Figure 1.

PAPPAS Is Caused by TBX4 Recessive Loss-of-Function Mutations

(A) Pedigrees of two consanguineous Iranian families segregating small patella syndrome in heterozygous TBX4 individuals (gray) and posterior amelia with pelvis and pulmonary hypoplasia syndrome (PAPPAS) in homozygous fetuses (black). The identified mutation and the genotype of available family members are indicated in green. Squares, circles, diamonds, and triangles denote males, females, unknown gender individuals, and fetuses, respectively. Open symbols are used for unaffected family members, and deceased individuals are indicated by a diagonal slash through the symbol. Wt, wildtype; mut, mutant; SB, stillbirth; TOP, termination of pregnancy; SAB, spontaneous abortion.

(B) Photographs and X-rays of affected fetus IV:5 in Family 1 with a germline homozygous p.Y113X TBX4 mutation. Scale bars: 1 cm.

(C) Phylogenetic alignment performed with Clustal O, and position of germline recessive TBX4 mutations in the conserved T-box domain of the transcription factor TBX4. NLS, nuclear localization signal.

(D) TBX4 and ACTB RT-PCR analysis on cDNA extracted from forelimb tissues of a control and the F1-IV:5 fetuses indicating complete absence of the endogenous TBX4 transcript in the mutant fetus. bp, base pairs.

(E) Xenopus tropicalis crispants show severe hindlimb dysplasia after injection of tbx4_gRNA1 pre-complexed with Cas9 protein in one ventral cell of a four-cell-stage embryo (blue), thereby targeting the ventral lineage unilaterally, i.e. primarily the lateral plate mesoderm and the epidermis. A representative post-metamorphic animal (NF stage 66) injected on the right side is shown; the number of toes is indicated. Scale bar: 0.5 cm, ventral view.