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. Typical GLI3-related Greig cephalopolysyndactyly syndrome (GLI3-GCPS) is characterized by macrocephaly, widely spaced eyes associated with increased interpupillary distance, preaxial polydactyly with or without postaxial polydactyly, and cutaneous syndactyly. Developmental delay, intellectual disability, or seizures appear to be uncommon manifestations (~<10%) of GLI3-GCPS and may be more common in individuals with large (>300 kb) deletions that encompass GLI3. Approximately 20% of individuals with GLI3-GCPS have hypoplasia or agenesis of the corpus callosum. The diagnosis of GLI3-GCPS is established in a proband with typical clinical findings and either a heterozygous pathogenic variant in GLI3 or a deletion of chromosome 7p14.1 involving GLI3 identified by molecular genetic testing. Treatment of manifestations: Elective surgical repair of polydactyly with greatest priority given to correction of preaxial polydactyly of the hands; for polydactyly of the feet, the cosmetic benefits and easier fitting of shoes can be outweighed by potential orthopedic complications. Syndactyly that is more than minimal is typically repaired surgically. Occupational and physical therapy to improve motor skills; developmental and educational support as needed; standardized treatment for seizures as needed. Surveillance: Monitoring for evidence of increased rate of head growth or neurologic concerns and the need for brain MRI. Physical medicine and occupational and physical therapy assessments of mobility and self-help skills at each visit or as needed; assessment of developmental progress and educational needs at each visit or as needed; monitor those with seizures at each visit. GLI3-GCPS is inherited in an autosomal dominant manner. Some individuals diagnosed with GLI3-GCPS have the disorder as the result of a genetic alteration inherited from a parent. The proportion of individuals with GLI3-GCPS caused by de novo genetic alteration is unknown. If the causative genetic alteration in the proband is a chromosome 7p14.1 deletion, the parents of the proband are at risk of having a balanced chromosome rearrangement and should be offered chromosome analysis. The risk to the sibs of a proband depends on the clinical/genetic status of the proband's parents: if a parent of the proband is affected and/or is known to have the genetic alteration identified in the proband, the risk to the sibs is 50%; if a parent has a balanced structural chromosome rearrangement, the risk to sibs is increased and depends on the specific chromosome rearrangement and the possibility of other variables. Each child of an individual with GLI3-GCPS has a 50% chance of inheriting the causative genetic alteration. If the GLI3-GCPS-causing genetic alteration has been identified in an affected family member (or a balanced structural chromosome rearrangement involving 7p14.1 is identified in a parent), prenatal and preimplantation genetic testing are possible.

Polydactyly is a prevalent congenital anomaly with an incidence of 2.14 per 1000 live births in China. GLI family zinc finger 3 (GLI3) is a classical causative gene of polydactyly, and serves as a pivotal transcription factor in the hedgehog signaling pathway, regulating the development of the anterior-posterior axis in limbs. Three pedigrees of polydactyly patients were enrolled from Hunan Province, China. Pathogenic variants were identified by whole-exome sequencing (WES) and Sanger sequencing. Three variants in GLI3 were identified in three unrelated families, including a novel deletion variant (c.1372del, p.Thr458GlnfsTer44), a novel insertion-deletion (indel) variant (c.1967_1968delinsAA, p.Ser656Ter), and a nonsense variant (c.2374 C > T, p.Arg792Ter). These variants were present exclusively in patients but not in healthy individuals. We identified three pathogenic GLI3 variants in polydactyly patients, broadening the genetic spectrum of GLI3 and contributing significantly to genetic counseling and diagnosis for polydactyly.

GLI3 gene mutations can result in various forms of polysyndactyly, such as Greig cephalopolysyndactyly syndrome (GCPS, MIM: #175700), Pallister-Hall syndrome (PHS, MIM: #146510), and isolated polydactyly (IPD, MIM: #174200, #174700). Reports on IPD-associated GLI3 mutations are rare. In this study, a novel GLI3 mutation was identified in a Chinese family with IPD. We report a family with six members affected by IPD. The family members demonstrated several special phenotypes, including sex differences, abnormal finger joint development, and different polydactyly types. We identified a novel frameshift variant in the GLI3 gene (NM_000168.6: c.1820_1821del, NP_000159.3: p.Tyr607Cysfs*9) by whole-exome sequencing. Further analysis suggested that this mutation was the cause of polydactyly in this family. The discovery of this novel frameshift variant in our study further solidifies the relationship between IPD and GLI3 and expands the previously established spectrum of GLI3 mutations and associated phenotypes.

We present two genetic causes of polyhydramnios that were challenging to diagnose due to their rarity and complexity. In view of the severe implications, we wish to highlight these rare genetic conditions when obstetricians consider differential diagnoses of polyhydramnios in the third trimester. Patient 1 is a 34-year-old Asian woman who was diagnosed with polyhydramnios at 28 weeks' gestation. First trimester testing, fetal anomaly scan, and intrauterine infection screen were normal. Subsequent antenatal ultrasound scans revealed macroglossia, raising the suspicion for Beckwith-Wiedemann syndrome. Chromosomal microarray analysis revealed a female profile with no pathological copy number variants. The patient underwent amnioreduction twice in the pregnancy. The patient presented in preterm labor at 34 weeks' gestation but elected for an emergency caesarean section. Postnatally, the baby was noted to have a bell-shaped thorax, coat hanger ribs, hypotonia, abdominal distension, and facial dysmorphisms suggestive of Kagami-Ogata syndrome. Patient 2 is a 30-year-old Asian woman who was diagnosed with polyhydramnios at 30 weeks' gestation. She had a high-risk first trimester screen but declined invasive testing; non-invasive prenatal testing was low risk. Ultrasound examination revealed a macrosomic fetus with grade 1 echogenic bowels but no other abnormalities. Intrauterine infection screen was negative, and there was no sonographic evidence of fetal anemia. She had spontaneous rupture of membranes at 37 + 3 weeks but subsequently delivered by caesarean section in view of pathological cardiotocography. The baby was noted to have inspiratory stridor, hypotonia, low-set ears, and bilateral toe polysyndactyly. Further genetic testing revealed a female profile with a pathogenic variant of the GLI3 gene, confirming a diagnosis of Greig cephalopolysyndactyly syndrome. These cases illustrate the importance of considering rare genetic causes of polyhydramnios in the differential diagnosis, particularly when fetal anomalies are not apparent at the 20-week structural scan. We would like to raise awareness for these rare conditions, as a high index of suspicion enables appropriate counseling, prenatal testing, and timely referral to pediatricians and geneticists. Early identification and diagnosis allow planning of perinatal care and birth in a tertiary center managed by a multidisciplinary team.

Many genetic testing methodologies are biased towards picking up structural variants (SVs) that alter copy number. Copy-neutral rearrangements such as inversions are therefore likely to suffer from underascertainment. In this study, manual review prompted by a virtual multidisciplinary team meeting and subsequent bioinformatic prioritisation of data from the 100K Genomes Project was performed across 43 genes linked to well-characterised skeletal disorders. Ten individuals from three independent families were found to harbour diagnostic inversions. In two families, inverted segments of 1.2/14.8 Mb unequivocally disrupted GLI3 and segregated with skeletal features consistent with Greig cephalopolysyndactyly syndrome. For one family, phenotypic blending was due to the opposing breakpoint lying ~45 kb from HOXA13 In the third family, long suspected to have Marfan syndrome, a 2.0 Mb inversion disrupting FBN1 was identified. These findings resolved lengthy diagnostic odysseys of 9-20 years and highlight the importance of direct interaction between clinicians and data-analysts. These exemplars of a rare mutational class inform future SV prioritisation strategies within the NHS Genomic Medicine Service and similar genome sequencing initiatives. In over 30 years since these two disease-gene associations were identified, large inversions have yet to be described and so our results extend the mutational spectra linked to these conditions.