ELISA10 was low (12 mg/L; normal range =30–3000 mg/L). MBL activity measured by C4 cleavage through the MBL pathway11 was undetectable at ,10% (normal range =35%–135%). MBL deficiency was also found in the patient’sparents.Genotypingofthe polymorphic sites 52, 54, and 57 in the coding sequence of MBL2,thegene coding for MBL, revealed a 57 (A/C) het- erozygous polymorphism in codon 57 of exon 1. This structural variant was asso- ciated with homozygous and heterozy- gous variations in the promoter region at 2550 (L/L) and 2221 (X/Y), respec- tively12 (Figure 2). No mutation was ob- served in codons 52 and 54. On the basis of the different haplotypes described by Garred et al.,13 our patient likely harbors the LXA/LYC haplotypes, which have been associated with MBL deficiency. The father has the same genotype as his son. The mother bears a 54 (A/B) het- erozygous polymorphism in exon 1 (no mutation was observed for 52 and 57 co- dons) and homozygous and heterozy- gous variations in the promoter region at 2550 (L/L) and 2221 (X/Y), respec- tively, defining the LXA/LYB haplotype. These results led us to sequence the MBL2 gene in 77 consecutive patients with IMN. We found two patients harboring the LYC/LYC haplotype and the LYB/LYB hap- lotype, respectively, and two patients with compound heterozygous mutations reveal- ing the already–described haplotypes LYB/ LYC and HYD/LYB (Figure 2). These ge- notypes have been shown to be associ- ated with MBL deficiency.13 These four patients had typical nephrotic PLA2R– related IMN with predominance of IgG4 and very weak or absent IgG1 and C1q in Figure 2. MBL genetic study. (A) Schematic representation of the major MBL2 isoform and the immune deposits. As observed in genetic polymorphism.