Clinical, Immunological and Genetic Analyses of ALPS

本项目的研究成果包括： 1)该项目使我们能够研究最大的ALPS患者队列，ALPS是第一批免疫失调遗传性疾病之一。基于这些患者超过18年随访的ALPS自然史研究已经完成，正在生成一份手稿，总结150例ALPS-FAS患者的临床和分子发病机制的关键特征，中位随访时间为13年。这包括验证新的生物标志物以及建立新的疾病治疗模式。阐明了fas介导的细胞凋亡在淋巴细胞稳态和淋巴瘤发生中的作用。 2)该项目还通过在14名患者中鉴定影响RAS通路的突变来鉴定ALPS样疾病的新遗传原因，也称为Ras相关的白细胞增生性疾病（RALD）： RALD：患有由NRAS和KRAS中的体细胞突变引起的这种ALPS样综合征的患者目前被单独分类为ALPS相关的细胞凋亡疾病。这些具有体细胞NRAS和KRAS突变的患者表现出自身免疫现象、巨大脾肿大、中度淋巴结病和正常或仅轻微升高的TCR α/β + DNT细胞。他们的淋巴结组织病理学也不是典型的ALPS-FAS。此外，这些患者表现出骨髓室异常，慢性持续性单核细胞增多，类似于其他无症状的年轻患者中的青少年粒单核细胞白血病（JMML）。 3)以下是我们根据存在的特定分子缺陷为ALPS患者设计的当前分类方案： ALPS-FAS：TNFRSF 6（肿瘤坏死因子受体超家族6）基因突变，编码蛋白质CD 95（Fas）。 ALPS-sFas：体细胞突变体：双阴性T（DNT）细胞群中的TNFRSF 6基因缺陷。 ALPS-FASLG：TNFSF 6基因突变，编码蛋白质CD 95配体（Fas配体）。 ALPS-CASP 10：CASP 10基因突变，编码caspase-10。 ALPS-U：迄今为止尚未确定的相关突变。 4)最近，我们阐明了导致ALPS的FAS蛋白功能紊乱的原因是基于在一些患者中影响蛋白质的细胞外部分的突变引起的单倍缺陷。这是一种新出现的独特的遗传功能障碍机制。在NCBI的支持下，我们已经实现了现有致病性FAS突变数据库的基于网络的出版物，迄今为止，FAS突变是ALPS的最常见原因，其是公开可用的，并且可以通过参考NCBI NIH ALPS网站<http://www.ncbi.nlm.nih.gov/lovd/home.php? select_db=FAS>. 5)表征了我们队列中第二大ALPS患者亚组的病理生理学和临床表型，FAS基因的体细胞突变主要限于其ALPS特征细胞，也称为双阴性T淋巴细胞。 6)在ALPS相关淋巴结病患者中扩展PET扫描作为成像模式的使用，作为监测疑似ALPS相关淋巴系统癌症（淋巴瘤）患者的工具。我们在大约10%的患者中发现了与ALPS-FAS相关的淋巴瘤。在过去20年对这些患者的纵向随访中，对淋巴瘤及其早期诊断和治疗进行了持续的关键监测。 7)使用新兴的基因组和细胞生物学工具，继续在ALPS和未确定的遗传缺陷患者亚组中寻找新的遗传突变。目前，大量分子病因未知的患者正在接受全外显子组DNA测序和分析。 8)最近，ALPS临床小组正在重新定位，以解决新的免疫性疾病类别，这涉及到招募新的小组成员以及为护理和辅助支持人员提供遗传学和分子生物学方面的特殊服务。 9)继续努力通过评价淋巴细胞亚群中Fas介导的细胞死亡来简化细胞凋亡测定技术，以便该检测程序可以容易地适用于更多的临床实验室进行患者评价。 10)ALPS和另一种称为HLH（噬血细胞性淋巴组织细胞增多症）的血液学疾病的临床表现中的混杂因素已被确定，建议临床医生了解这两种疾病中的区别性生物标志物。

Research accomplishments of this project include: 1) This project allows us to study the largest cohort of patients with ALPS, one of the first genetic disorders of immunedysregulation. ALPS natural history study based on follow up of these patients over 18 yeas has been completed and a manuscript is being generated summarizing the critical features of the clinical and molecular pathogenesis in 150 patients with ALPS-FAS with a median followup of 13 years. This included the validation of new biomarkers as well as establishing new modes of treatment for the disorder. It elucidates the role of fas mediated apoptosis in lymphocyte homeostasis and lymphoma genesis. 2)This project has also led to identifying new genetic causes of ALPS like disorders by identification of mutations affecting RAS pathway in 14 patients, otherwise known as Ras Associated Leukoproliferative Disorder (RALD) : RALD: Patients with this ALPS like syndrome caused by somatic mutations in NRAS and KRAS are currently classified separately as ALPS related apoptosis disorders. These patients with somatic NRAS and KRAS mutations present with autoimmune phenomena, massive splenomegaly, modest lymphadenopathy and normal or only marginally elevated TCR alpha/beta+ DNT cells. Their lymph node histopathology is also not typical of ALPS-FAS. Additionally, these patients show abnormalities of the myeloid compartment, with chronic persistent monocytosis, mimicking juvenile myelomonocytic leukemia (JMML) in otherwise asymptomatic young patients. 3) Provided below is the current classification scheme that we have devised for ALPS patients based on the particular molecular defect present: ALPS-FAS : mutations in the TNFRSF6 (tumor necrosis factor receptor superfamily 6) gene, encodes the protein CD95 (Fas). ALPS-sFas: somatic mutant: TNFRSF6 gene defect in the double negative T (DNT) cell population. ALPS-FASLG: mutations in TNFSF6 gene, encodes the protein CD95 ligand (Fas ligand). ALPS-CASP10: mutations in CASP10 gene, encodes caspase-10. ALPS-U: associated mutation unidentified to date. 4) Recently we clarified that the cause of disordered FAS protein function leading to ALPS is based on haploinsuffiiciency caused by mutations affecting the extracellular portion of the protein in some patients. This is a newly emerging unique mechanism of genetic dysfunction. With support from NCBI we have implemented a web based publication of the existing databases of pathogenic FAS mutations, by far the commonest cause of ALPS, which is publicly available and can be used for diagnostic help by referring to NCBI NIH ALPS website <http://www.ncbi.nlm.nih.gov/lovd/home.php?select_db=FAS>. 5) Characterized the pathophysiology and clinical phenotype of the second largest subgroup of ALPS patients in our cohort with somatic mutations in the FAS gene mostly limited to their ALPS signature cells, also known as double negative T lymphocytes. 6) Extended the use of PET scans as an imaging modality in patients with ALPS associated lymphadenopathy as a tool to monitor patients with suspected ALPS associated cancer of the lymphoid system (lymphoma). We have identified lymphomas associated with ALPS-FAS in approximately 10% of our patients. Ongoing critical surveillance for lymphoma and its early diagnosis and treatment has been pursued over the last 20 years of longitudinal follow up of these patients. 7) Continued search for new genetic mutations in the subgroup of patients with ALPS and undetermined genetic defects using emerging genomic and cell biology tools. Currently a large group of patients with unknown molecular etiologies are being subjected to whole exome DNA sequencing and analysis. 8) More recently the ALPS Clinical group is being repositioned to tackle new classes of immunological disorders which has involved the recruitment of new members of the team as well as special inservices on genetics and molecular biology for the nursing and ancillary support staff. 9) Continued efforts to streamline the techniques of apoptosis assay by evaluating Fas mediated cell death in lymphocyte subsets so that this test procedure can be readily adapted in more clinical laboratories for patient evaluation. 10) Confounding factors in the clinical presentation of ALPS and another hematological condition called HLH (Hemophagocytic lymphohistiocytosis) have been identified and clinicians are being advised to be aware of the distinguishing biomarkers in both conditions.