Report on the Laboratory
Sonalee Laboratory for Marfan syndrome and Marfan-related disorders
Dr Gavin Arno, PhD
Department of Cardiac & Vascular Sciences
St George’s University of London
Synopsis
Marfan syndrome is an autosomal dominant connective tissue disorder characterised by manifestations mainly in cardiovascular, ocular, skeletal, and pulmonary systems. The cardiovascular system seems to be the most seriously affected, with dilatation of the aortic root that may progress to aortic dissection, principal cause of the shorter life expectancy of untreated Marfan patients. The incidence of this disease is about 1/5,000 worldwide, without regard to ethnicity or geography, and at least 25% of cases are sporadic, representing fresh mutations. Marfan syndrome is caused by the gene FBN1 on chromosome 15. FBN1 encodes for a connective tissue microfibril named fibrillin-1. FBN1 mutations have been characterised at the molecular level in patients affected by Marfan syndrome and Marfan-related disorders, thus demonstrating that this gene can cause, by itself, different clinical manifestations.
Service
Starting from January 2003 an improved chromatography-based method (dHPLC) has been in use in the Sonalee Laboratory for mutation screening giving us a higher sensitivity, conservatively estimated at about 91%, whereas the former SSCA technique has an estimated sensitivity of 60-80%.
The dHPLC technique permits a more flexible approach to the number of patients analysed at the same time, allowing us to cut the time necessary to screen clustered samples from patients, reducing the time-gap between commencement and final report. At the moment, it is estimated that it would take about 8 weeks from sample reception to final report in the absence of a waiting list. The analysis is carried out by an experienced operator on a batch of 11 patients, thus projecting an estimate of about 90 patients per year.
The service provided includes the screening of all 65 exons of the gene, the sequencing of all the putative mutation sites and the interpretation of the results in accordance with published literature.
This graph shows the DNA sequence of a small fragment of the gene (FBN1) we are analysing
Finally, the samples where a mutation has not been identified with SSCA are being re-analysed using the more powerful dHPLC technique.
During the last 10 years the complete FBN1 screening analysis of 780 patients has been carried out. To date, 297 mutations have been identified, giving a ratio of about 38% in the mixed population of patients referred to the Sonalee Laboratory with diagnosis of classic Marfan syndrome, incomplete Marfan syndrome or Marfan-related disorders, the latter not always linked to the FBN1 gene. Our results are comparable with what is reported by other groups in consecutive series of patients, where a relatively low ratio in the heterogeneous group of patients with marfanoid habitus has been identified. The reason for this low yield of mutations is that the marfanoid group includes conditions clinically overlapping with Marfan but genetically linked to other loci than fibrillin-1. It is important to differentiate these conditions, because they have a completely different prognosis, so that not identifying a mutation in their cases helps to address the management, treatment and genetic counselling.
In addition, 460 relatives of patients for whom a mutation has been identified, here or at another facility, were tested for the presence of that particular mutation. Prenatal tests have also been carried out at the Sonalee Laboratory. Postnatal tests for babies are available via a simple saliva sample. Preventive management for mutation carriers is arranged and there is reassurance for non-carrier relatives.
We now offer mutation screening in TGFBR2in probands where no mutation in FBN1 can be identified if they fulfil the clinical criteria for this (presence of ascending aortic aneurysm, no ectopia lentis). Mutations in this gene have been identified in Loeys-Dietz syndrome, familial ascending aortic aneurysm and Marfan syndrome.
Methods
Starting from genomic DNA, we amplify all the 65 exons of FBN1 by Polymerase Chain Reaction (PCR). A set of PCR primers that allow routine amplification of all the 65 exons of the FBN1gene, including flanking splice sites, is used, so that we are able to investigate the presence of mutations in the coding regions as well as in the splice junction sequences.
DHPLC analysis is carried out using a WAVE Nucleic Acid Fragment Analysis system. The PCR samples are injected into a DNASep Column and run using predetermined protocols and conditions.
When we observe an abnormality, the positive fragment is sequenced to characterise the putative mutation.
Direct sequencing analysis of the amplified exon is performed using the same oligonucleotides utilised for the PCR as primers. The sequence analysis is performed using the BigDye Terminator kit technique and an ABI 310 Genetic Analyzer.
The putative mutation identified is searched for amongst all the other patients and checked for in our database of FBN1genomic variants observed in 50 controls, to determinate if it could be considered either a recurrent mutation or a polymorphism. In order to confirm the association of the mutation with the pathological condition under study and to provide genetic counselling, all available members of the proband’s family are tested for the mutation.
Wherever possible, the identified mutation is then tested in the genomic DNA extracted from a second sample (either blood or saliva sample) from the proband, in order to confirm the presence of the mutation in two independently extracted samples.
All the necessary techniques are set up in the Sonalee Laboratory, where the required skills are present to carry out the analysis.