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Expression of an LDL receptor allele with two different mutations (E256K and I402T)

Ekström, Ulf LU ; Abrahamson, Magnus LU ; Sveger, Tomas LU ; Sun, X M; Soutar, A K and Nilsson-Ehle, Peter LU (2000) In Molecular Pathology 53(1). p.31-36
Abstract
AIMS: To investigate the disease causing event in patients with familial hypercholesterolaemia, carrying two mutations each, E256K in exon 6 and I402T in exon 9, of the gene encoding the low density lipoprotein (LDL) receptor. It was not known whether the mutations were positioned in cis or trans, or if they were each pathogenic separately or only when present together. METHODS: Polymerase chain reaction, denaturing gradient gel electrophoresis and sequencing were used to characterise the LDL receptor locus of the patients and family members. The different LDL receptor mutants, constructed in vitro by oligonucleotide directed mutagenesis, were expressed in LDL receptor deficient Chinese hamster ovary (CHO1d1A7) cells, to determine the... (More)
AIMS: To investigate the disease causing event in patients with familial hypercholesterolaemia, carrying two mutations each, E256K in exon 6 and I402T in exon 9, of the gene encoding the low density lipoprotein (LDL) receptor. It was not known whether the mutations were positioned in cis or trans, or if they were each pathogenic separately or only when present together. METHODS: Polymerase chain reaction, denaturing gradient gel electrophoresis and sequencing were used to characterise the LDL receptor locus of the patients and family members. The different LDL receptor mutants, constructed in vitro by oligonucleotide directed mutagenesis, were expressed in LDL receptor deficient Chinese hamster ovary (CHO1d1A7) cells, to determine the effects of the mutations on LDL receptor function. RESULTS: The two mutations were located on the same allele of the LDL receptor gene. All mutant constructs resulted in the production of a detectable protein in CHO cells. The cells expressing only the I402T mutation, or the combination of I402T and E256K mutations, were seriously affected in mediating uptake and degradation of LDL. Contrary to initial predictions, the cells expressing only the E256K mutation showed essentially the same binding, uptake, and degradation of 125I labelled LDL as cells transfected with normal LDL receptor cDNA. These results suggest that the pathogenic mutation in the patients heterozygous for the E256K/I402T allele is the I402T mutation, and that E256K alone is a rare sequence variation, which does not affect LDL receptor protein function. E256K was not detected either in DNA from a healthy population or in DNA from other hypercholesterolaemic patients studied. CONCLUSIONS: Despite the information available on the structure-function relations between the LDL receptor and LDL receptor like proteins, predictions about the disease causing potential of a mutation are not reliable. These results suggest that the I402T mutation is pathogenic and that the substitution of E256K alone is a rare sequence variation, without a detectable phenotype modulating effect. (Less)
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author
organization
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type
Contribution to journal
publication status
published
subject
in
Molecular Pathology
volume
53
issue
1
pages
31 - 36
publisher
BMJ Publishing Group
external identifiers
  • pmid:10884919
  • scopus:0033959557
ISSN
1366-8714
language
English
LU publication?
yes
id
be50d762-7d6b-4b75-824b-c2fc4e7a697e (old id 1118194)
alternative location
http://mp.bmj.com/cgi/content/abstract/53/1/31
date added to LUP
2008-06-17 13:53:48
date last changed
2017-04-09 04:19:17
@article{be50d762-7d6b-4b75-824b-c2fc4e7a697e,
  abstract     = {AIMS: To investigate the disease causing event in patients with familial hypercholesterolaemia, carrying two mutations each, E256K in exon 6 and I402T in exon 9, of the gene encoding the low density lipoprotein (LDL) receptor. It was not known whether the mutations were positioned in cis or trans, or if they were each pathogenic separately or only when present together. METHODS: Polymerase chain reaction, denaturing gradient gel electrophoresis and sequencing were used to characterise the LDL receptor locus of the patients and family members. The different LDL receptor mutants, constructed in vitro by oligonucleotide directed mutagenesis, were expressed in LDL receptor deficient Chinese hamster ovary (CHO1d1A7) cells, to determine the effects of the mutations on LDL receptor function. RESULTS: The two mutations were located on the same allele of the LDL receptor gene. All mutant constructs resulted in the production of a detectable protein in CHO cells. The cells expressing only the I402T mutation, or the combination of I402T and E256K mutations, were seriously affected in mediating uptake and degradation of LDL. Contrary to initial predictions, the cells expressing only the E256K mutation showed essentially the same binding, uptake, and degradation of 125I labelled LDL as cells transfected with normal LDL receptor cDNA. These results suggest that the pathogenic mutation in the patients heterozygous for the E256K/I402T allele is the I402T mutation, and that E256K alone is a rare sequence variation, which does not affect LDL receptor protein function. E256K was not detected either in DNA from a healthy population or in DNA from other hypercholesterolaemic patients studied. CONCLUSIONS: Despite the information available on the structure-function relations between the LDL receptor and LDL receptor like proteins, predictions about the disease causing potential of a mutation are not reliable. These results suggest that the I402T mutation is pathogenic and that the substitution of E256K alone is a rare sequence variation, without a detectable phenotype modulating effect.},
  author       = {Ekström, Ulf and Abrahamson, Magnus and Sveger, Tomas and Sun, X M and Soutar, A K and Nilsson-Ehle, Peter},
  issn         = {1366-8714},
  language     = {eng},
  number       = {1},
  pages        = {31--36},
  publisher    = {BMJ Publishing Group},
  series       = {Molecular Pathology},
  title        = {Expression of an LDL receptor allele with two different mutations (E256K and I402T)},
  volume       = {53},
  year         = {2000},
}