Identification of SPRED1 deletions using RT‐PCR, multiplex ligation‐dependent probe amplification and quantitative PCR

Abstract

Legius syndrome, is a recently identified autosomal dominant disorder caused by loss of function mutations in the SPRED1 gene, with individuals mainly presenting with multiple café-au-lait macules (CALM), freckling and macrocephaly. So far, only SPRED1 point mutations have been identified as the cause of this syndrome. To determine if copy number changes (CNCs) are a cause of Legius syndrome, we have used a Multiplex Ligation-dependent Probe Amplification (MLPA) assay covering all SPRED1 exons in a cohort of 510 NF1-negative patients presenting with multiple CALMs with or without freckling, but no other NF1 diagnostic signs. Four different deletions were identified by MLPA and confirmed by quantitative PCR, reverse transcriptase PCR and/or array CGH: a deletion of exon 1 and the SPRED1 promoter region in a proband and two first-degree relatives; a deletion of the entire SPRED1 gene in a sporadic patient; a deletion of exon 2-6 in a proband and her father; and an ∼6.6 Mb deletion on chromosome 15 that spans SPRED1 in a sporadic patient. Deletions account for ∼10% of the 40 detected SPRED1 mutations in this cohort of 510 individuals. These results indicate the need for dosage analysis to complement sequencing-based SPRED1 mutation analyses.

A shared molecular mechanism underlies the human rasopathies Legius syndrome and Neurofibromatosis-1

“…The majority of reported Spred1 mutations produce a premature stop codon, resulting in C-terminally truncated protein products (Brems et al 2007;Messiaen et al 2009;Pasmant et al 2009;Spurlock et al 2009;Muram-Zborovski et al 2010;Denayer et al 2011;Laycock-van Spyk et al 2011;Spencer et al 2011). As the SPR domain has previously been reported to target Spred1 to the plasma membrane via interactions with phospholipids and caveolin-1 (Lim et al 2002;Nonami et al 2005), we surmised that the truncated mutants were unable to function as a result of mislocalization.…”

“…Thus far, ;12 missense mutations in SPRED1 have been characterized as pathogenic in Legius syndrome (11 point mutations and one four-amino-acid in-frame deletion), five of which are used in this study (Supplemental Table S1; Brems et al 2007;Messiaen et al 2009;Pasmant et al 2009;Spurlock et al 2009;Denayer et al 2011;Spencer et al 2011). Of these 12 missense mutations, nine localize to the EVH1 domain.…”

“…Initial experiments revealed that Spred1 mutations were loss-of-function mutations, incapable of inhibiting the Ras/MAPK pathway (Brems et al 2007). SPRED1 mutations account for at least 2% of the pathogenic mutations associated with patients clinically diagnosed with NF1 (Brems et al 2007;Messiaen et al 2009;Pasmant et al 2009;Spurlock et al 2009;Muram-Zborovski et al 2010;Denayer et al 2011;Laycock-van Spyk et al 2011;Spencer et al 2011).…”

The SPRED1 Variants Repository for Legius Syndrome

“…This database was developed to serve as a reference for all sequence variations currently reported in the SPRED1 gene as well as to provide as much phenotype information as possible (Calderon et al 2007; Crockett et al 2010; Margraf et al 2009; Wooderchak et al 2010). To date, 78 variants have been described in the SPRED1 gene, of which 49 have been classified as disease causing or pathogenic, 1 as suspected pathogenic, 7 as suspected benign, 8 as benign polymorphisms, and 13 with uncertain significance (Brems et al 2007; Denayer et al 2011; Messiaen et al 2009; Muram-Zborovski et al 2010; Pasmant et al 2009; Spencer et al 2011; Spurlock et al 2009). …”

Jaffe–Campanacci syndrome, revisited: detailed clinical and molecular analyses determine whether patients have neurofibromatosis type 1, coincidental manifestations, or a distinct disorder

“…All NF1 mutation-negative patients (except for KUL-22) with JCS and/or NOF also underwent comprehensive SPRED1 genetic testing, as previously described. 8,18 In addition, direct sequencing of GNAS1 exon 8 was performed after amplification using two pairs of primers: 8-1f: 5′-ggactctgag ccctctttcc-3′, 8-1r: 5′-ggactggggtgaatgtcaag-3′ and 8-2f: 5′-gagcg atcaggtgtgcaaaa-3′, 8-2r: 5′-cagagggactggggtgaatg-3′. In addition, clinical and genetic data on five individuals diagnosed with JCS at the Center for Human Genetics from the University Hospitals Leuven (KUL) was reviewed and included.…”