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Multiple osteochondromas (MO) is an autosomal dominant disorder and the most common genetic skeletal dysplasia, characterized by the growth of bone outgrowths capped by cartilage, called osteochondromas. Most MO cases are caused by mutations in the exostosin-1 ( EXT1 ) and exostosin-2 ( EXT2 ) genes. Only 5% of MO-causative variants are represented by single or multiple exon deletions; to date, no pathogenic large duplication has been described in the literature. In the present study, we describe the novel in-tandem intragenic duplication c.(1128_1202)_(1284+29_1344)dup involving exon 4 of EXT1 (NM_000127.2), detected in a three-generation family with MO. The variant has been detected by MLPA (multiplex ligation-dependent probe amplification) and then confirmed with qPCR (quantitative PCR). Our finding expands the spectrum of MO-causing variants describing a pathogenic large duplication, underlying the importance of quantitative analysis in patients with negative sequencing.

Stüve-Wiedemann syndrome (SWS), a rare autosomal recessive disorder, characterized by diminutive size, curvature of the elongated bones, bent fingers, episodes of heightened body temperature, respiratory distress or periods of breath-holding, and challenges with feeding, especially causes fatality in infants. SWS is an outcome of potential missense mutations in the leukemia inhibitory factor receptor gene reflected as numerous amino acid mutations at protein level. Employing in silico tools and techniques like mutational screening with Pred_MutHTP, I-Mutant2.0, PANTHER.db, PolyPhen, to classify mutations as deleterious/destabilizing, in conjunction with experimental data analysis, P136A and S279P emerged as 'effect'-causing mutations. Pre-existing knowledge suggests, SWS progression is effectuated conformationally altered and dysfunctional LIFR, unable to bind to LIF and further form the LIF/LIFR/gp130 signalling complex. To gain functional insights into the effect of the said mutations on the wild type protein, an all-atom, explicit, solvent molecular dynamics simulation was performed following docking approaches. Consequently, referring to the RMSD, RMSF, protein dynamic network analysis, energy landscape plots and domain motion analysis, it was revealed that unbound LIFR_WT was more prone to LIF binding as usual whereas the mutants exhibited considerable domain closure to inhibit LIF binding. We conducted binding affinity analysis via MM/GBSA and dissociation constant estimation after LIFR-LIF docking and found the WT_complex to be more stable and compact as a whole when compared to the flexible mutant complexes thus being associated with SWS. Our study offers a route for understanding molecular level implications upon LIFR mutations which opens an avenue for therapeutic interventions., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Background: Multiple osteochondromas is genetic disorder characterized by the formation of multiple benign cartilage-capped bone tumors, named osteochondromas, during skeletal development. The most feared complication is the secondary peripheral chondrosarcoma, a malignant cartilaginous neoplasm that arises from the chondroid cap of pre-existent osteochondromas. We conducted a retrospective cohort study on patients diagnosed and followed up from 1960 to 2019 to describe the clinical and pathological features of individuals affected by peripheral chondrosarcoma in multiple osteochondromas, to evaluate follow up information and individual outcome and to compare the results with literature. Data, including age, gender, site, histological grade, cartilage cap thickness, surgical treatments, surgical margins, genotype mutational status as well as treatment details were captured from the hospital electronic health records and from Registry of Multiple Osteochondromas. In addition, a complete histological review of all hematoxylin and eosin (H&E)-stained sections has been performed by expert pathologists., Results: One hundred five of the screened cases were included in the present study. The age at diagnosis of SPC ranges from 13 to 63, with median age at diagnosis of 34 years. The site most frequently affected by malignant degeneration was the pelvis (46 patients, 44%) with higher incidence in male patients (32 males vs.14 females). The second one was lower limbs (including femur, fibula, or tibia), identified in 35 patients. Histological information - available for 103 patients - showed: 59 patients with grade 1; 40 patients had a grade 2 and 4 patients had a grade 3. The most common surgical treatment was the complete resection, followed by debulking, amputation and partial resection. Most of cases did not have recurrence of the disease. Outcome in disease-free survival highlights that a worse course of the disease was associated with histological grade 2 or 3, and partial resection surgery. In most of analyzed cases (94%) a pathogenic variant was identified., Conclusions: In conclusion, the present study gives an overview of the secondary peripheral chondrosarcomas, confirming that this disease represents an impacting complication for multiple osteochondromas patients and suggests that malignant transformation can occur also in younger patient, in a not irrelevant number of cases., (© 2024. The Author(s).)

Background: The Pediatric Outcomes Data Collection Instrument (PODCI) is a validated quality-of-life questionnaire with 6 domains designed to provide a standardized method of measuring outcomes in pediatric musculoskeletal conditions. To our knowledge there are no reports on its use in children with multiple hereditary exostosis (MHE)., Questions/purposes: Most published studies on MHE patients have described the efficacy of specific surgical techniques or the specification of deformities. Little is known about the general health status of pediatric patients, the severity of pain, loss of function, and how MHE influences the activities of daily life. We aim to assess the functional levels of MHE pediatric patients with PODCI questionnaire., Patients and Methods: As a cross-sectional study, we prospectively administered PODCI to 34 pediatric patients diagnosed with MHE and their families. The score distributions were compared with values published earlier for children and adolescents without musculoskeletal disorders using the Student and Welch t tests. Parents and adolescents' reports were compared using Wilcoxon signed rank test. Physical examination and PODCI score relation were evaluated by Spearman test., Results: Children with MHE have significantly lower scores ( P <0,05) in comparison with unaffected children in all domains using the Student and Welch t test. Parents score differs from children score with statistically relevance in pain and comfort domain ( P <0,5). The Spearman test showed a negative correlation between physical examination and PODCI score with statistical significance., Conclusions: These results point towards PODCI's capacity in evaluating functional outcomes of pediatric patients with MHE., Level of Evidence: Diagnostic Study, Level III., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2023 Wolters Kluwer Health, Inc. All rights reserved.)

Only a limited number of genetic diseases are diagnosable in archaeological individuals and none have had causal mutations identified in genome-wide screens. Two individuals from the Gaelic Irish Medieval burial ground of Ballyhanna, Co. Donegal, showed evidence of bone tumours consistent with the autosomal dominant condition multiple osteochondromas. Genome sequencing of the earlier individual uncovered a missense mutation in the second exon of EXT1, a specific lesion that has been identified in several modern patients. The later individual lacked this but displayed a novel frameshift mutation leading to a premature stop codon and loss of function in the same gene. These molecular confirmations of a paleopathological diagnosis within a single rural ancient context are surprisingly disjunct, given the observation of clusters of this disease in modern isolated populations and a de novo mutation rate of only 10%., (© 2022. The Author(s), under exclusive licence to European Society of Human Genetics.)

Background and Objectives : Hereditary multiple exostoses (HME) is a disease characterized by cartilage-capped bony protuberances at the site of growth plates of long bones. Functional mutations in the exostosin genes ( EXT1 and EXT2 ) are reported to affect the hedgehog signalling pathways leading to multiple enchondromatosis. However, the exact role of each EXT protein in the regulation of heparan sulphate (HS) chain elongation is still an enigma. In this study, a Pakistani family with HME is investigated to find out the genetic basis of the disease. Materials and Methods : Genotyping of eight members of the family by amplifying microsatellite markers, tightly linked to the EXT1 and EXT2 genes. Results : The study revealed linkage of the HME family to the EXT1 locus 8q24.1. Sanger sequencing identified a heterozygous deletion ( c.247Cdel ) in exon 1 of EXT1 , segregating with the disease phenotype in the family. In silico analysis predicted a shift in the frame causing an early stop codon (p.R83GfsX52). The predicted dwarf protein constituting 134 amino acids was functionally aberrant with a complete loss of the catalytic domain at the C-terminus. Interestingly, an alternative open reading frame 3 (ORF3) caused by the frame shift is predicted to encode a protein sequence, identical to the wild type and containing the catalytic domain, but lacking the first 100 amino acids of the wild-type EXT1 protein. Conclusion : Consequently, haploinsufficiency could be the cause of HME in the investigated family as the mutated copy of EXT1 is ineffective for EXT-1/2 complex formation. The predicted ORF3 protein could be of great significance in understanding several aspects of HME pathogenesis.

Multiple Osteochondromatosis (MO, MIM 133700 & 133701), an autosomal dominant O-glycosylation disorder (EXT1/EXT2-CDG), can be associated with a reduction in skeletal growth, bony deformity, restricted joint motion, shortened stature and pathogenic variants in two tumor suppressor genes, EXT1 and EXT2. In this work, we report a cross-sectional study including 35 index patients and 20 affected family members. Clinical phenotyping of all 55 affected cases was obtained, but genetic studies were performed only in 35 indexes. Of these, a total of 40% ( n = 14) had a family history of MO. Clinical severity scores were class I in 34% ( n :18), class II in 24.5% ( n :13) and class III in 41.5% ( n :22). Pathogenic variants were identified in 83% (29/35) probands. We detected 18 (62%) in EXT1 and 11 (38%) in EXT2 . Patients with EXT1 variants showed a height z-score of 1.03 SD lower than those with EXT2 variants and greater clinical severity (II-III vs. I). Interestingly, three patients showed intellectual impairment, two patients showed a dual diagnosis, one Turner Syndrome and one hypochondroplasia. This study improves knowledge of MO, reporting new pathogenic variants and forwarding the worldwide collaboration necessary to promote the inclusion of patients into future biologically based therapeutics.

Osteochondromatosis is a benign proliferative disorder characterized by cartilage-capped bony protuberances. In humans and most mammals, variants in the EXT1 or EXT2 gene are strongly correlated with the etiology of osteochondromatosis. However, in cats, osteochondromatosis has only been associated with feline leukemia virus infection. In this study, to explore other factors involved in the etiology of feline osteochondromatosis, we examined the EXT1 and EXT2 genes in a feline leukemia virus-negative cat with osteochondromatosis. Genetic analysis revealed a heterozygous single base pair duplication in exon 6 of the EXT1 gene (XM&#95;023248762.2:c.1468dupC), leading to a premature stop codon in the EXT1 protein. Notably, this frameshift variant is recognized as one of the most common pathogenic variants in human osteochondromatosis. Our data suggest for the first time that genetic variants can have etiologic roles in osteochondromatosis in cats, as in humans and other animals., (© 2022 Stichting International Foundation for Animal Genetics.)

Competing Interests: The authors declare no conflicts of interest.

Hereditary multiple exostoses (HME) is a rare, pediatric disorder characterized by osteochondromas that form along growth plates and provoke significant musculoskeletal problems. HME is caused by mutations in heparan sulfate (HS)-synthesizing enzymes EXT1 or EXT2. Seemingly paradoxically, osteochondromas were found to contain excessive extracellular heparanase (Hpse) that could further reduce HS levels and exacerbate pathogenesis. To test Hpse roles, we asked whether its ablation would protect against osteochondroma formation in a conditional HME model consisting of mice bearing floxed Ext1 alleles in Agr-CreER background (Ext1 f/f ;Agr-CreER mice). Mice were crossed with a new global Hpse-null (Hpse -/- ) mice to produce compound Hpse -/- ;Ext1 f/f ;Agr-CreER mice. Tamoxifen injection of standard juvenile Ext1 f/f ;Agr-CreER mice elicited stochastic Ext1 ablation in growth plate and perichondrium, followed by osteochondroma formation, as revealed by microcomputed tomography and histochemistry. When we examined companion conditional Ext1-deficient mice lacking Hpse also, we detected no major decreases in osteochondroma number, skeletal distribution, and overall structure by the analytical criteria above. The Ext1 mutants used here closely mimic human HME pathogenesis, but have not been previously tested for responsiveness to treatments. To exclude some innate therapeutic resistance in this stochastic model, tamoxifen-injected Ext1 f/f ;Agr-CreER mice were administered daily doses of the retinoid Palovarotene, previously shown to prevent ectopic cartilage and bone formation in other mouse disease models. This treatment did inhibit osteochondroma formation compared with vehicle-treated mice. Our data indicate that heparanase is not a major factor in osteochondroma initiation and accumulation in mice. Possible roles of heparanase upregulation in disease severity in patients are discussed., (© 2022 Orthopaedic Research Society. Published by Wiley Periodicals LLC.)

Background: Despite the new next-generation sequencing (NGS) molecular approaches implemented the genetic testing in clinical diagnosis, copy number variation (CNV) detection from NGS data remains difficult mainly in the absence of bioinformatics personnel (not always available among laboratory resources) and when using very small gene panels that do not meet commercial software criteria. Furthermore, not all large deletions/duplications can be detected with the Multiplex Ligation-dependent Probe Amplification (MLPA) technique due to both the limitations of the methodology and no kits available for the most of genes., Aim: We propose our experience regarding the identification of a novel large deletion in the context of a rare skeletal disease, multiple osteochondromas (MO), using and validating a user-friendly approach based on NGS coverage data, which does not require any dedicated software or specialized personnel., Methods: The pipeline uses a simple algorithm comparing the normalized coverage of each amplicon with the mean normalized coverage of the same amplicon in a group of "wild-type" samples representing the baseline. It has been validated on 11 samples, previously analyzed by MLPA, and then applied on 20 patients with MO but negative for the presence of pathogenic variants in EXT1 or EXT2 genes. Sensitivity, specificity, and accuracy were evaluated., Results: All the 11 known CNVs (exon and multi-exon deletions) have been detected with a sensitivity of 97.5%. A novel EXT2 partial exonic deletion c. (744-122)-?&#95;804+?del -out of the MLPA target regions- has been identified. The variant was confirmed by real-time quantitative Polymerase Chain Reaction (qPCR)., Conclusion: In addition to enhancing the variant detection rate in MO molecular diagnosis, this easy-to-use approach for CNV detection can be easily extended to many other diagnostic fields-especially in resource-limited settings or very small gene panels. Notably, it also allows partial-exon deletion detection., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Corsini, Pedrini, Patavino, Gnoli, Lanza and Sangiorgi.)

Background: Stuve-Wiedemann syndrome (SWS) is a rare and severe genetic disease characterized by skeletal anomalies and dysautonomic disturbances requiring appropriate care. Peer support is mandatory to fill the lack of clinical recommendations in such rare diseases. We report a new case and provide the first systematic review of all previous published cases., Objective: To better describe the timeline of SWS and to improve paediatric management., Data Sources: SWS English publications available on Pubmed until 31/03/2021., Study Selection: Case description combining typical osteo-articular and dysautonomic involvement (with 2 items by categories required for children < 2 years and 3 items > 2 years)., Data Extraction: Demographic, clinical, genetics and outcome data., Results: In our cohort of 69 patients, the median age at report was 32 months. Only 46% presented antenatal signs. Mortality rate is higher during the first 2 years (42% < 2 years; 10% > 2 years) mainly due to respiratory failure, pulmonary arterial hypertension appearing to be a poor prognosis factor (mortality rate 63%). After 2 years, orthopaedic symptoms significantly increase including joint mobility restriction (81%), spinal deformations (77%) and fractures (61%)., Conclusions: Natural history of SWS is marked by a high mortality rate before 2 years due to dysautonomic disturbances. A specialized multidisciplinary approach is needed to address these early mortality risks and then adapt to the specific, mainly orthopaedic, needs of patients after 2 years of age. Further research is required to provide clinical guidelines and improve pre-natal counselling., (© 2022. The Author(s).)

The aim of the present study was to report a clinical survey of hereditary multiple exostoses (HME) in a large Chinese pedigree, and the identification of a novel deletion mutation of exostosin glycosyltransferase 2 ( EXT‑2 ) gene. A patient with multiple exostoses with huge cartilage‑capped tumors in scapula, knees and ankles received surgery in Department of Orthopedics (Shanghai Changhai Hospital). A total of 20 family members were recruited to the study, with seven members (five male; two female) diagnosed as HME. The family members of the patients with HME were examined, clinical data and peripheral blood samples were collected, and their DNA was sequenced. The incidence of HME in this family pedigree was 35%. Exostoses were most frequently in the tibiae with occurrence in six patients, followed by ribs, femurs, radii, fibulae, scapulae and humeri. DNA sequencing of peripheral blood revealed a novel deletion mutation, c.824‑826delGCA, in exon 5 of the EXT‑2 gene, which was observed in all the patients with HME, but not in the healthy family members. Several characteristics of HME in the pedigree were observed, such as susceptibility of male gender, decreased average age of onset and height and increased severity of clinical symptoms with generations.

Background: Hereditary multiple exostosis (HME) is an autosomal dominant skeletal disorder characterized by the development of multiple cartilage-covered tumors on the external surfaces of bones (osteochondromas). Most of HME cases result from heterozygous loss-of-function mutations in EXT1 or EXT2 gene., Methods: Clinical examination was performed to diagnose the patients: Whole exome sequencing (WES) was used to identify pathogenic mutations in the proband, which is confirmed by Sanger sequencing and co-segregation analysis: qRT-PCR was performed to identify the mRNA expression level of EXT1 in patient peripheral blood samples: minigene splicing assay was performed to mimic the splicing process of EXT1 variants in vitro., Results: We evaluated the pathogenicity of EXT1 c.1056 + 1G > T in a Chinese family with HME. The clinical, phenotypic, and genetic characterization of patients in this family were described. The variant was detected by whole-exome sequencing (WES) and confirmed by Sanger sequencing. Sequencing of the RT-PCR products from the patient's blood sample identified a large deletion (94 nucleotides), which is the whole exome 2 of the EXT1 cDNA. Splicing assay indicated that the mutated minigene produced alternatively spliced transcripts, which cause a frameshift resulting in an early termination of protein expression., Conclusions: Our study establishes the pathogenesis of the splicing mutation EXT1 c.1056 + 1G > T to HME and provides scientific foundation for accurate diagnosis and precise medical intervention for HME., (© 2022 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals LLC.)

Multiple osteochondromas (MO) is a rare disorder, characterized by benign osteocartilaginous tumors (osteochondromas), arising from the perichondrium of bones. The osteochondromas increase during growth, frequently causing deformities and limitations. Our study aims to analyze the data captured by the Registry of Multiple Osteochondromas, to refine Istituto Ortopedico Rizzoli (IOR) Classification, providing a representative picture of the phenotypic manifestations throughout the lifespan. We conducted a single-institution cross-sectional study. Patients were categorized according to IOR Classification, which identifies three patients' classes on the presence/absence of deformities and/or limitations. The present dataset was compared with our previously published data, to refine the classification. Nine hundred sixty-eight patients were included: 243 children (<10 years), 136 adolescents (10-15 years), and 589 adults. Of the entire population, half patients presented at least one deformity, and one quarter reported at least one limitation. Compared with our previous study, the amount of children was more than doubled and the percentage of mild/moderate cases was notably increased, giving a better disease overview throughout the lifespan and suggesting a different cut-off for dividing Class II in subclasses. We confirmed that MO is characterized by phenotypic heterogeneity, suggesting that an early classification of the disease may offer a useful tool to follow disease pattern and evolution, to support clinical practice, and to propose timely interventions., (© 2021 The Authors. American Journal of Medical Genetics Part A published by Wiley Periodicals LLC.)

Objectives: To discuss the mutational features and their relationships with disease in a family with hereditary multiple osteochondroma (HMO) from Guangxi Province (GXBB-1 family), China., Methods: Genomic DNA and total mRNA were extracted from peripheral blood cells of GXBB-1 family members. Whole elements of the EXT1 gene and its transcript, including exons, introns, exon-intron boundaries, and coding sequence (CDS) clones, were amplified and sequenced. Allele-specific PCR was used to confirm the position and type of mutation., Results: All patients from the GXBB-1 family harbored the cosegregating heterozygous c.1056+1G>A mutation located in EXT1 at an exon-intron boundary. Another three single-nucleotide polymorphisms (SNPs) were also detected in the patients, including IVS2+1G>A in intron 2, c.1844 T>C [p.Pro (CCT) 614Pro (CCC)] in exon 3, and c.2534G>A [p.Glu (GAG) 844Glu (GAA)] in exon 9. The latter two SNPs were synonymous variations., Conclusions: The heterozygous c.1056+1G>A mutation cosegregated with the phenotype, indicating that it is a pathogenic mutation in the GXBB-1 family. This mutation is reported for the first time in Chinese HMO patients. IVS2+1G>A and c.2534G>A have no relationship with the occurrence of disease. However, c.1844 T>C and c.1056+1G>A are linked, and their interaction needs to be further studied. c.1844T>C is a new SNP that has not been reported internationally., Competing Interests: The authors declare no conflicts of interest, financial, or otherwise., (Copyright © 2021 Guangzhi Yuan et al.)

Background: Multiple osteochondroma (MO), an autosomal dominant genetic disease, is caused by heterozygous mutations in the EXT1 and EXT2 genes. Approximately 80% of pathogenic mutations are nonsense/missense mutations, small indels, and splicing mutations. Splicing mutations, particularly at the 3' and 5' splice sites, disrupt normal mRNA processing and cause exon skipping or aberrant splicing, ultimately resulting in protein truncation and loss of function. Methods: Polymerase chain reaction (PCR) and Sanger sequencing were applied to detect subtle mutations in a Chinese family with MO, the pathogenicity of a splicing variant was predicted by bioinformatics and further verified using a minigene splicing assay. Results: A novel and heterozygous splicing mutation, c.626 + 2&#95;626 + 5delTAGG, was identified in the EXT2 gene of the proband and the father by PCR and Sanger sequencing, whereas the unaffected mother and brother had wild-type alleles at the same site. Bioinformatics predicted that the 5' splicing site of exon 3 in the EXT2 gene was destroyed due to this mutation. A hybrid minigene splicing assay (HMSA) indicated that the mutation disturbed the normal splicing of the EXT2 gene mRNA and led to a deletion of 79 bp at the 5' end of exon 3, which resulted in aberrant splicing of exon 3 and introduced an earlier stop codon in the EXT2 gene. Conclusion: A novel splicing mutation was identified that produced the MO phenotype through aberrant splicing in a Chinese family. This observation, expands our knowledge of the spectrum of molecular pathogenic mechanisms leading to aberrant mRNA splicing.

Objective: To explore the genetic basis for a pedigree affected with hereditary multiple osteochondroma (HMO)., Methods: Peripheral blood samples were collected from the proband and members of his pedigree with informed consent. Following extraction of genomic DNA, all coding exons and flanking intronic sequences (-10 bp) of the EXT1 and EXT2 genes were subjected to targeted capture and next generation sequencing (NGS). Suspected variant was verified by Sanger sequencing., Results: A heterozygous nonsense variant (c.1911C>A) was found in exon 10 of the EXT1 gene in the proband and his affected father but not in a healthy sister and normal controls. The variant was classified as a pathogenic based on the guidelines of the American College of Medical Genetics and Genomics (PVS1+PM2+PP1). Bioinformatic analysis predicted that the c.1911C>A variant may be disease-causing via nonsense-mediated mRNA decay and anomalous splicing., Conclusion: The c.1911C>A variant probably underlay the disease in this pedigree. Discovery of this variant enriched the variant spectrum of HMO.

Background: Multiple osteochondromas (MO) are an autosomal-dominant disease characterized by the growth of multiple cartilage-capped prominences in the growth plate region of the metaphysis in long and flat bones. Materials and Methods: To detect genetic mutations related to MO, a three-generation Chinese family with MO was evaluated using whole exome sequencing for mutation screening. The candidate pathogenic mutation was validated by Sanger sequencing. Results: A novel frameshift (NM&#95;000401.3:c.1321del:p.Leu441TrpfsTer28) in exon 8 of the exotosin 2 ( EXT2 ) gene was identified in two affected individuals. Codons 441 and 468 in the EXT2 gene are highly conserved among vertebrates as demonstrated by multiple sequence alignment. The c.1321 del C resulted in an amino acid change at codon 441, which generated a premature stop codon at position 468, causing complete loss of the glycosyltransferase domain. Conclusions: A novel frameshift mutation c.1321delC detected in the EXT2 gene may help in prenatal genetic screening and early diagnosis of MO.

Backgrounds: Exostosin-1 (EXT1) and exostosin-2 (EXT2) cause multiple osteochondromas (MO). In this study, we investigated the correlation between forearm deformity and mutant EXTs in Japanese families with MO., Methods: We evaluated 112 patients in 71 families with MO. Genomic DNA was isolated from peripheral blood leucocytes. Of these, 28 patients were selected and underwent radiography for their forearms since they had gross forearm deformities. We measured the radial articular angle (RAA), ulna variance (UV), carpal slip (CS), and percentage of radial bowing (%RB) to compare between patients with mutant EXT1 or EXT2 and those with missense or other mutations using Student's t-test., Results: Twenty-two (78.6%) and 6 (11.4%) out of 28 patients had mutations in EXT1 and EXT2, respectively. Nine (32.1%) and 19 (67.9%) of the 28 patients had missense and other mutations, respectively. The mean age of patients with EXT1 and EXT2 were 25.9 ± 20.3 and 33.5 ± 25.4 years, respectively and those with missense mutation and other mutations were 28.7 ± 27.0 and 24.6 ± 17.0 years, respectively. There were no significant differences in RAA, UV, and RB between patients harbouring mutant EXT1 or EXT2 (RAA, 40.1 ± 8.7 and 31.5 ± 13.9°; UV, -2.7 ± 5.7 and -3.1 ± 3.7 mm; %RB, 8.6 ± 1.5 and 8.3 ± 2.0%). CS was significantly greater in patients with mutant EXT1 than that in those with mutant EXT2 (EXT1, 44.1 ± 16.8%; EXT2, 18.6 ± 14.0%). There were no significant differences in RAA, UV, CS and %RB between patients with missense and other mutations., Conclusions: Patients with mutant EXT1 displayed greater CS than patients with mutant EXT2, indicating that patients with MO harbouring EXT1 mutations sustain more severe ulnar drift deformities than those with EXT2 mutations., Competing Interests: Declaration of Competing Interest The authors describe that there are no conflicts of interest to declare., (Copyright © 2020 The Japanese Orthopaedic Association. Published by Elsevier B.V. All rights reserved.)

Stuve-Wiedemann syndrome (SWS; MIM 601559) is a rare autosomal recessive disease caused by mutations in the leukemia inhibitor factor receptor gene (LIFR). Common clinical and radiological findings are often observed, and high neonatal mortality occurs due to respiratory distress and hyperthermic episodes. Despite initially considered as a lethal disorder during the newborn period, in recent years, several SWS childhood survivors have been reported. We report a detailed clinical and radiological characterization of four unrelated childhood SWS molecularly confirmed patients and review 22 previously reported childhood surviving cases. We contribute to the definition of the childhood survival phenotype of SWS, emphasizing the evolving phenotype, characterized by skeletal abnormalities with typical radiological findings, distinctive dysmorphic features, and dysautonomia. Based on the typical features and clinical course, early diagnosis is possible and crucial to plan appropriate management and prevent potential complications. Genetic confirmation is advisable in order to improve genetic counseling to the patients and their families., (© 2020 Wiley Periodicals LLC.)

Background: Hereditary Multiple Exostoses (HME), also known as Multiple Osteochondromas (MO) is a rare genetic disorder characterized by multiple benign cartilaginous bone tumors, which are caused by mutations in the genes for exostosin glycosyltransferase 1 (EXT1) and exostosin glycosyltransferase 2 (EXT2). The genetic defects have not been studied in the Saudi patients., Aim of Study: We investigated mutation spectrum of EXT1 and EXT2 in 22 patients from 17 unrelated families., Methods: Genomic DNA was extracted from peripheral leucocytes. The coding regions and intron-exon boundaries of both EXT1 and EXT2 genes were screened for mutations by PCR-sequencing analysis. Gross deletions were analyzed by MLPA analysis., Results: EXT1 mutations were detected in 6 families (35%) and 3 were novel mutations: c.739G > T (p. E247*), c.1319delG (p.R440Lfs*4), and c.1786delA (p.S596Afs*25). EXT2 mutations were detected in 7 families (41%) and 3 were novel mutations: c.541delG (p.D181Ifs*89), c.583delG (p.G195Vfs*75), and a gross deletion of approximately 10 kb including promoter and exon 1. Five patients from different families had no family history and carried de novo mutations (29%, 5/17). No EXT1 and EXT2 mutations were found in the remaining four families. In total, EXT1 and EXT2 mutations were found in 77% (13/17) of Saudi HME patients., Conclusion: EXT1 and EXT2 mutations contribute significantly to the pathogenesis of HME in the Saudi population. In contrast to high mutation rate in EXT 1 (65%) and low mutation rate in EXT2 (25%) in other populations, the frequency of EXT2 mutations are much higher (41%) and comparable to that of EXT1 among Saudi patients. De novo mutations are also common and the six novel EXT1/EXT2 mutations further expands the mutation spectrum of HME.

Aim: To detect mutations in the EXT1 and EXT2 genes in four Chinese families with hereditary multiple osteochondromas (HMO). HMO is an autosomal dominant disorder characterized by the overgrowth of multiple cartilage-capped bones in the metaphysis of long bones and flat bones. Methods: Polymerase chain reaction-based amplification followed by DNA sequencing of the complete coding sequences of EXT1 and EXT2 was performed for four Chinese families with HMO. Results: The mutant allele was found in six patients: three mutations were found in EXT1 and two in EXT2 . A novel frameshift mutation, which generates a premature stop codon at codon 586 and causes partial loss of the glycosyltransferase domain, was detected in exon 9 of EXT1 (F579Yfs*8). We hypothesize that F579Yfs*8 is a pathogenic mutation. Two novel missense mutations (G339S and V545D) were found in EXT1 . The variant c.1634T>A (V545D) is apparently benign. In addition we found a novel deletion mutation in EXT2 , c.856&#95;864 del TTCCTCCTG, which results in the deletion of 286Phe, 287Leu, and 288Leu, that is likely pathogenic. Finally, we identified a likely benign variant in exon 13 of EXT2 . c.2035-41T>C (rs3740878). Conclusions: We found three novel, potentially pathogenic mutations in EXT1 and EXT2 , including a novel frameshift mutation. More importantly, our study results have expanded the spectrum of EXT mutations conducive to the genetic diagnosis and counseling of patients with HMO.

Stüve-Wiedemann syndrome (SWS) is a rare genetic disorder characterized by skeletal dysplasia and severe dysautonomia, evidencing a difficult airway approach and likely increased malignant hyperthermia susceptibility. Developmental dysmorphism classically worsens with age, therefore translating in a poor prognosis. In this article, we describe a case of a 27-year-old woman diagnosed with SWS proposed for abscess drainage under dissociative anesthesia. This patient has outlived the life expectancy described for SWS, acknowledging the importance of reporting this rare adult clinical case in what SWS anesthetic management is concerned.

Although the main causative genes for hereditary multiple exostoses (HME) are exostosin (EXT)‑1 and EXT‑2, there are numerous patients with HME without EXT‑1 and EXT‑2 mutations. The present study aimed to identify novel candidate genes for the development of HME in patients without EXT‑1 and EXT‑2 mutations. Whole‑exome sequencing was performed in a Chinese family with HME and without EXT‑1 and EXT‑2 mutations, followed by a combined bioinformatics pipeline including annotation and filtering processes to identify candidate variants. Candidate variants were then validated using Sanger sequencing. A total of 1,830 original variants were revealed to be heterozygous mutations in three patients with HME which were not present in healthy controls. Two mutations [c.C1849T in solute carrier family 20 member 2 (SLC20A2) and c.G506A in leucine zipper and EF‑hand containing transmembrane protein 1 (LETM1)] were identified as possible causative variants for HME through a bioinformatics filtering procedure and harmful prediction. Sanger sequencing results confirmed these two mutations in all patients with HME. A mutation in SLC20A2 (c.C1849T) led to a change in an amino acid (p.R617C), which may be involved in the development of HME by inducing metabolic disorders of phosphate and abnormal proliferation and differentiation in chondrocytes. In conclusion, the present study revealed two mutations [SLC20A2 (c.C1849T) and LETM1 (c.G506A) in a Chinese family with HME. The mutation in SLC20A2 (c.C1849T)] was more likely to be involved in the development of HME.

Although at first glance chondrodysplasia and aneurysmal thoracic aortopathy seem oddly dissimilar, recent lines of evidences indicate that they share molecular similarities. Chondrodysplasias are a group of skeletal disorders characterized by genetic defects in hyaline cartilage. Aneurysmal thoracic aortopathy is the pathological enlargement of the thoracic aorta due to wall weakness, along with its ensuing life-threatening complications (i.e., aortic dissection and/or rupture). Extracellular matrix dysregulation, abnormal TGF-β signaling, and, to a more limited extent, endoplasmic reticulum stress emerge as common disease processes. In this review we provide a comprehensive overview of the genetic and pathomechanistic overlap as well as of how these commonalities can guide treatment strategies for both disease entities., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Aims: Identification of genetic mutations linked to hereditary multiple osteochondromas (HMO) is crucial for understanding the molecular mechanisms leading to disease pathogenesis. In this study, we investigated four patients and eight healthy individuals from a family with HMO. Methods: Clinical HMO data and Sanger sequences of the coding regions of the exostosin glycosyltransferase 1 ( EXT1 ) gene (18q24.11) and the EXT2 gene (11p12) of all 12 members of the family were analyzed. Results: A novel nonsense mutation in the EXT2 gene (c.526C>T; p.Gln176*) was detected, which was present in all four patients but absent in their healthy relatives. This mutation encodes a stop codon that results in a truncated EXT2 protein that consists of only 176 amino acids and lacks the remaining 522 amino acids at its C-terminus, missing the entire glycosyltransferase domain. Conclusions: Association of a truncated EXT2 protein with HMO provides new insights into exostosis pathogenesis, highlighting potential roles of the EXT2 gene and its glycosyltransferase domain. Further research is required to understand the mechanisms underlying the development of exostosis.

Objective: To detect variants of EXT1 and EXT2 genes among five pedigrees affected with multiple osteochondromas and provide prenatal diagnosis for the families based on the results., Methods: The EXT1 and EXT2 genes of the probands were analyzed by targeted next generation sequencing (NGS). Suspected pathological variants were validated by Sanger sequencing in the probands, their family members and 200 unrelated healthy controls. Multiple ligation-dependent probe amplification (MLPA) was used to confirm the presence of gross deletions. Prenatal diagnosis was provided for 2 couples carrying pathogenic or likely pathogenic variants., Results: Five variants were detected in the pedigrees, which included EXT1 exon 2-3 deletion, c.1468dupC (p.Leu490ProfsX31), c.2084delC (p.Pro695LeufsX11), and EXT2 c.187delT (p.Phe63SerfsX29) and c.1362T>G (p.Tyr454X). Among these, EXT1 exon 2-3 deletion, c.2084delC (p.Pro695LeufsX11) and EXT2 c.187delT (p.Phe63SerfsX29) were unreported previously. The three novel variants were not found among unaffected members of the pedigree and the 200 healthy controls. Upon prenatal diagnosis, the two fetuses were found to carry the same variants of the the probands., Conclusion: Pathological variants of the EXT1 and EXT2 genes probably underlie the multiple osteochondromas among the 5 pedigrees. Prenatal diagnosis based on the results can effectively reduce the birth of further offspring affected with the disease.

Background: Hereditary multiple osteochondromas (HMO) is a genetic condition characterized by the presence of multiple osteochondromas, usually at the lateral side of the most active growth plate of a long bone. These lesions may persist, be asymptomatic during childhood, and may increase in number and size until growth plates close. Therefore, diagnosis of HMO in children and young people can be challenging; while short stature can be more evident at the onset of puberty, asymptomatic ostheocondromas can progress into different degrees of orthopedic deformity. Moreover, multiple complications may arise due to the presence of osteochondromas, including tendon and compression muscle pain, neurovascular disorders, obstetric problems, scoliosis and malignant transformation into secondary peripheral chondrosarcoma in adulthood., Case Presentation: We report the case of a girl admitted to our Institute for growth delay. While laboratory tests, including growth hormone stimulation test, were normal, left hand X-ray revealed multiple osteochondromas, suggestive for HMO. The genetic test for EXT1 and EXT2 genes confirmed the radiological diagnosis, with a mutation inherited from the mother who displayed the same radiological abnormalities along with recurrent limb pain episodes., Conclusions: HMO is a genetic condition whose diagnosis can be challenging, especially in females. Every pediatricians should consider a skeletal dysplasia in case of unexplained growth delay and a skeletal survey might be fundamental in reaching a diagnosis.

Objective: To find novel potential gene mutations other than EXT1 and EXT2 mutations, to expand the mutational spectrum of EXT and to explore the correlation between clinical outcome and genotype in patients with hereditary multiple exostoses (HME)., Methods: The study recruited seven families diagnosed with multiple osteochondromas (MO). Family histories and clinical information were collected in detail through comprehensive physical and image examination. Patients with deformities and functional limitations were classified as "severe" and the remaining without functional limitations were classified as "mild," in accordance with previous study. Whole-exome sequencing (WES) was performed on a total of 13 affected individuals, 1 available unaffected relative, and 10 healthy unrelated individuals. Sanger sequencing was used to validate the screened mutations. Finally, the structural change in protein caused by pathogenic mutations was analyzed using information from the relevant database online and we attempted to correlate clinical phenotype with genotype in patients with HME., Results: Other than EXT1 and EXT2, no novel potential gene mutations were found through WES. We identified nine heterozygous mutations in EXT1 or EXT2. Of these mutations, four have not been reported previously. These are c.996delT in exon 2 of EXT1 (family 1), c.544C > T in exon 3 of EXT2 (family 2), c.1171C > T in exon 7 of EXT2 (family 5), and c.823 - 824delAA in exon 5 of EXT1 (family 7). The other five mutations have already been reported in previous works. It was surprising that we found two mutation sites, in exon 2 and exon 5, respectively, of EXT1 in 1 patient diagnosed with MO, when his father had two mutation sites, in exon 6 and exon 5, respectively, of EXT1 and EXT2 (family 4). In addition, 1 patient showed degeneration, while his father only exhibited slight symptoms (family 7). In our study, among 51 affected patients in seven families, the sex ratio (male vs female) was 58.9% (n = 30) vs 41.2% (n = 21). Male patients seemed to show more severe symptoms compared to females, but because the sample was small, we did not obtain statistically significance results., Conclusion: Whole-exome sequencing to screen pathogenic gene mutations was applied successfully. Although no third-gene mutation associated with HME was found, a total of nine mutations across EXT1 and EXT2 were identified, four of which are novel. Our results expand the mutational spectrum of EXT and can be used in genetic counseling and prenatal diagnosis for patients with MO., (© 2020 The Authors. Orthopaedic Surgery published by Chinese Orthopaedic Association and John Wiley & Sons Australia, Ltd.)

Multiple hereditary exostoses (MHE) and enchondromatosis are rare multifocal benign disorders usually causing skeletal deformities appearing already in childhood. MHE is a dominant autosomal inherited disorder characterized by multiple osteochondromas (exostoses) growing outward from the metaphyses of long bones as well as from flat bones. They may cause reduced joint motion and pain due to tendon, muscle, and nerve compression. Enchondromatosis (or Ollier's disease) is a noninherited disorder characterized by the presence of multiple intraosseous enchondromas located asymmetrically in the skeleton and with a wide variation regarding location, size, and number ranging from the involvement of a single hand to the involvement of the entire skeleton. It can occur together with soft-tissue hemangiomas in Maffucci's syndrome. Clinical problems caused by the enchondromas are mainly related to skeletal deformities causing malalignment and restricted motion of joint. In both disorders, there is a risk of malignant transformation as well as secondary degenerative joint changes., Competing Interests: Declaration of Competing Interest None., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

WAGR 11p13 deletion syndrome is associated with abnormalities including (W) ilms tumor, (A) niridia, (G) enitourinary abnormalities, and growth and mental (R) etardation (WAGR). Potocki-Schaffer syndrome is a contiguous gene syndrome associated with deletions in 11p11.2, principal features of which are multiple exostoses, parietal foramina development delay, mental retardation, and facial dysmorphism. In some cases, males may have enlarged anterior fontanels and genital abnormalities. Each of these syndromes is very rare. Here we report a patient with both WAGR and Potocki-Shaffer syndromes who presented with aniridia, nystagmus, macular dysplasia, enlarged anterior fontanel, mental retardation, ptosis, low-set ears, micrognathia, and atrial septal defect at 6 months old. SNP array revealed a large (26.25 Mb)deletion: arr[hg19]11p15.1p11.2(18742043-44991839)× 1. Genetic testing allowed for diagnosis of this patient at a very young age. In addition to the postnatal phenotype of the patient, we found one prenatal symptom of these syndromes is oligohydramnios, which when present might indicate advanced prenatal diagnosis. This made the possibility of prenatal diagnosis for these syndromes.

Potocki-Shaffer syndrome (PSS) is a rare contiguous gene deletion syndrome caused by heterozygous deletion of 11p11.2p12. Typical features described in patients with PSS include developmental delay, intellectual disability, multiple cartilaginous exostoses, biparietal foramina, craniofacial abnormalities, and genitourinary anomalies. While hypertension has been noted in three patients with PSS, it has not been described in most patients with this syndrome. This report details the evaluation and treatment of a teenager with PSS who presented on several occasions during childhood with elevated blood pressure measurements. The renin level was elevated, likely indicating a secondary cause for the HTN. The patient's BP responded to monotherapy with Angiotensin Converting Enzyme Inhibitor (ACEI)., (Copyright © 2019. Published by Elsevier Masson SAS.)

Objective: To identify pathogenic variations of EXT1 and EXT2 genes in two Chinese pedigrees affected with hereditary multiple exostosis (HME)., Methods: Genomic DNA was extracted from peripheral blood samples using a phenol-chloroform method. PCR and Sanger sequencing was conducted to amplify the exons and the flanking intronic regions of the EXT1 and EXT2 genes., Results: DNA sequencing has revealed a heterozygous missense variation c.812A>G (p.Tyr271Cys) in the exon 1 of EXT1 in pedigree 1, and a heterozygous frameshift variation c.1431dup (p.Ser478Leufs*43) in the exon 6 of EXT1 in the proband from pedigree 2. Both variations have co-segregated with the disease phenotype, which was also consistent with previous report., Conclusion: Two heterozygous pathogenic variations underlying HME have been identified. The result has facilitated genetic counseling and prenatal diagnosis for the affected pedigrees.

Hereditary multiple osteochondromas (HMO) is a rare autosomal dominant skeletal disorder, caused by heterozygous variants in either EXT1 or EXT2, which encode proteins involved in the biogenesis of heparan sulphate. Pathogenesis and genotype-phenotype correlations remain poorly understood. We studied 114 HMO families (158 affected individuals) with causative EXT1 or EXT2 variants identified by Sanger sequencing, or multiplex ligation-dependent probe amplification and qPCR. Eighty-seven disease-causative variants (55 novel and 32 known) were identified including frameshift (42%), nonsense (32%), missense (11%), splicing (10%) variants and genomic rearrangements (5%). Informative clinical features were available for 42 EXT1 and 27 EXT2 subjects. Osteochondromas were more frequent in EXT1 as compared to EXT2 patients. Anatomical distribution of lesions showed significant differences based on causative gene. Microscopy analysis for selected EXT1 and EXT2 variants verified that EXT1 and EXT2 mutants failed to co-localize each other and loss Golgi localization by surrounding the nucleus and/or assuming a diffuse intracellular distribution. In a cell viability study, cells expressing EXT1 and EXT2 mutants proliferated more slowly than cells expressing wild-type proteins. This confirms the physiological relevance of EXT1 and EXT2 Golgi co-localization and the key role of these proteins in the cell cycle. Taken together, our data expand genotype-phenotype correlations, offer further insights in the pathogenesis of HMO and open the path to future therapies., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Objective: To detect EXT1 and EXT2 gene mutations in two pedigrees affected with hereditary multiple exostosis (HME)., Methods: The coding regions and exon/intron boundaries of the EXT1 and EXT2 genes were analyzed by targeted next-generation sequencing (NGS). Suspected mutations were confirmed by Sanger sequencing of the probands, their family members and 200 unrelated healthy controls. Gross deletion was confirmed by quantitative PCR (qPCR) analysis and multiple ligation-dependent probe amplification (MLPA) analysis., Results: Two mutations were detected in the pedigrees, which included EXT2 gene c.337&#95;338insG mutation in pedigree 1 and deletion of entire EXT1 in pedigree 2. Analysis of sequencing data revealed that a novel heterozygous mutation (c.337&#95;338insG) in EXT2 gene in proband 1 and his father. The same mutation was not found among healthy family members and 200 unrelated healthy controls. As shown by NGS and MLPA analysis, proband 2 carried a heterozygous deletion of entire EXT1 gene. The same deletion was also found in her mother by qPCR., Conclusion: Mutations of the EXT1 and EXT2 genes probably underlie the HME in both pedigrees. NGS combined with Sanger sequencing, qPCR and MLPA is effective for attaining the diagnosis.

Multiple osteochondromas (MOs) or hereditary multiple exostoses is a rare autosomal-dominant disease characterized by growths of MOs, which are benign cartilage-capped bone tumors that grow away from the growth plates. Almost 90% of MOs have a molecular explanation and 10% are unexplained. MOs are genetically heterogeneous with two causal genes on 8q24.11 (EXT1) and 11p12 (EXT2), with a higher frequency in EXT1. MO is a very rare genetic disorder, and the genotype-phenotype of MO with EXT2 mutation has not been well investigated in Korea. We present the clinical radiographic and molecular analysis of a four-generation Korean family with 11 MO-affected members (seven males and four females). The affected members from the third generation available for molecular analysis and their detailed medical histories showed moderate-to-severe phenotypes (clinical classes II-III), including bony deformities and limb misalignment with pain requiring surgical correction. The x-rays showed MOs in multiple sites. A novel EXT2 frameshift mutation (c.590delC, p.P197Qfs*73) was revealed by targeted exome sequencing in the affected members of this family. In this article, we not only expand the phenotypic-genotypic spectrum of MOs but also highlight the phenotypic heterogeneity in a family with the same mutation. In addition, we compiled the mutation spectrum of EXT2 from a literature review and identified that exon 2 of EXT2 is a mutation hot spot. Early medical attention with diagnosis of MO through careful examination of the clinical manifestations and genetic analysis can provide the opportunity to establish coordinated multispecialty management of the patient., (© 2019 John Wiley & Sons Ltd/University College London.)

Rationale: Hereditary multiple exostoses (HMEs) is an autosomal dominant skeletal disorder., Patient Concerns: Six probands of the 6 unrelated Han Chinese families were identified as having HME. These patients had exostoses at multiple sites and significantly affected joints malformation and movement., Diagnoses: Hereditary multiple exostoses., Interventions: To detect the genetic mechanism of HME in 6 unrelated Chinese families, whole-exome sequencing (WES) and multiplex ligation-dependent probe amplification (MLPA) were used after genomic DNA was isolated from peripheral blood leucocytes. Point mutations identified by these methods were verified by Sanger sequencing after PCR amplification., Outcomes: Six mutations in the EXT1 and EXT2 genes were identified, including a heterozygous deletion mutation from exon 2 to exon 8 (Family 1), a c.448C>T, p.(Gln150X) heterozygous nonsense mutation (Family 4), a c.1057-2A>T heterozygous splicing substitution (Family 5), and a c.1468dupC, p.(Leu490fs519X) (Family 6) heterozygous duplication mutation in the EXT1 gene in addition to a heterozygous deletion mutation from exon 2 to exon 3 (Family 2) and a c.1197C>G, p.(Tyr399X) heterozygous nonsense mutation (Family 3) in the EXT2 gene., Lessons: Overall, we identified 5 novel mutations and 1 recurrent mutation in the EXT1 and EXT2 genes in 6 Chinese families with HME. Our findings expand the mutational spectrum of the EXT1 and EXT2 genes and are useful for genetic counseling and prenatal diagnosis.

Aims: Multiple exostoses (MO), also referred to as hereditary multiple exostoses (HME), is an autosomal dominant inherited skeletal disorder that has been found to be associated with mutations in the EXT1 and EXT2 genes. In the present study, we report a Chinese family with HME and our mutational analyses of the EXT1 and EXT2 genes in affected and unaffected individuals., Methods: All exons of the EXT1 and EXT2 genes in seven family members were polymerase chain reaction amplified from blood and sequenced., Results: A heterozygous mutation (c.1056G>T) was identified in exon 2 of the EXT1 gene in the proband and other affected family members; this mutation was not found in the unaffected family members., Discussion: This c.1056G>T mutation is located in the exostosin domain of the EXT1 protein and leads to an amino acid change of Glutamine (Gln) to Histidine (His) at position 352. Homology searches reveal that Gln352 is highly conserved in many species and may play an essential role in the normal function of the EXT1 protein., Conclusions: This study contributes to a better understanding of the genetic basis of HME, expands the known mutational spectrum of EXT1, and provides a reference for genetic counseling and prenatal diagnosis of this family.

Potocki-Shaffer syndrome (PSS) is a contiguous gene syndrome caused by 11p11.2 deletions. PSS is clinically characterized by intellectual disability, craniofacial anomalies, enlarged parietal foramina, and multiple exostoses. PSS occasionally shows autism spectrum disorder, epilepsy, and overgrowth. Some of the clinical features are thought to be associated with haploinsufficiency of two genes in the 11p11.2 region; variants affecting the function of ALX4 cause enlarged parietal foramina and EXT2 lead to multiple exostoses. However, the remaining clinical features were still yet to be linked to specific genetic alterations. In this study, we identified de novo truncating variants in an 11p11.2 gene, PHF21A, in three cases with intellectual disability and craniofacial anomalies. Among these three cases, autism spectrum disorder was recognized in one case, epilepsy in one case, and overgrowth in two cases. This study shows that PHF21A haploinsufficiency results in intellectual disability and craniofacial anomalies and possibly contributes to susceptibility to autism spectrum disorder, epilepsy, and overgrowth, all of which are PSS features.

Background: We describe a patient presenting with pachygyria, epilepsy, developmental delay, short stature, failure to thrive, facial dysmorphisms, and multiple osteochondromas., Methods: The patient underwent extensive genetic testing and analysis in an attempt to diagnose the cause of his condition. Clinical testing included metaphase karyotyping, array comparative genomic hybridization, direct sequencing and multiplex ligation-dependent probe amplification and trio-based exome sequencing. Subsequently, research-based whole transcriptome sequencing was conducted to determine whether it might shed light on the undiagnosed phenotype., Results: Clinical exome sequencing of patient and parent samples revealed a maternally inherited splice-site variant in the doublecortin (DCX) gene that was classified as likely pathogenic and diagnostic of the patient's neurological phenotype. Clinical array comparative genome hybridization analysis revealed a 16p13.3 deletion that could not be linked to the patient phenotype based on affected genes. Further clinical testing to determine the cause of the patient's multiple osteochondromas was unrevealing despite extensive profiling of the most likely causative genes, EXT1 and EXT2, including mutation screening by direct sequence analysis and multiplex ligation-dependent probe amplification. Whole transcriptome sequencing identified a SAMD12-EXT1 fusion transcript that could have resulted from a chromosomal deletion, leading to the loss of EXT1 function. Re-review of the clinical array comparative genomic hybridization results indicated a possible unreported mosaic deletion affecting the SAMD12 and EXT1 genes that corresponded precisely to the introns predicted to be affected by a fusion-causing deletion. The existence of the mosaic deletion was subsequently confirmed clinically by an increased density copy number array and orthogonal methodologies CONCLUSIONS: While mosaic mutations and deletions of EXT1 and EXT2 have been reported in the context of multiple osteochondromas, to our knowledge, this is the first time that transcriptomics technologies have been used to diagnose a patient via fusion transcript analysis in the congenital disease setting., (© 2019 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals, Inc.)

The molecular mechanism of hereditary multiple exostoses (HME) remains ambiguous and a limited number of studies have investigated the pathogenic mechanism of mutations in patients with HME. In the present study, a novel heterozygous splice mutation (c.1284+2del) in exostosin glycosyltransferase 1 (EXT1) gene was identified in a three‑generation family with HME. Bioinformatics and TA clone‑sequencing indicated that the splice site mutation would result in exon 4 skipping. Reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) revealed that the expression levels of wild‑type EXT1/EXT2 mRNA in patients with HME were significantly decreased, compared with normal control participants (P<0.05). Abnormal EXT1 transcript lacking exon 4 (EXT1‑DEL) and full‑length EXT1 mRNA (EXT1‑FL) were overexpressed in 293‑T cells and Cos‑7 cells using lentivirus infection. RT‑qPCR demonstrated that the expression level of EXT1‑DEL was significantly increased, compared with EXT1‑FL (17.032 vs. 6.309, respectively; P<0.05). The protein encoded by EXT1‑DEL was detected by western blot analysis, and the level was increased, compared with EXT1‑FL protein expression. Immunofluorescence indicated that the protein encoded by EXT1‑DEL was located in the cytoplasm of Cos‑7 cells, which was consistent with the localization of the EXT1‑FL protein. In conclusion, the present study identified a novel splice mutation that causes exon 4 skipping during mRNA splicing and causes reduced expression of EXT1/EXT2. The mutation in EXT1‑DEL generated a unique peptide that is located in the cytoplasm in vitro, and it expands the mutation spectrum and provides molecular genetic evidence for a novel pathogenic mechanism of HME.