The structural basis of alpha-tropomyosin linked (Asp230Asn) familial dilated cardiomyopathy.

Recently, linkage analysis of two large unrelated multigenerational families identified a novel dilated cardiomyopathy (DCM)-linked mutation in the gene coding for alpha-tropomyosin (TPM1) resulting in the substitution of an aspartic acid for an asparagine (at residue 230). To determine how a single amino acid mutation in α-tropomyosin (Tm) can lead to a highly penetrant DCM we generated a novel transgenic mouse model carrying the D230N mutation. The resultant mouse model strongly phenocopied the early onset of cardiomyopathic remodeling observed in patients as significant systolic dysfunction was observed by 2months of age. To determine the precise cellular mechanism(s) leading to the observed cardiac pathology we examined the effect of the mutation on Ca2+ handling in isolated myocytes and myofilament activation in vitro. D230N-Tm filaments exhibited a reduced Ca2+ sensitivity of sliding velocity. This decrease in sensitivity was coupled to increase in the peak amplitude of Ca2+ transients. While significant, and consistent with other DCMs, these measurements are comprised of complex inputs and did not provide sufficient experimental resolution. We then assessed the primary structural effects of D230N-Tm. Measurements of the thermal unfolding of D230N-Tm vs WT-Tm revealed an increase in stability primarily affecting the C-terminus of the Tm coiled-coil. We conclude that the D230N-Tm mutation induces a decrease in flexibility of the C-terminus via propagation through the helical structure of the protein, thus decreasing the flexibility of the Tm overlap and impairing its ability to regulate contraction. Understanding this unique structural mechanism could provide novel targets for eventual therapeutic interventions in patients with Tm-linked cardiomyopathies.

Roux-en-Y venting jejunostomy in pancreatic transplantation: a novel approach to monitor rejection and prevent anastomotic leak.

INTRODUCTION: Pancreatic transplantation (PTx) with portal venous delivery of insulin and enteric drainage of the exocrine secretion is more physiologic than bladder-systemic (BS) drainage. With portal-enteric (PE) PTx, the diagnosis of acute rejection (AR) requires a percutaneous biopsy. The roux-en-y (RNY) venting jejunostomy in patients with PEPTx offers a novel approach to monitor rejection and prevent anastomatic leaks. METHODS: From January 1996 to December 1998, we performed 17 simultaneous kidney/pancreas transplants (SKPTx). The initial 4 patients underwent BS drainage and the subsequent 13 patients underwent RNY venting jejunostomy with PE drainage. All patients were treated with quadruple therapy. There were 9 males, 14 patients were Caucasian with a mean age of 32 yr (range 30-54 yr), and a mean pre-transplantation duration of diabetes of 25 yr. Six patients underwent endoscopic donor duodenal biopsy through the jejunostomy to rule out clinically suspected AR. Gastrograffin was inserted into the jejunostomy to examine the integrity of anastamosis when indicated. In 9 out of 13 patients, the venting jejunostomy was taken down 9-12 months post-transplantation after allograft function was stable. RESULTS: Actual patient, kidney, and pancreas graft survival rates were 100, 100 and 94%, respectively, after a mean follow-up of 16 months. Renal allografts functioned immediately in 89% of patients. The mean length of hospital stay was 19 d. Four (23%) patients (2 with BS drainage and 2 with PE drainage) suffered an AR episode in the first month, and 4 (23%) patients had five AR from 3-36 months post-transplantation. Other complications were post-operative bleeding in 3 patients, wound infection in 2 patients and a proximal duodenal stump leak in 1 patient. In patients with clinical rejection, endoscopy through the venting jejunostomy showed inflamed, friable doudenal mucosa and doudenal biopsy findings were compatible with AR. CONCLUSION: These preliminary results suggest that RNY venting jejunostomy with PE drainage can be used safely to diagnose and monitor pancreas AR and to diagnose and prevent anastamotic leaks. This technique will be even more useful to visualize transplanted duodenal mucosa, collect pancreatic secretions (amylase) for analysis and perform endoscopic retrograde cholangiopancreatography if needed to obtain pancreatic biopsies.

The two-stage mutation model in retinal hemangioblastoma.

BACKGROUND: The two-stage mutation model involving successive inactivation of both alleles of a tumor suppressor gene was originally proposed by Knudson, who analyzed the age incidence curves for unilateral and bilateral retinoblastoma, and suggested that hereditary tumors arise by a single somatic event superimposed on a defective genetic background and sporadic tumors by a two-stage somatic process. In this study, the age-incidence curve of patients with retinal hemangioblastoma with and without associated von Hippel-Lindau disease were analyzed. METHODS: We reviewed the literature between 1964 and 1998 to find all reported cases of retinal hemangioblastoma and classified patients in a type A group (n = 223) when associated with von Hippel-Lindau disease and a type B group (n = 30) when not associated with von Hippel-Lindau disease. We analyzed and compared the age incidence of these two groups. RESULTS: There was a statistically significant difference between the mean age at diagnosis of retinal hemangioblastoma in the two groups, i.e., 48.4 +/- 16.6 years for type B patients and 24.9 +/- 12.0 years for type A patients (p < 0.0001). The age incidence curve for type A retinal hemangioblastoma fit a first-order equation (log S = 0.411-0.034t) with r = 0.97, indicating a single somatic mutation, whereas the age incidence curve for type B retinal hemangioblastoma fit a second-order equation (log S = 0.184-2.25 x 10(-4)t2) with r = 0.97, indicating two somatic mutations. CONCLUSIONS: Type B (sporadic) retinal hemangioblastoma may arise from two separate somatic mutations inactivating both alleles at the von Hippel-Lindau locus, whereas patients with von Hippel-Lindau disease (type A) inherit a defective allele and require only one additional somatic mutation.