Haematologica, Vol 92, Issue 1, 133-134 doi:10.3324/haematol.10585
Copyright © 2007 by Ferrata Storti Foundation
Maria D’Apolito* ,
Agnese Marrone° ,
Veronica Servedio* ,
Pietro Vajro# ,
Luigia De Falco° ,
Achille Iolascon°,
The aim of this study was to identify new pathogenic variations of the UGT1A1 gene in 11 patients diagnosed with neonatal unconjugated hyperbilirubinemia. We describe two cases in which clinically unapparent heterozygotic mutations in the UGT1A1 gene may become evident in combination with certain environmental conditions or additional genetic defects.
We identified a total of 12 sequence variations, seven of which are described for the first time.
Copyright © 2007 by Ferrata Storti Foundation
Red Cell Disorders |
Seven novel mutations of the UGT1A1 gene in patients with unconjugated hyperbilirubinemia
* Dipartimento di Scienze Mediche e del Lavoro, Università degli Studi di Foggia, Italy;
° Dept of Biochemistry and Biomedical Technologies University Federico II, CEINGE, Advanced Biotechnologies, Napoli, Italy;
# Dipartimento di Pediatria Università degli Studi di Napoli Federico II, Napoli
Correspondence: Achille Iolascon, Chair of Medical Genetics, Dept. of Biochemistry and Biomedical Technologies, University Federico II, Naples, CEINGE, Advanced Biotechnologies 80145 Naples. E-mail: iolascon{at}dbbm.unina.it
ABSTRACT
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The aim of this study was to identify new pathogenic variations of the UGT1A1 gene in 11 patients diagnosed with neonatal unconjugated hyperbilirubinemia. We describe two cases in which clinically unapparent heterozygotic mutations in the UGT1A1 gene may become evident in combination with certain environmental conditions or additional genetic defects.
Key words: Bilirubin, jaundice, Crigler-Najjar syndrome, Gilbert syndrome, bilirubin UDP-glucuronosyltransferase; UGT1A1.
Genetic alterations of the UGT1A1 gene result in Crigler-Najjar syndrome type I (CN1) and type II (CN2) and Gilbert’s syndrome, autosomal recessive conditions that are characterized by non-hemolytic unconjugated hyperbilirubinemia.1–3 Several other disorders associated with hemolysis caused by premature destruction of erythrocytes are also characterized by hyperbiliribinemia.4,5 Therefore, the expression of UGT1A1 may be a major factor responsible for bilirubin variability (a modifier gene) in inherited hemolytic diseases.
The aim of this study was to analyze the mutation pattern causing unconjugated hyperbilirubinemia in 11 unrelated patients with neonatal hyperbilirubinemia. For the analysis of mutations of UGT1A1 genes, the coding and promoter region and the splicing sites were analyzed by direct sequencing.6
Nine patients had a clinical diagnosis of CN1 (patients #1 to #4) and CN2 (patients #5 to #9). Two subjects (patients #10 and #11) presented with transient hyperbilirubinemia caused by hemolysis. Patient #10 had neonatal kernicterus associated with hyperbilirubinemia due to a skull hematoma. Phototherapy and phenobarbital were started in this neonate, whose bilirubin levels decreased shortly afterwards. The phototherapy was suspended when the bilirubin concentration was under 10 mg/dL following resorption of the hematoma. Patient #11, affected by glucose-6-phosphate dehydrogenase (G6PD) deficiency, showed increased bilirubin production due to hemolysis. Direct sequencing of G6PD revealed a change from cytosine to thymine at base position 563 (in exon 6) causing a change from serine to phenylalanine in amino acid position 188. Results of the analysis of UGT1A1 are summarized in Table 1.
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Table 1. The patients’ clinical features and mutations.
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Two out of the four CN1 patients had a frameshift mutation, p.Q239fsX256, a recurrent change already found in a cohort of Italian patients.7 Patients #3 and #4 showed novel variations. Patient #3 had a homozygous tri-nucleotide deletion c.513_515 del CTT causing a deletion of leucine codon at position 172 and the substitution of a phenylalanine residue by a leucine at position 171 [p.L172delF171L]. Although, this mutation eliminates a single amino acid (leucine), we believe that the substitution of a phenylalanine in a conserved diphenylalanine region of an aglycone binding domain may abolish the enzymatic activity of UGT1A1. Patient #4 was heterozygous for two novel mutations: a c.652insT insertion causing a frameshift and a C
T transition at nucleotide 847 in exon 1 that introduces a stop codon (p. Q283X).
The milder phenotype of CN2 patients is usually associated with homozygosity or compound heterozygosity for missense mutations. In addition to known causative alterations (p.N279Y, p.W461R), two missense mutations were identified in heterozygous subjects affected by CN2. In patient # 8, a novel missense mutation c.1328 TC was found in exon 5 which affected residue 443 (p. L443P) involving a polar amino acid substituition. In patient #9 a TA heterozygous transition at nucleotide 1060 of the UGT1A1 gene resulted in the substitution of a tryptophan residue by arginine at position 354 (p.W354R) in the carboxy-terminal domain of the UGT1A1 protein. Recently, missense p.W354R was reported to be caused by substitution of T to C at nucleotide 1060.7 These results suggest that nucleotide 1060 in UGT1A1 may be a mutational hot spot. In our cohort, all the individuals affected by CN2, except patient #7, were heterozygous for one UGT1A1 coding region variation. However, the in trans presence of a c.-41_- 40dupTA polymorphism [(TA)7] can explain the CN2 phenotype. This polymorphism can play a role in enhancing the effect of the heterozygous coding mutation.
Here, we describe two cases in which clinically unapparent heterozygotic mutations in UGT1A1 may become evident when combined with certain environmental conditions or additional genetic defects. In patient #10 a novel missense mutation, c.1108 AG, was detected in a heterozygous state. This change caused an amino acid substitution (p.I370V) in the catalytic core of the UGT1A1 protein. This patient’s severe hyperbilirubinemia and kernicterus were consequences of the imbalance between bilirubin production and conjugation. In patient #11, affected by G6PD deficiency, a novel heterozygous small deletion of one nucleotide at position 210 (c.210delC) of the UGT1A1 gene was found in combination with the Mediterranean variant, c.563 CT of the G6PD gene. This represents an example of co-inherited modifying gene causing clinical heterogeneity in monogenic disorders. The expression of UGT1A1 is a major modifying factor in inherited hemolytic diseases being responsible for a large proportion of the bilirubin variability in these conditions.
In conclusion, the identification of these novel mutations in the UGT1A1 gene, increasing the mutational spectrum of UGT1A1 allelic variants, contributes to a better understanding of the molecular pathology of disorders characterized by unconiugated hyperbilirubinemia.
References
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  F. M Petit, M. Hebert, V. Gajdos, A. Mollet-Boudjemline, and P. Labrune Comments on Seven novel mutations of the UGT1A1 gene in patients with unconjugated hyperbilirubinemia by D'Apolito et al. Haematologica, July 1, 2007; 92(7): e80 - e80. [Full Text] [PDF]
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