Rapid detection of polymorphisms in exons 10, 11 and 12 of the low density lipoprotein receptor gene (LDLR) and their use in a clinical genetic diagnostic setting

A molecular genetic service for diagnosing individuals with familial hypercholesterolaemia (FH) in the United Kingdom

“…8 To confirm SSCPs due to polymorphisms, restriction enzyme assays or forced site' assays were used. 39,40 Results…”

“…49 In addition to these criteria, strict measures were taken to assure that the nucleotide change was a true result by confirming the sequence change in a second PCR product and if possible a second sample from the patient, although the latter was not strictly necessary since tests were carried out under EQA guidelines, where sample transfer and genetic test set up must be observed by a second scientist (http:// www.cmgs.org). A number of polymorphisms are known to exist in LDLR (FH website) and although most resulted in recognisable SSCP band shift patterns, in a clinical genetic diagnosis setting a confirmation assay 39,40 was always used to *Indicates mutations not previously described.…”

“…Any failures were repeated. SSCP patterns due to known polymorphisms were excluded using direct assays 39,40 and any remaining SSCP band shifts were characterised by sequencing. Sequence alterations were confirmed by a direct assay if available or by repeating the sequencing reaction on a different PCR product.…”

I705 variant in the low density lipoprotein receptor gene has no effect on plasma cholesterol levels

“…We have set up a clinical genetic diagnostic service for FH7 8 and the I705 variant was identified in a subject with a clinical diagnosis of possible FH who was referred for FH genetic testing (data not shown). To investigate the pathogenicity of the amino acid substitution at codon 705, we have determined the frequency of the I705 variant in 2287 healthy UK men and examined the effect of this variant on plasma lipid levels.…”

“…PCR conditions were as described previously, the Nsi I enzyme was added directly to the PCR product, and the products (139 bp uncut = T705 and 115 bp + (non-detected) 14 bp cut) were separated on a 7.5% MADGE9and stained with ethidium bromide. The I705 carriers were then analysed for the 1061-8C variation in intron 7 by a natural Ear I restriction digest, after amplification of exon 8 (197 bp) with primers FH119 (FH website) and FH27 (FH website) and reaction conditions as previously described 8. A restriction site was lost in the rare C allele but a control constant cut site exists for confirmation of digestion.…”