EXT1 Research Articles

Novel EXT1 variants cause divergent symptom severity in multiple cartilaginous exostoses: a family report

Abstract Background Multiple cartilaginous exostoses (MCE) are a rare genetic disorder characterized by multiple osteochondromas in the metaphysis of long bones. Case Presentation. We report a 15-year-old male patient, his father, and his brother, all presenting varied severity of the same symptom. The patient exhibited symptoms since infancy, with osteochondromas affecting mobility and causing joint deformities. Whole exome sequencing identified a pathogenic NM_000127.3(EXT1):c.1056 + 2 T > G and a likely pathogenic NM_000127.3(EXT1):c.1047G > T variant in the EXT1 gene, both novel. Conclusions Despite three decades since the discovery of the EXT1 gene, MCE diagnosis may be delayed until adolescence or adulthood. This report contributes to clinical understanding, emphasizing the importance of early diagnosis, genetic analysis, and potential treatment approaches, to mitigate long-term deformities and complications in MCE patients.

The Missing Piece of the Puzzle: Unveiling the Role of PTPN11 Gene in Multiple Osteochondromas in a Large Cohort Study

This study is aimed at investigating the clinical and genetic characteristics of 244 unrelated probands diagnosed with multiple osteochondromas (MO). The diagnosis of MO typically involves identifying multiple benign bone tumors known as osteochondromas (OCs) through imaging studies and physical examinations. However, cases with both OCs and enchondromas (ECs) may indicate the more rare condition metachondromatosis (MC), which is assumed to be distinct disease. Previous cohort studies of MO found heterozygous loss-of-function (LoF) variants only in the EXT1 or EXT2 genes, with DNA diagnostic yield ranging from 78 to 95%. The PTPN11 gene, which is causative for MC, was not previously investigated as a gene candidate for MO. In this study, we detected a total of 177 unique single nucleotide and copy number variants in three genes across 220 probands, consisting of 80 previously reported and 97 novel variants. Specifically, we identified five cases with OCs and no ECs as well as four cases with MC carrying LoF variants in the PTPN11 gene and two additional cases with ECs harboring variants in the EXT1/2 genes. These findings suggest a potential overlap between the MO and MC both phenotypically and genetically. These findings highlight the importance of expanding genetic testing beyond the EXT1 and EXT2 genes in MO cases, as other genes such as PTPN11 may also be causative. This can improve the accuracy of diagnosis and treatment for individuals with MO and MC. It is essential to determine whether MO and MC represent distinct diseases or if they encompass a broader clinical spectrum.

EXT2: a novel prognostic and predictive biomarker for head and neck squamous cell carcinoma

ObjectiveThis study focused on EXT2, a member of the EXT family involved in heparan sulfate synthesis, to evaluate its potential as a prognostic and predictive biomarker in head and neck squamous cell carcinoma (HNSCC). Materials and MethodsThe present study used The Cancer Genome Atlas Head-Neck Squamous Cell Carcinoma (TCGA-HNSC) dataset-based UALCAN databases to analyze the EXT2 expression and its clinicopathological features. In addition, we recruited 51 oral squamous cell carcinoma patients (OSCC), the most common HNSCC subtype, to validate the EXT2 mRNA expression analysis. In addition, we identified the role of EXT2 in prognosis using a Kaplan-Meier plot and immune signature using the tumor infiltration level. Furthermore, functional roles were analyzed using the EXT2 gene and protein networks. ResultsThe expression of EXT2 mRNA was significantly upregulated in OSCC tumors, which is consistent with the UALCAN-based results. EXT2 protein was also significantly overexpressed in HNSCC samples and was correlated with clinicopathological features. High EXT2 expression is associated with poor survival outcomes in HNSCC patients. Functional analysis of EXT2 using in silico tools revealed its involvement in critical pathways, including Wnt signaling, proteoglycans in cancer, and cellular responses to fibroblast growth and inflammation. ConclusionThese findings highlight the potential of EXT2 as a prognostic and predictive biomarker of HNSCC.

Transcriptome profiling reveals the genes involved in tuberous root expansion in Pueraria (Pueraria montana var. thomsonii)

BackgroundPueraria is a dry root commonly used in Traditional Chinese Medicine or as food and fodder, and tuberous root expansion is an important agronomic characteristic that influences its yield. However, no specific genes regulating tuberous root expansion in Pueraria have been identified. Therefore, we aimed to explore the expansion mechanism of Pueraria at six developmental stages (P1–P6), by profiling the tuberous roots of an annual local variety “Gange No.1” harvested at 105, 135, 165, 195, 225, and 255 days after transplanting.ResultsObservations of the tuberous root phenotype and cell microstructural morphology revealed that the P3 stage was a critical boundary point in the expansion process, which was preceded by a thickening diameter and yield gain rapidly of the tuberous roots, and followed by longitudinal elongation at both ends. A total of 17,441 differentially expressed genes (DEGs) were identified by comparing the P1 stage (unexpanded) against the P2–P6 stages (expanded) using transcriptome sequencing; 386 differential genes were shared across the six developmental stages. KEGG pathway enrichment analysis showed that the DEGs shared by P1 and P2–P6 stages were mainly involved in pathways related to the “cell wall and cell cycle”, “plant hormone signal transduction”, “sucrose and starch metabolism”, and “transcription factor (TF)”. The finding is consistent with the physiological data collected on changes in sugar, starch, and hormone contents. In addition, TFs including bHLHs, AP2s, ERFs, MYBs, WRKYs, and bZIPs were involved in cell differentiation, division, and expansion, which may relate to tuberous root expansion. The combination of KEGG and trend analyses revealed six essential candidate genes involved in tuberous root expansion; of them, CDC48, ARF, and EXP genes were significantly upregulated during tuberous root expansion while INV, EXT, and XTH genes were significantly downregulated.ConclusionOur findings provide new insights into the complex mechanisms of tuberous root expansion in Pueraria and candidate target genes, which can aid in increasing Pueraria yield.

Genetic Susceptibility to Atrial Fibrillation Identified via Deep Learning of 12-Lead Electrocardiograms.

Artificial intelligence (AI) models applied to 12-lead ECG waveforms can predict atrial fibrillation (AF), a heritable and morbid arrhythmia. However, the factors forming the basis of risk predictions from AI models are usually not well understood. We hypothesized that there might be a genetic basis for an AI algorithm for predicting the 5-year risk of new-onset AF using 12-lead ECGs (ECG-AI)-based risk estimates. We applied a validated ECG-AI model for predicting incident AF to ECGs from 39 986 UK Biobank participants without AF. We then performed a genome-wide association study (GWAS) of the predicted AF risk and compared it with an AF GWAS and a GWAS of risk estimates from a clinical variable model. In the ECG-AI GWAS, we identified 3 signals (P<5×10-8) at established AF susceptibility loci marked by the sarcomeric gene TTN and sodium channel genes SCN5A and SCN10A. We also identified 2 novel loci near the genes VGLL2 and EXT1. In contrast, the clinical variable model prediction GWAS indicated a different genetic profile. In genetic correlation analysis, the prediction from the ECG-AI model was estimated to have a higher correlation with AF than that from the clinical variable model. Predicted AF risk from an ECG-AI model is influenced by genetic variation implicating sarcomeric, ion channel and body height pathways. ECG-AI models may identify individuals at risk for disease via specific biological pathways.

Hereditary multiple exostoses caused by a chromosomal inversion removing part of EXT1 gene

BackgroundHereditary multiple exostoses (HME) is an autosomal dominant skeletal disorder characterized by the development of multiple, circumscript and usually symmetric bony protuberances called osteochondromas. Most HME are caused by EXT1 and EXT2 loss of function mutations. Most pathogenic mutations are nonsense followed by missense mutations and deletions.Case presentationHere we report on a patient with a rare and complex genotype resulting in a typical HME phenotype. Initial point mutation screening in EXT1 and EXT2 genes by Sanger sequencing did not reveal any pathogenic variants. The patient along with the healthy parents was subsequently referred for karyotype and array-Comparative Genomic Hybridization (CGH) analyses.Chromosomal analysis revealed two independent de novo apparently balanced rearrangements: a balanced translocation between the long arms of chromosomes 2 and 3 at breakpoints 2q22 and 3q13.2 and a pericentric inversion with breakpoints at 8p23.1q24.1. Both breakpoints were confirmed by Fluorescence In Situ Hybridization (FISH). Subsequently, array-CGH revealed a novel heterozygous deletion within the EXT1 gene at one of the inversion breakpoints, rendering the inversion unbalanced. The mode of inheritance, as well as the size of the deletion were further investigated by Quantitative Real-time PCR (qPCR), defining the deletion as de novo and of 3.1 kb in size, removing exon 10 of EXT1. The inversion in combination with the 8p23.1 deletion most likely abolishes the transcription of EXT1 downstream of exon 10 hence resulting in a truncated protein.ConclusionsThe identification of a rare and novel genetic cause of HME, highlights the importance of additional comprehensive investigation of patients with typical clinical manifestations, even when EXT1 and EXT2 mutation analysis is negative.

Giant Osteochondroma of Tibia in a Case of Hereditary Multiple Exostoses: A Case Report

Osteochondromas, aka exostoses, are benign neoplasms of the cartilage. Genetic mutations of the EXT1 and EXT2 genes leads to a syndrome causing multiple osteochondromas, namely the hereditary multiple exostoses. Among the complications related to multiple osteochondromas, malignant degeneration into chondrosarcoma has the worst prognosis. Sudden enlargement of size of an osteochondroma or sudden onset of pain at the site of previous osteochondroma in a skeletally mature patient is concerning for malignant transformation. However, the size criterion does not apply for skeletally immature individuals, as observed in the case report here. The imaging of an uncommonly large osteochondroma of the extremity in a skeletally immature patient with hereditary multiple exostoses has been described here. Normal thickness of the cartilage cap (&lt;3cm) proved the benignity of the lesion on imaging, thereby nullifying the size criterion in the skeletally immature. Keywords: Hereditary multiple exostoses, Osteochondroma, Cartilage cap, Heparan sulphate, Bayonet deformity, Chondrosarcoma.

The Clinical and Molecular Spectrum of Trichorhinophalangeal Syndrome Types I and II in a Turkish Cohort Involving 22 Patients.

Trichorhinophalangeal syndrome is a rare autosomal dominant disorder characterized by distinctive craniofacial and skeletal abnormalities. This study aimed to delineate the trichorhinophalangeal syndrome phenotype and to compare the clinical and molecular findings between trichorhinophalangeal syndrome types I and II. A total of 22 trichorhinophalangeal syndrome patients aged 0.9-45 years from 17 families were enrolled. Nineteen patients were diagnosed with trichorhin ophalangeal syndrome I and 3 with trichorhinophalangeal syndrome II. Genetic analyses were made by TRPS1 sequencing and/or chromosomal microarray analyses. A novel frameshift variant (c.531_532del), a known missense variant, and whole-gene deletions were the pathogenic TRPS1 variants detected in trichorhinophalangeal syndrome I. Three trichorhinophalangeal syndrome II patients had large deletions with variable breakpoints involving the TRPS1-EXT1 interval. All patients had the typical craniofacial findings of trichorhinophalangeal syndrome such as a pear-shaped nose, long philtrum, and thin upper lip, as well as cone-shaped epiphyses. Sparse hair and eyebrows (20/22), short metacarpals and metatarsals (20/22), and small hands (19/22) were common. While craniofacial and limb abnormalities were similar in trichorhinophalangeal syndrome I and II, 3 of 19 trichorhinophal angeal syndrome I patients had mild, and 2 of 3 trichorhinophalangeal syndrome II patients had severe intellectual disability. Three trichorhinophalangeal syndrome II patients including the patient with the EXT1 deletion beginning from exon 2 had exostoses. In trichorhinophal angeal syndrome II, although microdeletion sizes and facial or skeletal features were not correlated, patients with larger deletions had severe intellectual disability. This study has expanded the existing knowledge on the phenotype-genotype spectrum in trichorhinophalangeal syndrome. We suggest including the EXT1 gene partially in the minimal critical region for trichorhinophalangeal syndrome II.

Haploinsufficiency of EXT1 and Heparan Sulphate Deficiency Associated with Hereditary Multiple Exostoses in a Pakistani Family.

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.

Population genetic structure analysis and identification of backfat thickness loci of Chinese synthetic Yunan pigs.

Yunan is a crossed lean meat pig breed in China. Backfat thickness is the gold standard for carcass quality grading. However, over 14years after breed registration, the backfat of Yunan thickened and the consistency of backfat thickness decreased. Meanwhile, no genetic study has been ever performed on Yunan population. So, in this study we collected all the 120 nucleus individuals of Yunan and recorded six backfat traits of them, carried out population genetic structure analysis, selection signals analysis and genome-wide association study of Yunan pigs with the help of their founder population Duroc and Chinese native Huainan pigs, to determine the genomic loci on backfat of Yunan. Genetic diversity indexes suggested Yunan pigs had no inbreeding risk while population genetic structure showed they had few molecular pedigrees and were stratified. A total of 71 common selection signals affecting growth and fat deposition were detected by F ST and XP-CLR methods. 34 significant loci associated with six backfat traits were detected, among which a 1.40Mb region on SSC4 (20.03-21.43Mb) were outstanding as the strong region underlying backfat. This region was common with the results of selection signature analysis, former reported QTLs for backfat and was common for different kinds of backfat traits at different development stage. ENPP2, EXT1 and SLC30A8 genes around were fat deposition related genes and were of Huainan pig's origin, among which Type 2 diabetes related gene SLC30A8 was the most reasonable for being in a 193.21Kb haplotype block of the 1.40Mb region. Our results had application value for conservation, mating and breeding improvement of backfat thickness of Yunan pigs and provided evidence for a human function gene might be reproduced in pigs.

RF30 | PSAT134 Autosomal Dominant Hereditary Multiple Osteochondromas Misdiagnosed As Paget's Disease of Bone

Abstract Background Autosomal dominant hereditary multiple osteochondromas is a rare genetic disorder with variable clinical manifestations. We present the case of a patient misdiagnosed with Paget's disease of bone found to have hereditary multiple osteochondromas. Clinical Case We present a 55-year-old male with history of chronic back and hip pain who presented to endocrinology clinic for evaluation of Paget's disease of bone. He was diagnosed with Paget's disease at age 16 after a bike accident resulting in knee pain where a "knot" was removed from his knee. He reported multiple "knots" in bones throughout adulthood but never sought treatment. He had history of elevated alkaline phosphatase on prior chemistries. He developed debilitating back, hip, and thigh pain resulting in immobility and suffered vertebral osteomyelitis from MRSA requiring laminectomy at age 52. On physical exam, massive maxillary and mandibular tori were noted along with bilateral cataracts and decreased muscle strength in upper and lower extremities. He underwent evaluation by oral surgery for extensive tooth decay and was recommended for extraction of teeth #12, 14, 15, and 17 with restorative needs for teeth #2, 4, 5, 13, 28, 30, 32. Labs notable for serum calcium 10.1 (ref: 8.4-10.5 mg/dl), phosphorus 4.2 (ref: 2.3-4.7 mg/dl), magnesium 1.8 (ref: 1.6-2.6mg/dl), PTH 34 (ref: 16-77 pg/ml), 25-OH Vit D 28 (ref: 25-80 ng/ml), 1,25 dihydroxyvitamin D 45.9 (ref: 19.9-79.3pg/ml), alkaline phosphatase 199 (ref: 40-150 unit/L), bone-specific alkaline phosphatase 52.5 (ref: 6.5 - 20.1 mcg/L). 24-hr urine calcium 0.06 g/24hr (ref: 0.04-0.35) from an 850ml urine volume. Procollagen type 1 n-terminal propeptide 179 (ref 22-105 UG/L). Prior MRI right hip showed "abnormal appearance of pelvic bony ring and proximal femurs with heterogenous marrow signal and cortical thickening suspicious for Paget's disease", as well as a 1.2cm circumscribed cystic focus within proximal right femoral diaphysis. Prior bone scan demonstrated diffusely uptake in pelvis, uptake in proximal right femur, focally intense uptake in L5 vertebrae. Plain film imaging demonstrated severe diffuse degenerative changes in cervical, thoracic and lumbar vertebrae and sessile osteochondromas arising from both scapulae, mild thoracic levoscoliosis. Genetic testing demonstrated loss-of-function mutation in the EXT-1 gene, diagnostic of hereditary multiple osteochondromas. Currently, there are no FDA-approved treatments for osteochondromas though studies involving biologic therapy are ongoing. This patient was referred to orthopedic oncology and OMFS for dental extraction and decision for conservative treatment with observation. Conclusion This case demonstrates how the rare entity of hereditary multiple osteochondromas can have variable clinical presentations leading to its misdiagnosis and highlights the need for ongoing research into non-surgical treatment options. Presentation: Saturday, June 11, 2022 1:00 p.m. - 3:00 p.m., Monday, June 13, 2022 12:36 p.m. - 12:41 p.m.

A case of a large pedunculated-type osteochondroma from late medieval Ilok, eastern Croatia: Bioarchaeological, paleoradiological and histological study

Osteochondroma or osteocartilaginous exostosis is one of the most common benign tumors of the bone. Causes for the disease are yet unknown, but there are indications that they may be linked to abnormality in the growth plate and possibly mutation in EXT1, EXT2 and EXT3 genes. Cases of reported osteochondromas range from prehistoric to contemporary examples, are not limited geographically, and no evidence for sex predominance has been reported.Here we present a unique case of large pedunculated-type osteochondroma on the right fibula of an adult female skeleton from the medieval site of Ilok-Krstbajer in eastern Croatia. In order to gain more insight into this pathological change we used holistic approach consisting of a combination of techniques that have not been used previously in the analysis of neoplasms from archaeological settings.The cauliflower-shaped growth is 50 mm long in sagittal and 57.41 mm in transverse diameter with the tumor exhibiting a bulbous, rough superior surface, and a flat, smoother inferior surface. The gross morphology of the tumor together with radiological and histopathological features support the diagnosis of a proximal fibular osteochondroma making it the first such case in an archaeological population. Based on archaeological context and similar clinical cases it seems that the presented osteochondroma did not have major impact on the life-quality of a woman affected by this pathology. The procedure used in this study is minimally invasive and highly accurate, and as such sets new analytical criteria for studies of ancient bone neoplasms.

An Easy-to-Use Approach to Detect CNV From Targeted NGS Data: Identification of a Novel Pathogenic Variant in MO Disease.

BackgroundDespite 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.AimWe 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.MethodsThe 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.ResultsAll 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)-?_804+?del —out of the MLPA target regions— has been identified. The variant was confirmed by real-time quantitative Polymerase Chain Reaction (qPCR).ConclusionIn 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.

A frameshift variant in the EXT1 gene in a feline leukemia virus-negative cat with osteochondromatosis.

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_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.

Secondary peripheral chondrosarcoma arising in solitary osteochondroma: variables influencing prognosis and survival

BackgroundSecondary peripheral chondrosarcomas arising in solitary osteochondromas is an unusual complication, reported in small series. In this study, we aimed to present our experience with this rare variant of chondrosarcoma and compare results with already published data in order to determine prognostic factors for overall and disease-free survival.MethodsThe case study includes retrospective data from patients diagnosed at a single institution from 1943 to 2019. Clinical data were collected reviewing all available medical records from first to last follow-up visits. To exclude the presence of the Multiple Osteochondroma Hereditary Syndrome, few patients, with a suspect of a familial form of the disease, were evaluated for the presence of germline heterozygous variants in EXT1 and EXT2 genes. Results were summarized using descriptive statistics and statistical analysis were performed to reveal associations between variables.ResultsTwo hundred and fourteen secondary peripheral chondrosarcomas that arose exclusively from solitary osteochondromas diagnosed in a multidisciplinary setting at the IRCCS Istituto Ortopedico Rizzoli were retrospectively identified, 66.4% males and 33.6% females with a median age at diagnosis of 38 years. The local recurrence rate was 17.3%, while the metastases one was 5.1%. Besides age, a high histologic grade is the only factor associated with worse 5-year and 10-year overall survival (log-rank p = 0.0005, HR = 3.74; 95% CI 1.69–8.26). Moreover, high histological grade (HR = 3.75; 95% CI = 1.69–8.34; p = 0.001) and surgical debulking (HR = 3.71; 95% CI = 1.57–8.79; p = 0.003) were associated with a significantly worse disease-free survival.ConclusionsOur study confirm the low-grade behavior of secondary peripheral chondrosarcomas and demonstrate that the best choice of treatment for those arising in solitary osteochondromas is the wide surgical excision, when possible. Location per se is not a factor that affects prognosis, while the accurate histological grade assessment is correlated with the tumor aggressiveness and a long term follow up is necessary for this rare variant of chondrosarcoma.

Mutations in the heparan sulfate backbone elongating enzymes EXT1 and EXT2 have no major effect on endothelial glycocalyx and the glomerular filtration barrier

In this study, the effect of heterozygous germline mutations in the heparan sulfate (HS) glycosaminoglycan chain co-polymerases EXT1 and EXT2 on glomerular barrier function and the endothelial glycocalyx in humans is investigated. Heparan sulfate (HS) glycosaminoglycans are deemed essential to the glomerular filtration barrier, including the glomerular endothelial glycocalyx. Animal studies have shown that loss of HS results in a thinner glycocalyx. Also, decreased glomerular HS expression is observed in various proteinuric renal diseases in humans. A case report of a patient with an EXT1 mutation indicated that this could result in a specific renal phenotype. This patient suffered from multiple osteochondromas, an autosomal dominant disease caused by mono-allelic germline mutations in the EXT1 or EXT2 gene. These studies imply that HS is indeed essential to the glomerular filtration barrier. However, loss of HS did not lead to proteinuria in various animal models. We demonstrate that multiple osteochondroma patients do not have more microalbuminuria or altered glycocalyx properties compared to age-matched controls (n = 19). A search for all Dutch patients registered with both osteochondroma and kidney biopsy (n = 39) showed that an EXT1 or EXT2 mutation does not necessarily lead to specific glomerular morphological phenotypic changes. In conclusion, this study shows that a heterozygous mutation in the HS backbone elongating enzymes EXT1 and EXT2 in humans does not result in (micro)albuminuria, a specific renal phenotype or changes to the endothelial glycocalyx, adding to the growing knowledge on the role of EXT1 and EXT2 genes in pathophysiology.

An early presentation of osteochondroma: a rare case

The most prevalent benign bone tumor, osteochondroma, accounts for 30 to 35% of all benign bone tumors. It most frequently affects teenagers and young adults, with a 1-in-50,000 incidence rate. Osteochondroma only has a 1% chance of developing into a malignant condition like chondrosarcoma. Osteochondroma has an autosomal dominant pattern, and prehypertrophic chondrocytes in the growth plate are affected by the EXT gene mutation. Osteochondroma patients typically have their knee joint affected and typically report painless swelling around major joints. These swellings result from the long bone´s metaphysis´s excessive development of cartilage and bone. This swelling could result in mechanical symptoms like compression of the neurovascular system. The mother of a 3-year-old kid brought him to the orthopaedics outpatient department with complaints of painless swelling in the right wrist, left scapula, and left ankle that had been reported two months prior. Her 5-year-old sibling has a similar, painless swelling that is only modest in size. The patient is being handled conservatively and recommended to keep regular follow-ups every three months because they currently have no additional complaints. The state and prognosis of the disease have been disclosed to the patient´s parents. Operative management needs to wait till the skeleton is fully developed.

Hereditary Multiple Exostoses-A Review of the Molecular Background, Diagnostics, and Potential Therapeutic Strategies.

Hereditary multiple exostoses (HMEs) syndrome, also known as multiple osteochondromas, represents a rare and severe human skeletal disorder. The disease is characterized by multiple benign cartilage-capped bony outgrowths, termed exostoses or osteochondromas, that locate most commonly in the juxta-epiphyseal portions of long bones. Affected individuals usually complain of persistent pain caused by the pressure on neighboring tissues, disturbance of blood circulation, or rarely by spinal cord compression. However, the most severe complication of this condition is malignant transformation into chondrosarcoma, occurring in up to 3.9% of HMEs patients. The disease results mainly from heterozygous loss-of-function alterations in the EXT1 or EXT2 genes, encoding Golgi-associated glycosyltransferases, responsible for heparan sulfate biosynthesis. Some of the patients with HMEs do not carry pathogenic variants in those genes, hence the presence of somatic mutations, deep intronic variants, or another genes/loci is suggested. This review presents the systematic analysis of current cellular and molecular concepts of HMEs along with clinical characteristics, clinical and molecular diagnostic methods, differential diagnosis, and potential treatment options.

Dysplasia Epiphysealis Hemimelica (Trevor’s Disease) in Children, Two New Cases: Diagnosis, Treatment, and Literature Review

Dysplasia epiphysealis hemimelica (DEH), also known as Trevor’s disease, is a rare nonhereditary skeletal disorder affecting one side of the epiphyses or the epiphyses-equivalents. It is often misdiagnosed for traumatic injuries, infections, or other tumors because of the nonspecific clinical features. The diagnosis is mostly based on radiographic involvement of one half of the epiphysis displaying an overgrowth; it is hard to distinguish between DEH and osteochondroma on the gross hystopathological exam. There are few immunohistochemical markers, as well as genetic tests, for EXT1 and EXT2 gene expression that can reveal a more accurate diagnosis. No evidence of malignant changes has been reported and no hereditary transmission or environmental factor has been incriminated as an etiological factor. The natural history of the disease is continuous growth of the lesions until skeletal maturity. Without treatment, the joint might suffer degenerative modification, and the patient can develop early onset osteoarthritis. In the present paper, we report two new cases of DEH of the ankle. The aim of this paper is to consider Trevor’s disease when encountering tumoral masses in the epiphyses of pediatric patients and to present our treatment approach and results.

P–562 PGT-M with de novo mutations : how to deal with it?

Abstract Study question How to technically deal with the PGT-M set-up in case of de novo mutations in female or male affected patients with dominant disease due to de novo mutations. Summary answer PGT-M was performed for three couples carrying de novo mutations using direct and linkage analysis on sperm or polar bodies to define haplotypes and phase. What is known already Couples with a de novo mutation in a gene causing AD disease, such as FGFR3 (achondroplasia), NF1 (neurofibromatosis) and EXT2 (multiple exostosis) cannot undergo PGT-M via standard techniques like karyomapping, as the absence of affected relatives makes phasing impossible. However, linkage analysis combined with direct mutation analysis allows on haploid cells from the mutation carrier, such as sperm or polar bodies (PB), allows the correct association of a haplotype and the disease-causing mutation. Flanking informative STRs must be positioned at &amp;lt; 1 Mb of the gene, in order to minimize the risk of recombination during meiosis. Study design, size, duration Couples underwent pre-test counselling with a geneticist and an IVF specialist. Pathogenic variants were identified and their absence from the couples’ parents confirmed. Four to six informative STRs were identified. For males we analysed 20–50 isolated sperm to define the haplotypes and the phase, before starting with the stimulation cycle; for females, we needed to wait after the oocyte pick-up and the biopsy of PBs. Point mutations are identified by SNaPshot, deletions by multiplexed STS. Participants/materials, setting, methods The 3 couples in the study presented in IVF centres, requesting PGT-M for either male or female AD disease. They had genetic testing reports from other laboratories. For FGFR3 and NF1, the described variants were confirmed. The patient with multiple exostosis came with a negative genetic result for EXT1 and EXT2 genes, but after diagnostic-quality NGS (Blueprint Genetics, Finland) we identified an EXT2 deletion. Diagnostic multiplex PCR was then performed on embryos or polar bodies. Main results and the role of chance The setup started with the confirmation of the mutations in the 3 couples and the confirmation of the de novo status. Four to six informative STRs were then identified for each couple. Multiplex PCR containing the STRs and the SNAPSHOT analysis for the point mutations was developed. To identify the phase and the disease-carrying haplotype in male carriers, we performed a multiplex PCR on 20–50 spermatozoa. In the female patient with NF1, the haplotype and the phase were determined on the polar bodies; the mutation was on her paternal allele, as predicted genetically. Prior to PGT, we evaluated the robustness of each multiplex on 20 to 50 single leukocytes of the couple. Each couple had at least one embryo not carrying the risk haplotype, suitable for transfer. The couples with NF1 and achondroplasia both delivered a healthy, unaffected baby. The pregnancy is ongoing in the couple with the EXT2 variant. PGT-M is now easily handled for standard situations, with semiautomated protocols that do not need extensive setups. De novo mutations however present a unique challenge, because of the impossibility in most cases of determining the phase of the disease-causing variant. We present a patient-centric approach with individualized protocols. Limitations, reasons for caution Allele drop-out could lead to misdiagnosis of the embryo. To avoid that, 6 flanking STRs (3 proximal and 3 distal) and genotyping of the variant should be performed. When possible, it is good practice to pre-define the different haplotypes with the parents of the patients. Wider implications of the findings: The increasing number of laboratories offering off-the-shelf testing with NGS panels and semi-automated PGT can fulfil demand for routine situations. However in more complex cases, diagnostic-quality NGS and individualized PGT-M programmes are needed. These cases also remind us that PGT-M requires extensive multidisciplinarity to maximize the chance of successful outcome. Trial registration number Not applicable