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Genetic factors and phenotypic variability in Marfan syndrome and abdominal aortic aneurysm Genetic factors play an important role in the development of many diseases. Genetic diseases are classified as polygenic or monogenic. Polygenic diseases are caused by interactions between a number of genes and the environment and do not follow a predictable pattern of inheritance although they are clustered in families. Monogenic diseases are caused by a single gene and influences of the environment are usually less important. Since the genetic factors involved in producing phenotypic variability in apparently monogenic familial diseases may well be functioning in polygenic diseases, the identification of these genetic factors is an important way to understand both monogenic and polygenic diseases. This thesis presents a study of two diseases affecting the structure of the aorta, one monogenic (Marfan syndrome) and one polygenic (abdominal aortic aneurysm). Both are likely to involve abnormalities of proteins of the extracellular matrix which provide strength and elasticity to the aortic wall. Marfan syndrome (MFS) is inherited in an autosomal dominant fashion with a prevalence of 1 in 10,000 individuals. The manifestations of MFS appear in many parts of the body, most severely in the cardiovascular system (aortic root dilatation and dissection), the skeletal system (severe chest deformities, long arms, legs and fingers and scoliosis, elbow contracture) and the ocular system (ectopia lentis). MFS was mapped to chromosomal location 15q21.1 in the fibrillin 1 (FBNl) gene. This gene is about 110 kb long and contains 65 exons. It encodes a protein fibrillin, which is a major component of 10-nm microfibrils and is thought to provide a scaffold for attachment of elastin. The protein is made up of domains with homology to calcium-binding or noncalcium- binding epidermal growth factor domains (cbEGF or ncbEGF) and transforming growth factor protein (TGF To date, all families with classical MFS have been linked to this gene (total worldwide LCD score >200) and 137 FBN1 mutations have been reported. One hundred and two of these are located in the cbEGF domains and eight are in the ncbEGF domains. The most common type of mutation is a substitution of one of the six cysteines which are important for inter-cysteine binding in EGF-like domains. In the first part of this thesis a screen of 39 unrelated individuals with varying manifestations of MFS was undertaken, to identify exons which might contain a disease-causing mutation. Twenty-four exons (about one third of the fibrillin gene) were screened and single strand conformation (SSC) variants were found in nine cases. None of these variants was seen in DNA from 30 healthy controls. The FBNl mutation identified at nucleotide 1622 in exon 13 in a typical Marfan patient results in the substitution of phenylalanine for cysteine at codon 541 (C541F). This affects the second cysteine of a cbEGF domain. The exon 13 mutation was not found in the clinically normal parents, suggesting the mutation was sporadic. This finding supports the view that about 25% subjects with MFS do not have a family history of the condition. The mutation described for exon 18 occurs in one of the cbEGF domains and involves an A to G transition at base 2261, resulting in substitution of cysteine for a tyrosine at amino acid residue 754 (Y754C). It has not been reported previously, but is likely to have major effect since it leads to an uneven number of cysteines. In this large family, 25 members have subluxated lenses although only 21 fit the revised criteria for Marfan syndrome. There is considerable variability in organ involvement among those carrying the SSC variant or the linked microsatellite allele, indicating that the Y754C mutation alone does not accoimt for the range of disease manifestations. It seems that fibrillin genotype is not the sole determinant of the clinical phenotype. SSC variation was detected in exon 25 for a patient with a severe juvenile of form MFS. This SSC variant was confirmed to be a G to A transition at base 3131, resulting in substitution of cysteine with tyrosine at amino acid residue 1044 (C1044Y). Again, this mutation leads to an uneven number of cysteines in a cbEGF domain, allowing formation of illegitimate sulphydryl bonds. The mutation is unique to this family, in which the parents are clinically normal. This variant is located in a region of the FBNl gene where mutations for neonatal MFS are clustered. The severe phenotype of this patient associated with a mutation in this region suggests a critical fimction for these domains of the fibrillin protein. Exon 25 is the first of a sequence of 12 contiguous cbEGF domains, the longest such sequence in the fibrillin molecule. Mutations in this region may be disruptive to microfibril formation since this region may have a unique flinction in the polymerization of the protein into stable microfibrils. A deletion of one of the TTTTA repeats in the intron 28 region of FBN 1 was detected in two different MFS families. This mutation does not involve intron/exon boundaries and no RNA splicing site is created by this variant. It is likely that the mutation does not produce any change in the protein structure. In one family, the variant was only detected in affected individuals, indicating that it is linked with the disease in this family. This variant was not found in the 30 healthy controls, indicating that the frequency in the Australian population is quite low. Further patients and controls need to be studied to evaluate the role of this variant in MFS. Aortic rupture is a significant problem for subjects with abdominal aortic aneurysm (AAA). Patients with AAA are also at risk for the development of aneurysms in other parts of the aorta and other arteries. Early detection of AAA is very important in reducing morbidity and mortality. AAA has a strong familial tendency, suggesting that genetic factors must play a role in its aetiology. Even though considerable progress toward understanding the cellular and biochemical events leading to aortic dilatation and rupture has been made, there has been relatively little progress in understanding the genetic factors underlying the pathogenesis of aortic aneurysms. The role of four candidate genes, coding for proteins of the extracellular matrix, in the development of AAA was investigated. These proteins provide strength and elasticity to the aorta. The role of the FBNl gene in aortic dilatation and dissection in MFS is conclusive and it was proposed for a role in AAA. Other candidate genes for AAA code for collagens and elastin. Several types of collagen have been isolated from blood vessels. Collagens type I and III are the major components in the arterial wall. In the second part of this thesis, 308 AAA patients and 935 siblings were recruited. Healthy controls (55 male and 55 female), age = 65 8 (mean SD) and neonatal controls (40 males and 40 females) were also identified. The prevalence of hypertension, cigarette smoking, chronic obstructive lung disease, elevated cholesterol and diabetes in AAA subjects was evaluated. For the siblings of AAA patients, 58% (539) were still alive and 42% (396) were dead. Fourteen of the dead siblings had died from aortic rupture. Fourteen of the siblings who were alive had aneurysms with 10 previously having had a surgical repair. Seventy-four siblings of subjects with AAA were able to be screened. Four had mild aortic dilation and six had moderate to severe aortic dilatation. There were 38 affected siblings from 29 families plus 21 individuals whose mother (8) or father (13) had AAA but there was no known sibling affected. Thus fifty (17%) of the index cases had at least one affected first degree relative. These clinical results suggest that AAA is a familial condition and a positive family history may be a significant risk factor in the development of AAA. The importance of screening the first degree relatives of AAA patients was confirmed by this study. Genes encoding the alpha 2 chain of collagen type I and the alpha 1 chain of collagen type III (C0L1A2 and C0L3A1 respectively) as well as those for elastin and fibrillin were investigated to evaluate their role in the development of AAA. Microsatellite alleles were scored in the 308 index cases. A significant difference was detected in the frequency of the COL1A2 gene allele 3 between female patients and male patients and between female patients and female controls. The results suggest that COLl A2 allele 3 may protect female subjects from developing AAA. More female subjects need to be collected to verify this hypothesis. No difference was found between AAA patients and controls for the C0L3A1, fibrillin and elastin genes, suggesting that these genes are unlikely to have a significant role in the development of AAA. A comparison was made between normotensive and hypertensive AAA patients in the fibrillin gene and no difference was found, unlike a previous study. The relatively fewer female subjects compared to male subjects with AAA and the modest number of siblings of subjects with AAA who were able to be screened, limit the conclusions which can be drawn from the study. In particular confirmation of a protective role for the COLl A2 gene allele 3 for females will form the basis of future work. Overall the pathogenesis of AAA is likely to be multifactorial. The high prevalence of risk factors for atherosclerosis in subjects with AAA does not enable further classification of the relationship between atherosclerosis arterial disease and AAA. The results of the first and second parts of this thesis do not support the suggestion that monogenic aortic aneurysm disease provides simple pointers to genetic factors that might be involved in the polygenic condition of AAA. Although both FBNl and C0L3AI have been implicated in monogenic disease of the aorta these genes do not appear to be significant contributors to common AAA. The important genes involved in the development of AAA remain unidentified. The completion of the Human Genome Project will assist in identifying further candidate genes and enhance the understanding of genetic factors in AAA and other polygenic conditions.