genomic variation in the map3k5 gene is associated with β-thalassemia disease severity and...

469ISSN 1462-241610.2217/PGS.13.31 © 2013 Future Medicine Ltd Pharmacogenomics (2013) 14(5), 469–483

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Genomic variation in the MAP3K5 gene is associated with b‑thalassemia disease severity and hydroxyurea treatment efficacy

Hemoglobinopathies, particularly b‑thalas‑semia and sickle cell disease (SCD), are major health problems, in which quantitative or quali‑tative defects in hemoglobin (Hb) production occur, respectively. Under normal circum‑stances, different types of Hb molecules are produced during embryonic, fetal, and adult life. At birth, fetal Hb (HbF), in particular, rep‑resents 80–90% of the total Hb synthesized, but it gradually decreases to approximately 1% by 10 months in infancy, as its synthesis is restricted to a small subset of erythrocytes termed ‘F cells’ [1]. However, the exact levels of HbF, and, thus, the F‑cell population in adults may vary considerably among different popu‑lations [2,3], and it has been shown that F‑cell and HbF levels are determined by a very strong genetic component, contributing to the trait by an estimated 89% [4].

The first studies, aiming to identify regula‑tors of HbF production, were conducted on individuals with heterocellular hereditary per‑sistence of HbF (HPFH) – that is, an increase of HbF levels unevenly distributed among F cells – and suggested the absence of linkage between the determinant of the HbF levels and the b‑globin gene cluster [5]. Later, while

seeking for genetic elements associated with elevated HbF levels in healthy adults, several cis‑acting variants on the b‑globin gene com‑plex were unraveled, including: the XmnI‑Gg (HBG2) gene promoter polymorphism [6]; the 4‑bp deletion of the HBG1 gene [7]; and variations in the DNaseI hypersensitive site 2 of the b‑locus control region [8,9]. In addition, regions unlinked to the b‑locus (trans‑acting), such as quantitative trait loci (QTLs) on Xp22 [10] and 6q23 [11] became known soon after. Initially, a study on an extensive, inbred kin‑dred of Asian–Indian origin with heterocellu‑lar HPFH revealed that a key locus controlling HPFH resides on chromosome 6q, which was fine‑mapped to 6q22.3–23.1 [11]. Among the first positional candidate genes in the 6q23 region, assumed to possibly explain this QTL, were the MYB proto‑oncogene and the HBS1L gene, as well as MAP3K5 [12]. In addition, genes within this region were shown to be associated with response to hydroxyurea (HU) treatment, based on elevated HbF levels in SCD patients. However, the mechanism by which this chro‑mosome 6q22–23 QTL influences HbF lev‑els in the context of HU treatment remains unknown [13].

Aim: In this study we explored the association between genetic variations in MAP3K5 and PDE7B genes, residing on chromosome 6q23, and disease severity in b‑hemoglobinopathy patients, as well as the association between these variants with response to hydroxyurea (HU) treatment. Furthermore, we examined MAP3K5 expression in the context of high fetal hemoglobin (HbF) and upon HU treatment in erythroid progenitor cells from healthy and KLF1 haploinsufficient individuals. Materials & methods: For this purpose, we genotyped b‑thalassemia intermedia and major patients and healthy controls, as well as a cohort of compound heterozygous sickle cell disease/b‑thalassemia patients receiving HU as HbF augmentation treatment. Furthermore, we examined MAP3K5 expression in the context of high HbF and upon HU treatment in erythroid progenitor cells from healthy and KLF1 haploinsufficient individuals. Results: A short tandem repeat in the MAP3K5 promoter and two intronic MAP3K5 gene variants, as well as a PDE7B variant, are associated with low HbF levels and a severe disease phenotype. Moreover, MAP3K5 mRNA expression levels are altered in the context of high HbF and are affected by the presence of HU. Lastly, the abovementioned MAP3K5 variants are associated with HU treatment efficacy. Conclusion: Our data suggest that these MAP3K5 variants are indicative of b‑thalassemia disease severity and response to HU treatment.

Original submitted 24 September 2012; Revision submitted 4 February 2013

KEYWORDS: b‑thalassemia n haplotype n hydroxyurea n MAP3K5 n PDE7B n pharmacogenomics n sickle cell disease n transcription profiling

Christina Tafrali, Arsinoi Paizi, Joseph Borg, Milena Radmilovic, Marina Bartsakoulia, Emily Giannopoulou, Olga Giannakopoulou, Maja Stojiljkovic-Petrovic, Branka Zukic, Konstantinos Poulas, Eleana F Stavrou, Polyxeni Lambropoulou, Alexandra Kourakli,

Alexander E Felice, Adamantia Papachatzopoulou, Sjaak Philipsen, Sonja Pavlovic, Marianthi Georgitsi & George P Patrinos**Author for correspondence: University of Patras, School of Health Sciences, Department of Pharmacy, University Campus, Rion, GR-26504, Patras, Greece Tel.: +30 2610 969834 [email protected] For a full list of affiliations, please see page 483

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ReseaRch aRticle Tafrali, Paizi, Borg et al.

Pharmacogenomics (2013) 14(5)470 future science group

MAP3K5, also known as ASK1, is a mem‑ber of the MAPK family and, as such, a part of the MAPK pathway. This signaling cascade is one of the most important mechanisms for the cytoplasmic transduction of extracellu‑lar signals. MAP3K5 is activated by various stresses and by proinflammatory cytokines, like TNF‑a, which contribute to cellular apopto‑sis. PDE7B is a strong candidate gene given its high affinity and specificity for cAMP, which may play a role in fetal‑to‑adult globin gene switching [14].

In continuing the global effort of scruti‑nizing the 6q23 region for variants possibly accounting for the modulation of HbF produc‑tion, we investigated a possible association of SNPs residing within the MAP3K5 and PDE7B genes with elevated HbF levels in b‑thalassemia intermedia or major patients and normal (non‑thalassemic) individuals. We also examined a cohort of heterozygous SCD/b‑thalassemia patients who had undergone HU therapy, in order to clarify whether there is a correlation of these SNPs with HU treatment response in patients of Hellenic origin.

Materials & methods�n Subjects

We recruited 92 adult b‑thalassemia major patients, 11 b‑thalassemia intermedia patients, previously described [15,16], and 94 ethnically matched, adult, healthy (nonthalassemic) donors of western Greek origin for genotyp‑ing. The b‑thalassemia intermedia patients have long survival and have maintained a total amount of Hb between 7.7 and 11.2 g/dl, with‑out regular transfusions or without transfu‑sions at all, and, thus, these cases are typically hard to identify. In addition, these particular cases have been extensively analyzed in the past, and the elevated amount of HbF pro‑duced in them (ranging between 22 and 98%) can neither be attributed to mutations leading to a‑thalassemia, nor to nondeletional HPFH or deletional HPFH [15,16]. By contrast, b‑thal‑assemia major patients have profound anemia and are transfusion‑dependent cases, despite bearing HBB gene mutations also identified among the intermedia cases.

A lso, an independent sample of 35 SCD/b‑thalassemia compound heterozygous patients of western Greek origin, receiving HU as treatment regimen [17], was also recruited. For patients entering the HU treatment regi‑men, HbF levels are measured before the initial HU administration (i.e., baseline HbF) and

are, then, monitored weekly during the first month, every 15 days during the second month, then once per month for the first year and then once every 2–3 months (Supplementary table 1; www.futuremedicine.com/doi/suppl/10.2217/pgs.13.31). The increase in HbF levels among the patients that respond to HU treatment is not immediate and HbF rises gradually until a plateau is reached, which occurs between the fourth and 12th week of treatment. Patients are monitored occasionally for toxicity caused by HU and if no side effects occur, they carry on receiving the drug for life. SCD/b‑thalas‑semia compound heterozygous patients with plateau HbF levels below 20% after HU treat‑ment in vivo were considered ‘nonresponders’, while patients with plateau HbF levels rising over 20% after HU treatment in vivo were considered as ‘responders’. This cutoff point was chosen to make our ana lysis more strin‑gent. Informed consent was obtained from all patients in the study and was endorsed by the University of Patras and University of Patras Hospital ethical committees.

In addition, eight healthy adults of Maltese origin (low HbF status) and four KLF1 haploin‑sufficient cases with HPFH, bearing the KLF1 p.K288X mutation (high HbF status), previ‑ously reported [18], were used for differential gene‑expression ana lysis. Erythroid progenitor cells were isolated and cultured as previously described, with and without the presence of HU [19]. HbF quantification by HPLC was per‑formed using the VARIANT™ b‑thalassemia Short Program (Bio‑Rad, CA, USA).

�n Analysis of raw Affymetrix data & heat map generationData sets involving the comparison between healthy adults versus KLF1+/- individuals car‑rying the KLF1 p.K288X mutation (adult Mal‑tese participants) were already available from our group [18,19] and were used to determine the mRNA expression profile of candidate genes. From the Maltese participants, we selected the expression profiles from subjects that showed the lowest and highest HbF levels ex vivo (i.e., HbF level measurements conducted on the erythroid progenitor cells, isolated and cultured as described in [19]), considering HbF levels before and after HU treatment [19]. Qual‑ity filtering of probe sets and differential gene‑expression ana lysis was performed as previously described [19]. Shortlisted probe sets were clus‑tered using Cluster 3.0 [20] and visualized using Java TreeView Version 1.1.6r2 [21].

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MAP3K5 & b‑thalassemia disease severity & hydroxyurea treatment efficacy ReseaRch aRticle

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�n SNP & tagSNP selection & linkage disequilibrium ana lysisSNP selection was based on previous reports that these variants were significantly associated with HbF levels in response to HU treatment, mostly in SCD patients, addressed either in terms of HbF percentage, in the case of MAP3K5, as well as absolute HbF levels (g/dl), in the case of PDE7B [13]. This region presents particular interest given the fact that it has been previously linked as a QTL to altered HbF levels in SCD patients [11,22,23].

TagSNPs across the MAP3K5 and PDE7B genes were selected using the tagSNP picker program via the HapMap project (release 27, phase II + III, February 2009, NCBI36 assem‑bly) [101] and the linkage disequilibrium TAG SNP Selection (TagSNP) program hosted at the National Institute of Environmental Health Sci‑ences website [24,102]. For tagSNP selection for the CEU population, an aggressive multimarker tagging method was selected, with an R2 cutoff value of 0.8 and a minor allele frequency cutoff value of 0.05.

The linkage disequilibrium between ana‑lyzed variants in MAP3K5 and a MAP3K5 promoter short tandem repeat (STR) polymor‑phism was interrogated by utilizing the HapMap phase II + III (release 28, August 2010) data set for Caucasians (CEU) [25,101] and visualized on HaploView 4.2 [103].

�n Genotype ana lysisGenomic DNA was extracted from peripheral blood leukocytes [19], and determination of human HBB gene mutations was performed as previously described [26]. The primers used and the amplification conditions of the fragments bearing the rs9376230 and rs9483947 polymor‑phisms in the MAP3K5 gene (Figure 1), the frag‑ments bearing the rs2327669, rs11154849, and rs9376173 SNPs, variants in the PDE7B gene (Figure 1), as well as the proximal promoter region of MAP3K5, are provided in Supplementary table 2. Amplification was carried out according to the KAPA2G™ Fast HotStart protocol (KAPA Biosystems, MA, USA); detailed information per SNP amplification conditions is available upon request. For rs9483947 and rs9376173 SNPs, PCR products were subjected to PvuII (New England Biolabs, MA, USA) restriction endonuclease ana lysis at 37°C for 3 h, and HaeII (New England Biolabs, MA, USA) restriction endonuclease ana lysis at 37°C for 4.5 h, respec‑tively. Restriction fragments were visualized by 2% agarose gel electrophoresis after ethidium

bromide staining. For the rs11154849 SNP, an amplification‑refractory mutation system (PCR‑ARMS) assay, was developed, with two alterna‑tive forward primers hybridizing exclusively either with the wild‑type or the mutant allele. For the rs2327669 SNP, a tetraprimer PCR‑ARMS assay was developed according to Ye and coworkers [27,104], with two different primer sets used simultaneously, targeting and amplifying either the wild‑type or the mutant allele. For the rs9376230 SNP, PCR products were puri‑fied with PureLink® PCR purification columns (Life Technologies, NY, USA) and subjected to DNA sequence ana lysis on an ABI 3130 genetic analyzer using the BigDye® Terminator chem‑istry v.3.1, according to manufacturer instruc‑tions (Applied Biosystems, CA, USA). The geno typing method for each polymorphism, summarized in Supplementary table 2, was selected based on the presence or absence of restriction sites that could be targeted, the feasibility of developing a PCR‑ARMS assay based on the sur‑rounding sequence of each SNP and the presence of nearby polymorphic sites that would have to be taken into consideration for proper primer design; direct sequencing was eventually opted for only in the case of one variant for which other methods failed to produce satisfactory results.

A MAP3K5 promoter fragment that includes a STR (GCGCG)

4/5 variant, residing between posi‑

tions ‑51 and ‑27 (from transcription start site; Figure 1), was PCR‑amplified, and the products were purified and sequenced as described above.

�n Cell culture, transfection & reporter assayA MAP3K5 promoter fragment spanning 279 bp from positions ‑202 to +77 (from the transcription start site) was subcloned into the pCAT® (plas‑mid vector carrying CAT) basic reporter vector (Promega, WI, USA), containing either the four, (GCGCG)

4, or five, (GCGCG)

5, repeats. All

experimental procedures, including cloning, cell culture and transfection of K562 cells, as well as functional CAT assays, were performed as pre‑viously described by our group [28,29]. Results represent mean value ± standard deviation of independent experiments.

�n Statistical ana lysisHardy–Weinberg equilibrium and geno type/allele frequencies were evaluated using the c2 or Fisher’s exact tests and a p‑value of <0.05 was considered statistically significant. For the func‑tional CAT assays the statistical significance was determined by the Student’s t‑test, with



two‑tailed, paired samples, and a difference of p < 0.05 was considered significant.

Results�n Lack of the rs9483947 & rs9376230

SNPs in the MAP3K5 gene is associated with high HbF levelsIn this study, we wanted to evaluate the possible association between two polymorphic variants residing in intron 1 of MAP3K5 gene, namely rs9483947 and rs9376230 (Figure 1), with disease severity, by comparing genotype frequencies in b‑thalassemia intermedia (mild phenotype) versus b‑thalassemia major (severe pheno‑type) patients of Hellenic origin and healthy (non thalassemic) controls. In other words, we assessed whether these variants can be associated with a milder disease form, attributed to elevated HbF levels.

Even though the distribution of the genotypes of both SNPs in MAP3K5 did not differ signifi‑cantly among b‑thalassemia major and controls

(Figure 2a & 2b), we observed a complete lack of the homozygous mutant genotype (C/C for both SNPs) in the b‑thalassemia intermedia cases (Figure 2a & 2b). However, owing to small sample sizes, this difference did not reach statistical significance. For rs9483947, the homozygous mutant C/C genotype is not rare in CEU, since it varies between 10% in the HapMap CEU cohort, 13% in the Hellenic (nonthalassemic) controls (present study) and up to almost 15% in Toscans of Italy, who represent another CEU population [101]. Genotypic details for CEU indi‑viduals regarding rs9376230 are largely lacking from HapMap [101] and the 1000 Genomes [105] projects, but in the Hellenic control population, the homozygous mutant rs9376230 C/C geno‑type is present with a frequency of 13.8%, a fre‑quency almost equal to that of rs9483947. Inter‑estingly, we observed a lack of the homo zygous mutant genotypes among the b‑thalassemia intermedia samples for both MAP3K5 variants, but we cannot, at this point, conclude whether

HBS1L MYB PDE7B MAP3K5

∼632.5 kb

∼137.6 kb

∼361.5 kbForwardstrand

Reversestrand

Chr 6q23.3

PDE7B

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8394

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(GC

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G) 4/

5

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Ex1

Ex2

Ex1

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Figure 1. MAP3K5 and PDE7B locus on chromosomal region 6q23.3. Exons are represented by cylinders; the 5´‑UTR is shown by lighter cylinders and the coding exons by darker cylinders. Large genomic regions have been omitted for simplicity (dashed lines). SNPs are indicated by single triangles; the promoter short tandem repeat is indicated by a triple triangle. The distance between MAP3K5 and PDE7B variants is indicated in kb. The scheme follows the orientation by which the gene is organized on the forward or reverse chromosomal strand (orientation indicated by arrow). HBS1L and MYB loci are indicated upstream of PDE7B. All genomic locations and distances are based on Ensembl (Human Release 29, October 2012) [106]. Chr: Chromosome; Ex: Exon.



these particular MAP3K5 SNPs are associated with disease severity. Larger sample sizes need to be further analyzed to confirm this finding.

�n Correlation of the rs9483947 & rs9376230 SNPs in the MAP3K5 gene with response to HU treatment efficacySubsequently, we addressed the question whether the rs9483947 and rs9376230 SNPs could be considered as potential pharmacogenomic mark‑ers to predict HU therapy efficacy in compound SCD/b‑thalassemia patients. Previously, these variants had been significantly associated with HU response in SCD patients receiving HU,

in relation to percentage elevated HbF levels (p = 0.034 for rs9483947 and p = 0.036 for rs9376230) [13]. To this end, we compared allele and genotype frequencies in 35 SCD/b‑thalas‑semia compound heterozygote patients of Hel‑lenic origin, who had been systematically receiv‑ing HU and recorded for changes in HbF levels prior to and after HU treatment. We opted to cluster the samples as ‘nonresponders’ (lower than 20% HbF levels upon HU treatment) and ‘responders’ (higher than 20% HbF levels upon HU treatment), in order to make the comparison more stringent. This dichotomization has been previously applied in the study of a novel, recently identified HU target gene, namely KLF10 [19].

T/T T/C C/CGenotype Genotype

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MAP3K5 rs9376230

MAP3K5 rs9483947

Figure 2. Correlation of the rs9483947 and rs9376230 SNPs in the MAP3K5 gene with disease severity and with response to hydroxyurea treatment efficacy. (A) Correlation of the rs9483947 SNP with disease severity; (B) correlation of rs9376230 with disease severity; (C) correlation of rs9483947 with response to hydroxyurea treatment efficacy; and (D) correlation of rs9376230 with response to hydroxyurea treatment efficacy. The rare C/C genotypes are not detected among the b‑thalassemia intermedia cases with the milder phenotype, yet this difference did not reach statistical significance. On the other hand, the C/C genotypes are more common in the group of ‘responders’ to hydroxyurea treatment, a group of patients that seem to benefit more from hydroxyurea treatment (p = 0.026 for rs9483947 and p = 1.4 × 10‑4 for rs9376230). Numbers of individuals per group are indicated in parentheses.



In this comparison we observed a complete lack of the homozygous mutant C/C genotype for both SNPs in the group of nonresponders, whereas this genotype was present in the group of responders, with a frequency of 15.4 and 30% for rs9483947 and rs9376230, respec‑tively (Figure 2C & 2D). Our findings on com‑pound SCD/b‑thalassemia patients support a strong association for rs9483947 (p = 0.026) and rs9376230 (p = 1.4 × 10‑4) with HU treatment response based on the increase of HbF levels in responders. The frequency of the homozygous mutant C/C genotype for rs9376230 in the group of nonresponders deviates significantly from the frequency observed in the healthy, eth‑nically matched population (p = 0.017), and so does the homozygous mutant C/C genotype for rs9483947 (p = 0.01), indicating that this group of patients, at least, represents a separate cohort of individuals who are likely to benefit less from HU treatment.

�n Lack of rs2327669 SNP in the PDE7B gene is associated with high HbF levelsAnother gene of interest for exploring associa‑tion with HbF levels, is PDE7B, first, given its chromosomal location on the 6q22.3–23.2 QTL region (Figure 1) and, second, for having been previously associated with baseline HbF levels [22], as well as an increase in the absolute levels (g/dl) of HbF upon HU treatment in SCD patients [13] and, third, for PDE7B being

specifically involved in the cAMP pathway, shown to affect the HBG1 and HBG2 genes and HbF expression in adult b‑thalassemia interme‑dia cases [14]. Three PDE7B polymorphisms were studied in our b‑thalassemia intermedia and b‑thalassemia major samples versus healthy individuals, in order to explore whether they associate with a milder disease phenotype in Hellenic b‑thalassemia patients with abnormally high HbF.

Of interest is rs2327669, an intronic PDE7B variant, and a tagSNP for the CEU population, which was previously shown to correlate with a significant change (p = 0.041) in absolute HbF levels (g/dl) after HU treatment under a reces‑sive genetic model [13]. We observed a complete lack of the homozygous mutant C/C genotype in the b‑thalassemia intermedia cohort, com‑pared with b‑thalassemia major cohort and controls (Figure 3). The homozygous mutant C/C genotype is relatively common among CEU (18.6% according to HapMap [101]) and the Hellenic healthy population does not consider‑ably differ from the CEU cohort (12.8% in con‑trols; Figure 3); thus, it is of interest that the lack of rs2327669 associates with a milder disease phenotype and higher HbF levels, much like we observed for the rs9483947 and rs9376230 SNPs in the MAP3K5 gene, even though the results did not reach statistical significance between b‑thalassemia major and b‑thalassemia intermedia cases (p = 0.6). Neither of the other PDE7B SNPs analyzed showed patterns of geno‑type frequencies deviating from the expected (data not shown).

�n Differential MAP3K5 expression in low & high HbF‑expressing cells & upon HU treatmentUpon total RNA hybridization to the Affyme‑trix Human Genome U133 plus 2.0 (Affymetrix Inc., CA, USA) arrays, data were normalized and a comparison was performed among the samples from the healthy Maltese adults (low HbF status) and the four KLF1 haploinsufficient cases, bearing the KLF1 p.K288X mutation (high HbF status). A total of 244 probes repre‑senting 182 unique genes were identified in the context of low versus high HbF levels (Figure 4a) [18], whereas a total of 236 probes representing 175 unique genes were identified when compar‑ing these two groups of samples upon HU treat‑ment [19]. The lists of differentially expressed genes between the groups interrogated were gen‑erated based on a twofold change cutoff. We can hypothesize that these genes may be involved

G/G G/C C/C

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Controls (n = 94)

Figure 3. Genotyping analysis of the rs2327669 SNP in the PDE7B gene in b‑thalassemia major and b‑thalassemia intermedia samples versus healthy controls. The rare C/C genotype is absent in patients with high fetal hemoglobin levels and a milder phenotype; however, no statistical significance was reached. Numbers of individuals per group are indicated in parentheses.



in the developmental regulation of HbF and act either directly or indirectly, ultimately lead‑ing to increased HBG1 synthesis and, hence, high HbF levels. Interestingly, one of these genes, which is upregulated (>twofold change) in a high HbF context (HPFH individuals), is MAP3K5 (p = 0.03 for probe 203836_s_at and

p = 0.006 for probe 203837_at as indicated in Figure 4b & 4C, respectively). MAP3K5 was upreg‑ulated under the influence of HU in both groups but did not reach the predefined cutoff point of twofold change (fold change = 1.6; p = 0.012 for probe 203836_s_at and p = 0.007 for probe 203837_at) (Figure 4b & 4C).

Control Control_HU HPFH HPFH_HU

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Figure 4. Differential MAP3K5 gene‑expression analysis from human erythroid progenitor cells of Maltese individuals with and without the KLF1 p.K288X mutation. (A) Heat map with the 244 significantly differentially expressed probe sets between control (low fetal hemoglobin) versus HPFH (high fetal hemoglobin) samples [18]. Downregulated genes are shown in red, whereas upregulated genes are shown in green. (B) Differential MAP3K5 gene expression without (control and HPFH) and with (control_HU and HPFH_HU) the influence of HU (ex vivo), as depicted by the probe 203836_s_at. (C) Differential MAP3K5 gene expression without (control and HPFH) and with (control_HU and HPFH_HU) the influence of HU (ex vivo), as depicted by the probe 203837_at. HPFH: Hereditary persistence of fetal hemoglobin; HU: Hydroxyurea.



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MAP3K5 haplotype

Figure 5. Haplotype block analysis showing linkage disequilibrium between the examined MAP3K5 polymorphisms associated with low fetal hemoglobin levels. The rs9376230 polymorphism could not be depicted in linkage disequilibrium (LD) analysis, since no HapMap genotype data exist [101]. This variant is situated 3 kb upstream of rs9483947 and these two SNPs are known to be in perfect linkage (1000GENOMES:phase_1_CEU; [105]). The MAP3K5 promoter short tandem repeat polymorphism could not be depicted in LD analysis, since no HapMap genotype data exist; however, its most proximal variation (rs3734552) is shown, which is situated 1.07 kb downstream of the repeat. The value within each red diamond represents the degree of LD between polymorphisms, measured as D’ (A) or r2 (B), whereas light purple diamonds represent lack of LD.



�n Correlation of the MAP3K5 promoter STR with high HbF levelsA short 5́ ‑GCGCG‑3´ tandem repeat is present in the proximal promoter region of MAP3K5 (Figure 1). First, we sought to explore its frequency in the Hellenic population, since no frequency data currently exist for the CEU population. The repeat was identified in either four or five copies in tandem, with allele frequencies of 67.5 and 32.5% for the four‑ and five‑repeat alleles, respectively. We did not detect alleles bearing less than four or more than five repeats in our study sample.

Interestingly, in b‑thalassemia intermedia patients, this STR seems to be in nearly perfect linkage with the examined MAP3K5 polymor‑phisms (Figure 5a & 5b): The five‑repeats allele is linked with the rare rs9483947 C allele (Figure 5C) and the rare rs9376230 C allele (Figure 5D), forming a (GCGCG)

5–C–C haplotype, which is present

only in heterozygosity, with a frequency of 31.8%, whereas the wild‑type haplotype (GCGCG)

4–T–

A is present with a frequency of 68.2% (Figure 5e). The frequency of the rare (GCGCG)

5–C–C hap‑

lotype in b‑thalassemia intermedia patients is in concordance with its frequency among healthy controls (30.8%). However, in b‑thalassemia major patients, the (GCGCG)

5–C–C haplotype

is much more common, with a frequency of 60%, which statistically deviates significantly from b‑thalassemia intermedia patients (p = 0.04) and control samples (p = 0.003; Figure 5e). Thus, even though the intronic MAP3K5 SNPs can‑not be strongly associated with disease severity when considered individually, they seem to create a haplotype that is significantly enriched among the severe (b‑thalassemia major) cases.

�n Functional characterization of the MAP3K5 promoter STR variant on gene transcriptionSince the MAP3K5 promoter lacks a typical TATA or CAAT box transcription element, we wanted to assess the role of a GC‑rich polymorphic motif on its proximal promoter region, residing between positions ‑27 and ‑51 from transcription start site. We performed reporter assays, with constructs bearing either

four, (GCGCG)4, or five, (GCGCG)

5, repeats,

transfected in K562 cells, and measured reporter gene transcription, as previously described [28,29]. As shown in Figure 6, the presence of an extra GCGCG repeat results in a 6% decrease of MAP3K5 gene expression, when comparing it with the effect of four repeats.

DiscussionThe protective effect of HbF in SCD was first observed in affected newborns of African origin by Watson [30]. The regulation of HBG1 and HBG2 gene expression has, ever since, been of particular interest because it provides a rational basis for molecular strategies to reactivate the HbF genes in the adult in order to compensate for the absent or defective b‑globin chains.

A QTL region, namely 6q22.3–23.1, was previously shown to be associated with elevated HbF levels [11], and positional candidate genes, such as MYB and HBS1L in particular [12], have been the focus of intense research ever since. Also, previous studies have shown that genetic variation in the intergenic HBS1L–MYB region explains a significant percentage (~20%) of the overall trait variance in healthy European CEU [31], and 3–7% of the trait variance in African–American and Brazilian patients with SCD [32]. The intergenic HBS1L–MYB region contains a distal regulatory locus that could be important in hematopoiesis by controlling MYB expression, as well as several binding sites for the fundamen‑tal erythroid transcription factor GATA‑1 [33], and an enhancer‑like element that can modulate HbF levels via GATA‑1 binding [34]. Moreover, missense variants in the MYB gene were shown to further account for the heritable variation of HbF levels observed in SCD patients [35].

As it has also been suggested by others, the function of this HbF QTL is not necessarily mediated through direct transcriptional regula‑tion of MYB [34]. In spite of the 6q22–23 locus having been extensively studied for more than a decade now, with the main focus being on the MYB and HBS1L genes, as well as their inter‑genic region, insufficient attention had been paid in regard to other positional candidate genes in this QTL. For this reason, we set off

Figure 5. Haplotype block analysis showing linkage disequilibrium between the examined MAP3K5 polymorphisms associated with low fetal hemoglobin levels (cont.). The plots were generated by HaploView 4.2 [103]. (C) Schematic graph showing linkage between the MAP3K5 promoter repeat and rs9483947 SNP. (D) Schematic graph showing linkage between the MAP3K5 promoter repeat and rs9376230 SNP. (E) Haplotype ana lysis for MAP3K5 variants among different groups of samples. Please see color figure at www.futuremedicine.com/doi/pdf/10.2217/pgs.13.31. *Statistical significance (p = 0.04); **statistical significance (p = 0.003). 4R: Four repeats; 5R: Five repeats.

www.futuremedicine.com/doi/pdf/10.2217/pgs.13.31

www.futuremedicine.com/doi/pdf/10.2217/pgs.13.31



In addressing the role of one such promoter variant, we initially thought that an increasing number of GCGCG repeats would alter the promoter’s conformation, potentially interfer‑ing with the recruitment of alternative regula‑tory proteins, much like it has been observed with several repeat polymorphisms in other genes of pharmacogenomic interest. Prelimi‑nary functional experiments provided sup‑porting evidence in favor of this hypothesis in the case of MAP3K5, since the presence of five repeats only slightly decreases gene transcrip‑tion (Figure 6). We propose, however, consider‑ing our combined genotypic data (Figure 5e), that the (GCGCG)

5–C–C haplotype may confer a

disadvantage in MAP3K5 gene expression, with yet to be identified mechanisms. Alternatively, as explained before, this haplotype may high‑light the presence and, thus, segregation of a still unknown genetic variant linked to the haplo‑type, which could be identified by resequencing the whole‑genomic MAP3K5 locus, including regulatory regions, across the different patient groups.

There is increasing evidence that polymorphic elements like STRs and variable number tandem repeat (VNTR) polymorphisms differentially affect gene transcription (reviewed in [36]). One such example, which already serves as a phar‑macogenomic marker in clinical practice, is the UGT1A1 (TA)

n promoter STR polymorphism.

UGT1A1 encodes a Phase II drug‑metaboliz‑ing enzyme that inactivates the active SN‑38 metabolite of the anticancer drug irinotecan. It has been shown that UGT1A1 enzymatic activity is inversely correlated with the number of TA repeats [37], and a strong association for the (TA)

7 allele with irinotecan‑induced severe

toxicity in (TA)7 homozygous patients has been

established [38]. Two other genes whose promoter variants seem to affect their transcription levels are TYMS and TPMT. A higher number of a 28‑bp‑long VNTR in the TYMS promoter is correlated with higher mRNA levels and higher promoter activities in reporter gene assays [39,40], as well as with poorer disease outcome, but also with less drug‑related toxicity, in patients receiv‑ing 5́ ‑fluoro uracil or methotrexate, two drugs that target TYMS [40,41]. Last, but not least, our group has previously shown that a VNTR poly‑morphism in the TPMT gene promoter affects gene transcription in a VNTR architecture‑dependent manner and that acute lymphoblastic leukemia patients undergoing 6‑mercaptopurine treatment display a VNTR architecture‑depen‑dent response to 6‑mercaptopurine [28,29].

to explore the associations of polymorphic vari‑ants residing in the MAP3K5 and PDE7B genes with b‑thalassemia disease severity in patients of Hellenic origin. Our data show that the presence of the homozygous mutant C/C genotypes from two intronic MAP3K5 SNPs, together with a promoter STR variant, define an 11.3 kb haplo‑type block that is significantly more common in b‑thalassemia major patients, presenting with a severe disease phenotype. This finding warrants further investigation, in larger sample sizes, and of different ethnic origin, to clearly establish this association with disease severity. Moreover, an intronic PDE7B SNP associates with b‑thalassemia major and lower HbF levels.

Despite the fact that the variants analyzed by Ma and co‑workers were selected as tagSNPs based on genotype data from the Yoruban sam‑ple of the HapMap project (i.e., sub‑Saharan Africans), a comparison of the HapMap geno‑typic data between Yorubans and CEU for rs9483947 does not reveal significant differ‑ence in the genotype frequencies (p = 0.26), thus our results can be compared with their data [13]. However, a similar comparison cannot be performed for rs9376230, for which genotypic data across different ethnic groups are lacking in HapMap [101]. Regarding the PDE7B rs2327669 variant, however, results should be regarded with caution considering that CEU and Yor‑uban populations (as in [13]) vary significantly in the genotype frequencies (p = 1.3 × 10‑9).

The potential effects of the MAP3K5 and PDE7B SNPs in the amelioration of disease severity and response to HU treatment are expected to be indirect and, to date, remain largely elusive, since MAP3K5, in particular, has not been clearly associated with mecha‑nisms governing erythropoiesis. Because of their intronic location and the lack of an obvi‑ous mechanistic explanation for this associa‑tion, it can be only speculated, at this point, that these variants may be linked with other genetic elements that confer altered functional capabilities to either MAP3K5 mRNA or pro‑tein. However, MAP3K5 is a very large gene, spanning a region of 235.47 kb, comprising of 30 exons, and in‑depth resequencing of its cod‑ing and regulatory elements would be the only way to fully address this question for different groups of b‑thalassemia patients or responders versus nonresponders to HU treatment. In addi‑tion, it would be interesting to investigate the role of MAP3K5 gene promoter variants on the effects of MAP3K5 gene expression and response to HU treatment in the future.



HU is a well‑established agent for augment‑ing HbF levels in b‑hemoglobinopathy patients, and is the only one currently approved by the US FDA. HU is a ribonucleotide reductase inhibitor that, by interfering with the synthesis of DNA in the dividing late erythroid progeni‑tors, leads to a transient arrest of hematopoiesis; this is a phenomenon that enhances the prema‑ture commitment of early progenitor cells that still possess the primitive HbF program [42]. HU has been successfully used to induce HbF levels in SCD patients [43], and a number of tar‑get genes that respond to HU therapy in vivo in SCD patients have been reported, includ‑ing MAP3K5 and PDE7B [13]. Indeed, we also observed a significant association between two MAP3K5 variants and a better response to HU, as assessed by comparing HbF levels before and after drug treatment, in compound heterozygous SCD/b‑thalassemia cases of Hellenic origin. The homozygous mutant C/C genotypes for both SNPs were more common among respond‑ers versus nonresponders. These results, to our knowledge, are replicated for the first time in a patient cohort of compound heterozygotes with SCD/b‑thalassemia receiving HU. We believe that our findings provide further support to the notion that these variants may be associated with elevated HbF levels upon HU treatment, since these variants have been previously associated with a significant change in HbF levels upon HU treatment in SCD patients, when applying a recessive model of inheritance [13]. What is more, we observed a significant upregulation of MAP3K5 gene expression in cultured erythroid progenitor cells from haploinsufficient KLF1 p.K288X Maltese HPFH cases, who have abnor‑mally elevated HbF levels. MAP3K5 seems to be upregulated with and without the presence of HU (Figure 4b &4C); however, the significant fluctuation observed on the effect of HU when MAP3K5 gene is detected by either 203836_s_at or 203837_at probe warrants further experimen‑tation, in order to confirm the transcription pro‑file results. Overall, HbF induction seems to be associated with an increased MAP3K5 expres‑sion and potentially with an alteration in the p38 and the JNK pathway in which MAP3K5 is a significant upstream player.

MAP3K5 is a member of the p38 and the JNK MAPK pathway, involved in various sig‑naling cascades activated by survival and mito‑genic signals, or as a response to various cellular stresses [44]. In many cell types, MAP3K5 causes apoptosis in response to various stresses via the activation of the p38 MAPK [45], whereas in

other cell types it induces differentiation and survival, again via p38 activation (reviewed in [44,46]). Histone hyperacetylation by activation of the p38 MAPK pathway is linked to HBG1 and HBG2 gene induction via apicidin‑medi‑ated stimulation of HbF synthesis; apicidin is a potent histone deacetylase inhibitor, which, like 5‑azacytidine and HU, was shown to stimulate HbF synthesis in K562 erythroleukemia cells, with specificity for HBG1 and HBG2 mRNA expression via the p38 and not the ERK‑ or the JNK–MAPK pathways [47]. It seems plausible that HU may act on a similar mechanism to activate the p38–MAPK pathway, on a cascade initiated after the HU‑induced cellular stress is ‘identified’ by MAP3K5 molecules.

PDE7B encodes for a cAMP‑specific phos‑phodiesterase, which hydrolyzes cAMP to non‑cAMP molecules, therefore functioning as an important regulator of the cAMP sig‑naling in cells [48]. The cAMP pathway can block the HbF gene expression in K562 cells by increasing c‑Myb expression, unlike in adult erythroid progenitor cells that lack significant c‑Myb expression [49–51]. By contrast, cAMP production in adult human erythroid progeni‑tor cells is required for full induction of HbF, and of HBG1 and HBG2 mRNA, by HU [51].

90

95

100

105

110

115

pBL–CAT5 pBL–MAP3K5prom_4R pBL–MAP3K5prom_5R

Reporter constructs

Rep

ort

er g

ene

exp

ress

ion

leve

l (%

)

Figure 6. Functional analysis of the MAP3K5 promoter repeat polymorphism (GCGCG)4/5, using the CAT reporter assay. The empty vector pBL–CAT5 was used as a positive control in order to compare the effect of the construct bearing the four (pBL–MAP3K5prom_4R) and five (pBL‑MAP3K5prom_5R) repeats on reporter gene transcription. For details on the assay and the normalization approach used, please refer to [28,29]. Error bars are shown.



Moreover, the cAMP‑dependent pathway, the activity of which is augmented by multiple cytokines, such as erythropoietin, stem cell fac‑tor and TGF‑b, which are found increased in b‑thalassemia intermedia patients, plays a role in inducing HbF gene expression in adult eryth‑roblasts from b‑thalassemia intermedia patients [14]. These data suggest that the cAMP‑specific pathway may be involved in HbF induction in a controllable manner depending on the different erythroid cell populations (i.e., highly activated pathway in fetal liver erythroblasts, attenuated in adult erythroblasts) [14,49], and may play a role in fetal‑to‑adult globin gene switching, potentially via the involvement of other tran‑scription factors bound either to globin gene promoters or the locus control region [14]. It remains to be seen whether variants linked to the PDE7B rs2327669 polymorphism have a functional effect on phosphodiesterase 7B that could, potentially, interfere with cAMP

availability and the cAMP‑specific pathway, as well as whether, in the case of a defective PDE7B enzyme, other phosphodiesterases can compensate for its role in controlling cAMP levels.

Future perspectiveIt has been long known that HU treatment can induce HbF and, thus, improve laboratory parameters and ameliorate clinical complica‑tions of SCD; however, the exact mechanisms of action are still incompletely defined, and the reasons for HbF response being highly variable upon HU treatment remain largely elusive. Only a very limited number of studies have so far addressed the effect of HU on differential gene expression in erythroid progenitor cells, in an effort to identify potential markers of HU response. The genes that have been correlated with HbF, to date, include ARG1, ARG2, FLT1, HAO2 and NOS1, alongside BCL11A and the

Executive summary

Background

� Regulation of fetal globin gene expression, and thus, fetal hemoglobin (HbF) production has attracted great interest over the last 50 years.

� Elevated HbF levels in healthy adults have been associated with cis‑ as well as trans‑acting genetic elements, including quantitative trait loci on chromosomes Xp22, 6q23 and 2p16.1.

� Genetic variants on 6q23 genes, namely MAP3K5 and PDE7B, have been previously associated with elevated HbF levels in the context of response to hydroxyurea (HU) treatment in sickle cell disease patients.

Materials & methods

� We have genotyped b‑thalassemia major and b‑thalassemia intermedia patients, as well as healthy controls, in order to explore the possible association between b‑thalassemia disease severity and genetic variants in the MAP3K5 and PDE7B genes.

� For assessing the role of MAP3K5 genetic variants in relation to HU treatment efficacy, we utilized a group of sickle cell disease/b‑thalassemia compound heterozygous patients, categorized as ‘responders’ and ‘nonresponders’, based on HbF levels measured upon HU treatment.

� MAP3K5 mRNA‑expression levels were explored in a group of healthy versus KLF1 haploinsufficient Maltese adults, whose erythroid progenitor cells were grown with or without the addition of HU in the culture media.

� The effect of a short tandem repeat (STR) promoter variant on MAP3K5 gene transcription was functionally assessed by CAT assays with constructs bearing different repeat variants.

Results

� The role of MAP3K5 gene variants with HbF levels and disease severity: two MAP3K5 SNPs, namely rs9483947 and rs9376230, and a promoter 5‑bp STR, form a haplotype that defines a region of 11.3 kb, which is associated with low HbF levels and a severe b‑thalassemia phenotype.

� Differential MAP3K5 gene expression in the context of elevated HbF and upon HU treatment: MAP3K5 gene expression is significantly upregulated in the context of high HbF (hereditary persistence of fetal hemoglobin in Maltese individuals), and it also emerges as a HU target, given its upregulated expression in erythroid progenitor cells that were cultured in the presence of HU.

� Effect of a MAP3K5 promoter STR polymorphism on gene transcription: the presence of an extra copy of the GCGCG repeat in the proximal MAP3K5 gene promoter leads to decreased MAP3K5 gene expression, when comparing transcription levels conferred by the five versus the four repeats, but its significance in modulating disease severity as a sole determinant warrants further investigation.

Conclusion

� We have identified an 11.3 kb haplotype block on chromosomal region 6q23 that is associated with disease severity in b‑thalassemia patients. This region is upstream of the recently identified HBS1L–MYB locus, which affects fetal globin gene expression.

� Furthermore, we have shown that increased MAP3K5 gene expression is associated with elevated HbF levels and HU treatment. Whether alterations in MAP3K5 gene expression affect the p38 MAPK pathway, in which MAP3K5 acts as a significant upstream player, or an alternative pathway, in human erythroid cells, deserves further investigation.



ReferencesPapers of special note have been highlighted as:n of interestnn of considerable interest

1 Patrinos GP, Grosveld FG. Pharmacogenomics and therapeutics of hemoglobinopathies. Hemoglobin 32(1–2), 229–236 (2008).

2 Rochette J, Craig JE, Thein SL. Fetal hemoglobin levels in adults. Blood Rev. 8(4), 213–224 (1994).

3 Boyer SH, Dover GJ, Serjeant GR et al. Production of F cells in sickle cell anemia: regulation by a genetic locus or loci separate from the beta‑globin gene cluster. Blood 64(5), 1053–1058 (1984).

4 Garner C, Tatu T, Reittie JE et al. Genetic influences on F cells and other hematologic variables: a twin heritability study. Blood 95(1), 342–346 (2000).

nn� Large-scale twin heritability study among healthy individuals providing concrete evidence that F-cell levels (producing baseline levels of fetal hemoglobin [HbF]) are indeed genetically determined and that heritability explains the trait variance by 89%.

5 Gianni AM, Bregni M, Cappellini MD et al. A gene controlling fetal hemoglobin expression in adults is not linked to the non‑alpha globin cluster. EMBO J. 2(6), 921–925 (1983).

6 Gilman JG, Huisman TH. DNA sequence variation associated with elevated fetal G gamma globin production. Blood 66(4), 783–787 (1985).

7 Gilman JG, Johnson ME, Mishima N. Four base‑pair DNA deletion in human A gamma globin‑gene promoter associated with low A gamma expression in adults. Br. J. Haematol. 68(4), 455–458 (1988).

8 Oner C, Dimovski AJ, Altay C et al. Sequence variations in the 5´ hypersensitive site‑2 of the locus control region of beta S chromosomes are associated with different levels of fetal globin in hemoglobin S homozygotes. Blood 79(3), 813–819 (1992).

9 Merghoub T, Perichon B, Maier‑Redelsperger M et al. Dissection of the association status of two polymorphisms in the beta‑globin gene cluster with variations in F‑cell number in non‑anemic individuals. Am. J. Hematol. 56(4), 239–243 (1997).

10 Dover GJ, Smith KD, Chang YC et al. Fetal hemoglobin levels in sickle cell disease and normal individuals are partially controlled by an X‑linked gene located at Xp22.2. Blood 80(3), 816–824 (1992).

11 Craig JE, Rochette J, Fisher CA et al. Dissecting the loci controlling fetal haemoglobin production on chromosomes 11p and 6q by the regressive approach. Nat. Genet. 12(1), 58–64 (1996).

nn� Seminal study addressing whether HbF production is a multigenic trait, revealing that, in addition to the known genetic factors residing on the chromosome 11 b-globin locus, chromosome 6q harbors a novel locus regulating HbF levels.

12 Game L, Close J, Stephens P et al. An integrated map of human 6q22.3–q24 including a 3‑Mb high‑resolution BAC/PAC

contig encompassing a QTL for fetal hemoglobin. Genomics 64(3), 264–276 (2000).

13 Ma Q, Wyszynski DF, Farrell JJ et al. Fetal hemoglobin in sickle cell anemia: genetic determinants of response to hydroxyurea. Pharmacogenomics J. 7(6), 386–394 (2007).

n� One of the first studies to examine the pharmacogenomics of hydroxyurea, providing evidence for genetic determinants of hydroxyurea response in sickle cell disease patients.

14 Bailey L, Kuroyanagi Y, Franco‑Penteado CF et al. Expression of the gamma‑globin gene is sustained by the cAMP‑dependent pathway in beta‑thalassaemia. Br. J. Haematol. 138(3), 382–395 (2007).

15 Papachatzopoulou A, Kourakli A, Makropoulou P et al. Genotypic heterogeneity and correlation to intergenic haplotype within high HbF beta‑thalassemia intermedia. Eur. J. Haematol. 76(4), 322–330 (2006).

16 Papachatzopoulou A, Kaimakis P, Pourfarzad F et al. Increased gamma‑globin gene expression in beta‑thalassemia intermedia patients correlates with a mutation in 3´HS1. Am. J. Hematol. 82(11), 1005–1009 (2007).

17 Giannopoulou E, Bartsakoulia M, Tafrali C et al. A single nucleotide polymorphism in the HBBP1 gene in the human b‑globin locus is associated with a mild b‑thalassemia disease phenotype. Hemoglobin 36(5), 433–445 (2012).

very recently identified KLF10 [13,19,52,53]. Con‑tinuous and meticulous research is necessary in unraveling the mechanisms of action of HU and the reasons behind the observed differential drug response, in an effort to be able to provide SCD and b‑thalassemia patients with improved and tailor‑made therapy in the near future. Fur‑ther investigations are required in additional ethnic groups and larger patient cohorts to unravel the role of the MAP3K5 gene not only as a trans‑acting factor regulating HBB gene expression and HbF levels in adults but also as a potential pharmacogenomic marker for HU treatment efficacy.

AcknowledgementsA Athanassiadou and L Psiouri are gratefully acknowl-edged for their expertise and assistance with the samples of hemoglobinopathy patients, respectively. The authors are indebted to Z Zagoriti for her assistance with healthy control samples.

Financial & competing interests disclosureThis work was funded by a long-term EMBO fellowship (ALTF 71-2011) to J Borg and a Research Promotion Foun-dation of Cyprus grant (RPF ΠΔΕ046_02) and a European Commission grant (FP7-200754) to GP Patrinos. This work has been also funded by the FP7-REGPOT-2011-1 ‘SEE-DRUG’ project. The authors have no other relevant affilia-tions or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.

Ethical conduct of research The authors state that they have obtained appropriate insti-tutional review board approval or have followed the princi-ples outlined in the Declaration of Helsinki for all human or animal experimental investigations. In addition, for investi-gations involving human subjects, informed consent has been obtained from the participants involved.

ReseaRch aRticle Tafrali, Paizi, Borg et al.ReseaRch aRticle


18 Borg J, Papadopoulos P, Georgitsi M et al. Haploinsufficiency for the erythroid transcription factor KLF1 causes hereditary persistence of fetal hemoglobin. Nat. Genet. 42(9), 801–805 (2010).

19 Borg J, Phylactides M, Bartsakoulia M et al. KLF10 gene expression is associated with high fetal hemoglobin levels and with response to hydroxyurea treatment in b‑hemoglobinopathy patients. Pharmacogenomics 13(13), 1487–1500 (2012).

20 de Hoon MJ, Imoto S, Nolan J, Miyano S. Open source clustering software. Bioinformatics 20(9), 1453–1454 (2004).

21 Saldanha AJ. Java Treeview – extensible visualization of microarray data. Bioinformatics 20(17), 3246–3248 (2004).

22 Wyszynski DF, Baldwin CT, Cleves MA et al. Polymorphisms near a chromosome 6q QTL area are associated with modulation of fetal hemoglobin levels in sickle cell anemia. Cell. Mol. Biol. (Noisy-le-grand) 50(1), 23–33 (2004).

n� Thorough medium-throughput study of genetic elements residing in the 6q22.3–q23.2 quantitative trait locus, including MAP3K5 and PDE7B genes, revealing variations that may modulate baseline HbF levels in sickle cell disease patients.

23 Creary LE, Ulug P, Menzel S et al. Genetic variation on chromosome 6 influences F cell levels in healthy individuals of African descent and HbF levels in sickle cell patients. PLoS ONE 4(1), e4218 (2009).

24 Xu Z, Taylor JA. SNPinfo: integrating GWAS and candidate gene information into functional SNP selection for genetic association studies. Nucleic Acids Res. 37(Web Server issue), W600–W605 (2009).

25 Barrett JC, Fry B, Maller J, Daly MJ. Haploview: analysis and visualization of LD and haplotype maps. Bioinformatics 21(2), 263–265 (2005).

26 Papachatzopoulou A, Kourakli A, Stavrou EF et al. Region‑specific genetic heterogeneity of HBB mutation distribution in south‑western Greece. Hemoglobin 34(4), 333–342 (2010).

27 Ye S, Dhillon S, Ke X, Collins AR, Day IN. An efficient procedure for genotyping single nucleotide polymorphisms. Nucleic Acids Res. 29(17), e88 (2001).

28 Zukic B, Radmilovic M, Stojiljkovic M et al. Functional analysis of the role of the TPMT gene promoter VNTR polymorphism in TPMT gene transcription. Pharmacogenomics 11(4), 547–557 (2010).

29 Kotur N, Stankovic B, Kassela K et al. 6‑mercaptopurine influences TPMT gene

transcription in a TPMT gene promoter variable number of tandem repeats‑dependent manner. Pharmacogenomics 13(3), 283–295 (2012).

30 Watson J. The significance of the paucity of sickle cells in newborn Negro infants. Am. J. Med. Sci. 215(4), 419–423 (1948).

31 Thein SL, Menzel S, Peng X et al. Intergenic variants of HBS1L–MYB are responsible for a major quantitative trait locus on chromosome 6q23 influencing fetal hemoglobin levels in adults. Proc. Natl Acad. Sci. USA 104(27), 11346–11351 (2007).

32 Lettre G, Sankaran VG, Bezerra MA et al. DNA polymorphisms at the BCL11A, HBS1L–MYB, and beta‑globin loci associate with fetal hemoglobin levels and pain crises in sickle cell disease. Proc. Natl Acad. Sci. USA 105(33), 11869–11874 (2008).

33 Wahlberg K, Jiang J, Rooks H et al. The HBS1L–MYB intergenic interval associated with elevated HbF levels shows characteristics of a distal regulatory region in erythroid cells. Blood 114(6), 1254–1262 (2009).

34 Farrell JJ, Sherva RM, Chen ZY et al. A 3‑bp deletion in the HBS1L–MYB intergenic region on chromosome 6q23 is associated with HbF expression. Blood 117(18), 4935–4945 (2011).

35 Galarneau G, Palmer CD, Sankaran VG, Orkin SH, Hirschhorn JN, Lettre G. Fine‑mapping at three loci known to affect fetal hemoglobin levels explains additional genetic variation. Nat. Genet. 42(12), 1049–1051 (2010).

36 Georgitsi M, Zukic B, Pavlovic S, Patrinos GP. Transcriptional regulation and pharmacogenomics. Pharmacogenomics 12(5), 655–673 (2011).

37 Beutler E, Gelbart T, Demina A. Racial variability in the UDP‑glucuronosyl transferase 1 (UGT1A1) promoter: a balanced polymorphism for regulation of bilirubin metabolism? Proc. Natl Acad. Sci. USA 95(14), 8170–8174 (1998).

38 Hoskins JM, Goldberg RM, Qu P, Ibrahim JG, McLeod HL. UGT1A1*28 genotype and irinotecan‑induced neutropenia: dose matters. J. Natl Cancer Inst. 99(17), 1290–1295 (2007).

39 Horie N, Aiba H, Oguro K, Hojo H, Takeishi K. Functional analysis and DNA polymorphism of the tandemly repeated sequences in the 5´‑terminal regulatory region of the human gene for thymidylate synthase. Cell Struct. Funct. 20(3), 191–197 (1995).

40 Pullarkat ST, Stoehlmacher J, Ghaderi V et al. Thymidylate synthase gene

polymorphism determines response and toxicity of 5‑FU chemotherapy. Pharmacogenomics J. 1(1), 65–70 (2001).

41 Krajinovic M, Costea I, Chiasson S. Polymorphism of the thymidylate synthase gene and outcome of acute lymphoblastic leukaemia. Lancet 359(9311), 1033–1034 (2002).

42 Weatherall DJ. Towards molecular medicine; reminiscences of the haemoglobin field, 1960–2000. Br. J. Haematol. 115(4), 729–738 (2001).

43 Platt OS. Hydroxyurea for the treatment of sickle cell anemia. N. Engl. J. Med. 358(13), 1362–1369 (2008).

44 Matsukawa J, Matsuzawa A, Takeda K, Ichijo H. The ASK1–MAP kinase cascades in mammalian stress response. J. Biochem. 136(3), 261–265 (2004).

45 Tobiume K, Matsuzawa A, Takahashi T et al. ASK1 is required for sustained activations of JNK/p38 MAP kinases and apoptosis. EMBO Rep. 2(3), 222–228 (2001).

46 Hattori K, Naguro I, Runchel C, Ichijo H. The roles of ASK family proteins in stress responses and diseases. Cell Commun. Signal. 7, 9 (2009).

47 Witt O, Monkemeyer S, Rönndahl G et al. Induction of fetal hemoglobin expression by the histone deacetylase inhibitor apicidin. Blood 101(5), 2001–2007 (2003).

48 Conti M, Jin SL. The molecular biology of cyclic nucleotide phosphodiesterases. Prog. Nucleic Acid Res. Mol. Biol. 63, 1–38 (1999).

49 Inoue A, Kuroyanagi Y, Terui K, Moi P, Ikuta T. Negative regulation of gamma‑globin gene expression by cyclic AMP‑dependent pathway in erythroid cells. Exp. Hematol. 32(3), 244–253 (2004).

50 Kuroyanagi Y, Kaneko Y, Muta K et al. cAMP differentially regulates gamma‑globin gene expression in erythroleukemic cells and primary erythroblasts through c‑Myb expression. Biochem. Biophys. Res. Commun. 344(3), 1038–1047 (2006).

51 Keefer JR, Schneidereith TA, Mays A, Purvis SH, Dover GJ, Smith KD. Role of cyclic nucleotides in fetal hemoglobin induction in cultured CD34+ cells. Exp. Hematol. 34(9), 1151–1161 (2006).

52 Ware RE, Despotovic JM, Mortier NA et al. Pharmacokinetics, pharmacodynamics, and pharmacogenetics of hydroxyurea treatment for children with sickle cell anemia. Blood 118(18), 4985–4991 (2011).

53 Flanagan JM, Steward S, Howard TA et al. Hydroxycarbamide alters erythroid gene expression in children with sickle cell anaemia. Br. J. Haematol. 157(2), 240–248 (2012).

MAP3K5 & b‑thalassemia disease severity & hydroxyurea treatment efficacy ReseaRch aRticleReseaRch aRticle


�n Websites101 The HapMap Project.

http://hapmap.ncbi.nlm.nih.gov/index.html.en

102 The National Institute of Environmental Health Sciences. http://snpinfo.niehs.nih.gov/snpinfo/snptag.htm

103 HaploView 4.2 – Broad Institute. www.broadinstitute.org/scientific‑community/science/programs/medical‑and‑population‑genetics/haploview/haploview

104 PRIMER1: primer design for tetra‑primer ARMS‑PCR. http://primer1.soton.ac.uk/primer1.html

105 The 1000 Genomes Project. www.1000genomes.org

106 The Ensembl. www.ensembl.org

Affiliations � Christina Tafrali

University of Patras, School of Health Sciences, Department of Pharmacy, University Campus, Rion, Patras, Greece

� Arsinoi Paizi University of Patras, School of Health Sciences, Department of Pharmacy, University Campus, Rion, Patras, Greece

� Joseph Borg University of Malta, Faculty of Medicine & Surgery, Laboratory of Molecular Genetics, Msida, Malta

and University of Malta, Faculty of Health Sciences, Department of Applied Medical Science, Msida, Malta and Department of Cell Biology, Erasmus MC, Rotterdam, The Netherlands

� Milena Radmilovic Institute of Molecular Genetics & Genetic Engineering, University of Belgrade, Belgrade, Serbia

� Marina Bartsakoulia University of Patras, School of Health Sciences, Department of Pharmacy, University Campus, Rion, Patras, Greece

� Emily Giannopoulou University of Patras, School of Health Sciences, Department of Pharmacy, University Campus, Rion, Patras, Greece

� Olga Giannakopoulou University of Patras, School of Health Sciences, Department of Pharmacy, University Campus, Rion, Patras, Greece

� Maja Stojiljkovic-Petrovic Institute of Molecular Genetics & Genetic Engineering, University of Belgrade, Belgrade, Serbia

� Branka Zukic Institute of Molecular Genetics & Genetic Engineering, University of Belgrade, Belgrade, Serbia

� Konstantinos Poulas University of Patras, School of Health Sciences, Department of Pharmacy, University Campus, Rion, Patras, Greece

� Eleana F Stavrou University of Patras, Faculty of Medicine, Laboratory of General Biology, Patras, Greece

� Polyxeni Lambropoulou University of Patras Hospital, Faculty of Medicine, Department of Internal Medicine, Hematology Unit, Patras, Greece

� Alexandra Kourakli University of Patras Hospital, Faculty of Medicine, Department of Internal Medicine, Hematology Unit, Patras, Greece

� Alexander E Felice University of Malta, Faculty of Medicine & Surgery, Laboratory of Molecular Genetics, Msida, Malta

� Adamantia Papachatzopoulou University of Patras, Faculty of Medicine, Laboratory of General Biology, Patras, Greece

� Sjaak Philipsen Department of Cell Biology, Erasmus MC, Rotterdam, The Netherlands

� Sonja Pavlovic Institute of Molecular Genetics & Genetic Engineering, University of Belgrade, Belgrade, Serbia

� Marianthi Georgitsi University of Patras, School of Health Sciences, Department of Pharmacy, University Campus, Rion, Patras, Greece

� George P Patrinos University of Patras, School of Health Sciences, Department of Pharmacy, University Campus, Rion, Patras, Greece



http://snpinfo.niehs.nih.gov/snpinfo/snptag.htm

http://snpinfo.niehs.nih.gov/snpinfo/snptag.htm

www.broadinstitute.org/scientific-community/science/programs/medical-and-population-genetics/haploview/haploview



http://primer1.soton.ac.uk/primer1.html

genomic variation in the map3k5 gene is associated with β-thalassemia disease severity and...

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