DataSheet_1_An ex vivo test to investigate genetic factors conferring susceptibility to atypical haemolytic uremic syndrome.docx

IntroductionComprehensive genetic analysis is essential to clinical care of patients with atypical haemolytic uremic syndrome (aHUS) to reinforce diagnosis, and to guide treatment. However, the characterization of complement gene variants remains challenging owing to the complexity of functional studies with mutant proteins. This study was designed: 1) To identify a tool for rapid functional determination of complement gene variants; 2) To uncover inherited complement dysregulation in aHUS patients who do not carry identified gene variants.
MethodsTo address the above goals, we employed an ex-vivo assay of serum-induced C5b-9 formation on ADP-activated endothelial cells in 223 subjects from 60 aHUS pedigrees (66 patients and 157 unaffected relatives).
ResultsSera taken from all aHUS patients in remission induced more C5b-9 deposition than control sera, independently from the presence of complement gene abnormalities. To avoid the possible confounding effects of chronic complement dysregulation related to aHUS status, and considering the incomplete penetrance for all aHUS-associated genes, we used serum from unaffected relatives. In control studies, 92.7% of unaffected relatives with known pathogenic variants exhibited positive serum-induced C5b-9 formation test, documenting a high sensitivity of the assay to identify functional variants. The test was also specific, indeed it was negative in all non-carrier relatives and in relatives with variants non-segregating with aHUS. All but one variants in aHUS-associated genes predicted in-silico as likely pathogenic or of uncertain significance (VUS) or likely benign resulted as pathogenic in the C5b-9 assay. At variance, variants in putative candidate genes did not exhibit a functional effect, with the exception of a CFHR5 variant. The C5b-9 assay in relatives was helpful in defining the relative functional effect of rare variants in 6 pedigrees in which the proband carried more than one genetic abnormality. Finally, for 12 patients without identified rare variants, the C5b-9 test in parents unmasked a genetic liability inherited from an unaffected parent.
DiscussionIn conclusion, the serum-induced C5b-9 formation test in unaffected relatives of aHUS patients may be a tool for rapid functional evaluation of rare complement gene variants. When combined with exome sequencing the assay might be of help in variant selection, to identify new aHUS-associated genetic factors.

引言
全面的基因分析对于非典型溶血性尿毒症综合征（atypical haemolytic uremic syndrome, aHUS）患者的临床诊疗至关重要，可辅助明确诊断并指导治疗方案制定。然而，由于突变蛋白功能研究的复杂性，补体基因变异的功能表征仍颇具挑战。本研究旨在达成两大目标：1）开发一种可快速对补体基因变异进行功能鉴定的工具；2）揭示未携带已明确基因变异的aHUS患者体内的遗传性补体失调。

方法
为达成上述研究目标，我们针对60个aHUS家系的223名受试者（66名患者及157名未患病亲属），采用了一项基于二磷酸腺苷（adenosine diphosphate, ADP）激活的内皮细胞上血清诱导补体膜攻击复合物C5b-9形成的离体实验。

结果
与对照血清相比，所有处于缓解期的aHUS患者血清均可诱导更多的C5b-9沉积，且该效应与补体基因异常的存在与否无关。为避免aHUS相关慢性补体失调可能带来的混杂效应，同时考虑到所有aHUS关联基因均存在不完全外显的情况，我们选用未患病亲属的血清开展实验。对照研究显示，携带已知致病变异的未患病亲属中，92.7%的血清诱导C5b-9形成检测呈阳性，证实该实验对功能变异的鉴定具有较高灵敏度。同时该检测亦具备良好特异性：所有非携带者亲属以及携带与aHUS不共分离变异的亲属的检测结果均为阴性。在aHUS关联基因中，除1个变异外，其余所有经计算机模拟预测为可能致病、意义未明变异（variant of uncertain significance, VUS）或可能良性的变异，在C5b-9实验中均表现为致病变异。与之不同的是，推定候选基因中的变异除CFHR5变异外，均未表现出功能效应。在6个先证者携带多种遗传异常的家系中，针对亲属的C5b-9实验有助于明确罕见变异的相对功能效应。最终，对于12例未检出罕见变异的患者，通过对其父母开展C5b-9检测，揭示了源自未患病父母的遗传易感性。

讨论
综上，针对aHUS患者未患病亲属的血清诱导C5b-9形成检测，可作为一种快速评估罕见补体基因变异功能的工具。若将该实验与外显子组测序联合应用，将有助于变异筛选，进而识别新的aHUS关联遗传因素。