Intended purpose
The SALSA MLPA probemixes P065 Marfan Syndrome-1 and P066 Marfan Syndrome-2 are in vitro diagnostic (IVD)
1 or research use only (RUO) semi-quantitative assays
2 for the detection of deletions or duplications in the
FBN1 gene, in order to confirm a potential cause for and clinical diagnosis of Marfan syndrome and other
FBN1-related disorders. P065 Marfan Syndrome-1 can also be used for the detection of deletions or duplications in the
TGFBR2 gene, in order to confirm a potential cause for and clinical diagnosis of
TGFBR2-related disorders. Both assays are for use with genomic DNA isolated from human peripheral whole blood specimens and are also intended for molecular genetic testing of at-risk family members.
The detection of copy number variations (CNVs) in
FBN1 requires the use of both P065 Marfan Syndrome-1 and P066 Marfan Syndrome-2. CNVs detected with P065 Marfan Syndrome-1 and P066 Marfan Syndrome-2 should be confirmed with a different technique. In particular, CNVs detected by only a single probe always require confirmation by another method. Most defects in the
FBN1 and
TGFBR2 genes are point mutations, none of which will be detected by MLPA. It is therefore recommended to use this assay in combination with sequence analysis.
Assay results are intended to be used in conjunction with other clinical and diagnostic findings, consistent with professional standards of practice, including confirmation by alternative methods, clinical genetic evaluation, and counselling, as appropriate. The results of this test should be interpreted by a clinical molecular geneticist or equivalent.
These devices are not intended to be used for standalone diagnostic purposes, pre-implantation or prenatal testing, population screening, or for the detection of, or screening for, acquired or somatic genetic aberrations.
1 Please note that these probemixes are for in vitro diagnostic (IVD) use in the countries specified at the end of this product description. In all other countries, the products are for research use only (RUO).
2 To be used in combination with a SALSA MLPA Reagent Kit and Coffalyser.Net analysis software.
Clinical background
Marfan syndrome is a systemic disorder of connective tissue that mainly affects the ocular, skeletal and cardiovascular systems. It has a penetrance of 100%, but a high degree of clinical variability, with phenotypes ranging from isolated features of Marfan syndrome to neonatal presentation of severe and progressive disease in multiple organ systems. The major causes of morbidity and mortality relate to the cardiovascular system. However, if proper management is executed, the life expectancy of a patient with Marfan syndrome approximates that of the general population. Marfan syndrome is an autosomal dominant disease with a prevalence of 1:5,000 - 1:10,000 that is caused by mutations in the
FBN1 gene (Dietz et al. 1991; Lee et al. 1991). In ~90-93% of patients diagnosed with Marfan syndrome a mutation is detected by sequencing of the
FBN1 coding region and flanking intronic regions. In ~5% of the patients, the pathogenic variant identified is a large deletion or duplication (Baetens et al. 2011; Hilhorst-Hofstee et al. 2011; Mannucci et al. 2020; Rand-Hendriksen et al. 2007; Stengl et al. 2020). In a small percentage of patients (~2-5%), no mutation in
FBN1 is identified. More information is available at
https://www.ncbi.nlm.nih.gov/books/NBK1335/.
Several Marfan-related disorders have been recognized that display a specific subset of the phenotypic features found in Marfan syndrome and that can also be caused by mutations in the
FBN1 gene. These
FBN1-related disorders include MASS phenotype (
mitral valve,
aortic,
skin and
skeletal features), ectopia lentis syndrome and familial
thoracic
aortic
aneurysm and
dissection (familial TAAD). In addition, there are also Marfan-related disorders that are caused by mutations in
TGFBR2. These
TGFBR2-related disorders include Loeys-Dietz syndrome and familial TAAD. Loeys-Dietz syndrome is a systemic disorder of connective tissue that has a large overlap in clinical features with Marfan syndrome. The most common clinical features involve the vascular, skeletal, craniofacial, cutaneous, allergic/inflammatory and ocular systems. In ~55-60% of patients diagnosed with Loeys-Dietz syndrome a pathogenic variant in
TGFBR2 is identified. Sequence analysis detects ~100% of these pathogenic variants. Large rearrangements of
TGFBR2 have thus far not been reported in patients with Loeys-Dietz syndrome features. There is one report of a deletion encompassing the entire
TGFBR2 gene, but this individual thus far lacked aortic involvement and did not show clear features of Loeys-Dietz syndrome (Campbell et al. 2011). More information about Loeys-Dietz syndrome is available at
https://www.ncbi.nlm.nih.gov/books/NBK1133/.
Probemix content
The SALSA MLPA Probemix P065-C1 Marfan Syndrome-1 contains 52 MLPA probes with amplification products between 130 and 504 nucleotides (nt). The SALSA MLPA Probemix P066-C1 Marfan Syndrome-2 contains 49 MLPA probes with amplification products between 130 and 490 nt.
The P065-C1 and P066-C1 probemixes contain 34 probes and 36 probes for the
FBN1 gene, respectively. Together, these probemixes cover each exon of
FBN1 by at least one probe. Exon 2, 65 and 66 are covered by two probes and one probe upstream of
FBN1 is present. In addition, the P065-C1 probemix contains nine probes for the
TGFBR2 gene. Each exon of
TGFBR2 is covered by one probe, with the exception of exon 1, which is covered by two probes. The P065-C1 and P066-C1 probemixes contain nine and thirteen reference probes, respectively. These reference probes detect autosomal chromosomal locations. Complete probe sequences and the identity of the genes detected by the reference probes are available online (
www.mrcholland.com).
These probemixes contains nine quality control fragments generating amplification products between 64 and 105 nt: four DNA Quantity fragments (Q-fragments), two DNA Denaturation fragments (D-fragments), one Benchmark fragment, and one chromosome X and one chromosome Y-specific fragment. More information on how to interpret observations on these control fragments can be found in the MLPA General Protocol and online at
www.mrcholland.com.