ORIGINAL ARTICLE

Identification and molecular characterization of a novel55-kb deletion recurrent in southern Italy: the ItalianGc(Acdb)°-thalassemia†

Giuseppina Lacerra1, Romeo Prezioso1, Gennaro Musollino1, Giulio Piluso2, Lucia Mastrullo3,4,Maria De Angioletti1,5,6

1Istituto di Genetica e Biofisica “Adriano Buzzati-Traverso”, Consiglio Nazionale delle Ricerche, Napoli; 2Dipartimento di Patologia Generale,

Seconda Universit�a di Napoli, Napoli; 3U.O.C. Ematologia, P.O. San Giovanni Bosco, ASL NA1, Napoli; 4U.O.C. Ematologia, P.O. San Gennaro,

ASL NA1, Napoli; 5ICCOM Consiglio Nazionale delle Ricerche, Sesto Fiorentino, Firenze; 6Core Research Laboratory – Istituto Toscano Tumori,

Firenze, Italy

Abstract

Objectives: To characterize the molecular basis of a b-thalassemia defect in subjects with mild

microcytosis associated with normal Hb A2 and increased levels of Hb F. Methods: Six subjects from

three apparently unrelated families from Campania (southern Italy) have been investigated using DNA

restriction analysis, inverse PCR, cloning, sequencing, multiplex ligation-dependent probe amplification

(MLPA), quantitative real-time PCR, and gap-PCR. Results: We have identified a novel 55-kb b-globin gene

cluster deletion in three unrelated families: the Italian Gc(Acdb)°-thalassemia. This deletion removes most of

the b-globin cluster. The 5′ breakpoint was within the Ac-globin exon 2, and the 3′ breakpoint was within a

160-bp palindrome: the breakpoint-flanking regions present a microhomology (5′-TGGG-3′) that, together

with the palindromic structure, may have contributed to the recombination. Conclusions: Large deletions

of b-globin gene cluster are usually found in single families. Here, we report about the novel Italian Gc(Acdb)°-thalassemia we have found in three families. Twenty years ago, the characterization of the first

family was challenging, whereas that of the other families has taken advantage of nowadays techniques.

The relatively high frequency of this novel deletion in southern Italy suggests that it should be tested,

together with the Sicilian (db)°-thalassemia, in Italian and Mediterranean families with microcytosis, normal

Hb A2, and increased Hb F levels.

Key words thalassemia; hemoglobin; Hb F; Gc(Acdb)°-thalassemia; novel deletion

Correspondence Maria De Angioletti, Core Research Laboratory – Istituto Toscano Tumori, Viale Pieraccini 6 – 50139 Firenze, Italy

and ICCOM – National Research Council of Italy, Sesto Fiorentino, Firenze, Italy. Tel: +39 055 4271543; Fax +39 055 4271280; e-mail:

maria.deangioletti@ittumori.it

†This article is dedicated to the memory of Clementina Carestia: her enthusiasm and effort has made this research possible.

Accepted for publication 26 December 2012 doi:10.1111/ejh.12066

Deletions and rearrangements in the b-globin gene clusterthat remove the d- and b-globin genes but leave one[Gc(Acdb)°-thalassemia] or both the c-globin genes [(db)°-thalassemia] may result in a variety of conditions character-ized by microcytosis associated with variable increase inhemoglobin (Hb) F in the adults (1, 2). These deletionsmay be clinically relevant because their interaction withb°-thalassemia mutations results in phenotypes ranging frommild to severe. Usually, these deletions are found in single

families, but few of them may be relatively frequent in dis-tinct areas: for example, the Sicilian (db)°-thalassemia inItaly (3). In the past, the characterization of these deletionshas been challenging for the technical difficulties; however,nowadays, their identification has been simplified by vari-ous techniques: multiplex ligation-dependent probe amplifi-cation (MLPA) (4), quantitative real-time PCR (qRT-PCR)(5), high-resolution CGH array (6), and next-generationsequencing (7).

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European Journal of Haematology 90 (214–219)

Twenty years ago, we identified in a family from southernItaly a 55-kb deletion, the Italian Gc(Acdb)°-thalassemia(Lacerra G & De Angioletti M, unpublished data), by South-ern blot analysis and inverse PCR. Recently, we have identi-fied the same deletion in two unrelated families from thesame geographic area using the MLPA, qRT-PCR, andsequencing of a specific gap-PCR amplicon.

Materials and methods

Patients and hematological data

Patient blood samples, collected after a signed informedconsent obtained according to our institutional procedures,were referred to our institution from local hematologicalservices (Campania, southern Italy). Blood counts and Hbanalysis were obtained using standard methods. The globinbiosynthetic ratio was calculated as previously described(8).

DNA restriction analysis and inverse PCR

Peripheral blood DNA was digested with few restrictionenzymes and subjected to Southern blot with c-, wb-, d-,and b-globin probes for restriction fragment length polymor-phisms (RFLP) analysis and deletion mapping (9). Thebreakpoints of the deletion were characterized by inversePCR and sequencing (see Supplementary text and Table S1).

MLPA analysis and qRT-PCR

SALSA-MLPA-KIT P102-HBB (MRC-Holland, Amsterdam,the Netherlands) for the analysis of the b-globin gene clusterwas used according to manufacturer recommendations.MLPA products were separated by ABI-3130XL GeneticAnalyzer (Applied Biosystems, Foster City, CA, USA),quantified with the Coffalyser software (MRC-Holland), andcompared with a DNA pool from normal subjects. qRT-PCRwas performed with the ABI 7900HT System and the PowerSYBR-Green PCR-Master mix (Applied Biosystems) withprimers chosen outside repeated sequences (see Table S2).The beta-2-microglobulin was used as reference gene.

Breakpoint characterization

The genomic DNA encompassing the deletion breakpointswas amplified by gap-PCR using TripleMasterPCR System(Eppendorf, Hamburg, Germany) and then sequenced (Sup-plementary Table S1). The primers used for the specific gap-PCR were E (5′-AACAAGGCAAAGGCTATAAAAAAAA-3′) and F (5′-CAACTGCTGTGTTCTTTTGTTCAGA-3′) forthe deletion fragment and G (5′-TCAAATGTATCATGCCTCTTTGCACC-3′) and H (5′-TGGAGTCAAGGCTGAGAA-GATGCAGGA-3′) for the internal control.

Results

Family A

Twenty years ago, an 8-year-old boy was referred to our Insti-tution from a local hematological service for the occasionalfinding of hypochromic and microcytic red blood cells(RBCs). In the proband, normal levels of Hb A2 were associ-ated with increased levels of Hb F (17%) that was almost allof Gc type (Fig. 1A) and with non-a- / a-globin chain biosyn-thetic ratio of 0.5, indicating a b-thalassemic biosyntheticimbalance. A similar phenotype was found in his father, butnot in his mother (Table 1). The molecular characterizationwas performed with the technique available at that time:Southern blot analysis followed by inverse PCR, cloning, andsequencing (for details see Supplementary Text 1). We foundthat the 5′ breakpoint was in the Ac-globin exon 2 (coordinates5270746-43), and the 3′ breakpoint was at 31 kb from theb-globin gene (coordinates 5215688-85) (Figs 1B and 2A).The sequence, compared with the reference, showed the pres-ence of the C?T polymorphism (rs112800089) downstreamthe breakpoint (Fig. 1B). The length of this novel deletion, theItalian Gc(Acdb)°-thalassemia, is 55.050 bp.

Families B and C

Recently, an occasional laboratory control revealed hypochro-mic and microcytic RBC with normal Hb A2 and increasedHb F levels in a 31-year-old woman. Similar findings werepresent in her mother and her brother, but not in her father(Table 1). This phenotype suggested a b-globin gene clusterdeletion. The Sicilian (db)°-thalassemia (3, 10, 11) wasexcluded by a specific gap-PCR (Supplementary Table S1).The deletion of this family has been studied by MPLA andqRT-PCR: powerful techniques that nowadays are availableto characterize unknown deletion. The MLPA showedreduced signals from the wb, d, and b genes and normal sig-nals from the c and e genes (Fig. 2A,B), confirming the pres-ence of a large deletion. By qRT-PCR with primers probingthe regions not tested by MLPA (Fig. 2C and SupplementaryTable S2), we have been able to restrict the possible break-point region: one breakpoint was localized between the qRT-PCR amplicons g and f, and the other was localized betweenthe qRT-PCR amplicon b and the MLPA fragment 16(Fig. 2A,C). Eventually, the final characterization wasperformed by sequencing a long-range gap-PCR amplicon(Supplementary Table S1): the sequence of the deletion wasidentical to that found in family A.After this finding, we found the Italian Gc(Acdb)°-thalasse-

mia by a specific gap-PCR (Fig. 2D) in another unrelatedmale patient (family C Table 1) with microcytosis andincreased Hb F levels and without the Sicilian (db)°-thalas-semia deletion (3, 10, 11). Both families B and C were fromCampania.

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Table 1 Hematological data and biosynthetic ratio of the family members

Family A Family B Family C

I-11 I-2 II-1 I-1 I-2 II-12 II-2 I-1

Sex/Age (years) M/36 F/32 M/8 M/64 F/58 F/31 M/28 M/25

RBC (1012/L) 5.8 4.8 5.1 5.3 6.2 4.3 6.5 6.7

Hb (g/dL) 13.4 13.9 11.4 15.0 13.0 10.7 14.5 14.0

Ht (%) 41 41 35 46 43 32 46 45

MCV (fL) 71 86 69 87 70 74 70 67

MCH (pg) 23 29 23 29 22 25 22 21

MCHC (g/dL) 34 33 33 33 31 34 32 31

Hb A2 (%) 2.4 2.7 2.7 2.4 2.3 2.5 2.5 2.8

Hb F (%) 12.0 <1 17.0 0.5 17.7 16.5 9.9 12.5

Serum iron (lg/dL) 105 65 85 82 80 87 112 160

Ferritin (ng/mL) 68 27 27 73 59 22 155 nd

Transferrin (mg/dL) 320 257 245 nd nd 365 nd nd

b/a biosynthetic ratio 0.58 1.02 0.48 nd nd nd nd nd

non-a/a biosynthetic ratio 0.59 1.02 0.50 nd nd nd nd nd

Italian Gc(Acdb)°-thalassemia pos neg pos neg pos pos pos pos

nd, not determined.1The father was heterozygote for the -a3.7 thalassemia mutation, which explains the milder hematological phenotype, the higher b/a biosynthetic

ratio and the lower Hb F values, in comparison with the son.2Sideropenic anemia; pos Italian Gc(Acdb)°-thalassemia carrier; neg not carrier of the Italian Gc(Acdb)°-thalassemia.

A

B

Figure 1 (A) Globin chain separation by reverse-phase HPLC in the proband of family A. The a-, b-, and Gc-globin chains are indicated. The posi-

tion of the ‘missing’ Ac-globin chain is indicated by an arrow. (B) Electropherogram of the sequence of the deletion breakpoint. The sequence

from Italian Gc(Acdb)°-thalassemia deletion breakpoint (Del) is aligned with the corresponding normal 5′ (N 5′) and 3′ (N 3′) sequences. The nucleo-

tide numbering of the normal sequences according to reference sequence GRCh37/hg19 is shown. The C?T polymorphism (rs112800089) is

indicated with an asterisk.

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The analysis of the deletion-specific amplicon revealed anidentical sequence in all families: the 3′-arm of the deletionincludes some polymorphisms and peculiar rearrangements

in the respect of the reference sequence GRCh37/hg19, butit is identical to the sequence NW_001838021.1 (Figure S2).This suggests that the deletional event happened on a

A

B C

D E

Figure 2 (A) Scheme of the b-globin gene cluster with location of the MLPA probes (indicated with numbers) and of the quantitative real-time

PCR fragments (indicated with small letter in italic; the j fragment is at 3′ arm of depicted region). The positions of the 160-bp palindrome (filled

arrowhead) and of the enhancer (empty diamond) at 3’ of the palindrome are reported. Genes are indicated by filled rectangles; pseudogene is

indicated by an empty rectangle; DNA hypersensitive sites (HS) are indicated by empty triangles. Three deletions with 3’ breakpoints localized in

the palindrome are shown. (B) MLPA analysis of the deletion. Ratio between the values obtained from the proband of family B and normal sub-

jects: the position of the different probes on the b-globin gene cluster is indicated on the x-axis. In black are shown the probes with half dosage

and in gray, the probe with full dosage. (C) Quantitative real-time PCR analysis (primers in supplementary Table S2) probing the regions not

tested by MLPA: five fragments (black diamonds) show half dosage, and 4 fragments (gray diamonds) show full dosage. The localization of

MLPA fragments 16 and 1–15 is shown. (D) Gap-PCR for the screening of carriers for the Italian Gc(Acdb)°-thalassemia. The specific deletion

amplicon was 1914 bp (primers F and E: Supplementary Table S1); the internal control amplicon was 867 bp (primers G and H: Supplementary

Table S1). Lanes 1–3: probands of the families A, B, C; Lane 4: Sicilian (db)°-thalassemia carrier; Lanes 5–6: normal controls; Lane 7: reagent con-

trol. (E) Sequence of the central portion of the 160-bp palindrome. An arrow indicates the middle point of the palindrome. The microhomology

found in the Italian Gc(Acdb)°-thalassemia (TGGG), in the HPFH-3 (CCACT), in the Belgian Gc(Acdb)°-thalassemia (G), and in the Chilean (ecdb)°-thal-

assemia (A) is in boxes.

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chromosome, likely frequent in this geographic area, thatdiffers from reference sequence GRCh37/hg19.

Discussion

Twenty years ago, we have characterized a deletion of theb-globin gene locus: the Italian Gc(Acdb)°-thalassemia that,recently, we have found in other two families. Nowadays,technologies have simplified the identification and the char-acterization of thalassemic deletions that still provide insightinto the mechanisms that produce them and into the controlof the fetal–adult Hb switch. The identical breakpointsequence and the origin from the same geographic region(Campania, southern Italy) suggest that these apparentlyunrelated families might have a common ancestor.The Italian Gc(Acdb)°-thalassemia is associated with

increased Hb F levels (10–18%) of Gc-type. Several mecha-nisms may contribute to this increased Hb F levels: (i) thedeletion enhances c-globin gene transcription because of thereduced competition of the deleted d- and b-globin genepromoters for the activity of locus control region (12); (ii)the deletion juxtaposes an enhancer element, localizedimmediately downstream the Italian Gc(Acdb)°-thalassemia3′ breakpoint (13), to the Gc-globin gene; and (iii) the dele-tion of a binding site for BCL11A, a repressor of c-globinexpression in adult life, localized between Ac- and d-globingenes (14).The 3′ breakpoint of this deletion is within few bases from

the breakpoints of several other deletions (Fig. 2E): theBelgian Gc(Acdb)°-thalassemia (15), the HPFH-3 (16), theChilean (ecdb)°-thalassemia (17), and the Pakistani I(ecdb)°-thalassemia (18). This suggests that this region isprone to rearrangement: in fact, all these breakpoints arelocated close to the center of a 160-bp palindromicsequences (Fig. 2A,E). The finding is not surprising becausepalindromic sequences can form stem-loop structures by in-trastrand base pairing in single-stranded DNA that couldfacilitate a breakage event contributing to various genomicrearrangements including deletions (19, 20), and it is knownthat palindromic sequences are mainly cleaved at their center(21). In addition, the sequences closely flanking the break-points of Italian Gc(Acdb)°-deletion present a microhomolo-gy (5′-TGGG-3′), which could increase the probability ofnon-homologous end-joining process (20). Similar regions ofmicrohomology are present in other deletions with the break-point within the 160-bp palindrome (Fig. 2E).Usually, large b-globin cluster deletions are present in sin-

gle families. However, few of them may account for most ofthe cases in distinct geographic areas (22): for example, theSicilian (db)°-thalassemia (3, 23), the Italian HPFH-4 (24),the Spanish (db)°-thalassemia (25), the Turkish/Macedonian(db)°-thalassemia (26), and the Turkish Gc(Acdb)°-thalasse-mia (11) in some Mediterranean regions.

Our series of 316 b-thalassemic families from southernItaly includes 14 families with microcytosis, normal Hb A2,and increased Hb F levels. We have characterized theirmolecular lesion (unpublished results): 11 showed the Sicil-ian (db)°-thalassemia (the more frequent in Italy) and 3showed the Italian Gc(Acdb)°-thalassemia.This finding is important for genetic counseling: in fact,

the relatively high frequency of the novel Italian Gc(Acdb)°-thalassemia in patients with microcytosis, normal Hb A2,and increased Hb F levels from southern Italy suggests thatit should be tested, together with the Sicilian (db)°-thalasse-mia, in patients from Italy (and also from Mediterraneanarea) with this clinical phenotype.

Acknowledgements

We thank the patients and their families for the collabora-tion. We also thank Rosario Notaro for the helpful sugges-tions and the fruitful discussions. This work has beensupported by grants from Ministero Istruzione, Universit�a eRicerca (MIUR), Legge 488/92, Cluster C02, Project 2. Avery preliminary molecular characterization of the Italian Gc(Acdb)°-globin deletion in family A has been presented inthe 6th ‘International Conference on Thalassaemia and theHaemoglobinopathies’ (Malta, April 1997).

Conflict of interest

The authors do not have any competing interests.

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Supporting Information

Additional Supporting Information may be found in theonline version of this article:Data S1. Supplementary Results, Methods and Figure S1.Table S1. Name, position and sequence of primers.Table S2. Primers and position of amplicons for quantita-

tive Real time-PCR.Figure S2. Multiple alignment of 3′ arm of the Italian

deletion.

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