Evaluation of patients with ALPS-like syndromes

该项目继续产生新定义的综合征，其特征是慢性淋巴结病、脾肿大和自身免疫现象。最初，这涉及具有涉及编码NRAS和KRAS的基因的功能获得性突变的ALPS样患者。这些导致RAS-RAF-ERK通路的过度活化和促凋亡蛋白BIM的抑制。这又导致在IL-2撤除后缺陷性T细胞凋亡，但正常T细胞和B细胞凋亡响应于星形孢菌素、依托泊苷、顺铂和放射以及正常Fas途径诱导的凋亡。低水平的BIM也影响T细胞再活化诱导的凋亡。这些初步观察结果已导致鉴定出12名具有NRAS或KRAS突变的体细胞功能获得的患者，其中许多患者在儿童早期呈现具有急性粒单核细胞白血病（AMML）特征的表型，但与AMML不同，它不是侵袭性或恶性疾病。这种疾病现在被命名为RAS相关的白细胞增生性疾病（RALD）。我们正在研究潜在的标记物，以更好地确定如何区分这种体细胞NRAS突变障碍与导致JMML的相同突变。 最近，我们描述了另外两种与ALPS具有某些特征的遗传性疾病，评估的患者来自NIH排除ALPS的患者队列。在这些新的综合征中包括一名儿童，其蛋白激酶C δ功能缺陷纯合子缺失，导致淋巴结病、B细胞过度增殖、CD 5 +/CD 20 + B细胞扩增、免疫球蛋白增加、自身抗体产生和缺陷型B细胞凋亡以及与NK细胞功能缺陷相关的慢性EBV病毒血症。通过PKC δ敲低，B细胞过度增殖的离体结果可以在正常单个核细胞中重现，并且通过插入野生型PKC δ，患者EBV转化的B细胞可以部分地从离体PMA诱导的凋亡缺陷中拯救。该患者定义了单基因凋亡缺陷的新实体，其主要影响B细胞，并且还干扰正常NK细胞的细胞毒性（后者目前机制未知），其似乎增加对慢性EBV病毒血症的易感性。重要的是，该患者对雷帕霉素治疗反应显著，巨脾肿大显著减少，B细胞活化减弱。最近，我们发现了一组PI 3激酶p110杂合突变患者的特征，这些突变导致该蛋白功能的增加，并与鼻窦炎感染、淋巴积聚和自身免疫的风险增加有关。这些患者的临床表型已扩展到包括高IgM综合征样表现，此外，这些患者发生淋巴恶性肿瘤的风险增加。我们目前正在与诺华公司合作，启动一项使用该公司开发的PI 3激酶p110特异性抑制剂的临床试验，并已完成1期试验，证明其对人体无毒性。FDA已经对这项拟议的合作试验进行了初步审查，预计将于2015年初开始。免疫学服务将提供特定的流式细胞术和细胞内功能检测，作为本临床试验监查计划的一部分。 最后，正在对排除ALPS的转诊队列中的其他患者样本（其中ALPS和RALD已被排除）进行基因组DNA水平的新免疫缺陷或上述缺陷的评价。

This project has continued to generate newly defined syndromes characterized by chronic lymphadenopathy, splenomegaly and autoimmune phenomena. Initially this involved ALPS-like patietns with gain-of-function mutations involving the genes that encode NRAS and KRAS. These resulted in the hyperactivation of the RAS-RAF-ERK pathway and suppression of the pro-apoptotic protein BIM. This in turn resulted in defective T cell apoptosis after IL-2 withdrawal, but normal T cell and B cell apoptosis in response to staurosporine, etoposide, cisplatin and radiation as well as a normal Fas pathway induction of apoptosis. The low levels of BIM also affected the apoptosis induced by T-cell reactivation. These intial observations have lead to identifying 12 patients with somatic gain of function NRAS or KRAS mutations with many presenting early in childhood with a phenotype that shares features of acute myelomonocytic leukemia (AMML) but it is not an aggressive or malignant disorder unlike AMML. This disroder is now named RAS Associated Leukoproliferative Disorder (RALD). We are workng on potential markers that better define how to distinguish this somatic NRAS mutation disorder from the same mutatons that result in JMML. Most recently we have characterized two additional genetic disorders that share certain features with ALPS and the patients evaluated came from the cohort of patients refered to the NIH to rule out ALPS. Included among these new syndromes is a child with a homozygous loss of function defect in protein kinase C delta that causes lymphadenopathy, B cell hyperproliferation, expansion of CD5+/CD20+ B cells, increased immunoglobulins, autoantibody production and defective B cell apoptosis as well as chronic EBV viremia associated with a defect in NK cell function. The ex vivo findings of B cell hyperproliferrration could be recapitulated in normal mononuclear cells by PKC delta knock down and the patient EBV transsformed B cells could be partially rescued from the ex vivo PMA induced apoptotic defect by insertion of wild-type PKC delta. This patient defines a new entity in monogenic apoptotic defects that primarily affects B cells and also interferes with normal NK cell cytotoxicity (the latter for at present an unknown mechanism) that appears to increase the susceptibility to chronic EBV viremia. Importantly this patient responded dramatically to rapamycin therapy with marked decrease in the massive splenomegaly and dimiinished B cell activation. Most recently, we have characterized a group of patients with heterozygous mutations in PI3 kinase p110 that result in a gain of function of this protein associated with incresaed risk for sinopulmonary infections, lymphoaccumulation and autoimmunity. The clinical phenotype of these patients has expanded to include a hyper IgM syndrome like presenation and in addition these patients have an increased risk for lymphoid malignancy. We are currently working with Novartis on the initiation of a clinical trial used a specific inhibitor of PI3 kinase p110 that the company developed and has completed phase 1 trials demonstrating its lack of toxicity in humans. The FDA has already provided preliminary review of this proposed cooperative trial and it is anticipated that this will start in early 2015. The Immunology Service will provide specific flow cytometric and intracellular functional testing as part of the monitoring program for this clinical trial. Finally, additional patient samples within the referral cohort to rule out ALPS, in whom ALPS and RALD have been ruled out, are being evaluated for either new immunologic defects at the genomic DNA level or for the defects noted above.