The use of molecular testing for the diagnosis of genetic disorders can be very complicated. This is certainly true for Marfan syndrome. Input from a geneticist or genetic counselor may be necessary to achieve a full understanding of the capabilities and limitations of genetic testing for Marfan syndrome. This booklet will help guide the discussion between patients and health care professionals.
The Promise of Molecular Testing
In 1991 it was recognized that mutations in the gene (FBN1) encoding the protein fibrillin-1 on chromosome 15 cause Marfan syndrome. In theory, this finding had the potential to lead to an efficient genetic diagnostic test that would accurately identify all affected people. Such a test could be used to identify individuals with Marfan syndrome before they were born or before they showed obvious manifestations of the condition, perhaps allowing initiation of more effective preventative therapies. It was also possible that molecular testing could predict outcome, allowing for more aggressive intervention in people destined to have earlier or more severe complications. Finally, such testing could increase reproductive options in families with Marfan syndrome, and could have important financial and psychosocial implications.
The reality is that genetic testing for the Marfan syndrome has not fulfilled its promise yet. At this time, there is limited application for molecular testing and a comprehensive clinical evaluation remains the most effective way to make a diagnosis of Marfan syndrome.
The main points emphasized in this section are summarized below:
- In the vast majority of cases, the diagnosis of Marfan syndrome can be established by comprehensive clinical evaluation alone.
- There is no “Marfan test” that can be used independent of clinical findings to definitively establish or exclude the diagnosis of Marfan syndrome.
- All of the medical problems associated with the Marfan syndrome can be diagnosed and treated effectively, even if the specific diagnosis of Marfan syndrome remains uncertain.
- Many of the manifestations of Marfan syndrome are common in the general population, while other manifestations are also components of other disorders that may or may not have the same genetic cause.
- The clinical variability seen in individuals with a documented fibrillin-1 gene mutation limits the ability to predict outcome.
- Proper interpretation of genetic testing results requires correlation with accurate clinical information. Inappropriate interpretation can result in misleading or even dangerous recommendations.
Molecular Testing: Challenges in Marfan Syndrome
Below you will find a detailed description of current molecular diagnostic techniques and a discussion of their use and limitations. It will become obvious that the promise of molecular testing for Marfan syndrome has been realized, but only to an extent. The limitations in the use of genetic tests for Marfan syndrome derive from several factors: 1) none of the current methods used to find mutations in the fibrillin-1 gene identify all mutations that cause Marfan syndrome; 2) mutations in the fibrillin-1 gene can cause conditions other than Marfan syndrome (see table 1) so it can be difficult to predict what condition to expect when a mutation is found; and 3) family members with the same mutation causing Marfan syndrome can show wide variation in the timing of onset and severity of many of the complications (i.e. knowing the mutation has little predictive value for people who are known to be affected).
|Table 1: Conditions that overlap with Marfan Syndrome |
|Condition Fibrillin-1 ||Overlap with Marfan Syndrome ||Mutation |
|Congenital contractural |
|Mitral valve prolapse Skeletal findings ||No |
|Skin and skeletal findings Aortic aneurysm / |
tear (selected types only)
|Familial aortic aneurysm ||Aortic |
enlargement and tear Variable skeletal findings
|Homocysteinuria ||Mitral valve prolapse Eye lens dislocation Skin and skeletal |
Isolated ectopia lentis
|Eye lens dislocation Common |
|Marfanoid habitus ||Skeletal findings ||At least sometimes |
|MASS phenotype ||Mitral valve prolapse |
Myopia Borderline aortic enlargement Skin and skeletal findings
|At least sometimes |
|Mitral valve prolapse Variable skeletal |
|At least sometimes |
|Shprintzen-Goldberg syndrome ||Aortic |
enlargement Skin and skeletal findings
Methods of Molecular Diagnosis
To understand how genetic tests are used, it is important to understand how Marfan syndrome is inherited. Marfan syndrome is a dominant condition meaning that a child only has to inherit one abnormal copy of the fibrillin-1 gene to develop the condition. Everyone has two copies of the fibrillin-1 gene, one that is inherited from each parent. Each individual with Marfan syndrome has one normal copy and one that carries a mutation that causes the condition. This means that each child of an affected individual has a 50% chance of inheriting the abnormal copy and developing Marfan syndrome. If they inherit the normal copy from their affected parent, then they are not at risk for Marfan syndrome. Importantly, about 25% of cases of Marfan syndrome are due to a new mutation that occurs during the formation of the sperm or egg cell that contributes to conception. This would produce a child who is the first person in their family with Marfan syndrome. The children of this individual would be at 50% risk to inherit Marfan syndrome.
The oldest method of molecular diagnosis for Marfan syndrome is called segregation or linkage analysis. It uses sites of normal variation in and around the fibrillin-1 gene to follow the different copies of the gene that are being passed from generation to generation in a given family. In this manner, despite knowing nothing about the precise mutation causing Marfan syndrome, one can determine which copy of the gene is passed along with the disease. If a young individual has inherited this copy from an affected parent, then it is concluded that they are predisposed to develop Marfan syndrome. In the example shown in figure 1A, the affected individual (blackened symbol) in generation I passed Marfan syndrome to each affected child along with the number "1" copy of the fibrillin-1 gene. Each child that received his number "2" copy does not have Marfan syndrome. The status of children in the next generation can be assigned by determining if they inherited the copy that segregates with disease from their affected parent. This type of analysis works best in large families in which multiple individuals with established and classic Marfan syndrome are willing to participate. Occasionally linkage analysis is not possible because of the inability to distinguish between the different copies of the gene segregating in the family (figure 1B). Fortunately, this is rare due to the large number of markers available for use. Linkage analysis can not be used to determine the status of a single individual with suspected Marfan syndrome (figure 1C). It is less useful, and can be misleading, in small families or in atypical disorders that might not be caused by a mutation in the fibrillin-1 gene (figure 1D). This type of analysis makes the important assumption that Marfan syndrome is caused only by mutations in the fibrillin-1 gene. While there is considerable evidence that the vast majority of cases are caused by fibrillin-1 gene mutations, the possibility that another gene can cause Marfan syndrome can not be excluded. In this rare event, segregation analysis using markers in and around the fibrillin-1 gene could give erroneous conclusions, especially in small families.
Even if the condition is caused by a mutation in FBN1, this test cannot establish the diagnosis of Marfan syndrome. It will simply indicate whether an individual is predisposed to develop the condition seen in the extended family. Linkage analysis can be used for both presymptomatic and prenatal diagnosis. The accuracy of the method is absolutely dependent upon proper diagnosis in participating family members, mandating comprehensive clinical evaluations. The test can generally be performed within a few weeks after the clinical information and samples have been received. The cost varies in different laboratories and may not be covered by insurance in some circumstances.
Figure 1 Pedigrees showing the outcome of segregation analysis in different clinical situations. 1A, large family with 3 generations (I-III) with multiple individuals with classic Marfan syndrome (blackened symbols). Males are squares and females are circles. A horizontal line connecting a male and a female indicates couples who have had children; a vertical line coming down from a couple indicates their children. Numbers below each symbol indicate different copies of the fibrillin-1 gene. The box indicates the copy of the gene that is passed along with Marfan syndrome. The data indicate that the girl in question in generation III (?) inherited the copy of the fibrillin-1 gene (#1) that predisposes to Marfan syndrome from her affected mother. 1B, example of an uninformative analysis. It is impossible to distinguish between the two copies of the fibrillin-1 gene in the affected father in generation I. One cannot determine which copy was inherited by each of his 3 children. 1C, we have learned nothing about the status of a child with features of a connective tissue disorder (gray symbol, ?) due to the absence of an affected parent. 1D, while multiple family members with a nonspecific connective tissue disorder (striped symbols) share a common copy of the fibrillin-1 gene (box), linkage analysis may tell us nothing about the status of a child in question (?) if the condition is not caused by a mutation in this gene. Linkage analysis is most conclusive when a copy of the fibrillin-1 gene can be shown to consistently cosegregate with disease in a large family (as in 1A).
Direct screening for fibrillin-1 gene mutations is a cumbersome and inefficient process due to the large size of the gene, the division of the gene into 65 separate coding regions (exons), the wide distribution of mutations along the length of the gene, and the lack of common mutations. When considering the use of mutation detection a number of important questions need to be considered:
- Does the absence of detection of a mutation exclude the diagnosis of Marfan syndrome? No, current best estimates suggest that a mutation is not found in about 10-30% of individuals with clear Marfan syndrome.
- Does the presence of a fibrillin-1 gene mutation establish the diagnosis of Marfan syndrome? No, FBN1 mutations can be found in many disorders that have some clinical overlap with Marfan syndrome. In addition, some sequence changes in the gene are innocent in that they do not disrupt protein function or cause a clinical disorder.
- Does the type or location of the mutation predict whether someone has or will develop Marfan syndrome? Only to an extent. Selected types of mutations (e.g. cysteine substitutions, splicing mutations, deletions or insertions) are very likely to cause Marfan syndrome, but a full range of clinical severity can be observed. Other types of mutations (e.g. nonsense mutations, frameshifts, other missense substitutions) have been associated with many different conditions. There is also no clearly defined relationship between the location of a mutation and the condition that will result.
Thus, while mutation identification currently has some limited prognostic value, it has not been proven to reliably guide clinical management. Presymptomatic or prenatal diagnosis is possible in selected families in which the disease-causing mutations have previously been identified. The initial identification of a mutation in a family can be an expensive process. Health insurance plans differ in whether mutation detection is covered. Once a mutation has been identified, the screening of additional family members can be performed reliably and quickly.
The fibrillin-1 gene directs cells to make fibrillin-1 protein. Many of the mutations that result in Marfan syndrome cause a decrease in the quantity and quality of fibrillin-1 that is deposited in the connective tissue matrix outside of the cell. Immunohistochemical analysis seeks to discriminate Marfan syndrome from other disorders by examining the amount of fibrillin-1 protein that is deposited outside of cultured skin cells using a labeled (glowing) antibody that specifically recognizes fibrillin-1. In another technique (pulse-chase analysis), the fibrillin-1 protein made by cultured skin cells is allowed to incorporate a radioactive label. Subsequent analysis of the cells can detect abnormalities of protein production, secretion from the cell and incorporation into the matrix. Both methods require a skin biopsy, with the analysis performed in a specialized laboratory that has expertise in test execution and interpretation. Both methods can yield either a false-positive or false-negative result in certain situations. Currently, protein-based molecular testing for Marfan syndrome is only offered on a research basis.
Marfan syndrome is a clinical diagnosis based on family history and the observation of specific physical manifestations. In selected circumstances, molecular testing can serve as a useful adjunct to comprehensive clinical evaluation for the diagnosis of Marfan syndrome or selected related disorders. Current methods are best used to determine if an individual or fetus carries a genetic predisposition to develop the condition present in family members.
There is no "Marfan test" that can be used to independently establish or exclude the diagnosis of Marfan syndrome. Improperly applied or interpreted molecular tests can be misleading and even dangerous. For example, an individual with nonspecific connective tissue findings and absent or borderline aortic enlargement might equate the failure to find a fibrillin-1 gene mutation with a clean bill of health. In fact, this individual may have Marfan syndrome with a missed fibrillin-1 gene mutation or may have another disorder that predisposes to aortic enlargement and tear that is unrelated to mutations in the fibrillin-1 gene. In either case, the absence of clinical follow-up would be life-threatening.
In the vast majority of people with true Marfan syndrome the diagnosis can be established by clinical evaluation alone. All individual manifestations of Marfan syndrome can be adequately treated in the absence of a specific diagnosis. The same treatment regimens apply regardless of the diagnosis. There are no "hidden" manifestations of Marfan syndrome that cannot be recognized and addressed after a comprehensive clinical evaluation including routine and noninvasive diagnostic tests. Currently, molecular diagnostic testing cannot exclude the presence of Marfan syndrome or another serious connective tissue disorder, offer definitive information regarding outcome, or alter treatment of the physical problems that brought the individual to clinical attention. The rational and accurate application of these methods will require individualized assessment and counseling by individuals who are familiar with Marfan syndrome and are aware of the uses and limitations of molecular diagnosis.
Laboratories that provide molecular testing for Marfan syndrome as a clinical service are listed on GeneTests at: http://www.genetests.org. Select the "Laboratory Directory" and search for Marfan syndrome. Click on "Testing" to get a list of laboratories and the services they provide.