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Verheij syndrome (VRJS) is a rare genetic disorder characterized by a range of developmental issues and physical abnormalities. This condition is caused by mutations or deletions in the PUF60 (poly-U-binding factor 60 kDa) gene, which is located on the long arm of chromosome 8, specifically in the q24.3 region. We present a unique case of an 11-year-old girl child with VRJS. The child presented with absence seizures. She was noted to have short stature, spina bifida of the lower cervical vertebrae, and a smaller right kidney on ultrasonography. This case expands the phenotypic spectrum of VRJS by demonstrating a milder presentation, highlighting the importance of a high index of suspicion for the diagnosis, even in atypical presentations. Whole exome sequencing identified the causative mutation, confirming the diagnosis. Growth hormone therapy was initiated for short stature but discontinued due to the subsequent development of idiopathic intracranial hypertension. Additionally, this report represents the first documented case of VRJS in India, emphasizing the importance of global data sharing and collaboration for improving the understanding and management of rare genetic disorders.

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Craniofacial microsomia (CFM), also known as the oculo-auriculo-vertebral spectrum, is a congenital disorder characterized by hypoplasia of the mandible and external ear due to tissue malformations originating from the first and second branchial arches. However, distinguishing it from other syndromes of branchial arch abnormalities is difficult, and causal variants remain unidentified in many cases. In this report, we performed an exome sequencing analysis of a Brazilian family with CFM. The proband was a 12-month-old boy with clinical findings consistent with the diagnostic criteria for CFM, including unilateral mandibular hypoplasia, microtia, and external auditory canal abnormalities. A heterozygous de novo nonsense variant (c.713C>G, p.S238*) in PUF60 was identified, which was predicted to be pathogenic in silico. PUF60 has been reported as a causal gene in Verheij syndrome, but not in CFM. Although the boy showed craniofacial abnormalities and developmental delay that overlapped with Verheij syndrome, the facial asymmetry with unilateral hypoplasia of the mandible observed in this case did not match the previously reported phenotypes of PUF60 variants. Our findings expand the phenotypic range of PUF60 variants that cover CFM and Verheij syndrome.

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Heterozygous variants in the Poly(U) Binding Splicing Factor 60kDa gene (PUF60) have been associated with Verheij syndrome, which has the key features of coloboma, short stature, skeletal abnormalities, developmental delay, palatal abnormalities, and congenital heart and kidney defects. Here, we report five novel patients from unrelated families with PUF60-related disorders exhibiting novel genetic and clinical findings with three truncating variants, one splice-site variant with likely reduced protein expression, and one missense variant. Protein modeling of the patient's missense variant in the PUF60 AlphaFold structure revealed a loss of polar bonds to the surrounding residues. Neurodevelopmental disorders were present in all patients, with variability in speech, motor, cognitive, social-emotional and behavioral features. Novel phenotypic expansions included movement disorders as well as immunological findings with recurrent respiratory, urinary and ear infections, atopic diseases, and skin abnormalities. We discuss the role of PUF60 in immunity with and without infection based on recent organismic and cellular studies. As our five patients showed less-severe phenotypes than classical Verheij syndrome, particularly with the absence of key features such as coloboma or palatal abnormalities, we propose a reclassification as PUF60-related neurodevelopmental disorders with multi-system involvement. These findings will aid in the genetic counseling of patients and families.

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Klippel-Feil syndrome (KFS) has a genetically heterogeneous phenotype with six known genes, exhibiting both autosomal dominant and autosomal recessive inheritance patterns. PUF60 is a nucleic acid-binding protein, which is involved in a number of nuclear processes, including pre-mRNA splicing, apoptosis, and transcription regulation. Pathogenic variants in this gene have been described in Verheij syndrome due to either 8q24.3 microdeletion or PUF60 single-nucleotide variants. PUF60-associated conditions usually include intellectual disability, among other findings, some overlapping KFS; however, PUF60 is not classically referred to as a KFS gene. Here, we describe a 6-year-old female patient with clinically diagnosed KFS and normal cognition, who harbors a heterozygous de novo variant in the PUF60 gene (c.1179del, p.Ile394Serfs*7). This is a novel frameshift variant, which is predicted to result in a premature stop codon. Clinically, our patient demonstrates a pattern of malformations that matches reported cases of PUF60 variants; however, unlike most others, she has no clear learning difficulties. In light of these findings, we propose that PUF60 should be considered in the differential diagnosis of KFS and that normal cognition should not exclude its testing.

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PURPOSE: Ovarian cancer (OC) is the leading cause of death from gynecological malignancies, and its etiology and pathogenesis are currently unclear. Recent studies have found that PUF60 overexpressed in various cancers. However, the exact function of PUF60 in global RNA processing and its role in OC has been unclear. METHODS: The expression of PUF60 and its relationship with clinical characteristics were analyzed by multiple database analysis and immunohistochemistry. Phenotypic effects of PUF60 on ovarian cancer cell proliferation and metastasis were examined by in vitro cell proliferation assay, migration assay, and in vivo xenograft models and lung metastasis models. RNA immunoprecipitation, seahorse analyses, RNA stability assay were used to study the effect of PUF60 on the stability of oxidative phosphorylation (OXPHOS)-related genes in OC. RESULTS: We report PUF60 is highly expressed in OC with frequent amplification of up to 33.9% and its upregulation predicts a poor prognosis. PUF60 promotes the proliferation and migration of OC cells both in vitro and in vivo. Mechanistically, we demonstrated that silencing of PUF60 enhanced the stability of mRNA transcripts involved in OXPHOS and decreased the formation of processing bodies (P-bodies), ultimately elevating the OXPHOS level. CONCLUSION: Our study unveils a novel function of PUF60 in OC energy metabolism. Thus, PUF60 may serve as a novel target for the treatment of patients with OC.

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Verheij syndrome (VRJS) is a craniofacial spliceosomopathy with a wide phenotypic spectrum. Haploinsufficiency of the poly-uridine binding splicing factor 60 gene (PUF60) and its loss-of-function (LOF) variants are involved in VRJS. We evaluated a human fetus with congenital heart defects and preaxial polydactyly. Clinical data were obtained from the medical record. Whole-exome sequencing (WES) was used to explore the potential genetic etiology, and the detected variant verified using Sanger sequencing. Functional studies were performed to validate the pathogenic effects of the variant. Using trio-WES, we identified a novel PUF60 variant (NM_078480.2; c.1678 T > A, p.*560Argext*204) in the pedigree. Bioinformatic analyses revealed that the variant is potentially pathogenic, and functional studies indicated that it leads to degradation of the elongated protein and subsequently PUF60 LOF, producing some VRJS phenotypes. These findings confirmed the pathogenicity of the variant. This study implicates PUF60 LOF in the etiopathogenesis of VRJS. It not only expands the PUF60 variant spectrum, and also provides a basis for genetic counseling and the diagnosis of VRJS. Although trio-WES is a well-established approach for identifying the genetic etiology of rare multisystemic conditions, functional studies could aid in verifying the pathogenicity of novel variants.

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Alternative splicing (AS) has been implicated in cell cycle regulation and cancer, but the underlying mechanisms are poorly understood. The poly(U)-binding splicing factor 60 (PUF60) is essential for embryonic development and is overexpressed in multiple types of cancer. Here, we report that PUF60 promotes mitotic cell cycle and lung cancer progression by controlling AS of the cell division cycle 25C (CDC25C). Systematic analysis of splicing factors deregulated in lung adenocarcinoma (LUAD) identifies that elevated copy number and expression of PUF60 correlate with poor prognosis. PUF60 depletion inhibits LUAD cell-cycle G2/M transition, cell proliferation, and tumor development. Mechanistically, PUF60 knockdown leads to exon skipping enriched in mitotic cell cycle genes, including CDC25C. Exon 3 skipping in the full-length CDC25C results in nonsense-mediated mRNA decay and a decrease of CDC25C protein, thereby inhibiting cell proliferation. This study establishes PUF60 as a cell cycle regulator and an oncogenic splicing factor in lung cancer.

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PUF60-related developmental disorder (also referred to as Verheij syndrome), resulting from haploinsufficiency of PUF60, is associated with multiple congenital anomalies affecting a wide range of body systems. These anomalies include ophthalmic coloboma, and congenital anomalies of the heart, kidney, and musculoskeletal system. Behavioral and intellectual difficulties are also observed. While less common than other features associated with PUF60-related developmental disorder, for instance hearing impairment and short stature, identification of specific anomalies such as ophthalmic coloboma can aid with diagnostic identification given the limited spectrum of genes linked with this feature. We describe 10 patients with PUF60 gene variants, bringing the total number reported in the literature, to varying levels of details, to 56 patients. Patients were recruited both via locally based exome sequencing from international sites and from the DDD study in the United Kingdom. Eight of the variants reported were novel PUF60 variants. The addition of a further patient with a reported c449-457del variant to the existing literature highlights this as a recurrent variant. One variant was inherited from an affected parent. This is the first example in the literature of an inherited variant resulting in PUF60-related developmental disorder. Two patients (20%) were reported to have a renal anomaly consistent with 22% of cases in previously reported literature. Two patients received specialist endocrine treatment. More commonly observed were clinical features such as: cardiac anomalies (40%), ocular abnormalities (70%), intellectual disability (60%), and skeletal abnormalities (80%). Facial features did not demonstrate a recognizable gestalt. Of note, but remaining of unclear causality, we describe a single pediatric patient with pineoblastoma. We recommend that stature and pubertal progress should be monitored in PUF60-related developmental disorder with a low threshold for endocrine investigations as hormone therapy may be indicated. Our study reports an inherited case with PUF60-related developmental disorder which has important genetic counseling implications for families.

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BACKGROUND: Verheij syndrome (VRJS) is a rare microdeletion syndrome of chromosome 8q24.3 that is characterized by severe growth retardation, microcephaly, vertebral anomalies, joint laxity/dislocation, psychomotor retardation, cardiac and renal defects, and dysmorphic facial features. Pathogenic variants of PUF60 (Poly-U Binding Splicing Factor 60 kDa) have been found to cause VRJS. Here we present a Turkish patient with Verheij syndrome who has typical facial dysmorphic features and renal and cardiac abnormalities, scoliosis, tethered cord, and mild intellectual disability. METHODS: This is a case report of a 11-year-old female child who presented with Verheij syndrome. Blood samples were collected from the patient and the family. We performed whole exome sequencing was used to identify potential genetic mutations. We also used 3-dimensional protein structure analysis to identify the effect of the mutation. RESULTS: A de-novo in-frame variant (c.449_457delCAAAGGGGG; p.Ala150_Phe152del) of the PUF60 gene was identified by whole exome sequencing. According to ACMG guidelines in 2015, the mutation is classified as pathogenic and it has been reported in the clinvar database. Results of in-silico prediction software tools predicted the mutation was pathogenic. Protein structure analysis showed that the three residues affected by the in-frame deletion form could lead to impaired stability and function of the PUF60 protein. CONCLUSIONS: To date, 25 patients have been reported with PUF60 mutations in the medical literature. In this article, we report a patient with VRJS who had the unusual findings of tethered cord syndrome and renal abnormalities. As far as we know, this is the first patient from Turkey who has been diagnosed with Verheij syndrome.

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PUF60 (Poly (U) binding splicing factor 60 kDa), a nucleic acid-binding protein, has been shown to regulate transcription and links to tumorigenesis in various cancers. However, its biological role and function in glioblastoma remain unknown. In this study, we found that PUF60 is highly expressed in glioblastoma and correlated with poor prognosis. Furthermore, PUF60 knockdown significantly decreased the proliferation of glioblastoma cells in vitro and in vivo. Mechanistically, PUF60 could reduce the ubiquitination level of EGFR by transcriptionally regulating STUB1, an E3 ubiquitin ligase of EGFR, which lead to the activation of the EGFR-AKT pathway. Collectively, our study reveals the oncogenic role of PUF60 in glioblastoma and provides a potential therapeutic target for glioblastoma treatment.

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Verheij syndrome (VRJS) is a rare craniofacial spliceosomopathy presenting with craniofacial dysmorphism, multiple congenital anomalies and variable neurodevelopmental delay. It is caused by single nucleotide variants (SNVs) in PUF60 or interstitial deletions of the 8q24.3 region. PUF60 encodes a splicing factor which forms part of the spliceosome. To date, 36 patients with a sole diagnosis of VRJS due to disease-causing PUF60 SNVs have been reported in peer-reviewed publications. Although the depth of their phenotyping has varied greatly, they exhibit marked phenotypic heterogeneity. We report 10 additional unrelated patients, including the first described patients of Khmer, Indian, and Vietnamese ethnicities, and the eldest patient to date, with 10 heterozygous PUF60 variants identified through exome sequencing, 8 previously unreported. All patients underwent deep phenotyping identifying variable dysmorphism, growth delay, neurodevelopmental delay, and multiple congenital anomalies, including several unique features. The eldest patient is the only reported individual with a germline variant and neither neurodevelopmental delay nor intellectual disability. In combining these detailed phenotypic data with that of previously reported patients (n = 46), we further refine the known frequencies of features associated with VRJS. These include neurodevelopmental delay/intellectual disability (98%), axial skeletal anomalies (74%), appendicular skeletal anomalies (73%), oral anomalies (68%), short stature (66%), cardiac anomalies (63%), brain malformations (48%), hearing loss (46%), microcephaly (41%), colobomata (38%), and other ocular anomalies (65%). This case series, incorporating three patients from previously unreported ethnic backgrounds, further delineates the broad pleiotropy and mutational spectrum of PUF60 pathogenic variants.

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Circadian genes such as Clock, Bmal1, Cryptochrome1/2, and Period1/2/3 constitute the precise circadian system. ClockΔ19 is a commonly used mouse model harboring a circadian clock gene mutation, which lacks the EXON-19-encoded 51 amino acids. Previous reports have shown that ClockΔ19 mice have severe metabolic abnormalities. Here, we report that the mitochondria of ClockΔ19 mice exhibit excessive fission and dysfunction. We also demonstrate that CLOCK binds to the RNA-binding protein PUF60 through its EXON 19. Further, we find that PUF60 directly maintains mitochondrial homeostasis through regulating Drp1 mRNA stability, while the association with CLOCK can competitively inhibit this function. In ClockΔ19 mice, CLOCKΔ19 releases PUF60, leading to enhanced Drp1 mRNA stability and persistent mitochondrial fission. Our results reveal a direct post-transcriptional role of CLOCK in regulating mitochondrial homeostasis via Drp1 mRNA stability and that the loss of EXON 19 of CLOCK in ClockΔ19 mice leads to severe mitochondrial homeostasis disorders.

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The mechanisms underlying self-renewal of embryonic stem cells (ESCs) hold great value in the clinical translation of stem cell biology and regenerative medicine research. To study the mechanisms in ESC self-renewal, screening and identification of key genes maintaining ESC self-renewal were performed by a genome-wide CRISPR-Cas9 knockout virus library. The mouse ESC R1 were infected with CRISPR-Cas9 knockout virus library and cultured for 14 days. The variation of single guide RNA (sgRNA) ratio was analyzed by high-throughput sequencing, followed by bioinformatics analysis to profile the altered genes. Our results showed 1375 genes with increased sgRNA ratio were found to be mainly involved in signal transduction, cell differentiation, and cell apoptosis; 2929 genes with decreased sgRNA ratio were mainly involved in cell cycle regulation, RNA splicing, and biological metabolic processes. We further confirmed our screen specificity by identifying Puf60, U2af2, Wdr75, and Usp16 as novel positive regulators in mESC self-renewal. Meanwhile, further analysis showed the relevance between Puf60 expression and tumor. In conclusion, our study screened key genes maintaining ESC self-renewal and successfully identified Puf60, U2af2, Wdr75, and Usp16 as novel positive regulators in mESC self-renewal, which provided theoretical basis and research clues for a better understanding of ESC self-renewal regulation.

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Aging has many effects on the female reproductive system, among which decreased oocyte quality and impaired embryo developmental potential are the most important factors affecting female fertility. However, the mechanisms underlying oocyte aging are not yet fully understood. Here, we selected normal reproductively aging female mice and constructed a protein expression profile of metaphase II (MII) oocytes from three age groups. A total of 187 differentially expressed (DE) proteins were identified, and bioinformatics analyses showed that these DE proteins were highly enriched in RNA splicing. Next, RNA-seq was performed on 2-cell embryos from these three age groups, and splicing analysis showed that a large number of splicing events and genes were discovered at this stage. Differentially spliced genes (DSGs) in the two reproductively aging groups versus the younger group were enriched in biological processes related to DNA damage repair/response. Binding motif analysis suggested that PUF60 might be one of the core splicing factors causing a decline in DNA repair capacity in the subsequent development of oocytes from reproductively aging mice, and changing the splicing pattern of its potential downstream DSG Cdk9 could partially mimic phenotypes in the reproductively aging groups. Taken together, our study suggested that the abnormal expression of splicing regulation proteins in aged MII oocytes would affect the splicing of nascent RNA after zygotic genome activation in 2-cell embryos, leading to the production of abnormally spliced transcripts of some key genes associated with DNA damage repair/response, thus affecting the developmental potential of aged oocytes.

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Background/Aim: The aim of the study was to report the case of a 5-month-old boy with a complex prenatal and neonatal symptomatology diagnosed with a "de novo" pathogenic variant of PUF60 gene. Case Report: Our hospital, undertook the antenatal and postnatal care of a 27-year-old pregnant lady. This was her second baby with a previously healthy boy. During her routine first-trimester anomaly scan, increased nuchal translucency was noticed. Multiple anomalies were seen throughout her subsequent antenatal visits. This triggered a sequence of tests, examinations and differential diagnosis. The final diagnosis was made at 5 months postpartum following the result of the whole exome sequence, which described a variant of unknown clinical significance (VUS, class 3 variant) in the PUF60 gene. We are mindful that changing the classification of a variant of unknown significance is challenging and requires supporting and robust criteria. Considering clinical symptomatology produced by the pathogenic mutation in the PUF gene, the identified c.1640A>G variant may be categorized as likely pathogenic. Conclusion: Our case adds new insights on the pathology and the underlying process involved in the PUF60 variant spectrum disorders. It also highlights the limits of current prenatal tests.

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Cancer cells employ alternative splicing (AS) to acquire splicing isoforms favouring their survival. However, the causes of aberrant AS in breast cancer are poorly understood. In this study, the METABRIC (Molecular Taxonomy of Breast Cancer International Consortium) data were analysed with univariate feature selection. Of 122 analysed spliceosome components, U2SURP, PUF60, DDX41, HNRNPAB, EIF4A3, and PPIL3 were significantly associated with breast cancer survival. The top 4 four genes, U2SURP, PUF60, DDX41, and HNRNPAB, were chosen for further analyses. Their expression was significantly associated with cancer molecular subtype, tumour stage, tumour grade, overall survival (OS), and cancer-specific survival in the METABRIC data. These results were verifiable using other cohorts. The Cancer Genome Atlas data unveiled the elevated expression of PUF60, DDX41, and HNRNPAB in tumours compared with the normal tissue and confirmed the differential expression of the four genes among cancer molecular subtypes, as well as the associations of U2SURP, PUF60, and DDX41 expression with tumour stage. A meta-analysis data verified the associations of U2SURP, PUF60, and HNRNPAB expression with tumour grade, the associations of PUF60, DDX41, and HNRNPAB expression with OS and distant metastasis-free survival, and the associations of U2SURP and HNRNPAB expression with relapse-free survival. Experimentally, we demonstrated that inhibiting the expression of the four genes separately suppressed cell colony formation and slowed down cell growth considerably in breast cancer cells, but not in immortal breast epithelial cells. In conclusion, we have identified U2SURP, PUF60, DDX41, and HNRNPAB are spliceosome-related genes pivotal for breast cancer survival.

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Abnormal expression or mutation of RNA splicing proteins are widely observed in human cancers. Here, we identified poly(U) binding splicing factor 60 (PUF60) as one of the most differentially expressed genes out of 97 RNA splicing proteins between normal and bladder cancer tissues by bioinformatics analysis of TCGA bladder cancer expression data. The expression of PUF60 was significantly higher in tumor tissues, while high PUF60 expression was associated with malignant phenotypes of bladder cancer and shorter survival time. Moreover, we identified aurora kinase A (AURKA) as a new downstream target of PUF60 in bladder cancer cells. PUF60 knockdown significantly inhibited cell viability and colony formation capacity in bladder cancer cells, whereas AURKA overexpression reversed this inhibition effect. Overexpression of PUF60 significantly promoted cell viability and colony formation in bladder cancer cells, while treatment with AURKA specific inhibitor reversed this promotive effect. Mechanistically, PUF60 specifically bound to the AURKA promoter, thereby activating its transcription and expression. Furthermore, we showed that there was a significant positive correlation between PUF60 and AURKA expression in bladder cancer tissues, and PUF60 and AURKA expression contributed to tumor progression and malignant phenotypes in the patients with bladder cancer. Collectively, these results indicate that the PUF60/AURKA axis plays a key role in regulating tumorigenesis and progression of bladder cancer, and may be a potential prognostic biomarker and therapeutic target for bladder cancer patients.

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Background: Abnormal transcriptional upregulation of telomerase reverse transcriptase (TERT) plays a dominant role in telomerase activation in various cancers. TERT promoter mutations (TPMs) have been identified as a key mechanism in TERT upregulation. However, the mechanism of TERT upregulation in cancers with low frequency of TPMs are not fully elucidated so far. Methods: The expression of PUF60 and TERT was detected by real-time PCR, western blot and immunohistochemistry. TERT promoter binding proteins were identified by streptavidin-agarose pulldown assay and mass spectrum (MS) analysis. The role of PUF60/TERT in renal cancer was evaluated on cell growth in vitro and in vivo. Results: In this study, we identify the regulation mechanism of TERT in renal cell carcinoma (RCC) cells which have rare TPMs but exert significant upregulation of TERT. We found that TERT was highly expressed in RCC tumor tissues, and elevated TERT expression was associated with poor prognosis for patients. We also detected the relatively rare TPM status in both RCC tumor tissues and RCC cell lines. Mechanistically, PUF60, a RNA binding protein, was identified as a novel TERT regulator which bound to the TERT and transcriptionally upregulated TERT expression in RCC cells. The in vitro and in vivo experiments also demonstrated that PUF60 could promote RCC cell growth through activation of TERT expression in a TPM status independent way. Furthermore, we showed that there was a strong correlation of the expression of PUF60 and TERT in RCC tumor tissues and RCC cell lines, and the patients with high expression of PUF60 and TERT had significantly shorter survival. Conclusions: Collectively, these results indicated that PUF60 transcriptionally upregulated TERT expression to promote RCC growth and progression in a TPM status independent way, suggesting that the PUF60/TERT signaling pathway may serve as potential prognostic biomarkers and therapeutic targets for RCC.

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The PUF60 gene encodes a ubiquitously expressed essential splicing factor that is recruited to the U2snRNA complex. The complex binds to the 3' splice site of exons in specific target genes and regulates the inclusion or exclusion of such exons. Recently, pathogenic variants of PUF60 have been shown to cause a relatively specific and potentially recognizable pattern of malformation referred to as Verheij syndrome. Here, we report a 12-year-old female patient with a de novo mutation in PUF60 whose phenotype was representative of the milder end of the phenotypic spectrum of Verheij syndrome; the de novo mutation was a frameshift mutation p.(Ser558Cysfs*21) that resulted in the addition of 21 extra amino acids at the carboxy end of the protein. Among the frequent features of Verheij syndrome, the patient exhibited coloboma, cervical spinal segmentation defects, and borderline intellectual functioning, but lacked cardiac abnormalities, deafness, and urogenital abnormalities. The results of RNA analysis using peripheral blood showed the escape of the mutant allele from nonsense-mediated mRNA decay, possibly accounting for the mild phenotype in the presently reported patient. Based on our clinical observations, we inferred that two embryologic processes, closure of the ocular plate and cervical spinal segmentation, are particularly susceptible to deficient PUF60-mediated splicing regulation, compared with other embryogenetic processes leading to the central nervous system, heart, ear, and kidney.

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