Expanding the phenotype of SPARC-related osteogenesis imperfecta: clinical findings in two patients with pathogenic variants in SPARC and literature review.

BACKGROUND: Secreted protein, acidic, cysteine rich (SPARC)-related osteogenesis imperfecta (OI), also referred to as OI type XVII, was first described in 2015, since then there has been only one further report of this form of OI. SPARC is located on chromosome 5 between bands q31 and q33. The encoded protein is necessary for calcification of the collagen in bone, synthesis of extracellular matrix and the promotion of changes to cell shape. METHODS: We describe a further two patients with previously unreported homozygous SPARC variants with OI: one splice site; one nonsense pathogenic variant. We present detailed information on the clinical and radiological phenotype and correlate this with their genotype. There are only two previous reports by Mendozo-Londono et al and Hayat et al with clinical descriptions of patients with SPARC variants. RESULTS: From the data we have obtained, common clinical features in individuals with OI type XVII caused by SPARC variants include scoliosis (5/5), vertebral compression fractures (5/5), multiple long bone fractures (5/5) and delayed motor development (3/3). Interestingly, 2/4 patients also had abnormal brain MRI, including high subcortical white matter changes, abnormal fluid-attenuated inversion in the para-atrial white matter and a large spinal canal from T10 to L1. Of significance, both patients reported here presented with significant neuromuscular weakness prompting early workup. CONCLUSION: Common phenotypic expressions include delayed motor development with neuromuscular weakness, scoliosis and multiple fractures. The data presented here broaden the phenotypic spectrum establishing similar patterns of neuromuscular presentation with a presumed diagnosis of 'myopathy'.

Arterial complications in classical Ehlers-Danlos syndrome: a case series.

BACKGROUND: The Ehlers-Danlos syndromes (EDS) are a group of connective tissue disorders with several recognised types. Patients with a type of EDS have connective tissue abnormalities resulting in a varying degree of joint hypermobility, skin and vascular fragility and generalised tissue friability. Classical EDS (cEDS) typically occurs as a result of dominant pathogenic variants in COL5A1 or COL5A2. The cardinal features of cEDS are hyperextensible skin, atrophic scarring and joint hypermobility. Arterial complications are more characteristically a feature of vascular EDS although individual cases of arterial events in cEDS have been reported. METHODS: A cohort of 154 patients with a clinical diagnosis of cEDS from the UK was analysed. RESULTS: Seven patients (4.5%) with a diagnosis of cEDS (four pathogenic, one likely pathogenic and two variants of uncertain significance in COL5A1) who had experienced arterial complications were identified. Arterial complications mostly involved medium-sized vessels and also two abdominal aortic aneurysms. No unique clinical features were identified in this group of patients. CONCLUSION: There is a possible increased risk of arterial complications in patients with cEDS, although not well-defined. Clinicians need to be aware of this possibility when presented with a patient with an arterial complication and features of cEDS. Long-term management in families with cEDS and a vascular complication should be individually tailored to the patient's history and their family's history of vascular events.

Rough endoplasmic reticulum expansion: a consistent finding in a patient cohort with vascular Ehlers-Danlos Syndrome and Osteogenesis Imperfecta.

Vascular Ehlers-Danlos Syndrome (vEDS) and Osteogenesis Imperfecta (OI) are two forms of connective tissue disorders. Previously, transmission electron microscopy of skin biopsies was routinely performed on all patients who were clinically suspected to have vEDS. At present, molecular genetics using genomic DNA extracted from a blood sample is the first line investigation for these patients. However, when variants of uncertain clinical significance are identified on genetic testing and individuals do not have the classical features of OI or vEDS, additional phenotypic information obtained from a skin biopsy can be valuable for contributing to the evidence for re-classifying pathogenicity of variants.We present a cohort of six patients with molecularly confirmed vEDS and one patient with a severe form of OI, who each had expanded (or dilated), protein-filled, rough endoplasmic reticulum identified on transmission electron microscopy. The patients were identified through retrospective screening of medical records, and biopsies were taken between 1999-2016. We discuss the potential role for assessing rough endoplasmic reticulum expansion as a useful tool to allow further phenotyping of these individuals.

Epigenetic resetting of human pluripotency.

Much attention has focussed on the conversion of human pluripotent stem cells (PSCs) to a more naïve developmental status. Here we provide a method for resetting via transient histone deacetylase inhibition. The protocol is effective across multiple PSC lines and can proceed without karyotype change. Reset cells can be expanded without feeders with a doubling time of around 24 h. WNT inhibition stabilises the resetting process. The transcriptome of reset cells diverges markedly from that of primed PSCs and shares features with human inner cell mass (ICM). Reset cells activate expression of primate-specific transposable elements. DNA methylation is globally reduced to a level equivalent to that in the ICM and is non-random, with gain of methylation at specific loci. Methylation imprints are mostly lost, however. Reset cells can be re-primed to undergo tri-lineage differentiation and germline specification. In female reset cells, appearance of biallelic X-linked gene transcription indicates reactivation of the silenced X chromosome. On reconversion to primed status, XIST-induced silencing restores monoallelic gene expression. The facile and robust conversion routine with accompanying data resources will enable widespread utilisation, interrogation, and refinement of candidate naïve cells.

Classical-like Ehlers-Danlos syndrome: a clinical description of 20 newly identified individuals with evidence of tissue fragility.

PURPOSE: Currently, 31 patients with classical-like EDS (clEDS) due to tenascin-X deficiency have been reported in the literature. We report on the clinical and molecular characteristics of 20 additional patients with clEDS to expand knowledge and to enable improved management of this rare genetic disorder. METHODS: Patients diagnosed with clEDS by the national EDS service in the UK (n = 21) and abroad (n = 1) were asked for consent for publication of their clinical and molecular data. RESULTS: Of 22 patients, 20 consented. All patients had typical features of clEDS: joint hypermobility, easy bruising, and skin hyperextensibility without atrophic scars. Importantly, 3/20 patients experienced gastrointestinal complications consisting of small or large bowel ruptures and one esophageal rupture. Other notable observations included two separate occurrences of spontaneous compartment syndrome, suspicion of nonaccidental injury due to significant bruising, and significant clinical variability regarding the debilitating effect of joint dislocations. CONCLUSIONS: We propose a predisposition to tissue fragility, particularly of the gastrointestinal tract in patients with clEDS. As such, clinical and molecular confirmation of this diagnosis is essential. It is recommended to follow up these patients closely to understand the natural history to develop better recommendations for management.

Clinical features, molecular results, and management of 12 individuals with the rare arthrochalasia Ehlers-Danlos syndrome.

Arthrochalasia Ehlers-Danlos syndrome (aEDS) is a rare autosomal dominant connective tissue disorder that is characterized by congenital bilateral hip dislocations, severe generalized joint hypermobility, recurrent joint (sub)luxations, and skin hyperextensibility. To date, 42 patients with aEDS have been published. We report 12 patients with aEDS from 10 families with 6 unpublished individuals and follow-up data on 6 adult patients. The clinical features are largely comparable with patients reported in the literature. Most (n = 10) patients had variants leading to (partial) loss of exon 6 of the COL1A1 or COL1A2 genes. One patient did not have a previously reported likely pathogenic COL1A1 variant. Data regarding management were retrieved. Hip surgery was performed in 5/12 patients and 3/12 patients underwent spinal surgery. As much as 4/12 patients were wheelchair-bound or unable to walk unaided. Fractures were present in 9/12 individuals with 1 patient requiring bisphosphonate treatment. Echocardiograms were performed in 10 patients and 2 individuals showed an abnormality likely unrelated to aEDS. One patient gave birth to two affected children and went through preterm labor requiring medication but had no additional complications. Of the eight adults in our cohort, the majority entered a career. Our data point toward a genotype-phenotype relationship with individuals with aEDS due to pathogenic COL1A1 variants causing complete or partial loss of exon 6 being more severely affected regarding musculoskeletal features. There is a significant lack of knowledge with regard to management of aEDS, particularly in adulthood. As such, systematic follow-up and multidisciplinary treatment is essential.

Atypical COL3A1 variants (glutamic acid to lysine) cause vascular Ehlers-Danlos syndrome with a consistent phenotype of tissue fragility and skin hyperextensibility.

PURPOSE: The Ehlers-Danlos syndromes (EDS) are a group of rare inherited connective tissue disorders. Vascular EDS (vEDS) is caused by pathogenic variants in COL3A1, most frequently glycine substitutions. We describe the phenotype of the largest series of vEDS patients with glutamic acid to lysine substitutions (Glu>Lys) in COL3A1, which were all previously considered to be variants of unknown significance. METHODS: Clinical and molecular data for seven families with three different Glu>Lys substitutions in COL3A1 were analyzed. RESULTS: These Glu>Lys variants were reclassified from variants of unknown significance to either pathogenic or likely pathogenic in accordance with American College of Medical Genetics and Genomics guidelines. All individuals with these atypical variants exhibited skin hyperextensibility as seen in individuals with classical EDS and classical-like EDS and evidence of tissue fragility as seen in individuals with vEDS. CONCLUSION: The clinical data demonstrate the overlap between the different EDS subtypes and underline the importance of next-generation sequencing gene panel analysis. The three different Glu>Lys variants point toward a new variant type in COL3A1 causative of vEDS, which has consistent clinical features. This is important knowledge for COL3A1 variant interpretation. Further follow-up data are required to establish the severity of tissue fragility complications compared with patients with other recognized molecular causes of vEDS.

Feeder-free culture of human pluripotent stem cells drives MDM4-mediated gain of chromosome 1q.

Culture-acquired variants in human pluripotent stem cells (hPSCs) hinder their applications in research and clinic. However, the mechanisms that underpin selection of variants remain unclear. Here, through analysis of comprehensive karyotyping datasets from over 23,000 hPSC cultures of more than 1,500 lines, we explored how culture conditions shape variant selection. Strikingly, we identified an association of chromosome 1q gains with feeder-free cultures and noted a rise in its prevalence in recent years, coinciding with increased usage of feeder-free regimens. Competition experiments of multiple isogenic lines with and without a chromosome 1q gain confirmed that 1q variants have an advantage in feeder-free (E8/vitronectin), but not feeder-based, culture. Mechanistically, we show that overexpression of MDM4, located on chromosome 1q, drives variants' advantage in E8/vitronectin by alleviating genome damage-induced apoptosis, which is lower in feeder-based conditions. Our study explains condition-dependent patterns of hPSC aberrations and offers insights into the mechanisms of variant selection.

The isochromosome 20q abnormality of pluripotent cells interrupts germ layer differentiation.

Chromosome 20 abnormalities are some of the most frequent genomic changes acquired by human pluripotent stem cell (hPSC) cultures worldwide. Yet their effects on differentiation remain largely unexplored. We investigated a recurrent abnormality also found on amniocentesis, the isochromosome 20q (iso20q), during a clinical retinal pigment epithelium differentiation. Here we show that the iso20q abnormality interrupts spontaneous embryonic lineage specification. Isogenic lines revealed that under conditions that promote the spontaneous differentiation of wild-type hPSCs, the iso20q variants fail to differentiate into primitive germ layers and to downregulate pluripotency networks, resulting in apoptosis. Instead, iso20q cells are highly biased for extra-embryonic/amnion differentiation following inhibition of DNMT3B methylation or BMP2 treatment. Finally, directed differentiation protocols can overcome the iso20q block. Our findings reveal in iso20q a chromosomal abnormality that impairs the developmental competency of hPSCs toward germ layers but not amnion, which models embryonic developmental bottlenecks in the presence of aberrations.

Epigenetic dynamics during capacitation of naïve human pluripotent stem cells.

Human pluripotent stem cells (hPSCs) are of fundamental relevance in regenerative medicine. Naïve hPSCs hold promise to overcome some of the limitations of conventional (primed) hPSCs, including recurrent epigenetic anomalies. Naïve-to-primed transition (capacitation) follows transcriptional dynamics of human embryonic epiblast and is necessary for somatic differentiation from naïve hPSCs. We found that capacitated hPSCs are transcriptionally closer to postimplantation epiblast than conventional hPSCs. This prompted us to comprehensively study epigenetic and related transcriptional changes during capacitation. Our results show that CpG islands, gene regulatory elements, and retrotransposons are hotspots of epigenetic dynamics during capacitation and indicate possible distinct roles of specific epigenetic modifications in gene expression control between naïve and primed hPSCs. Unexpectedly, PRC2 activity appeared to be dispensable for the capacitation. We find that capacitated hPSCs acquire an epigenetic state similar to conventional hPSCs. Significantly, however, the X chromosome erosion frequently observed in conventional female hPSCs is reversed by resetting and subsequent capacitation.

Diagnosis and management of vascular Ehlers-Danlos syndrome: Experience of the UK national diagnostic service, Sheffield.

The UK National Diagnostic Service for Ehlers-Danlos Syndromes (EDS) was established in 2009 for the rare types of EDS. Vascular EDS (vEDS) is an inherited connective tissue disorder caused by pathogenic variants in the COL3A1 gene. Associated tissue fragility affects multiple organ systems, increasing the risk of blood vessel dissection and rupture, with potentially fatal consequences. The diagnosis of vEDS has improved with advances in genetic testing, however this is most often suspected following an acute event. We provide data on the clinical features of vEDS for 180 patients (full cohort) seen in our service with confirmed molecular diagnoses. Increased awareness of this rare condition will prompt genetic testing essential to confirm the diagnosis. Outcomes are improved by early diagnosis followed by appropriate management. Fragile connective tissues make invasive procedures potentially dangerous, particularly in an emergency setting. Lifestyle advice from a young age can help acceptance and understanding of the diagnosis and inform choices. There is currently limited evidence for the use of drug therapy to reduce vascular events. We report on the incidence of vascular events in 126 patients (statistical analysis cohort) in our care and the use of medication. Our retrospective data showed that those patients on a long-term angiotensin II receptor blocker and/or beta-blocker had fewer vascular events than those not on cardiac medication who received the same lifestyle and emergency care advice.

An exemplary model of genetic counselling for highly specialised services.

With genomic testing being increasingly integrated into every day clinical practice and a wide range of practitioners ordering genetic tests, it is important that the scope of the genetic counselling role continues to evolve alongside these changes. We present an exemplary role for genetic counsellors in a highly specialised service within England's National Health Service for people who have or are suspected to have rare genetic types of Ehlers Danlos syndrome. The service employs genetic counsellors and consultants from the fields of genetics and dermatology. The service also works closely with other specialists and related charities and patient organisations. The genetic counsellors in the service provide routine genetic counselling such as diagnostic and predictive testing, but their role also includes the writing of patient literature and emergency and well-being resources, delivering workshops and talks, and the development of qualitative and quantitative research on the patient experience. Data from such research has informed the development of patient self-advocacy and supportive resources, raised awareness amongst healthcare professionals and enhanced the standard of care and outcomes for patients. The service aims to be an example of innovation and accessibility and provides a model that can be potentially adopted by other highly specialised services of rare genetic diseases.

In Vivo Tumorigenicity of the 20q11.21 Amplicon in an Engraftment Model of hPSCs and Differentiated Liver Cells.

Human pluripotent stem cells (hPSCs) are a promising source of somatic cells for clinical applications and disease modelling. However, during culture they accumulate genetic aberrations such as amplification of 20q11.21 which occurs in approximately 20% of extensively cultured hPSC lines and confers a BCL2L1-mediated survival advantage. During the production of the large number of cells required for transplantation and therapy these aberrations may become unavoidable which has important safety implications for therapies and may also impact upon disease modelling. Presently, these risks are poorly understood; whilst it is apparent that large-scale genetic aberrations can pose an oncogenic risk, the risks associated with smaller, more insidious changes have not been fully explored. In this report, the effects of engraftment of human embryonic stem cells (hESCs) and hESC-derived hepatocyte-like cells (HLCs) with and without amplification of the 20q11.21 minimal amplicon and isochromosome 20q (i20q) in SCID-beige mice are presented. The cells were tracked in vivo using a luminescent reporter over a period of approximately four months. Intrasplenic injection of hESCs showed greater engraftment potential and the formation of more severely disruptive lesions in the liver and spleen of animals injected with cells containing 20q11.21 compared with i20q and wild type. HLCs with 20q11.21 engrafted more successfully and formed more severely disruptive lesions than wild type cells or cells with i20q. These results reinforce the notion that karyotyping of therapeutic hPSC is required for transplant, and suggest that screening for known common aberrations is necessary. Further work to identify commonly arising genetic aberrations should be performed and routine screening for hPSCs intended for therapeutic use should be used.

Characterizing the Genetic Stability of Human Naïve and Primed Pluripotent Stem Cells.

The presence of genetic changes in human pluripotent stem cells (hPSCs) can affect their behavior and impact on the utility of hPSC-based applications in research and clinic. The spectrum of spontaneously arising genetic abnormalities in hPSCs is wide and ranges from numerical and structural chromosomal anomalies down to point mutations. The detection of genetic changes in hPSCs is confounded by the fact that no single method detects all types of abnormalities with the same accuracy and sensitivity, therefore necessitating the use of a combination of different methods. Here, we provide detailed protocols for two methods commonly utilized for the detection of genetic changes in naïve and primed hPSCs: karyotyping by G-banding and fluorescent in situ hybridization (FISH).

Multi-exon COL5A1 deletion in a child with classical Ehlers-Danlos syndrome: A case report expanding the allelic spectrum and showing evidence of parental gonosomal mosaicism.

Classical Ehlers-Danlos syndrome (cEDS) is a rare inherited autosomal dominant connective tissue disorder with core clinical features including skin hyperextensibility, abnormal scarring, and generalized joint hypermobility. Classical EDS is predominantly caused by small pathogenic variants in the genes COL5A1 and COL5A2 and occasionally by a COL1A1 point mutation p.(Arg312Cys), while gross deletions or duplications are uncommon. Gonosomal mosaicism is thought to be exceedingly rare with only two cases reported in the literature. We report a child with cEDS due to a rare gross deletion of exons 2-65 in the COL5A1 gene, inherited from an unaffected mosaic father. The level of mosaicism in the father was approximately 43% in leucocyte cells and 30% in DNA extracted from skin. Our results expand the allelic spectrum of cEDS variants and suggest that parental mosaicism needs to be considered in patients with suspected cEDS, given its implication for genetic counseling.

Single Nucleotide Polymorphism (SNP) Arrays and Their Sensitivity for Detection of Genetic Changes in Human Pluripotent Stem Cell Cultures.

Human pluripotent stem cells (hPSCs) can be grown in culture indefinitely, making them a valuable tool for use in basic biology, disease modeling, and regenerative medicine. However, over prolonged periods in culture, hPSCs tend to acquire genomic aberrations that confer growth advantages, similar to those seen in some cancers. Monitoring the genomic stability of cultured hPSCs is critical to ensuring their efficacy and safety as a therapeutic tool. Most commonly employed methods for monitoring of hPSC genomes are cytogenetic methods, such as G-banding. Nonetheless, such methods have limited resolution and sensitivity for detecting mosaicism. Single nucleotide polymorphism (SNP) array platforms are a potential alternative that could improve detection of abnormalities. Here, we outline protocols for SNP array whole-genome screening of hPSCs. Moreover, we detail the procedure for assessing the SNP array's sensitivity in detecting low-level mosaic copy-number changes. We show that mosaicism can be confidently identified in samples only once they contain 20% variants, although samples containing 10% variants typically display enough variation to warrant further investigation and confirmation, for example by using a more sensitive targeted method. Finally, we highlight the advantages and limitations of SNP arrays, including a cost comparison of SNP arrays versus other commonly employed methods for detection of genetic changes in hPSC cultures. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: DNA sample preparation for SNP arrays Basic Protocol 2: SNP array hybridization, washing, and scanning Basic Protocol 3: SNP array data analysis Support Protocol: Assessment of SNP array sensitivity for detection of mosaicism.

Non-collagen pathogenic variants resulting in the osteogenesis imperfecta phenotype in children: a single-country observational cohort study.

BACKGROUND/OBJECTIVES: In England, children (0-18 years) with severe, complex and atypical osteogenesis imperfecta (OI) are managed by four centres (Birmingham, Bristol, London, Sheffield) in a 'Highly Specialised Service' (HSS OI); affected children with a genetic origin for their disease that is not in COL1A1 or COL1A2 form the majority of the 'atypical' group, which has set criteria for entry into the service. We have used the data from the service to assess the range and frequency of non-collagen pathogenic variants resulting in OI in a single country. METHODS: Children with atypical OI were identified through the HSS OI service database. All genetic testing for children with OI in the service were undertaken at the Sheffield Diagnostic Genetics Service. Variant data were extracted and matched to individual patients. This study was done as part of a service evaluation project registered with the Sheffield Children's Hospital Clinical Governance Department. RESULTS: One hundred of 337 children in the HSS met the 'atypical' criteria. Eighty have had genetic testing undertaken; 72 had genetic changes detected, 67 in 13 genes known to be causative for OI. The most frequently affected genes were IFITM5 (22), P3H1 (12), SERPINF1 (8) and BMP1 (6). CONCLUSION: Among children with more severe forms of OI (approximately one-third of all children with OI), around 20% have pathogenic variants in non-collagen genes. IFITM5 was the most commonly affected gene, followed by genes within the P3H1 complex. These data provide additional information regarding the likelihood of different genetic origins of the disease in children with OI, which may influence clinical care.

Proteomics analysis of epithelial cells reprogrammed in cell-free extract.

The functional reprogramming of a differentiated cell to a pluripotent state presents potential beneficial applications in regenerative medicine. We report here the proteomic profile of 293T epithelial cells reprogrammed to a pluripotent state using undifferentiated embryonal carcinoma (NCCIT) cellular extracts. 293T cells were reversibly permeabilized with streptolysin O, incubated in an extract of NCCIT cells or a control extract of 293T cells for 1 h, resealed with CaCl(2), and cultured. OCT4 and SOX2 gene expression were up-regulated in NCCIT extract-treated cells relative to control cells, whereas there was no alteration in DNMT3B gene expression. Thirty percent of NCCIT extract-treated cells were positive for SSEA-4, and karyotyping confirmed their 293T origin, excluding the possibility of contamination from NCCIT cells. Two-dimensional PAGE revealed approximately 400 protein spots for each cell type studied. At least 10 protein spots in the proteome of NCCIT extract-treated cells had an expression profile similar to that of NCCIT and remained unaltered in control cells. Using tandem mass spectrometry, we identified these proteins, which include 78-kDa glucose-regulated protein precursor and tropomyosin alpha-3 chain. This investigation provides the first evidence that proteins are altered in a specific manner in NCCIT extract-treated cells. This is the first report on the proteomic characterization of the nuclear reprogramming process.

Pinacidil enhances survival of cryopreserved human embryonic stem cells.

Human embryonic stem cells (hESCs) can be maintained as undifferentiated cells in vitro and induced to differentiate into a variety of somatic cell types. Thus, hESCs provide a source of differentiated cell types that could be used to replace diseased cells of a tissue. The efficient cryopreservation of hESCs is important for establishing effective stem cell banks, however, conventional slow freezing methods usually lead to low rates of recovery after thawing cells and their replating in culture. We have established a method for recovering cryopreserved hESCs using pinacidil and compared it to a method that employs the ROCK inhibitor Y-27632. We show that pinacidil is similar to Y-27632 in promoting survival of hESCs after cryopreservation. The cells exhibited normal hESC morphology, retained a normal karyotype, and expressed characteristic hESC markers (OCT4, SSEA3, SSEA4 and TRA-1-60). Moreover, the cells retained the capacity to differentiate into derivatives of all three embryonic germ layers as demonstrated by differentiation through embryoid body formation. Pinacidil has been used for many years as a vasodilator drug to treat hypertension and its manufacture and traceability are well defined. It is also considerably cheaper than Y-27632. Thus, the use of pinacidil offers an efficient method for recovery of cryopreserved dissociated human ES cells.