Kariminejad A et al. (2019), Homozygous Null TBX4 Mutations Lead to Posterio...

XB-ART-56511

Am J Hum Genet 2019 Dec 05;1056:1294-1301. doi: 10.1016/j.ajhg.2019.10.013.

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Homozygous Null TBX4 Mutations Lead to Posterior Amelia with Pelvic and Pulmonary Hypoplasia.

Kariminejad A , Szenker-Ravi E , Lekszas C , Tajsharghi H , Moslemi AR , Naert T , Tran HT , Ahangari F , Rajaei M , Nasseri M , Haaf T , Azad A , Superti-Furga A , Maroofian R , Ghaderi-Sohi S , Najmabadi H , Abbaszadegan MR , Vleminckx K , Nikuei P , Reversade B .

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The development of hindlimbs in tetrapod species relies specifically on the transcription factor TBX4. In humans, heterozygous loss-of-function TBX4 mutations cause dominant small patella syndrome (SPS) due to haploinsufficiency. Here, we characterize a striking clinical entity in four fetuses with complete posterior amelia with pelvis and pulmonary hypoplasia (PAPPA). Through exome sequencing, we find that PAPPA syndrome is caused by homozygous TBX4 inactivating mutations during embryogenesis in humans. In two consanguineous couples, we uncover distinct germline TBX4 coding mutations, p.Tyr113∗ and p.Tyr127Asn, that segregated with SPS in heterozygous parents and with posterior amelia with pelvis and pulmonary hypoplasia syndrome (PAPPAS) in one available homozygous fetus. A complete absence of TBX4 transcripts in this proband with biallelic p.Tyr113∗ stop-gain mutations revealed nonsense-mediated decay of the endogenous mRNA. CRISPR/Cas9-mediated TBX4 deletion in Xenopus embryos confirmed its restricted role during leg development. We conclude that SPS and PAPPAS are allelic diseases of TBX4 deficiency and that TBX4 is an essential transcription factor for organogenesis of the lungs, pelvis, and hindlimbs in humans.

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Species referenced: Xenopus
Genes referenced: actb fgf10 hhip mmut rspo2 shox2 sms tbx3 tbx4 tbx5
gRNAs referenced: tbx4 gRNA1 tbx4 gRNA2

???displayArticle.disOnts??? Holt-Oram syndrome [+]
???displayArticle.omims??? HOLT-ORAM SYNDROME; HOS [+]
Phenotypes: Xtr.wt +tbx4 CRISPR (Fig, S2.) [+]

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	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.
	Figure 2 The Heterozygous TBX4 Parents Present with SPS X-ray and photographs of the indicated family members in Family 1 (A) and Family 2 (B). White arrows point to abnormal ischio-pubic junction, and arrowheads point to hypoplastic patellae in both heterozygous parents of the two families. Note the short fourth and fifth rays in the feet of both parents in Family 1 and the mother in Family 2, and the wide gap between the first and second toes in both parents of Family 2.
	Figure S1: Segregation analysis of causative germline TBX4 mutations. Sanger sequencing of TBX4 mutations in available family members of Family 1 (a) and Family 2 (b). wt: wildtype, mut: mutant.
	Figure S2: Phenotype of mildly affected Xenopus tropicalis animals. Representative images of an NF stage 66 adult animal, that had been unilaterally/ventrally injected with Tbx4_gRNA2 and Cas9 protein at the 4-cell stage, stained with alizarin red/alcian blue. Note the dislocated joints (black arrowheads) both at the hip and knee, and the shorter femur of the hindlimb in the injected side (right). Scale bars: 0.2 cm.

References [+] :

Agarwal, Tbx5 is essential for forelimb bud initiation following patterning of the limb field in the mouse embryo. 2003, Pubmed