Electron microscopy in the diagnosis of Ehlers-Danlos syndromes: correlation with clinical and genetic investigations.

BACKGROUND: The Ehlers-Danlos syndromes (EDS) consist of 13 subtypes with overlapping features including joint hypermobility, skin and vascular fragility and generalized connective tissue friability. As DNA analysis has become the gold standard for investigation of EDS, transmission electron microscopy (TEM) in clinical practice is decreasing. However, owing to the use of next-generation sequencing, the frequency of variants of uncertain significance (VUS) identified using DNA analysis is increasing. We hypothesized that TEM can provide evidence for or against pathogenicity of VUS. OBJECTIVES: The aim of this study was to evaluate the role of TEM in the diagnosis of EDS subtypes. METHODS: Data were collected from patients who underwent a skin biopsy between October 2012 and March 2017 at the London EDS National Diagnostic Service. TEM biopsies were categorized as 'normal' or 'abnormal' according to the description and conclusion in the TEM reports. Definitive diagnoses were reached via a combination of clinical features, structural and functional studies and DNA investigations. RESULTS: The analysis included 177 patients, comprising 30 abnormal and 147 normal TEM reports. A definitive diagnosis of monogenic EDS subtypes was made in 24 patients. Overall, 17 of these 24 patients (71%) had an abnormal biopsy report and seven (29%) had a normal biopsy report. No TEM findings were specifically associated with any EDS subtype, although collagen flowers were present in most patients with a genetically confirmed diagnosis of classical EDS. CONCLUSIONS: TEM analysis of collagen structure may have the potential to provide evidence for or against the pathogenicity of a VUS, but more work is needed to establish a clear role for TEM in this process. What's already known about this topic? Collagen fibril abnormalities can be seen in several Ehlers-Danlos syndrome (EDS) subtypes. What does this study add? This study provides clinical data, transmission electron microscopy (TEM) data and molecular data of one of the largest groups of patients suspected to have a monogenetic EDS subtype. No TEM findings were specifically associated with an EDS subtype. There was a higher percentage (71%) of abnormal biopsy findings in patients with a definitive diagnosis of a monogenetic EDS subtype and where a class 4/5 genetic variant was present.

Bone formation by heterodimers through non-viral gene delivery of BMP-2/6 and BMP-2/7.

Non-viral gene delivery is a safe technique to release sustained physiologic dosages of bone morphogenetic protein (BMP). Co-delivery of multiple BMPs can result in the formation of more potent BMP heterodimers. In this study, non-viral co-delivery of BMP-2/6 and BMP-2/7, as a mean to produce heterodimers, was assessed. Goat MSCs were non-virally transfected with plasmid DNA encoding BMP isoforms (pBMP) known to be relevant for osteogenesis: BMP-2, -6 or -7. As a result, BMP-2, -6 and -7 were produced and detectable for up to 14 d and their combined delivery (pBMP-2 with pBMP-6 or pBMP-7) was used to create BMP-2/6 and BM-2/7 heterodimers. Formation and secretion of the heterodimer proteins was validated by sandwich enzyme-linked immunosorbent assay (ELISA). Produced BMPs and heterodimers were biologically active, as confirmed by differentiation of reporter cells and MSCs. To assess bone formation, transfected MSCs were seeded on to ceramic scaffolds and implanted subcutaneously into nude mice. Bone formation was significantly enhanced in the pBMP-2/6 condition and a trend for more bone formation was observed in the pBMP-2/7 and pBMP-6 homodimer condition. No bone was found in the pBMP-2, pBMP-7 or control condition. In conclusion, simultaneous delivery of pBMP-2 with pBMP-6 or -7 resulted in the production of heterodimers that were beneficial for bone formation as compared to BMP homodimers. Combination of BMP sequences could reduce the need for high BMP protein dosages and might enhance prolonged availability of the growth factors.

Two patients with Ehlers-Danlos syndrome type VIII with unexpected hoarseness.

Ehlers-Danlos syndrome (EDS) encompasses a genetically and clinically heterogeneous group of connective tissue disorders, characterized by joint hypermobility, skin hyperextensibility and tissue fragility. It is a rare condition, and inheritance is either autosomal dominant or recessive. Previously grouped into 11 different subtypes, with increasing knowledge of the underlying molecular defects, it was reclassified in 1997 into 6 major groups, with type VIII excluded from this classification. Type VIII EDS is a very rare subtype, characterized by severe, early-onset periodontitis, skin fragility and abnormal scarring. Voice abnormalities have occasionally been described in other forms of the condition, and may be due to defects in the collagen of the vocal ligament. We report two cases of patients with EDS type VIII and hoarseness.

A combined vascular surgical and clinical genetics approach to diffuse aneurysmal disease.

We report two patients who presented with extensive aneurysmal disease, in association with minimal external physical signs. Patient 1 remained genetically undiagnosed despite multiple structural, biochemical and genetic investigations. He made a good recovery following surgery for popliteal and left axillary artery aneurysms. Patient 2 was diagnosed with vascular type Ehlers-Danlos syndrome, associated with a high degree of tissue and blood vessel fragility, and is being managed conservatively. Early multidisciplinary assessment of such patients facilitates accurate diagnosis and management.

Radioimmunotherapy in the pi-BCL1 B cell lymphoma model: efficacy depends on more than targeted irradiation alone.

We report the in vivo radioimmunotherapy (RIT) of a new variant of the BCL1 syngeneic mouse B-cell lymphoma model, pi-BCL1, using a panel of monoclonal antibodies (MoAb) reactive with B cell-associated antigens (CD19, CD22, CD40, MHC II, and idiotype). MoAb were radiolabeled with 131I or used in conjunction with external beam irradiation. When administered early in disease (day 4) 131I-anti-MHC II MoAb produced long term disease free survivors as a result of targeted irradiation alone; the equivalent unlabelled MoAb was non-therapeutic. In contrast, 131I-anti-CD40, and 131I-anti-Idiotype (Id) MoAb administered at day 4 despite targeting irradiation and having intrinsic therapeutic activity as unconjugated antibodies, protected mice for approximately 30 days. The 131I-anti-CD19 and anti-CD22 were therapeutically inactive. Treating later in the disease (day 14, after tumor inoculation) permitted study of the efficacy in the presence of an increased tumor load. An increased tumor burden brought about an expected reduction in therapeutic activity with 131I-anti-MHC II, but surprisingly, 131I anti-CD40 and 131I-anti-Id were able to produce prolonged disease free survival in most mice. This unexpected potency of 131I-anti-CD40 and 131I-anti-Id late in disease appeared to result from the direct cytotoxic action induced by these MoAb. Mechanisms by which these two MoAb operate, in producing long-term disease free survival in animals with advanced disease appear different. Our therapeutic results have important implications for the selection of reagents for RIT and demonstrate that successful treatment with such agents may involve more than simple targeting of irradiation.

High-throughput synthesis and direct screening for the discovery of novel hydrolytic metal complexes.

A 'combinatorial catalysis' strategy was utilized to both rapidly synthesized organometallic complexes and to screen them for catalytic activity in chemical reactions. The application of this strategy has yielded several metal-complexes that catalyze the hydrolysis of carboxylic acid esters efficiently.

Liquid-phase combinatorial synthesis: in search of small-molecule enzyme mimics.

The applications, advantages and recent advances in liquid-phase combinatorial chemistry using poly-(ethyleneglycol) as a soluble polymer support are reviewed. Our recent efforts towards the synthesis of peptide-based catalysts on polyethyleneglycol are reported. The screening of libraries of peptides for catalysis is discussed.