Incidence of β-thalassemia carrier on 1495 couples in preconceptional period

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Author(s): D. Dell’Edera et al. Article title: Incidence of β-thalassemia carrier on 1495 couples in preconceptional periodArticle no: DJMF 735998Enclosures: 1) Query sheet 2) Article proofs 3) Track changes manuscript showing language editing

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RID Given Names Surname Suix

1. Domenico Dell’Edera

2. Annunziata Anna Epifania

3. Antonio Malvasi

4. Elena Pacella

5. Andrea Tinelli

6. Antonio Capalbo

7. Maria Brigida Lioi

8. Giancarlo Di Renzo

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The Journal of Maternal-Fetal and Neonatal Medicine, 2012; Early Online: 1–4

© 2012 Informa UK, Ltd.

ISSN 1476-7058 print/ISSN 1476-4954 online

DOI: 10.3109/14767058.2012.735998

Objective: This research, conducted on 1495 couples in precon-ceptional period, demonstrates how the study of globular resistance of erythrocytes (GRO) is not a first choice test and not useful as other more accurate tests to identify subjects with β-thalassemia trait. Instead, the complete blood count (CBC) and the evaluation of HbA, HbA2 and HbF by high pres-sure liquid chromatography (HPLC) are essential. Methods: Each couple arrived in our laboratory to screen for β thalassemia. In case of patients with positive (240) or doubtful (112) results, we studied β-globin gene. Results: Of the 2990 subjects examined, we found 280 subjects with β-thalassemia trait (9.36%). During biochemical tests, among 112 subjects with doubtful – normal GRO or altered GRO – results, 40 of them resulted positive for the molecular analysis, while 72 of them did not show muta-tions in β-globin genes. The 2710 samples with non-carriers of β-thalassemia trait presented as mean evaluation of HbA2 2.6%, while the 280 subjects with β-thalassemia trait presented as mean evaluation of HbA2 4.8%. Molecular study showed that the β thalassemia phenotype is caused by a small number of muta-tions, whose regional distribution is typical. Conclusions: In the presence of thalassemic parameters in the CBC, the accurate and precise quantification of hemoglobin HbA2 is essential for the diagnosis of β-thalassemia trait. DNA mutation analysis provides the most effective way to detect primary gene mutations. The mutations identified in this work can be identified with a simple and inexpensive kit. This means, in economic terms, a significant savings for health spending.

Keywords: β-thalassemia carrier, globular resistance of erythrocytes (GRO), hemoglobin subtypes, HbA2 levels

Introduction

β thalassemia is an inherited hemoglobin disorder resulting from impaired production of β-globin chains of the hemoglobin tetramer [1–4].

he β thalassemic [5,6] defect can be observed in two modali-ties: complete lack (β0 thalassemia) or reduced synthesis (β+ thal-assemia) of the β chains. he consequence is the alteration of the erythropoiesis and of the life of the red cells.

he “carriers of beta thalassemia” do not show any objective clinical sign or can show a moderate skin paleness related to the low degree of anemia. he full blood examination shows an intermediate anemia featuring hemoglobin (Hb) values of about 1 g/dl lower than the normal value. he number of red blood cells can be normal or – more oten – higher. he mean cell volume (MCV) and mean cell hemoglobin (MCH) are lower (normal range: MCV ≥ 80 l; MCH ≥ 27 pg). he values of HbA2 are higher [7,8] (normal range: Hb ≥ 12 g/dl in women; Hb ≥ 13 g/dl in men; HbA2 ≥ 2% and ≤3.2%; HbF ≤ 1.5%). In the presence of thalassemic parameters in the CBC, the accurate and precise quantiication of hemoglobin HbA2 is essential for the diagnosis of β-thalassemia trait.

Population screening, genetic counseling, prenatal diagnosis and option of terminating afected pregnancies remain the main-stay strategy to devise a control program and investigating the underlying molecular defects in β-thalassemia is an important prerequisite for such programs.

his work, conducted on 1495 couples in preconceptional period (total number: 2990 subjects) in the province of Matera (South Italy: people of Mediterranean area) (total population: 203.827), demonstrates how the study of globular resistance and osmotic fragility of erythrocytes (GRO) or “Simmel test” is not necessary to identify subjects with β-thalassemia trait. In some cases, the GRO is misleading, so is not a irst choice test and not useful as other more accurate tests to identify subjects with β-thalassemia trait.

Instead, the complete blood count (CBC) and the evaluation of hemoglobin by high pressure liquid chromatography (HPLC) (HbA, HbA2 and HbF) tests are essential.

Furthermore, DNA mutation analysis provides the most efec-tive way to detect primary gene mutations as well as gene–gene interactions that can inluence the overall phenotype. Just as important as the laboratory investigations is the family work up. Oten, the irst and most helpful clue to gene–gene interactions comes from the family study.

Material and methods

Over the past 5 years, we have studied 1495 couples in preconceptional period (2990 samples) in the province of Matera

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ORIGINAL ARTICLE

Incidence of β-thalassemia carrier on 1495 couples in preconceptional period

Domenico Dell’Edera1, Annunziata Anna Epifania1, Antonio Malvasi2, Elena Pacella3, Andrea Tinelli4, Antonio Capalbo5, Maria Brigida Lioi6 & Giancarlo Di Renzo7

1Unit of Cytogenetic and Molecular Genetics, “Madonna delle Grazie” Hospital, Matera, Italy, 2Obstetrics and Gynecology Department, “Santa Maria” Hospital, Bari, Italy, 3Department of Ophthalmology, Sapienza University, Rome, Italy, 4Obstetrics and Gynecology Department, “V. Fazzi” Hospital, Lecce, Italy, 5Genera Center for Reproductive Medicine, Clinica Valle Giulia, Rome, Italy, 6Department of Animal Production Sciences, University of Basilicata, Potenza, Italy, and 7Department of Obstetrics and Gynecology, Centre for Perinatal and Reproductive Medicine, University of Perugia, Perugia, Italy

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Correspondence: Dr. Domenico Dell’Edera, Unit of Cytogenetic and Molecular Genetics, Hospital “Madonna delle Grazie”, Postal code: 75100 Matera, Italy. Tel: 0835253439; 3392048017; 3295832594. E-mail: [email protected]

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2 D. Dell’Edera et al.

he Journal of Maternal-Fetal and Neonatal Medicine

(South Italy). Each couple arrived in our laboratory to screen for β thalassemia. To each person, we applied the following tests ater a venous blood samples (in EDTA-K3):

• Blood ilms for erythrocytes morphology. • Detection of red cell indices with automated cell counter Sysmex

XE 2100 (Dasit Cornaredo, Milan, Italy): counts the number of red blood cells (RBC), hemoglobin (Hb), mean corpuscular volume (MCV), mean corpuscular hemoglobin levels (MCH), mean corpuscular hemoglobin concentration (MCHC).

• Globular resistance and osmotic fragility of erythrocytes (GRO).

• High performance liquid chromatography (HPLC) to quantify hemoglobin subtypes in the blood samples through Tosoh HPLC G7 system (Tosoh Bioscience S.r.l. – Turin, Italy) [9].

• Monitoring of iron serum and ferritin serum.

A molecular study of β globin genes was performed in patients with carriers of the β-thalassemia trait. Molecular analysis involves the following steps:

• DNA isolation starting from 25 µl of blood, using the extrac-tion kit of Promega Italy S.r.l. (DNA IQTM System, cod.C6701).

• Polymerase chain reaction (PCR) and reverse-hybridization. he procedure includes two steps: PCR ampliication using biotinylated primers and hybridization of ampliication prod-ucts to a test strip containing allele-speciic oligonucleotide probes immobilized as an array of parallel lines. Bound bioti-nylated sequences are detected using streptavidin-alkaline phosphatase and color substrates. he ampliication and the reverse hybridization on a strip have been obtained with the use of commercial kits produced by Nuclear Laser Medicine (cod. AC006: genetic test aimed at the check of 23 mutations in the β-globin genes. he mutations analysed are listed in Table I).

Results

From the results obtained from the biochemical study, the sample was divided into three groups (Table II). In subjects carrying the β-thalassemia trait and those are not carriers of β-thalassemia trait, but with doubts blood indices, we proceeded to molecular study of β-globin gene. Molecular analysis of β-globin genes was performed on subjects belonging to Groups 2 and 3. Of the 2990 subjects examined, 280 subjects were found to be carries of β-thalassemia trait (9.36%) [10] (240 belonging to the second group, 40 belonging to the third group).

In addition, only in a small country in the province of Matera (Calciano), the presence of two subjects heterozygous for the presence of “Hemoglobin Sickler (HbS)” has been reported. he study shows an high incidence of iron deiciency anemia (12.4%) caused by poor eating habits (Table III) [11]. he 2710 samples non-carriers of β-thalassemia trait presented as mean evaluation of HbA2 2.6%. he 280 subjects with β-thalassemia trait presented as the mean evaluation of HbA2 4.8% (Figure 1). HbA2 determi-nation plays a key role in screening programs for β thalassemia because a small increase in this fraction is the most important marker of β thalassemia heterozygous carriers.

From the results presented in this work, it is shown that the proportion of HbA2 in non-carriers of β-thalassemia trait (more than 1 year of age) is between 2.0 and 3.2%.

Some report normal values of HbA2 up to 3.5%. his threshold value, based on our experience, seems too high.

his must be taken into account to avoid to miss a possible diagnosis of β-thalassemia carrier for a person who has HbA2 values at the lower limit of the concentration ranges for people carriers of this syndrome.

So, it is more correct to classify a subject that has a value of HbA2 intermediate between those of non-carriers (upper limit of HbA2 of 3.3%) and the lower limit of the carriers of β-thalassemia trait (HbA2 equal to 3.8%).

Table I. β globin gene mutation investigated.

Mutation Nucleotide exchange Gene defect

-101 C > T β+

-92 C > T β+

-87 C > G β+

-30 T > A β+

Codon 5 -CT β0

Codon 6 G > A (HbC) –

Codon 6 A > T (HbS) –

Codon 6 -A β0

Codon 8 -AA β0

Codon 8/9 G > C β0

Codon 30 G > A β0

IVS1.1 G > A β0

IVS1.2 T > A β0

IVS1.5 G > C β+

IVS1.6 T > C β+

IVS1.110 G > A β0

IVS1.116 T > G β0

IVS1-25 25 bp del β0

Codon 39 C > T β0

Codon 44 -C β0

IVS2.1 G > A β0

IVS2.745 C > G β+

IVS2.844 C > G β+

Table II. Classiication of the subjects based on the blood parameters.

Group 1. Normal subjects (total subjects: 2638)

GRO normal.

Normal erythrocyte morphology.

HbA2 < 3.2%.

Group 2. Individuals carrying the β-thalassemia trait (total subjects: 240)

Altered GRO.

Altered erythrocyte morphology.

HbA2 > 4%.

Group 3. Individuals not carrying the β-thalassemia trait (total subjects: 112)a

Altered GRO and/or normal GRO.

Altered and/or normal erythrocyte morphology.

HbA2 between 3.4 and 4.8.

GRO, globular resistance and osmotic fragility of erythrocytes.aOf the 112 subjects who underwent molecular diagnosis of β-globin genes, 72 were

false positives.

Table III. Subjects with iron deiciency anemia.

Total number of subjects examined 2990

Number of non-carriers of β-thalassemia trait, but with iron deiciency anemia

Men 167 (45%)

371 (=12.4%)

Women 204 (55%)

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Incidence of β-thalassemia carrier 3

© 2012 Informa UK, Ltd.

It follows that the total error must not exceed ±0.25% of HbA2 as an absolute value and ±7.0% (0.25/3.55 × 100) as a relative value, to avoid the possibility of classifying at the same time a person who had a HbA2 equal to 3.55% both as a healthy subject and as a carrier of β-thalassemia trait [12].

hrough the analysis of molecular β-globin gene done on β-thalassemia carriers, we have obtained the following results: 175 (=62.5%) patients were carriers of the mutation in codon 39 (C > T); 44 (=15.71%) patients were carriers of the mutation IVS-I-nt110 (G > A); 19 (=6.78%) patients were carriers of the mutation IVS-I-nt6 (T > C); 25 (=8.93%) patients were carriers of the mutation IVS-1-nt1 and 14 (=5%) patients were carriers of the mutation “silent β-101”. In this last group of patients, the molecular diagnosis has been very useful, because the hemato-logical picture alone would not show the mutation in the gene β globin (GRO is normal and HbA2 is 3.2%). his mutation is known as “silent” because of the extreme weakness with which the picture is expressed biochemically.

Molecular examination showed that three patients (=1.1%) did not have one of the 23 mutations listed in this paper. To identify the mutation was used gene sequencing [mutations detected: codon 121 (G > T), codon 17 (A > T), IVS2.848 (C > A)].

It can be seen that the molecular analysis of β-globin genes is useful in cases where the biochemical parameters cannot be exhaustive. A detailed list of the most common β-thalassemia mutations according to the ethnic distribution and severity can be found on the following website: http://globin.cse.psu.edu/globin/html/huisman.

Conclusions

From the above, it is clear that the β thalassemia is an inher-ited disease, widespread in the province of Matera (Italy) as in almost all Mediterranean countries, caused by multiple muta-tions in the gene for β globin. herefore, the common objec-tive is the development of new analytical techniques for early detection of mutations that can alter the function of β-globin genes. his work shows how the study of globular resistance and osmotic fragility of erythrocytes (GRO) or “Simmel test” is not a test of irst choice and not useful as other more accurate tests existing. Conversely, the measure of HbA2 is important for the study of microcytic anemias and of hemoglobinopathies with particular reference to the recognition of the carrier for β thalas-semia. Table IV shows a lowchart that could be used to identify individuals carrying β-thalassemia trait. Another algorithm can be found at the following website: http://guideline.gov/content.aspx?id=13350.

his work shows that in the province of Matera (Italy), it was observed that the β thalassemia phenotype is caused by a small number of mutations, whose regional distribution is typical.

he knowledge of the frequency of mutations prevalent in β-globin genes in the province of Matera allows the use of commercial kits studying the most frequent mutations in the region. In economic terms, it will have a signiicant savings because, in most cases, the study of the mutations shown in this work will be suicient without performing the sequencing of the entire gene β globin.

Currently, in the Basilicata region (Italy), there is an extensive program of health education, for both the medical health profes-sionals, but also to the entire population, which aims to provide the necessary information about the geographical risk, clinical course and modes of transmission of the disease and thalassemia screening methods available.

β-thalassemia carrier screening is of great importance, and identiication of carriers couple allows correct genetic counseling and aware family planning to at risk couples. Knowing mutations of both carrier spouses allows, through prenatal molecular diag-nosis, to understand the genetic constitution of the fetus.

Declaration of Interest: he authors report no conlicts of interest.

References

1. Myers RM, Tilly K, Maniatis T. Fine structure genetic analysis of a beta-globin promoter. Science 1986;232:613–618.

2. Rowley PT, Lipkin M Jr, Fisher L. Screening and genetic counseling for beta-thalassemia trait in a population unselected for interest: comparison of three counseling methods. Am J Hum Genet 1984;36:677–689.

3. Petrou M. Screening for beta thalassaemia. Indian J Hum Genet 2010;16:1–5.

4. Wajcman H, Préhu C, Bardakdjian-Michau J, Promé D, Riou J, Godart C, Mathis M, et al. Abnormal hemoglobins: laboratory methods. Hemoglobin 2001;25:169–181.

5. hein SL. Genetic modiiers of beta-thalassemia. Haematologica 2005;90:649–660.

6. Kan YW, Chang JC. Molecular diagnosis of hemoglobinopathies and thalassemia. Prenat Diagn 2010;30:608–610.AQ6

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Figure 1. HbA2 distribution in the sample. he non-carriers of β-thalassemia trait (2710 subjects) have a mean of HbA2 of 2.6%, while the carriers of β-thalassemia traits (280 subjects) have a mean of HbA2 of 4.8%.

Table IV. Diagnostic procedure to query the status of a healthy carrier of β thalassemia: lowchart.

First level (basic screening) Second level

In case of:

• Isolated elevation of HbA2.

• Suspected β-thalassemia silent.

• Genetic conditions not resolved in step 1.

• Persistence of diagnostic doubts.

• Molecular identiication of hemoglobin variants

1. Tests of routine 2. Test

• Blood count performed with electronic cell counter (CBC).

• Molecular analysis of the globin genes

• Determination of HbA, HbA2 and HbF by HPLC

• Qualitative and quantitative evaluation of hemoglobin variants by HPLC (e.g. HbE, HbC, HbS)

• Determination of blood serum ferritin.

2. Further investigation

In case of hemoglobin variants:

• Qualitative evaluation of Hb variants by electrophoresis.

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he Journal of Maternal-Fetal and Neonatal Medicine

7. Pornprasert S, Kasemrad C, Sukunthamala K. Diagnosis of thalassemia on dried blood spot samples by high performance liquid chromatography. Hemoglobin 2010;34:486–494.

8. Mosca A, Paleari R, Ivaldi G, Galanello R, Giordano PC. he role of haemoglobin A(2) testing in the diagnosis of thalassaemias and related haemoglobinopathies. J Clin Pathol 2009;62:13–17.

9. Kalleas C, Tentes I, Margaritis D, Anagnostopoulos K, Toli A, Pendilas D, Bourikas G, et al. Efect of HbS in the determination of HbA(2) with the TOSOH HLC-723G7 analyzer and the HELENA Beta-hal Quik column kit. Clin Biochem 2007;40:242–247.

10. Dell’Edera D, Pacella E, Epifania AA, Benedetto M, Tinelli A, Mazzone E, Laterza F, Malvasi A. Importance of molecular biology in the characterization of beta-thalassemia carriers. Eur Rev Med Pharmacol Sci 2011;15:79–86.

11. Passarello C, Giambona A, Cannata M, Vinciguerra M, Renda D, Maggio A. Iron deiciency does not compromise the diagnosis of high HbA(2) ß thalassemia trait. Haematologica 2012;97:472–473.

12. Desirable Speciications for Total Error, Imprecision, and Bias, derived from intra- and inter-individual biologic variation. Available at: http://www.westgard.com/biodatabase1.htm. AQ9

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ORIGINAL ARTICLE

Incidence of �-thalassemia carrier[AU: Please check and approve the running head or

provide an alternative.]

D. Dell’Edera et al.

Incidence of beta �-thalassemia carrier on 1495 couples in

preconceptional period.

Authors:

Domenico Dell’Edera1, Annunziata Anna Epifania

1, Antonio Malvasi

2, Elena Pacella

3,

Andrea Tinelli4, Antonio Capalbo

5, Maria Brigida Lioi

6 and & Giancarlo Di Renzo

7.

1Unit of Cytogenetic and Molecular Genetics –, “Madonna delle Grazie” Hospital,

Matera, Italy.,

2Obstetrics and Gynecology Department -, “Santa Maria” Hospital, Bari, Italy.,

3Department of OphthalmologyOphtalmology, Sapienza University, Rome, Italy.,

4Obstetrics and Gynecology Department -, “V. Fazzi” Hospital, Lecce, Italy.,

5Genera Center for Reproductive Medicine, Clinica Valle Giulia, Rome, Italy.,

6Department of Animal Production Sciences, University of Basilicata, Potenza, Italy.,

and

7Department of Obstetrics and Gynecology, Centre for Perinatal and Reproductive

Medicine, University of Perugia, Perugia, Italy.[AU: Please carefully check the authors’

names and affiliations.]

Correspondence to:

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Dr. Domenico Dell’Edera,

Unit of cytogenetic Cytogenetic and molecular Molecular geneticsGenetics,

Hospital “Madonna delle Grazie”,

Postal code: 75100 Matera, Italy.

Tel. : 0835253439 – ; 3392048017 – ; 3295832594.

eE-mail: [email protected]

Abstract

Objective. : This paperresearch, conducted on 1495 couples in preconceptional period,

demonstrates how the study of Globular globular resistance of erythrocytes (GRO) is not

a first choice test and not useful as other more accurate tests to identify subjects with

�beta -thalassemia trait.

Instead, the complete blood count (CBC) and the evaluation of HbA, HbA2 and HbF by

high pressure liquid chromatography (HPLC) are essential.

Methods. : Each couple arrived in our laboratory to screen for �beta thalassemia. In case

of patients with positive (240) or doubtful (112) results, we studied beta �-globin gene.

Results. : Of the 2990 subjects examined, we found 280 subjects with �beta-thalassemia

trait (9,.36%). During biochemical tests, among 112 subjects resulted with doubtful ( –

normal GRO normal or altered GRO); – results[AU: Please check the edit made to the

sentence “During biochemical tests…”], 40 of them resulted positive for the molecular

analysis, while 72 of them did n’t not show mutations in beta �-globin genes. The 2710

samples with non-carriers of beta �-thalassemia trait, presented as as mean evaluation of

HbA2 2.,6%, while the 280 subjects with beta �-thalassemia trait presented ass mean

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evaluation of HbA2 4,.8%. Molecular study showed that the β thalassemia phenotype is

caused by a small number of mutations, whose regional distribution is typically.

Conclusions. : In the presence of thalassemic parameters in the CBC, the accurate and

precise quantification of hemoglobin HbA2 is essential for the diagnosis of �-thalassemia

trait. DNA mutation analysis provides the most effective way in which to detect primary

gene mutations. The mutations identified in this work can be identified with a simple and

inexpensive kit. This means, in economic terms, a significant savings for health spending.

Keywords: �-thalassemia carrier, globular resistance of erythrocytes (GRO),

haemoglobinhemoglobin subtypes, beta thalassemia carrier, HbA2 levels, Globular

resistance of erythrocytes (GRO).

Introduction

�Beta- thalassemia is an inherited hemoglobin disorder resulting from impaired

production of �beta-globin chains of the hemoglobin tetramer [1–4]1,2,3,4

.

The β thalassemic [5,6] defect can be observed in two modalities: complete lack

(β0 thalassemia) or reduced synthesis (β+

thalassemia) of the �beta chains. The

consequence is the alteration of the erythropoiesis and of the life of the red cells.

The “carriers of beta thalassemia” do n’t not show any objective clinical sign, or

can show a moderate skin paleness related to the low degree of anemia. The full blood

examination shows an intermediate anemia featuring haemoglobinhemoglobin (Hb)

values of about 1 gr/dl lower than the normal value[AU: Please check the change from

“norm” to “normal value” for more readability.]. The number of red blood cells can be

normal, or – more often – higher. The mean cell volume (MCV) and mean cell

hemoglobin (MCH) are lower (normal range: MCV � 80 fl; MCH � 27 pg). The values of

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HbA2 are higher [7,8] (normal range: Hb � 12 gr/dl in women; Hb � 13 gr/dl in men;

HbA2 � 2% and �3.2%; HbF � 1.5%). In the presence of thalassemic parameters in the

CBC, the accurate and precise quantification of hemoglobin HbA2 is essential for the

diagnosis of �-thalassemia trait.

Population screening, genetic counseling, prenatal diagnosis and option of

terminating affected pregnancies remain the mainstay strategy to devise a control

program and investigating the underlying molecular defects in �-thalassemia is an

important prerequisite for such programs.

This work, conducted on 1495 couples in preconceptional period (total number:

2990 subjects) in the province of Matera (South Italy: people of Mediterranean area)

(total population: 203.827), demonstrates how the study of Globular globular resistance

and osmotic fragility of erythrocytes (GRO) or “Simmel test” is not necessary to identify

subjects with beta �-thalassemia trait. In some cases, the GRO is misleading, so is not a

first choice test and not useful as other more accurate tests to identify subjects with beta

�-thalassemia trait.

Instead, the complete blood count (CBC), ) and the evaluation of hemoglobin by

high pressure liquid chromatography (HPLC) (HbA, HbA2 and HbF) tests are essential.

Furthermore, DNA mutation analysis provides the most effective way in which to

detect primary gene mutations as well as gene-–gene interactions that can influence the

overall phenotype. Just as important as the laboratory investigations is the family work

up. Often, the first and most helpful clue to gene gene–gene interactions comes from the

family study.

Material and methods

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Over the past 5 years, we have studied 1495 couples in preconceptional period (2990

samples) in the province of Matera (South Italy). Each couple arrived in our laboratory to

screen for �beta thalassemia. To each person, we applied the following tests after a

venous blood samples (in EDTA-K3):

ο• blood Blood films for erythrocytes morphology.

ο• Detection of red cell indices with automated cell counter Sysmex XE 2100

(Dasit Cornaredo – , Milan, Italy): counts the number of red blood cells

(RBC), hemoglobin (Hb), mean corpuscular volume (MCV), mean

corpuscular hemoglobin levels (MCH), Mean mean corpuscular

hemoglobin concentration (MCHC).

ο• Globular resistance and osmotic fragility of erythrocytes (GRO).

ο• High performance liquid chromatography (HPLC) to quantify hemoglobin

subtypes in the blood samples through Tosoh HPLC G7 system (Tosoh

Bioscience S.r.l. – Turin, Italy[9]). [9].

ο• Monitoring of iron serum and ferritin serum.

A molecular study of β globin genes was performed in patients with carriers of the β β-

thalassemia trait. Molecular analysis involves the following steps:

ο• DNA isolation starting from 25 µl of blood, using the extraction kit of

Promega Italy S.r.l. (DNA IQTM System, cod.C6701).

ο• Polymerase chain reaction (PCR) and reverse-hybridization. The

procedure includes two steps: PCR amplification using biotinylated

primers and hybridization of amplification products to a test strip

containing allele-specific oligonucleotide probes immobilized as an array

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of parallel lines. Bound biotinylated sequences are detected using

streptavidin-alkaline phosphatase and color substrates. The amplification

and the reverse hybridization on a strip have been obtained with the use of

commercial kits produced by Nuclear Laser Medicine (cod. AC006:

genetic test aimed at the check of 23 mutations in the β- globin genes. The

mutations analysed are listed in Table I).

Results

From the results obtained from the biochemical study, the sample was divided into three

groups (Table II). In subjects carrying the beta �-thalassemia trait and those are not

carriers of beta �-thalassemia trait, but with doubts blood indices, we proceeded to

molecular study of beta �-globin gene. Molecular analysis of �beta-globin genes was

performed on subjects belonging to group Groups 2 and 3. Of the 2990 subjects

examined, were found 280 subjects were found to be carries ofwith �beta-thalassemia

trait (9,.36%[10]) [10] (240 belonging to the second group, 40 belonging to the third

group).

In addition, only in a small country in the province of Matera (Calciano) ), has

reported the presence of two subjects heterozygous for the presence of “Hemoglobin

Sickler (HbS)” has been reported. The study shows an high incidence of iron deficiency

anemia (12,.4%) caused by poor eating habits (Table III[11]) [11]. The 2710 samples

non-carriers of beta �-thalassemia trait presented aass mean evaluation of HbA2 2,.6%.

The 280 subjects with beta �-thalassemia trait presented as as thethe mean evaluation of

HbA2 4,.8% (Figure 1). HbA2 determination plays a key role in screening programs for �

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thalassemia because a small increase in this fraction is the most important markers of �

thalassemia heterozygous carriers.

From the results presented in this work, is it is shown that the proportion of HbA2

in non-carriers of �beta-thalassemia trait (more than 1 year of age) is between 2.0% and

3.2%.

Some report normal values of HbA2 up to 3.5%. This threshold value, based on

our experience, it seems too high.

This must be taken into account to avoid to miss a possible diagnosis of �beta-

thalassemia carrier for a person who has HbA2 values at the lower limit of the

concentration ranges for people carriers of this syndrome.

So, it is more correct to classify a subject that has a value of HbA2 intermediate

between those of non-carriers (upper limit of HbA2 of 3.3%) and the lower limit of the

carriers of �beta-thalassemia trait (HbA2 equal to 3,.8%).

It follows that the total error must not exceed ± 0.25% of HbA2 as an absolute

value and ± 7.0% (0.25 / /3.55 x × 100) as a relative value, to avoid the possibility of

classifying at the same time a person who had a HbA2 equal to 3.55% both as a healthy

subject and as a carrier of �beta-thalassemia trait [12].

Through the analysis of molecular beta �-globin gene done on �beta- thalassemia

carriers, we have obtained the following results: 175 (=62,.5%) patients were carriers of

the mutation in codon 39 (C > T); 44 (=15,.71%) patients were carriers of the mutation

IVS-I-nt110 (G > A); 19 (=6,.78%) patients were carriers of the mutation IVS-I-nt6 (T >

>C); 25 (=8,.93%) patients were carriers of the mutation IVS-1-nt1 and 14 (=5%) patients

were carriers of the mutation “silent β-101”. In this last group of patients, the molecular

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diagnosis has been very useful, because the hematological picture alone only, would not

show the mutation in the gene �beta globin (GRO is normal and HbA2 is 3.2%). This

mutation is known as “silent” because of the extreme weakness with which the picture is

expressed biochemically.

Molecular examination showed that three patients (=1,.1%) did not have one of

the 23 mutations listed in this paper. To identify the mutation was used gene sequencing

[mutations detected: codon 121 (G > >T), codon 17 (A > >T), IVS2.848 (C > >A)].[AU:

Please check the clarity of the sentence “To identify the mutation was used gene

sequencing..”]

It can be seen that the molecular analysis of �beta- globin genes is useful in cases

where the biochemical parameters cannot’t be exhaustive. A detailed list of the most

common �beta- thalassemia mutations according to the ethnic distribution and severity

can be found on the Internetfollowing website:

http://globin.cse.psu.edu/globin/html/huisman.

Conclusions

From the above, it is clear that the � thalassemia is an inherited disease, widespread in the

province of Matera (Italy) as in almost all Mediterranean countries, caused by multiple

mutations in the gene for � globin. Therefore, the common objective is the development

of new analytical techniques for early detection of mutations that can alter the function of

�beta- globin genes. This work shows how the study of Globular globular resistance and

osmotic fragility of erythrocytes (GRO) or “Simmel test” is not a test of first choice and

not useful as other more accurate tests existing. Conversely, the measure of HbA2 is

important for the study of microcytic anemias and of haemoglobinopathies with

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particular reference to the recognition of the carrier for �beta thalassemia. In Table IV IV

shows ait is proposed a flowchart that could be used to identify individuals carrying of

thecarrying �beta-thalassemia trait[AU: Please check the edit made to the sentence.].

Another algorithm can be found at the following website:

http://guideline.gov/content.aspx?id=13350.

This work shows that, in the province of Matera (Italy), it was observed that the �

thalassemia phenotype is caused by a small number of mutations, whose regional

distribution is typically[AU: Please check the change of the word from “typically” to

“typical” in the main text and in the abstract].

The knowledge of the frequency of mutations prevalent in �beta- globin genes in

the province of Matera allows the use of commercial kits studying the most frequent

mutations in the region. In economic terms, it will have a significant savings because, in

most cases, will be sufficient the study of the mutations shown in this work will be

sufficient, without performing the sequencing of the entire gene �beta globin.

Currently, in the Basilicata region (Italy) ), there is an extensive program of health

education, for both the medical health professionals, but also to the entire population,

which aims to provide the necessary information about the geographical risk, clinical

course and modes of transmission of the disease and thalassemia screening methods

available.

�Beta- thalassemia carrier screening is of great importance, and identification of

carriers couple allows correct genetic counseling and aware family planning to at risk

couples. Knowing mutations of both carrier spouses allows, through prenatal molecular

diagnosis, to understand the genetic constitution of the fetus.

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Funding

None.

Conflict of interestDeclaration of Interest:

The authors reported no conflicts of interest.

References

1. Myers RM, Tilly K, Maniatis T. Fine structure genetic analysis of a beta-globin

promoter. Science 1986;232:613–618.

2. Rowley PT, Lipkin M Jr, Fisher L. Screening and genetic counseling for beta-

thalassemia trait in a population unselected for interest: comparison of three

counseling methods. Am J Hum Genet 1984;36:677–689.

3. Petrou M. Screening for beta thalassaemia. Indian J Hum Genet 2010;16:1–5.

4. Wajcman H, Préhu C, Bardakdjian-Michau J, Promé D, Riou J, Godart C, Mathis

M, et al. Abnormal hemoglobins: laboratory methods. Hemoglobin 2001;25:169–

181.

5. Thein SL. Genetic modifiers of beta-thalassemia. Haematologica 2005;90:649–

660.

6. Kan YW, Chang JC. Molecular diagnosis of hemoglobinopathies and thalassemia.

Prenat Diagn 2010;30:608–610.

7. Pornprasert S, Kasemrad C, Sukunthamala K. Diagnosis of thalassemia on dried

blood spot samples by high performance liquid chromatography. Hemoglobin

2010;34:486–494.

8. Mosca A, Paleari R, Ivaldi G, Galanello R, Giordano PC. The role of

haemoglobin A(2) testing in the diagnosis of thalassaemias and related

haemoglobinopathies. J Clin Pathol 2009;62:13–17.

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9. Kalleas C, Tentes I, Margaritis D, Anagnostopoulos K, Toli A, Pendilas D,

Bourikas G, et al. Effect of HbS in the determination of HbA(2) with the TOSOH

HLC-723G7 analyzer and the HELENA Beta-Thal Quik column kit. Clin

Biochem 2007;40:242–247.

10. Dell’Edera D, Pacella E, Epifania AA, Benedetto M, Tinelli A, Mazzone E,

Laterza F, Malvasi A. Importance of molecular biology in the characterization of

beta-thalassemia carriers. Eur Rev Med Pharmacol Sci 2011;15:79–86.

11. Passarello C, Giambona A, Cannata M, Vinciguerra M, Renda D, Maggio A. Iron

deficiency does not compromise the diagnosis of high HbA(2) ß thalassemia trait.

Haematologica 2012;97:472–473.

12. Desirable Specifications for Total Error, Imprecision, and Bias, derived from

intra- and inter-individual biologic variation. Available at:

http://www.westgard.com/biodatabase1.htm.[AU: Please check the inserted

details of ref. 12.]

Table I. � globin gene mutation investigated.

Mutation Nucleotide exchange Gene defect

-101 C > T �+

-92 C > T �+

-87 C > G �+

-30 T > A �+

Codon 5 -CT �0

Codon 6 G > A (HbC) --–

Codon 6 A > T (HbS) –--

Codon 6 -A �0

Codon 8 -AA �0

Codon 8/9 G > C �0

Codon 30 G > A �0

IVS1.1 G > A �0

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IVS1.2 T > A �0

IVS1.5 G > C �+

IVS1.6 T > C �+

IVS1.110 G > A �0

IVS1.116 T > G �0

IVS1-25 25 bp del �0

Codon 39 C > T �0

Codon 44 -C �0

IVS2.1 G > A �0

IVS2.745 C > G �+

IVS2.844 C > G �+

Table II: . classification Classification of the subjects based on the blood parameters.

Group 1. Normal Subjects subjects (total subjects: 2638)

GRO* normal.

normal erythrocyte morphology.

HbA2 <3.2%.

• GRO normal.

• Normal erythrocyte morphology.

• HbA2 < 3.2%.

Group 2. Individuals carrying the �beta-thalassemia trait (total subjects: 240)

GRO* alterate.

altered erythrocyte morphology.

HbA2>4%.

• Altered GRO.

• Altered erythrocyte morphology.

• HbA2 > 4%.

Group 3. Individuals not carrying the �beta-thalassemia trait (total subjects: 112)**.)a

GRO* alterate and/or normal.

alterate and/or normal erythrocyte morphology.

HbA2 between 3.4 and 4.8.

• Altered GRO and/or normal GRO.

• Altered and/or normal erythrocyte morphology.

• HbA2 between 3.4 and 4.8.

GRO, globular resistance and osmotic fragility of erythrocytes.

aOf the 112 subjects who underwent molecular diagnosis of �-globin genes, 72 were false positives.

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Table III: s. Subjects with iron deficiency anemia.

Total number of subjects examined 2990

Men 167 (45%)

371 (=12,.4%)

Number of non-carriers of �beta-

thalassemia trait, but with iron

deficiency anemia Women 204 (55%)

Table IV. Diagnostic procedure to query the status of a healthy carrier of �beta

thalassemia: flowchart.

First level (basic screening) •Second level

In case of:

Isolated elevation of HbA2.

Suspected �beta- thalassemia silent.

Genetic conditions not resolved in step 1.

Persistence of diagnostic doubts.

molecular Molecular identification of hemoglobin variants

1. Tests of routine 2. Test

blood Blood count performed with electronic cell

counter (CBC).

Molecular analysis of the globin genes

Determination of HbA, HbA2 and HbF by HPLC

Qualitative and quantitative evaluation of

hemoglobin variants by HPLC (e.g. eg.: HbE, HbC,

HbS)

Determination of blood serum ferritin.

2. Further investigation

In case of hemoglobin variants:

qualitative Qualitative evaluation of Hb variants

by electrophoresis.

Figure 1. HbA2 distribution in the sample. The non-carriers of �beta-thalassemia trait

(2710 subjects) have a mean of HbA2 of 2.6%, while the carriers of �beta-thalassemia

traits (280 subjects) have a mean of HbA2 of 4.8%.

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Documents

Incidence of β-thalassemia carrier on 1495 couples in preconceptional period