Background: 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. Methods: 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. Results: 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. Conclusion: 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.

Spondylometaphyseal Dysplasia, Kozlowski Type (SMDK) is an autosomal dominant skeletal disorder characterized by marked scoliosis, platyspondyly, overfaced pedicles, and mild metaphyseal changes. Pathogenic variants in TRPV4, which encodes a calcium-permeable nonselective cation channel, are known to underlie SMDK. In this study, we identified a previously unreported missense variant in NM_021625.5( TRPV4 ): c.2354G > C (p.Trp785Ser), in a patient clinically diagnosed with SMDK. This variant affects a highly conserved residue and is predicted to alter protein conformation. Functional validation through cellular experiments revealed that the p.W785S substitution markedly reduces agonist-induced calcium influx and membrane currents, indicating a loss-of-function effect on TRPV4 channel activity. This deviates from the typical gain-of-function paradigm observed in most TRPV4-related skeletal dysplasias and may explain the relatively milder phenotype in our case. Our findings establish p.W785S as a novel pathogenic variant and highlight loss of TRPV4 activity as an alternative mechanism contributing to disease pathogenesis in SMDK.

Objective: Osteogenesis imperfecta (OI) is a group of connective tissue disorders with significantly clinical and genetic heterogeneity, which is characterized by low bone mineral density, recurrent fractures and skeletal deformities. This study aimed to conduct clinical and genetic analyses in a Chinese OI cohort to expand the spectrum of pathogenic variants and provide evidence for precise genetic counseling and prenatal genetic diagnosis. Methods: A total of 77 Chinese families with clinically suspected OI were enrolled in this study. Clinical assessments at enrollment included physical examinations, X-ray imaging, and bone mineral density testing. Whole exome sequencing (WES) combined with Sanger sequencing was used to detect candidate pathogenic variants. Variant pathogenicity was evaluated via bioinformatics analysis and familial co-segregation analysis. In this OI cohort, the spectra of pathogenic variants, clinical phenotypes, and genotype-phenotype correlations were analyzed. Results: A 100% detection rate for pathogenic variants was achieved in the 77 families, with 79 variants identified in total. Among the 79 variants, 21 (26.6%) were novel variants founded across six OI-associated genes. Interestingly, apart from the correlation between different pathogenic genes and clinical phenotypes, we also discovered that the severity and phenotype of patients associated with the location of pathogenic variants within the type I collagen domain, exhibiting an aggravating trend from the amino terminus to the carboxyl terminus. Conclusion: Based on previous studies of large OI cohorts, we expanded the spectrum of pathogenic variants by identifying 21 novel ones. Meanwhile, we discovered that the location of pathogenic variants, particularly missense variants, in type I procollagen is correlated with the clinical manifestations and severity of patients. These findings will provide important evidence for the precise diagnosis and genetic counseling of the disease.

Objectives: Congenital insensitivity to pain with anhidrosis (CIPA) is a rare autosomal recessive disorder caused by mutations in NTRK1 that is characterized by pain insensitivity, anhidrosis, and recurrent fever. While genetic testing is the gold standard for CIPA diagnosis, the complexity of NTRK1 variants poses major challenges. Conventional sequencing that is limited to the coding regions of NTRK1 results in misdiagnoses or missed diagnoses in approximately 57% of patients. Accordingly, to improve the diagnostic efficiency of CIPA, we integrated whole-genome sequencing (WGS) with functional assays to identify deep intronic variants in NTRK1 . Methods: All 18 probands were initially screened using polymerase chain reaction (PCR) and Sanger sequencing covering all exons and canonical splice sites of NTRK1 . For patients with only one identified pathogenic allele, WGS was performed to detect potential deep intronic variants. Candidate variants were functionally validated using reverse transcription PCR (RT-PCR) and T cloning sequencing to evaluate their effects on pre-mRNA splicing. Results: Total 23 pathogenic variants including 11 novel variants in NTRK1 were identified in 18 unrelated families with CIPA. Functional assays confirmed that five of these variants disrupted the normal splicing of NTRK1 , resulting in multiple aberrant splicing patterns, including two exon-skipping events (c.428 + 273A>T, c.850 + 5G>A), three intron retentions (c.2187 + 389C>T, c.2188–459G>T, c.287 + 4A>C), and one pseudoexon insertion (c.2188–459G>T). Conclusion: This study expands the spectrum of pathogenic variants in NTRK1 and improves the genetic diagnosis of CIPA. The functional characterization of five novel non-canonical splicing variants provides deeper insight into the molecular pathogenesis of this disorder and establishes a foundation for future precision medicine approaches in CIPA.

Background: Progressive osseous heteroplasia (POH) is an ultrarare disorder characterized by heterotopic ossification that progresses from the skin and subcutaneous tissues into deeper connective tissues. Most affected individuals harbor heterozygous inactivating variants in GNAS , whereas postzygotic mosaic variants have rarely been described. Evidence to guide surgical management remains limited. Methods: We retrospectively characterized two children with clinically diagnosed POH who underwent genetic testing and surgical treatment. Whole-exome sequencing was performed on peripheral blood and, when available, resected tissue, followed by PCR-Sanger sequencing and T-vector cloning. Case 1 underwent clinical and radiographic follow-up for 5 years; follow-up for Case 2 was limited to family-provided radiographs obtained at 2 years. Results: Case 1 harbored a possible postzygotic mosaic GNAS variant, NM_000516.7:c.-1_2del, with variant allele frequencies of 14.7% in peripheral blood and 40.56% in bone lesion tissue. The predominantly localized wrist lesion was macroscopically excised. A small radiographic recurrence appeared by the second postoperative year but progressed slowly thereafter; active wrist flexion was 5–10 ° at 5 years. Case 2 harbored NM_000516.7:c.565_568del (p. (Asp189MetfsTer14)), detected as a heterozygous variant in peripheral blood. The lesion diffusely involved neurovascular structures, tendon sheaths, the joint capsule, and adjacent bones, precluding complete excision. Family-provided radiographs at 2 years showed an extent of ossification broadly similar to that before surgery; no in-person clinical assessment was available. Conclusion: These cases provide additional clinical, molecular, and longitudinal information on POH and illustrate the importance of lesion distribution and resectability when interpreting surgical outcomes. The available postoperative findings cannot establish a causal relationship between variant status and prognosis because the patients differed in baseline disease extent, completeness of excision, follow-up duration, and outcome ascertainment. A possible association between mosaic burden and lesion distribution remains a hypothesis that requires evaluation in larger cohorts.