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

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.