Beta-Thalassemia mutations in subjects with borderline HbA2

values

M.Usman Javed1, Mavra Fatima2, Ayisha Imran3, A.S.Chughtai4, N.A. Malik5

Abstract

Background: Thalassemia is an inherited hemoglobinopathy in which there is imbalance in

the synthesis of chains of Hemoglobin molecule, thus causing anemia. This inherited disorder
is very common in Mediterranean region, South Asia and Middle East. In Pakistan, there’s a
culture of consanguous marriages so there is a really high incidence of children who suffer
from thalassemias

Objective : To determine the types of beta thalassemia mutations in subjects with borderline
Hemoglobin A2 (HbA2).

Materials and methods : A cross-sectional study was conducted at Chughtai Lab ,Lahore on
16 samples as it is a pilot study in Pakistan. The duration of this study is six months i.e from -
--- to -----.

Results : In this study, the mean age of patients was 22.19±16.55years. There were 42 (60%)
males and 28 (40%) females. The mean HbA2 of patients was 3.39±0.28%. In this study, IVS
mutations were observed in 23 (32.9%) cases, out of which 1.1 (G>T) was observed in 15
(21.4%) cases, 1.110 (G>A) in 8 (11.4%) cases, 2.1 (G>A) in 4 (5.7%) cases while 1.5 (G>C)
in 8 (11.4%) cases. CODON mutations were observed in 23 (32.9%) cases, out of which 44 (-
C) was observed in 8 (11.4%) cases, 15 (TGG>TAG) in 4 (5.7%) cases, 5(-CT) in 8 (11.4%)
cases, 6 (A>T) in 8 (11.4%) cases while 8 (-AA) in 3 (4.3%) cases. CAP occurred in 16
(22.2%) cases and all had +1 (A>C).

Conclusion : Subjects with borderline HbA2 were found to have beta thalassemia mutations
of which most common was IVS mutations alongwith few others mentioned above in the
results. However the effect of mutations on clinical course and treatment of such subjects is
yet to be determined.

Keywords : Beta thalassemia, hemoglobinopathies, mutations, Hemoglobin A2 (HbA2)

Introduction

Thalassemia, the most common hereditary anemia is characterized by defects in the synthesis
of one or more of the globin chains that form haemoglobin (Hb) tetramer. β-thalassemia, one
of the genetic hemoglobinopathies with autosomal recessive pattern of inheritance, is known
to be caused by more than 200 point mutations observed at the molecular level.1

Over 30 million people are carrying the defective gene.2 It's particularly associated with
people of Mediterranean, Indian subcontinent, and Middle East origin. An estimated 5000-
9000 children with β-thalassemia are born per year, although no documentary registry is
available in Pakistan. The estimated carrier rate is 5-7%, with 9.8 million carriers in the total
population.3

Clinically β-thalassemia’s are divided into β-thalassemia major which is the homozygous
state, and β- thalassemia trait or minor, which corresponds to the heterozygous state. Other
relatively uncommon forms are also present. In β- thalassemia major there is severe
transfusion dependent anemia while in β- thalassemia trait there is mild to moderate
microcytic hypochromic anaemia.4

Beta-thalassemia has an overall carrier frequency of more than 5% in Pakistan.5 Accurate

determination of the carrier phenotype is essential for detecting couples at risk for producing
offspring with hemoglobinopathy. Heterozygous β-thalassemia is usually silent at the clinical
level.6 The diagnostic characteristic of β- thalassemia trait is an elevated level of HbA2 on
Haemoglobin electrophoresis or high performance liquid chromatography 000(HPLC).HPLC
is a sensitive and precise method for identification of Hb A2, Hb F and abnormal
hemoglobin. It has become the method of choice for thalassemia screening because of its
speed and reliability, but it is expensive and cannot be used routinely.7

Iron deficiency modulates the synthesis of HbA2, resulting in reduced HbA2 levels in
patients with iron deficiency anemia. The diagnosis heterozygous beta-thalassemia is based
on a raised HbA2 level. Patients with beta-thalassemia and concomitant iron deficiency can
show normal HbA2 levels.8

OBJECTIVE

To detect Beta-Thalassemia mutations by using Polymerase Chain Reaction in subjects with

borderline HbA2 levels using Capillary electrophoresis.

MATERIALS AND METHODS : A cross-sectional study was conducted at Chughtai Lab

,Lahore on 16 samples as it is a pilot study in Pakistan. The duration of this study is six
months i.e from ---- to -----. Sample size of total 16 cases (it’s a pilot study in Pakistan)
cases was calculated with 95% confidence level, 6% margin of error and taking anticipated
proportion i.e. 7% in patients with borderline HbA2. Data collection will be done by using a
proforma. Samples were collected by non-probability, consecutive sampling technique. Both
males and females of age more than 6 months and cases with HbA2 levels between 3-4%
(borderline) were included in this study. However , patients having iron deficiency anemia
and patients who had recent blood transfusion (within 1 month) were excluded from this
study. All samples received in hematology department of Chughtai Lab, Lahore for
Hemoglobin electrophoresis meeting the inclusion criteria were studied. 3 ml of blood
samples will be collected in EDTA vaccutainers, 1 ml of which will be used to extract the
DNA and 2 ml for Hb electrophoresis and estimation of complete blood count. Blood
samples collected will be subjected to complete blood count (CBC) and red blood cell (RBC)
indices using automatic cell counter Sysmex XP-300 Peripheral smears will be prepared and
stained with Giemsa stain for microscopic examination. Samples will be saved to run on
Sebia and DNA extraction. Ferritin levels will be done to rule out iron deficiency anemia.
DNA will be extracted using a commercial kit according to manufacturer’s instructions. An
ARMS (Amplification refractory mutation system) Polymerase chain reaction (PCR) protocol
will be followed to detect any mutations : G>C at IVS-I-5, G>T at IVS-I-1, codons 8/9 (+G),
codons 41/42 (-TTCT) and 619 bp deletion), sequence alteration of C>T at position -101,
C>G at position -87,T>A at -30, -CT at cd5, G>A (HbC) at cd6, A>T (HbS) at cd6, -A at
cd6, -AA at cd8, +G at cd8/9, TGG/TGA at cd15, G>T(Hb Knossos) at cd27, G>A at IVS
1.1, G>C at IVS 1.5, T>C at IVS 1.6, G>A at IVS 1.110, T>G at 1.116, G>C at 1.130, C>T
at cd39, -C at cd44, G>A at IVS 2.1, C>G at 2.745, C>A at 2.848. Data was entered in to
SPSS version ------ and analyzed. Quantitative variables like age, hemoglobin, MCV, MCH,
RBC count, HbA2 levels will be analyzed by means and standard deviation. Qualitative
variables like gender and patients having mutations will be presented as frequency or
percentages.

RESULTS

In this study, the mean age of patients was 22.19±16.55years. There were 42 (60%) males
and 28 (40%) females. The mean HbA2 of patients was 3.39±0.28%, the mean RBC was
4.94±0.56mg/dl, mean hemoglobin level was 12.84±1.14mg/dl. The mean hematocrit level
was 42.17±3.62%, mean MCV was 85.09±6.59fl, mean MCH was 26.23±1.51pg/cell while
mean MCHC was 30.14±1.26g/dl. Table 1

In this study, IVS mutations were observed in 23 (32.9%) cases, out of which 1.1 (G>T) was
observed in 15 (21.4%) cases, 1.110 (G>A) in 8 (11.4%) cases, 2.1 (G>A) in 4 (5.7%) cases
while 1.5 (G>C) in 8 (11.4%) cases. CODON mutations were observed in 23 (32.9%) cases,
out of which 44 (-C) was observed in 8 (11.4%) cases, 15 (TGG>TAG) in 4 (5.7%) cases, 5(-
CT) in 8 (11.4%) cases, 6 (A>T) in 8 (11.4%) cases while 8 (-AA) in 3 (4.3%) cases. CAP
occurred in 16 (22.2%) cases and all had +1 (A>C). Table 2

Table 1: Characteristics of patients

N 70
Dear madam, total number of
Age (years) 22.19±16.55
samples are 16 as it’s a pilot study
Gender (M:F) 42 (60%) : 28 (40%)
in Pakistan.
HbA2 3.39±0.28
RBC 4.94±0.56 Its only a comparison of borderline
HB 12.84±1.14 HbA2 levels and PCR, so in my
HCT 42.17±3.62 opinion there is no need to
elaborate CBC findings like Hb,
MCV etc (but u know better than
me madam)/

IVS 1.5 mutations are in 5 patients

 MCV 85.09±6.59
MCH 26.23±1.51
MCHC 30.14±1.26

Table 2: Distribution of mutations

Mutations Frequency Percent

1.1 (G>T) 15 21.4
1.110 (G>A) 8 11.4
IVS
2.1 (G>A) 4 5.7
1.5(G>C) 8 11.4
44 (-C) 8 11.4
15 (TGG>TAG) 4 5.7
Codon 5(-CT) 8 11.4
6 (A>T) 8 11.4
8 (-AA) 3 4.3
CAP +1 (A>C) 16 22.2

DISCUSSION :

Some carriers are difficult to identify because the level of HbA2 is not in the typical carrier
range (i.e, HbA2 = 3.8%-6.0%). These atypical carriers have borderline HbA2 values (ie,
HbA2 levels between normal and β-thalassemia carrier levels, 3.3%-3.8%).1-3 The prevalence
of borderline A2 carriers in populations with high frequency of β-thalassemia has been
reported in 2.2% to 3.0% in one study and up to 16.7% in another study.2, 3 Borderline
HbA2 levels associated with reduced mean corpuscular volume (MCV) and mean corpuscular
hemoglobin (MCH) are generally the consequence of mild β+-thalassemia mutations
(i.e., HBB c.92 + 6 T → C), co-inherited δ and β-thalassemia, β-promoter mutations −92
(HBB c.-142 C → T), or coexisting iron deficiency anemia.1-5 Borderline HbA2 with normal
MCV and MCH may be an outlier value of the normal HbA2 distribution in the non-carrier
population or the effect of genetic determinants able to increase HbA2 levels. The genetic
determinants so far identified are the triplication of the α-globin genes, β-promoter mutations
(HBB c.-151 C → T), and some HBD and HBB gene variants.9

CONCLUSION :

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