title: Rapid and efficient FBN1 mutation detection using automated sample preparation and direct sequencing as the primary strategy.
abstract: Mutations in the fibrillin-1 (FBN1) gene cause Marfan syndrome (MFS) and the other type-1 fibrillinopathies. Finding these mutations is a major challenge considering that the FBN1 gene has a coding region of 8,600 base pairs divided into 65 exons. Most of the more than 600 known mutations have been identified using a mutation scanning method prior to sequencing of fragments with a suspected mutation. However, it is not obvious that these screening methods are ideal, considering cost, efficiency, and sensitivity. We have sequenced the entire FBN1 coding sequence and flanking intronic sequences in samples from 105 patients with suspected MFS, taking advantage of robotic devices, which reduce the cost of supplies and the quantity of manual work. In addition, automation avoids many tedious steps, thus reducing the opportunity for human error. Automated assembling of PCR, purification of PCR products, and assembly of sequencing reactions resulted in completion of the FBN1 sequence in half of the time needed for the manual protocol. Mutations were identified in 69 individuals. The mutation detection rate (76%), types, and genetic distribution of mutations resemble the findings in other MFS populations. We conclude that automated sequencing using the robotic systems is well suited as a primary strategy for diagnostic mutation identification in FBN1.
authors.affiliation: Department of Medical Genetics, Ulleval University Hospital, Oslo, Norway.
authors.forename: Lena
authors.initials: L
authors.lastname: Tjeldhorn
authors.forename: Svend
authors.initials: S
authors.lastname: Rand-Hendriksen
authors.forename: Kristina
authors.initials: K
authors.lastname: Gervin
authors.forename: Kristin
authors.initials: K
authors.lastname: Brandal
authors.forename: Elin
authors.initials: E
authors.lastname: Inderhaug
authors.forename: Odd
authors.initials: O
authors.lastname: Geiran
authors.forename: Benedicte
authors.initials: B
authors.lastname: Paus
mesh_terms: D053000:Analytic Sample Preparation Methods; D001331:Automation; D002648:Child; D004252:DNA Mutational Analysis; D000071838:Fibrillin-1; D000071837:Fibrillins; D006801:Humans; D008382:Marfan Syndrome / Q000175:diagnosis / Q000235:genetics; D008840:Microfilament Proteins / Q000235:genetics*; D009154:Mutation; D017422:Sequence Analysis, DNA / Q000379:methods*; D013997:Time Factors; D054392:Adipokines
chemical_list: D000071838:Fibrillin-1; D000071837:Fibrillins; D008840:Microfilament Proteins; C000605722:FBN1 protein, human; D054392:Adipokines