Pyrosequencing for detection of mutations in the connexin 26 (GJB2) and mitochondrial 12S RNA (MTRNR1) genes associated with hereditary hearing loss

Hereditary hearing loss (HHL) is one of the most common congenital disorders and is highly heterogeneous. Mutations in the connexin 26 (CX26) gene (GJB2) account for about 20% of all cases of childhood deafness, and approach 50% in documented recessive cases of non-syndromic hearing loss. In addition, a single mitochondrial DNA mutation, mt1555A>G, in the 12S rRNA gene (MTRNR1), is associated with familial cases of progressive deafness. Effective screening of populations for HHL necessitates rapid assessment of several of these potential mutation sites. Pyrosequencing links a DNA synthesis protocol for determining sequence to an enzyme cascade that generates light whenever pyrophosphate is released during primer strand elongation. We assessed the ability of Pyrosequencing to detect common mutations causing HHL. Detection of the most common CX26 mutations in individuals of Caucasian (35delG), Ashkenazi (167delT), and Asian (235delC, V37I) descent was confirmed by Pyrosequencing. A total of 41 different mutations in the CX26 gene and the mitochondrial mt1555A>G mutation were confirmed. Genotyping of up to six different adjacent mutations was achieved, including simultaneous detection of 35delG and 167delT. Accurate and reproducible results were achieved taking advantage of assay flexibility and experimental conditions easily optimized for a high degree of standardization and cost-effectiveness. The standardized sample preparation steps, including target amplification by PCR and preparation of single-stranded template combined with automated sequence reaction and automated genotype scoring, positions this approach as a potentially high throughput platform for SNP/mutation genotyping in a clinical laboratory setting. .     

Lymphocytotoxins in epileptic patients receiving phenytoin.

The present study was undertaken to determine the prevalence and nature of lymphocytotoxic antibodies in patients receiving phenytoin. Of forty-six sera examined, lymphocytotoxins and antinuclear antibodies were detected in fourteen and eleven samples respectively. Antibodies to double-stranded RNA (poly-A-poly-U) were found in six out of thirty-eight sera studied. A highly significant correlation was found between the presence of lymphocytotoxins and antinuclear antibodies (chi2 = 9.72; P less than 0.01). Lymphocytotoxins were found to be of the IgM class, to have activity against T and B cells, and to be dependent on the presence of Ca and Mg ions for activity. These findings may have pathogenetic importance in the genesis of the altered immune state of patients reveiving phenytoin.     

Blood pressure response to low level static contractions

Summary The present study re-examines the 15% MVC concept, i.e. the existence of a circulatory steady-state in low intensity static contractions below 15% of maximal voluntary contraction (MVC). Mean arterial blood pressure was studied during static endurance contractions of the elbow flexor and extensor muscles at forces corresponding to 10% and 40% MVC. Mean value for endurance time at 10% MVC was significantly longer for flexion [111.3 (SD 56.1) min] than for extension [18.1 (SD 7.5) min;n = 7]. At 40% MVC the difference in mean endurance time disappeared [2.3 (SD 0.7) min for elbow flexion and 2.3 (SD 0.7) min for elbow extension]. Mean arterial blood pressure exhibited a continuous and progressive increase during the 10% MVC contractions indicating that the 15% MVC concept would not appear to be valid. The terminal blood pressure value recorded at the point of exhaustion in the 10% MVC elbow extension experiment was identical to the peak pressure attained in the 40% MVC contraction. For the elbow flexors the terminal pressor response was slightly but significantly lower at 10% MVC [122.3 (SD 10.1) mmHg, 16.3 (SD 1.4) kPa] in comparison with 40% MVC [130.4 (SD 7.4) mmHg, 17.4 (SD 1.0) kPa]. When the circulation to the muscles was arrested just prior to the cessation of the contraction, blood pressure only partly recovered and remained elevated for as long as the occlusion persisted, indicating the level of pressure-raising muscle chemoreflexes. Based on blood pressure recordings obtained during the occlusion, it is suggested that the slight reduction in terminal pressor response seen in the 10% MVC elbow flexion experiment was due to a reduced chemoreflex drive characteristic of a slow twitch muscle group during prolonged low force contractions.     

Hypothermic Machine Preservation in Liver Transplantation Revisited: Concepts and Criteria in the New Millennium

To overcome the present shortage of liver donors by expansion of the existing donor pool and possibly lengthening of the storage time, hypothermic machine perfusion of the liver as a dynamic preservation method is revisited. The three most important aspects are defined to be the type of preservation solution, the characteristics of perfusion dynamics, and the oxygen supply. Reviewing hypothermic liver machine perfusion experiments, the University of Wisconsin machine preservation solution is the solution most used. It is also found that nothing conclusive can be said about the optimal perfusion characteristics, since either perfusion pressure or perfusion flow is reported. The best estimation is perfusion of the liver in a physiological manner, i.e. pulsatile arterial perfusion and continuous portal venous perfusion. The applied pressures could be chosen to be somewhat lower than physiological pressures to prevent possible endothelial cell damage. Oxygen supply is necessary to achieve optimal preservation of the liver. The minimal amount of partial oxygen pressure required is inversely related to the normalized flow. Incorporating these features in a system based on existing standard surgical and organ sharing procedures and which is able to work stand-alone for 24 h, weighing less than 23 kg, could successfully implement this technique into every day clinical practise.     

Near-haploid clones in a malignant fibrous histiocytoma

Near-haploid solid tumors are very rare. In a storiform-pleomorphic malignant fibrous histiocytoma (MFH) of bone, we found three cell populations: one with a near-haploid, a second with a near-diploid, and a third with a near-tetraploid chromosome number. The near-haploid cells had few structural rearrangements: i(12p) and t(13q21q) in one clone, and these two and an additional t(19;?)(p11;?) in another clone. One structurally normal copy of all chromosomes was also present, except that the only chromosome 13 was involved in the t(13q21q). There were also two near-diploid clones, one without the t(19;?) and one with a single copy of this derivative chromosome. This is the first MFH reported to have a near-haploid modal chromosome number, and also the first tumor with i(12p) among bone and soft tissue tumors.     

Trisomy 5 and loss of the Y chromosome as the sole cytogenetic anomalies in a cavernous hemangioma/angiosarcoma

Cytogenetic analysis of a cavernous hemangioma with transition to angiosarcoma revealed the mosaic karyotype 47, XY,+5/46, X,-Y,+5/45, X,-Y/46, XY. No cytogenetically analyzed hemangiomas or angiosarcomas have been reported before.     

Metal content of neutrophil granules is altered in chronic inflammation

The mass fraction of certain elements was measured in isolated granulocytes and isolated granulocyte granule fractions from patients with active inflammatory arthritides (N=6) and healthy controls (N=6). The patients had significantly increased amounts of Ca in the granulocytes, in the specific and light azurophil granules, but normal Ca amounts in the dense azurophil granules. Sr was below the detection limit in the granulocytes and granule fraction from controls, but it appeared in high concentrations in the granulocytes and all granule fractions from the patients. The patients had considerably increased granulocyte amounts of Mn but only slightly increased Mn concentrations in the specific granules. Mn was not detectable in azurophil granules from patients and controls. A prominent accumulation of Fe was seen in the granulocytes from the patients, together with an Fe accumulation in the specific granules. Fe was below the detection limit in azurophil granules from patients and controls. The patients had reduced granulocyte Zn and reduced amounts of Zn in the dense and light azurophil granules but normal Zn amounts in the specific granules. The results obtained indicate that (1) the granulocyte accumulation of Ca, Sr, and Fe observed during chronic inflammation is associated with corresponding granule accumulation of these metals; (2) the considerable Mn accumulation in granulocytes during inflammation is not localized in their granules; and (3) the granule subpopulations differ in their capacity to store certain metals.     

Monosomy and trisomy of 15q24→qter in a family with a translocation t(6;15)(P25;q24)

A child with multiple anomalies, including growth retardation, a left-sided diaphragmatic hernia with lung hypoplasia, and cerebral malformations is described. Cytogenetic investigation demonstrated a deletion of the distal part of one chromosome 15, del(15)(q24qter), an aberration not previously described. Family studies revealed that the mother had a balanced translocation, t(6;15)(p25;q24). Two of her subsequent pregnancies resulted in abortions after prenatal diagnosis: one fetus was trisomic for 15q24→qter, while the other had monosomy 15q24→qter and a left-sided diaphragmatic hernia similar to the first child.     

Antibodies to β2 microglobulin in the sera of patients with systemic lupus erythematosus

The present study reports the detection of antibodies to β₂ microglobulin in the sera of patients with systemic lupus erythematosus (SLE). Using a Farr-type ammonium sulphate precipitation assay, test sera were reacted with ¹²⁵Iβ₂ microglobulin, and immunoglobulins precipitated by 50% saturated ammonium sulphate. Increased β₂ microglobulin binding activity (normal values: mean±2 sd = 35.5 ±7.8) was detected in 18 of 42 SLE sera. Anti-HLA sera did not reveal increased binding activity, suggesting that the antibody in SLE serum was directed toward free β₂ microglobulin. Direct validation was done by reacting ¹²⁵Iβ₂ microglobulin with 4 SLE sera having increased ¹²⁵Iβ₂ microglobulin binding activity, and subjecting the reactants to sucrose density gradient ultracentrifugation. Two peaks were obtained, one corresponding to free β₂ microglobulin, and the other to 7S material complexed to β₂ microglobulin. Normal sera demonstrated only one peak corresponding to unbound β₂ microglobulin. Assays of β₂ microglobulin binding activity on protein fractions obtained by Sephadex G200 column chromatography also showed the presence of increased binding activity with 7S fractions. Using a double antibody assay, the 7S material reactive to β₂ microglobulin was demonstrated to be IgG. It was also shown that sera with abnormal β₂ microglobulin binding activity had higher titres of antinuclear antibody compared to those lacking such activity (t = 3.18; P<0.01), indicating the pathogenetic relationship of this antibody to increased disease activity. This antibody may be responsible for some of the abnormalities of cell-mediated function previously described in SLE patients.