1. A novel A–>G mutation in intron I of the hepatic lipase gene leads to alternative splicing resulting in enzyme deficiency
- Author
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David N. Nevin, Silvia Santamarina-Fojo, Klaus A. Dugi, Korbinian Brand, and John D. Brunzell
- Subjects
Genetics ,Splice site mutation ,Alternative splicing ,Mutant ,Intron ,Cell Biology ,QD415-436 ,Biology ,Molecular biology ,Biochemistry ,Exon ,Endocrinology ,RNA splicing ,Gene ,Minigene - Abstract
We have identified the underlying molecular de- fect in a patient with hepatic lipase (HL) deficiency present- ing with hypertriglyceridemia and premature cardiovascular disease. DNA sequencing of polymerase chain reaction (PCR) amplified DNA and digestion with BsrI established homozy- gosity for an A + G mutation in intron I of the patient's hepatic lipase gene. This mutation introduces an additional AG motif within a potential branch lariat signal located 13 bp upstream of the native 3' splice site. Two minigene constructs (normal and mutant) consisting of exons 1 and 2 as well as 192 bp of intron I of HL were generated by the overlap PCR extension method and transfected in human 293 cells. Se- quence analysis of reverse transcribed, amplified cDNA gen- erated from total RNA isolated from transfected cells demon- strated the presence of abnormally spliced products containing 13 and 78 additional bases as well as the accumu- lation of unspliced mRNA. No normally spliced mRNA was identified. Thus, the A + G mutation disrupts normal splic- ing of intron I and generates a new AG site that is utilized as an alternative 3' splice signal leading to the most prominent RT-PCR product in vitro. Translation of these alternatively spliced products leads to premature termination resulting in the synthesis of a truncated, non-functional enzyme. The absence of normal HL protein in post heparin plasma of this patient was confirmed by Western blotting. DNA restriction analysis demonstrated that all four of the proband's children, who exhibit HL activity levels between those of the HL-defi- cient father and the mother with normal HL activity, are heterozygotes for the splice site mutation. Thus, our studies establish the functional significance of a novel mutation in the HL gene of a patient presenting with HL defi- ciency.-Brand, K., K. A. Dugi, J. D. Brunzell, D. N. Nevin, and S. Santamarina-Fojo. A novel A + G mutation in intron 1 of the hepatic lipase gene leads to alternative splicing result- ing in enzyme deficiency. J. Lipid Res. 1996. 37: 1213-1223.
- Published
- 1996