Glenda Durrheim

Phenotypic Variability and Unusual Clinical Severity of Congenital Long-QT Syndrome in a Founder Population

Background— In the congenital long-QT syndrome (LQTS), there can be a marked phenotypic heterogeneity. Founder effects, by which many individuals share a mutation identical by descent, represent a powerful tool to further understand the underlying mechanisms and to predict the natural history of mutation-associated effects. We are investigating one such founder effect, originating in South Africa in approximately ad 1700 and segregating the same KCNQ1 mutation (A341V). Methods and Results— The study population involved 320 subjects, 166 mutation carriers (MCs) and 154 noncarriers. When not taking β-blocker therapy, MCs had a wide range of QTc values (406 to 676 ms), and 12% of individuals had a normal QTc (≤440 ms). A QTc >500 ms was associated with increased risk for cardiac events (OR=4.22; 95% CI, 1.12 to 15.80; P=0.033). We also found that MCs with a heart rate <73 bpm were at significantly lower risk (OR=0.23; 95% CI, 0.06 to 0.86; P=0.035). This study also unexpectedly determined that KCNQ1-A341V is associated with greater risk than that reported for large databases of LQT1 patients: A341V MCs are more symptomatic by age 40 years (79% versus 30%) and become symptomatic earlier (7±4 versus 13±9 years, both P<0.001). Accordingly, functional studies of KCNQ1-A341V in CHO cells stably expressing IKs were conducted and identified a dominant negative effect of the mutation on wild-type channels. Conclusions— KCNQ1-A341V is a mutation associated with an unusually severe phenotype, most likely caused by the dominant negative effect of the mutation. The availability of an extended kindred with a common mutation allowed us to identify heart rate, an autonomic marker, as a novel risk factor.

Thy-1 associated PP85-90 is a potential docking site for SH2 domain-containing signal transduction molecules.

Thy‐1, a glycosylphosphatidylinositol (GPI)‐anchored glycoprotein expressed at high levels on thymocytes, has been implicated in positive and negative signal transduction. We show that Thy‐1 associates with a protein of 85–90kDa, which is prominently phosphorylated in vitro as well as in vivo following the stimulation of thymocytes with pervanadate. pp85–90 is not identical to known proteins that are phosphorylated following T cell activation. The SH2 domains of fyn, csk, phosphatidylinositol 3′‐kinase, rasGAP, vav and lck bind to pp85–90 with varying affinities. The SH2 domains of ZAP70, SHP‐1 and PLCγ1 and the SH3 domains of lck, vav and HS1 did not bind to pp85–90. The molecular weight, iso‐electric point, efficient phosphorylation by fyn and lck and preferential binding to the SH2 domain of fyn compared to that of lck indicate that Thy‐1‐associated pp85–90 may be identical to a recently cloned, fyn‐associated transmembrane adaptor protein, PAG‐85.

Isolation of myosin messenger ribonucleoprotein particles which contain a protein fraction affecting myosin synthesis

Abstract Myosin mRNP particles were purified using metrizamide buoyant density gradient centrifugation. This mRNP particle contains only the 26S myosin mRNA and, therefore, is a purified mRNP with regard to mRNA composition. A protein fraction, eluting from DEAE-cellulose between 0.15M KC l and 0.28M KC l , was iso=lated from the purified myosin mRNP particle. A significant stimulation of myosin heavy chain synthesis is observed when the isolated fraction is added to an unfractionated wheat germ cell-free system containing myosin mRNA. This protein fraction was found to have no effect on the translation of TMV RNA. In addition, this protein fraction does not bind TMV RNA, but is associated with myosin mRNA during translation.

Long QT syndrome in South Africa: the results of comprehensive genetic screening.

Abstract Congenital long QT syndrome (cLQTS) is a genetic disorder predisposing to ventricular arrhythmia, syncope and sudden death. Over 700 different cLQTS-causing mutations in 13 genes are known. The genetic spectrum of LQTS in 44 South African cLQTS patients (23 known to carry the South African founder mutation p.A341V in KCNQ1) was established by screening for mutations in the coding regions of KCNQ1, KCNH2, KCNE1, KCNE2 and SCN5A, the most frequently implicated cLQTS-causing genes (five-gene screening). Fourteen disease-causing mutations were identified, eight (including the founder mutation) in KCNQ1, five in KCNH2 and one in KCNE1. Two mutations were novel. Two double heterozygotes were found among the 23 families (8.5%) carrying the founder mutation. In conclusion, cLQTS in South Africa reflects both a strong founder effect and a genetic spectrum similar to that seen in other populations. Consequently, five-gene screening should be offered as a standard screening option, as is the case internationally. This will disclose compound and double heterozygotes. Fivegene screening will most likely be even more informative in other South African sub-populations with a greater genetic diversity.

Structural alterations in chromatin during myogenesis in the chicken

SummaryDifferentiation of mononucleated myoblasts to multinucleated myotubes is accompanied by hypertrophy achieved by co-ordinated synthesis of muscle proteins. This process may be achieved by co-ordinated synthesis and translation of new mRNA or gradual accumulation of constitutively synthesized mRNA, followed by co-ordinated translational activation. If the former process occurs, many structural alterations should occur in chromatin, whereas in the latter scenario, no chromatin changes will be necessary. The results of our investigation into chromatin structure of myoblast and myotube nuclei show that according to techniques used, viz. chromatin solubilization by nucleases, thermal denaturation, in vitro transcription, nucleosome sizing, there are major structural changes in chromatin during muscle cell differentiation. Since these alterations were detectable at a fairly gross level, many genes must be affected which could account for the increase in RNA and proteins observed in myotubes. This evidence argus in favour of new mRNA synthesis for rapid translation, rather than a gradual accumulation of mRNA followed by co-ordinated translation.

POSITIVE AND NEGATIVE MODULATION OF H-RAS TRANSFORMING POTENTIAL BY MUTATIONS OF PHENYLALANINE-28

Conserved amino-acids of H-ras from residues 25 to 34 were mutated in human H-ras cDNA with a pre-existing valine-12 activating mutation ([V12]p21), and built into SV40-driven expression vectors. The influence of the introduced mutations was initially screened by transfection of Rat-1 cells to score foci of transformed cells. Nonconservative mutations of amino-acids 25 (tryptophan for glutamine), 27 (asparagine for histidine) and 34 (alanine for proline) did not abrogate the transforming potential of [V12]p21. The conservative mutation of phenylalanine-28 to tryptophan ([V12W28]p21) was also still transforming. Significantly, in the absence of the valine-12 activating mutation, tryptophan-28-ras ([W28]p21) was weakly transforming while, in contrast, [V12D28]p21 was unable to transform Rat-1 cells and retarded cell growth. Analysis of the binding and dissociation of GTP and GDP to normal and mutated p21 expressed in Escherichia coli showed that [V12D28]p21 and [D28]p21 do not bind GTP. The dissociation rate of both GTP and GDP bound to [W28]p21 is increased, suggesting a mechanism for its transforming potential in Rat-1 cells. These studies illustrate the importance of phenylalanine-28 in guanine nucleotide binding by p21h-ras. The mutations described could be valuable tools in investigations of cellular signal transduction involving small GTP-binding proteins.

The translation of chick skeletal muscle poly (A)-containing mRNA in primary heart muscle cells in culture.

Abstract Chick skeletal muscle mRNA (40 μg/0.8 ml) in phosphate-buffered saline was added to 60 mm petri dishes containing a monolayer of primary heart muscle cells. After 30 minutes absorption the cultures were supplemented with complete medium and the incubation continued in a humidified CO2-incubator. Sucrose density gradient analysis of the absorbed RNA showed no degradation. This skeletal muscle mRNA was translated in primary heart muscle cells in culture into functional proteins as indicated by the linear increase in the accumulation of acetylcholine receptors as well as a similar increase in creatine kinase activity. In addition, the synthesis of the three unique light chains of skeletal muscle myosin (SLC1, SLC2, SLC3) in these primary heart muscle cells was also demonstrable.