Regulation of iron homeostasis through the erythroferrone-hepcidin axis in sickle cell disease.

Sickle cell disease (SCD) has a distinct pattern of transfusional iron overload (IO) when compared to transfusion-dependent ß-thalassaemia major (TDT). We conducted a single institution prospective study to evaluate plasma biomarkers of iron regulation and inflammation in patients with SCD with IO (SCD IO cases, n = 22) and without IO (SCD non-IO cases, n = 11), and non-SCD controls (n = 13). Hepcidin was found to be inappropriately low, as evidenced by a significantly higher median hepcidin/ferritin ratio in non-SCD controls compared to SCD IO cases (0·3 vs. 0·02, P < 0·0001) and SCD non-IO cases (0·3 vs. 0·02, P < 0·0001), suggesting that certain inhibitory mechanism (s) work to suppress hepcidin in SCD. As opposed to the SCD non-IO state, where hepcidin shows a strong significant positive correlation with ferritin (Spearman ρ = 0·7, P = 0·02), this correlation was lost when IO occurs (Spearman ρ = -0·2, P = 0·4). Although a direct non-linear correlation between erythroferrone (ERFE) and hepcidin did not reach statistical significance both in the IO (Spearman ρ = -0·4, P = 0·08) and non-IO state (Spearman ρ = -0·6, P = 0·07), patients with highest ERFE had low hepcidin levels, suggesting that ERFE contributes to hepcidin regulation in some patients. Our results suggest a multifactorial mechanism of hepcidin regulation in SCD.

Characteristics and potential biomarkers of adult sickle cell patients with chronic pain.

OBJECTIVES: In this study, we investigated the evolution of chronic pain in sickle cell patients (SCD) as an age-dependent phenomenon and studied the frequency of vaso-occlusive episode frequency, opioid use, quantitative sensory testing (QST), and biomarkers of chronic pain (CP). METHODS: We undertook a cross-sectional study of the evolution of CP in SCD. A total of 72 subjects (age 15-66) were enrolled. VOE frequency, presence of CP hydroxyurea (HU) therapy, opioid use, and laboratory parameters were collected. QST was performed, and plasma tryptase, substance P, and NGF (Nerve Growth Factor) levels were assayed. RESULTS: There was an age-dependent increase in frequency of CP, VOEs, opioid use, and Von Frey monofilament values. CP patients had significantly higher opioid use (daily morphine equivalents) (52.8 mg vs 6.94 mg, P = .009), suggesting a correlation between opioid use and hyperalgesia. NGF levels were also significantly higher (P = .051). Our results confirm previous observations of an age-dependent increase in the proportion of patients with CP and support the contributing role of mast cell activation and neurogenic inflammation. CONCLUSIONS: This is the first study of NGF as a possible biomarker of CP in SCD. If confirmed, this could provide a diagnostic marker and therapeutic target for CP in SCD.

Characteristics of sickle cell patients with frequent emergency department visits and hospitalizations.

Vaso-occlusive episodes (VOEs) are a hallmark of sickle cell disease (SCD), and account for >90% of health care encounters for this patient population. The Cooperative Study of Sickle Cell Disease, a large study enrolling >3000 patients, showed that the majority of SCD patients (80%) experienced 0-3 major pain crises/year. Only a small minority (~5%) experienced ≥6 VOEs/year. Our study sought to further understand this difference in VOE frequency between SCD patients. We analyzed 25 patients (13M/12F, mean age of 28.8) with ≥6 ED visits or hospitalizations/year (high utilizers), and compared these with 9 patients (6M/3F, mean age of 37.6) who had ≤2 ED visits or hospitalizations/year (low utilizers). All subjects were given a demographic survey along with questionnaires for depression, anxiety, and Health Locus of Control. Each subject then underwent quantitative sensory testing (QST) with three different modalities: pressure pain sensitivity, heat and cold sensitivity, and Von Frey monofilament testing. Laboratory and clinical data were collected through subjects' medical records. CBC and chemistry analysis showed high utilizers had higher WBC (p<0.01), ANC (p<0.01), total bilirubin (p = 0.02), and lower MCV (p = 0.03). Opioid use (morphine equivalents) over the past 6 months was significantly higher in the high utilizer group (12125.7 mg vs 2423.1 mg, p = 0.005). QST results showed lower pressure pain threshold at the ulna (224.4 KPa vs 338.9 KPa, p = 0.04) in the high utilizer group. High utilizers also had higher anxiety (9.0 vs 4.6, p = 0.04) and depression scores (10.0 vs 6.0, p = 0.051). While the low utilizer group had higher education levels with more associate and bachelor degrees (p = 0.009), there was no difference in income or employment. These data show that many biological and psychosocial factors contribute to high health care utilization in SCD. A multi-disciplinary and multi-faceted approach will be required to address this complex problem.

Preemptive Genotyping of CYP2C8 and CYP2C9 Allelic Variants Involved in NSAIDs Metabolism for Sickle Cell Disease Pain Management.

Interindividual variability in analgesic effects of nonsteroidal anti-inflammatory drugs prescribed for sickle cell disease (SCD) pain is attributed to polymorphisms in the CYP2C8 and CYP2C9 enzymes. We described CYP2C8 and CYP2C9 genotype/phenotype profiles and frequency of emergency department (ED) visits for pain management in an African American SCD patient cohort. DNA from 165 unrelated patients was genotyped for seven CYP2C8 and 15 CYP2C9 alleles using the iPLEX ADME PGx multiplexed panel. CYP2C8*1 (0.806),*2 (0.164), *3 (0.018), and *4 (0.012) alleles were identified. Genotype frequencies were distributed as homozygous wild type (66.7%), heterozygous (27.8%), and homozygous variant/compound heterozygous (5.4%), respectively. CYP2C9*1 (0.824), *2 (0.027), *3 (0.012), *5 (0.009), *6 (0.009), *8 (0.042), *9 (0.061), and *11(0.015) were observed with extensive (68.5%), intermediate (18.1%) and poor predicted metabolizers (0.6%), respectively. Fifty-two and 55 subjects, respectively had at least one variant CYP2C8 or CYP2C9 allele. Although the distribution of the CYP2C9 (p = 0.0515) phenotypes was marginally significantly in high and low ED users; some CYP2C8 and CYP2C9 allelic combinations observed in 15.2% (25) of the cohort are associated with higher risks for analgesic failure. CYP2C8 and CYP2C9 preemptive genotyping could potentially enable clinicians to identify patients with impaired metabolic phenotypes.

Human platelet 12-lipoxygenase: naturally occurring Q261/R261 variants and N544L mutant show altered activity but unaffected substrate binding and membrane association behavior.

The single nucleotide polymorphism (SNP) R261Q in the human platelet 12-lipoxygenase has been correlated with several human diseases. To understand better the biological performance we have compared enzymatic properties of the recombinant enzymes: 'wild-type' as Q261 and R261 variants with a single Q261R mutation at the enzyme periphery and N544L mutant with an altered active site. The R261 variant does not follow the same kinetics such as WT-Q261 showing a lag phase, a slower accumulation of product, following a different time-course without reaching plateau characteristic for the Q261 variant. The N544L substitution in the active site almost eradicates enzymatic activity proving that asparagine is as important for catalysis as the conserved histidines and C-terminal isoleucine. All three enzymes have comparable substrate binding and membrane association behavior. We conclude that the naturally occurring SNP, causing single mutation at a location distant to the active site, can alter the protein-protein association of this oligomeric enzyme making impact on kinetic properties of an allosteric mechanism and molecular recognition/signaling at a submembrane frontier.

Mortality in sickle cell patients on hydroxyurea therapy.

The efficacy of hydroxyurea (HU) and its role in the reduction in mortality in sickle cell patients has been established. Nevertheless, many patients still die of complications of this disease while on HU. Of the 226 patients treated with HU at our center, 38 died (34 of sickle cell-related causes). Acute chest syndrome (ACS) was the most common (35%) cause of death. Deceased and surviving patients did not differ significantly in average HU dose, baseline fetal hemoglobin (Hb F), or maximum Hb F response. However, the deceased patients were significantly older when HU was instituted, were more anemic, and more likely to have BAN or CAM haplotypes. They also had significantly higher serum blood-urea-nitrogen (BUN) and creatinine levels. Sickle cell patients who die while on HU therapy may represent a subgroup of older patients, possibly with more severe disease and more severe organ damage. Such patients need early identification and prompt HU institution.