Molecular basis of altered red blood cell membrane properties in Southeast Asian ovalocytosis: role of the mutant band 3 protein in band 3 oligomerization and retention by the membrane skeleton

Southeast Asian ovalocytosis (SAO) is an asymptomatic trait characterized by rigid, poorly deformable red cells that resist invasion by several strains of malaria parasites. The underlying molecular genetic defect involves simple heterozygous state for a mutant band 3 protein, which contains a deletion of amino acids 400 through 408, linked with a Lys 56-to-Glu substitution (band 3-Memphis polymorphism). To elucidate the contribution of the mutant SAO band 3 protein to increased SAO red blood cell (RBC) rigidity, we examined the participation of the mutant SAO band 3 protein in increased band 3 attachment to the skeleton and band 3 oligomerization. We found first that SAO RBC skeletons retained more band 3 than normal cells and that this increased retention preferentially involved the mutant SAO band 3 protein. Second, SAO RBCs contained a higher percentage of band 3 oligomer-ankyrin complexes than normal cells, and these oligomers were preferentially enriched by the mutant SAO protein. At the ultrastructural level, the increased oligomer formation of SAO RBCs was reflected by stacking of band 3-containing intramembrane particles (IMP) into longitudinal strands. The IMP stacking was not reversed by treating SAO RBCs in alkaline pH (pH 11), which is known to weaken ankyrin-band 3 interactions, or by removing the cytoplasmic domain of band 3 from SAO membranes with trypsin. Finally, we found that band 3 protein in intact SAO RBCs exhibited a markedly decreased rotational mobility, presumably reflecting the increased oligomerization and the membrane skeletal association of the SAO band 3 protein. We propose that the mutant SAO band 3 has an increased propensity to form oligomers, which appear as longitudinal strands of IMP and exhibit increased association with membrane skeleton. This band 3 oligomerization underlies the increase in membrane rigidity by precluding membrane skeletal extension, which is necessary for membrane deformation.     

Detection of genomic deletions of PKP2 in arrhythmogenic right ventricular cardiomyopathy

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited myocardial disease that predominantly affects the right ventricle and is associated with ventricular arrhythmias that may lead to sudden cardiac death. Mutations within at least seven separate genes have been identified to cause ARVC, however a genetic culprit remains elusive in approximately 50% of cases. Although negative genetic testing may be secondary to pathogenic mutations within undiscovered genes, an alternative explanation may be the presence of large deletions or duplications involving known genes. These large copy number variants may not be detected with standard clinical genetic testing which is presently limited to direct DNA sequencing. We describe two cases of ARVC possessing large deletions involving plakophilin‐2 (PKP2) identified with microarray analysis and/or multiplex ligation‐dependent probe amplification (MLPA) that would have been classified as genotype negative with standard clinical genetic testing. A deletion of the entire coding region of PKP2 excluding exon 1 was identified in patient 1 and his son. In patient 2, MLPA analysis of PKP2 revealed deletion of the entire gene with subsequent microarray analysis demonstrating a de novo 7.9 Mb deletion of chromosome 12p12.1p11.1. These findings support screening for large copy number variants in clinically suspected ARVC cases without clear disease causing mutations following initial sequencing analysis.     

Radiotherapy for benign disease; assessing the risk of radiation-induced cancer following exposure to intermediate dose radiation

Most radiotherapy (RT) involves the use of high doses (>50 Gy) to treat malignant disease. However, low to intermediate doses (approximately 3–50 Gy) can provide effective control of a number of benign conditions, ranging from inflammatory/proliferative disorders (e.g. Dupuytren''s disease, heterotopic ossification, keloid scarring, pigmented villonodular synovitis) to benign tumours (e.g. glomus tumours or juvenile nasopharyngeal angiofibromas). Current use in UK RT departments is very variable. This review identifies those benign diseases for which RT provides good control of symptoms with, for the most part, minimal side effects. However, exposure to radiation has the potential to cause a radiation-induced cancer (RIC) many years after treatment. The evidence for the magnitude of this risk comes from many disparate sources and is constrained by the small number of long-term studies in relevant clinical cohorts. This review considers the types of evidence available, i.e. theoretical models, phantom studies, epidemiological studies, long-term follow-up of cancer patients and those treated for benign disease, although many of the latter data pertain to treatments that are no longer used. Informative studies are summarized and considered in relation to the potential for development of a RIC in a range of key tissues (skin, brain etc.). Overall, the evidence suggests that the risks of cancer following RT for benign disease for currently advised protocols are small, especially in older patients. However, the balance of risk vs benefit needs to be considered in younger adults and especially if RT is being considered in adolescents or children.     

Long‐term effectiveness of recommended boosted protease inhibitor‐based antiretroviral therapy in Europe

Objectives

The aim of the study was to evaluate the long‐term response to antiretroviral treatment (ART) based on atazanavir/ritonavir (ATZ/r)‐, darunavir/ritonavir (DRV/r)‐, and lopinavir/ritonavir (LPV/r)‐containing regimens.

Methods

Data were analysed for 5678 EuroSIDA‐enrolled patients starting a DRV/r‐, ATZ/r‐ or LPV/r‐containing regimen between 1 January 2000 and 30 June 2013. Separate analyses were performed for the following subgroups of patients: (1) ART‐naïve subjects (8%) at ritonavir‐boosted protease inhibitor (PI/r) initiation; (2) ART‐experienced individuals (44%) initiating the new PI/r with a viral load (VL) ≤500 HIV‐1 RNA copies/mL; and (3) ART‐experienced patients (48%) initiating the new PI/r with a VL >500 copies/mL. Virological failure (VF) was defined as two consecutive VL measurements >200 copies/mL ≥24 weeks after PI/r initiation. Kaplan–Meier and multivariable Cox models were used to compare risks of failure by PI/r‐based regimen. The main analysis was performed with intention‐to‐treat (ITT) ignoring treatment switches.

Results

The time to VF favoured DRV/r over ATZ/r, and both were superior to LPV/r (log‐rank test; P < 0.02) in all analyses. Nevertheless, the risk of VF in ART‐naïve patients was similar regardless of the PI/r initiated after controlling for potential confounders. The risk of VF in both treatment‐experienced groups was lower for DRV/r than for ATZ/r, which, in turn, was lower than for LPV/r‐based ART.

Conclusions

Although confounding by indication and calendar year cannot be completely ruled out, in ART‐experienced subjects the long‐term effectiveness of DRV/r‐containing regimens appears to be greater than that of ATZ/r and LPV/r.