Fetal hemoglobin in sickle cell anemia: Molecular characterization of the unusually high fetal hemoglobin phenotype in African Americans
Authors: Idowu Akinsheye, Nadia Solovieff, Duyen Ngo, Anita Malek, Paola Sebastiani, Martin H. Steinberg, David H.K. Chui
Journal: American Journal of Hematology (2011)

Fetal hemoglobin (HbF) is a major modifier of disease severity in sickle cell anemia (SCA). Three major HbF quantitative trait loci (QTL) are known: theXmn Isite upstream of Gγ- globin gene(HBG2)on chromosome 11p15,BCL11Aon chromosome 2p16, andHBS1L-MYBintergenic polymorphism (HMIP) on chromosome 6q23. However, the roles of these QTLs in patients with SCA with uncharacteristically high HbF are not known. We studied 20 African American patients with SCA with markedly elevated HbF (mean 17.2%). They had significantly higher minor allele frequencies (MAF) in two HbF QTLs,BCL11A, and HMIP, compared with those with low HbF. A 3-bp (TAC) deletion in complete linkage disequilibrium (LD) with the minor allele of rs9399137 in HMIP was also present significantly more often in these patients. To further explore other genetic loci that might be responsible for this high HbF, we sequenced a 14.1 kb DNA fragment between the Aγ-(HBG1) and δ-globin genes (HBD). Thirty-eight SNPs were found. Four SNPs had significantly higher major allele frequencies in the unusually high HbF group. In silico analyses of these four polymorphisms predicted alteration in transcription factor binding sites in 3. HbF inhibits deoxy-HbS polymerization. Patients with elevated HbF have fewer vaso-occlusive complications and prolonged survival [1]. Three major HbF QTL are known. The C>T polymorphism (rs7482144) at nucleotide –158 upstream of HBG2 is associated with increased HbF in some patients with SCA [2]. Polymorphisms in intron 2 of BCL11A represented by rs766432 was associated with HbF in healthy Northern Europeans [3], African Americans with SCA [4, 5], Chinese with β-thalassemia trait, and Thai's with HbE-β thalassemia [5]. BCL11A polymorphisms correlate highly with HbF levels in SCA, accounting for 7–12% of the HbF variance [6]. The HMIP polymorphisms are distributed in three LD blocks [7]. HMIP block 2 represented by rs9399137 is most significantly associated with HbF expression and might function as a distal regulatory element [8, 9]. We studied a selected group of 20 African American patients with SCA with exceptionally high HbF (mean 17.2%), which differed by more than four times the standard deviation of 30 other patients with low HbF (mean 5.0%; Table I). All study subjects' HBB underwent nucleotide sequencing to ascertain that they were HbS homozygotes. Multiplex ligation-dependent probe amplification was carried out to ensure that they did not harbor hereditary persistence of HbF (HPFH) 1, HPFH 2, Black (δβ)0- and Black (Aγδβ)0-thalassemia deletions [10]. Furthermore, their HBG2 and HBG1 promoters were also sequenced to be certain that they did not have promoter HPFH single nucleotide mutations [11, 12]. They were unlikely to be on hydroxyurea based on their MCV being less than 100 fL. In addition, we conducted a subset analyses in 56 patients with unusually high HbF (mean 20.7%), which differed by more than 11 times the standard deviation of 489 patients with low HbF (mean 3.1%; Table II). These patients were selected from 1,086 subjects from the Cooperative Study of Sickle Cell Disease (CSSCD), who were previously investigated in a genome-wide association study (GWAS) of HbF [13]. The MAF of rs7482144, also known as the Xmn I site, on chromosome 11p15 in the unusually high HbF group (10%) is not significantly different from that in the low HbF group (8%) as shown in Table III. The SNP rs5006884, a missense mutation (CTC>TTC or Leu172Phe) in OR51B6 (http://www.ncbi.nlm.nih.gov/projects/SNP/snp_ref.cgi?rs=5006884) on chromosome 11p15 was reported to be associated with HbF in SCA in a GWAS [13]. In this study, the MAF of rs5006884 in the unusually high HbF group (2%) is actually less than that in the low HbF group (10%), even though the difference is not statistically significant (Table III). It should be recognized that the small sample size in this study does not afford sufficient power to discern possible MAF differences if present at these two SNPs between these two groups of patients. The SNP rs7482144 did not reach genome-wide significance in previous GWAS of HbF in the full CSCCD cohort [13]. In this study with subsets from the CSCCD cohort, rs7482144 has a significantly higher MAF in the unusually high HbF group (30%) compared to the low HbF group (10%), P = 0.002. The MAF of rs5006884 in OR51B6 in the high HbF group (22%) is significantly higher than that in the low HbF group (10%), P = 0.00055. This SNP reached genome-wide significance in the GWAS of the full CSSCD cohort; however, the mean HbF level in homozygotes for this variant was only 10.6%. The present subset analysis shows that for patients with SCA with unusually high HbF the frequency of this mutation is comparable with the general population (Table III). To examine the BCL11A QTL, three SNPs, rs766432, rs4671393, and rs11886868 were chosen for genotyping [6]. These three SNPs are in strong LD. Only data on rs766432 that is most highly correlated with HbF in patients with SCA are presented. The MAF in the unusually high HbF group (45%) is significantly higher than that in the low HbF group (25%), P = 0.05 (Table III). The MAF in the subgroups from CSCCD were similar: 47% in the high and 20% in the low HbF groups, P = 6.399 × 10−10. The allele C of this SNP was associated with increased levels of HbF [13] and the mean HbF levels in subjects homozygous for the C allele was 8.26%. The present subset analysis in the CSSCD subgroups shows that in patients with unusually high HbF the frequency of this mutation is almost twice that of the full cohort. The QTL in HMIP is best represented by rs9399137 [9]. The MAF of rs9399137 among patients with SCA of African descent without European admixture was reported to be 1–2% [14, 15]. A GWAS on over 800 African American patients with SCA did not detect genome-wide significance of association of this QTL with HbF [13]. The functional motif for this QTL is most likely a 3-bp (TAC) deletion which is in complete LD with the minor allele of rs9399137 [9]. The frequency of this 3-bp deletion is 23% in non-African HapMap populations but only 5% in Africans [9]. In this study, the MAF of rs9399137 in the African American patients with SCA with unusually high HbF is 18%, significantly higher than that with low HbF (3%), P = 0.02 (Table III). Furthermore, the 3-bp deletion as reported by Farrell et al [9] was found for the first time in these African American patients with SCA and it is in complete LD with the minor allele of rs9399137. Among the subset of CSSCD patients, the MAF of rs9399137 in the unusually high HbF group (9%) is also significantly higher than that in the low HbF group (3%), P = 0.006 (Table III). These results raise the possibility that some African American patients with SCA with markedly elevated HbF might have inherited the minor allele of chromosome 6q23 QTL due to European genetic admixture. The minor T allele of rs7775698 tags either an ancestral T nucleotide found mostly in African populations, or a 3-bp deletion often found in European and Chinese populations [9]. In this, the MAF of rs7775698 in the high HbF group (40%) is higher than that in the low HbF group (21%). However, the difference is not statistically significant (P = 0.07). Among the patients with CSCCD, the MAF of rs7775698 in the high HbF group is 16% compared with 19% in the low HbF group (Table III). We found a 2-bp (CC) deletion plus an (A) insertion 19 bp downstream of rs9399137. This deletion/insertion was present in 33% of chromosome 6 in both high and low HbF groups. It is unlikely that it plays a significant functional role in modulating HbF expression. Its relatively high frequency in both groups makes it a probable haplotype marker in African American patients with SCA. Two of the three known HbF major QTLs are present in the unusually high HbF patient study group at a frequency significantly higher than those with low HbF. Their cumulative effect was estimated by assigning to each minor allele at each locus a score of 1, and adding the scores to generate the number of minor allele present (NOMAP) [16]. There was an average of two minor alleles in the unusually high HbF group compared with one in the low HbF group (P = 0.001) as shown in Fig. 1. Correlation between NOMAP (Number of HbF QTL Minor Allele Present) and HbF levels in the unusually high and low HbF groups. The presence of either one minor allele, T in rs7482144 (Xmn I), C in rs766432 (BCL11A), or 3-bp deletion in HMIP is counted as 1. To explore other possible genetic loci that may modulate HbF expression, we undertook nucleotide sequencing of a 14.1 kb DNA fragment between HBG1 and HBD in patients with 15 high and 15 low HbF. This DNA fragment was chosen because BCL11A binds to this intergenic region and it also encompasses the Corfu deletion that in homozygotes is characterized by markedly elevated HbF [17, 18]. Thirty eight SNPs were found in both the high and low HbF groups (Supporting Information Table I). SNP rs10128558 as described by Galarneau et al [19] was present in our study groups and found to be in complete LD with Xmn I polymorphism. Its MAF in the high HbF group (10%) is not different from the low HbF group (8%). Based on in silico analysis by TFSEARCH (threshold score 85), 22 of the 38 SNPs are associated with alterations in transcription factor binding including erythroid-specific transcription factors. In addition, four SNPs have significantly higher major allele frequency in the high compared to the low HbF groups (P < 0.05). Three of these four SNPs result in alteration in transcription factor binding sites (Supporting Information Table I). This study based on a small cohort of carefully selected African American patients with SCA with unusually high HbF revealed that the MAF for rs766432 (BCL11A), rs9399137 and 3-bp deletion both within HMIP are much higher than that found in patients with low HbF. These findings also raise the possibility that some African American patients with markedly elevated HbF might have inherited the minor allele of chromosome 6q23 QTL due to European genetic admixture. Validation of these findings in a larger patient cohort and further functional investigations into these and other polymorphisms are warranted. Blood samples referred to the Hemoglobin Diagnostic Reference Laboratory for DNA-based diagnostics at the Boston Medical Center were selected for this study. The samples were collected between 2003 and 2008. Patients younger than 5-year-old, the time at which HbF levels stabilized [13] and patients with MCV greater than 100 fL were excluded. Nucleotide sequencing of the HBB and promoters of HBG2 and HBG1 was done after PCR amplification. The presence of HBB deletions was excluded by multiplex ligation dependent probe amplification [20]. This study was approved by the Boston University School of Medicine Institutional Review Board. We used data from the CSSCD. From 1,086 cases, who underwent GWAS [13], we selected 56 patients with unusually high HbF and 489 patients with low HbF for subset analyses. The Xmn I polymorphism (rs7482144) was genotyped by polymerase chain reaction (PCR) of the HBG2 promoter, followed by restriction enzyme digestion analysis. Genotyping of SNPs in BCL11A was done by a TaqMan SNP genotyping assay (Applied BioSystems, Foster City, CA) according to the manufacturer's instruction. Predesigned probes were ordered for genotyping analyses: rs766432 (C__1025980_10), rs11886868 (C__11363852_10), rs4671393 (C__25926414_10). Amplification was done with 5 μl of 2X TaqMan Universal PCR master mix, 0.5 μl of 40X primer and TaqMan probe dye mix, and between 10–50 ng of DNA. Cycling conditions consisted of 10 min at 95°C, followed by 40 cycles 15 sec at 92°C, 1 min at 60°C. Allelic discrimination is performed on Applied BioSystem RT-PCR system. Genotyping of SNPs in HMIP, rs9399137, rs7775698, and 3-bp (TAC) deletion, and SNP rs5006884 in OR51B6 was done by PCR, followed by nucleotide sequencing using the BigDye terminator cycle sequencing kit from Applied BioSystems. Short (500–600 bp) and overlapping fragments of DNA covering the 14.1 kb region were amplified by PCR. All PCR reactions were performed in a total volume of 20 μl. Master mix concentrations, and cycling conditions were optimized based on the region being amplified. Usually, each reaction contained 100–250 ng of DNA, 1X PCR buffer (Applied BioSystems), 2 mM MgCl2, 200 μM dNTP, 1 ng of each primer, and 0.5U AmpliTaq polymerase. Cycling conditions consisted of 5 min at 94°C, followed by 30 cycles of 40 sec at 94°C, 40 sec at 55°C, and 3 min at 72°C and a 7-min elongation step at 72°C. Association between HbF and the minor alleles of each QTL was statistically analyzed by the Fisher exact test performed in R (www.r-project.org). Comparison of hematological parameters and correlation between NOMAP and HbF in both groups was examined using paired T-test. An overall significance level of 0.05 was set for all statistical analyses. The authors sincerely thank Drs. Hong-yuan Luo, Richard M. Sherva, and John J. Farrell for advice throughout the course of this study. The support and encouragement of Dr. Katya Ravid was very much appreciated. Additional Supporting Information may be found in the online version of this article. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article. Idowu Akinsheye*, Nadia Solovieff , Duyen Ngo*, Anita Malek*, Paola Sebastiani , Martin H. Steinberg*, David H.K. Chui*, * Division of Hematology/Oncology, Department of Medicine, Boston University School of Medicine, Boston, Massachusetts, Department of Biostatistics, Boston University School of Public Health, Boston, Massachusetts.