Mutations and polymorphisms in hemoglobin genes and the risk of pulmonary hypertension and death in sickle cell disease

Abstract

Pulmonary hypertension is a common complication of sickle cell disease (SCD) and a risk factor for early death. Hemolysis may participate in its pathogenesis by limiting nitric oxide (NO) bioavailability and producing vasculopathy. We hypothesized that hemoglobin mutations that diminish hemolysis in SCD would influence pulmonary hypertension susceptibility. Surprisingly, coincident alpha-thalassemia (Odds Ratio [OR]=0.95, 95% CI=0.46-1.94, P=NS) was not associated with pulmonary hypertension susceptibility in homozygous SCD. However, pulmonary hypertension cases were less likely to have hemoglobin SC (OR=0.18, 95% confidence interval [CI]=0.06-0.51, P=0.0005) or Sbeta(+) thalassemia (OR=0.25, 95% CI=0.06-1.16, P=0.10). These compound heterozygotes may be protected from pulmonary hypertension because of reduced levels of intravascular hemolysis, but develop this complication at a lower rate possibly due to the presence of non-hemolytic risk factors such as renal dysfunction, iron overload and advancing age. Despite this protective association, patients with SC who did develop pulmonary hypertension remained at significant risk for death during 49 months of follow-up (Hazard Ratio=8.20, P=0.0057).

Figure 1. Overview of Chromosome 11 Markers

The region of Chromosome 11 investigated in this study is schematically represented. At the top of the figure, chromosomal coordinates in the human genome are presented (March 2006 build, hg18). Below these coordinates, the methods utilized to type specific markers are presented (either sequencing, Taqman, or RFLP). Finally, the locations of the individual globin genes are illustrated on the blue line with exons presented as larger blocks on the line with the gene name presented above the exon blocks. The sequenced region of the HBB gene (with the 3 exons represented as blocks on the line) is expanded below with the locations of 29 individual single nucleotide variants (including 4 new variants that are not presently included in the dbSNP database).

Figure 2. Haplotypes of the β Globin Locus on Chromosome 11 in Adults with Sickle Cell Disease

(A) Pattern of pairwise linkage disequilibrium (LD) for 17 SNP markers in 260 unrelated patients with SCD. Red squares indicate strong LD and values within each square are LD measurements by LOD score. The outlined blocks represent haplotype blocks. The sickle and hemoglobin C mutations are markers 6 and 7, respectively. (B) The haplotypes of the β globin locus observed in 260 adults with SCD as the blocks defined in panel A. Lines connecting each haplotype block are in bold when observed with a frequency of greater than 5% and the fraction adjacent to each block is the observed haplotype frequency. Note that the hemoglobin C mutation is carried on a nearly unique haplotype across the 4 blocks which is distinguished from all other haplotypes by markers 7 (HbC) and 15 (SNP_02). (C) Age corrected P values for each single marker association comparing PH cases defined by echocardiography to controls among 260 patients with SCD. P values are presented as the |Log₁₀ P value| where P=0.05 is 1.3 (indicated by red dashed line). (D) Age corrected P values for each single marker association comparing PH cases defined by NTproBNP to controls among 177 patients with SCD.

Figure 3. Kaplan-Meier Survival for Sickle Cell Anemia (HbSS) and Hemoglobin SC By Presence or Absence of Pulmonary Hypertension

(A) Survival data published by Gladwin et al. and updated by Kato et al. was reanalyzed according the tricuspid jet velocity (TRV) for those with homozygous SCD (HbSS, n=176) from the first 252 consecutive patients screened with SCD during 49 months of follow-up.4,5 The presence of pulmonary hypertension (TRV ≥ 2.5 m/s) in HbSS is a significant risk factor for mortality (Hazard Ratio = 4.81, 95% CI 1.73-14.19, Logrank test P=0.0029). (B) Survival data for 41 patients with Hemoglobin SC with and without PH. PH is a significant risk factor for mortality in Hemoglobin SC (Hazard Ratio = 8.20, 95% Confidence Interval 2.71-352.10, Logrank test P=0.0057).