【作者】 王琳；

【导师】 罗红；

【作者基本信息】 中南大学 ， 临床医学（专业学位）， 2024， 硕士

【摘要】 背景:原发性纤毛运动障碍(Primary Ciliary Dyskinesia,PCD)是一种以基因突变所致纤毛运动障碍为特征的罕见的遗传性疾病。RSPH4A作为编码纤毛放射辐头部的基因之一,普遍表达于纤毛及精子鞭毛,是导致PCD的基因之一。RSPH4A蛋白在纤毛运动中发挥着重要作用。既往研究表明RSPH4A双等位基因突变与女性不孕相关,但目前尚无研究证实RSPH4A双等位基因突变与弱畸精子症相关。目的:明确携带RSPH4A双等位基因突变PCD患者的临床表型,以及携带RSPH4A双等位基因突变男性PCD患者是否存在弱畸精子症。方法:(1)获取患者的临床信息;(2)进行DNA萃取、随后进行全外显子区域的测序,并利用双脱氧链终止测序法证实RSPH4A基因中的变异,借助生信分析软件推测RSPH4A变异位点的致病性;(3)巴氏染色分析精子鞭毛形态与计算机辅助精子软件分析精子运动特征;(4)高速视频显微成像(High-speed video microscopy,HSVM)观察及分析患者和健康对照的鼻纤毛摆动模式和摆动频率;(5)通过免疫荧光(Immunofluorescence,IF)技术,检测鼻纤毛和精子鞭毛RSPH4A及放射辐头部结构蛋白RSPH1的表达情况。结果:(1)共收集了三个携带RSPH4A突变的PCD患者的家系,三位先证者均表现为超过10年的咳嗽、咳痰和鼻塞,肺部高分辨CT均提示支气管扩张,其中患者F1-III-1和患者F2-IV-2经鼻呼出气一氧化氮(Nasal nitricoxide,n NO)流速明显降低。两位女性患者F2-IV-2和F3-IV-5均表现为不孕;男性患者F1-III-1的精液分析结果提示前向运动精子比例低于正常参考值,精子的巴氏染色结果提示为精子鞭毛多发形态异常。(2)全外显子测序提示患者F1-III-1携带RSPH4A新复合杂合突变c.2T>C,p.(Met1Thr)和c.1774＿1775del,p.(Leu592Aspfs*5),患者F2-IV-2携带RSPH4A新纯合突变c.2T>C,p.(Met1Thr),患者F3-IV-5携带RSPH4A新纯合突变c.351dup T,p.(Pro118Serfs*2),以上突变均经双脱氧链终止测序法验证。四大生信分析软件SIFT、Polyphen 2、PROVEAN和CADD预测上述突变均为致病性突变。(3)HSVM提示患者F1-III-1与患者F3-IV-5的鼻纤毛呈旋转摆动模式,患者F1-III-1的纤毛摆动频率较健康对照减慢(患者F1-III-1 6.47(四分位距,5.85-10.25)Hz VS健康对照11.35(四分位距,9.9-12.8)Hz,P值<0.05),患者F3-IV-5的纤毛摆动频率与健康对照大致相同(患者F3-IV-5 11.23(四分位距,9.89-12.08)Hz VS健康对照11.35(四分位距,9.9-12.8)Hz,P值>0.05)。(4)IF结果显示患者F3-IV-5的鼻纤毛和患者F1-III-1的精子鞭毛中,RSPH4A及RSPH1表达均缺失。结论:携带RSPH4A双等位基因突变可能导致男性PCD患者不育,表现为弱畸精子症,我们的研究可能有助于扩宽PCD和弱畸精子症相关的候选基因图谱,并有助于更全面的遗传咨询。更多还原

【Abstract】 Background:Primary Ciliary Dyskinesia(PCD)is a rare inherited disease in which genetic mutations impair cilia function.As an important encoding gene of radial spokes in cilia,RSPH4A is ubiquitously found in both cilia and sperm flagella,and it contributes to the causation of PCD.The RSPH4A protein plays a crucial role in maintaining the normal functioning of motile cilia.Previous studies have demonstrated the RSPH4A biallelic mutation was associated with female infertility,but asthenoteratozoospermia related to the biallelic mutation of the RSPH4A has not been reported.Objective:To clarify the clinical phenotype of PCD patients carrying RSPH4A biallelic mutations,and to determine whether male PCD patient carrying RSPH4A mutations present asthenoteratozoospermia.Methods:(1)Collecting clinical data of patients.(2)DNA extraction,whole exome sequencing and Sanger sequencing were used to verify the RSPH4A variants,and bioinformatics analysis was employed to predict the pathogenicity of the RSPH4A mutations.(3)Papanicolaou staining and computer-aided sperm analysis were performed to analyze the morphology and motility of the sperm of patient F1-III-1.(4)High-speed video microscopy analysis(HSVM)was performed to observe and analyze the beating pattern and frequency of the nasal cilia of patients and healthy control.(5)Utilizing immunofluorescence(IF)methodology,we conducted an examination of expression patterns of RSPH4A in both nasal cilia and sperm flagella,as well as RSPH1,a constituent protein of the radial spoke head.Results:(1)Three families of PCD patients with RSPH4A mutations were collected.All three patients presented over 10 years of history of chronic cough,yellowish purulent and daily nasal congestion.The high-resolution computed tomography(HRCT)of three patients all showed bronchiectasis.The nasal nitric oxide level was markedly reduced both in patient F1-III-1 and patient F2-IV-2.Both female patients F2-IV-2and patient F3-IV-5 presented infertility.The semen analysis indicated that the proportion of progressively motile sperm was below the standard reference range and papanicolaou staining revealed morphological abnormalities in the sperm of patient F1-III-1.(2)Whole exome sequencing showed that the patient F1-III-1 carried novel compound heterozygous RSPH4A variants c.2T>C,p.(Met1Thr)and c.1774＿1775del,p.(Leu592Aspfs*5),patient F2-IV-2 carried novel homozygous RSPH4A variant c.2T>C,p.(Met1Thr)and patient F3-IV-5carried novel homozygous RSPH4A variant c.351dup T,p.(Pro118Serfs*2).The above mutations were confirmed through Sanger sequencing.Bioinformatics tools,including SIFRT,Polyphen 2,PROVEAN,and CADD predicted these mutations to be pathogentic.(3)Rotational movement of the cilia was observed in patient F1-III-1and patient F3-IV-5.HSVM of nasal brush biopsy samples indicated that the nasal epithelial ciliary beat frequency of patient F1-III-1 was slower than the healthy control(F1-III-1 6.47(IQR,5.85-10.25)Hz VS normal control 11.35(IQR,9.9-12.8)Hz,P<0.05),and the nasal epithelial ciliary beat frequency of patient F3-IV-5 was not significantly different from the healthy control(patient F3-IV-5 11.23(IQR,9.89-12.08)Hz VS normal control 11.35(IQR,9.9-12.8)Hz,P>0.05).(4)IF revealed the absence of RSPH4A and RSPH1 expression in the nasal cilia of patient F3-IV-5 and sperm flagella of patient F1-III-1.Conclusion:It is confirmed that male patient with PCD carrying RSPH4A biallelic mutation present asthenoteratozoospermia and our study may help to expand the candidate gene spectrum related to PCD and asthenoteratozoospermia,and contribute to more comprehensive genetic counseling.更多还原