Abstract
To explore the genetic causes of 3 male infertility patients with acephalospermia and the outcome of assisted reproductive technology. Clinical diagnosis, sperm morphology examination, sperm transmission electron microscopy examination were performed on 3 patients, and the whole exome sequencing technology was used for screening, Sanger sequencing verification, mutation pathogenicity analysis, and protein sequence homology comparison. Assisted reproductive technology was implemented to assist pregnancy treatment. The 3 patients were all sporadic infertile men, aged 25, 42 and 26 years, and there was no obvious abnormality in the general physical examination. Male external genitalia developed normally, bilateral testicles were normal in volume, and bilateral epididymis and spermatic vein were palpated without nodules, cysts, and tenderness. Repeated semen analysis showed that a large number of immature sperm could be seen, and they had the ability to move. The SUN5 gene of the 3 male infertile patients was a case of homozygous missense mutation c.7C>T (p.Arg3Trp), a case of compound heterozygous missense mutation c.1067G>A (p.Arg356His) and nonsense mutation c.216G>A (p.Trp72*) and a case of homozygous missense mutation c.1043A>T (p.Asn348Ile), of which c.7C>T (p.Arg3Trp) and c.1067G>A (p.Arg356His) were new variants that had not been reported. SIFT, Mutation Taster and PolyPhen-2 software function prediction results were all harmful, the nonsense mutation c.216G>A (p.Trp72*) led to the premature termination of peptide chain synthesis which might have a greater impact on protein function. The homology regions in the protein sequence homology alignment were all highly conserved.The 3 male patients and their spouses obtained 4 biological offspring through intracytoplasmic sperm injection, all of which were boys, and one of them was a twin.Three male infertile patients might be caused by SUN5 gene mutations. Such patients could obtain their biological offspring through assisted reproductive technology. It was still necessary to pay attention to the genetic risk of ASS, it was recommended that both men and women conduct genetic counseling and screening at the same time. In clinical diagnosis, whole exome sequencing technology could be used to perform auxiliary examinations to determine the treatment plan and assisted reproductive methods as soon as possible to reduce the burden on the family and society. The newly discovered mutation sites of SUN5 gene provided clues and directions for elucidating the pathogenic mechanism, and at the same time expanded the pathogenic mutation spectrum of ASS.
Keywords: Acephalic spermatozoa syndrome, SUN5 gene, Whole exome sequencing, Gene mutation, Assisted reproductive technology
畸形精子症是男性不育的常见原因之一[1]，其中无头精子症(acephalic spermatozoa syndrome，ASS)是一种严重导致男性不育的罕见畸形精子症，其特征是精液中含有无头精子尾部和一些松散的精子头部，主要表现为头颈部连接处异常[2-3]，精子在电子显微镜下通常显示精子头部和尾部之间缺少植入窝和基板[4]。ASS有2种亚型：一种类型表现为没有细胞核的小“针头样”精子, 另一种类型是头颈部错误连接的精子[3, 5]。从临床上疾病的家族聚集性和罕见的近亲家系导致发病等遗传特征分析，考虑ASS可能为常染色体隐性遗传性疾病，已有研究表明SUN5双等位基因变异可导致ASS[6]。由于目前报道的ASS病例数较少，SUN5基因的致病变异谱尚不完整，基因型与表型之间的相关性仍不明确。本研究回顾性分析河南省人民医院生殖中心发现的3例SUN5基因变异导致的ASS，其中有2个位点是未见报道的新发变异。结合临床表型、精子形态学检查、精子透射电镜检查，同时使用全外显子组测序(whole exome sequencing，WES)技术筛查、Sanger测序验证和变异致病性分析进行研究，探索SUN5基因引起ASS的发病机制及辅助生殖助孕治疗的结局。
1. 病例资料
1.1. 临床资料和方法
回顾性分析2015年8月至2019年5月就诊于河南省人民医院生殖中心男科的3例男性不育患者，包括家族史、临床表型、实验室检查及辅助生殖技术助孕等临床信息。
1.2. 精液分析和精子形态学检测
禁欲3~7 d后手淫法取精液于干燥的无菌容器内，置37 ℃恒温操作台上静置60 min以上，待精液液化后采用西班牙全自动精子质量分析仪分析。使用“拉薄”技术直接涂片，采用世界卫生组织(World Health Organization, WHO)第五版改良巴氏染色法染色。参照WHO第五版精子形态判读标准，每份样本由专业检测人员人工镜下分析至少200条精子，计算正常形态精子率、头部、颈部、尾部畸形率。
1.3. 透射电镜观察精子超微结构
取患者及正常对照组的精液样本，3 000×g离心10 min，弃上清，加入2.5%(体积分数)的戊二醛固定24 h以上，磷酸盐缓冲液洗涤4次，每次10 min，1%(体积分数)锇酸固定90 min，磷酸盐缓冲液洗涤4次，每次10 min，然后依次用质量分数为30%、50%、70%、90%丙酮洗涤，每次10 min，用100%丙酮洗涤3次，每次10 min，加入812树脂，37 ℃过夜聚合，用莱卡EMUC7超薄切片机切片，取切片在日本JEOLJEM-1400透射电镜下观察和拍照。
1.4. 基因组DNA提取和全外显子组测序与Sanger测序验证
在患者知情同意下留取血液2 mL，通过基因组DNA提取试剂盒(北京全式金生物技术公司)提取样本DNA。将基因组DNA打断成随机片段，纯化后进行全基因组外显子捕获和富集, 并制备基因组DNA文库，然后使用Illumina Hiseq 2500测序仪进行高通量测序，结合OMIM、HGMD、1000Genome、GenomAD等数据库收录的致病基因和变异的信息对检出的变异进行分析。利用蛋白质功能预测软件(SIFT、Mutation Taster和PolyPhen-2软件)对该基因变异致病性进行预测分析，对检出的可能致病变异进行Sanger测序验证。
1.5. 蛋白序列同源性比对
查找Uniprot和Ensembl数据库中该基因的人类及其他哺乳动物的蛋白序列，使用DNAMAN软件进行蛋白序列同源性比对。
1.6. 辅助生殖技术助孕治疗
3例男性不育患者及配偶均签字同意接受辅助生殖技术助孕治疗。对其配偶实施促排卵治疗，待卵细胞发育成熟后经阴道超声引导下行取卵术。取卵日男方手淫取精后送体外受精(in vitro fertilization, IVF)实验室镜检，挑选形态相对正常的成熟精子进行卵胞浆内单精子注射(intracytoplasmic sperm injection，ICSI)治疗。受精卵在培养箱中培养3~5 d，选择发育良好的胚胎进行移植，如果因卵巢过度刺激综合征等因素取消新鲜移植周期，则实施冷冻胚胎的复融移植，观察并记录相应的胚胎实验室数据，随访并记录女方移植后的妊娠结局。
1.7. 临床诊断与家系分析
3例患者均为散发的不育男性，年龄分别为25、42、26岁，3例患者的一般体格检查无明显异常。男性外生殖器发育正常，双侧睾丸体积正常，触诊双侧附睾及精索静脉无结节、囊肿和压痛。多次精液分析均提示可见大量幼稚精子，具有活动能力。性激素水平正常，染色体核型为46XY，生精基因AZF未见缺失，且患者父母均否认出现类似病史，否认近亲结婚，家族中也未发现表型类似成员。
1.8. 精子改良巴氏染色涂片
3例患者的精子经改良巴氏染色后显微镜下观察均可见大量无头精子，99%无头畸形，0.9%颈部畸形(图 1)。
图 1.
正常形态精子和1例患者经改良巴氏染色的无头精子形态
Morphology of normal sperm and a patient of acephalous sperm with modified pasteurization stain
A, sperm morphology of normal control group; B, C, D, sperm morphologies of a patient (modified Pap staining, ×200)
1.9. 透射电子显微镜观察精子超微结构
透射电子显微镜下正常的精子超微结构包括顶体、细胞核、近远端中心粒、线粒体鞘以及“9+2”尾丝结构等(图 2A), 一个无头精子尾部包括近远端中心粒、节段柱、线粒体鞘等结构(图 2B), 一个无尾的精子包含细胞核结构但缺乏植入窝、基板和尾部(图 2C), 一个精子首尾异常分离表明, 头部包含核和几乎正常形态的顶体，尾部有正常排列的轴索，但缺乏核尾极的植入窝和基板(图 2D)。
图 2.
正常精子超微结构和1例患者的精子超微结构
Ultrastructure of normal sperm and the ultrastructure of a patient's sperm
A, sperm ultrastructure of the normal contral group; B, C, D, sperm ultrastructures of a patient (×20 000, 80 kV)
1.10. 全外显子组测序及变异的Sanger验证
3例患者均存在SUN5(NM_080675.3)基因变异，分别为1例纯合错义变异位点c.7C>T(p.Arg3Trp)、1例错义变异位点c.1067G>A(p.Arg356His)和无义变异位点c.216G>A(p.Trp72*)的复合杂合变异、1例纯合错义变异c.1043A>T(p.Asn348Ile)。Sanger测序验证结果表明患者携带的基因变异分别来自父母双方。
1.11. 变异致病性预测分析
对3个错义变异位点c.7C>T(p.Arg3Trp)、c.1067G>A(p.Arg356His)、c.1043A>T(p.Asn348Ile)的致病性进行预测，SIFT、Mutation Taster和PolyPhen-2软件功能预测结果均为有害(表 1); 而无义变异位点c.216G>A(p.Trp72*)导致肽链合成提前终止，对蛋白功能影响可能较大。
表 1.
3位患者SUN5基因变异的致病性评价
Pathogenicity prediction of SUN5 gene mutation in 3 patients
| Cases | Mutations | Amino acid change | Zygosity | SIFT | Mutation Taster | Polyphen-2 |
| Hom, Homozygous; Het, Heterozygous; NA, not available. | ||||||
| 1 | c.7C>T | p.Arg3Trp | Hom | Damaging | Disease causing | Probably damaging |
| 2 | c.1067G>A | p.Arg356His | Het | Damaging | Disease causing | Probably damaging |
| c.216G>A | p.Trp72* | Het | NA | Disease causing | NA | |
| 3 | c.1043A>T | p.Asn348Ile | Hom | Damaging | Disease causing | Probably damaging |
1.12. 变异位点的保守性分析
3例患者检出的错义变异位点p.Arg3Trp、p.Arg356His和p.Asn348Ile在人类和其他哺乳动物同源蛋白的相同位点高度保守，发生的错义变异极有可能对蛋白功能有害。
1.13. 辅助生殖技术助孕结局
3例男性患者及配偶同意接受ICSI助孕治疗，均成功生育血亲后代(表 2)。
表 2.
3例患者的基本参数和ICSI实验室数据
Basic parameters and ICSI laboratory data of 3 patients
| Cases | Male age/years | MⅡ egg, n | D3 embryo, n | Transferred embryos, n | Clinical pregnancy | Live birth, n |
| ICSI, intracytoplasmic sperm injection; MⅡ, metaphase Ⅱ. | ||||||
| 1 | 25 | 9 | 6 | 1 | Yes | 1 |
| 2 | 42 | 7 | 3 | 2 | Yes | 1 |
| 3 | 26 | 12 | 3 | 2 | Yes | 2 |
2. 讨论
目前，不孕不育是影响全球人类健康的主要医学和社会问题。大约8%~18%的育龄夫妇面临生育问题[7]，而男性因素、尤其是异常的精子发生和射精障碍占59%。精子形态与其功能密切相关，精子形态上的任何缺陷都可能会导致其功能异常[8]。无头精子症早期也被称为针形精子症或断头精子症，大部分精子头部滞留在睾丸内。在精液中大部分精子都是不包含遗传物质的尾部，实际上是完全无法自然受精的[9]。
人的SUN5基因全长约22.7 kb，包含13个外显子，定位于20q11.21，编码的SUN5蛋白含有379个氨基酸，有一个SUN结构域，特异表达于睾丸组织[10], 定位在成熟精子头颈部连接区域。SUN5基因是第一个被人类发现的ASS的致病基因，随后又相继发现致病基因PMFBP1、TSGA10。SUN蛋白有5种，所有的SUN蛋白都存在于生精细胞中，其表达和定位受到严格的控制。SUN5蛋白是在减数分裂的早期产生的，其特征是C-末端为保守的SUN结构域，N-末端为跨膜区域，中间为卷曲螺旋区域，一般通过与KASH(Klarsicht, ANC-1, and Syne/Nesprin homology)结构域结合形成核骨架和细胞骨架的连接子(linker of nucleoskeleton and cytoskeleton，LINC)复合体[11]，参与细胞核的锚定[12]、迁移[13]和精子发生等过程[14]。SUN5蛋白定位于核膜及植入窝区域，可能参与头尾黏连作用(将耦合装置与精子头部细胞核膜连接)。在附睾的精子中，SUN5蛋白位于头部与尾部之间的交界处，通过高尔基体的转运完成翻译后修饰[15]。SUN5基因的变异导致了SUN5蛋白严重减少或缺失，某些变异可以改变SUN5蛋白在精子头尾交界处的分布，从而导致头尾之间被截断。Zhu等[6]研究结果表明，双等位基因SUN5的变异能够导致人无头精子症，从而引起男性不育，为常染色体隐性疾病。动物研究也发现SUN5定位于精子颈部，锚定精子头和尾部。经过小鼠模型中的精子超微结构观察发现，SUN5缺失后，尽管精子头尾连接耦合装置(head to tail coupling apparatus, HTCA)可以正常组装，但在精子变形之初HTCA就从精子核膜植入窝处分离，随着精子不断延长，最终HTCA连同精子尾巴一起从精子头部脱落，产生没有受精功能的无头精子。
目前，SUN5基因变异相关的ASS均符合常染色体隐性遗传规律，等位基因上存在两个有害变异即可能致病。本文报道的3例ASS均存在SUN5基因上的致病变异，分别为两个纯合变异和一个复合杂合变异，包括两个未报道的新发错义变异位点c.7C>T(p.Arg3Trp)和c.1067G>A(p.Arg356His)，两个已报道的致病变异位点c.216G>A(p.Trp72*)和c.1043A>T(Asn348Ile)[6]，以上位点在正常人群中变异率极低，其中c.216G>A(p.Trp72*)变异位点为无义变异，编码第72位氨基酸Trp的密码子变为终止密码子，预测可能导致肽链的合成中断或发生无义介导的mRNA降解，造成严重的蛋白功能缺失[6]; 第348和356位氨基酸位于SUN5蛋白的SUN结构域内，且第356位氨基酸存在已知的另一变异p.Arg356Cys，若该结构域内发生基因变异，可能严重破坏SUN5蛋白与其他相邻蛋白的相互作用，如KASH或LINC蛋白[6]。3个错义变异位点同源蛋白的氨基酸残基高度保守，保证蛋白质功能正常，变异致病性预测均为有害，以上结果均支持c.7C>T(p.Arg3Trp)和c.1067G>A(p.Arg356His)可能为ASS新发现的致病变异位点。综合以上遗传学分析得出，SUN5基因变异极有可能导致人类ASS的发生，同时新发现的变异位点也进一步丰富了SUN5基因在ASS患者中的致病变异谱。
ASS患者精子主要表现为头尾分离或头颈部异常，实际上无头精子不含遗传物质，但研究表明头尾部连接异常的精子可能不会影响受精率和临床妊娠，大多数ASS患者通过ICSI可以实现妊娠[16-17]。本研究3例患者的配偶均行该基因相应变异位点的验证，均未发现相同基因位点变异，通过ICSI治疗均成功生育亲生子代。即使SUN5基因变异患者通过ICSI治疗可具有良好的妊娠结局，仍然需要注意ASS的遗传风险，在进行辅助生殖前，建议男女双方同时进行遗传咨询和筛查。在临床诊断时可通过WES技术进行辅助检查，及早确定治疗方案和辅助生殖助孕方式，减轻家庭和社会的负担。新发现的SUN5基因变异位点为阐明致病机制提供了线索和方向，同时也扩大了ASS的致病变异谱。
Funding Statement
北京市自然科学基金(7182177)、国家重点研发计划(2018YFC1004202)、河南省医学科技攻关计划省部共建项目(SBGJ202001002)、河南省医学科技攻关计划省部共建项目(SBGJ202002003)
Supported by the Beijing Municipal Natural Science Foundation (7182177), National Key Research and Development Project (2018YFC1004202), the Joint Construction Project of Henan Medical Science and Technology Project (SBGJ202001002), the Joint Construction Project of Henan Medical Science and Technology Project (SBGJ202002003)
Contributor Information
洪 锴 (Kai HONG), Email: kenhong99@hotmail.com.
郭 海彬 (Hai-bin GUO), Email: 13673631683@163.com.
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