Binal N. Shah

Genetic variants and cell-free hemoglobin processing in sickle cell nephropathy

Intravascular hemolysis and hemoglobinuria are associated with sickle cell nephropathy. ApoL1 is involved in cell-free hemoglobin scavenging through association with haptoglobin-related protein. APOL1 G1/G2 variants are the strongest genetic predictors of kidney disease in the general African-American population. A single report associated APOL1 G1/G2 with sickle cell nephropathy. In 221 patients with sickle cell disease at the University of Illinois at Chicago, we replicated the finding of an association of APOL1 G1/G2 with proteinuria, specifically with urine albumin concentration (β=1.1, P=0.003), observed an even stronger association with hemoglobinuria (OR=2.5, P=4.3×10−6), and also replicated the finding of an association with hemoglobinuria in 487 patients from the Walk-Treatment of Pulmonary Hypertension and Sickle cell Disease with Sildenafil Therapy study (OR=2.6, P=0.003). In 25 University of Illinois sickle cell disease patients, concentrations of urine kidney injury molecule-1 correlated with urine cell-free hemoglobin concentrations (r=0.59, P=0.002). Exposing human proximal tubular cells to increasing cell-free hemoglobin led to increasing concentrations of supernatant kidney injury molecule-1 (P=0.01), reduced viability (P=0.01) and induction of HMOX1 and SOD2. HMOX1 rs743811 associated with chronic kidney disease stage (OR=3.0, P=0.0001) in the University of Illinois cohort and end-stage renal disease (OR=10.0, P=0.0003) in the Walk-Treatment of Pulmonary Hypertension and Sickle cell Disease with Sildenafil Therapy cohort. Longer HMOX1 GT-tandem repeats (>25) were associated with lower estimated glomerular filtration rate in the University of Illinois cohort (P=0.01). Our findings point to an association of APOL1 G1/G2 with kidney disease in sickle cell disease, possibly through increased risk of hemoglobinuria, and associations of HMOX1 variants with kidney disease, possibly through reduced protection of the kidney from hemoglobin-mediated toxicity.

APOL1,α-thalassemia, and BCL11A variants as a genetic risk profile for progression of chronic kidney disease in sickle cell anemia

Sickle cell anemia (SCA) is caused by a mutation in the β-globin gene that results in hemoglobin polymerization and affects approximately 1 in 500 African Americans and 25 million people worldwide. Chronic kidney disease (CKD) is observed in up to 58% of adults, and is associated with a 3-fold increased risk for early mortality in SCA. Multiple genetic modifiers of clinical complications in SCA have been reported, and combining these genetic factors into a risk profile may strengthen the predictive value as compared to the individual factors alone. Homozygosity or compound heterozygosity for APOL1 G1/G2 (G1=S342G/I384M substitutions, G2=N388/Y389 deletions) is observed in 10-15% of African Americans, and is the strongest genetic association for CKD in African Americans in general. APOL1 G1/G2 is also associated with proteinuria and albumin-

Gain-of-function EGLN1 prolyl hydroxylase (PHD2 D4E:C127S) in combination with EPAS1 (HIF-2α) polymorphism lowers hemoglobin concentration in Tibetan highlanders

Tibetans have lived at high altitude for generations and are thought to be genetically adapted to hypoxic environments. Most are protected from hypoxia-induced polycythemia, and a haplotype of EPAS1, encoding hypoxia-inducible factor (HIF-2α), has been associated with lower hemoglobin levels. We earlier reported a Tibetan-specific EGLN1 haplotype encoding PHD2 which abrogates HIF augmentation in hypoxia. We genotyped 347 Tibetan individuals from varying altitudes for both the Tibetan-specific EGLN1 haplotype and 10 candidate SNPs in the EPAS1 haplotype and correlated their association with hemoglobin levels. The effect of the EGLN1 haplotype on hemoglobin exhibited age dependency at low altitude, while at higher altitudes, it showed a trend to lower hemoglobin levels in the presence of the Tibetan-selected EPAS1 rs142764723 C/C allele. The observed gene-environment and gene-gene interactions and the moderate effect of the EGLN1 and EPAS1 haplotypes on hemoglobin indicate that other modifiers exist. It remains to be determined whether a blunting of erythropoiesis or other physiological consequences of HIF downregulation are the primary drivers of these genetic adaptations among Tibetans.Key messageMost Tibetans are protected from polycythemia while living in high altitude.An EGLN1 co-adapted haplotype, EGLN1 c.12C>G, c.380G>C is uniquely Tibetan.The Tibetan EPAS1 haplotype has introgressed from the Denisovan genome.While EGLN1 and EPAS1 genotypes lower Hb, this study indicates additional Hb modifiers.

Erythrocytic ferroportin reduces intracellular iron accumulation, hemolysis, and malaria risk

Irons grip on malaria Malaria parasites have coevolved with their human and mammalian hosts. These Plasmodium species invade the iron-rich environment of red blood cells. Zhang et al. found that the iron transporter ferroportin persists on the surface of mature mammalian red blood cells. Red blood cells are at risk of oxidative damage if their hemoglobin releases its iron; ferroportin is thus important to expel this iron. The authors also found that the transporter can deprive malaria parasites of the iron they need for proliferation. The Q248H mutation in the human ferroportin gene enhances ferroportin expression during development and seems to provide protection against malaria. This effect may explain the enrichment of the Q248H mutation among African populations. Science, this issue p. 1520 Ferroportin exports free iron from mature erythrocytes to protect cells from oxidative damage and the malaria parasite. Malaria parasites invade red blood cells (RBCs), consume copious amounts of hemoglobin, and severely disrupt iron regulation in humans. Anemia often accompanies malaria disease; however, iron supplementation therapy inexplicably exacerbates malarial infections. Here we found that the iron exporter ferroportin (FPN) was highly abundant in RBCs, and iron supplementation suppressed its activity. Conditional deletion of the Fpn gene in erythroid cells resulted in accumulation of excess intracellular iron, cellular damage, hemolysis, and increased fatality in malaria-infected mice. In humans, a prevalent FPN mutation, Q248H (glutamine to histidine at position 248), prevented hepcidin-induced degradation of FPN and protected against severe malaria disease. FPN Q248H appears to have been positively selected in African populations in response to the impact of malaria disease. Thus, FPN protects RBCs against oxidative stress and malaria infection.

Honokiol is a FOXM1 antagonist

Honokiol is a natural product and an emerging drug for a wide variety of malignancies, including hematopoietic malignancies, sarcomas, and common epithelial tumors. The broad range of activity of honokiol against numerous malignancies with diverse genetic backgrounds suggests that honokiol is inhibiting an activity that is common to multiple malignancies. Oncogenic transcription factor FOXM1 is one of the most overexpressed oncoproteins in human cancer. Here we found that honokiol inhibits FOXM1-mediated transcription and FOXM1 protein expression. More importantly, we found that honokiol’s inhibitory effect on FOXM1 is a result of binding of honokiol to FOXM1. This binding is specific to honokiol, a dimerized allylphenol, and was not observed in compounds that either were monomeric allylphenols or un-substituted dihydroxy phenols. This indicates that both substitution and dimerization of allylphenols are required for physical interaction with FOXM1. We thus demonstrate a novel and specific mechanism for FOXM1 inhibition by honokiol, which partially may explain its anticancer activity in cancer cells.

Association of circulating transcriptomic profiles with mortality in sickle cell disease

Sickle cell disease (SCD) complications are associated with increased morbidity and risk of mortality. We sought to identify a circulating transcriptomic profile predictive of these poor outcomes in SCD. Training and testing cohorts consisting of adult patients with SCD were recruited and prospectively followed. A pathway-based signature derived from grouping peripheral blood mononuclear cell transcriptomes distinguished 2 patient clusters with differences in survival in the training cohort. These findings were validated in a testing cohort in which the association between cluster 1 molecular profiling and mortality remained significant in a fully adjusted model. In a third cohort of West African children with SCD, cluster 1 differentiated SCD severity using a published scoring index. Finally, a risk score composed of assigning weights to cluster 1 profiling, along with established clinical risk factors using tricuspid regurgitation velocity, white blood cell count, history of acute chest syndrome, and hemoglobin levels, demonstrated a higher hazard ratio for mortality in both the training and testing cohorts compared with clinical risk factors or cluster 1 data alone. Circulating transcriptomic profiles are a powerful method to risk-stratify severity of disease and poor outcomes in both children and adults, respectively, with SCD and highlight potential associated molecular pathways.

Risk factors for vitamin D deficiency in sickle cell disease

Vitamin D deficiency (VDD), 25‐OHD levels <20 ng/ml, is prevalent among patients with sickle cell disease (SCD) and is linked to acute and chronic pain and bone fracture in this population. There is limited literature regarding VDD‐associated risk factors for SCD. We examined potential clinical and genomic parameters associated with VDD in 335 adults with SCD in a cross‐sectional study. VDD was present in 65% of adult SCD patients, and 25‐OHD levels independently and positively correlated with older age (P < 0·001) and vitamin D supplementation (P < 0·001). 25‐OHD levels were higher in SCD patients over 40 years of age compared to the general African‐American population. Both lower 25‐OHD levels and increased pain frequency were associated with increased expression of SLC6A5 encoding glycine transporter‐2 (GlyT2), a protein involved in neuronal pain pathways. Lower 25‐OHD levels were also associated with increased expression of CYP3A4, and with decreased expression of GC (also termed DBP) and VDR, three genes involved in vitamin D metabolism. We conclude that vitamin D supplementation should be an almost universal feature of the care of young adults with SCD, and that further research is warranted into genomic factors that regulate vitamin D metabolism in SCD.

Prospective study of thrombosis and thrombospondin-1 expression in Chuvash polycythemia.

In Chuvash polycythemia (CP), homozygous germline VHL results in augmented hypoxia sensing, elevated erythropoietin and hemoglobin, and increased morbidity and mortality from thrombosis, but the relative risks and molecular basis have not been prospectively evaluated. We enrolled 128 CP adults and 128 controls from Russia’s Chuvash Republic in an observational study from 20052009, in order to prospectively define the risk of complications and to provide mechanistic insights. We hypothesized that variation in the expression of hypoxia inducible factor (HIF)-regulated genes may contribute to increased thrombosis. During a median follow up of 8.8-years, CP was associated with a 9.6-fold increase in the rate of new thrombosis compared to controls, after adjustment for significant risk factors such as age and smoking. The probability of new thrombosis in CP did not increase with higher baseline hemoglobin concentration, but it increased with age, smoking, baseline therapeutic phlebotomy and higher expressions of THBS1, CXCL2 and EREG. Hypomorphic VHL impairs HIF-α degradation, leading to an increased transcription of many HIF-regulated genes, including erythropoietin, and to elevated hematocrit levels, thrombosis and early mortality, but not malignancy.Whether the increased risk of thrombosis is related to the elevated hemoglobin concentration has been unclear, and hypoxia itself has been implicated as a risk for thrombosis. The British Committee for Standards in Haematology recommends phlebotomy for polycythemia vera if the hematocrit is >45%, and for idiopathic erythrocytosis/polycythemia if the hematocrit is >54%, but its benefits for CP are unknown. The CP patients and controls in this study were matched by age, sex and place of residence within Russia’s Chuvash Republic, and underwent VHL genotyping. The ethics committees of the Chuvash State University and the Republic Clinical Hospital gave their approval. Written informed consent was obtained from all subjects according to the Declaration of Helsinki. Consistent with previous studies, CP adults had higher hemoglobin and erythropoietin concentrations, lower systemic blood pressure, body mass index, white blood cell count, and serum ferritin concentration, less history of hypertension and more reports of bleeding (Table 1). They were also more likely to be current smokers than controls. Twenty-seven patients aged 24-76 years and 3 controls aged 45-67 years had a history of thrombosis. CP patients with past thrombosis were on average 10 years older than those without (P=0.002). The subjects were followed up between 2015 and 2016

Associations of α-thalassemia and BCL11A with stroke in Nigerian, United States, and United Kingdom sickle cell anemia cohorts

Alpha-thalassemia and the BCL11A rs1427407 T allele are commonly observed in sickle cell anemia (SCA) patients and are associated with reduced hemolysis and higher hemoglobin F levels, respectively. We investigated whether a high-risk genetic profile, defined as SCA patients who did not inherit either α-thalassemia or the BCL11A rs1427407 T allele, had stronger associations with clinical and laboratory variables than the individual genetic components in the University of Ibadan cohort (n=249). We then replicated our findings in SCA cohorts from the University of Illinois at Chicago (UIC)(n=260) and Walk-Treatment of Pulmonary Hypertension and Sickle cell disease with Sildenafil Therapy (Walk-PHaSST)(n=387). High-risk was associated with higher reticulocytes (15.0% vs. 7.8%, P=0.08) and stroke history (6% vs. 1%, P=0.02) than standard risk patients and these associations were more significant than the individual genetic components in the University of Ibadan cohort. These findings were replicated in high-risk patients from UIC and Walk-PHaSST for reticulocytes (UIC: 13.5% vs. 11.8%, P=0.03; Walk-PHaSST: 9.6% vs. 8.2%, P=0.0003) and stroke history (UIC: 32% vs. 22%, P=0.07; Walk-PHaSST: 14% vs. 7%, P=0.01). On combined analysis, high-risk had strong associations with increased markers of hemolysis (hemoglobin β= -0.29, 95%CI: -0.50 to -0.09; P=0.006; reticulocyte% β=2.29, 95%CI: 1.31 to 3.25; P=1x10-5) and stroke history (OR=2.0, 95%CI: 1.3 to 3.0; P=0.0002), but no association with frequent vaso-occlusive crises (≥3/year). A high-risk genetic profile is associated with increased hemolysis and stroke history in three independent cohorts. This profile may help identify patients to prioritize for hydroxyurea and for closer monitoring strategies for stroke.

HMOX1 and acute kidney injury in sickle cell anemia

TO THE EDITOR: The pathophysiologic mechanisms, genetic predictors, and long-term consequences of acute kidney injury (AKI) on kidney function in sickle cell anemia (SCA) are not clear.[1][1][⇓][2][⇓][3][⇓][4]-[5][5] In the general population, AKI is associated with the subsequent development