Osteogenesis imperfecta (OI) is a genetically and clinically heterogeneous bone disorder, with more than 20 genes contributing to OI development. Previously, we identified WNT1 c.620G > A (p.Arg207His) mutation among Chinese patients with autosomal recessive OI (AR-OI). This study aims at investigating the causative role of WNT1 deficiency in OI and evaluate whether AAV-based gene therapy could ameliorate bone abnormalities. We generated and analyzed the Wnt1R207H/R207H rat model. The AAV9-Wnt1 virus was delivered via direct intraosseous injection into the femoral marrow cavity of the OI rats to evaluate its therapeutic potential. The homozygous Wnt1R207H/R207H rat recapitulated key features of AR-OI, including fractures, reduced bone mass, growth retardation, decreased survival rate, increased osteoclast numbers, diminished osteoblast function and mineralization capacity, compared with heterozygous and wild-type littermates. In vitro, Wnt1 overexpression in osteoblasts promoted osteoblast activity and bone mineralization. Furthermore, AAV9-Wnt1 treatment in OI rats resulted in significant recovery of bone density and mechanical strength, stimulation of osteoblast activity, suppression of osteoclast activity, and upregulation of Type I collagen expression. Our study demonstrates that WNT1 c.620G > A (p.Arg207His) is pathogenic, and confirms that AAV-mediated Wnt1 gene therapy represents a promising strategy for treating OI caused by WNT1 mutations.

Osteogenesis imperfecta (OI) is a group of monogenic skeletal diseases caused by defect of type I collagen. There is no specific treatment strategy and targeted therapy is urgently needed. Dickkopf-1 (DKK1), is a Wnt antagonist, and inhibition of DKK1 stimulates Wnt signaling and osteogenic capacity. Here, we used DKK1 neutralizing antibody (DKK1 nAb) to identify its effect on OI and clarify its effects on different types of OI. Mesenchymal stem cells (MSCs) derived from OI patients with variants in COL1A1, COL1A2, SERPINF1, and WNT1 were treated with or without DKK1 nAb and induced toward osteoblast differentiation in vitro. In vivo, two Sprague-Dawley OI rat models carrying a Col1a2 (c.1000G > A) or Wnt1 (c.620G > A) mutation were administered DKK1 nAb for 2 and 4 weeks to assess skeletal therapeutic effects. In vitro, DKK1 nAb promoted osteogenic differentiation and mineralization and upregulated the mRNA levels of COL1A1, COL1A2, CTNNB1, and RUNX2 in MSCs from some, though not all, OI patients. This regulatory effect is independent of the genotypes of OI patients. In vivo, a 2-week DKK1 nAb treatment exerted no significant improvement in skeletal parameters in either Col1a2 or Wnt1 mutant rats. While 4-week treatment showed modest improvement in trabecular bone in Col1a2 mutant OI rats, whereas no significant changes were detected in Wnt1 mutant OI rats. The DKK1 nAb demonstrates a modest therapeutic effect in OI, indicating its potential for some individuals, although the limited sample size necessitates caution in interpretation, and further validation in larger cohorts is warranted.

ObjectiveSplit-hand/foot malformation (SHFM) is a group of congenital birth defects affecting the hands and feet, significantly impairing patients quality of life. The TP63 gene encodes the p63 protein, heterozygous TP63 variants can cause SHFM. The aim of this study was to identify TP63 gene variants in three Chinese families with SHFM.MethodsThree Chinese families with SHFM enrolled in this study. Proband 1 and Proband two had familial history of SHFM, whereas Proband 3 was a sporadic case of this disease. Peripheral blood was collected from the proband and their available family members for genomic DNA extraction. Candidate pathogenic variants were identified through whole exome sequencing (WES), validated using PCR-based Sanger sequencing and bioinformatic analysis.ResultsWe found three different missense variants in the TP63 gene: c.2032G>C (p.Glu678Gln), c.956G>A (p.Arg319His), and c.689T>A (p.Val230Asp). Among them, c.689T>A (p.Val230Asp) is novel variant.ConclusionIn the present work, three TP63 gene variants were found in the families with SHFM, which expanded the variant spectrum of TP63. These findings not only provide further evidence for their heterogeneous role in limb and non-limb malformations but also broaden the genetic spectrum of associated disorders. These insights pave the way for improved prenatal genetic diagnosis and informed counseling.

ObjectivesCongenital 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.MethodsAll 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.ResultsTotal 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).ConclusionThis 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.

ObjectiveOsteogenesis 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.MethodsA 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.ResultsA 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.ConclusionBased 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.

This article reports a rare case of autoinflammatory disease presenting initially with skin induration and swelling after trauma as the initial manifestation, followed by progressive limb weakness. The patient was a middle-aged female who developed skin induration and swelling after trauma, which gradually progressed to limb weakness, dysarthria and bilateral facial paralysis, accompanied by livedo reticularis of the lower extremities, diffuse skin induration of the limbs, and beaded subcutaneous nodules in the right upper limb. The patient had a susceptibility to infection since childhood and a history of chronic livedo reticularis. Skin pathological examination revealed panniculitis. A comprehensive etiological screening for special infections and autoimmune diseases was completed with an unremarkable results, and whole-exome sequencing showed no abnormal findings. Following a multidisciplinary discussion combined with RNA sequencing results, the patient was diagnosed with an autoinflammatory disease, with a suspected type Ⅰ interferonopathy. Treatment with tofacitinib resulted in gradual improvement of clinical symptoms. This case highlights the importance of detailed medical history collection, systematic physical examination and multidisciplinary collaborative diagnosis and treatment, and underscores the pivotal role of molecular diagnosis in the confirmation of rare diseases. It can provide a reference for the clinical diagnosis and management of similar rare cases.

Idiopathic scoliosis (IS) is the most common form of spinal deformity with unclear pathogenesis. In this study, we first reanalyzed the loci associated with IS, drawing upon previous studies. Subsequently, we mapped these loci to candidate genes using either location-based or function-based strategies. To further substantiate our findings, we verified the enrichment of variants within these candidate genes across several large IS cohorts encompassing Chinese, East Asian, and European populations. Consequently, we identified variants in the EPHA4 gene as compelling candidates for IS. To confirm their pathogenicity, we generated zebrafish mutants of epha4a. Remarkably, the zebrafish epha4a mutants exhibited pronounced scoliosis during later stages of development, effectively recapitulating the IS phenotype. We observed that the epha4a mutants displayed defects in left-right coordination during locomotion, which arose from disorganized neural activation in these mutants. Our subsequent experiments indicated that the disruption of the central pattern generator (CPG) network, characterized by abnormal axon guidance of spinal cord interneurons, contributed to the disorganization observed in the mutants. Moreover, when knocked down efnb3b, the ligand for Epha4a, we observed similar CPG defects and disrupted left-right locomotion. These findings suggested that ephrin B3-Epha4 signaling is vital for the proper functioning of CPGs, and defects in this pathway could lead to scoliosis in zebrafish. Furthermore, we identified two cases of IS in NGEF, a downstream molecule in the EPHA4 pathway. Collectively, our data provide compelling evidence that neural patterning impairments and disruptions in CPGs may underlie the pathogenesis of IS.

Meniscus is vital for maintaining the anatomical and functional integrity of knee. Injuries to meniscus, commonly caused by trauma or degenerative processes, can result in knee joint dysfunction and secondary osteoarthritis, while current conservative and surgical interventions for meniscus injuries bear suboptimal outcomes. In the past decade, there has been a significant focus on advancing meniscus tissue engineering, encompassing isolated scaffold strategies, biological augmentation, physical stimulus, and meniscus organoids, to improve the prognosis of meniscus injuries. Despite noteworthy promising preclinical results, translational gaps and inconsistencies in the therapeutic efficiency between preclinical and clinical studies exist. This review comprehensively outlines the developments in meniscus tissue engineering over the past decade (Scheme 1). Reasons for the discordant results between preclinical and clinical trials, as well as potential strategies to expedite the translation of bench-to-bedside approaches are analyzed and discussed.

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.

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.

Background: Osteogenesis imperfecta (OI) is marked by clinical and genetic heterogeneity, and the genotype–phenotype correlation remains not very clear. We conducted a clinical and genetic study in a Chinese OI cohort to determine the spectra of phenotypes and pathogenic variants. Methods: In this study, 298 Chinese families were recruited from 2019 to 2024. Clinical phenotypes including fractures, short stature, skeletal deformities, blue sclera, dentinogenesis imperfecta, and hearing loss were recorded and analyzed. Next-generation sequencing combined with PCR-based techniques was used to detect candidate pathogenic variants. Variant pathogenicity was evaluated via conservation analysis, bioinformatics analysis, and functional studies at the cellular level. In this OI cohort, the spectra of pathogenic variants, clinical phenotypes, and genotype–phenotype correlations were analyzed. Results: Our OI cohort included 71 type I (23.83%), 122 type III (40.94%), 90 type IV (30.20%), and 15 type V (5.03%) probands. The cohort consisted of 196 children (65.77%) and 102 adults (34.23%). For the first time, phenotypic differences between different age groups were confirmed. In total, we identified 231 variants, including 47 novel pathogenic variants. Notable variants include two atypical splicing variants, one small deletion, two small duplications, one gross deletion, and one gross duplication. New genotype–phenotype correlations were observed: patients with SERPINF1 variants had the highest fracture frequency, followed by those with WNT1 variants, compared to patients with other gene variants. Conclusions: We performed the clinical and genetic analysis in a large Chinese OI cohort. The expanded spectra of genetic variants and clinical phenotypes were constructed by identifying 47 novel pathogenic variants and summarizing the skeletal and extra-skeletal manifestations. The current paper will provide important evidence for the precise diagnosis of the disease.

Objective To identify the causative variants in 5 Chinese families with tuberous sclerosis complex(TSC)to provide genetic counseling and prenatal diagnosis.Methods Genetic counseling and clinical diagnosis were performed in 8 patients from five unrelated TSC families by teleconsultation.With informed consent obtained from the participants,3 to 5 mL peripheral blood samples were collected from the probands and their family mem-bers for the extraction of genomic DNA.Candidate pathogenic variants were screened by panel sequencing(PS).The candidate pathogenic variants found in TSC1 and TSC2 by PS were validated by PCR-Sanger sequencing and bioinformatics analysis.Results All the pathogenic mutations were identified in the probands and their available family members.Causative variants in TSC1 or TSC2 were detected in all patients,including three reported variants and two novel variants.The two novel variants,TSC2:c.245G＞A and TSC2:c.235delG,which were predicted to cause the nonsense variant p.(Trp82∗)and the frameshift variant p.(Val79Lysfs27∗)respectively was believed to introduce premature stop codons.The analysis of family co-segregation and bioinformatics were identified as very positive factors for pathogenicity.Conclusions This result provides more evidences for the genetic counseling and prenatal diagnosis in these families and expand the spectrum of TSC2 pathogenic variants.

OBJECTIVE:To obtain skin-derived induced pluripotent stem cells (iPSCs) from an Osteogenesis imperfecta (OI) patient carrying WNT1c.677C>T mutation in order to provide a new cell model for investigating the underlying molecular mechanism and stem cell therapy for OI.METHODS:The pathogenic variant of the patient was identified by Sanger sequencing. With informed consent from the patient, skin tissue was biopsied, and primary skin fibroblasts were cultured. Skin fibroblasts were induced into iPSCs using Sendai virus-mediated non-genomic integration reprogramming method. The iPSC cell lines were characterized for pluripotency, differentiation capacity, and karyotyping assay.RESULTS:The patient was found to carry homozygous missense c.677C>T (p.Ser226Leu) mutation of the WNT1 gene. The established iPSC lines possessed self-renewal and capacity for in vitro differentiation. It also has a diploid karyotype (46,XX).CONCLUSION:A patient-specific WNT1 gene mutation (WNT1c.677C>T) iPSC line was established, which can provide a cell model for the study of OI caused by the mutation.

In recent years, with the development of gene testing and new drug research, the diagnosis and treatment of inherited diseases have made rapid progress, corresponding to higher requirements for genetics education. As a teacher of medical genetics, the author joined the course remodeling during last 10 years from a web-based study of genetic disorders to a “case-based learning” supported by flipped classroom in order to optimize teaching effects and learning outcomes. The result of this remodeling project proposes a new strategy to guide perspectives course design in future.

OBJECTIVE:To explore the genetic etiology of a fetus with cryptophthalmos detected by prenatal ultrasonography.METHODS:A fetus undergoing induced labor at 32nd gestational week due to absence of bilateral eye fissures detected by prenatal ultrasonography in January 2017 was selected as the study subject. Umbilical cord blood sample from the fetus and peripheral blood samples from its parents were collected for the extraction of genomic DNA. Pathogenic variants were screened through whole exome sequencing (WES) and verified by Sanger sequencing. Pathogenicity of candidate variants was verified by bioinformatic analysis and protein structure simulation. Based on the results of genetic testing, prenatal diagnosis was provided to the couple upon their subsequent pregnancy.RESULTS:The couple had four adverse pregnancies previously. The aborted fetus was the fifth, with fused bilateral upper and lower eyelids, poorly developed eyeballs, adhesion of the cornea with the upper eyelid, low-set ears, and abnormal plantar creases, and was diagnosed with cryptophthalmos. WES and Sanger sequencing revealed that the fetus has harbored compound heterozygous variants of the FREM2 gene, namely c.4537G>A (p.D1513N) and c.7292C>T (p.T2431M). Both variants were unreported associated with cryptophthalmos previously. Protein structure simulation showed that they may lead to loss of hydrogen bonds in the protein product. Based on the guidelines from the American College of Medical Genetics and Genomics (ACMG), both variants were predicted to be likely pathogenic (PM1_Supporting+PM2_Supporting+PM5+PP3+PP4; PM2_Supporting+PM3+PP3+PP4). The mother was performed prenatal diagnosis in her sixth pregnancy based on the variants detected in this family, and delivered a daughter with normal phenotype.CONCLUSION:The FREM2: c.4537G>A and c.7292C>T compound heterozygous variants probably underlay the pathogenesis of cryptophthalmos in this fetus. Above finding has enriched the mutational spectrum of the FREM2 gene.

Pathogenic variations in the NTRK1 can cause congenital insensitivity to pain with anhidrosis (CIPA), a rare autosomal recessive inherited neuropathy. The precise diagnosis of CIPA relies on the identification of pathogenic genotypes. Therefore, it is essential to expand the NTRK1 variation spectrum and improve molecular diagnosis methods. In this study, 74 probands with typical manifestations of CIPA but unknown genotypes were recruited. A comprehensive molecular genetic analysis was performed to identify variations in the NTRK1, using techniques including Sanger and next-generation sequencing, bioinformatic analysis, quantitative polymerase chain reaction (qPCR), gap-PCR, short tandem repeat (STR) genotyping, and reverse-transcription PCR. In addition, functional assays were conducted to determine the pathogenicity of variants of uncertain significance (VUS) and further characterized changes in glycosylation and phosphorylation of 14 overexpressed mutant vectors with variants at different domains in the TrkA protein, which is encoded by NTRK1. A total of 48 variations in the NTRK1 were identified, including 22 novel ones. When combined with data from another 53 CIPA patients examined in our previous work, this study establishes the largest genotypic and phenotypic spectra of CIPA worldwide, including 127 CIPA families. Moreover, functional studies indicated that the pathogenicity of VUS mainly affected insufficient glycosylation in the extracellular domain and abnormal phosphorylation in the intracellular domain. This study not only provides important evidence for precise diagnosis of CIPA but also further enriches our understanding of the pathogenesis of this disease.

Syndactyly type V (SDTY5) is an autosomal dominant extremity malformation characterized by fusion of the fourth and fifth metacarpals. In the previous publication, we first identified a heterozygous missense mutation Q50R in homeobox domain (HD) of HOXD13 in a large Chinese family with SDTY5. In order to substantiate the pathogenicity of the variant and elucidate the underlying pathogenic mechanism causing limb malformation, transcription-activator-like effector nucleases (TALEN) was employed to generate a Hoxd13Q50R mutant mouse. The mutant mice exhibited obvious limb malformations including slight brachydactyly and partial syndactyly between digits 2-4 in the heterozygotes, and severe syndactyly, brachydactyly and polydactyly in homozygotes. Focusing on BMP2 and SHH/GREM1/AER-FGF epithelial mesenchymal (e-m) feedback, a crucial signal pathway for limb development, we found the ectopically expressed Shh, Grem1 and Fgf8 and down-regulated Bmp2 in the embryonic limb bud at E10.5 to E12.5. A transcriptome sequencing analysis was conducted on limb buds (LBs) at E11.5, revealing 31 genes that exhibited notable disparities in mRNA level between the Hoxd13Q50R homozygotes and the wild-type. These genes are known to be involved in various processes such as limb development, cell proliferation, migration, and apoptosis. Our findings indicate that the ectopic expression of Shh and Fgf8, in conjunction with the down-regulation of Bmp2, results in a failure of patterning along both the anterior-posterior and proximal-distal axes, as well as a decrease in interdigital programmed cell death (PCD). This cascade ultimately leads to the development of syndactyly and brachydactyly in heterozygous mice, and severe limb malformations in homozygous mice. These findings suggest that abnormal expression of SHH, FGF8, and BMP2 induced by HOXD13Q50R may be responsible for the manifestation of human SDTY5.

Synpolydactyly (SPD) is mainly caused by mutations of polyalanine expansion (PAE) in the transcription factor gene HOXD13 and the involved cell types and signal pathway are still not clear possible pathways and single-cell expression characteristics of limb bud in HOXD13 PAE mice was analyzed in this study. We investigated a previous study of a mouse model with SPD and conducted weighted gene co-expression network analysis (WGCNA) using a single-cell RNA sequencing dataset from limb bud cells of SPD mouse model of HOXD13 + 7A heterozygote. Analysis of WGCNA revealed that synpolydactyly-associated Hoxd13 PAEs alter the immune response and osteoclast differentiation, and enhance DNA replication. Bmp4, Hand2, Hoxd12, Lnp, Prrx1, Gmnn, and Cdc6 were found to play potentially key roles in synpolydactyly. These findings evaluated the main genes related to SPD with PAE mutations in HOXD13 and advance our understanding of human limb development.

Synpolydactyly (SPD) is caused by mutations in the transcription factor gene HOXD13. Such mutations include polyalanine expansion (PAE), but further study is required for the phenotypic spectrum characteristics of HOXD13 PAE. We investigated four unrelated Chinese families with significant limb malformations. Three PAEs were found in the HOXD13 polyalanine coding region: c.172_192dup (p.Ala58_Ala64dup) in Family 1, c.169_192dup (p.Ala57_Ala64dup) in Family 2, and c.183_210dup (p.Ala62_Ala70dup) in Family 3 and Family 4. Interestingly, we identified a new manifestation of preaxial polydactyly in both hands in a pediatric patient with an expansion of seven alanines, a phenotype not previously noted in SPD patients. Comparing with the wild-type cells and mutant cells with polyalanine contractions (PACs), the HOXD13 protein with a PAE of nine-alanine or more was difficult to enter the nucleus, and easy to form inclusion bodies in the cytoplasm, and with the increase of PAE, the more inclusion bodies were formed. This study not only expanded the phenotypic spectrum of SPD, but also enriched our understanding of its pathogenic mechanisms.