MAB21L1variant details. (a) Schematic of MAB21L1 (top) and MAB21L2 (bottom) proteins. Newly identified heterozygous MAB21L1 coding variants are indicated by a red arrow; previously reported MAB21L1 and MAB21L2 recessive variants are indicated with a black arrow; previously reported MAB21L2 dominant variants are indicated with a gray arrow. Missense variants are bolded. (b) Pedigrees for Families 1–3 indicating MAB21L1 genotype and Sanger sequencing traces for the identified coding variants. (c) Alignment of MAB21L1 and related proteins showing conservation at and around the Arg51Leu, Arg62Cys, and Gly220Arg variants. Identical amino acids are shaded in gray; positions of variant amino acids are indicated with dark gray. Human MAB21L1 (NP_005575.1), human MAB21L2 (NP_006430.1), mouse Mab21l1 (NP_034880.1), chicken Mab21l1 (NP_989864.1), zebrafish mab21l1 (NP_694506.2), and Caenorhabditis elegans mab‐21 (NP_497940.2) are shown

Functional analyses of MAB21L1 and variant proteins. (a) Western blot analysis. Western blot analysis of N‐terminal FLAG‐tagged MAB21L1 wild‐type and Arg51Leu, Arg62Cys, and Gly220Arg variants. Constructs were expressed in HLE‐B3 cells. β‐actin was used as a loading control. The proteins correspond to their expected molecular weight (~41kDa MAB21L1 and ~42kDa β‐actin). (b) Enzyme‐linked immunosorbent assay. N‐terminal FLAG‐tagged MAB21L1 wild‐type and Arg51Leu, Arg62Cys, and Gly220Arg variants were transfected into HLE‐B3 cells. Cell lysates were assessed for FLAG‐tagged protein expression; protein levels of Arg51Leu and Arg62Cys were found to be significantly affected. (c) Immunocytochemistry. N‐terminal FLAG‐tagged MAB21L1 wild‐type and variants were transfected into HLE‐B3 cells and stained for FLAG (green) and 4ʹ,6‐diamidino‐2‐phenylindole, dihydrochloride (DAPI) (blue; cell nuclei). Wild‐type and variant proteins can be found within the cell nucleus, indicating no disruption in localization. (d) In vivo complementation assays. Proportion of phenotypically normal embryos at 24 hpf in the progeny of heterozygous mab21l2^Q48Sfs*5 crosses injected with wild‐type or variant Arg51Leu, Arg62Cys, or Gly220Arg MAB21L1 messenger RNA. UN, uninjected; hpf, hours postfertilization; 1‐MAB21L1‐WT; 2‐MAB21L1‐Arg51Leu; 3‐MAB21L1‐Arg62Cys; 4‐MAB21L1‐Gly220Arg. Statistical significance is indicated by asterisks; *p ≤ .05, **p ≤ .01, and ***p ≤ .001; error bars indicate SEM