Ciliopathies are rare genetic diseases marked by considerable phenotypic heterogeneity and overlap. Among the key mechanisms of cilium biology, its compartmentalization is achieved through gating complexes and active transport such as intraflagellar transport (IFT). Among the IFT components, IFT27 plays a role in BBSome-mediated transport of ciliary membrane proteins required for ciliary signaling. While this gene was first linked to Bardet-Biedl syndrome, we next expanded its phenotypic spectrum to a fetal lethal ciliopathy. Here, we identified a second fetal case with short ribs, polydactyly, hypodysplastic kidneys, imperforate anus, and situs inversus. Genome sequencing identified novel biallelic variants in IFT27. Functional analysis of tissues from both fetal cases revealed that all the identified variants lead to mRNA decay. Immunohistochemistry on fetal kidney sections showed that those variants are associated with altered ciliogenesis. Overall, we showed that complete loss of IFT27 function leads to a severe phenotypic spectrum overlapping with short ribs polydactyly and Pallister-Hall syndromes. In addition, our results argue for a role of IFT27 in ciliogenesis in humans.

The GLI3 gene, a pivotal component of the hedgehog (HH) signaling pathway, plays a fundamental role in the development and patterning of various body structures, including the brain and limbs. Mutations in the GLI3 gene, particularly in the C-terminal domain, are implicated in congenital anomalies such as Greig cephalopolysyndactyly syndrome and Pallister-Hall syndrome. Recent studies have also suggested an archaic human-type mutation in the C-terminal end, which altered downstream gene regulations and anatomical structures in mice. However, the biological effects of the disruption in the Gli3 C-terminal end have not been studied well. Here we report novel Gli3 mutant mice with nonsense mutations in the C-terminal end using CRISPR/Cas12a-mediated genome editing. Analysis of the genotype-phenotype correlations has revealed that the C-terminal end of Gli3 is critical for functional protein synthesis; therefore, the disruption of this region causes severe abnormalities in brain and digit formation. These results provide insight into the mechanisms by which GLI3 mutants can cause adverse consequences during human development or result in diverse phenotypes during evolution.

GLI family zinc finger 3 (GLI3) is a transcription factor involved in limb development. GLI3 gene variants have been shown to be associated with several human congenital limb malformations, including Greig cephalopolysyndactyly, Pallister-Hall syndrome, non-syndromic postaxial polydactyly (PAP-A/B), and preaxial polydactyly type IV (PPD-IV). The aim of this study was to identify GLI3 gene variants in ten Chinese families with limb malformations. Ten Chinese families with limb malformations were recruited. Variant screening in probands was then performed using NGS, with candidate pathogenic variants verified by polymerase chain reaction (PCR) combined with Sanger DNA sequencing. Variant pathogenicity was evaluated using bioinformatics, evolutionary conservation, and disease and mutant allele co-segregation approaches. The biological effects of missense variants were predicted by three-dimensional protein conformation analysis. Ten GLI3 variants were identified: two missense variants c.1063G>A (p.Val355Ile) and c.1489C>A (p.Leu497Ile), four nonsense variants c.2374C>T (p.Arg792*), c.2008C>T (p.Gln670*), c.1096 C>T (p.Arg366*), and c.2029C>T (p.Gln677*); three frameshift variants c.600delC (p.Tyr200*), c.1880_1881del (p.His627Argfs*48), and c.811_812delCT (p.Leu271Serfs*5); a large fragment deletion of NC_000007.14: g.42061081_42069739. Seven of these ten variants have never been recorded in the Human Gene Variant Database. Ten GLI3 variants were successfully identified in families with different limb malformations, indicating significant clinical and allelic heterogeneity of GLI3-related limb malformations. The present study expands the spectra of pathogenic variants and clinical manifestation for GLI3-related morphological disorder and provides solid evidence for genetic counseling and prenatal gene diagnosis in the affected families.

Pallister-Hall syndrome (PHS) is an extremely rare genetic disorder. It presents as a polymalformative syndrome affecting craniofacial structures, the central nervous system, limbs, various internal organs, and the endocrine system. It is considered a ciliopathy, as it is associated with loss-of-function variants in the GLI3 gene, a transcription factor essential for primary cilium signaling. The syndrome shows marked clinical heterogeneity depending on the type of genetic variant, which makes diagnosis challenging. It is usually suspected at an early age when a hypothalamic hamartoma is associated with polydactyly. Endocrine manifestations are often linked to the hamartoma, further complicating diagnosis. A 23-year-old Colombian patient presented with a history of hypothalamic hamartoma, gelastic seizures, postaxial polydactyly of hands and feet, and craniofacial dysmorphisms. Physical examination revealed dolichocephaly, bilateral epicanthus, broad nasal bridge, short and broad neck, mild cervical kyphosis, mild scoliosis, micrognathia, bilateral ulnar deviation of the fourth and fifth fingers, and overlapping toes on both feet. No genital anomalies were found. Neuropsychological evaluation reported a borderline intellectual quotient of 78. Whole-exome sequencing identified a de novo heterozygous pathogenic variant in GLI3 (c.2151del; p.(Gln71HisfsTer16)), confirmed by Sanger sequencing. We report the first case described in Colombia of a previously unreported truncating genetic variant. We performed a clinical-molecular correlation in a 23-year-old adult whose diagnosis of PHS was delayed until adulthood, years after the initial identification of a hypothalamic hamartoma, refractory gelastic seizures, polydactyly, and mild cognitive impairment. This case broadens the clinical spectrum regarding the viability of patients with PHS into adulthood, showing that it is not restricted to the severe neonatal or infantile presentations classically described.

Polydactyly is a prevalent limb deformity with an autosomal dominant inheritance pattern, manifesting in both syndromic and nonsyndromic forms. It exhibits significant etiological and clinical diversity. This study aims to identify the pathogenic cause in two patients with sub-PHS (sub-Pallister-Hall Syndrome) and PAP (postaxial polydactyly), respectively, from two Chinese pedigrees. Exome sequencing was performed on patients to screen for potential pathogenic variants. Subsequently, these variants were validated by Sanger sequencing. The c.3342dupC and c.4431dupT mutant plasmids were transfected into HEK293T cells, and the effects of GLI3 mutations on transcription and protein levels were analyzed via qRT-PCR and Western blot. Additionally, Swiss Model was utilized to predict the effects of mutations on protein tertiary structure. The mutations of GLI3 (NM_000168.6: c.3342dupC; p. A1115Rfs*14) (NM_000168.6: c.4431dupT; p. Glu1478Ter) were identified in affected individuals. These mutations were present exclusively in the patients and absent in the healthy individuals. No significant difference in transcription levels between the mutations and wild type was observed. Functional analysis revealed that the truncated variants p. A1115Rfs*14 and p. Glu1478Ter exhibited reduced molecular weight and potential functional impairment due to protein retention. The mutations p. A1115Rfs*14 and p. Glu1478Ter in GLI3 may account for sub-PHS and PAP in the two patients, respectively. This finding expands the mutation and phenotype spectrum associated with GLI3, providing valuable insights for the clinical diagnosis of polysyndactyly.