LETTER TO THE EDITOR

Hb Famagusta—analysis of a novel δ-globin chain variant[HBD:c.60C>A] in four families with diverse globin genotypes

Carsten W. Lederer & Eleni Pavlou & Christiana Makariou & Georgia Hadjilambi &Nicoletta Andreou & Michael Hadjigavriel & Annita Kolnagou & Maria Sitarou &

Soteroulla Christou & Marina Kleanthous

Received: 6 December 2013 /Accepted: 18 December 2013# Springer-Verlag Berlin Heidelberg 2014

Dear Editor,Detection of HbA2 levels is central to the diagnosis of thalas-semias, so that identification of δ-globin alleles in a popula-tion is critical to national disease prevention and control [1].Here, we report a novel δ-globin gene variant, HbA2[δN19K]or Hb Famagusta, distinguishable from normal δ-globin byhigh-performance liquid chromatography (HPLC) and cellu-lose acetate electrophoresis (CAE), presenting in a normalgenetic background and in combination with three indepen-dent α- and β-thalassemia mutations in four distinct GreekCypriot families, respectively.

Analyses were performed as detailed in ESMSupplementarymaterials and methods. All subjects have given full informedconsent for inclusion in this study. The index case (M1) wasdetected amongst study subjects analyzed between 2011 and2013 as part of the thalassemia prevention program of the

Republic of Cyprus (Table 1). M1 presented with hematologyindicative of thalassemia carrier status, while HPLC analysisshowed abnormally low HbA2 and an additional elution peakafter and baseline-separated from HbA2. Subsequent CAE con-firmed the presence of an additional hemoglobin species,HbA2′, concluded to be a novel variant.

Comprehensive globin sequence analysis and gap-PCR ofthe index case M1 revealed heterozygosity (a) for the commonα-globin MEDI deletion [2], (b) for a known single-nucleotidepolymorphism in normal β-globin [3], and (c) for a hithertounreported δ-globin mutation (Fig. 1a), thus diagnosing α-thalassemia carrier status and providing the molecular basisof the novel δ-globin variant. This increased the number ofδ-globin variants in Cyprus [4] to seven, with 12 variant orδ-thalassemia alleles in total. The detected HBD:c.60C>Amutation introduces a codon change AAC→AAA (asparagineto lysine) at residue 19 of the mature δ-globin protein, shiftingits predicted isoelectric point (pI) from 7.84 to 8.55. M2, thefather of M1, was then found to carry the mutation in anotherwise normal globin background and thus allowed detec-tion and separation of the novel variant in the basic heterozy-gote state by HPLC (Fig. 1b) and CAE (Fig. 1c). The mutationwas subsequently identified in fivemore individuals from threeadditional and apparently unrelated Cypriot families (Table 1)and has been deposited in the IthaGenes database with IthaID2292 (www.ithanet.eu; [5]). Common to three of the fourfamilies was ancestry from the Famagusta area of Cyprus,prompting the designation Hb Famagusta for the novelvariant. Of the seven carrier individuals, four were withoutadditional globin mutations, one also carried the αα/--MEDI,one the αα/-α3.7, and one the β-globin IVS-Ι-110(G>A)mutation.

The HbA2[δN19K] amino acid substitution affects asolvent-exposed turn between two α-helices of δ-globin, re-mote from any binding partner within the known structure ofthe HbA2 heterotetramer [6] (Fig. 1d), and changes a

Electronic supplementary material The online version of this article(doi:10.1007/s00277-013-1996-6) contains supplementary material, whichis available to authorized users.

C. W. Lederer (*) : E. Pavlou :M. KleanthousThe Cyprus Institute of Neurology and Genetics,Nicosia 1683, Cypruse-mail: Lederer@cing.ac.cy

C. Makariou :G. Hadjilambi :N. Andreou : S. ChristouMinistry of Health – Thalassemia Centre, Archbishop Makarios IIIHospital, Nicosia, Cyprus

M. HadjigavrielMinistry of Health – Thalassemia Centre, Limassol General Hospital,Limassol, Cyprus

A. KolnagouMinistry of Health – Thalassemia Centre, Paphos General Hospital,Paphos, Cyprus

M. SitarouMinistry of Health – Thalassemia Centre, Archbishop Makarios IIIHospital, Larnaca, Cyprus

Ann HematolDOI 10.1007/s00277-013-1996-6

Table 1 Study subjects. Seven individuals from four different Cypriot families (as indicated by alternating italics) are heterozygous for the novelδ-globin[N19K] variant

Subject Place ofresidence

RBC(106/μL)

Hb(g/dL)

MCV(fL)

MCH(pg)

CAE Genotype

HbA2

(%)HbA2′(%)a

HbA2+2′(%)

α-globingene

β-globingene

M1 Kaimakli (N) 6.98 14.0 60.5 20.3 1.3 0.5c 1.8 αα/--MEDI Ν/Nb

M2 Pallouriotissa (N) 5.19 14.9 86.3 28.7 1.4 0.9c 2.3 αα/αα N/N

F2 Kato Pafos (P) 4.45 13.0 88.3 29.2 1.3 0.8c 2.1 αα/αα N/Nb

F3 Kato Pafos (P) 5.74 11.6 62.7 20.2 3.1 1.8c 4.9 αα/αα N/IVS-I-110(G>A)

F1 Choletria (P) 4.93 12.3 74.8 24.9 1.3 0.5c 1.8 αα/-α3.7 N/Nb

M3 Aradippou (L) 4.79 14.4 91.2 30.1 1.4 0.9c 2.3 αα/αα N/N

F4 Larnaca (L) 4.61 13.3 88.5 28.9 1.5 0.9c 2.4 αα/αα N/N

a HbA2′ hemoglobin levels extremely significantly (p value=6.3×10−4 ) below those of same-person normal HbA2, by pairwise comparison across allHbA2′ carriers

N district of Nicosia, P district of Pafos, L district of Larnaca

αα/αα, N/N—diagnosed homozygous normal according to hematological sample parameters, respectively, for the α-globin locus after exclusion of the-α3.7 mutation and for the β-globin locusb Status confirmed by standard detection assays for β-globin mutationsc HbA2′ levels highly or extremely significantly below haploid-corrected HbA2 levels of the respective reference population, as specified in ESM Fig. S1

Fig. 1 Characterization of the HbA Famagusta mutation and variant. aSequencing revealed anAAA>AAC codon change (N19K, indicated by anarrow) for δ-globin underlying the observation of a novel variant. bCation-exchange HPLC for all carriers (displayed for M2) showed an abnormallylow level of HbA2 (at 2.91 min of retention time) and a baseline-separatedadditional peak (at 3.37 min) of unknown identity, indicated as HbA2′. cCellulose acetate electrophoresis confirmed the HPLC-based detection of a

novel hemoglobin variant, HbA2′ (displayed for M2). dHighlighting N19in the knownHbA2 structure (based on PDB 1si4 [6]) showed its marginal,non-helical position in the tetramer. Theα- and δ-globin chains (α1,α2andδ1, δ2) are displayed as flat ribbon diagrams in gray and blue, respectively,with random coils shown in cyan, turns in green, and residueN19 highlight-ed in mauve and displayed as a ball-and-stick diagram in each δ-globinchain. Heme groups are shown as stick diagrams in red

Ann Hematol

hydrophilic uncharged for a (hydrophilic) positively chargedresidue, consistent with our structure-based prediction of anunusually stable variant monomer (ESM Table S1) [7].Predicted structural changes of the variant tetramer againstHbA2 gave a root-mean-square deviation (RMSD) of 0.275 Å,which fell within the lower RMSD range predicted, e.g., forstructural variants of HbA [8]. Moreover, HbA2 and HbFamagusta had extremely similar predicted free energies(−34.2 vs. −41.0 kcal/mol) and stabilities (−58.42 vs.−65.3 kcal/mol), and consistent with nature and position ofthe amino acid change, predicted differences in energies forinteraction of α- and δ-globin monomers (−24.8 vs.−25.02 kcal/mol), and of α1δ1 and α2δ2 dimers (−14.6 vs.−14.5 kcal/mol) which were within the margin of error [9].Therefore, none of these predictions indicated inherent instabilityof either the variant δ-globin monomer or its heterotetramer. Yet,pairwise comparison of measured HbA2 and Hb Famagustalevels for all carriers showed extremely significant reduction ofvariant compared to normal HbA2 (p value=6.3×10

−4), indicat-ing lower stability of variant monomer or heterotetramer(Table 1). Haploid HbA2 levels for all carriers correspondedperfectly with averages of matched control populations (r2=0.999) from the national carrier database. Conversely, evaluatingHb Famagusta levels against the distribution of haploid-equivalent HbA2 levels of controls (ESM Fig. S1) establishedhighly to extremely significantly reduced variant stability foreach carrier and moreover showed that an inferred decrease offreeα-globin (fromβ/β0 over N/N andαα/-α3.7 toαα/--MEDI)was paralleled by exacerbated reduction ofHb Famagusta. Givennormal (haploid) levels of HbA2 in all backgrounds, the ratio ofHbA2′:HbA2 thus fell from 0.6 in β-thalassemia-carrier andnormal backgrounds to 0.4 in α-thalassemia carriers. Theseexperimental observations match two potentially concurrent sce-narios, specifically as follows: (a) δ-globin[N19K] has loweredaffinity toα-globin, so that at limiting concentrations ofα-globininα-thalassemia carriers, normal δ- andβ-globin outcompete thevariant as α-globin binding partners; and (b) δ-globin[N19K] asmonomer is less stable (inherently or through targeted proteoly-sis) than normal δ-globin and, while stabilized byα-globin in thevariant tetramer, is degraded more quickly than normal δ-globinat limiting α-globin levels. Scenario b and targeted degradationof the variant monomer would moreover reconcile measure-ments with structure-based predictions for Hb Famagusta.

Acknowledgments This work was in part funded by the EuropeanUnion FP7 THALAMOSS project (#306201). The authors thank allstudy subjects for their participation, Eleni Karitsi for technical assistance,and Coralea Stephanou and Petros Kountouris for critical revision of themanuscript.

Conflict of interest The authors declare that they have no conflict ofinterest.

References

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2. Baysal E, Kleanthous M, Bozkurt G, Kyrri A, Kalogirou E,Angastiniotis M, Ioannou P, Huisman TH (1995) alpha-Thalassaemiain the population of Cyprus. Br J Haematol 89(3):496–499

3. Spence SE, Pergolizzi RG, Donovan-Peluso M, Kosche KA, DobkinCS, Bank A (1982) Five nucleotide changes in the large interveningsequence of a beta globin gene in a beta+ thalassemia patient. NucleicAcids Res 10(4):1283–1294

4. Pavlou E, Phylactides M, Kyrri A, Kalogerou E, Makariou C,Georgiou I, Kleanthous M (2006) Delta-thalassemia in Cyprus.Hemoglobin 30(4):455–462

5. Lederer CW, Basak AN, Aydinok Y, Christou S, El-Beshlawy A,Eleftheriou A, Fattoum S, Felice AE, Fibach E, Galanello R,Gambari R, Gavrila L, Giordano PC, Grosveld F, Hassapopoulou H,Hladka E, Kanavakis E, Locatelli F, Old J, Patrinos GP, Romeo G,Taher A, Traeger-Synodinos J, Vassiliou P, Villegas A, Voskaridou E,Wajcman H, Zafeiropoulos A, Kleanthous M (2009) An electronicinfrastructure for research and treatment of the thalassemias and otherhemoglobinopathies: the Euro-Mediterranean ITHANET project.Hemoglobin 33(3):163–176. doi:10.1080/03630260903089177

6. Sen U, Dasgupta J, Choudhury D, Datta P, Chakrabarti A, ChakrabartySB, Chakrabarty A, Dattagupta JK (2004) Crystal structures of HbA2and HbE and modeling of hemoglobin delta 4: interpretation of thethermal stability and the antisickling effect of HbA2 and iden-tification of the ferrocyanide binding site in Hb. Biochemistry43(39):12477–12488. doi:10.1021/bi048903i

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Ann Hematol

Ann Hematol

DOI 10.1007/s00277-013-1996-6 ESM1 1

Hb Famagusta—analysis of a novel δ-globin chain variant

[HBD:c.60C>A] in four families with diverse globin genotypes

Carsten W. Lederer Eleni Pavlou Christiana Makariou Georgia Hadjilambi

Nicoletta Andreou Michael Hadjigavriel Annita Kolnagou Maria Sitarou

Soteroulla Christou Marina Kleanthous

Electronic supplementary material 1

Supplementary materials and methods

Hematological analyses

Hematological studies were performed using routine methods.

Hemoglobin analysis was performed by cation-exchange high

performance liquid chromatography (HPLC), using the β-

thalassemia VARIANTTM

2 program (Bio-Rad Life Sciences), and

by cellulose acetate electrophoresis (CAE), using standard

procedures (www.ithanet.eu/ithapedia, Cellulose acetate

electrophoresis; [1]) and equipment (Shandon).

Molecular analyses

Nucleotide and amino-acid numbers are given with RefSeq

NG_000007 as reference for genomic DNA positions and

NM_000519.3 as reference for human HBD cDNA and predicted

amino-acid sequences. By convention in the hemoglobin field and

contrary to guidelines of the Human Genome Variation Society

(HGVS), amino-acid references count the initiating methionine as

amino acid 0 (zero) and therefore differ by -1 from corresponding

HGVS position references.

Where indicated through abnormal hematological indices, samples

were investigated for α-globin deletions by gap-polymerase chain

reaction (PCR) (www.ithanet.eu/ithapedia, Gap-PCR; [1]) or for

β-globin mutations by Sanger sequencing of PCR-amplified

genomic DNA. Mutations routinely tested with abnormal

hematological parameters were --MEDI, --MEDII, –α20.5

, –α3.7

α–

5ntα, α

PolyA2α where α-thalassemia was indicated [2] and IVS-I-

110(G>A), IVS-II-745(C>G), IVS-I-6(T>C), IVS-I-1(G>A) where

β-thalassemia was indicated [3]. Similarly, mutations in δ-globin

were detected by sequencing of two overlapping PCR fragments.

The first of these fragments (amplified using primers δ1F: 5’-

TACATTCCACTATATTAGCC-3’ and δ6R: 5’

CAGTATTCTATGCCTCTCAT-3’) spanned 1161 bp including

5’ UTR , exon 1 and exon 2 and extending from -328 to +814

relative to the translational start site (TSS) as position (+1). The

second of these fragments (amplified using primers δ5F: 5’-

TGCATACCAGCTCTCACCTG-3’ and δ8R: 5’-

CAGGAACCTTCTTACACACC-3’) was a 947-bp fragment,

extending from +707 to +1653 relative to the TSS. Sequencing

reactions (Invitrogen, BigDye Terminator v.1.1 cycle sequencing

kit) were performed using primers δ1F, δ15S (5’-

AACCAACCTGCTCACTGGAG-3’) and δ17S (5’-

ATTTATGCTGATGGGAATAAC-3’) and were acquired using a

3130xl Genetic Analyzer (Applied Biosystems Hitachi).

Bioinformatics and statistical analyses

In silico protein-structure and stability analyses were based on

RCSB PDB entry 1si4 of the relaxed (R2) state of normal HbA2

[4]. The protein display and energy calculations for normal and

variant HbA2 were performed in YASARA [5], using the FoldX

forcefield plugin [6] and the POV-Ray rendering tool

(www.povray.org). Structure-dependent comparative energy and

stability calculations for tetra- and dimers by FoldX and for δ-

globin variant monomers by iStable [7] assumed a temperature of

37 °C and pH of 7. Predicted pI was calculated using the ExPasY

Compute pI/Mw tool [8].

The difference in steady-state HbA2 to Hb Famagusta levels for

the population of carrier individuals was assessed by pairwise two-

tailed t-test using Office 2010 Excel (Microsoft). The significance

of changes in haploid-equivalent normal HbA2 and in Hb

Famagusta was assessed for each test case against a reference

population of individuals matched for gender and α- and β-globin

status, assuming a normal distribution of control values and using

the Excel NORM.DIST function. The squared correlation

coefficient r2 for the correlation of same-sample HbA2 and HbA2′

levels were calculated using the Excel RSQ function. The HbA2

distribution for all samples (Fig. 5) was plotted as a scatter dot plot

with mean and standard deviation using Prism 5 (Graphpad

Software, Inc.). By convention and for any of the statistical tests

shown in this study, a p-value below 0.05, 0.01 and 0.001 is

referred to as representing a significant, highly significant and

extremely significant, respectively, difference of test values to

control values.

References

1. Lederer CW, Basak AN, Aydinok Y, Christou S, El-Beshlawy

A, Eleftheriou A, Fattoum S, Felice AE, Fibach E, Galanello

R, Gambari R, Gavrila L, Giordano PC, Grosveld F,

Hassapopoulou H, Hladka E, Kanavakis E, Locatelli F, Old J,

Patrinos GP, Romeo G, Taher A, Traeger-Synodinos J,

Ann Hematol

DOI 10.1007/s00277-013-1996-6 ESM1 2

Vassiliou P, Villegas A, Voskaridou E, Wajcman H,

Zafeiropoulos A, Kleanthous M (2009) An electronic

infrastructure for research and treatment of the thalassemias

and other hemoglobinopathies: the Euro-mediterranean

ITHANET project. Hemoglobin 33 (3):163-176.

doi:10.1080/03630260903089177

2. Baysal E, Kleanthous M, Bozkurt G, Kyrri A, Kalogirou E,

Angastiniotis M, Ioannou P, Huisman TH (1995) alpha-

Thalassaemia in the population of Cyprus. Br J Haematol 89

(3):496-499

3. Baysal E, Indrak K, Bozkurt G, Berkalp A, Aritkan E, Old JM,

Ioannou P, Angastiniotis M, Droushiotou A, Yuregir GT, et al.

(1992) The beta-thalassaemia mutations in the population of

Cyprus. Br J Haematol 81 (4):607-609

4. Sen U, Dasgupta J, Choudhury D, Datta P, Chakrabarti A,

Chakrabarty SB, Chakrabarty A, Dattagupta JK (2004) Crystal

structures of HbA2 and HbE and modeling of hemoglobin

delta 4: interpretation of the thermal stability and the

antisickling effect of HbA2 and identification of the

ferrocyanide binding site in Hb. Biochemistry 43 (39):12477-

12488. doi:10.1021/bi048903i

5. Krieger E, Koraimann G, Vriend G (2002) Increasing the

precision of comparative models with YASARA NOVA--a

self-parameterizing force field. Proteins 47 (3):393-402.

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forcefield. Bioinformatics 27 (12):1711-1712.

doi:10.1093/bioinformatics/btr254

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Ann Hematol

DOI 10.1007/s00277-013-1996-6 ESM2 1

Hb Famagusta—analysis of a novel δ-globin chain variant

[HBD:c.60C>A] in four families with diverse globin genotypes

Carsten W. Lederer Eleni Pavlou Christiana Makariou Georgia Hadjilambi

Nicoletta Andreou Michael Hadjigavriel Annita Kolnagou Maria Sitarou

Soteroulla Christou Marina Kleanthous

Electronic supplementary material 2 Supplementary tables and figures

Table S1 HbA2 stability predictions for different δ-globin-variants. All δ-globin variants so far identified in Cyprus [1] carry amino-acid

changes compared to the normal protein either in α-helices (T4I, G25D, A27S, R116C and L141P) or in a loop sequence at the interface

of the α1δ1-α2δ2 heterodimers (V98M). Structure-based meta-analysis by iStable [2] was used to predict the stability of the novel δ-

globin N19K variant monomer for comparison with other Cypriot variants. According to the calculated ΔΔG value, the novel δ-globin

variant is in Cyprus second in stability only to the δ-globin[T4I] mutation, which is positioned at the very start of the first δ-globin α-

helix towards the flexible N terminus.

δ-globin

variant

HGVS protein

name

dbSNP ID Codon

change

ΔΔG iStable confidence

score

Variant origin

and reference

T4I HBD:p.T5I rs35406175 ACT>ATT -0.046 0.593 Greek, Cypriot [1]

N19K HBD:p.N20K n/a AAC>AAA -0.568 0.871 Cypriot [this report]

G25D HBD:p.G26D rs34389944 GGT>GAT -1.449 0.716 Japanese, Cypriot [1]

A27S HBD:p.A28S rs35152987 GCC>TCC -1.624 0.938 Greek, Cypriot, Sardinian [3,4]

V98M HBD:p.V99M rs28933076 GTG>ATG -1.131 0.893 Black [5,1]

R116C HBD:p.R117C rs33971270 CGC>TGC -0.968 0.866 Cypriot, Greek [3,4]

L141P HBD:p.L142P rs33956485 CTG>CCG -2.371 0.856 Cypriot [3,4,1]

ΔΔG (free energy change value) – negative values indicate instability compared to the normal protein

Conf. – iStable confidence score, with a greater value indicating greater confidence in the predicted stability change,

which for all variants was lowered stability ().

References

1. Pavlou E, Phylactides M, Kyrri A, Kalogerou E, Makariou C,

Georgiou I, Kleanthous M (2006) Delta-thalassemia in

Cyprus. Hemoglobin 30 (4):455-462.

doi:N23370T6144926V7 [pii]

2. Capriotti E, Fariselli P, Casadio R (2005) I-Mutant2.0:

predicting stability changes upon mutation from the protein

sequence or structure. Nucleic Acids Res 33 (Web Server

issue):W306-310. doi:33/suppl_2/W306 [pii]

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Identification of four novel delta-globin gene mutations in

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Ann Hematol

DOI 10.1007/s00277-013-1996-6 ESM2 2

Fig. S1 Levels of Hb Famagusta vs. haploid-equivalent HbA2 in

reference populations. The levels of Hb Famagusta (red triangles)

as assessed by cellulose acetate electrophoresis were plotted

against the haploid-equivalent HbA2 levels of the reference

population (grey dots) matched for gender and α- and β-globin

genotype. Accordingly, for the four subjects carrying the CD 20

AAC>AAA mutation in the wild-type background (N/N), M2 and

M3 (M) were plotted separately from F2 and F4 (F). For each

genotype, the p value is given for the significance of Hb

Famagusta reduction compared to the distribution of haploid-

equivalent HbA2 levels in matching controls of sample size n.

p=5.2 x 10-3 (n=580)

N/IVS-I-110

p=5.7 x 10-3 (n=310)

N/N (M)

p≤6.0 x 10-3 (n=285)

N/N (F)

Genotype

p=2.2 x 10-6 (n=56)

αα/-α3.7

p=1.3 x 10-8 (n=176)

αα/--MEDI

% H

bA

2′ &

hap

loid

Hb

A2

0

1

2

3

4