title: Semiquantitative multiplex PCR: a useful tool for large rearrangement screening and characterization.
abstract: Methods presently employed for detection of large rearrangements have several drawbacks, such as the amount of sample and time required, technical difficulty, or the probability of false-negative carriers. Using the low-density-lipoprotein receptor (LDLR) gene, whose mutations are responsible for familial hypercholesterolemia (FH), we have developed a procedure to detect large rearrangements in this gene based on semiquantitative PCR, with important improvements as compared to previous methods. Our method covers the complete LDLR gene and introduces an internal control in the reaction. The procedure discriminates the four different large rearrangements (two deletions and two insertions) that we have used as positive mutation controls (Valencia-1 to -5). All altered exons from each rearrangement are identified. Furthermore, when families from probands carrying these large rearrangements (34 members) were analyzed, our results agreed with those obtained previously with Southern blot. We have also analyzed a sample of 110 unrelated FH probands and the method has correctly identified the two different large rearrangements present and insertions or deletions as small as 1 bp. In conclusion, the method we present allows the identification of large rearrangements affecting exons of the gene, including small insertions or deletions or complete gene deletion. In addition, it constitutes a first characterization step of rearrangements, and is easy to carry out fast, and can be applied to the analysis of any gene.
authors.affiliation: Fundación de Investigación Hospital Clínico Universitario de Valencia, Laboratorio de Estudios Genéticos, Hospital Clínico Universitario de Valencia, University of Valencia, Valencia, Spain.
authors.forename: Ana B
authors.initials: AB
authors.lastname: Garcia-Garcia
authors.forename: Sebastian
authors.initials: S
authors.lastname: Blesa
authors.forename: Sergio
authors.initials: S
authors.lastname: Martinez-Hervas
authors.forename: M Luisa
authors.initials: ML
authors.lastname: Mansego
authors.forename: Veronica
authors.initials: V
authors.lastname: Gonzalez-Albert
authors.forename: Juan F
authors.initials: JF
authors.lastname: Ascaso
authors.forename: Rafael
authors.initials: R
authors.lastname: Carmena
authors.forename: Jose T
authors.initials: JT
authors.lastname: Real
authors.forename: Felipe J
authors.initials: FJ
authors.lastname: Chaves
mesh_terms: D001055:Apolipoproteins B / Q000235:genetics; D002869:Chromosome Aberrations; D005091:Exons; D017353:Gene Deletion; D005820:Genetic Testing / Q000379:methods*; D006801:Humans; D006938:Hyperlipoproteinemia Type II / Q000175:diagnosis* / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D011973:Receptors, LDL / Q000235:genetics*
chemical_list: D001055:Apolipoproteins B; D011973:Receptors, LDL