PMC9315306_01
Clinical features of the two patients. The chest high-resolution computed tomography (HRCT) scan of P1 (F1:II-1) showed bronchiectasis, rhinosinusitis, and situs inversus. Radiographic image of P1 (F1:II-1) showed scoliosis with a Cobb's angle of 14 . The HRCT scan of P2 (F2:II-1) showed bronchiectasis, rhinosinusitis, situs inversus, and immature uterus. Radiographic image of P2 (F2:II-1) showed scoliosis with a Cobb's angle of 11 .
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F1
Upright radiograph revealed scoliosis with a Cobb's angle of 14  (Figure 1). HRCT findings were similar to P1, and upright radiograph revealed scoliosis with a Cobb's angle of 11  (Figure1). 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).
DNAAF4 variants were identified in two patients with PCD. (A) pedigree analysis of the two patients from two Han Chinese families, where family 2 is a consanguineous family. Circles indicate women. Squares indicate men. Solid symbols indicate patients. Crossed-out symbols mean that subjects had passed away. The arrows indicate the probands. (B) the two variants were validated by Sanger sequencing. Red arrows indicate the variant sites; (C) conservative analysis of the missense variant (C) 988C > T (p.Arg330Trp). Red box indicates mutant amino acid sites. (D) 3D mock structure of DNAAF4. The purple region is the CS domain, while green, yellow, and brick red indicate the three tetratricopeptide repeats, respectively. The variant p.(Arg330Trp) is indicated by red color. (E) DNAAF4 protein structure and the reported disease-causing variants of DNAAF4. Variants reported in this study are highlighted in red.
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F2
These two disease-causing variants were validated by Sanger sequencing (Supplementary Table S2; Figure2).
TEM, HE staining, and immunofluorescence analysis of the nasal ciliated cells and sperm. (A) TEM analysis indicated loss of IDAs and ODAs of P2 (F1:II-1) compared with the normal control. Scale bars, 100 nm; (B,C) immunofluorescence of nasal ciliated cells revealed the absence of DNAH5 and DNALI1 (red) of the two patients compared with normal control. Anti-acetylated tubulin monoclonal antibody was used to mark the ciliary axoneme. DNAH5 was used to label the outer dynein arm (ODA), DNALI1 was used to label the inner dynein arm (IDA), and DAPI was used to label the nuclei. Scale bars, 20 mum; (D) HE staining of sperm. Compared to normal controls, the patient's sperm had significant short, coiled, and irregular tails. (E) immunofluorescence analysis showed the absence of DNALI1 and DNAI1 (red) in the mutant sperm from P1 (F1: II-1) compared with the normal control. Scale bars, 10 mum.
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F3
HE staining showed irregular flagella, short, absent, coiled, and bent, indicating asthenoteratozoospermia (Figure3).