A germline mutation of CDKN2A and a novel RPLP1-C19MC fusion detected in a rare melanotic neuroectodermal tumor of infancy: a case report [aCGH]. Homo sapiens

Purpose: Melanotic neuroectodermal tumor of infancy (MNTI) is exceptionally rare and occurs predominantly in the head and neck (92.8% cases). A 2-month-old female patient presented with a mass arising in the fibula which was excised at our surgical center. This is only the eighth case of MNTI affecting an extremity, and the first reported in the fibula. To understand better the etiology of this MNTI, we used high-throughput sequencing technology to carry out an exhaustive genomic, transcriptomic and epigenetic characterization on the excised primary tumor and a derived cell line. Methods: RNA was extracted from the flash frozen tumor and paired-end RNA-Seq was performed to identify potential oncogenic fusion genes and to quantify gene expression in the MNTI transcriptome. Genomic DNA was extracted from the tumor and from a sample of the patient's blood and whole-exome sequencing was done to detect somatic and germline variants. Copy number variation in the MNTI was determined by comparative genomic hybridization (CGH) of DNA from the tumor and blood to a SNP array. A cell line was derived from the tumor and was tested for sensitivity to a panel of compounds targeting epigenetic regulators. Results: Whole-exome analysis indicated no somatic, non-synonymous coding mutations within the tumor, but a heterozygous, unique germline, loss of function mutation in CDKN2A (p16INK4A, D74A). SNP-array CGH revealed the tumor to be euploid, with no detectable gene copy number variants. Multiple chromosomal translocations were identified by RNA-Seq, and fusion genes included RPLP1-C19MC, potentially deregulating the C19MC cluster, an imprinted locus containing microRNA genes reactivated by gene fusion in embryonal tumors with multilayered rosettes. Since the presumed cell of origin of MNTI is from the neural crest, we also compared gene expression with a dataset from human neural crest cells (GEO acession: GSE28875) and identified 185 genes with significantly different expression. Consistent with the melanotic phenotype of the tumor, elevated expression of tyrosinase was observed. Other highly expressed genes encoded muscle proteins and modulators of the extracellular matrix. The derived MNTI cell line was sensitive to inhibitors of lysine demethylase, but not to compounds targeting other epigenetic regulators. Conclusions: In the absence of somatic copy number variations or mutations, the fully transformed phenotype of the MNTI may have arisen in infancy because of the combined effects of a germline CDKN2A mutation, tumor promoting somatic fusion genes and epigenetic deregulation. Very little is known about the etiology of MNTI and this report advances knowledge of these rare tumors by providing the first comprehensive genomic, transcriptomic and epigenetic characterization of a case. Overall design: Genomic DNA was extracted from the tumor and whole-exome and copy number comparative analyses were performed relative to a reference sample of genomic DNA from the patient’s blood (germline DNA). This dataset represents aCGH samples.

### 研究目的
婴儿型黑色素性神经外胚层瘤（Melanotic neuroectodermal tumor of infancy, MNTI）极为罕见，约92.8%的病例发生于头颈部。本次研究对象为一名2月龄女性患者，其腓骨处出现肿物，并于本手术中心完成切除。这是第8例累及肢体的MNTI，也是首例报道的腓骨部位MNTI。为深入阐明该病例的发病机制，我们采用高通量测序（high-throughput sequencing）技术，对切除的原发肿瘤及一株衍生细胞系开展了全面的基因组、转录组与表观基因组特征分析。

### 研究方法
从新鲜冷冻的肿瘤组织中提取RNA，进行双端RNA测序（paired-end RNA-Seq），以识别潜在的致癌融合基因并量化MNTI转录组的基因表达水平。从肿瘤组织及患者血液样本中提取基因组DNA，实施全外显子组测序（whole-exome sequencing）以检测体细胞及生殖系变异。通过肿瘤与血液DNA的SNP阵列（SNP array）比较基因组杂交（comparative genomic hybridization, CGH），确定MNTI的拷贝数变异情况。从肿瘤组织中构建细胞系，并针对一组靶向表观遗传调控因子的化合物进行敏感性测试。

### 研究结果
全外显子组分析显示，肿瘤组织内未检测到体细胞非同义编码突变，但存在CDKN2A（p16INK4A, D74A）的杂合性独特生殖系功能丧失突变。SNP阵列CGH结果显示，肿瘤为整倍体，未检测到可检出的基因拷贝数变异。RNA测序鉴定出多条染色体易位，融合基因包括RPLP1-C19MC，该融合可能导致C19MC基因簇失调——该印记基因座包含在伴多层菊形团的胚胎性肿瘤中因基因融合而激活的微小RNA（microRNA）基因。鉴于MNTI的假定起源细胞为神经嵴细胞，我们还将基因表达谱与人类神经嵴细胞数据集（基因表达汇编（Gene Expression Omnibus, GEO）登录号：GSE28875）进行比对，鉴定出185个表达存在显著差异的基因。与肿瘤的黑色素表型一致，我们观察到酪氨酸酶（tyrosinase）的表达上调。其他高表达基因编码肌肉蛋白及细胞外基质调控因子。衍生的MNTI细胞系对赖氨酸去甲基化酶抑制剂敏感，但对其他靶向表观遗传调控因子的化合物无响应。

### 研究结论
在未检测到体细胞拷贝数变异或突变的情况下，该MNTI的完全转化表型可能在婴儿期发生，其诱因是生殖系CDKN2A突变、促瘤体细胞融合基因及表观遗传失调的共同作用。目前对MNTI的发病机制所知甚少，本研究通过首次对一例病例开展全面的基因组、转录组和表观基因组特征分析，增进了对这类罕见肿瘤的认知。

### 整体实验设计
本研究从肿瘤组织中提取基因组DNA，并以患者血液的基因组DNA（生殖系DNA）作为参照样本，开展全外显子组及拷贝数比较分析。本数据集包含基于阵列的比较基因组杂交（array-based comparative genomic hybridization, aCGH）样本。