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Abstract：
BACKGROUND: Primary ciliary dyskinesia (PCD) is an autosomal recessive hereditary disease characterized by recurrent respiratory infections. In clinical manifestations, DNAH5 (NM_001361.3) is one of the recessive pathogenic genes. Primary familial brain calcification (PFBC) is a neurodegenerative disease characterized by bilateral calcification in the basal ganglia and other brain regions. PFBC can be inherited in an autosomal dominant or recessive manner. A family with PCD caused by a DNAH5 compound heterozygous variant and PFBC caused by a MYORG homozygous variant was analyzed.
METHODS: In this study, we recruited three generations of Han families with primary ciliary dyskinesia combined with primary familial brain calcification. Their clinical phenotype data were collected, next-generation sequencing was performed to screen suspected pathogenic mutations in the proband and segregation analysis of families was carried out by Sanger sequencing. The mutant and wild-type plasmids were constructed and transfected into HEK293T cells instantaneously, and splicing patterns were detected by Minigene splicing assay. The structure and function of mutations were analyzed by bioinformatics analysis.
RESULTS: The clinical phenotypes of the proband (II10) and his sister (II8) were bronchiectasis, recurrent pulmonary infection, multiple symmetric calcifications of bilateral globus pallidus and cerebellar dentate nucleus, paranasal sinusitis in the whole group, and electron microscopy of bronchial mucosa showed that the ciliary axoneme was defective. There was also total visceral inversion in II10 but not in II8. A novel splice variant C.13,338 + 5G > C and a frameshift variant C.4314delT (p. Asn1438lysfs *10) were found in the DNAH5 gene in proband (II10) and II8. c.347_348dupCTGGCCTTCCGC homozygous insertion variation was found in the MYORG of the proband. The two pathogenic genes were co-segregated in the family. Minigene showed that DNAH5 c.13,338 + 5G > C has two abnormal splicing modes: One is that part of the intron bases where the mutation site located is translated, resulting in early translation termination of DNAH5; The other is the mutation resulting in the deletion of exon76.
CONCLUSIONS: The newly identified DNAH5 splicing mutation c.13,338 + 5G > C is involved in the pathogenesis of PCD in the family, and forms a compound heterozygote with the pathogenic variant DNAH5 c.4314delT lead to the pathogenesis of PCD.
METHODS: In this study, we recruited three generations of Han families with primary ciliary dyskinesia combined with primary familial brain calcification. Their clinical phenotype data were collected, next-generation sequencing was performed to screen suspected pathogenic mutations in the proband and segregation analysis of families was carried out by Sanger sequencing. The mutant and wild-type plasmids were constructed and transfected into HEK293T cells instantaneously, and splicing patterns were detected by Minigene splicing assay. The structure and function of mutations were analyzed by bioinformatics analysis.
RESULTS: The clinical phenotypes of the proband (II10) and his sister (II8) were bronchiectasis, recurrent pulmonary infection, multiple symmetric calcifications of bilateral globus pallidus and cerebellar dentate nucleus, paranasal sinusitis in the whole group, and electron microscopy of bronchial mucosa showed that the ciliary axoneme was defective. There was also total visceral inversion in II10 but not in II8. A novel splice variant C.13,338 + 5G > C and a frameshift variant C.4314delT (p. Asn1438lysfs *10) were found in the DNAH5 gene in proband (II10) and II8. c.347_348dupCTGGCCTTCCGC homozygous insertion variation was found in the MYORG of the proband. The two pathogenic genes were co-segregated in the family. Minigene showed that DNAH5 c.13,338 + 5G > C has two abnormal splicing modes: One is that part of the intron bases where the mutation site located is translated, resulting in early translation termination of DNAH5; The other is the mutation resulting in the deletion of exon76.
CONCLUSIONS: The newly identified DNAH5 splicing mutation c.13,338 + 5G > C is involved in the pathogenesis of PCD in the family, and forms a compound heterozygote with the pathogenic variant DNAH5 c.4314delT lead to the pathogenesis of PCD.
摘要：
背景：原发性纤毛运动障碍（PCD）是一种常染色体隐性遗传性疾病，其特征是反复呼吸道感染。在临床表现中，DNAH5(NM_001361.3)是隐性致病基因之一。原发性家族性脑钙化（PFBC）是一种神经退行性疾病，其特征是基底神经节和其他脑区的双侧钙化。PFBC可以以常染色体显性或隐性方式遗传。分析了具有由DNAH5复合杂合变体引起的PCD和由MYORG纯合变体引起的PFBC的家族。
方法：在本研究中，我们招募了三代患有原发性纤毛运动障碍合并原发性家族性脑钙化的汉族家庭。收集他们的临床表型数据，我们进行了下一代测序以筛选先证者中可疑的致病突变,并通过Sanger测序进行了家族分离分析.构建突变体和野生型质粒,并瞬时转染HEK293T细胞,并通过Minigene剪接法检测剪接模式。通过生物信息学分析对突变的结构和功能进行分析。
结果：先证者（II10）和他的妹妹（II8）的临床表型为支气管扩张，反复肺部感染,双侧苍白球和小脑齿状核的多个对称钙化，整个组的鼻窦炎，支气管粘膜电镜显示睫状轴突缺损。II10中也有总内脏倒置，而II8中没有。新型剪接变体C.13,3385G>C和移码变体C.4314delT（p。Asn1438lysfs*10)在先证者(II10)和II8的DNAH5基因中发现。c.347_348dupCTGGCCTTCCGC纯合插入变异在先证者的MYORG中发现。这两个致病基因在家族中共同分离。Minigene显示DNAH5c.13,338+5G>C有两种异常剪接模式:一种是突变位点所在的部分内含子碱基被翻译,导致DNAH5的早期翻译终止;另一种是导致外显子76缺失的突变。
结论：新发现的DNAH5剪接突变c.13,338+5G>C参与了PCD家族的发病过程，并与致病变体DNAH5c.4314delT形成复合杂合子导致PCD的发病机理。
方法：在本研究中，我们招募了三代患有原发性纤毛运动障碍合并原发性家族性脑钙化的汉族家庭。收集他们的临床表型数据，我们进行了下一代测序以筛选先证者中可疑的致病突变,并通过Sanger测序进行了家族分离分析.构建突变体和野生型质粒,并瞬时转染HEK293T细胞,并通过Minigene剪接法检测剪接模式。通过生物信息学分析对突变的结构和功能进行分析。
结果：先证者（II10）和他的妹妹（II8）的临床表型为支气管扩张，反复肺部感染,双侧苍白球和小脑齿状核的多个对称钙化，整个组的鼻窦炎，支气管粘膜电镜显示睫状轴突缺损。II10中也有总内脏倒置，而II8中没有。新型剪接变体C.13,3385G>C和移码变体C.4314delT（p。Asn1438lysfs*10)在先证者(II10)和II8的DNAH5基因中发现。c.347_348dupCTGGCCTTCCGC纯合插入变异在先证者的MYORG中发现。这两个致病基因在家族中共同分离。Minigene显示DNAH5c.13,338+5G>C有两种异常剪接模式:一种是突变位点所在的部分内含子碱基被翻译,导致DNAH5的早期翻译终止;另一种是导致外显子76缺失的突变。
结论：新发现的DNAH5剪接突变c.13,338+5G>C参与了PCD家族的发病过程，并与致病变体DNAH5c.4314delT形成复合杂合子导致PCD的发病机理。
