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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
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


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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:05 Apr 2016 12:49
Number of Pages:66

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