Duplications involving the long range HMX1 enhancer are associated with human isolated bilateral concha-type microtia
Duplications involving the long range HMX1 enhancer are associated with human isolated bilateral concha-type microtia
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DOI：
10.1186/s12967-020-02409-6
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发表时间：
2020-06-17
影响因子：
7.4
通讯作者：
Zhang, Xue
中科院分区：
文献类型：
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作者：
Si, Nuo;Meng, Xiaolu;Zhang, Xue
背景小耳畸形是一种先天性耳畸形，其严重程度从轻度结构异常到外耳完全缺失不等。外耳型小耳畸形被认为是一种轻度形式。H6家族同源盒1转录因子基因（HMX 1）在颅面结构发育中起重要作用。Hmx 1下游进化保守增强子区（ECR）的拷贝数变异（CNVs）与耳和眼异常相关，在不同动物中已有报道，但在人类中尚未报道。到目前为止，除了HOXA 2的突变外，还没有报道引起孤立性人类小耳畸形的遗传缺陷。在这里，我们招募了5个中国家庭孤立的双侧外耳型小耳畸形，并试图确定潜在的遗传causes. MethodsSingleNucleotide多态性（SNP）阵列进行映射的疾病位点和检测CNVs的基因组规模主要是在最大的家庭（F1）。进行全基因组测序以筛选候选疾病基因座中的所有SNV和CNV。然后进行阵列比较基因组杂交（aCGH）以检测其他四个家族F2-F5中的CNV。使用定量实时聚合酶链反应（qPCR）验证和确定含有HMX 1-ECR区域的已鉴定CNV的程度。通过gap-PCR和桑格测序鉴定F1和F2中的精确断裂点。双荧光素酶测定用于检测增强子功能。qPCR检测61例其他类型白内障患者的HMX 1-ECR CNVs。结果F1的连锁分析和单倍型分析将疾病位点定位在4p16.1上的1.9Mb区间，该区间包含HMX 1及其下游ECR区域。全基因组测序在HMX 1或候选疾病位点内的其他基因的编码区中未检测到潜在致病性SNV，但在HMX 1和CPZ之间的基因间区域中检测到94.6 Kb重复。aCGH和qPCR也显示了其他4个家系中HMX 1下游基因间区的共分离重复。21.8 Kb的最小重叠区域包含与Hmx 1的小鼠ECR共有的核心序列。荧光素酶分析证实了人序列中的增强子功能，并证明HOXA 2可以增加其增强子活性。在61例其他类型的小耳畸形患者的HMX 1-ECR区域中未检测到CNVs。结论涉及长距离HMX 1增强子的重复与人孤立性双侧外耳型小耳畸形相关。我们增加了人类HMX 1-ECR拷贝数变异与耳畸形相关的证据，与其他物种一样。这项研究还提供了人类功能保守非编码元件（CNE）的另一个例子。
BackgroundMicrotia is a congenital anomaly of ear that ranges in severity from mild structural abnormalities to complete absence of the outer ears. Concha-type microtia is considered to be a mild form. The H6 family homeobox 1 transcription factor gene (HMX1) plays an important role in craniofacial structures development. Copy number variations (CNVs) of a downstream evolutionarily conserved enhancer region (ECR) of Hmx1 associated with ear and eye abnormalities have been reported in different animals, but not yet in human. To date, no genetic defects responsible for isolated human microtia has been reported except for mutations in HOXA2. Here we recruited five Chinese families with isolated bilateral concha-type microtia, and attempt to identify the underlying genetic causes.MethodsSingle Nucleotide polymorphism (SNP) array was performed to map the disease locus and detect CNVs on a genome scale primarily in the largest family (F1). Whole genome sequencing was performed to screen all SNVs and CNVs in the candidate disease locus. Array comparative genomic hybridization (aCGH) was then performed to detect CNVs in the other four families, F2-F5. Quantitative real-time polymerase chain reaction (qPCR) was used to validate and determine the extent of identified CNVs containing HMX1-ECR region. Precise breakpoints in F1 and F2 were identified by gap-PCR and sanger sequencing. Dual-luciferase assays were used to detect the enhancer function. qPCR assays were also used to detect HMX1-ECR CNVs in 61 patients with other types mictrotia.ResultsLinkage and haplotype analysis in F1 mapped the disease locus to a 1.9 Mb interval on 4p16.1 containing HMX1 and its downstream ECR region. Whole genome sequencing detected no potential pathogenic SNVs in coding regions of HMX1 or other genes within the candidate disease locus, but it detected a 94.6 Kb duplication in an intergenic region between HMX1 and CPZ. aCGH and qPCRs also revealed co-segregated duplications in intergenic region downstream of HMX1 in the other four families. The 21.8 Kb minimal overlapping region encompassing the core sequences consensus with mouse ECR of Hmx1. Luciferase assays confirmed the enhancer function in human sequences, and proved that HOXA2 could increase its enhancer activity. No CNVs were detected in HMX1-ECR regions in 61 patients with other type of microtia.ConclusionDuplications involving long range HMX1 enhancers are associated with human isolated bilateral concha-type microtia. We add to evidences in human that copy number variations in HMX1-ECR associates with ear malformations, as in other species. This study also provides an additional example of functional conserved non-coding elements (CNEs) in humans.