Fig 2: A homozygous KASH5 mutation was identified from Pakistani patients suffering from reproductive failure and born to a consanguineous marriage. (A) The pedigree of the family. Squares represent males, circles represent females, diamonds indicate offspring with the numbers inside indicating the number of children, and the slash symbols denote deceased family members. Solid squares and circles indicate affected male and female patients, respectively. Parallel slash lines indicate consanguineous marriages. Red arrows indicate members receiving whole exome sequencing. Blue arrows indicate probands. (B) Representative images of H&E-stained testicular sections from a control man diagnosed with obstructive azoospermia and patient IV:4. Spg, spermatogonia; Spc, spermatocyte; Spz, spermatozoon. Scale bars denote 50 µm. (C) Sequencing chromatograms of peripheral blood gDNA from the patients and their father (III:2), brother (IV:1) and sisters (IV:7 and IV:8). The red arrow indicates the KASH5 mutation (c.1270_1273del). (D) The gene structure and protein domains of KASH5 (Ensembl transcript ID: ENST00000447857.8). Vertical bars indicate exons and slashed lines represent introns. The non-mutant KASH5 protein (562 amino acids) comprises five domains: N-terminal domain, coiled-coil domain, the C-terminal domain, transmembrane (TM) domain, and the leucine-rich (LR) domain. The mutation (g.49412966_49412969del) locates in exon 17, which causes a frameshift within the C-terminal domain. Lines indicate the mutation positions at the genomic and protein levels (predicted).

Fig 3: Diminished interaction between the predicted truncated KASH5 and SUN1. (A) Immunofluorescence staining using the anti-FLAG antibodies in HEK293T cells transfected with plasmids expressing FLAG-KASH5-WT or FLAG-KASH5-MUT proteins. Scale bars, 10 µm. (B) Co-immunoprecipitation using the anti-FLAG antibody with HEK293T cells transfected with plasmids expressing GFP-SUN1 proteins and plasmids expressing FLAG-KASH5-WT or FLAG-KASH5-MUT proteins, followed by western blotting analyses using anti-GFP or anti-FLAG antibodies. Cells transfected with GFP-empty plasmids serves as a negative co-IP control. Arrows indicate the bands corresponding to the proteins of interest.