Results
A total of 206 infants, consisting of 114 males (55.3%), with the mean
age of 8.2 months (SD 2.0, range 6-12 months), were enrolled randomly
after informed consent. Interestingly, we found 97 individuals (47%)
with some form of thal minor, 39 with α-thal (18.9% of the total
population), 45 with β-globin mutation mainly Hb E (21.8%), and 13 with
combined α and β globin abnormalities (6.3%). None of these individuals
had the genotype found in thal diseases such as HbH disease or Hb
E/β-thal; therefore, they were all classified as thal minor and were
subsequently used as a group for further analysis. Details of all
comprehensive genotype data are shown in Table 1 . Infants with
thal minor had no history of blood transfusion and no
hepatosplenomegaly.
We found no significant difference in all clinical characteristics: age,
gender, growth and nutrition parameters, and the iron markers such as
serum ferritin, TS, and hepcidin levels. The exception was having a
family history of anemia or thal being more common in infants with thal
minor (43.3%) than those without thal (12.8%), as shown inTable 2. In our logistic regression analysis, having a history
of anemia or thal showed an increased risk of thal minor in infants: odd
ratio 5.18 (95% CI: 2.60-10.33), P =0.001. Interestingly, the
number of infants with thal minor with IDA at first diagnosis was higher
than those with normal globin genotypes (32.0% vs. 20.2%);
moreover, the number of infants with normal iron status and ID were
significantly different among infants with and without thal minor
(P =0.037) (Table 2). To determine the effects of iron
status (normal iron, ID, or IDA) on hematological parameters, we
performed subgrouping analysis within the groups of infants with and
without thal minor (Table 3 ). There were significant
differences in Hb, hematocrit (Hct), mean corpuscular volume (MCV), mean
corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration
(MCHC), and red blood cell distribution width (RDW) within both groups
suggesting the significant role of iron in determining Hb, Hct and red
blood cell indices. With the exception of increased RBC counts and RDW,
other RBC parameters were lowest in IDA, followed by ID and normal iron.
Of note, infants with thal minor who had IDA (N=31) displayed the
statistically lowest values in MCV, MCH, and highest RDW (Table
2), suggesting co-inheritance of globin mutations can have epistatic
effects on hematology on top of the primary effects of iron status.
In this regard, we further compared the group of infants with normal
iron and IDA to see the effects of thal minor (see Supplementary
Table 1 ). Coinheritance of thal had significant effects on Hb, Hct,
RBC, MCV, MCH, RDW but not MCHC (suppl. Table 1) only in those
with normal iron status. However, we found only RBC and MCV to be
significantly different in infants who already had IDA, suggesting this
epistatic effect was operative on those only two parameters. In
addition, we found an effect of iron on significantly decreased levels
of Hb A2 in infants without thal minor but not in those with thal. On
the other hand, the basal Hb F in infants with thal minor was generally
higher than those without thal minor, suggesting a delay in globin
switching, one of the consequences of globin
abnormality.39,40 Finally, the iron status was not
significantly associated with the levels of persistent Hb F expression
within the groups of both infants with and without thal minor
(Table 3 ).
To determine the effects of thal minor on hepcidin expression, we
compared serum hepcidin, serum ferritin, and TS among healthy infants
(no thal and normal iron status) with those having different types of
thal minor as shown in Figure 1 . Details of measurements in
each group are shown in Supplementary Table 2A (with
iron-replete) and 2B (with iron deplete). The levels of
hepcidin tended to be slightly lower in those with thal minor and lowest
in those with combined α and β globin mutations (Figure 1C).This finding was consistent with a slightly increasing trend of serum
ferritin (Figure 1A) and TS (Figure 1B) in those with
combined thal minors, although there were no statistically significant
differences. We also found no differences in these parameters as a group
(normal vs . thal minor) and by different genders (male vs .
female) (see Supplementary Figure 1 ).
The primary diagnosis of infants with IDA using Hb levels, SF and TS
values in our study was further confirmed in the majority of those by
the therapeutic response to iron therapy. Thirty-four out of 53 IDA
infants with (n=31) and without thal minor (n=22) who can be reached by
telephone appointments have received iron therapy (4-6 mg/kg/day) for
8-12 weeks, and all showed therapeutic response. Their hematology during
fellow up visits revealed a significant increase in all RBC parameters
compared to the baseline within their groups (P <0.05).
Interestingly, IDA infants without thal minor (n=18) had slightly
greater increment for Hb and MCV after iron therapy than IDA infants
with a thal minor (n=16); however, there were no statistically
significant differences between the groups (P =0.099 and 0.278,
respectively). The mean Hb increment after iron therapy was 1.7 g/dL (SD
1.1, range 0.1-4.1) vs 1.1 (SD 0.9, range 0.2-3.1 g/dL) and mean MCV
increment 4.9 fL (SD 3.7, range 0.3-11.8 fL) vs 3.5 (SD 3.4, range
0-9.5) fL, in IDA infants without and with thal minor respectively. This
result has confirmed our diagnosis of IDA in both groups at the
baseline. (Supplementary Table 3 )