Background
Methods
Study population
DNA extraction and whole genome amplification
Determining SNPs in the HMOX1 promoter
Allele specific PCRs
|
Sequences
|
---|---|
Fw | 5′-ACTGGCACTCTGCTTTATGTGTGA-3′ |
Rw A(−413) | 5′-GGAGGCAGCGCTGCTCAGAGTAAT-3′ |
Rw (−413)T | 5′-GGAGGCAGCGCTGCTCAGAGTAAA-3′ |
Conditions |
95°C 15 min, 35 cycles: (94°C 30 sec, 60°C 30 sec, 72°C 30 sec), 72° 10 min
|
Restriction fragment length polymorphism
| |
Fw | 5′-TTATTTTATATTTTGTAGAGCC-3′ |
Rw | 5′-AGATGATTCATACAGTCCTTTC-3′ |
Conditions |
94°C 15 min, 45 cycles: (94°C 30 sec, 49°C 30 sec, 72°C 3 min), 72° 10 min
|
(GT)
n
repeat length polymorphism
| |
Fw (5’fam)
| 5′-AGAGCCTGCAGCTTCTCAGA-3′ |
Rw | 5′-ACAAAGTCTGGCCATAGGAC-3′ |
Conditions |
95°C 15 min, 30 cycles: (95°C 30 sec, 64°C 30 sec, 72°C 30 sec), 72° 10 min
|
Determining the HMOX1 promoter (GT)n repeat length polymorphism
Statistical analysis
Results
Population demographics
Controls (n=287)
|
Uncomplicated malaria (n=101)
|
Severe anaemia (n=63)
|
Cerebral malaria (n=132)
| |
---|---|---|---|---|
Age (years)
| ||||
Mean ± SD | 8.01 ± 3.97 | 5.51 ± 3.32 | 2.92 ± 2.62 | 4.81 ± 2.77 |
Minimum/Maximum | 0-15 | 0.5-14 | 0-12 | 0.5-13 |
Sex (n)
| ||||
Male | 145 (50.52%) | 55 (54.46%) | 40 (63.49%) | 69 (52.27%) |
Female | 142 (49.48%) | 46 (45.54%) | 23 (36.51%) | 63 (47.72%) |
Parasitaemia (p/ul)
| ||||
Median | - | 52.000 | 50.265 | 97.157 |
Percentiles 25 and 75 | - | 24.941-121.912 | 17.730-114.295 | 33.788-212.900 |
Haemoglobin (g/dl)
| ||||
Mean ± SD | - | 10.49 ± 1.89 | 4.10 ± 1.17 | 7.56 ± 2.26 |
Minimum/Maximum | - | 6.80-17.5 | 1.80-11.20 | 0.80-13.40 |
Allele and genotype frequencies of the SNPs in the HMOX1 promoter
T(−413)A Alleles and genotypes N (%)
|
Controls (n=281)
|
Uncomplicated malaria (n=95)
|
Severe anaemia (n=61)
|
Cerebral malaria (n=119)
| |
---|---|---|---|---|---|
Alleles
|
A
| 175 (31.1) | 56 (29.5) | 35 (28.7) | 70 (29.4) |
T
| 387 (68.9) | 134 (70.5) | 87 (71.3) | 168 (70.6) | |
Genotypes
|
A/A
| 26 (9.3) | 11 (11.6) | 5 (8.2) | 12 (10.1) |
A/T
| 123 (43.8) | 34 (35.8) | 25 (41.0) | 46 (38.7) | |
T/T
| 132 (47.0) | 50 (52.6) | 31 (50.8) | 61 (51.3) | |
G(−1135)A Alleles and genotypes N (%)
|
Controls (n=238)
|
Uncomplicated malaria (n=78)
|
Severe anaemia (n=55)
|
Cerebral malaria (n=107)
| |
Alleles
|
G
| 397 (83.4) | 123 (78.9) | 91 (82.7) | 179 (83.6) |
A
| 79 (16.6) | 33 (21.2) | 19 (17.3) | 35 (16.4) | |
Genotypes
|
G/G
| 179 (75.2) | 57 (73.1) | 42 (76.4) | 81 (75.7) |
G/A
| 39 (16.4) | 9 (11.5) | 7 (12.7) | 17 (15.9) | |
A/A
| 20 (8.4) | 12 (15.4) | 6 (10.9) | 9 (8.4) |
Allele and genotype frequencies of the (GT)n repeat length polymorphism in the HMOX1 promoter
Microsatellites Alleles & genotypes N (%)
|
Controls (n=564)
|
Uncomplicated malaria (n=95)
|
Severe anaemia (n=58)
|
Cerebral malaria (n=128)
| |
---|---|---|---|---|---|
Alleles
|
S
| 114 (20.2) | 41 (21.6) | 23 (19.8) | 48 (18.8) |
M
| 210 (37.2) | 68 (35.8) | 40 (37.5) | 100 (39.1) | |
L
| 240 (42.6) | 81 (42.6) | 53 (45.7) | 108 (42.2) | |
Genotypes
|
S/S
| 12 (4.3) | 6 (6.3) | 3 (5.2) | 6 (4.7) |
S/M
| 41 (14.5) | 12 (12.6) | 6 (10.3) | 23 (18.0) | |
S/L
| 49 (17.4) | 17 (17.9) | 11 (19.0) | 13 (10.2) | |
M/M
| 44 (15.6) | 17 (17.9) | 5 (8.6) | 20 (15.6) | |
M/L
| 81 (28.7) | 22 (23.2) | 24 (41.4) | 37 (28.9) | |
L/L
| 55 (19.5) | 21 (22.1) | 9 (15.5) | 29 (22.7) |
Analysis of the combination of the A(−413)T and (GT)n repeat length polymorphisms
(GT)n
|
T(−413)A
|
N (%)
|
OR (95% CI)
|
P value
|
---|---|---|---|---|
SS | AA | 0 (0.0) | - | - |
SS | AT | 3 (0.6) | 1.77 (0.15-21.34) | 0.65 |
SS | TT | 23 (4.3) | 0.63 (0.22-1.84) | 0.40 |
SM | AA | 3 (0.6) | <0.001 (<0.001- > 999.99) | 1.0 |
SM | AT | 54 (10.0) | 1.01 (0.46-2.22) | 1.0 |
SM | TT | 22 (4.1) | 1.12 (0.42-3.37) | 0.75 |
SL | AA | 2 (0.4) | <0.001 (<0.001- > 999.99) | 1.0 |
SL | AT | 21 (3.9) | 0.39 (0.09-1.58) | 0.19 |
SL | TT | 65 (12.1) | 0.70 (0.33-1.50) | 0.36 |
MM | AA | 37 (6.9) | 0.66 (0.27-1.58) | 0.35 |
MM | AT | 27 (5.0) | 0.70 (0.24-2.04) | 0.52 |
MM | TT | 18 (3.4) | 0.59 (0.14-2.45) | 0.47 |
ML | AA | 9 (1.7) | 1.43 (0.28-7.21) | 0.66 |
ML | AT | 90 (16.7) | 1.30 (0.66-2.57) | 0.45 |
ML | TT | 57 (10.6) | 0.71 (0.31-1.62) | 0.42 |
LL | AA | 2 (0.4) | <0.001 (<0.001- > 999.99) | 0.99 |
LL | AT | 25 (4.7) | 0.13 (0.02-1.08) | 0.06 |
LL | TT | 80 (14.9) | Reference | - |