A novel intronic mutation results in the use of a cryptic splice acceptor site within the coding region of UGT1A1, causing Crigler-Najjar syndrome type 1

Baljit S. Sappal, Siddhartha S. Ghosh, Benjamin Shneider, Ajit Kadakol, Jayanta Roy-Chowdhury, Namita Roy Chowdhury

Research output: Contribution to journalArticle

11 Citations (Scopus)

Abstract

Crigler-Najjar syndrome type 1 (CN-1) is characterized by severe unconjugated hyperbilirubinemia due to an inherited deficiency of hepatic bilirubin uridinediphosphoglucuronate glucuronosyltransferase (UGT1A1), inherited as an autosomal recessive characteristic. CN-1 is potentially lethal because of the risk of bilirubin encephalopathy (kernicterus). Genetic lesions of the coding region of the UGT1A1 gene are known to cause CN-1. Here, we report a CN-1 patient who has a novel G > A mutation at the splice acceptor site in intron 4 (IVS4-1 G > A) on one allele, and a T > A substitution followed by a 13-nt deletion in exon 2 (877T > A 878-890del) of the other allele. As the UGT1A1 gene is expressed specifically in the liver, structural analysis of the expressed UGT1A1 mRNA requires liver biopsy. To use a noninvasive approach to determine the effect of the splice site mutation on splicing of the RNA transcript, we amplified the relevant region of the genomic DNA by long-range polymerase chain reaction (PCR). The amplicon was cloned in an expression plasmid and transfected into COS-7 cells. The expressed mRNA was amplified by reverse-transcription-primed PCR. Nucleotide sequence determination of the amplicon showed that the splice acceptor site mutation caused splicing of the 3′-end of exon 4 to a cryptic splice site within exon 5. This resulted in deletion of the first 7 nucleotides of exon 5, causing a frameshift and premature truncation of UGT1A1, with consequent inactivation of the enzyme.

Original languageEnglish (US)
Pages (from-to)134-142
Number of pages9
JournalMolecular Genetics and Metabolism
Volume75
Issue number2
DOIs
StatePublished - 2002

Fingerprint

Crigler-Najjar Syndrome
RNA Splice Sites
Exons
Kernicterus
Mutation
Polymerase chain reaction
Bilirubin
Liver
Nucleotides
Genes
Alleles
RNA Splicing
Polymerase Chain Reaction
Glucuronosyltransferase
Messenger RNA
Hyperbilirubinemia
Biopsy
COS Cells
Transcription
Structural analysis

Keywords

  • Bilirubin
  • Crigler-Najjar syndrome
  • Splicing abnormality
  • UDP-glucuronosyltransferase

ASJC Scopus subject areas

  • Biochemistry
  • Genetics
  • Endocrinology, Diabetes and Metabolism

Cite this

A novel intronic mutation results in the use of a cryptic splice acceptor site within the coding region of UGT1A1, causing Crigler-Najjar syndrome type 1. / Sappal, Baljit S.; Ghosh, Siddhartha S.; Shneider, Benjamin; Kadakol, Ajit; Roy-Chowdhury, Jayanta; Chowdhury, Namita Roy.

In: Molecular Genetics and Metabolism, Vol. 75, No. 2, 2002, p. 134-142.

Research output: Contribution to journalArticle

Sappal, Baljit S. ; Ghosh, Siddhartha S. ; Shneider, Benjamin ; Kadakol, Ajit ; Roy-Chowdhury, Jayanta ; Chowdhury, Namita Roy. / A novel intronic mutation results in the use of a cryptic splice acceptor site within the coding region of UGT1A1, causing Crigler-Najjar syndrome type 1. In: Molecular Genetics and Metabolism. 2002 ; Vol. 75, No. 2. pp. 134-142.
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AB - Crigler-Najjar syndrome type 1 (CN-1) is characterized by severe unconjugated hyperbilirubinemia due to an inherited deficiency of hepatic bilirubin uridinediphosphoglucuronate glucuronosyltransferase (UGT1A1), inherited as an autosomal recessive characteristic. CN-1 is potentially lethal because of the risk of bilirubin encephalopathy (kernicterus). Genetic lesions of the coding region of the UGT1A1 gene are known to cause CN-1. Here, we report a CN-1 patient who has a novel G > A mutation at the splice acceptor site in intron 4 (IVS4-1 G > A) on one allele, and a T > A substitution followed by a 13-nt deletion in exon 2 (877T > A 878-890del) of the other allele. As the UGT1A1 gene is expressed specifically in the liver, structural analysis of the expressed UGT1A1 mRNA requires liver biopsy. To use a noninvasive approach to determine the effect of the splice site mutation on splicing of the RNA transcript, we amplified the relevant region of the genomic DNA by long-range polymerase chain reaction (PCR). The amplicon was cloned in an expression plasmid and transfected into COS-7 cells. The expressed mRNA was amplified by reverse-transcription-primed PCR. Nucleotide sequence determination of the amplicon showed that the splice acceptor site mutation caused splicing of the 3′-end of exon 4 to a cryptic splice site within exon 5. This resulted in deletion of the first 7 nucleotides of exon 5, causing a frameshift and premature truncation of UGT1A1, with consequent inactivation of the enzyme.

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