Abstract

Although β thalassemia is considered to be a classic monogenic disease, it is clear that there is considerable clinical variability between patients who inherit identical β globin gene mutations, suggesting that there may be a variety of genetic determinants influencing different clinical phenotypes. It has been suggested that variations in the structure or amounts of a highly expressed red cell protein (alpha hemoglobin stabilizing protein [AHSP]), which can stabilize free α globin chains in vitro, could influence disease severity in patients with β thalassemia. To address this hypothesis, we studied 120 patients with Hb E-β thalassemia with mild, moderate, or severe clinical phenotypes. Using gene mapping, direct genomic sequencing, and extended haplotype analysis, we found no mutation or specific association between haplotypes of AHSP and disease severity in these patients, suggesting that AHSP is not a disease modifier in Hb E-β thalassemia. It remains to be seen if any association between AHSP and clinical severity is present in other population groups with a high frequency of β thalassemia. (Blood. 2004;103:3296-3299)

Introduction

Although β thalassemia results from reduced synthesis of the β globin chains of adult hemoglobin (α2β2), the pathophysiology is predominantly determined by excess free α globin chains which precipitate and cause oxidative damage in developing red cells (causing dyserythropoiesis) and in mature red cells (causing hemolysis).1,2  Up to a certain threshold, the erythron can cope with and compensate for excess α globin chains by known and unknown mechanisms. For example, individuals with the β thalassemia trait have somewhat abnormal red cells but no significant anemia.1  By contrast, most patients who are compound heterozygotes or homozygotes for β thalassemia have severe forms of anemia (thalassemia intermedia or major).1  However, within this group of patients who have a significant degree of anemia there is still considerable variability in hematologic and clinical severity and this is thought to be determined primarily by variations in the amounts of free α chains.1,3  In general, patients who inherit mild forms of thalassemia (β++ and β+) tend to be less severely affected than those with β0 thalassemia.4,5  Similarly, patients who inherit 2 α genes6  (-α/-α and --/αα) or 3 α genes7  (-α/αα) are often less severely affected than those with 4 (αα/αα), 5 (αα/ααα), or more α genes.8  Finally, those patients who coinherit any increased propensity to produce fetal (γ) globin chains in adult life9  (eg, the Xmn I+ allele) also tend to be less severely affected since γ chains can combine with excess α globin to produce fetal hemoglobin (Hb F; α2γ2), adding to the pool of functional hemoglobin and reducing unpaired α globin chains. However, even taking all of these factors into consideration it still appears that there are additional, as-yet-unidentified genetic determinants of the clinical outcome of patients with β thalassemia (reviewed in Weatherall3 ).

Recently, it has been shown that a highly expressed protein called alpha hemoglobin stabilizing protein (AHSP) can act as a chaperone for free α chains and prevent their precipitation.10,11  Mice lacking AHSP have abnormal red cell production and lifespan, thought to be caused by a relatively small excess of unchaperoned α globin chains.10  Furthermore, the phenotype of mice with β thalassemia intermedia is exacerbated by concomitant loss of AHSP.12  Based on these observations it has been proposed that alleles altering the levels or function of AHSP might account for some of the clinical variability observed in patients with β thalassemia.10,13  Here, we have fully characterized the AHSP gene in 120 Thai patients with clinically mild, moderate, and severe forms of Hb E-β thalassemia (a subtype of β thalassemia) to determine whether there are common alleles of AHSP in this population that modify the clinical presentation of β thalassemia. The influence of distinct alleles of AHSP were directly compared with the previously determined genetic determinants of severity, including the interacting β thalassemia alleles, the presence of α thalassemia, and the coinheritance of the high γ expression (Xmn I+) allele.

Study design

Clinical analysis

We recruited and obtained informed consent from 120 patients with Hb E-β thalassemia who have mild, moderate, or severe clinical phenotypes based on criteria previously defined by the Thalassemia International Federation14  (TIF). These criteria are onset of anemia, baseline levels of hemoglobin, symptoms of anemia, degree of splenomegaly and/or requirement to undergo splenectomy, transfusion requirement and/or treatment regimen (occasional transfusion, hypertransfusion with iron chelation or stem cell transplantation), and linear growth and height development during the follow-up period. In this study the average follow-up was 7.6 ± 0.79 years. This study was approved by a local ethical committee at Siriraj Hospital, Thailand.

Laboratory studies

The molecular basis of β thalassemia was determined using reverse dot-blot (RDB) hybridization,15  which simultaneously identifies 17 previously characterized β thalassemia mutations common in Thailand. DNA from patients with negative RDB results was subsequently analyzed by direct genomic sequencing of the β globin genes (2.4 kb). In addition, gap-polymerase chain reaction (PCR) analyses were performed to identify 3 previously characterized β globin deletions found in Thailand (3.5 kb, 619 bp, and 105 bp).1,16,17  Coinheritance of either deletional or nondeletional forms of α thalassemia and the Xmn I polymorphism were analyzed by standard protocols1,18  in all individuals. Two sets of PCR primers were used to amplify a 1.4-kb fragment of AHSP gene from genomic DNA. They are as follows: AHSP-F, 5′-tgcacagagagattcacgcacc-3′ (12179-12200) and AHSP-R, 5′-gcactggtctttattgaggtgtcag-3′ (13048-13072), producing an 895-bp coding sequence at annealing temperature 58°C; and AHSP-PF, 5′-cacgcttacaggtggcttatctg-3′ (11698-11720) and AHSP-PR, 5′-gtcacgattttcccaggttgg-3′ (12271-12251), for 574 bp of promoter and upstream region encompassing a polythimidine tract ((T)n) repeat at annealing temperature 55°C (coordinates according to the GenBank accession number AC106 730.2 are shown in parentheses). PCR products were sequenced using fluorescent-labeled dideoxyterminators (Applied Biosystems, Foster City, CA). The promoter region containing (T)n repeat PCRs were subcloned using pGEM-T Vector plasmid (Promega, Madison, WI) and DH5-α-competent cells (Invitrogen, Paisley, UK) before sequencing. Mapping analysis of AHSP loci on chromosome 16p was performed in genomic DNA from 30 patients (10 with mild and 20 with moderate and severe phenotypes) who have identical globin genotypes (β41/42E, αα/αα, Xmn I +/-). In each case, DNA was digested with HindIII and BglII and hybridized with AHSP genomic probe (895-bp PCR product).

Results and discussion

We successfully identified the β globin mutations in all patients studied; 115 had β0 or severe β+ alleles and 5 had mild β+ alleles (Table 1). Most individuals with Hb E-mild β+ thalassemia had a mild phenotype (n = 4) therefore they were excluded from further study. A summary of the β globin mutations, hematologic parameters, and clinical features in the 3 different clinical categories are presented in Table 1 and Table 2.

Table 1.

Identification of β thalassemia mutations in 120 patients with Hb E-β thalassemia


β globin mutation
 

No.
 

%
 
β0 or severe β+ thalassemias   
Codon 41/42 (-TTCT)   62   51.2  
Codon 17 (A/T)   18   15.0  
IVSII-654 (C/T)   18   15.0  
IVSI-5 (G/C)   5   4.1  
CD 71/72 (+A)   4   3.3  
IVSI-1 (G/T)   3   2.5  
CD 35 (C/A)   2   1.6  
CD 27/28 (+C)   1   0.8  
CD 95 (+A)   1   0.8  
Initiation codon (T/G)   1   0.8  
Mild β+ thalassemias   
−28 (A/G)   1   0.8  
CD19 (A/G)   2   1.6  
3.48-kb deletion   2   1.6  
Total
 
120
 
100
 

β globin mutation
 

No.
 

%
 
β0 or severe β+ thalassemias   
Codon 41/42 (-TTCT)   62   51.2  
Codon 17 (A/T)   18   15.0  
IVSII-654 (C/T)   18   15.0  
IVSI-5 (G/C)   5   4.1  
CD 71/72 (+A)   4   3.3  
IVSI-1 (G/T)   3   2.5  
CD 35 (C/A)   2   1.6  
CD 27/28 (+C)   1   0.8  
CD 95 (+A)   1   0.8  
Initiation codon (T/G)   1   0.8  
Mild β+ thalassemias   
−28 (A/G)   1   0.8  
CD19 (A/G)   2   1.6  
3.48-kb deletion   2   1.6  
Total
 
120
 
100
 

Most mutations are β0 or severe β+ thalassemia (96%). An initiation codon mutation (ATG → AGG), originally identified in a Chinese patient with β thalassemia major, was found for the first time in Thailand in this study.

Table 2.

Baseline hematology and demographic data in Hb E-β thalassemia patients with different clinical severity


 

Hb E-β thalassemia, n = 116
 
  
Parameters
 
Mild, n = 33
 
Moderate, n = 18
 
Severe, n = 65
 
No. of males (%)   17 (51)   11 (61)   26 (40)  
Age, y   9.46 ± 5.03   10.23 ± 4.78   8.06 ± 4.36  
Hb, g/dL   8.69 ± 0.90   6.81 ± 1.22   6.07 ± 0.98  
Hct, %   27.73 ± 2.63   22.96 ± 3.79   20.34 ± 2.94  
MCV, fL   60.43 ± 6.22   61.87 ± 5.44   66.57 ± 8.23  
MCH, pg   18.9 ± 2.70   19.22 ± 3.49   18.2 ± 2.97  
Retic, %   5.7 ± 2.06   7.9 ± 2.19   10.4 ± 5.13  
RDW, %   28.33 ± 3.18   32.57 ± 2.98   35.26 ± 4.44  
Hb F, g*  4.42 ± 1.51   3.35 ± 0.59   2.97 ± 0.85  
Hepatomegaly (%)   7 (21)   9 (50)   33 (51)  
Splenomegaly (%)   14 (42)   12 (66.7)   51 (78.5)  
Hx transfusion (%)   5 (15)  9 (50)   60 (92) 
Splenectomy (%)
 
2 (6)
 
1 (5.5)
 
14 (21.5)
 

 

Hb E-β thalassemia, n = 116
 
  
Parameters
 
Mild, n = 33
 
Moderate, n = 18
 
Severe, n = 65
 
No. of males (%)   17 (51)   11 (61)   26 (40)  
Age, y   9.46 ± 5.03   10.23 ± 4.78   8.06 ± 4.36  
Hb, g/dL   8.69 ± 0.90   6.81 ± 1.22   6.07 ± 0.98  
Hct, %   27.73 ± 2.63   22.96 ± 3.79   20.34 ± 2.94  
MCV, fL   60.43 ± 6.22   61.87 ± 5.44   66.57 ± 8.23  
MCH, pg   18.9 ± 2.70   19.22 ± 3.49   18.2 ± 2.97  
Retic, %   5.7 ± 2.06   7.9 ± 2.19   10.4 ± 5.13  
RDW, %   28.33 ± 3.18   32.57 ± 2.98   35.26 ± 4.44  
Hb F, g*  4.42 ± 1.51   3.35 ± 0.59   2.97 ± 0.85  
Hepatomegaly (%)   7 (21)   9 (50)   33 (51)  
Splenomegaly (%)   14 (42)   12 (66.7)   51 (78.5)  
Hx transfusion (%)   5 (15)  9 (50)   60 (92) 
Splenectomy (%)
 
2 (6)
 
1 (5.5)
 
14 (21.5)
 

All variances (mean ± standard deviation [SD]) were compared using either a 2-tailed student t test or the χ2 test as appropriate. P < .05 was taken to be statistically significant. Hx transfusion indicates that the patient's transfusion history includes occasional transfusion and regular transfusion.

*

Hb F levels (g) are calculated from the proportion of Hb F (%) from cellulose acetate electrophoresis and total hemoglobin levels. This result suggests a significant contribution of Hb F in ameliorating the clinical phenotype in the mild group (4.42 g vs 2.97 g in severe group, P < .05).

Although some patients received transfusion and/or even splenectomy (n = 2) from other hospitals before being referred to our center, our re-evaluation for their clinical severity indicated that they fell into the mild group.

Hematology data in 30 patients (46%) in the severe group who received high transfusion with iron chelation regimen were collected and averaged before the treatment started.

As expected, despite being compound heterozygotes for β0 (or severe β+) and βE mutations, approximately 50% of mildly affected patients coinherited either α thalassemia (-α or αTα) or were homozygous for the Xmn I polymorphism (+/+) (Table 3). None of these determinants were found in the severe group and only one patient (βCD17E, Xmn I +/-) who fell into the moderate group coinherited an α thalassemia allele (αCSα) (Table 3).

Table 3.

Genotypes of α thalassemia, Xmn I, and AHSP in patients with Hb E-β thalassemia


 

Previously known disease-modifying genotypes
 
  
 

AHSP haplotypes
 
    
Phenotypes
 
−α/αα
 
αTα/αα
 
Xmn I +/+
 
αα/αα
 
I/I
 
I/II
 
II/II
 
I allele frequency
 
II allele frequency
 
Hb E-β thalassemia          
Mild (all), n = 33 (%)   8 (24)   4 (12)   6 (18)   21 (51)   12 (36.4)   17 (51.6)   4 (12)   41 (62.2)   25 (37.8)  
Mild (unexplained), n = 17* (%)   0   0   0   17   5 (29.4)   10 (59)   2 (11.6)   20 (59)   14 (41)  
Moderate, n = 18 (%)   0   1 (6)   0   17 (94)   7 (39)   9 (50)   2 (11)   23 (64)   13 (36)  
Severe, n = 65   0   0   0   65 (100)   28 (43)   26 (40)   11 (17)   82 (63)   48 (37)  
Controls          
General population, n = 211 (%)   NA   NA   NA   NA   133 (63.1)   71 (33.6)   7 (3.3)   337 (80)   85 (20)  
Surin controls, n = 30 (%)
 
NA
 
NA
 
NA
 
NA
 
11 (36.7)
 
12 (40)
 
7 (23.3)
 
34 (57)
 
26 (43)
 

 

Previously known disease-modifying genotypes
 
  
 

AHSP haplotypes
 
    
Phenotypes
 
−α/αα
 
αTα/αα
 
Xmn I +/+
 
αα/αα
 
I/I
 
I/II
 
II/II
 
I allele frequency
 
II allele frequency
 
Hb E-β thalassemia          
Mild (all), n = 33 (%)   8 (24)   4 (12)   6 (18)   21 (51)   12 (36.4)   17 (51.6)   4 (12)   41 (62.2)   25 (37.8)  
Mild (unexplained), n = 17* (%)   0   0   0   17   5 (29.4)   10 (59)   2 (11.6)   20 (59)   14 (41)  
Moderate, n = 18 (%)   0   1 (6)   0   17 (94)   7 (39)   9 (50)   2 (11)   23 (64)   13 (36)  
Severe, n = 65   0   0   0   65 (100)   28 (43)   26 (40)   11 (17)   82 (63)   48 (37)  
Controls          
General population, n = 211 (%)   NA   NA   NA   NA   133 (63.1)   71 (33.6)   7 (3.3)   337 (80)   85 (20)  
Surin controls, n = 30 (%)
 
NA
 
NA
 
NA
 
NA
 
11 (36.7)
 
12 (40)
 
7 (23.3)
 
34 (57)
 
26 (43)
 

Nine α thalassemia alleles (—SEA, —THAI, —FIL, —MED, —(α)20.5, − α3.7, − α4.2, αCSα [Hb Constant Spring], and αPSα [Hb Paksé]) were analyzed using a PCR-based technique.18  NA indicates not applicable.

*

In the mild group (n = 33), 16 cases have either α thalassemia or Xmn I (+/+) or both (n = 2); therefore, 17 cases remain unexplained for their milder phenotype.

To determine whether AHSP might play a role in modifying the clinical severity of patients with β thalassemia, we sequenced the AHSP and its promoter region in 50 individuals with identical β globin genotypes (β41/42E, βCD17E, and β654E), normal α globin genotypes (αα/αα), and Xmn I polymorphism (+/- or -/-) including representatives from each of the different severity groups (17 mild, 13 moderate, and 20 severe). No nucleotide mutation that might obviously alter structure or function of AHSP was identified. However, we did find several single nucleotide polymorphisms (SNPs; Figure 1). These polymorphic sites have been noted previously when we sequenced a number of control samples (n = 20) from different ethnic origins to establish the genomic sequence of the AHSP gene (data not shown). These SNPs form distinct haplotypes with different numbers of (T)n repeats at the promoter (haplotypes I and II; Figure 1). By using associations at coordinate 12 390 (I with G and II with A), we could identify these 2 haplotypes using a mismatch PCR restriction fragment length polymorphism (RFLP) for a Mae III site (supplemental information is available through the Blood website; see the Supplemental Document link at the top of the online article). We analyzed the haplotype in all patients (mild, moderate, and severe) but found no statistically significant association between a particular haplotype and disease severity (χ2 test).

Figure 1.

Genomic structure of the AHSP gene and its single nucleotide polymorphisms (SNPs). The AHSP gene is located on chromosome 16 (Genbank accession number AC106 730.2), and the figure demonstrates the genomic structure of this gene containing 3 exons (black boxes). Five polymorphic sites (4 SNPs and one (T)n repeat) were identified, and these allow the construction of 2 major haplotypes, I and II. The minor haplotype Ia is similar to haplotype I for the T repeats18  and other SNPs except an A/G polymorphism in the intervening sequence (IVS) I (coordinate number 12 347 according to Genbank accession number AC106 730.2). Approximately 1.4 kb of the AHSP gene (shaded box) was sequenced in 50 individuals with Hb E/β thalassemia.

Figure 1.

Genomic structure of the AHSP gene and its single nucleotide polymorphisms (SNPs). The AHSP gene is located on chromosome 16 (Genbank accession number AC106 730.2), and the figure demonstrates the genomic structure of this gene containing 3 exons (black boxes). Five polymorphic sites (4 SNPs and one (T)n repeat) were identified, and these allow the construction of 2 major haplotypes, I and II. The minor haplotype Ia is similar to haplotype I for the T repeats18  and other SNPs except an A/G polymorphism in the intervening sequence (IVS) I (coordinate number 12 347 according to Genbank accession number AC106 730.2). Approximately 1.4 kb of the AHSP gene (shaded box) was sequenced in 50 individuals with Hb E/β thalassemia.

Interestingly, the allele frequency of haplotype II is higher in the patient groups compared with a group of nonthalassemic controls obtained in Bangkok, which represents the general Thai population. This might reflect different ethnic origins of these 2 groups. It has been proposed that Hb E has a single origin in Southeast Asia in the area adjoining Thailand, Cambodia, and Laos (the so-called “Hb E Triangle”).19  Hence, we analyzed 30 healthy nonthalassemic individuals (normal Hb A-A2) from Surin, a province located in the Hb E-Triangle where 50% of population are carriers of Hb E, to determine whether the AHSP haplotype II might occur at a high frequency in this region. This population has AHSP allele frequencies (0.43) and haplotype combinations that are more similar to our patient groups (Table 3), suggesting that both Hb E and AHSP haplotype II may occur independently at high frequencies in this subpopulation. This finding highlights the importance of obtaining properly selected control groups for genetic-association studies in Southeast Asians, since the population admixture might give rise to a false-positive association if the origin of the subgroup analyzed is not clearly identified.

Finally, we excluded the possibility of any large deletions, duplications, or rearrangements, which may result in any differences in AHSP expression in these cases by comprehensive gene mapping analysis (data not shown).

The amounts of free α globin chains appear to be a major factor contributing to differences in hematologic and clinical severity in patients with β thalassemia. This study confirmed that coinheritance of α thalassemia alleles and increased γ globin expression (associated with the Xmn I+ polymorphism) ameliorate clinical severity.20  AHSP is a good candidate for modifying β thalassemia since it selectively binds and stabilizes free α chains11  and is highly expressed in hemoglobin-synthesizing erythroid precursors.10  The ability of reduced AHSP gene dosage to exacerbate the β thalassemia phenotype was demonstrated recently in mice.12  Moreover, Galanello et al21  reported that severity of β thalassemia is concordant with reduced AHSP mRNA expression in 2 unrelated Sardinian families. The current study demonstrates that the AHSP gene does not modulate clinical severity in Thai patients with Hb E-β thalassemia. However, since we were unable to examine AHSP protein or mRNA levels, it remains possible that reduced AHSP expression by genetic or epigenetic factors unlinked to its gene impact the severity of β thalassemia in the patients described here. In order to fully define the role of AHSP as a modifier of β thalassemia, it will be important to further examine its gene inheritance and expression in various patient populations.

Prepublished online as Blood First Edition Paper, January 8, 2004; DOI 10.1182/blood-2003-11-3957.

Supported by the Medical Research Council, Oxford, United Kingdom (V.V. and D.R.H.), and by the Siriraj-Thalassemia Research Program, Bangkok, Thailand (W.C. and V.S.T.).

The online version of the article includes a data supplement.

The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 U.S.C. section 1734.

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