Basic demographic data
Between December 2016 and March 2019, 60 SCD patients were recruited for the study. Fifty-one patients (51/60; 85%) were SBT patients and 9 (9/60; 15%) were homozygous Hb SS patients. Homozygosity was confirmed in all 9 patients by typing the sickle mutation rs334 (T>A) at chr11:5227002 (GRCh38.p12) by NGS. There were 30 male and 30 female participants. Thirty-seven (72.55%) SBT patients had IVS 1-5 (G→C) mutation, 11(21.57%) had IVS 1-1 (G→A) mutation, 2 had CD-16 mutation and one SBT patient had CD 41/42 mutation. Even though IVS 1-5 (G→C) clinically behaves as an b0 mutation for all practical purposes SBT patients with IVS 1-5 (G→C) was classified separately as severe Hb S/b+ as it is known to produce some Hb A (n=37) (20-23). SBT patients identified with other b mutations were all unquestionably severe mutations and were classified as Hb S/b0 type (n=14). SCD patients in the present study were living in 10 out of the 25 districts of Sri Lanka. Geographically, two prominent patient clusters were noted and the Southern cluster comprising Hambantota and Monaragala districts accounting for 27 (45.0%) SCD patients was the dominant cluster (Fig 1). The SCD patients comprised of three ethnic groups; 52 (86.67%) were Sinhalese, 5 (8.33%) were Moors and 3 (5.0%) were Tamils. Non-parametric statistical methods were used since data were not normally distributed.
Haematological data
Basic haematological parameters of those who had not received a blood transfusion in the three months prior to blood sampling are summarized in table 1.
Table 1: Haematological parameters of Hb SS, Hb S/b+ severe and Hb S/b0 type patients
Parameter
|
Hb SS
|
Hb S/b+(severe) (n=31)
|
Hb S/b 0 (n=11)
|
p-value
|
Mean (SD)
|
Mean (SD)
|
Mean (SD)
|
|
|
Male (n=4)
|
Female (n=2)
|
Total
|
Male (n=16)
|
Female (n=15)
|
Total
|
Male (n=5)
|
Female (n=6)
|
Total
|
|
Hb (g/dl)
(13.0-18.0 – Male)
(11.5 – 16.5 Female)
|
8.4 (1.1)
|
8.7 (0.2)
|
8.5 (0.9)
|
8.3 (1.0)
|
8.3 (1.0)
|
8.3 (1.0)
|
8.0 (0.8)
|
8.3 (0.6)
|
8.1 (0.7)
|
0.787
|
Hb A2 (%)
(1.5 – 3.2 %)
|
1.7 (0.4)
|
1.4 (0.5)
|
1.6 (0.4)
|
4.4 (0.6)
|
4.2 (0.5)
|
4.3 (0.5)
|
4.9 (0.2)
|
4.4 (0.6)
|
4.6 (0.5)
|
<0.000*
|
Hb F (%)
(< 1.0%)
|
20.6 (1.8)
|
31.9 (0.5)
|
24.4 (6.0)
|
23.2 (6.4)
|
24.3 (6.4)
|
23.7 (6.4)
|
19.1 (5.7)
|
22.9 (8.7)
|
21.2 (7.4)
|
0.514
|
MCV (fl) (80 – 100)
|
84.2 (4.3)
|
85.0 (9.9)
|
84.5 (5.6)
|
69.3 (4.6)
|
68.2 (3.9)
|
68.8 (4.3)
|
67.8 (3.3)
|
69.2 (4.1)
|
68.6 (3.7)
|
0.001*
|
MCH (pg) (27 – 32)
|
29.6 (0.5)
|
29.9 (2.0)
|
29.7 (1.0)
|
22.1 (1.7)
|
22.3 (1.4)
|
22.2 (1.6)
|
21.0 (1.6)
|
22.0 (1.3)
|
21.5 (1.4)
|
<0.000*
|
MCHC (%) (33-35)
|
33.0 (0.5)
|
33.5 (0.7)
|
33.2 (0.6)
|
31.4 (0.8)
|
31.6 (1.0)
|
31.5 (0.9)
|
31.7 (1.1)
|
31.5 (0.6)
|
31.6
(0.8)
|
0.002*
|
Reticulocytes (%)
(0.5 – 1.5)
|
9.6 (2.1)
|
6.3 (5.5)
|
8.5 (4.5)
|
5.3 (1.6)
|
5.1 (1.7)
|
5.2 (1.6)
|
4.8 (3.0)
|
5.1 (2.4)
|
5.0 (2.6)
|
0.048*
|
Absolute Retic count (×1012/L)
(0.020-0.200)
|
0.2767 (0.1041)
|
0.1866 (0.1328)
|
0.2467 (0.1104)
|
0.2066 (0.0669)
|
0.1905 (0.0609)
|
0.1988 (0.0635)
|
0.1643 (0.0856)
|
0.1855 (0.0896)
|
0.1759 (0.0840)
|
0.244
|
WBC (×109/L)
(4.5 – 11.0)
|
14.0 (8.8)
|
6.7 (0.1)
|
11.6 (7.8)
|
8.7 (3.6)
|
10.5 (5.6)
|
9.59 (4.7)
|
10.2 (5.5)
|
12.2 (7.4)
|
11.34 (6.4)
|
0.863
|
PLT (×103/µL)
(150 – 450)
|
324.0 (178.0)
|
252.0 (111.8)
|
300 (151.2)
|
235.5 (155.0)
|
330.3 (151.5)
|
281.4 (158.3)
|
254.6 (42.8)
|
347.2 (199.5)
|
305.1 (151.6)
|
0.715
|
Hb, Haemoglobin; Hb A2, Adult Haemoglobin-2; Hb F, Foetal Haemoglobin; MCV, Mean Corpuscular Volume; MCH, Mean Corpuscular Haemoglobin; MCHC, Mean Corpuscular Haemoglobin Concentration; WBC, White Blood Cells; PLT, Platelets. p<0.05 of Kruskal Wallis H test was taken as significant
p value has been calculated with respect to total figures of the three groups (Hb SS, severe Hb S/b+ and Hb S/b0 type)
Clinical findings
Age at presentation of all SCD patients was highly variable, ranging from 4 months to 55 years (Mean 9.8 years; SD- 11.3 years). Most of the patients (51.7%; n=31) had presented with fever, whilst the next common presenting symptoms were joint pain and abdominal pain (26.7%; n=16). Icterus led to identification of the disease in a further 9 (15%) cases. Six more patients were incidentally diagnosed whilst investigating anaemia. A further three patients were diagnosed during pregnancy (including two Hb S/b0 patients and one severe Hb S/b+ patient). Three out of 9 (33.33%) Hb SS, 1/37 (2.7%) severe Hb S/b+ and 2/14 (14.3%) Hb S/b0 (p 0.012) patients were on regular blood transfusion (defined as > 8 transfusions / year). Based on clinical records it appeared that blood transfusions had mostly been given when haemoglobin concentration of the patient fell to 6g/dl or less, although this could not be ascertained with certainty. Incidentally, 12 (23.53%) of SBT patients (7 severe Hb S/b+ and 5 Hb S/b0 patients) never had any transfusion in their lifetime.
Clinical features of Hb SS, severe Hb S/b+ and Hb S/b0 and patients are summarized in table 2. Joint pains were the most common clinical symptom observed among all SCD patients. Ischemic cerebrovascular event had occurred in one severe Hb S/b+ and one Hb SS patient. Similarly, avascular necrosis of the hip was present in one Hb SS patient and one severe Hb S/b+ patient. Fisher’s exact test showed that the incidence of dactylitis was the only clinical feature which was significantly different between Hb SS, severe Hb S/b+ and Hb S/b0 patients (p 0.027). Nevertheless, none of the clinical features were significantly different between severe Hb S/b+ and Hb S/b0 patients. Genotype-phenotype associations were also assessed separately between Hb SS, severe Hb S/b+ and Hb S/b0 patients those who were on regular transfusions and those who were not. Nevertheless, Fisher’s exact test was unable to find any difference between the patients in the two transfusion categories. Splenectomy had been carried out in 1 / 9 Hb SS and 4/51 severe Hb S/b+ patients. The exact reason for splenectomy and its justification could not be deduced from the clinical records. Four of the splenectomized patients had undergone the surgery before the age of 20 years. Forty-one SCD patients (6 Hb SS, 23 severe Hb S/b+ and 12 Hb S/b0 patients) (68.3%) in our series had a history of at least one pain event (Joint/Abdominal/Chest) in their lifetime, while 19 SCD patients including three Hb SS individuals had not experienced any pain events. Cold weather (33.33%, n=20) was the most frequently identified precipitating factor for pain events among SCD individuals, followed by infections (26.32%, n=15). Thirteen (21.66%) SCD patients who had pain crisis reported no obvious precipitant factor for pain events. At the time of data collection 26 (43.33 %) SCD patients were taking Hydroxyurea. Nineteen (31.67%) SCD patients were on Folic acid only. Twenty-eight (48.7%) SCD patients were one penicillin prophylaxis. Six SCD patients (10%) were not on any medication.
Table 2: Summary of clinical features observed between severe Hb S/b+, Hb S/b0 and Hb SS groups
Clinical feature / presentation
|
severe Hb S/b+
group
n = 37
|
Hb S/b0 group
n = 14
|
Hb SS group
n = 9
|
Cumulative figure
N=60
|
p value1 between two SBT groups
|
p value2 between all three groups
|
Joint pain
|
20 (54.05%)
|
12 (85.70%)
|
7 (77.78%)
|
39 (65.0%)
|
0.053
|
0.080
|
Palpable spleen
|
26 (70.27%)
|
7 (50.0%)
|
3 (33.33%)
|
36 (60.0%)
|
0.204
|
0.086
|
Hospital admission due to pain
|
20 (54.05%)
|
10 (71.43%)
|
5 (55.56%)
|
35 (58.33%)
|
0.346
|
0.562
|
Jaundice
|
14 (37.84%)
|
9 (64.28%)
|
6 (66.67%)
|
29 (48.33%)
|
0.120
|
0.109
|
Major infections
|
13 (35.14%)
|
4 (28.57%)
|
4 (44.44%)
|
21 (35.0%)
|
0.749
|
0.744
|
Recurrent headaches
|
11 (29.73%)
|
1 (7.14%)
|
2 (22.22%)
|
14 (23.33%)
|
0.142
|
0.281
|
Pica
|
6 (16.22%)
|
3 (21.43%)
|
2 (22.22%)
|
11 (18.33%)
|
0.692
|
0.711
|
Abdominal pain
|
6 (16.22%)
|
4 (28.57%)
|
0
|
10 (16.67%)
|
0.432
|
0.217
|
Dactylitis
|
3 (8.11%)
|
3 (21.43%)
|
4 (44.44%)
|
10 (16.67%)
|
0.327
|
0.027*
|
Gallstones
|
6 (16.22%)
|
3 (21.43%)
|
1 (11.11%)
|
10 (16.67%)
|
0.692
|
0.888
|
Pallor
|
4 (10.81%)
|
2 (14.28%)
|
3 (33.33%)
|
9 (15.0%)
|
0.661
|
0.220
|
Acute chest syndrome
|
3 (8.11%)
|
3 (21.43%)
|
2 (22.22%)
|
8 (13.33%)
|
0.327
|
0.290
|
Vision impairment
|
8 (21.62%)
|
0
|
0
|
8 (13.33%)
|
0.088
|
0.074
|
Abdominal distension
|
3 (8.11%)
|
1 (7.14%)
|
1 (11.11%)
|
5 (8.33%)
|
1.000
|
1.000
|
Facial deformities
|
1 (2.70%)
|
2 (14.28%)
|
0
|
3 (5.0%)
|
0.179
|
0.189
|
Nocturnal enuresis
|
2 (5.40%)
|
1 (7.14%)
|
0
|
3 (5.0%)
|
1.000
|
1.000
|
Leg ulcers
|
2 (5.40%)
|
1 (7.14%)
|
0
|
3 (5.0%)
|
1.000
|
1.000
|
1Fisher’s exact test p<0.05 was taken as significant between two SBT groups
2Fisher’s exact test p<0.05 was taken as significant between all three groups
Genetic findings
Gap PCR for common α+ gene deletions found only 4 (6.67%) SBT patients with 3.7 kb deletions. None of the SCD patients had the 4.2 kb α gene deletion.
Haplotyping by traditional RFLP showed that the sickle mutation occurred on two main beta globin haplotypes in Sri Lanka. Namely Arab-Indian (AI) and Benin. Out of 18 β globin haplotypes among the 9 Hb SS patients, 14 were AI haplotype and 4 were Benin haplotype. Presence of AI haplotype and Benin haplotype in Sri Lanka was confirmed with NGS by typing 4 different SNPs (rs3834466, rs28440105, rs10128556 and rs968857) in 9 Hb SS patients as described previously (24). When looking at the genetic variants that moderate Hb F levels, rs6545816 in BCL11A was found at the highest allele frequency (88%) followed by rs7482144 in Xmn I-HBG2 (47%) (Table 3). Allelic discrimination plot of rs6545816 is shown in Fig 2.
Table 3: Presence and frequency of Hb F determining variants in Sri Lankan SCD patients
Locus
|
Variants
|
Position on chromosome
|
Allele change
|
Genotypes detected
|
Hb F boosting allele (Frequency)
|
Chromosome 2
|
|
|
|
|
|
BCL11A
|
rs6545816
|
60,568,365
|
A > C
|
CC, n= 37
|
C (88%)
|
|
|
|
|
AC, n= 14
|
|
|
rs1427407
|
60,571,547
|
G > T
|
GG, n= 43
|
T (12%)
|
|
|
|
|
GT, n= 14
|
|
Chromosome 6
|
|
|
|
|
|
HMIP-2A
|
rs66650371
|
135,460,326-135,460,328
|
In > Del
|
II, n= 52
DI, n= 5
|
Del (6%)
|
|
|
|
|
DD, n=1
|
|
HMIP-2B
|
rs9402686
|
135,469,509
|
G > A
|
GG, n= 52
|
A (4%)
|
|
|
|
|
GA, n= 4
|
|
Chromosome 11
|
|
|
|
|
|
Xmn I – HBG2
|
rs7482144
|
5,232,745
|
G > A
|
GG, n = 4
|
A (47%)
|
|
|
|
|
GA, n= 56
|
|