Abstract
Background
Gilbert syndrome is characterized by mild unconjugated hyperbilirubinemia. The high levels of bilirubin could be related to the co-inheritance of Gilbert syndrome determined either by mutations of the coding region or by variation in the (TA)n motifs of the promoter region of the bilirubin UGT1A1 gene. The co-inheritance of Gilbert syndrome has been reported to elevate bilirubin levels in beta thalassemia and sickle cell disease patients.
Aim
In this study, we have tried to investigate whether the variability in serum bilirubin levels found in transfusion-dependent beta thalassemia, beta thalassemia intermedia, and heterozygous beta thalassemia individuals could be related to the coexistence of Gilbert syndrome.
Methods
The promoter region (TA)n motifs of the bilirubin UGT1A1 gene were analyzed in 104 beta thalassemia individuals. The control group consisted of 50 healthy individuals.
Results
The analysis of the UGT1A1 promoter showed three (TA) motifs: (TA)5, (TA)6, and (TA)7. The frequency of genotype (TA)7/(TA)7 did not differ significantly between the groups studied. A significant difference was observed in mean serum bilirubin levels between individuals showing (TA)7/(TA)7 and (TA)6/(TA)6 genotypes and also between (TA)7/(TA)7 and (TA)6/(TA)7 genotypes among all groups studied. According to the beta genotype, no differences were observed between mean serum bilirubin levels in the three groups (β+/β+, β0/β+, and β0/β0).
Conclusion
These results indicate that the (TA)7/(TA)7 configuration is one of the factors responsible for hyperbilirubinemia and, therefore, seems to interfere with the clinical expression of homozygous beta thalassemia. This emphasizes the role played by co-inherited modifying genes on clinical heterogeneity of monogenic disorders.
Introduction
Homozygous beta thalassemia patients show a marked variability in serum unconjugated bilirubin levels. This variability may be related to either red cell destruction rate, ineffective erythropoiesis, or to bilirubin elimination capacity, which depends partially on bilirubin glucoronidation activity. However, in the absence of evident signs of hemolysis, it may be difficult to evaluate whether the hyperbilirubinemia is caused by the inherited hematological disorder or by other causes. The primary bilirubin catabolizing hepatic enzyme UDP glucoronosyl transferase 1A1 (UGT1A1) mediates the conjugation of bilirubin to a water-soluble form that is excreted in bile.Citation1 Gilbert syndrome, a benign form of unconjugated hyperbilirubinemia, is a manifestation of reduced activity of this enzyme.Citation2 The reduced activity of the enzyme could be a result of a genetic defect in the UGT1A1 gene. The commonly reported mutations leading to Gilbert syndrome are 211G → A(G71R), 524T → A(L175Q), 686C > A(P229Q), 1091C > T(P364L), 1352C > T(P451L), 1456T → G(Y486D)Citation3 or due to an additional dinucleotide TA insertion in the UGT1A1 promoter region A(TA) n TAA; the ‘n’ represents the different number of dinucleotide TA and it varies between 5 and 8.Citation4 The reference sequence consists of six TA repeats [(TA)6]; the additional TA dinucleotide leads to an elongated sequence [(TA)7]. However, the homozygosity of the (TA)7 configuration is associated with Gilbert syndrome.
The co-inheritance of Gilbert syndrome has been reported to elevate bilirubin levels in sickle cell disease,Citation5 beta thalassemia,Citation6 glucose-6-phosphate dehydrogenase (G6PD) deficiencyCitation7 HbE–beta thalassemia,Citation8 hereditary spherocytosis,Citation9 and has also been correlated with the response to hydroxyurea therapy in sickle cell disease.Citation10
Homozygous and intermediate beta thalassemia patients as well as beta thalassemia heterozygous individuals exhibit a marked variability in serum bilirubin levels due to reasons such as ineffective erythropoiesis, transfused red cell destruction rate, hemolysis, or bilirubin activation capacity associated with the activity of bilirubin glucuronidation. Both bilirubin production (hemolysis) and clearance (glucuronidation) are genetically determined in thalassemia patients. Defective glucuronidation in carriers of Gilbert alleles is known to aggravate jaundice in all hemolytic anemias, including thalassemia. The genotype–phenotype relationship in thalassemia is complex. Cholelithiasis occurs with a variable (2.3%–52%) incidence in homozygous beta thalassemia. The reported variation is partly related to age and partly to its clinical severity. It is more common in thalassemia intermedia than in thalassemia major. However, the reason for this variation is not completely understood.Citation11
In the present study, we tried to correlate the elevated bilirubin levels in a group of transfusion-dependent beta thalassemia patients, beta thalassemia intermedia patients, as well as beta thalassemia heterozygotes with the coexistence of different promoter UGT1A1*28 repeat genotypes. The results were compared to a healthy control group from the same ethnic origin.
Materials and methods
Peripheral blood samples were collected after informed consent from 79 beta thalassemia homozygous patients and 25 beta thalassemia carriers. Fifty age- and sex-matched healthy individuals were enrolled as the normal control group. The study was approved by the institutional ethics committee.
Red cell indices were measured on an automated Sysmex K 1000 blood cell counter (Sysmex, Transasia, Japan). HbA2 and HbF levels were measured by using cation exchange HPLC on the Variant Hemoglobin Testing System (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The serum bilirubin levels were measured on the autoanalyzer COBAS C 111 (Roche, Germany). Genomic DNA was isolated from peripheral blood leukocytes by using the QIAamp Blood DNA Mini Kit (Qiagen, Germany). The analysis of the A(TA)nTAA motif in the promoter region of the UGT1A1 gene was performed by polymerase chain reaction and gene scan analysis on the ABI 3130 Genetic Analyzer (Applied Biosystems Inc., Foster City, CA, USA).
Statistical analysis
Continuous variables were expressed as mean ± SD. The chi-square test was used for the categorical variables as needed. Statistical significance was set at P < 0.05. Statistical analysis was conducted by using Graph Pad In stat 2 software (Graph Pad Software Inc., La Jolla, CA, USA).
Results
Clinical analysis
Of the 79 beta thalassemia homozygotes, 40 patients had a severe clinical presentation requiring regular blood transfusions. They presented with pallor and icterus within 2 years of life (beta thalassemia major). The remaining 39 individuals showed a milder clinical presentation. They presented later in life (after 2 years) and most of them were untransfused; however, some required intermittent transfusions and were grouped as the thalassemia intermedia.
Molecular analysis
Thirteen different beta thalassemia mutations were encountered. Of these, five mutations were of β+ thalassemia [−86 (C → G), −87 (C → G), −88 (C → T), Cap site +1 (A → C), and IVS1 nt 5 (G → C)] and the remaining eight were of β0 thalassemia (CD8/9 (+G), CD15 (G → A), CD 30 (G → C), CD 41/42 (−CTTT), IVS1 nt 1 (G → T), 619 bp deletion, IVS2 nt 837 (T → G), CD 5(−CT)]. The analysis of the UGT1A1 promoter region showed three (TA) motifs: (TA)5, (TA)6, and (TA)7 that gave rise to four genotypes. They were (TA)5/(TA)6, (TA)6/(TA)6, (TA)6/(TA)7, and (TA)7/(TA)7. The frequencies of the genotypes in the promoter region of the UGT1A1 gene in the three groups of thalassemia individuals and in the healthy control group are shown in . The frequency of the reference motif (TA)6 was predominant among all the groups studied. Overall, the prevalence of the (TA)6 motif was 65.1%. The frequency of the (TA)7/(TA)7 genotype, associated with Gilbert syndrome, was similar in all groups except the thalassemia intermedia group (12% – control group, 12.5% – beta thalassemia major, 20.5% – beta thalassemia intermedia, and 12% – beta thalassemia heterozygotes). The overall prevalence of the (TA)7 motif was 34.0% and that of shorter (TA)5 allele was 0.9%.
Table 1. (TA)n genotype frequencies among beta thalassemia patients and normal controls
Thalassemia major
The increased serum bilirubin levels were observed in 28 thalassemia major patients. Among these patients, homozygosity for the (TA)7/(TA)7 genotype was observed in five patients. However, the (TA)7/(TA)7 genotype was not found in those with normal bilirubin levels. A significant difference was observed in the mean serum bilirubin level among patients with genotypes (TA)7/(TA)7 and (TA)6/(TA)6 (P < 0.003) and also among patients with genotypes (TA)7/(TA)7 and (TA)6/(TA)7 (P < 0.05) (). Eleven patients in this group were homozygous for β0 mutations (β0/ β0) while 21 were homozygous for β+ mutations (β+/β+) and the remaining eight were double heterozygotes(β0/β+). No differences were observed in mean serum bilirubin levels among the patients according to their beta genotypes (β0/β0, β0/β+, and β+/β+). The (TA)5/(TA)6 genotype was seen in three thalassemia major cases. The mean serum bilirubin level among these patients was (1.17 ± 0.1 mg/dl).
Table 2. Mean bilirubin levels (mg/dl) among the study group according to the UGT1A1genotype
Thalassemia intermedia
Increased serum bilirubin levels were observed in all thalassemia intermedia patients (mean 4.2 ± 1.95 mg/dl). Mean serum bilirubin levels were significantly higher in patients with (TA)7/(TA)7 genotype than in patients with (TA)6/(TA)6 (P < 0.004) and (TA)6/(TA)7 (P < 0.05) (). Nine patients in this group were homozygous for β0 mutations (β0/ β0) while 16 were homozygous for β+ mutations (β+/ β+) and the remaining 14 were double heterozygotes (β0/β+). Here also, we did not observe any difference in the mean serum bilirubin level according to the beta genotypes.
The mean serum bilirubin levels for each of the three promoter genotypes in the thalassemia homozygous patients were higher than those in the corresponding genotypes of the control group (). The serum bilirubin levels, (TA)n configurations, and beta genotypes in beta thalassemia homozygote patients are shown in ().
Table 3. Combined effect of (TA)n configurations and beta genotypes on mean bilirubin levels in beta thalassemia homozygote patients
Beta thalassemia heterozygotes and controls
Two percent of the individuals from the control group showed the presence of shorter (TA)5 repeats [(TA)5/(TA)6] (). Lower serum bilirubin levels (mean 0.16 ± 0.08 mg/dl) were seen in this group. The frequency of the other three genotypes was similar in both beta thalassemia heterozygotes and control group. The mean serum bilirubin levels in both the groups were significantly higher in individuals with the (TA)7/(TA)7 genotype than the (TA)6/(TA)7 or (TA)6/(TA)6 genotype (P < 0.003) (). There was a considerable overlap seen in the mean serum bilirubin levels in the beta thalassemia heterozygotes and the control group in the corresponding 3 UGT1A1 promoter genotypes. Ten beta thalassemia heterozygotes showed the presence of β0 mutations and 15 showed the presence of β+ genotypes. No difference was seen in the mean serum bilirubin level according to the beta genotypes.
Discussion
The serum bilirubin level is a function of numerous factors that may modify bilirubin production and excretion. Among these is the red cell lifespan which influences bilirubin production, conjugation, and transport which explains bilirubin removal from the body. Hyperbilirubinemia and a propensity to gallstone formation is a complication of thalassemia. Gallstones are much more common in thalassemia intermedia than in thalassemia major because of ineffective erythropoiesis and peripheral hemolysis.Citation11 The rapid turnover of the red cells is responsible for this phenomenon that might be contributing in increasing the disease severity. Homozygosity for the extended (TA)7 sequence has been found to be an important risk factor for hyperbilirubinemia and cholelithiasis in patients with HbE–beta thalassemia and thalassemia intermedia.Citation8 Edison et al.Citation12 showed that the (TA)7 motif in the promoter region of the UGT1A1 gene is associated with hyperbilirubinemia in homozygous HbE patients. In the study of Yao et al.Citation13 in healthy individuals of Caucasian and Asian descent, the (TA)6/(TA)6 genotype was reported to be significantly more prevalent in Asians than in Caucasians. In our study, among the four genotypic combinations, the overall prevalence of the (TA)6/(TA)6 genotype was higher (46.1%). Similar results among healthy Indian adults (43.3%) were reported by D'Silva et al..Citation14
The (TA) repeat genotype influences the serum bilirubin levels. The extended motif (TA)7 is associated with Gilbert syndrome. The frequency of the (TA)7/(TA)7 genotype, associated with Gilbert syndrome, was similar in all the four groups studied. Tzetis et al.Citation15 studied Gilbert syndrome in association with thalassemia. They reported that the frequency of the (TA)7/(TA)7 genotype, associated with Gilbert syndrome, was similar in all the groups; however, the mean serum bilirubin levels did show variation with different genotypes and were high in patients showing the (TA)7 motif. They concluded that the (TA)7/(TA)7 genotype (Gilbert syndrome) is a modifying factor of bilirubin in thalassemic patients. Similar results were also reported by Galanello et al.Citation16 The mean serum bilirubin level among the thalassemia intermedia patients in our study was elevated (4.2 ± 1.95 mg/dl). In all the four groups studied, we observed significantly higher mean serum bilirubin levels among individuals with the (TA)7/(TA)7 genotype as against (TA)6/(TA)6 and (TA)6/(TA)7 genotype. Our findings are consistent with other molecular studies conducted by Kalotychou et al.Citation17 and Galanello et al.Citation6 Serum bilirubin levels among all the beta thalassemia homozygote patients carrying the (TA)7/(TA)7 genotype were three- to four-fold higher than the corresponding values in the control group. Pignatti-Borgna et al.Citation18 showed that thalassemia minor, at least in women, is a risk factor for cholelithiasis and the (TA)7 motif whether in homozygosity or heterozygosity further increases this risk. In our group of patients there was considerable overlap seen in the mean serum bilirubin levels in beta thalassemia heterozygotes and the control group in the corresponding 3 UGT1A1 promoter genotypes. Maruo et al.Citation19 suggested that a combined effect of two polymorphisms, (TA)7 and T-3279G, is the cause of Gilbert syndrome. An increase in the number of (TA) repeats was reported to be the cause of hyperbilirubinemia by Raijmakers et al.,Citation20 and Sampietro et al.Citation21 in their study that included subjects of beta thalassemia and G6PD deficiency observed that nearly 80% of the patients with increased bilirubin levels were either heterozygous or homozygous for the (TA)7 motif and that Gilbert syndrome accounted for a large proportion of the variability of the bilirubin levels. A study of Rawa et al. (2012) also reported a statistically significant difference in the (TA)6/(TA)6 genotype distribution between healthy individuals and patients with G6PD deficiency (P = 0.041).Citation22 In the present study, the (TA)7 repeat pattern was found to contribute to increase in the bilirubin levels and co-inheritance of Gilbert syndrome did not influence the severity of beta thalassemia. When beta thalassemia major patients were sorted by their thalassemia genotype, no significant difference was observed between the mean serum bilirubin level and the beta globin genotypes (β0/ β0, β0/β+, and β+/β+). Similar observations were reported by Tzetis et al.Citation15
UGT1A1 promoters with 5 and 8 TA repeats also exist but are extremely rare in Caucasians but common in people of African origin.Citation23 The extended repeats reduce the transcription and thereby the activity of the UGT1A1 enzyme. The rare (TA)8 motif was reported by Ostanek et al.Citation24 in the Slovenian population and by Tsezou et al.Citation4 in Caucasian population. The study conducted among healthy Polish individuals and patients with different types of hereditary haemolytic anemias showed the presence of uncommon genotypes, with shorter TA repeats (TA)5/(TA)6, (TA)5/(TA)7.Citation11 Among our study, 2% of the normal controls and 5% of the beta thalassemia major group showed presence of the (TA)5/(TA)6 genotype. These findings are consistent with the study conducted by D'Silva et al.Citation14 in healthy Indian adults. However, another study conducted in North Indian neonates did not report the (TA)5 allele among their neonatal and control groups. However, they reported the (TA)8 allele in 10% of their neonates.Citation25
In conclusion, much of the observed hyperbilirubinemia could be attributed to variation at the UGT1A1 locus and UGT1A1 testing helped to substantiate a genetic diagnosis, thereby, aiding in individual and family disease management. Although UGT1A1 variation plays a large role in hyperbilirubinemia, genetic assessment of UGT1A1 gene alone may not be comprehensive. Assessment of additional genes may also be useful to evaluate the genetic causes of hyperbilirubinemia. The role of increased bilirubin levels related to UGT1A1 promoter polymorphisms and gallstone formation is complex. The results of the present study indicate that the (TA)7/(TA)7 genotype associated with Gilbert syndrome is a modifying factor of bilirubin levels when co-inherited with beta thalassemia. This emphasizes the role played by co-inherited modifying genes on the clinical heterogeneity of monogenic disorders.
Author's Contribution
All the authors have contributed sufficiently to the project to be included as authors. P.S.D. performed the research and wrote the paper. A.H.N. designed the research study and helped in writing the paper. R.B.C. contributed to the designing of the research study. K.K.G. contributed to the data analysis.
Acknowledgement
This work is funded by the Indian Council of Medical Research (ICMR), New Delhi, India.
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