Laboratory Assessment of Patients with Systemic Mastocytosis

绝大多数肥大细胞增多症患者携带体细胞KIT突变。与生殖系KIT激活突变相关的肥大细胞增多症非常罕见。我们研究了一个新的生殖系KIT K509 I突变引起的系统性肥大细胞增多症患者的独特临床病理特征。我们试图研究生殖系KIT K509 I突变对人肥大细胞发育和功能的影响。 检测来源于CD 34（+）外周血祖细胞的原代人肥大细胞的生长、发育、存活和IgE介导的活化。此外，建立了稳定表达KIT K509 I突变的肥大细胞转导系统。 KIT K509 I活检的肥大细胞呈圆形，CD 25（-），分化良好。与健康受试者的祖细胞相比，在干细胞因子（SCF）中培养的KIT K509 I祖细胞扩增了10倍，并发育为成熟的高颗粒肥大细胞，抗原介导的脱粒增强。KIT K509 I祖细胞在SCF的情况下培养存活，但缺乏扩增，并发育成低颗粒肥大细胞。KIT K509 I肥大细胞转导系统显示SCF非依赖性存活依赖于邻近外显子连接处KIT的优先剪接。 我们的结论是，生殖系KIT突变与肥大细胞增多症驱动分化良好的肥大细胞表型不同的体细胞KIT D816 V疾病，其致癌潜力可能受到SCF和选择性KIT剪接。 在肥大细胞室中，KIT的生殖系和体细胞突变都已在罕见的家族性肥大细胞增多症患者中发现。在寻求表征患者的独特的，遗传性过敏表型的患者中提到的严重过敏性皮肤，气道，或胃肠道疾病或系统性肥大细胞增多症（SM）的评价，我们确定了9个特应性受试者血清基础类胰蛋白酶水平持续增加的克隆性肥大细胞疾病的证据。进行了全面的临床评价，总的和分馏血清类胰蛋白酶水平，在体外嗜碱性粒细胞活化进行了测定，并进行骨髓活检，在5个这些指数患者。发现基础血清类胰蛋白酶水平升高与不同的临床特征分离，并且注意到每个索引患者的多个家庭成员共享该表型的元素。血清类胰蛋白酶基础水平升高为常染色体显性遗传。在患有遗传性类胰蛋白酶血症的受试者中，在静息状态下测量的平均基础总血清类胰蛋白酶水平为21.6 ng/mL;然而，在18名受影响患者的亚组中，类胰蛋白酶分级进一步显示血清中的成熟类胰蛋白酶不可检测（<1 ng/mL），表明类胰蛋白酶原水平升高。与基础血清类胰蛋白酶水平升高分离的突出特征是与慢性和发作性肥大细胞脱粒一致的症状，33例受试者中有26例报告了发作性荨麻疹、潮红和/或痉挛性腹痛，通常与尿急、腹泻或两者相关。来自6个不同家族的33例受试者中有10例报告了速发过敏反应史。胃肠道表现，无论是慢性或发作性，是另一个突出的特点，在28/33例类胰蛋白酶水平升高的受试者。33例胰蛋白酶血症受试者中有31例出现特应性症状，33例受试者中有28例报告了环境过敏和哮喘。结缔组织异常存在于23 33例患者中，类胰蛋白酶水平升高，9个家庭中的8个，慢性肌肉骨骼疼痛存在于11 33例类胰蛋白酶血症患者中，9个家庭中的6个。33例受试者中有10例报告了自主神经功能障碍，表现为体位性直立性心动过速综合征，33例受试者中有17例被诊断为神经精神病。这5组临床特征（皮肤、结缔组织、胃肠道、特应性和神经精神）与类胰蛋白酶水平升高独立且显著相关。由于报告的一些症状，胰蛋白酶血症的受试者可能已与家族性表现的克隆性肥大细胞疾病，索引患者在9个家庭中的5个进行骨髓活检。与健康志愿者相比，观察到肥大细胞数量显著增加（P <0.05;平均值，9.6个细胞/高倍视野hpf;范围，4-19.4个细胞/hpf）（平均值，2.5个细胞/hpf;范围，1-5个细胞/hpf）。然而，没有一个患者符合世界卫生组织建立的SM或单克隆肥大细胞活化诊断标准。所有5例患者均不存在肥大细胞聚集体，不存在CD 2/CD 25的异常表达，KIT D816 V突变试验结果均为阴性。在单个患者中观察到梭形肥大细胞（>25%）。在体外嗜碱性粒细胞活化进行了评估，受影响的受试者从9个家庭。来自受影响受试者的嗜碱性粒细胞表现出降低的活化，如基于用抗IgE或N-甲酰基-甲硫氨酰亮氨酰-苯丙氨酸刺激后的CD 203 c平均荧光强度所测量的。然而，CD 203 c嗜碱性粒细胞群体中的CD 63诱导，尽管可变，但在患者和配对对照受试者之间没有显著差异。其他的遗传学研究正在进行中，随着这些家族中功能障碍的通路被阐明，我们可能会对高活动性结缔组织表型、特应性疾病、自主神经功能障碍和神经精神疾病及其与肥大细胞介质的界面有更多的了解。

The wast majority of patients with mastocytosis carry somatic KIT mutations. Mastocytosis associated with germline KIT activating mutations is exceedingly rare. We investigated the unique clinicopathologic features of a patient with systemic mastocytosis caused by a de novo germline KIT K509I mutation. We sought to investigate the effect of the germline KIT K509I mutation on human mast cell development and function. Primary human mast cells derived from CD34(+) peripheral blood progenitors were examined for growth, development, survival, and IgE-mediated activation. In addition, a mast cell transduction system that stably expressed the KIT K509I mutation was established. KIT K509I biopsied mast cells were round, CD25(-), and well differentiated. KIT K509I progenitors cultured in stem cell factor (SCF) demonstrated a 10-fold expansion compared with progenitors from healthy subjects and developed into mature hypergranular mast cells with enhanced antigen-mediated degranulation. KIT K509I progenitors cultured in the absence of SCF survived but lacked expansion and developed into hypogranular mast cells. A KIT K509I mast cell transduction system revealed SCF-independent survival to be reliant on the preferential splicing of KIT at the adjacent exonic junction. We concluded that germline KIT mutations associated with mastocytosis drive a well-differentiated mast cell phenotype distinct to that of somatic KIT D816V disease, the oncogenic potential of which might be influenced by SCF and selective KIT splicing. Within the mast cell compartment, both germline and somatic mutations in KIT have been identified in patients with rare familial presentations of mastocytosis. While seeking to characterize patients with unique, inherited allergic phenotypes among patients referred for evaluation of severe allergic skin, airway, or gastrointestinal disease or systemic mastocytosis (SM), we identified 9 atopic subjects with persistent increases in serum basal tryptase levels in the absence of evidence for a clonal mast cell disorder. Thorough clinical evaluations were undertaken, total and fractionated serum tryptase levels were obtained, in vitro basophil activation was assayed, and bone marrow biopsies were performed in 5 of these index patients. Increased basal serum tryptase levels were found to segregate with distinct clinical features, and it was noted that multiple family members of each index patient shared elements of this phenotype. An autosomal dominant inheritance pattern of increased basal total serum tryptase levels was revealed in all 9 families. The mean basal total serum tryptase level measured during a resting state was 21.6 ng/mL among subjects with inherited tryptasemia; however, tryptase fractionation in a subset of 18 affected patients further revealed that mature tryptase in serum was undetectable (<1 ng/mL), suggesting increase in protryptase levels. Prominent among features segregating with basal serum tryptase level increases were symptoms consistent with chronic and episodic mast cell degranulation, with 26 of 33 subjects reporting episodic urticaria, flushing, and/or cramping abdominal pain frequently associated with urgency, diarrhea, or both. A history of anaphylaxis was reported in 10 of 33 subjects from 6 different families. Gastrointestinal manifestations, whether chronic or episodic, were another prominent feature seen in 28 of 33 subjects with increased tryptase levels. Atopic symptoms were present in 31 of 33 tryptasemic subjects, with environmental allergies and asthma being reported among 28 of 33 affected subjects. Connective tissue abnormalities were present in 23 of 33 subjects with increased tryptase levels from 8 of 9 families, and chronic musculoskeletal pain was present among 11 of 33 tryptasemic subjects from 6 of 9 families. Autonomic dysfunction, manifesting as postural orthostatic tachycardia syndrome, was reported in 10 of 33 affected subjects, and a neuropsychiatric diagnosis was present in 17 of 33 affected subjects. These 5 clusters of clinical characteristics (cutaneous, connective tissue, gastrointestinal, atopic, and neuropsychiatric) were independently and significantly associated with increased tryptase levels. Because some of the reported symptoms in tryptasemic subjects could have been consistent with the familial presentation of a clonal mast cell disorder, index patients in 5 of 9 families underwent bone marrow biopsy. A significant increase in mast cell numbers was observed (P < .05; mean, 9.6 cells/high-power field hpf; range, 4-19.4 cells/hpf) compared with that seen in healthy volunteers (mean, 2.5 cells/hpf; range, 1-5 cells/hpf). However, none of the patients met the World Health Organization established criteria for a diagnosis of SM or monoclonal mast cell activation. Mast cell aggregates were not present, aberrant expression of CD2/CD25 was absent, and KIT D816V mutation test results were negative in all 5 patients. Spindled-shaped mast cells (>25%) were observed in a single patient. In vitro basophil activation was assessed in affected subjects from 6 of 9 families. Basophils from affected subjects demonstrated reduced activation, as measured based upon CD203c mean fluorescence intensity after stimulation with anti-IgE or N-formyl-methionylleucyl-phenylalanine. However, CD63 induction within the CD203c basophil population, although variable, was not significantly different between patients and paired control subjects. Additional genetic study is ongoing, and as the dysfunctional pathways in these families are elucidated, we might gain additional insights into the hypermobile connective tissue phenotype, atopic disease, autonomic dysfunction, and neuropsychiatric illness and their interface with mast cell mediators.