Mutation scanning the GJB1 gene with high-resolution melting analysis: implications for mutation scanning of genes for Charcot-Marie-Tooth disease

BACKGROUND: X-linked Charcot-Marie-Tooth type 1 disease has been associated with 280 mutations in the GJB1 [gap junction protein, beta 1, 32 kDa (connexin 32, Charcot-Marie-Tooth neuropathy, X-linked)] gene. High-resolution melting analysis with an automated instrument can be used to scan DNA for alterations, but its use in X-linked disorders has not been described. METHODS: A 96-well LightScanner for high resolution melting analysis was used to scan amplicons of the GJB1 gene. All mutations reported in this study had been confirmed previously by sequence analysis. DNA samples were amplified with the double-stranded DNA-binding dye LC Green Plus. Melting curves were analyzed as fluorescence difference plots. The shift and curve shapes of melting profiles were used to distinguish controls from patient samples. RESULTS: The method detected each of the 23 mutations used in this study. Eighteen known mutations provided validation of the high-resolution melting method and a further 5 mutations were identified in a blind study. Altered fluorescence difference curves for all the mutations were easily distinguished from the wild-type melting profile. CONCLUSION: High-resolution melting analysis is a simple, sensitive, and cost-efficient alternative method to scan for gene mutations in the GJB1 gene. The technology has the potential to reduce sequencing burden and would be suitable for mutation screening of exons of large multiexon genes that have been discovered to be associated with Charcot Marie Tooth neuropathy.     

Can we predict the success of a parasite to colonise an invasive host?

Parasitology Research - To understand whether a parasite can exploit a novel invasive host species, we measured reproductive performance (number of eggs per female per day, egg size, development...     

Chronic Hyperglycemia Reduces Surface Active Material Flux in Tracheal Fluid of Fetal Lambs

I tested the hypothesis that chronic hyperglycemia alters fetal lung maturation by continuous infusion of glucose (14±2 mg/kg per min, mean±SE) from 112 up to 145 d gestation into six chronically catheterized fetal lambs from which tracheal fluid could be collected. Serum glucose levels (32±2 mg/dl) and serum insulin levels (38±4 μU/ml) in these glucose-treated fetuses were significantly higher than serum glucose levels (18±2 mg/dl, P < 0.001) and serum insulin levels (12±3 μU/ml, P < 0.001) in six chronically catheterized control fetuses of the same gestational ages. Glucose infusion to the fetuses did not alter maternal serum glucose (60±3 mg/dl) or serum insulin levels (35±5 μU/ml). Arterial blood gases (pH 7.34±0.01, Po₂ 24.3±0.5 mmHg, Pco₂ 41.5±0.9 mmHg), oxygen saturation (73±2%), hematocrit (31±1%), and tracheal fluid flow (2.4±0.1 ml/g per h) in the glucose-treated fetuses were not significantly different from controls. Among the control fetuses, surface active material (SAM) began to appear in tracheal fluid at 123 d gestation and was present in all six fetuses by 129 d gestation, whereas SAM did not appear at all in tracheal fluid of four of the glucose-treated fetuses, and appeared in two at low levels after 142 d gestation. SAM flux in the glucose-treated fetuses (<1 μg/g per h) was statistically lower than SAM flux in the control fetuses (60±9 μg/kg per h, P < 0.001). Between 130 and 140 d gestation, tracheal fluid phospholipid content rose fourfold, mixed lecithin content rose ninefold, disaturated phosphatidylcholine content rose fourfold in the control fetuses, whereas little or no increase in these measurements occurred in the glucose-treated fetuses (all differences significant). I conclude that chronic hyperglycemia with secondary hyperinsulinemia reduces SAM flux in tracheal fluid of fetal lambs. The reduction in SAM flux is attributed to low surface active phospholipid content of the SAM. A similar mechanism may operate in utero to cause respiratory distress in infants of diabetic mothers whose maternal glucose homeostasis is poorly controlled.     

Pulse wave analysis to assess vascular compliance changes in stable renal transplant recipients

BACKGROUND: Calcineurin inhibitor drugs (cyclosporine and tacrolimus) given to renal transplant recipients to prevent rejection are associated with an increased incidence of hypertension. Reduced arterial compliance, which is a consequence of hypertension, is associated with an increased risk of cardiovascular disease and can be measured noninvasively using pulse wave analysis technology. The purpose of the study was to determine whether calcineurin inhibitor drugs have any effect on arterial compliance. METHODS: A total of 36 stable renal transplant recipients were evaluated using pulse wave analysis to determine large and small vessel compliance. Of the patients, 18 were receiving cyclosporine and 18 tacrolimus. Patients were matched for age and sex. RESULTS: No significant differences in systolic blood pressure, diastolic blood pressure, heart rate, or small vessel compliance were observed. There was a significant decrease in large vessel compliance in patients receiving tacrolimus versus those receiving cyclosporine, respectively (13.5 +/- 4.0 mL/mm Hg x 10 v 9.9 +/- 3.3 mL/mm Hg x 10; P =.005). CONCLUSIONS: Differences in large vessel compliance in renal transplant subjects may depend on the choice of calcineurin inhibitor. Specifically, decreased large vessel compliance in tacrolimus-treated subjects may be associated with an increased cardiovascular risk. This may be due to a difference in vascular collagen accumulation or to elastin loss in large elastic arteries.     

Treatment of dorsal perilunate dislocations and fracture–dislocations using a standardized protocol

Background

The aims of this study were to evaluate the associated injuries occurring with acute perilunate instability and to assess the clinical and radiographic outcomes of perilunate dislocations and fracture–dislocations treated with a combined dorsal and volar approach.

Methods

A total of 45 patients (46 wrist injuries) with perilunate dislocations and fracture–dislocations were prospectively evaluated. The size of the mid-carpal ligament tear, the location of the scapholunate ligament tear, and the presence of osteochondral fragments and of the dorsal radiocarpal ligament avulsions were recorded at injury. Final clinical and radiographic outcomes were evaluated in 25 cases (25 wrists) with a minimum of 6 months of follow-up.

Results

Intraoperative examination of the 46 cases with operative treatment showed the volar carpal ligament tear to be present 100 % of the time and to be an average length of 3.4 cm. Complete avulsion of the dorsal extrinsic radiocarpal ligaments was found in 65.2 % of cases. The scapholunate ligament was torn in 35 cases. Osteochondral fragments were found either volarly or dorsally in 74 % of the cases. The average flexion–extension arc was 82°, forearm rotation was 155°, and grip strength averaged 59 % of the uninjured hand. The average final scapholunate angle was 55° and the scapholunate gap was 2.2 mm.

Conclusion

Treatment of perilunate fracture–dislocations with a combined volar and dorsal approach results in reasonable and functional clinical results. The incidence of associated injuries with these carpal dislocations is high. Although the perilunate fracture–dislocations have a slightly better radiologic alignment than the dislocation group, the clinical outcome is similar.     

Systemic arterial compliance following ultra-marathon

There is a growing interest in training for and competing in race distances that exceed the marathon; however, little is known regarding the vascular effects of participation in such prolonged events, which last multiple consecutive hours. There exists some evidence that cardiovascular function may be impaired following extreme prolonged exercise, but at present, only cardiac function has been specifically examined following exposure to this nature of exercise. The primary purpose of this study was to characterize the acute effects of participation in an ultra-marathon on resting systemic arterial compliance. Arterial compliance and various resting cardiovascular indices were collected at rest from 26 healthy ultra-marathon competitors using applanation tonometry (HDI CR-2000) before and after participation in a mountain trail running foot race ranging from 120-195 km which required between 20-40 continuous hours (31.2±6.8 h) to complete. There was no significant change in small artery compliance from baseline to post race follow-up (8.5±3.4-7.7±8.2 mL/mmHgx100, p=0.65), but large artery compliance decreased from 16.1±4.4 to 13.5±3.8 mL/mmHgx10 (p=0.003). Participation in extreme endurance exercise of prolonged duration was associated with acute reductions in large artery compliance, but the time course of this effect remains to be elucidated.