The VACTERL association is a non-random cluster of congenital malformations involving six distinct conditions: vertebral defects (V), anal atresia (A), cardiac defects (C), tracheoesophageal malformation (TE), renal defects (R), and limb anomalies (L), and is diagnosed when a fetus exhibits three or more of these. Its prevalence is approximately 0.47-0.58 per 10,000 live births. This paper examines the effect of disruptions in the Sonic Hedgehog and cilia-associated signaling pathways, genetically related developmental variations, and maternal environmental factors on the development of VACTERL. In the SHH signaling pathway, we focus on the effects of Sonic Hedgehog ligands, GLI transcription factors, and factors influencing GLI activity (RAC1 and ZIC3), as well as downstream targets (FOXF1 and HOXD13) and other genes and proteins involved in the regulation of SHH signaling (FGF8 and LPP), in the pathogenesis of VACTERL. In this context, ZIC3, which was shown to play a major role in VACTERL pathogenesis in large-scale resequencing, and TRAP1, which was associated with VACTERL pathogenesis in whole-exome resequencing, were highlighted. We also examine the cilia-associated signaling pathways, particularly the role of IFT172 and candidate ciliopathy genes. In addition, we describe the influence of TRAP1, COL11A2, SALL4, WBP11, Copy Number Variants, and maternal environmental factors on VACTERL. We also discuss current diagnostic, therapeutic, and prognostic approaches including prenatal and postnatal treatment options. Furthermore, we highlight the advantages of thoracoscopic surgery over traditional open-surgical treatment while discussing the differential diagnosis of VACTERL from other neonatal malformations with similar symptoms, such as Townes-Brocks syndrome, Baller-Gerold syndrome, and CHARGE syndrome.

RECQL4 encodes a RecQ helicase, one of a family of DNA unwinding enzymes with roles in DNA replication, double-strand break repair, and genomic stability. Pathogenic variants in RECQL4 are clinically associated with 3 rare autosomal recessive conditions: Rothmund-Thomson syndrome type II, Baller-Gerold syndrome, and RAPADILINO syndrome. These 3 syndromes show overlapping growth retardation, low bone density, and skeletal defects affecting the arms and hands. Here, we take advantage of the ability to generate one-sided CRISPR knockdowns of recql4 in Xenopus laevis tadpoles. Tadpoles develop normally until feeding starts, after which growth slows on the edited side, leading to a curved posture, smaller eyes (microphthalmia), and reduced head size (microcephaly). Forelimb buds fail to develop, leading to complete absence of the forelimb on the edited side. Additionally, Meckel's cartilage (lower jaw) ossification is absent or reduced and the hyoid cartilage is smaller, but this is not due to deficiencies in cranial neural crest migration on the edited side. Knockdown of recql4 also results in hypoplastic vasculature, with reduced branching from the aorta on the edited side. Taken together, our results clearly show the utility of unilateral CRISPR editing in Xenopus for understanding the specific phenotypic developmental effects of mutations affecting cell proliferation.

RECQL4 is a member of the RecQ family of helicases, playing essential roles in DNA replication and maintaining genome integrity. Mutations in RECQL4 are linked to severe human diseases, including Rothmund-Thomson Syndrome, RAPIDALINO Syndrome, and Baller-Gerold Syndrome. However, we still do not fully understand its functions and genetic interactions. The role of the ATP-dependent helicase activity in RECQL4 remains controversial. To understand RECQL4's functions further, we conducted a genome-wide forward genetic screen using murine models that closely mimic the RECQL4 mutations found in patients with Rothmund-Thomson syndrome. Our goal was to identify loss-of-function alleles that could rescue the proliferation and viability defects associated with RECQL4 mutation. From our screening we identified the loss of KLHDC3, a substrate-binding subunit of the Cullin-RING ligase (CRL) E3, as the most significant rescue allele. KLHDC3 facilitates the ubiquitin-mediated destruction of proteins with specific C-terminal degron motifs. Its loss normalized cell proliferation and DNA replication rates in cells with mutated RECQL4. Further analysis revealed that the loss of KLHDC3 led to the stabilization of minute levels of a truncated RECQL4 protein. This RECQL4 fragment contained a neo-degron sequence specific for KLHDC3, formed after Cre-mediated recombination of the Recql4 fl allele. Although this rescue mechanism does not apply to human RECQL4 mutations, it shows that very low chromatin-bound levels of a truncated RECQL4 protein-comprising only the N-terminal 480 amino acids, including its Sld2-like domain but lacking the ATP-dependent helicase domain and the entire C-terminal portion-are sufficient to support DNA replication in mammalian cells. These results demonstrate that the ATPase activity and helicase domain of RECQL4 are not essential for DNA replication in mammals. Furthermore, our findings suggest that there are unlikely to be monogenic loss-of-function alleles that can rescue RECQL4 mutations. This demonstrates that RECQL4 is an essential and non-redundant regulator of DNA replication and cell viability and that this activity does not require the ATP dependent helicase activity.

Baller-Gerold syndrome (BGS, OMIM: 218600), RAPADILINO syndrome (OMIM 266280), and Rothmund-Thomson syndrome (RTS, OMIM 266280), which are caused in some cases by RECQL4 pathogenic variants, show autosomal recessive inheritance. Some refer to them collectively as RECQL4 syndromes. Most cases have been reported during infancy and childhood periods. However, there have been no reports of phenotypes resulting in a lethal course in the perinatal period. We identified two fetuses with biallelic RECQL4 pathogenic variants during the perinatal period. The two fetuses with RECQL4 syndrome showed structural abnormalities, including severely hypoplastic forearms and lower legs. One fetus also had severe pulmonary hypoplasia. One case resulted in neonatal death because of respiratory failure, and the other was artificially terminated during pregnancy. The RECQL4 pathogenic variants were identified by exome sequencing followed by Sanger sequencing. The biallelic RECQL4 pathogenic variants can induce a lethal skeletal disorder.