ADAMTS13 missense variants associated with defective activity and secretion of ADAMTS13 in a patient with non-cirrhotic portal hypertension.

BACKGROUND: Non-cirrhotic intrahepatic portal hypertension (NCIPH) is characterized by thrombotic microangiopathy of the portal venous system, low ADAMTS13 (a disintegrin-like and metalloproteinase with thrombospondin type 1 motifs-13), and high vWF (von Willebrand factor) levels. This study aimed to screen for ADAMTS13 mutations, focusing on the CUB domain, in these patients. METHODS: Prospectively recruited NCIPH patients and healthy volunteers underwent tests for plasma vWF-ADAMTS13 balance. Sanger sequencing of the CUB domain of ADAMTS13 was done in a subset of the NCIPH patients, and the detected mutation was screened for in all the study participants. Next-generation sequencing of clinically relevant exome and liver immunostaining for ADAMTS13 was done in patients with detected ADAMTS13 mutation. RESULTS: Plasma vWF-ADAMTS13 balance was significantly altered in 24 NCIPH patients (Child's class A:23, B:1) as compared to 22 controls. On initial sequencing of the CUB domain (17 cases and 3 controls), one NCIPH patient showed a rare missense variant (SNV) at position c.3829C >T resulting in p.R1277W (rs14045669). Subsequent RFLP analysis targeted to the R1277W variant did not detect this in any other NCIPH patient, nor in any of the 22 controls. The NCIPH patient with the R1277W variant had severe ADAMTS13 deficiency, consistently high vWF, other missense SNVs in ADAMTS13, vWF, and complement genes. Immunostaining of his liver biopsy revealed globules of ADAMTS13 within stellate cells. CONCLUSIONS: We report missense variants in ADAMTS13, vWF, and complement genes in a patient with NCIPH who had decreased secretion and activity of ADAMTS13 protein. Further studies are needed in NCIPH patients in this regard.

Dimethylformamide interferes with Coomassie dye staining of proteins on blue native gel electrophoresis.

Blue native gel electrophoresis (BN-PAGE) is used extensively for characterization of mitochondrial respiratory complexes and uses the binding of Coomassie brilliant blue G-250 to visualize proteins. Oxidative modification of sulfhydryl groups of such proteins can be evaluated by labeling with iodoacetamide conjugated to biotin (BIAM) and detected with streptavidin peroxidase on Western blots following BN-PAGE. However, dissolving BIAM in dimethylformamide, a recommended solvent, reduces Coomassie blue G staining to proteins during BN-PAGE. This interference is prevented by dissolving BIAM in dimethyl sulfoxide. Precautions in the use of the dye for protein staining subsequent to BIAM labeling are discussed.

Monocrotophos toxicity and bioenergetics of muscle weakness in the rat.

Acute organophosphate pesticide poisoning is a common medical emergency with high fatality in agricultural communities of Asia. Organophosphate compounds inhibit acetylcholinesterase and prolonged neuromuscular weakness is a major cause of morbidity and mortality of poisoning. Organophosphate pesticide induced muscle weakness may not only arise from inhibition of acetylcholinesterase but also from non-cholinergic pathomechanisms, particularly mitochondrial dysfunction, affecting the production of sufficient ATP for muscle function. This study examined whether muscle weakness in rats subject to monocrotophos toxicity (0.8LD50) was caused by inhibition of ATP synthesis, by oxidative phosphorylation and glycolysis, in addition to inhibition of muscle acetylcholinesterase. Severe muscle weakness in rats following monocrotophos administration was associated with inhibition of muscle acetylcholinesterase (30-60%) but not with reduced ATP production. The rats rapidly recovered muscle strength with no treatment. The ability of rats to spontaneously reactivate dimethoxy phosphorylated acetylcholinesterase and efficiently detoxify organophosphates may prevent severe inhibition of muscle acetylcholinesterase following acute severe monocrotophos poisoning. This may protect rodents against the development of prolonged muscle weakness induced by organophosphates.

Anaesthetic management of total craniopagus twins for magnetic resonance imaging and cerebral angiography.

We describe the anaesthetic management of 4-yr-old total craniopagus twins for radiological investigations. There are some unique anaesthetic problems associated with this condition. These include cross-circulation between the twins that results in induction of both the twins after the administration of i.v. induction agent to one twin and difficulty in mask ventilating both the twins simultaneously due to the angle between the heads; different arterial pressures in the two children complicate pharmacological management and underline the importance of physiological measures to control arterial pressure. Adequate preparation and teamwork is the keystone to the management of these patients.

Unusual presentation of a retroperitoneal lymphangioma.

A 3 year old child presented with hemorrhagic ascites of short duration. Based on clinical manifestation and investigations a differential diagnosis of peritoneal tuberculosis, pancreatic ascites or malignant ascites was considered. Laparotomy revealed a huge retroperitoneal cystic lymphangioma confirmed by histopathology. The unusual presentation of retroperitoneal tumours as pseudoascites is discussed.