Rapid and simultaneous detection of multiple mutations by pooled and multiplex single nucleotide primer extension: Application to the study of insulin-responsive glucose transporter and insulin receptor mutations in non-insulindependent diabetes
Rapid and simultaneous detection of multiple mutations by pooled and multiplex single nucleotide primer extension: Application to the study of insulin-responsive glucose transporter and insulin receptor mutations in non-insulindependent diabetes
The application of molecular scanning techniques to the detection of potentially pathogenic mutations in candidate genes in patients with non-insulin-dependent diabetes has revealed a number of molecular variants of uncertain pathophysiologic significance. The determination of the significance of such variants requires large-scale population studies of the prevalence of the mutant in affected and control groups. Herein, we describe two adaptations of the technique of single nucleotide primer extension (SNuPE) which allow the simultaneous examination of large numbers of alleles at multiple loci. The usefulness of these adaptations is illustrated by their application to the simultaneous detection of three point mutations, two in the tyrosine kinase domain of the insulin receptor and one in the insulin-responsive glucose transporter(GLUT4)in a highly insulin-resistant NIDDM population. By pooling genomic or amplified DNA and performing the SNuPE reactions with three primers of different length we could readily examine 300 alleles on a single 20 lane gel. Using pooled SNuPE, we also examined a large British Caucasian control population for the prevalence of GLUT4II e 383, a variant which has previously been reported only in NIDDM. GLUT4 II e 383 was detected in 2/42 of the highly insulin-resistant NIDDM subjects and 4/240 middleaged blood donors. Family studies and examination of the expressed mutant transporter will be necessary to establish whether this mutation is of functional significance. Pooled and multiplex SNuPE are powerful techniques with wide applicability to population genetic studies of specific mutaitions.
391-395
Krook, Anna
210b552c-3a13-4bda-ba82-28c45962729c
Stratton, Irene M.
772f25b9-23c0-4240-a3f6-1e76b03b172f
O'rahilly, Stephen
e5c7869f-10d1-4fdc-a564-e4907be415a3
September 1992
Krook, Anna
210b552c-3a13-4bda-ba82-28c45962729c
Stratton, Irene M.
772f25b9-23c0-4240-a3f6-1e76b03b172f
O'rahilly, Stephen
e5c7869f-10d1-4fdc-a564-e4907be415a3
Krook, Anna, Stratton, Irene M. and O'rahilly, Stephen
(1992)
Rapid and simultaneous detection of multiple mutations by pooled and multiplex single nucleotide primer extension: Application to the study of insulin-responsive glucose transporter and insulin receptor mutations in non-insulindependent diabetes.
Human Molecular Genetics, 1 (6), .
(doi:10.1093/hmg/1.6.391).
Abstract
The application of molecular scanning techniques to the detection of potentially pathogenic mutations in candidate genes in patients with non-insulin-dependent diabetes has revealed a number of molecular variants of uncertain pathophysiologic significance. The determination of the significance of such variants requires large-scale population studies of the prevalence of the mutant in affected and control groups. Herein, we describe two adaptations of the technique of single nucleotide primer extension (SNuPE) which allow the simultaneous examination of large numbers of alleles at multiple loci. The usefulness of these adaptations is illustrated by their application to the simultaneous detection of three point mutations, two in the tyrosine kinase domain of the insulin receptor and one in the insulin-responsive glucose transporter(GLUT4)in a highly insulin-resistant NIDDM population. By pooling genomic or amplified DNA and performing the SNuPE reactions with three primers of different length we could readily examine 300 alleles on a single 20 lane gel. Using pooled SNuPE, we also examined a large British Caucasian control population for the prevalence of GLUT4II e 383, a variant which has previously been reported only in NIDDM. GLUT4 II e 383 was detected in 2/42 of the highly insulin-resistant NIDDM subjects and 4/240 middleaged blood donors. Family studies and examination of the expressed mutant transporter will be necessary to establish whether this mutation is of functional significance. Pooled and multiplex SNuPE are powerful techniques with wide applicability to population genetic studies of specific mutaitions.
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Published date: September 1992
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We would like to thank Drs. David MoUer and Jeffrey Flier for helpful discussions, Dr. Robert C.Turner for the provision of data on the highly insulin-resistant UKPDS subjects, Dr George Miller for provision of the normal control subjects, Dr Anne Temple and Dr Steve Humphries for provision of their DNA and Mr Philip Saker for extraction of DNA from the UKPDS samples. Samples from Welsh subjects were kindly provided by Drs. Alan Recs, Ruth Morgan and David Owens. SO'R is supported by the Wellcome Trust.
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Local EPrints ID: 486939
URI: http://eprints.soton.ac.uk/id/eprint/486939
ISSN: 0964-6906
PURE UUID: d957f65b-3cf1-499a-896e-ef81cbfb4b25
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Date deposited: 08 Feb 2024 17:45
Last modified: 18 Mar 2024 04:01
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Author:
Anna Krook
Author:
Irene M. Stratton
Author:
Stephen O'rahilly
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