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Permanent URL to this publication: http://dx.doi.org/10.5167/uzh-10291

Matyas, G. Molecular bases of Marfan syndrome and related disorders. 2008, University of Zurich, Faculty of Medicine.

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Monogenic disorders are direct results of pathogenic mutations in one of the about 25,000 genes estimated in the human genome. In such cases, molecular genetic analysis of a single gene has the power to determine whether or not a person is affected by a disorder. However, one of the most difficult problems ahead is to discover genes and mutations that underlie monogenic disorders and to find out how singlegene defects contribute to clinical phenotypes. This work exemplifies molecular genetic testing for monogenic disorders by focusing on Marfan syndrome. Mutations in the human fibrillin-1 gene (FBN1) cause Marfan syndrome (MFS), an autosomal dominant connective tissue disorder. The knowledge about FBN1 mutations is important for early diagnosis, management, and genetic counseling. However, mutation detection in FBN1 is a challenge because the gene is very large in size and the mutations detected so far are scattered over all 65 exons. Conventional methods for large-scale detection of mutations are expensive, technically demanding, or time consuming. Recently, a high-capacity low-cost mutation detection method was introduced based on denaturing high-performance liquid chromatography (DHPLC). To assess the sensitivity and specificity of this method, we blindly screened 64 DNA samples of known FBN1 genotype exon-by-exon using exon-specific DHPLC conditions. Analysis of 682 PCR amplicons correctly identified 62 out of 64 known sequence variants. In three MFS patients of unknown FBN1 genotype, we detected two mutations and eight polymorphisms. In total, 20 mutations and two polymorphisms are described here for the first time. Our results demonstrate 1) that DHPLC is a highly sensitive (89-99%, P = 0.05) method for FBN1 mutation detection; but 2) that chromatograms with moderate and weak pattern abnormalities also show false positive signals (45-59%, P = 0.05); 3) that the difference in the chromatograms of heterozygous and homozygous amplicons is mostly independent of the type of sequence change; and 4) that DHPLC column conditions, additional base changes, and the amounts of injected PCR products influence significantly the shape of chromatograms. A strategy for FBN1 mutation screening is discussed. (Paper 1; Matyas et al. 2002a, Hum Mutat 19:443-456.) Current molecular genetic testing of FBN1 may miss mutations in the promoter region or in other noncoding sequences as well as partial or complete gene deletions and duplications. In Paper 2, we tested for copy number variations by successively applying multiplex ligation-dependent probe amplification (MLPA) and the Affymetrix Human Mapping 500 K Array Set, which contains probes for ~500,000 single-nucleotide polymorphisms (SNPs) across the genome. By analyzing genomic DNA of 101 unrelated individuals with MFS or related phenotypes, we identified FBN1 deletions in two patients with MFS. Our high-resolution approach narrowed down the deletion breakpoints. Subsequent sequencing of the junctional fragments revealed the deletion sizes of 26,887 and 302,580 bp, respectively. Surprisingly, both deletions affect the putative regulatory and promoter region of the FBN1 gene, strongly indicating that they abolish transcription of the deleted allele. This expectation of complete loss of function of one allele, i.e. true haploinsufficiency, was confirmed by transcript analyses. Our findings not only emphasize the importance of screening for large genomic rearrangements in comprehensive genetic testing of FBN1 but, importantly, also extend the molecular etiology of MFS by providing hitherto unreported evidence that true haploinsufficiency is sufficient to cause MFS. (Paper 2; Matyas et al. 2007, Hum Genet 122:23-32.) Very recently, heterozygous mutations in the genes encoding transforming growth factor beta receptors I (TGFBR1) and II (TGFBR2) have been reported in Loeys-Dietz aortic aneurysm syndrome (LDS). In addition, dominant TGFBR2 mutations have been identified in Marfan syndrome type 2 (MFS2) and familial thoracic aortic aneurysms and dissections (TAAD). In the past, mutations of these genes were associated with atherosclerosis and several human cancers. In Paper 3, we report a total of nine novel and one known heterozygous sequence variants in the TGFBR1 and TGFBR2 genes in nine of 70 unrelated individuals with MFS-like phenotypes who previously tested negative for mutations in the gene encoding the extracellular matrix protein fibrillin-1 (FBN1). To assess the pathogenic impact of these sequence variants, in silico analyses were performed by the PolyPhen, SIFT, and Fold-X algorithms and by means of a 3D homology model of the TGFBR2 kinase domain. Our results showed that in all but one of the patients the pathogenic effect of at least one sequence variant is highly probable. These deleterious alleles occurred de novo or segregated with the disease in the families, indicating a causative association between the sequence variants and clinical phenotypes. Since TGFBR2 mutations found in patients with MFS-related disorders cannot be distinguished from heterozygous TGFBR2 mutations reported in tumor samples, we emphasize the importance of segregation analysis in affected families. In order to be able to find the mutation that is indeed responsible for a MFSrelated phenotype, we also recommend that genetic testing for sequence alterations in TGFBR1 and TGFBR2 should be complemented by mutation screening of the FBN1 gene. (Paper 3; Matyas et al. 2006, Hum Mutat 27:760–769.) In Paper 4, we present a novel method for accurate quantification of single nucleotide polymorphism (SNP) variants in transcripts and pooled DNAs in a one-tube reaction. Our approach is based on single-nucleotide primer extension (SNuPE) and laserinduced fluorescence capillary electrophoresis (LIF-CE), and takes advantage of distinct mobilities of SNuPE products with different nucleotides incorporated at their 3’ ends. The method, called SNuPE-ONCE, was tested on two polymorphisms and five mutations that comprised the three most frequent (~70%) nucleotide changes in the human genome (C/T, A/G, and A/T). The usefulness of the method was demonstrated by analyzing nonsense-mediated mRNA instability in fibroblasts. Our data show 1) that the method provides highly reproducible relative allele frequencies (SD<0.017) with a good accuracy (e.g. for heterozygotes 0.500 �} 0.036, P = 0.01), depending on the sequence and the proportion of the SNP variants in the sample, and 2) that relative allele frequencies as low as 1% can be detected quantitatively and unambiguously. Our assay relies on a CE instrument available in many laboratories and offers a useful method for quantitative SNP genotyping as well as for a variety of expression studies. (Paper 4; Matyas et al. 2002b, Hum Mutat 19:58-68.) Taken together, mutation screening strategies and methods presented here are applicable and suitable for the most monogenic disorders, independently of the mode of their inheritance. In special cases, such as imprinting diseases and trinucleotide repeat expansion disorders, however, alternative methods, such as methylation sensitive assays and/or Southern blot, are needed to detect the underlying diseasecausing mutation.


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Item Type:Habilitation
Communities & Collections:04 Faculty of Medicine > Institute of Medical Molecular Genetics
Dewey Decimal Classification:570 Life sciences; biology
610 Medicine & health
Deposited On:20 Jan 2009 13:39
Last Modified:12 Apr 2012 03:21
Number of Pages:66

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