Report on the laboratory
Sonalee Laboratory for Marfan syndrome and Marfan-related disorders
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
When the Sonalee Laboratory was set up in 1999 the best technique available for mutation screening was Single Strand Conformation Analysis (SSCA), a gel-based method which is reliable and relatively simple to adapt to a large turnover of samples. However, SSCA is vastly time-consuming due to the large size of the gene under study. This affects the speed of the process and, consequently, the answer to the patient, thus contributing to an already distressing situation. Furthermore, tests carried out in the Sonalee Laboratory showed that an improved chromatography-based method (dHPLC) had a higher sensitivity, conservatively estimated at about 90%, whereas the SSCA technique has an estimated sensitivity of 60-80%.
This graph shows the DNA sequence of a small fragment of the gene (FBN1) we are analysing
Starting from January 2003 the new dHPLC set-up has been in use in the Sonalee Laboratory. The dHPLC technique permits a more elastic 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.
Finally, the samples where a mutation has not been identified with SSCA will eventually be re-analysed using the more powerful dHPLC technique.
During the last 6 years the complete FBN1 screening analysis of 510 patients had been carried out. To date, 189 mutations have been identified, giving a ratio of about 37% in 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 related to Marfan but genetically linked to other loci than fibrillin-1. It is important to discriminate 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, 302 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, with exclusion of the presence of the family mutation in 3 out of 3 foetuses. Postnatal tests for babies are available via a simple buccal swab. Preventive management for mutation carriers is arranged and there is reassurance for non-carrier relatives.
Methods
Starting from genomic DNA, we amplify all the 65 exons of FBN1 by Polymerase Chain Reaction (PCR) using a Thermal Cycler. A set of PCR primers that allow routine amplification of all the 65 exons of the FBN1 gene, 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 Terminators 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 FBN1 genomic 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 buccal 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.
Sonalee Laboratory list of publications
ELCIOGLU NH, AKALIN F, ELCIOGLU M, COMEGLIO P, CHILD AH
Neonatal Marfan syndrome caused by an exon 25 mutation of the fibrillin-1 gene
Genet Counsel 2004, 15, 219-225
COLLOD-BEROUD G, LE BOURDELLES S, ADES L, ALA-KOKKO L, BOOMS P, BOXER M, CHILD A, COMEGLIO P, DE PAEPE A, HYLAND JC, HOLMAN K, KAITILA I, LOEYS B, MATYAS G, NUYTINCK L, PELTONEN L, RANTAMAKI T, ROBINSON P, STEINMANN B, JUNIEN C, BEROUD C, BOILEAU C
Update of the UMD-FBN1 mutation database and creation of an FBN1 polymorphism database Hum Mutat 2003, 22(3):199-208
BEHAN WM, LONGMAN C, PETTY RK, COMEGLIO P, CHILD AH , BOXER M, FOSKETT P, HARRIMAN DG
Muscle fibrillin deficiency in Marfan's syndrome myopathy
J Neurol Neurosurg Psychiatry 2003, 74(5):633-638
COMEGLIO P, EVANS AL, BRICE GW, ANDERLID BM, CHILD AH
Gene symbol: FBN1. Disease: Marfan syndrome
Hum Genet 2003, 112(1):104
COMEGLIO P, EVANS AL, BRICE G, COOLING RJ, CHILD AH
Identification of FBN1 gene mutations in patients with ectopia lentis and marfanoid habitus
BR J Ophthalmol 2002, 86(12): 1359-1362
COMEGLIO P , EVANS AL, BRICE GW, CHILD AH
Detection of six novel FBN1 mutations in British patients affected by Marfan syndrome
Hum Mutat 2001, 18(6):546-547