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.

The pulmonary artery in patients with Marfan syndrome: a cross-sectional study

“…All the patients were assessed for all subsets of the Ghent criteria (Ghent 1) 1 by the same group of physicians. [10][11][12][13][14][15][16] For assessing fulfilment of the major cardiovascular criterion in the Ghent nosology, information was collected about previous surgery related to dilatation or dissection of the ascending aorta, and electrocardiographic evidence of dilatation of the ascending aorta was recorded. 1,13 The assessment of fulfilment of the Ghent criteria included sequencing of the entire coding region of the gene FBN1 and searching for large deletions or duplications.…”

“…1,13 The assessment of fulfilment of the Ghent criteria included sequencing of the entire coding region of the gene FBN1 and searching for large deletions or duplications. 12,13,16 mR or ct of the aorta and pulmonary artery This study was a substudy of the Norwegian Marfan Study. In this substudy, MR imaging of the aorta and the pulmonary artery was performed except when MR was contraindicated, in which case CT images were obtained instead.…”

“…8 This article presents data on pulmonary artery dimensions in the study population described in detail in previous studies. [10][11][12][13][14][15][16] All investigations in patients were carried out in the period [2003][2004]. Therefore all results reported in those studies are in accordance with the Ghent 1 criteria.…”

Dural Ectasia in Marfan Syndrome: A Case Control Study

“…The assessment included sequencing of the entire coding region of the gene FBN1 and a search for large deletions or duplications. 22,23 …”

Prevalence data on all Ghent features in a cross-sectional study of 87 adults with proven Marfan syndrome

“…16 -18 Details of the mutation analysis have been published. 19,20 As recommended by the authors of the Ghent criteria, the serum concentration of homocysteine was measured in all 105 individuals to exclude homocystinuria. 1 When all data were present, the status for each individual was assessed with regard to each organ system (major criteria fulfilled and organ systems involved).…”