Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, Minnesota.
Medical Genome Facility, Proteomics Core, Mayo Clinic, Rochester, Minnesota.
J Am Soc Nephrol. 2022 May;33(5):1033-1044. doi: 10.1681/ASN.2021111488. Epub 2022 Mar 23.
Membranous nephropathy (MN) is a common cause of proteinuria in patients receiving a hematopoietic stem cell transplant (HSCT). The target antigen in HSCT-associated MN is unknown.
We performed laser microdissection and tandem mass spectrometry (MS/MS) of glomeruli from 250 patients with PLA2R-negative MN to detect novel antigens in MN. This was followed by immunohistochemical (IHC)/immunofluorescence (IF) microscopy studies to localize the novel antigen. Western blot analyses using serum and IgG eluted from frozen biopsy specimen to detect binding of IgG to new 'antigen'.
MS/MS detected a novel protein, protocadherin FAT1 (FAT1), in nine patients with PLA2R-negative MN. In all nine patients, MN developed after allogeneic HSCT (Mayo Clinic discovery cohort). Next, we performed MS/MS in five patients known to have allogeneic HSCT-associated MN (Cedar Sinai validation cohort). FAT1 was detected in all five patients by MS/MS. The total spectral counts for FAT1 ranged from 8 to 39 (mean±SD, 20.9±10.1). All 14 patients were negative for known antigens of MN, including PLA2R, THSD7A, NELL1, PCDH7, NCAM1, SEMA3B, and HTRA1. Kidney biopsy specimens showed IgG (2 to 3+) with mild C3 (0 to 1+) along the GBM; IgG4 was the dominant IgG subclass. IHC after protease digestion and confocal IF confirmed granular FAT1 deposits along the GBM. Lastly, Western blot analyses detected anti-FAT1 IgG and IgG4 in the eluate obtained from pooled frozen kidney biopsy tissue and in the serum of those with FAT1-asssociated MN, but not from those with PLA2R-associated MN.
FAT1-associated MN appears to be a unique type of MN associated with HSCT. FAT1-associated MN represents a majority of MN associated with HSCT.
膜性肾病（MN）是接受造血干细胞移植（HSCT）的患者蛋白尿的常见原因。HSCT 相关 MN 的靶抗原尚不清楚。
我们对 250 例 PLA2R 阴性 MN 患者的肾小球进行激光微切割和串联质谱（MS/MS）检测，以检测 MN 中的新型抗原。随后进行免疫组织化学（IHC）/免疫荧光（IF）显微镜研究以定位新型抗原。使用血清和从冷冻活检标本洗脱的 IgG 进行 Western blot 分析，以检测 IgG 与新“抗原”的结合。
MS/MS 在 9 例 PLA2R 阴性 MN 患者中检测到一种新型蛋白原钙黏蛋白 FAT1（FAT1）。在所有 9 例患者中，MN 均在异基因 HSCT 后发生（Mayo 诊所发现队列）。接下来，我们对 5 例已知异基因 HSCT 相关 MN 的患者进行了 MS/MS（雪松西奈验证队列）。MS/MS 在所有 5 例患者中均检测到 FAT1。FAT1 的总光谱计数范围为 8 至 39（平均值±标准差，20.9±10.1）。所有 14 例患者均为 MN 的已知抗原（包括 PLA2R、THSD7A、NELL1、PCDH7、NCAM1、SEMA3B 和 HTRA1）阴性。肾活检标本显示 IgG（2 至 3+）伴肾小球基底膜（GBM）轻度 C3（0 至 1+）；IgG4 是主要的 IgG 亚类。蛋白酶消化后的 IHC 和共聚焦 IF 证实了 GBM 上颗粒状 FAT1 沉积物。最后，Western blot 分析在从合并的冷冻肾活检组织洗脱液和 FAT1 相关 MN 患者的血清中检测到抗-FAT1 IgG 和 IgG4，但在 PLA2R 相关 MN 患者的血清中未检测到。
FAT1 相关 MN 似乎是一种与 HSCT 相关的独特 MN 类型。FAT1 相关 MN 代表与 HSCT 相关的大多数 MN。
