http://informahealthcare.com/hemISSN: 0363-0269 (print), 1532-432X (electronic)

Hemoglobin, Early Online: 1–3! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/03630269.2014.964361

SHORT COMMUNICATION

A New b0 Frameshift Mutation, HBB: c.44delT (p.Leu14ArgfsX5),Identified in an Argentinean Family Associated with SecondaryGenetic Modifiers of b-Thalassemia

Carolina Pepe, Silvia Eandi Eberle, Alejandro Chaves, Berenice Milanesio, Fernando M. Aguirre, Vanesa AvalosGomez, Lilian Diaz, Adrian P. Mansini, Diego A. Fernandez, Gabriela Sciuccati, Andrea Candas, Carolina Cervio,Mariana Bonduel, and Aurora Feliu-Torres

Servicio de Hematologıa-Oncologıa, Hospital de Pediatrıa ‘‘Prof. Dr. Juan P. Garrahan’’, Buenos Aires, Argentina

Abstract

b-Thalassemia intermedia (b-TI) patients present with a wide spectrum of phenotypesdepending on the presence of primary, secondary, and tertiary genetic modifiers whichmodulate, by different mechanisms, the degree of imbalance between a and b chains. Here wedescribe a new b0 frameshift mutation, HBB: c.44delT (p.Leu14ArgfsX5), identified in fourmembers of a family, associated with secondary genetic modifiers in three of them. Thedifferent genotype present in this family was suspected after hematological analysis andthorough observation of blood smears highlighting their importance in the identification ofb-TI patients among members of the same family.

Keywords

b-Thalassemia intermedia (b-TI), geneticmodifiers, new mutation

History

Received 8 April 2014Accepted 2 May 2014Published online 30 September 2014

The clinical manifestations of b-thalassemia (b-thal) are

extremely variable in severity, ranging from the transfusion-

dependent b-thal major (b-TM) to the asymptomatic carrier

state. Somewhere in between these two different entities is

b-thal intermedia (b-TI), with a wide spectrum of phenotypes

and transfusion requirement (1). The phenotypic diversity of

b-TI depends on the existence of primary, secondary, and

tertiary genetic modifiers which modulate, by different

mechanisms, the degree of imbalance between a and bchains, finally determining the patient’s clinical and hemato-

logical manifestations (2).

The primary genetic modifiers are the mutations present on

the HBB gene, for which more than 800 genetic variants have

been previously described (3). These mutations are very

heterogeneous, with some of them associated with no b chain

production from the affected b allele (b0-thal) and others

allowing b chain synthesis at a reduced rate (b+-thal) (4). The

production of a and g chains is modulated by secondary

modifiers, such as coinheritance of a-thalassemia (a-thal),

presence of triplicate/quadruplicate a genes, and increased gchain production associated with more elevated levels of

Hb F. Hb F levels are regulated by three major loci: HBG2: c.-

211 C4T (Gg-158 C>T or XmnI polymorphism) on 11p15.4

inside the b-globin gene cluster, HBS1L-MYB intergenic

region on 6q23.3 and BCL11A on 2p16.1 (5).

A novel HBB frameshift mutation was identified in an

Argentinean family associated with secondary genetic modi-

fiers whose presence may ameliorate or worsen the b-thal

phenotype. The proband, a 19-year-old female of Spanish

ancestry, was born after an uncomplicated full-term preg-

nancy and delivery. She had suffered from anemia since

childhood receiving iron therapy on multiple occasions. On

admission to our institution, her physical examination was

unremarkable except for pallor. The proband’s family (her son

and two brothers) were also examined. Hematological data

were obtained with a Sysmex XS800i (Sysmex Corporation,

Kobe, Japan). Hemoglobin (Hb) electrophoresis was carried

out with a semiautomatic agarose-gel system at an alkaline

pH (Sebia, Lisses, Evry, France). Hb A2 was measured by

anion exchange chromatography (6) and Hb F according to

the method described by Betke et al. (7).

The HBB gene was amplified and sequenced from DNA

isolated from peripheral blood leukocytes using primers

previously described by Roldan et al. (8). Exonic and intronic

flanking regions were directly sequenced with the Sanger

method and an automated capillary sequencer ABI PRISM

3130 (Applied Biosystems, Buenos Aires, Argentina). The

most frequent deletions on the a-globin cluster [�a3.7

(rightward) and �a4.2 (leftward)] were analyzed with gap-

polymerase chain reaction (gap-PCR) using primers and a

strategy described by Chong et al. (9), with some modifica-

tions. Briefly, 100 ng of genomic DNA was amplified using

200 mM dNTPs, primers, 1� PCR buffer, 1�QSolution

Address correspondence to Dr. Aurora Feliu-Torres, Servicio deHematologıa-Oncologıa, Hospital de Pediatrıa ‘‘Prof. Dr. Juan P.Garrahan,’’ Combate de los Pozos 1881, (C1245AAM) Buenos Aires,Argentina. Tel: +54-11-4308-4300, Ext: 1301-1597. Fax: +54-11-4308-5325. E-mail: afeliu@garrahan.gov.ar

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(Qiagen, Buenos Aires, Argentina), and 1.25 U of Hot Star

Taq DNA polymerase (Qiagen) in 25 mL of reaction volume.

After amplification, 10 mL of product was analyzed by

electrophoresis in 1.0% agarose gel stained with ethidium

bromide. The presence of the anti-3.7 a-globin gene

triplication was analyzed using PCR as previously described

(10). The number of a genes present on the anti-3.7 allele, the

excess of a-globin genes generated by other mechanisms, and

the presence of different deletions on the a-globin gene

cluster were studied by multiplex ligation-dependent probe

amplification (MLPA) using the commercial kit Salsa MLPA

P140B HBA (MRC-Holland, Amsterdam, The Netherlands)

following the manufacturer’s instructions. The genetic variantGg-158 was analyzed by PCR-RFLP (restriction fragment

length polymorphism) using the XmnI enzyme (11).

Hematological and molecular findings are shown in

Table 1. Hematological results for all individuals suggested

a diagnosis of b-thal trait. In addition to anisocytosis,

microcytosis, and hypochromia, individuals with a more

severe phenotype (the proband, her son, and one of her

brothers) have moderate-to-severe poikilocytosis with promi-

nent basophilic stippling. Hb A2 percentage was increased in

all individuals.

A new mutation, HBB: c.44delT (p.Leu14ArgfsX5) was

identified in all members of the family analyzed. It results

from a single nucleotide deletion at codon 14. The deletion

determines a frameshift and the generation of a premature

termination codon (codon 18) (Figure 1). The resultant

mRNA could be targeted to nonsense-mediated decay or

could be translated to a very short peptide that would be

completely hydrolyzed by the proteolytic system of red blood

cells (RBCs), resulting in no protein being synthesized from

the affected allele (12,13).

An increased level of Hb F was observed in the proband’s

son, who is homozygous for the Gg-158 T allele. The other

family members with only one T allele have normal or slightly

increased Hb F levels. It has been described that the effect of

the �158 determinant is not consistent, as normal levels of

Hb F have been found both in b-thal heterozygotes and in non

thalassemic individuals. Nevertheless, in conditions of

erythropoietic stress, the Gg-158 genetic marker could lead

to increased Hb F production, playing a role in the

amelioration of the phenotype (14).

The analysis of the a-globin gene cluster revealed the

absence of deletions inside the cluster but the presence of an

extra a-globin gene in the proband, her son, and one of her

brothers. This extra a-globin gene increases the a/b-globin

chain imbalance impairing the phenotype of these three

patients.

In conclusion, we describe a new b0 mutation identified in

a heterozygous state in an Argentinean family. In the proband,

Table 1. Hematological and molecular studies (RBC: red blood cell; Hb: hemoglobin; MCV: mean corpuscular volume;MCH: mean corpuscular Hb).

Parameters Proband Son Brother Brother

Sex-Age (years) F-19 M-2 M-15 M-14RBC (1012/L) 4.95 5.49 5.99 5.64Hb (g/dL) 9.0 10.3 11.2 11.4MCV (fL) 64.0 54.5 55.1 62.5MCH (pg) 19.2 18.8 18.4 20.2Hb A2 (%) 5.0 4.4 5.7 6.0Hb F (%) 0.25 14.4 1.70 2.50Blood film:� anisocytosis [+ +] [+ +] [+ +] [+ +]� macrocytosis [+] – [+] [+]� microcytosis [+ + +] [+ +] [+ + +] [+ + +]� hypochromia [+ + +] [+ +] [+ + +] [+ + +]� basophilic stippling [+ +] [+ +] [+ +] [+]� elliptocytes [+ +] [+] [+ +] [+ +]� ovalocytes [+ +] [+] [+ +] [+ +]

Serum ferritin (ng/mL) 49.8 55.7 88.4 72.9b Genotype HBB: c.44delT/bA HBB: c.44delT/bA HBB: c.44delT/bA HBB: c.44delT/bA

a Genotype aaa/aa aaa/aa aaa/aa aa/aaGg Genotype HBG2: c.-211 C/T

(Gg -158C/T)HBG2: c.-211T/T

(Gg -158T/T)HBG2: c.-211C/T

(Gg -158C/T)HBG2: c.-211C/T

(Gg -158C/T)

Figure 1. DNA sequencing of the HBB gene in the proband, her son, andher brothers shows the frameshift generated by a T deletion at codon 14,resulting in the premature stop codon at position 18 of amino acidsequence.

2 C. Pepe et al. Hemoglobin, Early Online: 1–3

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one of her brothers, and her son, the b0 mutation was

coinherited with the anti-3.7 a-globin gene triplication, while

in the other brother it was coinherited with a normal set of

a-globin genes. The analysis of the hematological character-

istics denotes a milder disorder of RBCs in the brother with a

normal set of a-globin genes, highlighting the importance of

thorough observation of blood smears to detect phenotypic

differences among members of the same family in the

identification of patients with b-TI.

Declaration of interest

The authors report no conflicts of interest. The authors alone

are responsible for the content and writing of this article.

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DOI: 10.3109/03630269.2014.964361 A New �0 Mutation with Secondary Genetic Modifiers 3

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