UGT1A1 gene linkage analysis: application of polymorphic markers rs4148326/rs4124874 in the Iranian population

Document Type: Original Article


Division of Genetics, Department of Biology, Faculty of Sciences, University of Isfahan, Isfahan, Iran


Objective(s): Mutations in the UGT1A1 gene are responsible for hyperbilirubinemia syndromes including Crigler-Najjar type 1 and 2 and Gilbert syndrome. In view of the genetic heterogeneity and involvement of large numbers of the disease causing mutations, the application of polymorphic markers in the UGTA1 gene could be useful in molecular diagnosis of the disease.
Materials and Methods: In the present study, two polymorphic markers including rs4148326 and rs4124874 in the UGT1A1 gene region were characterized. The markers were selected using bioinformatics analysis of the UGT1A1 gene region and genotyped in 212 unrelated healthy individuals and 13 family trios in the Iranian population using Tetra-Primer ARMS PCR technique. The allele frequency and population status of the alleles were estimated using GENEPOP, FBAT, PowerMarker and Arlequin software.
Results: The results indicated that in the case of rs4148326 marker, allele frequency for T and C allele was 66.04% and 33.96%, respectively. For rs4124874 marker, allele frequency for G and T alleles was 39.4% and 60.6%, respectively. The values of heterozygosity index for the markers examined were 64.1 for rs4148326 and 72.1 for rs4124874, respectively. The haplotype estimation analysis of the markers resulted in three informative haplotypes with frequencies ≥0.05. Moreover, the results suggested the presence of linkage disequilibrium between two markers.
Conclusion: Altogether, the data suggested that rs4148326 and rs4124874 could be introduced as informative markers for molecular diagnosis of Crigler-Najjar type 1 and 2 and Gilbert syndrome in the Iranian population.


1. Borlak J, Thum T, Landt O, Erb K, Hermann R. Molecular diagnosis of a familial nonhemolytic hyperbilirubinemia (Gilbert's syndrome) in healthy subjects. Hepatology 2000; 32:792-795.

2. Sampietro M, Iolascon A. Molecular pathology of Crigler-Najjar type I and II and Gilbert's syndromes. Haematologica 1999; 84:150-157.

3. Kadakol A, Ghosh SS, Sappal BS, Sharma G, Chowdhury JR, Chowdhury NR. Genetic lesions of bilirubin uridine‐diphosphoglucuronate glucuronosyltransferase (UGT1A1) causing Crigler‐Najjar and Gilbert syndromes: Correlation of genotype to phenotype. Hum Mutat 2000; 16:297-306.

4. Zhou Y, Wang S-n, Li H, Zha W, Wang X, Liu Y, et al. Association of UGT1A1 variants and hyperbilirubinemia in breast-fed full-term Chinese infants. PloS One 2014; 9:e104251.

5. Canu G, Minucci A, Zuppi C, Capoluongo E. Gilbert and Crigler najjar syndromes: An update of the UDP-glucuronosyltransferase 1A1 gene mutation database. Blood Cells Mol Dis 2013; 50:273-280.

6. Costa E. Hematologically important mutations: Bilirubin UDP-glucuronosyltransferase gene mutations in Gilbert and Crigler–Najjar syndromes. Blood Cells Mol Dis 2006;36:77-80.

7. Moghrabi N, Clarke DJ, Boxer M, Burchell B. Identification of an A-to-G missense mutation in exon 2 of the UGT1 gene complex that causes Crigler-Najjar syndrome type 2. Genomics 1993; 18:171-173.

8. Bosma PJ, Chowdhury JR, Bakker C, Gantla S, de Boer A, Oostra BA, et al. The genetic basis of the reduced expression of bilirubin UDP-glucuronosyltransferase 1 in Gilbert's syndrome. New Engl J Med 1995; 333:1171-1175.

9. Seppen J, Bosma P, Roy Chowdhury J. Discrimination between Crigler-Najjar syndromes type I and II by expression of mutant bilirubin-UDP-glucuronosyltrans-ferase. J Clin Invest 1994; 94:2385.

10. Stenson PD, Mort M, Ball EV, Shaw K, Phillips AD, Cooper DN. The Human Gene Mutation Database: building a comprehensive mutation repository for clinical and molecular genetics, diagnostic testing and personalized genomic medicine. Hum Genet 2014; 133:1-9.

11. Francoual J, Trioche P, Mokrani C, Seboui H, Khrouf N, Chalas J, et al. Prenatal diagnosis of Crigler–Najjar syndrome type I by single‐strand conformation polymor-phism (SSCP). Prenatal Diag 2002; 22:914-916.

12. Kaniwa N, Kurose K, Jinno H, Tanaka-Kagawa T, Saito Y, Saeki M, et al. Racial variability in haplotype frequencies of UGT1A1 and glucuronidation activity of a novel single nucleotide polymorphism 686C> T (P229L) found in an African-American. Drug Metab Dispos 2005; 33:458-465.

13. Milton JN, Sebastiani P, Solovieff N, Hartley SW, Bhatnagar P, Arking DE, et al. A genome-wide association study of total bilirubin and cholelithiasis risk in sickle cell anemia. PloS One 2012; 7:e34741.

14. Kringen MK, Piehler AP, Grimholt RM, Opdal MS, Haug KBF, Urdal P. Serum bilirubin concentration in healthy adult north-europeans is strictly controlled by the UGT1A1 TA-repeat variants. PloS One 2014; 9:e90248.

15. Horsfall LJ, Hardy R, Wong A, Kuh D, Swallow DM. Genetic variation underlying common hereditary hyperbilirubinaemia (Gilbert’s syndrome) and respiratory health in the 1946 British birth cohort. J Hepatol 2014; 61:1344-1351.

16. Jones AG, Ardren WR. Methods of parentage analysis in natural populations. Mol Ecol 2003; 12:2511–2523.

17. Miller S, Dykes D, Polesky H. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res 1988; 16:1215.

18. Ye S, Dhillon S, Ke X, Collins AR, Day IN. An efficient procedure for genotyping single nucleotide polymor-phisms. Nucleic Acids Res 2001; 29:e88-e.

19. Chiapparino E, Lee D, Donini P. Genotyping single nucleotide polymorphisms in barley by tetra-primer ARMS-PCR. Genome 2004; 47:414-420.

20. Collins A, Ke X. Primer1: primer design web service for tetra-primer ARMS-PCR.  Open Bioinform J 2012; 6:55-58.

21. Newton C, Graham A, Heptinstall L, Powell S, Summers C, Kalsheker N, et al. Analysis of any point mutation in DNA. The amplification refractory mutation system (ARMS). Nucleic Acids Res 1989; 17:2503-2516.

22. Raymond M, Rousset F. GENEPOP (version 1.2): population genetics software for exact tests and ecumenicism. J Hered 1995; 86:248-249.

23. Schneider S, Roessli D, Excoffier L. Arlequin: a software for population genetics data analysis. User manual ver2; 2000; 2:2496-2497.

24. Rabinowitz D, Laird N. A unified approach to adjusting association tests for population admixture with arbitrary pedigree structure and arbitrary missing marker information. Hum Hered 2000; 50:211-223.

25. Liu K, Muse SV. PowerMarker: an integrated analysis environment for genetic marker analysis. Bioinformatics 2005; 21:2128-2129.

26. Lewontin R. The interaction of selection and linkage. II. Optimum models. Genetics 1964; 50:757.

27. Zhao JH. 2LD, GENECOUNTING and HAP: Computer programs for linkage disequilibrium analysis. Bioinformatics 2004; 20:1325-1326.

28. Zhao H, Nettleton D, Soller M, Dekkers J. Evaluation of linkage disequilibrium measures between multi-allelic markers as predictors of linkage disequilibrium between markers and QTL. Genet Res 2005; 86:77-87.

29. Browning SR, Browning BL. Haplotype phasing: existing methods and new developments. Nat Rev Genet 2011; 12:703-714.

30. Karolchik D, Hinrichs AS, Kent WJ. The UCSC genome browser. Curr Protoc Bioinformatics 2009;  33:1-.4.

31. Fazeli Z, Vallian S. Estimation haplotype frequency of BglII/EcoRI/VNTR markers at the PAH gene region in Iranian population. Int J Hum Genet 2009; 9:115-121.

32. Kraemer D, Scheurlen M. [Gilbert disease and type I and II Crigler-Najjar syndrome due to mutations in the same UGT1A1 gene locus]. Med Klin (Munich) 2002; 97:528-532.

33. Morioka I, Morikawa S, Yusoff S, Harahap ISK, Nishimura N, Yokoyama N, et al. Genetic disorders associated with neonatal jaundice. East J Med 2013; 15:155-162.

34. Yang J, Cai L, Huang H, Liu B, Wu Q. Genetic variations and haplotype diversity of the UGT1 gene cluster in the Chinese population. PloS One 2012; 7:e33988.

35. Judson R, Stephens JC, Windemuth A. The predictive power of haplotypes in clinical response. Pharmacogenomics 2000; 1:15-26.

36. Crawford DC, Nickerson DA. Definition and clinical importance of haplotypes. Annu Rev Med 2005; 56:303-320.

37. Wen SH, Tsai MY. Haplotype association analysis of combining unrelated case-control and triads with consideration of population stratification. Front Genet 2014; 5:103.v