Diagnostic yield of molecular autopsy in patients with sudden arrhythmic death syndrome using targeted exome sequencing.

AIMS: The targeted genetic screening of Sudden Arrhythmic Death Syndrome (SADS) probands in a molecular autopsy has a diagnostic yield of up to 35%. Exome sequencing has the potential to improve this yield. The primary aim of this study is to examine the feasibility and diagnostic utility of targeted exome screening in SADS victims, utilizing familial clinical screening whenever possible. METHODS AND RESULTS: To determine the feasibility and diagnostic yield of targeted exome sequencing deoxyribonucleic acid (DNA) was isolated from 59 SADS victims (mean age 25 years, range 1-51 years). Targeted exome sequencing of 135 genes associated with cardiomyopathies and ion channelopathies was performed on the Illumina HiSeq2000 platform. Non-synonymous, loss-of-function, and splice-site variants with a minor allele frequency <0.02% in the NHLBI exome sequencing project and an internal set of control exomes were prioritized for analysis followed by <0.5% frequency threshold secondary analysis. First-degree relatives were offered clinical screening for inherited cardiac conditions. Seven probands (12%) carried very rare (<0.02%) or novel non-sense candidate mutations and 10 probands (17%) had previously published rare (0.02-0.5%) candidate mutations-a total yield of 29%. Co-segregation fully confirmed two private SCN5A Na channel mutations. Variants of unknown significance were detected in a further 34% of probands. CONCLUSION: Molecular autopsy using targeted exome sequencing has a relatively low diagnostic yield of very rare potentially disease causing mutations. Candidate pathogenic variants with a higher frequency in control populations are relatively common and should be interpreted with caution.

Cholesterol crystal embolism leading to perforation of the gallbladder.

BACKGROUND: Cholesterol crystal embolism (CCE) from atherosclerotic arterial disease leading to perforation of the gallbladder is rare. We describe our experience of managing a patient with perforation of gallbladder caused by CCE. METHODS: A 64-year-old man was admitted to this hospital because of acute abdominal pain with clinical features suggestive of peritonitis. He was known to suffer from atherosclerotic peripheral arterial disease and had undergone aortobifemoral bypass 17 years ago. A CT scan showed a collection of peri-hepatic fluid. The gallbladder was normal in appearance but contained multiple calculi. At laparotomy, free bile was observed in the peritoneal cavity, leaking from a pin-hole size peroration of the fundus of the gallbladder. Hence cholecystectomy was performed. RESULTS: The patient made an uneventful recovery. Histological study of the gallbladder showed chronic cholecystitis and obliteration of the lumen of the mural arteries with cholesterol crystals within, indicating CCE. CONCLUSIONS: Although perforation of the gallbladder following CCE of its mural arteries is rare, the diagnosis should be considered in patients with abdominal pain and known atherosclerotic arterial disease. Management should include an early recognition of this condition, prompt institution of treatment, prevention of further insults by discontinuing or avoiding predisposing factors, and modification of cardiovascular risk factors.

Development of a reconstructed human skin model for angiogenesis.

We have previously shown that reconstructed human skin engineered from autologous keratinocytes, fibroblasts, and sterilized donor allodermis stimulates angiogenesis within 5-7 days when placed on well-vascularized wound beds in nude mice. When this reconstructed skin was used clinically in more demanding wound beds, some grafts were lost, possibly due to delayed vascularization. As this reconstructed skin lacks any endothelial cells, our aim in this study was to develop an angiogenic reconstructed skin model in which to explore strategies to improve angiogenesis both in vitro and in vivo. We report that culture of small-vessel human dermal microvascular endothelial cells (HuDMECs) was achieved using magnetic beads coated with an antibody to platelet cell adhesion molecule as a means of purifying the culture. Keratinocytes, fibroblasts, and HuDMECs could be cultured from the same skin biopsy. Initial studies culturing HuDMECs and other sources of endothelial cells with the tissue-engineered skin showed that these cells were capable of slowly entering the dermis under standard culture conditions in vitro. In conclusion, this provides us with a model in which to explore strategies for improving angiogenesis in vitro and also establishes the culture methodologies for the production of reconstructed skin containing autologous keratinocytes, fibroblasts, and endothelial cells.