PMC5225734_01
Family tree of patient
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F1
Her father and second brother also had cyanosis but were otherwise well while her mother and the other two siblings were normal (Figure 1(Fig. 1)).
(From right) Skin colour of patient, her mother, second brother and father
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F2
Family study was performed on the individuals with cyanosis (Figure 2(Fig. 2)).
(From right) Venous blood in ethylenediaminetetraacetic acid (EDTA) tube of healthy control (normal colour), patient (dark brown colour), patient's mother (normal colour), patient's second brother and patient's father (dark brown colour)
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F3
The venous blood samples were collected and showed distinct dark brown colour from the affected family members as opposed to normal colour from the healthy control and unaffected mother (Figure 3(Fig. 3)).
(From right) Venous blood's colour of healthy control (bright red) and patient (dark brown) with exposure of atmospheric oxygen for 10 minutes
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F4
In contrast, a drop of healthy control's venous blood turned to bright red after exposure of atmospheric oxygen (Figure 4(Fig. 4)) due to conversion of deoxygenated Hb to oxygenated Hb.
Abnormal band (X) was seen in between HbA2 and HbS at alkaline pH.
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F5
Hb electrophoresis at alkaline pH showed abnormal band in between HbA2 and HbS (Figure 5(Fig. 5)) while at acidic pH showed abnormal band in between HbA and HbS (Figure 6(Fig. 6)).
Abnormal band (X) was seen in between HbA and HbS at acidic pH.
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F6
Hb electrophoresis at alkaline pH showed abnormal band in between HbA2 and HbS (Figure 5(Fig. 5)) while at acidic pH showed abnormal band in between HbA and HbS (Figure 6(Fig. 6)).
HPLC of patient's haemoglobin showed an unknown peak at retention time of 4.84 min.
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F7
Brother: 3.7 %) at zone 4. High-performance liquid chromatography (HPLC) of Hb demonstrated an unknown peak at retention time of 4.84 min with the levels of 20 % in patient (Figure 7(Fig. 7)) and 16.7 % in brother but no result for father due to insufficient sample. Besides, there was slightly raised of HbA2 in three of them (Patient: HPLC 4.3 %, CE 4.7 %; Father: CE 4.2 %; Brother: HPLC 4.1 %, CE 4.4 %).
beta gene sequencing revealed a heterozygous point mutation of CAC>TAC on codon 92 at beta globin genes.
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F8
Deoxyribonucleic acid (DNA) analysis of deletional alpha thalassemia by multiplex polymerase chain reaction (PCR) showed alphaalpha/alphaalpha in patient but alphaalpha/-alpha3.7 in her father and brother. beta gene sequencing confirmed a point mutation of CAC>TAC on codon 92 in one of the beta genes in three of them (Figure 8(Fig. 8)).