Dear Editor:

Cartilage hair hypoplasia (CHH) is a primary immunodeficiency variably affecting both cellular and humoral immunity with skeletal dysplasias in a phenotypic continuum with anauxetic dysplasia (AD; Mäkitie and Vakkilainen 2012). Characteristic phenotypic features of CHH can include severe disproportionate metaphyseal dysplasia and light‐colored, sparse hair, combined immunodeficiency, Hirschsprung disease, bone marrow dysplasia and susceptibility to malignancies. The disorder is inherited in an autosomal recessive manner through mutations in the nontranslated ribonuclease mitochondrial RNA‐processing (RMRP) gene (reviewed in Mäkitie and Vakkilainen 2012). The most common mutation, n.70A > G, predominantly affects the Old Order Amish and Finnish, but numerous other mutations affecting other ethnicities have been identified in the promoter and coding regions. Flow cytometric workup is critical for the diagnosis, monitoring, therapy, and follow‐up of patients with combined immunodeficiencies, including CHH, severe combined immunodeficiency (SCID), and others. Much of the early immunologic work in CHH‐AD focused on humoral deficiency in children whose immunologic status was not identified at birth (reviewed in Mäkitie and Vakkilainen 2012). Recent flow cytometric analyses in CHH have separately demonstrated variable T‐cell lymphopenia with equally variable functional deficits.

We report the case of a female infant with prenatally detected severe long bone shortening and femoral bowing, concerning for possible thanatophoric dysplasia. Maternal medical history and family history was unremarkable. A 48‐gene prenatal skeletal dysplasia next‐generation sequencing panel performed on amniocentesis fluid did not reveal a pathogenic variant. The patient underwent an induced vaginal delivery at 37 weeks gestation and was born at 3084 g with profound skeletal dysplasia, short‐limb dwarfism, and Apgars of 4 and 9 at 1 and 5 min, respectively. Newborn screening demonstrated absent T‐cell receptor excision circles (TRECs) concerning for SCID. Radiographs performed shortly after birth demonstrated a normal‐appearing thymic shadow, shortened and bowed long bones, and platyspondyly. A postnatal 109‐gene skeletal dysplasia panel showed heterozygosity in RMRP (NR_003051.3: n.‐19_‐3dup and n.223C > T) in trans, consistent with CHH‐AD. RMRP was not included in the prenatal panel.

With the lack of TRECs on newborn screening, flow cytometric evaluation of the patient's peripheral blood for lymphocyte subsets was performed on the fifth day of life. Analysis of peripheral blood specimens was performed on a Becton Dickinson (BD, Franklin Lakes, NJ) Canto II flow cytometer configured with a 20 mW solid state 488 nm laser, a 17 mW HeNe 633 nm laser and a 30 mW solid state 405 nm laser. Whole blood specimens were prepared using BD FACS Lyse reagents on a BD wash station. Primary conjugated BD antibodies reported in this study included TCR‐alpha‐beta‐FITC (clone WT31), TCR‐gamma‐delta‐PE (11F2), CD3‐APC‐H7 (SK7), CD4‐PE‐Cy7 (SK3), CD7‐PE (M‐T701), CD8‐PerCP‐Cy5 (SK1), CD16‐V450 (3GB), CD19‐APC (SJ25C1), CD45‐V500 (2D1), CD62L‐APC (DREG‐56), and CD56‐PE‐Cy7 (NCAM16.2). CD45RA‐FITC (clone IM05844) and CD45RO‐PE (IM13076) were obtained from Immunotech Inc. (Ocala, FL). Kaluza software version 2.1 (Beckman Coulter, Miami, FL) was used to analyze the FCS files for antigen expression patterns.

Initial workup demonstrated an absolute lymphocyte count (ALC) of 800 cells/μl (reference 1500–6400 cells/μl; Figure 1a). Flow cytometric lymphocyte phenotyping demonstrated an absolute and relative T lymphopenia (23.92 CD3⁺ T‐cells/μl [957–3611 cells/μl]) composed of 20.66 cells/μl, or 86.40%, CD3⁺CD4⁻CD8⁻ double negative (DN) T‐cells which expressed neither the central memory marker CD45RO nor the marker of naïveté CD45RA (Figure 1b‐d). The CD8⁺ single‐positive (SP) T‐cells exclusively expressed CD45RA, but CD4⁺ SP cells favored the memory phenotype. There were normal NK‐ and B‐cell counts with increased relative numbers of NK‐cells but a normal maturation pattern for both NK‐ and B‐cells. The granulocytic and monocytic compartments were unremarkable by forward and side scatter, CD45, CD4, and CD56 expression, but these lineages were not specifically subjected to flow cytometric maturation evaluation.

Flow cytometry was performed again at week 4 which demonstrated a decreasing ALC of 700 cells/μl with continued T‐cell lymphopenia (50.00 cells/μl) and declining B‐ and NK‐cell counts. Remarkably, the DN T‐cell population continued to expand to 40.00 cells/μl, but 24.82 cells/μl, or 62.04% of these were CD45RA⁺ naïve cells. The memory cells among both the CD4⁺ and CD8⁺ T‐cells expanded as well. This trend continued through week 8. Further immunophenotyping revealed that 36.74 cells/μl, or 77.16% of the DN T‐cells expressed the gamma‐delta T‐cell receptor (TCR; Figure 1e‐f). At all stages, CD62L was also assessed; nearly 100% of CD45RA‐expressing T‐cells also expressed CD62L, and the CD45RO‐expressing T‐cells split into CD62L‐expressing central memory and CD62‐negative effector memory T‐cells (data not shown).

The patient underwent an unrelated bone marrow donor hematopoietic stem cell transplantation with reduced‐intensity conditioning at week 11 of life (day +0) with a second stem cell infusion from the same donor on day +14 due to unstable engraftment and concomitant bacteremia. Following these procedures, the patient underwent flow cytometric evaluation on day +34/48 (Month 4) and day +53/77 (Month 5), demonstrating an expansion of both naïve and memory CD4⁺ T‐cells and to a lesser extent CD8⁺ T‐cells, followed by B‐ and NK‐cells which is within the range of expected timeframes. A chimerism assay performed on transplant day +44/58 demonstrated a 100% CD3⁺ T‐cell donor component.

This patient's postnatal immunophenotypic findings appear to recapitulate in part the prenatal generation of thymocytes, referred to as “layered” immune constitution (reviewed in Rudd 2020). In brief, bone marrow‐derived common lymphocyte progenitor cells initially generate naïve CD45RA⁺ DN T‐cells in a thymus‐independent manner. Later, the progenitors establish definitive thymopoiesis, seeding the thymus to produce naïve CD45RA⁺ SP T‐cells. Initial waves of fetal thymic T‐cell production are characterized by gamma‐delta T‐cells, often with germline (invariant) VDJ sequences. Later waves have gamma‐delta TCR and then alpha‐beta TCR‐bearing T‐cells with high diversity. This patient's serial immunologic evaluation showed a postnatal wave of DN T‐cells, which were predominantly gamma‐delta TCR positive and which showed an increased ratio of CD45RA⁺ to CD45RO⁺ cells from weeks 4 to 8. This wave was then followed by a weak expansion of SP memory T‐cells prior to transplant. Since we did not sequence the TCR genes in this patient, the degree of VDJ heterogeneity in the DN T‐cells is unknown.

To our knowledge, this is among the earliest in life a patient with CHH‐AD spectrum has undergone flow cytometric evaluation for SCID and the first whose naïve and memory compartments have been serially followed in the short term. This patient is also the first reported to have the compound heterozygous variants of n.‐19_‐3dup and n.233C > T affecting RMRP. Two patients with an identical promoter duplication were previously reported but those patients' immune constitution statuses were not queried. In another study, other patients, including a set of siblings evaluated at birth, had moderate T‐lymphopenia at presentation, but the causative RMRP mutation was not identified until they were months to years old. Two patients with homozygous RMRP n.70A > G with no TRECs at birth had profound lymphopenia and minimal response to lymphocyte stimulation on serial evaluation, but their naïve and memory compartments were not quantified. The patient in this study is the first to document a near‐complete lack of naive T‐cells, including all CD4⁺ T‐cells, at birth; other studies indicate a progressive decline in T‐cell numbers and function after patients come to clinical attention (reviewed in Mäkitie and Vakkilainen 2012; additional specific citations available upon request).

As this is a single patient with a unique genotype, we cannot necessarily draw conclusions about the natural history of this patient's compound heterozygosity in the phenotypic expression of the CHH‐AD mutations. RMRP compound heterozygosity has profound to severe immunodeficiency, including a duplication or insertion in the RMRP promoter region (reviewed in Mäkitie and Vakkilainen 2012). This patient's serial flow cytometric analysis underscores the severity of combined immunodeficiency that can occur in the CHH‐AD spectrum. Further, the pattern of T‐cell reconstitution observed in this patient prior to transplant raises the possibility that delayed thymic maturation may play a role in CHH‐AD spectrum patients with SCID.

亲爱的编辑：

软骨毛发发育不全 (CHH) 是一种原发性免疫缺陷，其在伴有缺氧发育不良的表型连续体中影响细胞和体液免疫，伴有骨骼发育不良（AD；Mäkitie 和 Vakkilainen  2012）。CHH 的特征性表型特征可包括严重的不成比例的干骺端发育不良和浅色、稀疏的头发、联合免疫缺陷、先天性巨结肠、骨髓发育不良和对恶性肿瘤的易感性。该疾病通过非翻译核糖核酸酶线粒体 RNA 加工 ( RMRP ) 基因的突变以常染色体隐性方式遗传（综述于 Mäkitie 和 Vakkilainen  2012）。最常见的突变，n.70A > G，主要影响旧秩序阿米什人和芬兰人，但已经在启动子和编码区发现了许多影响其他种族的其他突变。流式细胞术检查对于联合免疫缺陷患者的诊断、监测、治疗和随访至关重要，包括 CHH、严重联合免疫缺陷 (SCID) 等。CHH-AD 的许多早期免疫学工作都集中在出生时未确定免疫状态的儿童的体液缺乏（Mäkitie 和 Vakkilainen  2012 年综述）。最近在 CHH 中进行的流式细胞术分析分别证明了可变的 T 细胞淋巴细胞减少和同样可变的功能缺陷。

我们报告了一例产前检测到严重的长骨缩短和股骨弯曲的女婴，这与可能的致死性发育不良有关。产妇病史和家族史无异常。对羊膜穿刺液进行的 48 基因产前骨骼发育不良下一代测序面板未发现致病性变异。患者在妊娠 37 周时接受了引产，出生时体重 3084 g，患有严重的骨骼发育不良、短肢侏儒症，1 分钟和 5 分钟时阿普加斯分别为 4 和 9。新生儿筛查显示与 SCID 相关的 T 细胞受体切除环 (TRECs) 缺失。出生后不久进行的 X 光片显示胸腺阴影正常，长骨缩短并呈弓形，呈扁平状。反式的RMRP (NR_003051.3: n.‐19_‐3dup 和 n.223C > T) ，与 CHH-AD 一致。产前检查不包括RMRP。

由于新生儿筛查中缺乏 TREC，因此在出生后第五天对患者外周血的淋巴细胞亚群进行流式细胞术评估。在配置有 20 mW 固态 488 nm 激光器、17 mW HeNe 633 nm 激光器和 30 mW 固态 405 nm 激光器的 Becton Dickinson (BD, Franklin Lakes, NJ) Canto II 流式细胞仪上进行外周血样本分析. 在 BD 清洗站上使用 BD FACS Lyse 试剂制备全血样本。本研究报道的主要偶联 BD 抗体包括 TCR-α-β-FITC（克隆 WT31）、TCR-γ-δ-PE（11F2）、CD3-APC-H7（SK7）、CD4-PE-Cy7（SK3）、 CD7-PE (M-T701)、CD8-PerCP-Cy5 (SK1)、CD16-V450 (3GB)、CD19-APC (SJ25C1)、CD45-V500 (2D1)、CD62L-APC (DREG-56) 和 CD56 -PE-Cy7 (NCAM16.2)。CD45RA-FITC (克隆 IM05844) 和 CD45RO-PE (IM13076) 购自 Immunotech Inc. (Ocala, FL)。Kaluza 软件版本 2.1 (Beckman Coulter, Miami, FL) 用于分析 FCS 文件的抗原表达模式。

初步检查显示绝对淋巴细胞计数 (ALC) 为 800 个细胞/μl（参考 1500-6400 个细胞/μl；图 1a）。流式细胞术淋巴细胞表型显示绝对和相对 T 淋巴细胞减少（23.92 CD3 ⁺ T 细胞/μl [957–3611 细胞/μl]）由 20.66 个细胞/μl 或 86.40%，CD3 ⁺ CD4 ^- CD8 ^-双阴性（DN ) 既不表达中央记忆标记 CD45RO 也不表达幼稚 CD45RA 标记的 T 细胞（图 1b-d）。CD8 ⁺单阳性 (SP) T 细胞仅表达 CD45RA，但 CD4 ⁺SP 细胞有利于记忆表型。NK 细胞和 B 细胞计数正常，NK 细胞的相对数量增加，但 NK 和 B 细胞的成熟模式正常。粒细胞和单核细胞的前向和侧向散射、CD45、CD4 和 CD56 表达不显着，但这些谱系没有专门进行流式细胞成熟度评估。

第 4 周再次进行流式细胞术，结果显示 ALC 下降 700 个细胞/μl，T 细胞淋巴细胞持续减少（50.00 个细胞/μl），B 细胞和 NK 细胞计数下降。值得注意的是，DN T 细胞群继续扩大到 40.00 个细胞/μl，但 24.82 个细胞/μl，或其中 62.04% 是 CD45RA ⁺幼稚细胞。^{CD4 +}和 CD8 ⁺中的记忆细胞T 细胞也扩大了。这种趋势持续到第 8 周。进一步的免疫表型分析显示，36.74 个细胞/μl，或 77.16% 的 DN T 细胞表达 gamma-delta T 细胞受体（TCR；图 1e-f）。在所有阶段，还评估了 CD62L；近 100% 的表达 CD45RA 的 T 细胞也表达 CD62L，表达 CD45RO 的 T 细胞分裂成表达 CD62L 的中央记忆 T 细胞和表达 CD62 的负效应记忆 T 细胞（数据未显示）。

由于植入不稳定和伴随的菌血症，患者在第 11 周（第 +0 天）接受了无关的骨髓供体造血干细胞移植，并在第 +14 天从同一供体进行了第二次干细胞输注，并进行了强度降低的调节。在这些程序之后，患者在第 +34/48 天（第 4 个月）和第 +53/77 天（第 5 个月）接受了流式细胞术评估，表明幼稚和记忆 CD4 ⁺ T 细胞的扩增以及较小程度的 CD8 ⁺ T 细胞，其次是在预期时间范围内的 B 细胞和 NK 细胞。在移植日 +44/58 进行的嵌合分析显示 100% CD3 ⁺ T 细胞供体成分。

该患者的产后免疫表型发现似乎部分概括了胸腺细胞的产前生成，称为“分层”免疫构成（在 Rudd  2020中进行了综述）。简而言之，骨髓来源的普通淋巴细胞祖细胞最初以不依赖胸腺的方式产生幼稚的 CD45RA ⁺ DN T 细胞。后来，祖细胞建立了明确的胸腺生成，播种胸腺以产生幼稚的 CD45RA ⁺SP T 细胞。胎儿胸腺 T 细胞产生的初始波以 γ-δ T 细胞为特征，通常具有种系（不变）VDJ 序列。后面的波有 gamma-delta TCR，然后是具有高度多样性的 alpha-beta TCR 轴承 T 细胞。该患者的系列免疫学评估显示出生后 DN T 细胞波，主要为 gamma-delta TCR 阳性，并显示从第 4 周到第 8 周 CD45RA ⁺与 CD45RO ⁺细胞的比率增加。随后是一个微弱的波移植前SP记忆T细胞的扩增。由于我们没有对该患者的 TCR 基因进行测序，因此 DN T 细胞中 VDJ 异质性的程度是未知的。

据我们所知，这是 CHH-AD 谱系患者生命中最早接受 SCID 流式细胞术评估的患者之一，也是第一个在短期内对其幼稚和记忆隔间进行连续跟踪的患者。该患者也是第一个报道具有影响RMRP的 n.‐19_‐3dup 和 n.233C > T 的复合杂合变体的患者。先前报道了两名具有相同启动子重复的患者，但未询问这些患者的免疫构成状态。在另一项研究中，包括一组出生时评估的兄弟姐妹在内的其他患者在就诊时患有中度 T 淋巴细胞减少症，但直到数月至数岁时才发现致病的RMRP突变。两名患有纯合RMRP的患者出生时没有 TREC 的 n.70A > G 有严重的淋巴细胞减少和对连续评估的淋巴细胞刺激的最小反应，但他们的幼稚和记忆隔间没有被量化。本研究中的患者是第一个记录在出生时几乎完全缺乏幼稚 T 细胞（包括所有 CD4 ^{+ T 细胞）的患者；}其他研究表明，在患者获得临床关注后，T 细胞数量和功能会逐渐下降（在 Mäkitie 和 Vakkilainen 2012中进行了综述；可根据要求提供其他具体引文）。

由于这是一名具有独特基因型的患者，我们不一定能得出关于该患者在 CHH-AD 突变表型表达中复合杂合性的自然史的结论。RMRP复合杂合性具有严重到严重的免疫缺陷，包括RMRP启动子区域的重复或插入（在 Mäkitie 和 Vakkilainen  2012中进行了综述）。该患者的连续流式细胞仪分析强调了 CHH-AD 谱系中可能发生的联合免疫缺陷的严重性。此外，在该患者移植前观察到的 T 细胞重建模式提出了延迟胸腺成熟可能在 CHH-AD 谱系 SCID 患者中发挥作用的可能性。