Introduction
Sickle cell disease (SCD) is a common genetic disorder among Africans. Individuals with the disease have variable clinical expression but homozygosity for the HbS gene, also known as sickle cell anaemia (SCA), is the most severe form [
1]. Children with SCA have chronic hemolysis, leading to accumulation of serum bilirubin and consequent gallstones [
2]. Bilirubin is a tetrapyrol that results from the breakdown of heme in red blood cells. At moderate levels, it protects against oxidative stress and inflammatory injuries, and some infectious diseases [
3‐
5]. However, excessive bilirubin levels, as seen in chronic hemolysis, have been linked to increased incidence of gallstones [
1,
2,
5]. In children with SCA, this risk increases with advancing age with a cumulative incidence of approximately 50% by adulthood and some of them may need cholecystectomy [
2,
6,
7].
Uridine diphosphate glucuronosyltransferase 1A isoform 1
(UGT1A1) is a member of the superfamily of phase II conjugating enzymes that aids the elimination of bilirubin, drugs and a vast variety of endogenous and exogenous substrates by adding a glucuronide moiety to the substrates [
8,
9]. Genetic mutations resulting in absence or severely reduced
UGT1A1 activity leads to Criggler-Najjar syndrome which is characterized by severely elevated serum bilirubin and increased risk of kernicterus [
10]. However, variations in (TA) n tandem repeat sequence within the TATA box promoter region affect
UGT1A1 gene expression and the activity of its (TA) n four alleles, namely; (TA) 5, 6, 7, and 8, leading to moderate elevation of serum bilirubin [
8,
9]. There is a negative association between the
UGT1A1 and repeat length of the four alleles attributable to the decreasing promoter activity acting via altered affinity for the TATA–binding protein [
8,
9]. The promoter activity leads to functional changes such that, genotype (TA)5/5 is considered to have ~ 20% increased expression i.e. high activity in comparison to genotype (TA) 6/6 known as the wild-type with normal activity. In addition, genotypes heterozygous and or homozygous for (TA)7 and (TA)8 alleles have approximately between 30 and 50% reduced expression i.e. low activity, compared to the wild-type [
8]. However, genotypes (TA)7/7, 7/8 & 8/8 have the lowest activity [
2,
8,
9] and (TA)7/7 has been described generally as genetic hallmark for Gilbert syndrome [
2,
11]. There is an inverse relationship between the serum bilirubin levels across these subgroups and the degree of genotype activity. To this end, individuals with low-activity genotypes have elevated levels of serum bilirubin and are, therefore, subjected to the modulating effects of higher serum bilirubin levels including susceptibility to gallstones [
2,
8,
9,
11].
Despite the huge burden of SCA in Africa [
1], with Nigeria having the highest burden of SCA in the world [
12], there is little understanding of the contributions of genetic modifiers of SCA phenotypes in the country. To the best of our knowledge, there are no data on the effects of
UGT1A1 polymorphisms on the clinical expression of Nigerian SCA patients. The aims of this study were to determine the distribution of
UGT1A1 (TA) n genotypes among a group of young SCA patients and healthy controls and also determine the influence of the
UGT1A1 (TA) n genotypes on the laboratory parameters and clinical events among the young SCA patients.
Results
The 101 patients with SCA consisted of 66 males and 35 females with median age of 9 and a range 2 - 21 years. The controls were made up of 19 sickle cell trait (HbAS) and 45 haemoglobin HbAA, median age of 8, range 2 - 18 years (p = 0.4260), and 41 males. The SCA patients have been on follow up for a median of 4 years, range 1–14 years.
UGT1A1 (TA) n alleles and genotypes
Four (TA) n alleles: (TA)5, 6, 7, and 8 were found with gene frequencies of 0.11, 0.43, 0.41 and 0.05 respectively. The alleles were associated with 10 genotypes: TA5/5, 5/6, 5/7, 5/8, 6/6, 6/7, 6/8, 7/7, 7/8, 8/8 (Table
1). The wild-type (normal) (TA) 6/6), low (TA) 7/7, 7/8, 8/8), intermediate (TA) 5/7,5/8, 6/7, 6/8), and high (TA) 5/5, 5/6), enzyme activity genotypes were found in 24.8, 24.8, 41.5%, & 8.9% patients and 20.3, 15.6, 61%, & 3.1% controls respectively. The low activity genotypes were found in 25 (24.7%) patients and 10 (15.6%) of the controls (
P = 0.1773). Homozygous (TA) n TA7/7 was found in 22 (21.7%) patients and 5 (7.8%) controls
p = 0.018 (Table
1). The observed genotype distributions of the patients and control group were not significantly different from the values expected under Hardy-Weinberg equilibrium (
x2 = 15.10, df = 9,
p = 0.09), and (
x2 = 11.86, df = 9,
p = 0.22), respectively.
Table 1
Allele and genotype frequencies of UGT1A1 promoter polymorphisms among participants
Allelotypes |
(TA) 5 | 18 (11.7) | 2 (6.7) | 10 (12.5) |
(TA)6 | 67 (43.5) | 16 (53.3) | 28 (35.0) |
(TA)7 | 61 (39.6) | 10 (33.3) | 37 (46.2) |
(TA)8 | 8 (5.2) | 2 (6.7) | 5 (6.3) |
UGT1A1 Genotypes |
TA5/5 | 0 (0) | 0 (0) | 1 (2.2) |
TA5/6 | 9 (8.9) | 0 (0) | 1 (2.2) |
TA5/7 | 6 (6.0) | 1 (5.2) | 8 (17.7) |
TA5/8 | 3 (2.9) | 0 (0) | 0 (0) |
TA6/6 | 25 (24.7) | 8 (42.1) | 5 (11.1) |
TA6/7 | 31 (30.7) | 7 (36.8) | 21 (46.7) |
TA6/8 | 2 (2.0) | 1 (5.2) | 1 (2.2) |
TA7/7 | 22 (21.7) | 1 (5.2) | 4 (8.9) |
TA7/8 | 2 (2.0) | 1 (5.2) | 4 (8.9) |
TA8/8 | 1 (1.0) | 0 (0) | 0 (0) |
UGT1A1 Genotypes by degree of Activity |
Low-Activity genotypes TA (7/7, 7/8, 8/8) | 25 (24.8) | 2 (10.5) | 8 (17.8) |
Intermediate-Activity genotypes (TA6/7, TA6/8), TA5/7, TA5/8, | 42 (41.5) | 9 (47.4) | 30 (66.7) |
Normal Activity genotypes i.e. (Wild Type) TA6/6 | 25 (24.8) | 8 (42.1) | 5 (11.1) |
High-Activity genotypes TA5/5, TA5/6, | 9 (8.9) | 0 (0) | 2 (4.4) |
Effects of UGT1A1 genotype on serum bilirubin and other laboratory parameters of patients
Both the total bilirubin and unconjugated bilirubin levels showed distinct quantitative patterns across the
UGT1A1 (TA) n genotype subgroups with the low-activity genotype group having the highest levels of serum bilirubin (
p < 0.0001). The LDH also showed a similar pattern (
p = 0.0002). However, this was not demonstrated in the other laboratory parameters (Table
2). The stratification of the individual
UGT1A1 (TA) n genotypes separately shows that (TA)7 and (TA) 8 alleles were associated with higher levels of both serum bilirubin and LDH in general (Additional file
2: Table S1).
Table 2
Influence of UGT1A1 (TA) n genotype on laboratory parameters of patients
Total Bilirubin (mg/dl) | 2.8 (1.2–8.1) | 1.8 (0.8–4.6) | 1.4 (0.4–3.8) | 1.4 (0.5–2.8) |
< 0.0001**
1
|
Unconjugated Bilirubin (mg/dl) | 1.8 (0.6–6.3) | 0.8 (0.1–3.3) | 0.6 (0.1–2.3) | 0.5 (0.3–1.6) |
< 0.0001**
1
|
LDH (IU/L) | 987 (296–1860) | 798 (215–1489) | 789 (340–1417) | 287 (197–800) |
0.0002**
2
|
AST (IU/L) | 46 (18–89) | 37 (8–89) | 42 (7–89) | 39 (18–89) | 0.4837** |
ALT (IU/L) | 25 (4–65) | 19 (7–77) | 20 (7–44) | 12 (4–34) | 0.216** |
Hb conc (g/dl) | 7.3 (6.3–10) | 7.5 (6.3–9.7) | 7.2 (6.2–10) | 7.9 (7–8.8) | 0.608** |
MCV (fl) | 80.6 (66.9–104.1) | 82.3 (60.3–10.2) | 77.3 (63.9–96.3) | 81 (55.9–115) | 0.642** |
RBC (× 1012/L) | 2.7 (1.9–4.1) | 2.7 (1.8–4.1) | 2.9 (1.8–4.8) | 2.8 (2.2–3.9) | 0.258** |
WBC (× 109/L) | 13 (8.5–26) | 13.4 (6.1–29.3) | 13.3 (7–25) | 12.2 (7.6–23.1) | 0.889** |
Platelet (× 109/L) | 367 (118–771) | 349 (159–601) | 361 (108–669) | 391 (135–832) | 0.955** |
HbF (%) | 9.7 (1.3–20.6) | 8.2 (1.7–24.4) | 10.7 (2.5–32) | 9.4 (0.9–28.5) | 0.86** |
HbS (%) | 80 (71–91.5) | 82 (44–91.5) | 80 (44–88.3) | 80 (65–89) | 0.90** |
HbA2 (%) | 1.6 (0.5–3.5) | 1.7 (0.2–3.8) | 1.5 (0.2–4.0) | 1.1 (0.3–3.1) | 0.3147** |
Effects of UGT1A1 (TA) n genotype on clinical events
Asymptomatic gallstones were found in 6 (5.9%) patients. Gallstones were significantly more common in patients with low-activity genotypes compared to all the other remaining genotype subgroups 5 (20%) vs 1(1.3%)
p = 0.0033, (Table
3). These were 2 females and 4 males; the two females were aged 10 and 13 years respectively, while the males were aged 10, 13, 15 and 16 years respectively. Four of the patients with gallstone had TA 7/7 genotypes, the remaining two each had TA 7/8, or TA 6/7. No significant relationship was found with the other clinical events.
Table 3
Influence of UGT1A1 (TA) n genotype on clinical events of patients
VOC rate per year | 2 (0–6) | 1.5 (0–6) | 1 (0–6) | 0 (0–6) | 0.2218* |
Overt Stroke | 1 | 2 | 1 | 0 | 1.000† |
No overt stroke | 24 | 40 | 24 | 9 |
Osteonecrosis | 1 | 2 | 2 | 0 | 1.000† |
No osteonecrosis | 24 | 40 | 23 | 9 |
Leg ulcer | 0 | 2 | 4 | 0 | 0.331† |
No Leg ulcer | 25 | 40 | 21 | 9 |
Gallstones | 5 | 1 | 0 | 0 |
0.0033†
|
No Gallstone | 20 | 41 | 25 | 9 |
Priapism (Male only event, N = 66) | | | | | |
Priapism | 2 | 3 | 0 | 0 | 0.594† |
No Priapism | 15 | 25 | 16 | 5 |
Comparison of laboratory parameters between patients with and without gallstones
There were significant differences between the serum bilirubin and HbF levels in patients with gallstones when compared with those without. No difference was observed in the LDH and age of the two groups. Furthermore, when those with gallstones were compared with age- and sex-matched patients within the same
UGT1A1 (TA) n genotype subgroup, only serum bilirubin and HbF showed significant differences between the two groups (Table
4).
Table 4
Comparison of parameters in patients with and without gallstones
Parameter | Patients with gallstones (N = 6) Median (Range) | Patients without gallstones (N = 95) Median (Range) | P value |
Total Bilirubin (mg/dl) | 6.4 (2.8–8.1) | 1.8 (0.4–6.7) | 0.0001* |
Unconjugated Bilirubin (mg/dl) | 4.7 (0.9–6.3) | 0.79 (0.1–5) | 0.0007* |
LDH (IU/L) | 1004 (592–1860) | 794 (197–1750) | 0.1263* |
HbF (%) | 4.7 (1.3–6.8) | 10.2 (0.9–32) | 0.0107* |
Hb (g/dl) | 7.1 (6.3–8.8) | 7.5 (6.2–10) | 0.4210* |
Age in years | 11.5 (8–16) | 9 (2–21) | 0.1368* |
Sex |
Male (n = 66) | 4 | 62 | 1.000† |
Female (n = 35) | 2 | 33 | |
Parameter | Patients with gallstones (N = 6) Median (Range) | Matched peers without gallstones within same UGT1A1 genotype activity group N = 10 Median (Range) | P value |
Total Bilirubin (mg/dl) | 6.4 (2.8–8.1) | 2.2 (1.9–3.2) |
0.0023*
|
Unconjugated Bilirubin (mg/dl) | 4.7 (0.9–6.3) | 1.2 (1.0–2.0) |
0.0020*
|
LDH (IU/L) | 1004 (592–1860) | 890 (340–1603) | 0.628* |
HbF (%) | 4.7 (1.3–6.8) | 14.7 (4.2–17.9) |
0.022*
|
Hb (g/dl) | 7.1 (6.3–8.8) | 8.0 (6.5–8.9) | 0.137* |
Relationship between UGT1A1 (TA) n genotypes and other parameters by multivariate analysis
Unconjugated bilirubin was significantly associated with the low activity UGT1A1 (TA) n genotypes (Adjusted Odd Ratio (1.08), 95% Confidence interval (1.034768–1.127873), P < 0.0001). Also, significant association was found with the total bilirubin when it was used in place of unconjugated bilirubin in the logistic regression model (Adjusted Odd Ratio (1.05), 95% Confidence interval (1.029172–1.089832), P < 0.0001). No association was found with the other laboratory parameters.
Discussion
There are gaps in the understanding of the impacts of genetic modifiers on SCA phenotype among African patients. Given the distinct segregation of genetic markers among different populations, it is pertinent that more studies are carried out among diverse ethnic cohorts to fully understand the impact of genetic polymorphisms in SCA.
This study confirms the variability of bilirubin levels based on the activity of the
UGT1A1 (TA) n genotypes as previously reported [
2,
4,
5,
8,
9]. However, we are not aware of any previous study that has described the stratification of LDH among SCA patients based on
UGT1A1 (TA) n genotype activity as found in this study. While the
UGT1A1 modulation of serum bilirubin levels is well understood [
2,
17,
18], the exact mechanism through which
UGT1A1 could be associated with LDH is not clear. However, it should be noted that they are both markers of hemolysis [
19]. Given the association of LDH with some phenotypes of SCA [
19,
20], there is need to further unravel the link between LDH and
UGT1A1 activity.
Gilbert syndrome (GBS) has been described in individuals with the TA7/7 genotype [
2,
4,
11,
17]. The proportion of patients with TA7/7 genotype in this study (21.7%) is higher than between 3 and 18% described among Europeans [
21,
22] and Brazilians of different descents [
23‐
25]. Similarly, it is higher than the 6% found among Kuwaiti SCA patients [
26] and the 5 to 11% among other Africans [
11,
22]. However, it is lower than the 32% described among the SCA patients in the USA [
27]. Nevertheless, the TA7/7 genotype prevalence in this study, is comparable to between 18.2 and 20.3% earlier described among Nigerians with non-SCD related illnesses [
28,
29]. However, the higher preponderance of TA7/7 among the patients compared to the controls in the present study is not clear but may be due to the low sample size.
Besides the TA7/7 genotype, the other
UGT1A1 (TA) n genotypes found in this study have been described among Africans [
9,
22]. These observations indicate that the
UGT1A1 (TA) n genotype is quite variable among Nigerians. It also confirms the suggestions that the expression of the
UGT1A1 (TA) n genotype variants is heterogeneous among Africans compared to Caucasians [
9,
21,
22].
Our finding that the low-activity
UGT1A1 (TA) n genotype was associated with gallstones confirms previous observations that SCA patients with the low-activity
UGT1A1 (TA) n genotypes especially the TA7/7, are at risk of developing gallstones [
2,
6,
7,
11,
17]. In addition to the TA7/7 genotype, other authors [
11,
30], have reported that some other low-activity
UGT1A1 (TA) n genotypes like TA7/8 and TA8/8 predispose SCA patients to gallstones as found in this study.
The proportion of patients with asymptomatic gallstones in this study (5.9%), is comparable to between 4 and 6% earlier reported among Nigerian children of similar age to our SCA cohorts [
31‐
33]. This is also similar to the 4% found in Ghana [
34], a close neighbour to Nigeria. However, it is lower than between 9 and 58% reported for some other African [
35‐
37], Italian [
38], American [
39], and Brazilian [
40] patients. These observations possibly highlight the variations in propensity to gallstone development among children with SCA from different backgrounds.
Despite the observation that the
UGT1A1 (TA) n low -activity genotype is a leading factor in hyperbilirubinemia and lithogenesis among SCA patients [
2,
6,
7,
41,
42], the impact of
UGT1A1 polymorphism on the phenotypic expression of the Nigerian SCA patients was unknown prior to this study as none of the previous studies from Nigeria examined the
UGT1A1 of the patients [
31‐
33]. There is therefore, the need to closely follow up these patients given that, results of follow-up studies have indicated higher prevalence of gallstones and its complications with increasing age of SCA patients [
2,
26,
30,
37,
40].
Beyond bilirubin metabolism and gallstone development, it has been speculated that individuals with the low-activity genotypes may be subjected to some other modulating effects of higher serum bilirubin levels that are often associated with these genotypes [
2,
8,
9,
11]. These other modulating effects include protection against oxidative stress, inflammatory injuries, and reduced susceptibility to infections [
2‐
5,
8,
9,
43]. We did not observe any association between the
UGT1A1 (TA) n genotypes and any of the other SCA downstream events/phenotypes examined (VOC, leg ulcer, priapism, overt stroke and osteonecrosis) that could be perturbed by both inflammatory and oxidative injuries in SCA. However, the small sample size of the study makes it difficult to draw any firm conclusion.
The major limitations of the present study are its hospital-based nature and the small sample. Despite these, it was able to confirm that UGT1A1 (TA) n genotypes are tightly associated with bilirubin and LDH levels, and the development of gallstones among young Nigerians with SCA. In addition, it also suggests that the pathway to elevated serum bilirubin and gallstone development, among our study cohort, may not be exclusively driven by hemolysis but also by UGT1A1 polymorphisms.
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