Voskarides Konstantinos, Stefanou Charalambos, Pieri Myrtani, Demosthenous Panayiota, Felekkis Kyriakos, Arsali Maria, Athanasiou Yiannis, Xydakis Dimitris, Stylianou Kostas, Daphnis Eugenios, Goulielmos Giorgos, Loizou Petros, Savige Judith, Höhne Martin, Völker Linus A, Benzing Thomas, Maxwell Patrick H, Gale Daniel P, Gorski Mathias, Böger Carsten, Kollerits Barbara, Kronenberg Florian, Paulweber Bernhard, Zavros Michalis, Pierides Alkis, Deltas Constantinos
Department of Biological Sciences and Molecular Medicine Research Center, University of Cyprus, Nicosia, Cyprus.
Department of Nephrology, Limassol General Hospital, Limassol, Cyprus.
PLoS One. 2017 Mar 23;12(3):e0174274. doi: 10.1371/journal.pone.0174274. eCollection 2017.
Recent data emphasize that thin basement membrane nephropathy (TBMN) should not be viewed as a form of benign familial hematuria since chronic renal failure (CRF) and even end-stage renal disease (ESRD), is a possible development for a subset of patients on long-term follow-up, through the onset of focal and segmental glomerulosclerosis (FSGS). We hypothesize that genetic modifiers may explain this variability of symptoms.
We looked in silico for potentially deleterious functional SNPs, using very strict criteria, in all the genes significantly expressed in the slit diaphragm (SD). Two variants were genotyped in a cohort of well-studied adult TBMN patients from 19 Greek-Cypriot families, with a homogeneous genetic background. Patients were categorized as "Severe" or "Mild", based on the presence or not of proteinuria, CRF and ESRD. A larger pooled cohort (HEMATURIA) of 524 patients, including IgA nephropathy patients, was used for verification. Additionally, three large general population cohorts [Framingham Heart Study (FHS), KORAF4 and SAPHIR] were used to investigate if the NEPH3-V353M variant has any renal effect in the general population.
Genotyping for two high-scored variants in 103 TBMN adult patients with founder mutations who were classified as mildly or severely affected, pointed to an association with variant NEPH3-V353M (filtrin). This promising result prompted testing in the larger pooled cohort (HEMATURIA), indicating an association of the 353M variant with disease severity under the dominant model (p = 3.0x10-3, OR = 6.64 adjusting for gender/age; allelic association: p = 4.2x10-3 adjusting for patients' kinships). Subsequently, genotyping 6,531 subjects of the Framingham Heart Study (FHS) revealed an association of the homozygous 353M/M genotype with microalbuminuria (p = 1.0x10-3). Two further general population cohorts, KORAF4 and SAPHIR confirmed the association, and a meta-analysis of all three cohorts (11,258 individuals) was highly significant (p = 1.3x10-5, OR = 7.46). Functional studies showed that Neph3 homodimerization and Neph3-Nephrin heterodimerization are disturbed by variant 353M. Additionally, 353M was associated with differential activation of the unfolded protein response pathway, when overexpressed in stressed cultured undifferentiated podocyte cells, thus attesting to its functional significance. Genetics and functional studies support a "rare variant-strong effect" role for NEPH3-V353M, by exerting a negative modifier effect on primary glomerular hematuria. Additionally, genetics studies provide evidence for a role in predisposing homozygous subjects of the general population to micro-albuminuria.
近期数据强调，薄基底膜肾病（TBMN）不应被视为良性家族性血尿的一种形式，因为在长期随访中，一部分患者可能会发展为慢性肾衰竭（CRF）甚至终末期肾病（ESRD），其发病机制是局灶节段性肾小球硬化（FSGS）。我们推测基因修饰因子可能解释了这种症状的变异性。
我们使用非常严格的标准，在硅芯片上对所有在裂孔隔膜（SD）中显著表达的基因中潜在有害的功能性单核苷酸多态性（SNP）进行筛选。在来自19个具有相同遗传背景的希腊塞浦路斯家族的成年TBMN患者队列中，对两个变异进行基因分型。根据是否存在蛋白尿、CRF和ESRD，将患者分为“重度”或“轻度”。一个更大的包含524名患者（包括IgA肾病患者）的汇总队列（HEMATURIA）用于验证。此外，三个大型普通人群队列[弗雷明汉心脏研究（FHS）、KORAF4和SAPHIR]用于研究NEPH3-V353M变异在普通人群中是否有任何肾脏影响。
对103名具有奠基者突变且被分类为轻度或重度受影响的成年TBMN患者进行两个高分变异的基因分型，结果表明与变异NEPH3-V353M（滤连蛋白）有关联。这一有前景的结果促使在更大的汇总队列（HEMATURIA）中进行检测，结果表明在显性模型下，353M变异与疾病严重程度相关（p = 3.0x10-3，调整性别/年龄后的OR = 6.64；等位基因关联：调整患者亲属关系后的p = 4.2x10-3）。随后，对弗雷明汉心脏研究（FHS）的6531名受试者进行基因分型，结果显示纯合353M/M基因型与微量白蛋白尿有关联（p = 1.0x10-3）。另外两个普通人群队列KORAF4和SAPHIR证实了这种关联，对所有三个队列（11258名个体）的荟萃分析具有高度显著性（p = 1.3x10-5，OR = 7.46）。功能研究表明，353M变异会干扰Neph3同二聚化以及Neph3-Nephrin异二聚化。此外，当在应激培养的未分化足细胞中过表达时，353M与未折叠蛋白反应途径的差异激活有关，从而证明了其功能意义。遗传学和功能研究支持NEPH3-V353M具有“罕见变异-强效应”作用，通过对原发性肾小球血尿发挥负性修饰作用。此外，遗传学研究为普通人群中纯合子个体易患微量白蛋白尿提供了证据。
