Proband 1 (P1, F1: II-1), a 19-year-old man, comes from a nonconsanguineous family-1. He had typical PCD symptoms, such as chronic cough and sputum production, dyspnea, hemoptysis, sinusitis, etc. In addition, he complained of abnormalities in his sense of smell and hearing. However, his fertility was unknown because he was not married. He denied any history of asthma, tuberculosis, measles pneumonia, or drowning. No relevant family history was reported. High-resolution CT (HRCT) showed sinusitis, bronchiectasis, and situs inversus. Upright radiograph revealed scoliosis with a Cobb's angle of 14  (Figure 1). Furthermore, the semen routine test showed that the percentage of motile sperm was 0. Proband 2 (P2, F2: II-1), a 37-year-old woman, comes from a consanguineous family-2. She had chronic cough and sputum, dyspnea, and sinusitis. She also had abnormalities in the sense of smell and hearing. In addition, she suffered from infertility. HRCT findings were similar to P1, and upright radiograph revealed scoliosis with a Cobb's angle of 11  (Figure1). The HRCT also indicated an immature uterus. Her forced expiratory volume in 1 second (FEV1)%prediction, forced vital capacity (FVC)%prediction, FEV1/FVC, and nasal NO were 36%, 50%, 60%, and 8.4 nL/min, respectively (Supplementary Tables S1-S3; Figure1).
We used the patient's peripheral blood to extract DNA for Whole exome sequencing (WES). After WES, 12.02 and 6.8 GB data were used for subsequent analysis. The percentages of bases with a coverage depth of more than 10x were 99.6% and 98.1%, respectively. Two variants, DNAAF4 (NM_130810.4): c.988C > T (p.Arg330Trp) in P1 and a novel variant DNAAF4 (NM_130810.4): c.733C > T (p.Arg245Ter) in P2, were identified. We classified the pathogenicity of the two variants according to ACMG. The results showed the variant in P1 was consistent with PM2+PM3+PP3+PP5 (likely pathogenic), and the variant in P2 met the criteria PVS1+PM2+PP5 (pathogenic). These two disease-causing variants were validated by Sanger sequencing (Supplementary Table S2; Figure2).
High-speed microscopy showed complete static of sperm flagella and a few residual beating of respiratory cilia in P1 and complete static of the respiratory cilia in P2 compared with normal control (Supplementary Videos S1-S3). Immunofluorescence showed the deficiency of DNAH5 and DNALI1, which labelled ODA and IDA, respectively, in the respiratory cilia of both probands. In addition, the DNALI1 and DNAI1 which respectively labelled the ODA and IDA in the sperm flagellum of P1 were also absent. TEM revealed the absence of ODA and IDA of P2 respiratory cilia. HE staining showed irregular flagella, short, absent, coiled, and bent, indicating asthenoteratozoospermia (Figure3).