目的

先天性小耳畸形是体表重大畸形，严重影响患者的外貌和心理健康，进行小耳畸形发病特征和相关遗传学研究意义重大。本研究探讨中国小耳畸形患者伴发畸形的发生情况和特点，分析之间的关联；经过对家族史的详细调查，了解小耳畸形患者的遗传特征；在小耳畸形患者中进行候选基因序列变异和拷贝数变异检测，对变异进行生物信息学分析。通过以上研究逐步深化对小耳畸形的认知，可作为今后小耳畸形分子机制的研究和防治的基础。

方法

1.在基于医院的研究中选取2014年12月-2016年2月间小耳畸形患者，对所有非综合征型小耳畸形患者672例进行详细地体格检查，并记录相关伴发畸形。将伴发畸形根据受影响的主要器官系统进行分类，采用SPSS 19.0统计学软件分析小耳畸形与伴发畸形间的关系。并与其他国家地区的小耳伴发畸形资料对比分析中国小耳群体患病特点。

2.对上述672个患者通过查体、直接询问及电话随访的方法获得患者的家系资料及耳部畸形发病情况并登记。主要调查对象为先证者、一级亲属、二级亲属、三级亲属、四级亲属，按照小耳畸形患者是否具有耳部畸形家族史背景分为家族史组和散发组，对数据进行统计学分析，探寻有价值的规律。

3. 随机选取200例散发小耳畸形患者对六个候选基因（HOXA1、HOXA2、EYA1、SIX1、SALL1、FGF3）进行序列变异和拷贝数变异检测，并利用生物信息学分析变异的危害。

结果

    1.本研究显示了男性（72%）、单侧（92.9%）和右侧（63%）多发，与文献中的结果一致。我们在293（43.6%）位患者中发现了一项及以上的伴发畸形，尤其是耳面颈系统、肌肉骨骼系统、心血管系统伴发率最高。耳廓发育程度越差伴发畸形率越高。小耳不同伴发畸形比例有显著异质性，但大体在器官系统畸形方面的权重是相符的，即伴发畸形最常累及的是耳面颈系统，接下来是肌肉骨骼系统，然后是心血管系统，后面是泌尿生殖系统，呼吸系统、神经系统、消化系统等较少累及。

2. 本研究中耳部畸形家族史阳性率为34.1%，其中一级亲属所占比例最高，二、三、四级亲属比例依次减少，符合遗传学规律，且父母两系背景在各级小耳畸形患者人数构成比无统计学差异。有33.8%小耳畸形患者伴发附耳/瘘管，且这部分患者比单纯小耳患者有更高的家族耳部畸形发生率。

3.在200例患者中，鉴定出1例患者HOXA2基因的错义突变（c.260C>T, p.A87V），此突变不存在于患者父母，为新发突变，在1000 human genome、 HGMD、ESP6500、 dbSNP和ExAC等数据库中未查到相关记录，经生物信息学分析该突变高度保守，推测其具有致病性。检测到两例患者存在EYA1基因的拷贝数缺失，推测为小耳畸形的致病原因。

结论

1.本研究是中国小耳患者中第一份有关伴发畸形的详细专题性研究，小耳患者的伴发畸形率较高，且临床异质性较大，为了探索小耳畸形病因及为患者提供更好的治疗，应加大未来对相关伴发畸形的研究力度。

2. 本研究结合其他耳部畸形多发性，将家属耳部畸形纳入研究并证实耳部畸形家族史发生率为34.1%，且存在垂直传递、隔代遗传和家族聚集的遗传特征。在研究小耳畸形遗传特征时不应忽略亲属中的其他耳畸形。虽然小耳畸形目前发病机制不明，但遗传因素不容忽视。

3.本研究分别对200例散发小耳患者六个候选基因进行了序列变异和拷贝数变异检测，成功地鉴定了一例患者存在HOXA2基因编码区的c.260C>T（p.A87V）突变，此突变位于exon1,为国际首次报道，扩增了小耳畸形HOXA2基因突变谱；并且在两例患者中检测到EYA1基因拷贝数缺失。这些发现可能是小耳畸形的致病因素，其发病机制，有待下一步研究验证。

Objective

Microtia is a major surface malformation that can seriously affect the patients’appearance and mental health.It is of great significance to study the characteristics of microtia and related genetics.The aim of this study is to have a better understanding of the abnormality phenotypes that co-occur with microtia and analyze if there is a singnificant relationship between microtia and other deformities in Chinese population. Carrying out a detailed survey of family history to understand the genetic characteristics of patients with microtia.Using candidate genes mutation and copy number variation detecting in microtia patients to detect new varions and carrying out bioinformatics analysis.Through the above research,we will gradually deepen the cognition of microtia,which can be used as the basis for the research and prevention of molecular mechanism of microtia in the future.

Methods

1.A total of 672 patients with microtia were collected from December 2014 to February 2016 in the hospital-based study. All patients were examed in detail and classified into three types of microtia,with associated anomalies detected and recorded. The cases with associated anomalies were sub-classified according to the main organ system affected.Spss version 19.0 was used to verify the relationship between microtia and associated anomalies.And comparing with other countries’ associated anomalies data,to analyze the charateristics of the Chinese patients.

2.Through the examination, we used direct inquiry and telephone follow-up method in the 672 patients above for obtaining and registering the information and family incidence of ear deformities. The main investigators were the probands, the first-degree relatives, the second-degree relatives, the third-degree relatives and the fourth-degree relatives.It was divided into familial group and sporadic group according to the background of the ear malformations of the patients’relatives to explore the potential value.

3. Randomly selecting 200 cases of patients with sporadic microtia. Mutation detection and copy number mutation detection of six candidate genes（HOXA1、HOXA2、EYA1、SIX1、SALL1、FGF3）were performed in the patients. The bioinformatic was used to analyze the variation.

Results

1.Showing preponderances of male (72%), unilateral (92.9%) and right-sided (63%),consistent with prior reports in the literature.We identfied that 293 patients (43.6%) were documented with  one or more associated anomalies,especially the ear, face and neck,musculoskeletal,card-iovascular system were the most common associated anomalies. The poorer one auricle developed，the higher the incidence of associated anomalies.These studies exhibited noteworthy heterogeneity in the proportion of associated congenital anomalies,but was largely consistent with the weight of main organ system malformations.For example,generally the highest incidence of concurrent malformations was ear,face and neck system, followed by musculoskeletal system, cardiovascular system,followed by urogenital system,ranked behind were respiratory system, nervous system, digestive system.

  2. In this study,the proportion of the patients who had auricle deformity familial history was 34.1%, among which the proportion of first-degree relatives was the highest, and the proportion of second, third and fourth-degree relatives decreased,according with the laws of genetics. Parents of two-line background levels of  patients with microtia constituted no significant difference in the ratio. There were 33.8% microtia patients associate with tags / fistula, and these patients had a higher incidence of familial history than the patients without tags / fistula.

3. In one of the 200 patients, a missense mutation (c.260C> T, p.A87V) of the HOXA2 gene was identified.The mutation was not present in the parents, and the mutation was not found in 1000 human genome, HGMD, the ESP6500 , dbSNP, ExAC database.Bioinformatics analysis of the mutation was highly conserved, and presumably pathogenic. Two copies of the EYA1 gene were found to be deletion presumed to be pathogenic.

Conclusion

1. This was the first detailed and specific study of associated anomalies with microtia  in Chinese patients.The incidence of associated deformities was high in patients with microtia,and existing  great clinical heterogeneity among them.We should pay more attention to investigate associated congenital anomalies for future studies exploring the etiology of microtia and provide better treatment for patients.

2. In this study, the occurrence of familial history of ear malformations was 34.1%, and there were genetic characteristics of vertical transmission, generation inheritance and family aggregation. Other ear deformities in relatives should not be overlooked when studying the genetic characteristics of microtia. Although the current pathogenesis of microtia was unknown, but genetic factors can not be ignored.

3.In this study,200 patients carried out candidate genes mutation and copy number variation detecting,and successfully identified the HOXA2 c.260C> T (p.A87V) mutation in the coding region of a unilateral sporadic patient. Differenced with previous studies in the  family cases, this mutation was located in exon1.Our data expanded the spectrum of HOXA2 mutations. And the deletion of EYA1 gene copy number were detected in two patients. These findings may be the causative factors, although not clear the specific mechanism,experimental verification was necessary.