Case Report: Minigene assays reveal a novel DNAAF6 intronic variant as the key etiology for primary ciliary dyskinesia

Yupeng Long¹Huixian Li²Haoyue Yu³Qian Yi³Xiong Meng³Xi Wang³Qinqin Ren³Dongyan Ding³Haidong Li³Fenglan Zhang²Hao Qiu²Xuemei Yang^3*

• ¹Laboratory and Blood Transfusion Department of Jiangbei Campus, The First Affiliated Hospital of Army Medical University (The 958th hospital of Chinese People’s Liberation Army), Chongqing, China
• ²Center for Clinical Genetics and Genomics, Dian Diagnostics Group Co., Ltd., Hangzhou, China
• ³Respiratory and Critical Care Medicine Department of Jiangbei Campus, The First Affiliated Hospital of Army Medical University (The 958th hospital of Chinese People’s Liberation Army), Chongqing, China

Background: Primary ciliary dyskinesia (PCD), a rare hereditary disorder characterized by impaired ciliary motility, is frequently linked to infertility. Elucidating PCD's genetic basis is critical for accurate diagnosis and clinical management. However, research on the pathogenicity of intronic variants in non-classical splice regions of DNAAF6—a newly identified PCD-associated gene in recent years—remains scarce. Case presentation: A proband with primary ciliary dyskinesia (PCD) and male infertility underwent whole exome sequencing (WES), revealing a novel hemizygous intronic variant in the DNAAF6 gene (NM_173494.2: c.515+3_515+6del, Clinvar Variation ID:4075425). Sanger sequencing confirmed this variant in his affected brother. A minigene splicing assay showed that the variant caused exon 6 skipping, leading to a frameshift. Transmission electron microscopy (TEM) analysis of sperm flagella indicated absent outer and inner dynein arms, resulting in flagellar immotility. Conclusion: This study identifies and confirms a novel pathogenic intronic variant of DNAAF6 in a Chinese PCD family, expanding the DNAAF6 mutational spectrum. It also emphasizes the critical role of minigene-based functional validation in interpreting variants of uncertain significance (VUS).