Case Reports1. A Late Presentation of Loeys-Dietz Syndrome: Beware of TGFβ Receptor Mutations in Benign Joint Hypermobility

Background: Thoracic aortic aneurysms (TAA) and dissections are not uncommon causes of sudden death in young adults. Loeys-Dietz syndrome (LDS) is a rare, recently described, autosomal dominant, connective tissue disease characterized by aggressive arterial aneurysms, resulting from mutations in the transforming growth factor beta (TGFβ) receptor genes TGFBR1 and TGFBR2. Mean age at death is 26.1 years, most often due to aortic dissection. We report an unusually late presentation of LDS, diagnosed following elective surgery in a female with a long history of joint hypermobility. Methods: A 51-year-old Caucasian lady complained of chest pain and headache following a dural leak from spinal anaesthesia for an elective ankle arthroscopy. CT scan and echocardiography demonstrated a dilated aortic root and significant aortic regurgitation. MRA demonstrated aortic tortuosity, an infrarenal aortic aneurysm and aneurysms in the left renal and right internal mammary arteries. She underwent aortic root repair and aortic valve replacement. She had a background of long-standing joint pains secondary to hypermobility, easy bruising, unusual fracture susceptibility and mild bronchiectasis. She had one healthy child age 32, after which she suffered a uterine prolapse. Examination revealed mild Marfanoid features. Uvula, skin and ophthalmological examination was normal. Results: Fibrillin-1 testing for Marfan syndrome (MFS) was negative. Detection of a c.1270G > C (p.Gly424Arg) TGFBR2 mutation confirmed the diagnosis of LDS. Losartan was started for vascular protection. Conclusions: LDS is a severe inherited vasculopathy that usually presents in childhood. It is characterized by aortic root dilatation and ascending aneurysms. There is a higher risk of aortic dissection compared with MFS. Clinical features overlap with MFS and Ehlers Danlos syndrome Type IV, but differentiating dysmorphogenic features include ocular hypertelorism, bifid uvula and cleft palate. Echocardiography and MRA or CT scanning from head to pelvis is recommended to establish the extent of vascular involvement. Management involves early surgical intervention, including early valve-sparing aortic root replacement, genetic counselling and close monitoring in pregnancy. Despite being caused by loss of function mutations in either TGFβ receptor, paradoxical activation of TGFβ signalling is seen, suggesting that TGFβ antagonism may confer disease modifying effects similar to those observed in MFS. TGFβ antagonism can be achieved with angiotensin antagonists, such as Losartan, which is able to delay aortic aneurysm development in preclinical models and in patients with MFS. Our case emphasizes the importance of timely recognition of vasculopathy syndromes in patients with hypermobility and the need for early surgical intervention. It also highlights their heterogeneity and the potential for late presentation. Disclosures: The authors have declared no conflicts of interes

Role of polysaccharide and lipid in lipopolysaccharide induced prion protein conversion

<p>Conversion of native cellular prion protein (PrP^c) from an α-helical structure to a toxic and infectious β-sheet structure (PrP^Sc) is a critical step in the development of prion disease. There are some indications that the formation of PrP^Sc is preceded by a β-sheet rich PrP (PrP^β) form which is non-infectious, but is an intermediate in the formation of infectious PrP^Sc. Furthermore the presence of lipid cofactors is thought to be critical in the formation of both intermediate-PrP^β and lethal, infectious PrP^Sc. We previously discovered that the endotoxin, lipopolysaccharide (LPS), interacts with recombinant PrP^c and induces rapid conformational change to a β-sheet rich structure. This LPS induced PrP^β structure exhibits PrP^Sc-like features including proteinase K (PK) resistance and the capacity to form large oligomers and rod-like fibrils. LPS is a large, complex molecule with lipid, polysaccharide, 2-keto-3-deoxyoctonate (Kdo) and glucosamine components. To learn more about which LPS chemical constituents are critical for binding PrP^c and inducing β-sheet conversion we systematically investigated which chemical components of LPS either bind or induce PrP conversion to PrP^β. We analyzed this PrP conversion using resolution enhanced native acidic gel electrophoresis (RENAGE), tryptophan fluorescence, circular dichroism, electron microscopy and PK resistance. Our results indicate that a minimal version of LPS (called detoxified and partially de-acylated LPS or dLPS) containing a portion of the polysaccharide and a portion of the lipid component is sufficient for PrP conversion. Lipid components, alone, and saccharide components, alone, are insufficient for conversion.</p

Shaking Alone Induces De Novo Conversion of Recombinant Prion Proteins to β-Sheet Rich Oligomers and Fibrils

<div><p>The formation of β-sheet rich prion oligomers and fibrils from native prion protein (PrP) is thought to be a key step in the development of prion diseases. Many methods are available to convert recombinant prion protein into β-sheet rich fibrils using various chemical denaturants (urea, SDS, GdnHCl), high temperature, phospholipids, or mildly acidic conditions (pH 4). Many of these methods also require shaking or another form of agitation to complete the conversion process. We have identified that shaking alone causes the conversion of recombinant PrP to β-sheet rich oligomers and fibrils at near physiological pH (pH 5.5 to pH 6.2) and temperature. This conversion does not require any denaturant, detergent, or any other chemical cofactor. Interestingly, this conversion does not occur when the water-air interface is eliminated in the shaken sample. We have analyzed shaking-induced conversion using circular dichroism, resolution enhanced native acidic gel electrophoresis (RENAGE), electron microscopy, Fourier transform infrared spectroscopy, thioflavin T fluorescence and proteinase K resistance. Our results show that shaking causes the formation of β-sheet rich oligomers with a population distribution ranging from octamers to dodecamers and that further shaking causes a transition to β-sheet fibrils. In addition, we show that shaking-induced conversion occurs for a wide range of full-length and truncated constructs of mouse, hamster and cervid prion proteins. We propose that this method of conversion provides a robust, reproducible and easily accessible model for scrapie-like amyloid formation, allowing the generation of milligram quantities of physiologically stable β-sheet rich oligomers and fibrils. These results may also have interesting implications regarding our understanding of prion conversion and propagation both within the brain and via techniques such as protein misfolding cyclic amplification (PMCA) and quaking induced conversion (QuIC).</p></div

Secondary structure composition of shaking-induced fibrils as determined from deconvolution and curve fitting of the FTIR amide I band.

<p>Secondary structure composition of shaking-induced fibrils as determined from deconvolution and curve fitting of the FTIR amide I band.</p

RENAGE of shaking-converted prions under various conditions.

<p>A) ShPrP ^90–232 oligomers are formed by shaking in pH 5.5, 6.2 and 7.4 buffers, at 350 rpm and 37°C. B) RENAGE after shaking at 350 rpm for 1 day with full length recMoPrP ^23–231 (lane 1), truncated recMoPrP ^90–231 (lane 2) and C-terminal domain recMoPrP ^120–231 (lane 3) and after 2 days with recMoPrP ^23–231 (lane 4), truncated recMoPrP ^90–231 (lane 5) and C-terminal domain recMoPrP ^120–231 (lane 6). Samples in panel B were shaken at 350 rpm and 37°C in pH 6.2 buffer. C) Shaking a 0.6 mL solution of recShPrP^90–232 in a 0.6 mL centrifuge tube without any air or bubbles for two weeks (lane 1) as compared to the same sample of 0.4 mL in a 1.5 mL centrifuge tube (i.e. with air), shaken for one week (lane 2).</p

Fourier transform infrared spectroscopy shows that shaking-induces conversion to oligomers with increased β-sheet structure, dominated by turns and loops.

<p>A) FTIR of oligomers formed by shaking-induced conversion (at 250 rpm and 37°C) of recMoPrP ^23–231 (black line) is drastically different from monomeric recMoPrP^c^23–231 (grey line). The absorbance spectra are shown in solid lines and the corresponding 2^nd derivative spectra are shown in dashed lines. B) Spectral deconvolution and component analysis of the fibril FTIR spectrum (solid line) is fit with Gaussian peaks to a deconvoluted spectrum (dashed line).</p

Shaking-induced fibrils have Proteinase K resistance.

<p>SDS-PAGE of recMoPrP^c^23–231 (panel A) and fibrils (panel B) without (PK-) and with PK at 1∶50, 1∶200 and 1∶400 (PK:PrP, g:g) shows that shaking-induced fibrils have 12, 13, 14 and 17 kDa resistance bands.</p

RENAGE indicates that shaking recPrP^c generates oligomers.

<p>A PrP PICUP ladder (lane 1) is used to size the oligomers formed by shaking recMoPrP ^90–231 in pH 5.5 buffer at 350 rpm and 37°C for 1 day (lane 2). Shaking-induced oligomers are predominantly a distribution of 8-mers to 13-mers. In comparison oligomers formed in urea and salt exhibit a bimodal size distribution of 7-mers to 12-mers (lane 3). Longer periods of shaking recMoPrP ^90–231 (shaking at 350 rpm, 37°C for 2 days) will also generate a fibril band and bimodal distribution of 8-mers to 12-mers and larger oligomers (>16-mers) (lane 4).</p

Secondary structure composition of shaking-induced oligomers as determined from deconvolution and curve fitting of the FTIR amide I band.

<p>Secondary structure composition of shaking-induced oligomers as determined from deconvolution and curve fitting of the FTIR amide I band.</p

Electron microscopy confirms the formation of oligomers and fibrils seen in RENAGE.

<p>Negative stain EM of shaking-induced prion oligomers (panel A) and fibrils (panel B). The oligomers shown here were formed from shaking recMoPrP ^90–231 at 350 rpm at room temperature for 1 day. The fibril sample was formed by shaking recShPrP ^90–232 at 350 rpm at 37°C for 5 days. The corresponding RENAGE analysis of the same sample is shown alongside the micrograph. The indicated scale bar = 100 nm.</p