背景

阵发性睡眠性血红蛋白尿症（PNH）是一种超级罕见病，国内缺乏大宗病例报道,尤其缺乏针对不同亚型的分析。PNH克隆扩增、PNH血栓形成的分子机制仍尚无定论。

目的

分析PNH患者的临床特征，并比较不同亚型PNH的特点。探索PNH克隆增殖及易发血栓的机制。

方法

1.收集2009年至2019年在北京协和医院就诊并随访2年以上的PNH患者的资料进行统计学分析。记录诊断时及随访过程中的实验室相关临床资料、药物治疗、克隆演变、疾病预后及女性PNH患者妊娠分娩等情况。

2.采集6例PNH患者外周血粒细胞，分选出CD59阳性和CD59 阴性细胞，并分别送检全外显子、全转录组测序，同时采集6例健康对照的外周血作为对照。筛出同时具有WES与转录组异常的基因进行GO富集，关注与增殖凋亡、免疫及血栓相关的基因。

3. 后续将以上基因在40例PNH患者外周血细胞WES测序的结果库中进行筛选，找出同时满足千人库频率低于0.001且在PNH中发生率≥10%的基因作为候选基因。将前述候选基因在另外40例PNH外周血样本中进行一代测序验证，最终确定目标基因。

4. 随后收集7例PNH患者外周血粒细胞，分选CD59 阳性和CD59阴性细胞，采用Q-PCR检测其目标基因的RNA表达， 用Western Blot、流式及质谱检测目标蛋白表达。

结果

1.研究共纳入512例患者，其中男性288例（56.3%），女性224例（43.7%），发病年龄中位数为33岁（9~80岁）。经典型PNH、合并骨髓衰竭型（BMF）/PNH和亚临床PNH患者比例分别为46.8%、51.6%和1.6%。经典型PNH患者临床症状更多与溶血相关，而出血症状则在BMF/PNH患者中更常见。与BMF/PNH患者相比，经典型PNH患者的PNH克隆、乳酸脱氢酶（LDH）水平更高，铁蛋白水平更低。虽然经典型PNH和BMF/PNH患者发生血栓比例相似（P=0.66），但BMF/PNH患者发生肾损害的几率更高（P<0.05）。BMF/PNH患者中使用免疫抑制剂（P<0.05）更多，而经典型PNH患者使用糖皮质激素、铁补充剂和抗凝剂（P<0.05）更多。对免疫抑制剂治疗无效患者比治疗有效患者的PNH克隆更大（66.89±35.01% vs 38.64±30.47%，P=0.001）；治疗效果稳定（诊断vs末次随访：38.64±30.47%vs 39.50±43.13%，P=0.829）及治疗无效（诊断vs末次随访：68.89±35.01%vs 96.00±1.00%，P=0.133）患者在随访过程中的PNH克隆稳定；起初治疗有效但随访过程中复发患者的复发后PNH克隆较复发前显著增加（43.53±30.96% vs 93.33±3.22%，P<0.01）。与BMF/PNH相比，经典型PNH发生髓系恶性肿瘤几率更低（P=0.02）。PNH患者主要死亡原因为血栓（29.6%）、出血（18.5%）和感染（18.5%），经典型PNH和BMF/PNH在死亡原因方面无差异。与BMF/PNH患者相比，经典型PNH患者的总生存较好，BMF/PNH中MDS/PNH的预后最差。

2. 经GO富集发现，同正常人相比，PNH患者T细胞活化相关基因上调。血小板脱颗粒，凝血、止血，白细胞增殖，血小板活化相关基因下调。对比CD59+和CD59-细胞，发现CD59+细胞中白细胞增殖、细胞凋亡调控、免疫活化及血小板活化、凝血调节相关基因较CD59-细胞下调。

3. 在初步筛选目的基因中，包括增殖凋亡相关基因416个，免疫相关基因716个，血栓相关基因共141个。这些基因经40例PNH患者WES测序结果库筛选后，共获得10个目标基因。再经Sanger测序验证靶点突变有 SELP（突变率12.5%）、NRP1 （10%）、SLC15A4（5%）、vWF（2.5%）、ABCA13（2.5%）和FLT1（2.5%），靶点外碱基突变有SWAP70（62.5%）和EpHB2（10%）。其中SWAP70、EPHB2、ABCA13、SLC15A4基因突变率在血栓组显著高于非血栓组（P<0.05）。 最终确定SELP、NRP1、vWF、FLT1、SWAP70和EpHB2这6个为目的基因。

4. 从Q-PCR和蛋白水平验证目的基因，发现PNH在RNA和蛋白水平均与健康对照有差异的基因为NRP1和EpHB2；仅在RNA水平与健康对照存在差异的是SELP；仅在蛋白水平与健康对照有差异的是FLT1和vWF；在RNA和蛋白质水平与健康对照均无差异，但在CD59+和CD59-之间存在差异的基因是SWAP70。以上差异基因中，EpHB2、NRP1 及FLT1与增殖、凋亡相关；SELP、vWF和EpHB2与血栓相关；SWAP70与免疫相关。EpHB2、NRP1、FLT1和 SWAP70在CD59+和CD59-细胞之间也有差异表达。质谱分析还发现CD59+细胞存在抗增殖蛋白（BTG1）表达，而CD59-细胞有存在细胞周期相关蛋白（INCA1、NPEPPS）表达。

结论

1.经典型PNH表现出更多与溶血相关的症状和并发症，并且PNH克隆更大。经典型PNH以支持治疗为主，而BMF/PNH以免疫抑制治疗为主，且免疫抑制治疗效果与PNH克隆大小相关。BMF/PNH比经典型PNH预后更差。

2.同正常人相比，PNH患者T细胞活化相关基因上调，增殖、出凝血相关基因下调。其中，CD59+细胞增殖和血栓调节基因下调更为明显。

3.筛选出的PNH高发基因，在RNA和（或）蛋白水平表达与正常人也不同，且在CD59+和CD59-细胞间表达存在差异，部分还在PNH血栓组高发。这些可能是PNH的血栓、CD59-细胞的增殖相关的重要基因。

4.CD59+细胞存在抗增殖蛋白表达，而CD59-细胞存在细胞周期相关蛋白表达，考虑是参与PNH克隆增殖的因素。

全文结论

PNH发病年龄轻，PNH发生血栓风险高且预后不佳，免疫抑制剂治疗效果与PNH克隆大小相关。PNH患者基因组存在增殖能力下调，免疫活化上调，凝血相关下调，且增殖凋亡、免疫及凝血功能相关基因、蛋白表达异常，且在不同细胞亚群中存在差异。在PIGA之外的一些基因的异常表达可能与PNH克隆增殖和血栓形成密切相关。

Background: Paroxysmal nocturnal hemoglobinuria (PNH) is a super rare disease. There lackes researches in characters of PNH with large population in China, especially analysis of PNH subtypes.And the molecular mechanism of PNH clonal amplification and thrombosis of PNH is still inconclusive.

Objective: This research aimed to summarize and analyze the clinical characteristics of PNH between different subtypes without complement inhibitor therapy in China, and to explore the mechanism of PNH clonal proliferation and thrombosis of PNH.

Methods:

1. PNH patients’ clinical data, who were treated in Hospital  from 2009 to 2019 and followed up for more than 2 years was cllected, including the current situation of domestic PNH patients from the aspects of laboratory and clinical characteristics, therapy, clonal evolution, prognosis and pregnancy and delivery of female PNH patients.

2. Peripheral blood of 6 PNH patients were collected for sorting out CD59 positive and CD59 negetive cells, and sequenceed by whole exon sequencing(WES)and whole transcriptome sequence respectively. At the same time, peripheral blood of 6 matched healthy controls was selected as transcriptome control. According to the transcriptome up-regulation and down-regulation of the genome and its association with WES, genes with simultaneous abnormal expression of transcriptome and abnormal WES were first selected. Selected genes were enriched by Gene Ontology (GO), and then genes related to proliferation, immunity and thrombosis were selected.

3. Peripheral blood of another 40 PNH patients was collected for WES, and the target genes that simultaneously meet the frequency less than 0.001 in 1000 Genomes and the incidence more than 10% in 40 patients were selected. Another 40 samples were sequenced by Sanger to verify the final target genes.

4. And 7 of 40 samples sequenced by Sanger method, whose peripheral blood were collected and sorted by flow cytometry to verify the target gene through quantitative polymerase chain reaction(Q-PCR), Western Blot, flow cytometry and mass spectrometry.

Results:

1. 512 patients were included in total, including 288 males (56.3%) and 224 females (43.7%). Median age was 33 (9 ~ 80) years old. The proportions of patients with classic PNH, combined with bone marrow failure (BMF/PNH) and subclinical PNH were 46.8%, 51.6% and 1.6%, respectively. Symptoms in patients with classic PNH are more likely to be associated with hemolysis, while bleeding is more common in patients with BMF/PNH. Compared with BMF/PNH patients, patients with classic PNH had higher PNH clone, lactate dehydrogenase (LDH) level and lower ferritin level. Although the incidence of thrombosis was similar in patients with classic PNH and BMF/PNH (P = 0.66), the incidence of renal deficiency was higher in BMF/PNH (P<0.05). BMF/PNH were more often treated with immunosuppresive therapy (P<0.05), while classic PNH were more treated with glucocorticoids, iron supplements and anticoagulants (P<0.05). Moreover, compared with patients who were effective in immunosuppresive therapy, PNH clones were larger in those of patients who were ineffective (66.89 ± 35.01% vs 38.64 ± 30.47%, P = 0.001). For patients with continuous effect(at diagnosis vs last follow-up:38.64±30.47% vs 39.50±43.13%, P= 0.829) and without effect( at diagnosis vs last follow-up:68.89±35.01% vs 96.00±1.00%, P=0.133), PNH clone was stable during follow-up. For patients who were effective at first but relapsed during follow-up, PNH clone after relapse were significantly higher than before therapy(43.53±30.96% vs 93.33±3.22%, P<0.01). Compared with BMF/PNH, classic PNH had lower incidence of myeloid malignancy (P = 0.02). The main causes of death in patients with PNH were thrombosis (29.6%), bleeding (18.5%) and infection (18.5%). There was no difference between classic PNH and BMF/PNH. Compared with BMF/PNH, classic PNH have better overall survival, and the prognosis of MDS/PNH is the worst.

2. Results of GO enrichment indicated that T cell activation related genes were up-regulated, while platelet degranulation, coagulation and hemostasis, leukocyte proliferation, and platelet activation related genes down-regulated in PNH patients when compared with healthy control. Genes related to leukocyte proliferation, apoptosis regulation, immune activation, platelet activation and coagulation regulation were down regulated in CD59 positive cells compared with CD59 negative cells.

3. Then 416 proliferation/apoptosis related genes, 716 immune related genes and 141 thrombus related genes were first distinguished. After screening these genes in WES sequencing results of 40 PNH cases, 10 target genes were then obtained. After Sanger sequencing verification, 6 target genes were finally determined, which were SELP (mutation incidence 12.5%), NRP1 (10%); SLC15A4 (5%), vWF (2.5%), ABCA13(2.5%) and FLT1 (2.5%). In addition, out of target base mutations SWAP70 (62.5%) and EpHB2 (10%) were also included. The abnormal frequencies of SWAP70, EpHB2, ABCA13 and SLC15A4 in thrombotic group were significantly higher than patients without thrombotic group (P <0.05).

4. Some target genes were verified by Q-PCR, Western Blot, flow cytometry respectively. It was found that NRP1 and EphB2 in PNH were different from healthy controls in both RNA and protein levels; SELP in PNH was different from healthy controls only in RNA level; FLT1 and vWF in PNH were different from healthy controls only in protein level; SWAP70 in PNH were similar in both RNA and protein levels to healthy controls, but SWAP70 in CD59 positive was lower than CD59 negative in protein expression level. Among the above differential genes, founction of FLT1, EpHB2 and NRP1 are related to proliferation and apoptosis; SELP, vWF and EpHB2 were associated with thrombosis; SWAP70 was associated with immunity. What’s more, FLT1, EpHB2, NRP1 and SWAP70 were also differentially expressed between CD59 positive and CD59 negative cells. Mass spectrometry analysis showed that anti proliferative protein (BTG1) was expressed in CD59 positive cells, while cycle related protein (INCA1, NPEPPS) was expressed in CD59 negative cells.

Conclusions:

1. Symptoms in patients with classic PNH are more likely to be associated with hemolysis, and PNH clone was larger. Classic PNH is mainly treated with supportive therapy, while BMF/PNH is mainly with immunosuppressive therapy, and the response to immunosuppressive therapy was related to size of PNH clone. BMF/PNH is easier to have clone evolution than classic PNH.

2. Compared with healthy controls, genes related to immune function such as T cell activation were up regulated, while leukocyte proliferation, platelet degranulation, coagulation, hemostasis, and platelet activation related genes were down regulated in PNH. Among them, down-regulation of proliferation and thrombolism related genes in CD59 positive cells were more obvious.

3. The selected high-frequency genes in PNH are also different from normal people in RNA and/or protein levels, and there are differences in the expression between CD59 positive and CD59 negetive cells, and some of them are also more frequent in PNH thrombosis group. These genes may be closely related to thrombosis of PNH and proliferation of CD59 negative cells.

4. Antiproliferative proteins expressed in CD59 positive cells and cell cycle related proteins expressed in CD59 negative cells.

Full text conclusion:

Age at diagnosis of PNH is young. Incidence of thrombosis is high and indicates poor prognosis. Effect of immunosuppressive therapy is related to PNH clone. The genome characteres of PNH display down-regulation of proliferation and coagulation, up-regulation of immune activation. What’s more, abnormal expression of genes and proteins also are different between CD59 positive and CD59 negative cells. The abnormal expression of some genes except for PIGA may be closely related to mechanism of clone proliferation and thrombosis.

Key words: paroxysmal nocturnal hemoglobinuria, clinical features, gene sequencing, transcriptome sequencing, protein expression