Study For Spectrum Of UGT1A1Variations Associated With Inherited Nonhemolytic Unconjugated Hyperbilirubinemia

Inherited unconjugated hyperbilirubinemia is categorized as Gilbert syndrome (GS),Crigler-Najjar syndrome type I (CNS-I) and type II (CNS-II).Asian GS is associated withmanyUGT1A1(UDP-glucuronosyltransferase1A1gene) variants.In Asian populations, thecompound homozygous UGT1A1G71R and Y486D variants are frequently observed incases with bilirubin levels greater than200μmol/L.To clarify linkage disequilibrium relationship of Chinese GS-associated variants andthespectrum of UGT1A1variations in Chinese CNS-II patients, We enrolled46unrelatedGilbert syndrome patients,8GS families and11Chinese kindreds with CNS-II(9of whomdisplayed bilirubin levels between104.3μmol/L and171.5μmol/L).DNA was extracted from the peripheral blood of46unrelated GS patients,8GSpatients and their family members,11unrelated CNS-II patients and their family members,and80healthy controls. The enhancer, promoter, and coding regions of UGT1A1wereamplified by polymerase chain reaction (PCR) and PCR products were directly sequencedby a dye terminating method.Results1) Ten variants were identified, including c.-3345delC, c.-3279T>G, c.-1352C>A,c.-40_-39insTA, c.211G>A (p.G71R), c.686C>A (p.P229Q), c.715C>T (p.Q239X),c.1091C>T (p.P364L), c.1253delT (p.M418fsX423), c.1456T>G (P.Y486D).2) All variants are Hardy-Weinberg equal.3) LD relationship exists in all variants;c.-3279T>G variant and c.-1352C>A arecomplete linkage disequilibrium (D’=1, r2=1).4) Twelve haplotypes are constructed from above variants(only list variantallele):H1(-3345delC/c.-1352A)，H2(c.-3279G)，H3(c.-1352A)，H4(c.-3279G/c.-40_-39insTA)，H5(c.-3279G/c.-40_-39insTA/c.686A)， H6(c.-1352A/c.211A)， H7(c.-3279G/c.1091T)， H8(c.-1352A/c.1456G)，H9(c.-1352A/c.211A/c.1456G)，H10(c.3279G/c.-40_-39insTA/c.715T)，H11(c.1352A/c.1253delT)，H12(c.-3279G/c.-40_-39insTA/c.1091T). The frequencies of H2andH3in controls are all greater than that in GS patients (0.282and0.098, P=3.160E-4;0.381and0.087, P=4.557E-7) and CNS-II patients (0.000and0.294, P=0.001;0.381and0.091, P=0.014).The frequencies of H4and H6in GSpatients are all greater than that in controls (0.359and0.131,P=2.271E-5;0.315and0.169; P=0.007). The frequencies of H7and H8in CNS-IIpatients are allgreater than that in controls (0.091and0.006, P=0.039;0.091and0.000, P=0.014). H9, H10,H11and H12exist only in CNS-IIpatients.5) Twenty seven diplotypes are constructed from all subjects. H2/H2is identified onlyin controls. The frequencies of H2/H3and H3/H3in controls are all greater than that inGSpatients (0.325and0.065, P=0.001;0.163and0.022, P=0.015), and the two diplotypesdon’t exist in CNS-II patients. The frequencies of H4/H4, H4/H6and H6/H7in GSpatientsare greater than that in controls (0.152and0.013, P=0.007;0.239and0.013, P=1.147E-4;0.273and0.000, P=1.184E-4); the frequency of H6/H6in GSpatients is greater than that incontrols (0.130and0.025, P=0.05). H9/H8、H9/H9、H10/H7、H11/H8、H12/4、H1/H6existonly in CNS-IIpatients.6) Twenty five percent of parents of6nuclear GS families in8GS families are also GSpatients (3/12).7) None of family members of11CNS-II probands are manifested with CNS-II;mothers of proband C3and C10, a sister of proband C7are GS patients.8) Genotypes of UGT1A1gene in9CNS-II patients whose bilirubin levels are less than200μmol/L are not combination of homozygous Y486D variant and G71R variant which isfrequently detected in CNS-II patients whose bilirubin levels are greater than200μmol/L.Conclusion1) The c.-3279T>G variant and c.-1352C>A variant are completely LD relationship,and the two variants don’t influence activity of UGTs.2) The c.-40_-39insTA variant and c.211G>A (p.G71R) variant are major pathogenicvariants of GS which is transmitted by mode of recessiveness.3) Most CNS-II patients are caused by UGT1A1variants which are located in sharedexons of UGT1A locus or can cause truncation of UGT1A1enzyme. 4) For CNS-II patients, different Bilirubin levels (greater or less than200μmol/L)indicate different spectrum of UGT1A1variations.