Genotype-phenotype correlation and molecular heterogeneity in pyruvate kinase deficiency

Pyruvate kinase (PK) deficiency is a rare recessive congenital hemolytic anemia caused by mutations in the PKLR gene. This study reports the molecular features of 257 patients enrolled in the PKD Natural History Study. Of the 127 different pathogenic variants detected, 84 were missense and 43 non-missense, including 20 stop-gain, 11 affecting splicing, five large deletions, four in-frame indels, and three promoter variants. Within the 177 unrelated patients, 35 were homozygous and 142 compound heterozygous (77 for two missense, 48 for one missense and one non-missense, and 17 for two non-missense variants); the two most frequent mutations were p.R510Q in 23% and p.R486W in 9% of mutated alleles. Fifty-five (21%) patients were found to have at least one previously unreported variant with 45 newly described mutations. Patients with two non-missense mutations had lower hemoglobin levels, higher numbers of lifetime transfusions, and higher rates of complications including iron overload, extramedullary hematopoiesis, and pulmonary hypertension. Rare severe complications, including lower extremity ulcerations and hepatic failure, were seen more frequently in patients with non-missense mutations or with missense mutations characterized by severe protein instability. The PKLR genotype did not correlate with the frequency of complications in utero or in the newborn period. With ICCs ranging from 0.4 to 0.61, about the same degree of clinical similarity exists within siblings as it does between siblings, in terms of hemoglobin, total bilirubin, splenectomy status, and cholecystectomy status. Pregnancy outcomes were similar across genotypes in PK deficient women. This report confirms the wide genetic heterogeneity of PK deficiency.     

A Randomized Controlled Trial of the Clinical Utility of Genotypic Resistance Testing in Patients with Limited Prior Exposure to Antiretroviral Drugs

Abstract

Purpose: To assess the clinical utility of genotypic resistance testing among HIV-1-infected patients with limited prior exposure to antiretroviral drugs. Method: Patients experiencing virological failure were randomly allocated to either centralized genotypic resistance testing or to no testing and were followed for a minimum of 1 year. Results: 55 patients were recruited from 14 centers in the United Kingdom. There were no demonstrable differences between the groups in terms of virological or immunological response. For patients allocated to resistance testing, there was an increased tendency to recycle previously used drugs. Conclusion: The study did not demonstrate a benefit of genotypic resistance testing in this population, although statistical power was low. However, testing did alter prescribing behavior, and clinical effects may become manifest in the longer term.     

Quality of life in children and adolescents 1-year after cure of Cushing syndrome: a prospective study

Objective  Cushing syndrome (CS) in children is associated with symptoms that may impair health related quality of life (HRQL). There are no prospective reports of HRQL in children with CS.
Methods  Prospective study of 40 children (mean age 13 ± 3·2 years) with CS evaluated prior to and 1-year post-treatment. The Child Health Questionnaire (CHQ) was used to assess HRQL; Wechsler Intelligence Scale for Children (WASI) was used to assess cognitive function, and patient-reported symptoms were assessed with a CS symptom checklist.
Results  Active CS was associated with low physical and psychosocial summary scores compared to US population data ( P <  0·001). Despite improvement from pre- to 1-year postcure, residual impairment remained in physical summary and function, and role-physical, global health and emotional impact (parent) scores. Incomplete recovery of adrenal function at 1-year post-treatment was associated with impaired scores. WASI IQ scores declined and a correlation was noted between age at first evaluation and IQ score changes. Most self-reported CS symptoms showed improvement, but forgetfulness, unclear thinking and decreased attention span did not improve after cure of CS.
Conclusion  CS in children and adolescents is associated with impaired HRQL, with residual impairment 1 year after cure. Our results also suggest that younger children are more likely to experience negative changes in cognitive function. HRQL is an important outcome measure in children and adolescents with CS and identification of factors that contribute to HRQL may help to diminish the physical and psychological burden of disease in this population of patients.     

Dual‐Dye Optical Mapping after Myocardial Infarction: Does the Site of Ventricular Stimulation Alter the Properties of Electrical Propagation?

Introduction: Myocardial infarction (MI) disrupts electrical conduction in affected ventricular areas. We investigated the effect of MI on the regional voltage and calcium (Ca) signals and their propagation properties, with special attention to the effect of the site of ventricular pacing on these properties.
Methods: New Zealand White rabbits were divided into four study groups: sham-operated (C, n = 6), MI with no pacing (MI, n = 7), MI with right ventricular pacing (MI + RV, n = 6), and MI with BIV pacing (MI + BIV, n = 7). At 4 weeks, hearts were excised, perfused, and optically mapped. As previously shown, systolic and diastolic dilation of the LV were prevented by BIV pacing, as was the reduction in LV fractional shortening.
Results: Four weeks after MI, optical mapping revealed markedly reduced action potential amplitudes and conduction velocities (CV) in MI zones, and these increased gradually in the border zone and normal myocardial areas. Also, Ca transients were absent in the infarcted areas and increased gradually 3–5 mm from the border of the normal zone. Neither BIV nor RV pacing affected these findings in any of the MI, border, or normal zones.
Conclusions: MI has profound effects on the regional electrical and Ca signals and on their propagation properties in this rabbit model. The absence of differences in these parameters by study group suggests that altering the properties of myocardial electrical conduction and Ca signaling are unlikely mechanisms by which BIV pacing confers its benefits. Further studies into the regional, cellular, and molecular benefits of BIV pacing are therefore warranted.     

Low Dopamine D2/D3 Receptor Availability is Associated with Steep Discounting of Delayed Rewards in Methamphetamine Dependence

Background:

Individuals with substance use disorders typically exhibit a predilection toward instant gratification with apparent disregard for the future consequences of their actions. Indirect evidence suggests that low dopamine D₂-type receptor availability in the striatum contributes to the propensity of these individuals to sacrifice long-term goals for short-term gain; however, this possibility has not been tested directly. We investigated whether striatal D₂/D₃ receptor availability is negatively correlated with the preference for smaller, more immediate rewards over larger, delayed alternatives among research participants who met DSM-IV criteria for methamphetamine (MA) dependence.

Methods:

Fifty-four adults (n = 27 each: MA-dependent, non-user controls) completed the Kirby Monetary Choice Questionnaire, and underwent positron emission tomography scanning with [¹⁸F]fallypride.

Results:

MA users displayed steeper temporal discounting (p = 0.030) and lower striatal D₂/D₃ receptor availability (p < 0.0005) than controls. Discount rate was negatively correlated with striatal D₂/D₃ receptor availability, with the relationship reaching statistical significance in the combined sample (r = -0.291, p = 0.016) and among MA users alone (r = -0.342, p = 0.041), but not among controls alone (r = -0.179, p = 0.185); the slopes did not differ significantly between MA users and controls (p = 0.5).

Conclusions:

These results provide the first direct evidence of a link between deficient D₂/D₃ receptor availability and steep temporal discounting. This finding fits with reports that low striatal D₂/D₃ receptor availability is associated with a higher risk of relapse among stimulant users, and may help to explain why some individuals choose to continue using drugs despite knowledge of their eventual negative consequences. Future research directions and therapeutic implications are discussed.     

In situ phosphorylation of the α subunit of eukaryotic initiation factor 2 in reticulocyte lysates inhibited by heme deficiency, double-stranded RNA, oxidized glutathione, or the heme-regulated protein kinase

Protein synthesis initiation in reticulocyte lysates is inhibited by heme deficiency, low levels of double-stranded RNA (dsRNA), oxidized glutathione (GSSG), or the purified kinase (HRI) that acts on the alpha polypeptide of eukaryotic initiation factor 2 (eIF-2alpha). The phosphoprotein profiles produced in lysates in response to these various conditions have been monitored directly in lysates after labeling for brief periods with pulses of [gamma-(32)P]ATP. The [(32)P]phosphoprotein profiles were analyzed by electrophoresis in sodium dodecyl sulfate/polyacrylamide slab gels under conditions in which the HRI and eIF-2alpha polypeptides were clearly distinguished. All four modes of inhibition produced a rapid phosphorylation of eIF-2alpha compared to control lysates, which displayed little or no phosphorylation of eIF-2alpha. In heme-deficient lysates, phosphorylation of eIF-2alpha occurred rapidly both before and after the shut-off of protein synthesis; the delayed addition of hemin to these lysates resulted in a decrease in the phosphorylation of eIF-2alpha and the subsequent restoration of protein synthesis. These data suggest that rapid turnover of phosphate occurs at the site(s) of eIF-2alpha phosphorylation. In lysates inhibited by heme deficiency, GSSG, or added HRI, the phosphorylation of eIF-2alpha was accompanied by the rapid in situ phosphorylation of HRI. The inhibition of initiation induced by dsRNA was accompanied by the phosphorylation of eIF-2alpha and a 67,000-dalton polypeptide but not HRI. These observations in situ indicate that (i) the phosphorylation of eIF-2alpha is the critical event in these inhibitions of protein chain initiation, and (ii) the phosphorylation of HRI is associated with its activation in heme deficiency.