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Identification of Promotor and Exonic Variations, and Functional Characterization of a Splice Site Mutation in Indian Patients with Unconjugated Hyperbilirubinemia.

Gupta N, Benjamin M, Kar A, Munjal SD, Sarangi AN, Dalal A, Aggarwal R - PLoS ONE (2015)

Bottom Line: Functional characterization of an exonic variation (c.1084G>A) located at a splice site revealed that it results in frameshift deletion of 31 nucleotides and premature truncation of the protein.Functional characterization of a splice site variation indicated that it leads to disordered splicing.These variations may explain UH in subjects who lacked homozygous A(TA)7TAA promoter alleles.

View Article: PubMed Central - PubMed

Affiliation: Department of Gastroenterology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, 226014, India.

ABSTRACT

Background: Mild unconjugated hyperbilirubinemia (UH), due to reduced activity of the enzyme uridine diphosphoglucuronate-glucuronosyltransferase family, polypeptide 1 (UGT1A1), is a common clinical condition. Most cases are caused by presence in homozygous form of an A(TA)7TAA nucleotide sequence instead of the usual A(TA)6TAA sequence in promoter region of the UGT1A1 gene. In some cases, other genetic variations have been identified which differ between populations. There is need for more data on such genetic variations from India.

Methods: DNA from subjects with unexplained persistent or recurrent UH was tested for the presence of TA promoter insertions. In addition, all five exons and splicing site regions of UGT1A1 gene were sequenced. Several bioinformatics tools were used to determine the biological significance of the observed genetic changes. Functional analysis was done to look for effect of a splice site mutation in UGT1A1.

Results: Of 71 subjects with UH (68 male; median age [range], 26 [16-63] years; serum bilirubin 56 [26-219] μM/L, predominantly unconjugated) studied, 65 (91.5%) subjects were homozygous for A(TA)7TAA allele, five (7.0%) were heterozygous, and one (1.4%) lacked this change. Fifteen subjects with UH had missense exonic single nucleotide changes (14 heterozygous, 1 homozygous), including one subject with a novel nucleotide change (p.Thr205Asn). Bioinformatics tools predicted some of these variations (p.Arg108Cys, p.Ile159Thr and p.Glu463Val) to be deleterious. Functional characterization of an exonic variation (c.1084G>A) located at a splice site revealed that it results in frameshift deletion of 31 nucleotides and premature truncation of the protein.

Conclusion: Our study revealed several single nucleotide variations in UGT1A1 gene in Indian subjects with UH. Functional characterization of a splice site variation indicated that it leads to disordered splicing. These variations may explain UH in subjects who lacked homozygous A(TA)7TAA promoter alleles.

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Related in: MedlinePlus

Molecular characterization of c.1084G>A mutation.(A) Results of RT-PCR performed on RNA isolated from transfected COS7 cells. Lane 1: empty pCAS2 vector, lane 2: pCAS_WT vector, and lane 3: pCAS_MT vector. (B) Sequence chromatograms of products of RT-PCR from COS7 transfectants.
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pone.0145967.g004: Molecular characterization of c.1084G>A mutation.(A) Results of RT-PCR performed on RNA isolated from transfected COS7 cells. Lane 1: empty pCAS2 vector, lane 2: pCAS_WT vector, and lane 3: pCAS_MT vector. (B) Sequence chromatograms of products of RT-PCR from COS7 transfectants.

Mentions: The only subject with UH who was homozygous for c.1084G>A (p.Gly362Ser) mutation was subjected to in vitro characterization of this mutation to determine whether it caused a defect in mRNA splicing, using pCAS2 minigene. Results indicated that the mutation results in only a minor difference in the size of mature transcript as compared to the control DNA sequence (Fig 4A). Sequencing of specific bands of RT-PCR from pCAS_WT and pCAS_MT revealed a 31-bp deletion from 3’-end of exon 3 in pCAS_MT, whereas pCAS_WT was efficiently spliced (Fig 4B). This result confirms that c.1084G>A mutation leads to abolition of the natural splice site, and the splicing machinery instead chooses another cryptic splice site at 1053 position. This leads to splicing 31 bp before the natural splice site, and causes a frameshift deletion. This mutation leads to p.Lys353Thr and forms a stop codon after three amino acids (p.Lys353ThrfsX3).


Identification of Promotor and Exonic Variations, and Functional Characterization of a Splice Site Mutation in Indian Patients with Unconjugated Hyperbilirubinemia.

Gupta N, Benjamin M, Kar A, Munjal SD, Sarangi AN, Dalal A, Aggarwal R - PLoS ONE (2015)

Molecular characterization of c.1084G>A mutation.(A) Results of RT-PCR performed on RNA isolated from transfected COS7 cells. Lane 1: empty pCAS2 vector, lane 2: pCAS_WT vector, and lane 3: pCAS_MT vector. (B) Sequence chromatograms of products of RT-PCR from COS7 transfectants.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4696816&req=5

pone.0145967.g004: Molecular characterization of c.1084G>A mutation.(A) Results of RT-PCR performed on RNA isolated from transfected COS7 cells. Lane 1: empty pCAS2 vector, lane 2: pCAS_WT vector, and lane 3: pCAS_MT vector. (B) Sequence chromatograms of products of RT-PCR from COS7 transfectants.
Mentions: The only subject with UH who was homozygous for c.1084G>A (p.Gly362Ser) mutation was subjected to in vitro characterization of this mutation to determine whether it caused a defect in mRNA splicing, using pCAS2 minigene. Results indicated that the mutation results in only a minor difference in the size of mature transcript as compared to the control DNA sequence (Fig 4A). Sequencing of specific bands of RT-PCR from pCAS_WT and pCAS_MT revealed a 31-bp deletion from 3’-end of exon 3 in pCAS_MT, whereas pCAS_WT was efficiently spliced (Fig 4B). This result confirms that c.1084G>A mutation leads to abolition of the natural splice site, and the splicing machinery instead chooses another cryptic splice site at 1053 position. This leads to splicing 31 bp before the natural splice site, and causes a frameshift deletion. This mutation leads to p.Lys353Thr and forms a stop codon after three amino acids (p.Lys353ThrfsX3).

Bottom Line: Functional characterization of an exonic variation (c.1084G>A) located at a splice site revealed that it results in frameshift deletion of 31 nucleotides and premature truncation of the protein.Functional characterization of a splice site variation indicated that it leads to disordered splicing.These variations may explain UH in subjects who lacked homozygous A(TA)7TAA promoter alleles.

View Article: PubMed Central - PubMed

Affiliation: Department of Gastroenterology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, 226014, India.

ABSTRACT

Background: Mild unconjugated hyperbilirubinemia (UH), due to reduced activity of the enzyme uridine diphosphoglucuronate-glucuronosyltransferase family, polypeptide 1 (UGT1A1), is a common clinical condition. Most cases are caused by presence in homozygous form of an A(TA)7TAA nucleotide sequence instead of the usual A(TA)6TAA sequence in promoter region of the UGT1A1 gene. In some cases, other genetic variations have been identified which differ between populations. There is need for more data on such genetic variations from India.

Methods: DNA from subjects with unexplained persistent or recurrent UH was tested for the presence of TA promoter insertions. In addition, all five exons and splicing site regions of UGT1A1 gene were sequenced. Several bioinformatics tools were used to determine the biological significance of the observed genetic changes. Functional analysis was done to look for effect of a splice site mutation in UGT1A1.

Results: Of 71 subjects with UH (68 male; median age [range], 26 [16-63] years; serum bilirubin 56 [26-219] μM/L, predominantly unconjugated) studied, 65 (91.5%) subjects were homozygous for A(TA)7TAA allele, five (7.0%) were heterozygous, and one (1.4%) lacked this change. Fifteen subjects with UH had missense exonic single nucleotide changes (14 heterozygous, 1 homozygous), including one subject with a novel nucleotide change (p.Thr205Asn). Bioinformatics tools predicted some of these variations (p.Arg108Cys, p.Ile159Thr and p.Glu463Val) to be deleterious. Functional characterization of an exonic variation (c.1084G>A) located at a splice site revealed that it results in frameshift deletion of 31 nucleotides and premature truncation of the protein.

Conclusion: Our study revealed several single nucleotide variations in UGT1A1 gene in Indian subjects with UH. Functional characterization of a splice site variation indicated that it leads to disordered splicing. These variations may explain UH in subjects who lacked homozygous A(TA)7TAA promoter alleles.

Show MeSH
Related in: MedlinePlus